PD-1/LAG3 BI-SPECIFIC ANTIBODIES, COMPOSITIONS THEREOF, AND METHODS OF MAKING AND USING THE SAME

Abstract

Provided herein are antibodies that selectively bind to LAG3 and its isoforms and homologs, and compositions comprising the antibodies. Also provided herein are antibodies that selectively bind to PD-1 and its isoforms and homologs, and compositions comprising the antibodies. In addition, provided herein are bi-specific antibodies and antigen binding constructs that selectively bind to LAG3 and/or PD-1, their isoforms and homologs, and compositions comprising the antibodies and antigen binding constructs. Also provided are methods of using the antibodies, such as therapeutic and diagnostic methods.

Description

FIELD

Provided herein are antibodies with dual binding specificity for lymphocyte-activation gene 3 (LAG3) and for programmed cell death protein (PD-1), also referred to as PD-1/LAG3 bi-specific antibodies. Also provided herein are antibodies with binding specificity for PD-1 or LAG3. In addition, provided herein are compositions comprising the antibodies, including pharmaceutical compositions, diagnostic compositions, and kits. Also provided are methods of making the bi-specific antibodies, and methods of using the bi-specific antibodies, for example, for therapeutic purposes, diagnostic purposes, and research purposes.

BACKGROUND

The lymphocyte activation gene 3 (LAG3) was discovered in 1990. Triebel et al., 1990, J. Exp. Med. 171:1393-4053. It was identified as selectively transcribed in activated natural killer (NK) cells and T lymphocytes. See id. The LAG3 protein was originally described as a type I membrane protein of 498 amino acids including a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. See id. The extracellular region has four Ig domains, and the whole protein has sequence similarity to CD4. See id.

LAG3 is selectively expressed in regulatory T cells, and its natural ligand is MHC class II. Huang et al., 2004, Immunity 21:503-513. Regulatory T cells are important for maintaining immune tolerance to limit autoimmunity and in regulating lymphocyte expansion. See id. They also suppress natural immune responses to parasites and viruses, and they have suppressed antitumor immunity induced by therapeutic vaccines. See id. Antibodies to LAG3 were shown to inhibit suppression by induced regulatory T cells. See id. Antibody targeting of LAG3 has been shown to enhance antitumor immunity in animal models of cancer. Pardoll, 2012, Nature Rev. Cancer 12:252-264; Jing et al., 2015, J. Immunother. Cancer 3:2-29. LAG3 is an immune checkpoint protein target for active drug development, and clinical trials have been proposed for antibodies to LAG3 for the treatment of solid tumors.

Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface protein molecule that belongs to the immunoglobulin superfamily. It is expressed on T and B lymphocytes and macrophages, and plays a role in cell fate and differentiation. See Ishida et al., EMBO J., 1992, 11:3887-3895, incorporated by reference in its entirety. Activation of PD-1 is thought to negatively regulate the immune response. See Blank et al., Cancer Immunol. Immunother., 2007, 56:739-745; and Freeman et al., J. Exp. Med., 2000, 192:1027-1034, each of which is incorporated by reference in its entirety.

PD-1 has two known ligands, PD-L1 and PD-L2, which are both members of the B7 family. See Freeman et al., supra; and Latchman et al., Nat. Immunol., 2001, 2:261-268, each of which is incorporated by reference in its entirety. The interaction between PD-1 and these ligands is thought to play a role in a variety of diseases, including cancer (see Ribas and Tumeh, Clin. Cancer Res., 2014, Jun. 26, PMID: 24970841), autoimmune disease (see Dai et al., Cell Immunol., 2014, 290:72-79), and infection (see Day et al., Nature, 2006, 443:350-354). Each of the references cited in the preceding sentence is incorporated by reference in its entirety. In particular, the engagement of PD-1 by one of its ligands is thought to inhibit T-cell effector functions in an antigen-specific manner.

In view of the role of PD-1 and LAG3 in multiple disease processes, there is a need for methods of modulating the immune regulation and downstream signaling processes activated by both LAG3 and PD-1. There is also a need for therapeutics that can specifically target cells and tissues that express LAG3 and/or PD-1.

SUMMARY

Provided herein are antibodies that selectively bind LAG3. In some embodiments, the antibodies bind human LAG3. In some embodiments, the antibodies also bind homologs of human LAG3. In some aspects, the homologs include a cynomolgus monkey homolog.

Also provided herein are antibodies that selectively bind PD-1. In some embodiments, the antibodies bind human PD-1. In some embodiments, the antibodies also bind homologs of human PD-1. In some aspects, the homologs include a cynomolgus monkey homolog.

Also provided herein are bi-specific antibodies or bi-specific antibody constructs that comprise a first binding domain that selectively binds LAG3, including human LAG3 or a homolog thereof, and a second binding domain that selectively binds PD-1, including human PD-1 or a homolog thereof.

In some embodiments, the antibodies comprise at least one CDR sequence defined by a consensus sequence provided in this disclosure. In some embodiments, the antibodies comprise an illustrative CDR, V_H, or V_L sequence provided in this disclosure, or a variant thereof. In some aspects, the variant is a variant with one or more conservative amino acid substitutions.

Also provided are compositions and kits comprising the antibodies. In some embodiments, the compositions are pharmaceutical compositions. Any suitable pharmaceutical composition may be used. In some embodiments, the pharmaceutical composition is a composition for parenteral administration.

This disclosure also provides methods of using the anti-LAG3 antibodies provided herein. In some embodiments, the method is a method of treatment. In some embodiments, the method is a diagnostic method. In some embodiments, the method is an analytical method. In some embodiments, the method is a method of purifying and/or quantifying LAG3.

In some embodiments, the antibodies are used to treat a disease or condition. In some aspects, the disease or condition is selected from a cancer, autoimmune disease, and infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a comparison of the Kabat and Chothia numbering systems for CDR-H1. Adapted from Martin A.C.R. (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains. In R. Kontermann & S. Dübel (Eds.), Antibody Engineering vol. 2 (pp. 33-51). Springer-Verlag, Berlin Heidelberg.

FIG. 2(A, B) provides an alignment of the V_H sequences provided herein. CDRs according to Chothia are outlined, and CDRs according to Kabat are underlined.

FIG. 3 provides an alignment of the V_L sequences provided herein. CDRs according to Chothia are outlined, and CDRs according to Kabat are underlined.

FIG. 4 is a graph illustrating the mean tumor volume for different antibody combinations tested in the MC38 murine tumor model to compare tumor growth control.

FIG. 5 includes two semi-logarithmic graphs that illustrate individual and mean serum concentration-time profiles in a cynomolgous PK study of a PD-1/LAG3 bi-specific antibody.

FIG. 6 includes two semi-logarithmic graphs that illustrate serum concentration-time profiles in a cynomolgous PK study of additional PD-1/LAG3 bi-specific antibodies.

FIG. 7 includes two graphs that illustrate immunogenicity assessments for the additional PD-1/LAG3 bi-specific antibodies in individual cynomolgous monkeys.

FIG. 8 includes two graphs and a table that provide a comparison of the cell binding affinities of three separate PD-1/LAG3 bi-specific antibodies for human PD-1 and cynomolgous PD-1.

FIG. 9 includes two graphs and a table that provide a comparison of the cell binding affinities of three separate PD-1/LAG3 bi-specific antibodies for human LAG3 and cynomolgous LAG3.

FIG. 10 includes two graphs and a table that provide a comparison of the ability of three separate PD-1/LAG3 bi-specific antibodies to inhibit binding between human PD-1 and PD-L1 or PD-L2.

FIG. 11 is a graph and table that provide a comparison of the ability of three separate PD-1/LAG3 bi-specific antibodies to inhibit binding between human LAG3 and MHC-class II.

FIG. 12 is a graph and table that provide a comparison of the ability of three separate PD-1/LAG3 bi-specific antibodies to inhibit binding to both human PD-1 and human LAG3 co-expressed on U2OS engineered cells.

FIG. 13 is a graph that provides a comparison between a mono-specific (“PD-1 stump”) control and three separate PD-1/LAG3 bi-specific antibodies in a PBMC CMV-antigen recall assay.

DETAILED DESCRIPTION

1. Definitions

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.

The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value±10%, ±5%, or ±1%. In certain embodiments, the term “about” indicates the designated value±one standard deviation of that value.

The terms “first” and “second” are intended to indicate two separate entities, but does not mean that one is before the other in time or space, unless otherwise noted.

The term “combinations thereof” includes every possible combination of elements to which the term refers to. For example, a sentence stating that “if α₂ is A, then α₃ is not D; α₅ is not S; or α₆ is not S; or combinations thereof” includes the following combinations when α₂ is A: (1) α₃ is not D; (2) α₅ is not S; (3) α₆ is not S; (4) α₃ is not D; α₅ is not S; and α₆ is not S; (5) α₃ is not D and as is not S; (6) α₃ is not D and α₆ is not S; and (7) α₅ is not S and α6 is not S.

The terms “LAG3” and “LAG3 antigen” are used interchangeably herein. LAG3 is also known by a variety of synonyms, including lymphocyte-activation gene 3, CD223, cluster of differenetiation 223, and FDC, among others. Unless specified otherwise, the terms include any variants, isoforms and species homologs of human LAG3 that are naturally expressed by cells, or that are expressed by cells transfected with an LAG3 gene. LAG3 proteins include, for example, human LAG3 (GI: 15928632; SEQ ID NO: 1). In some embodiments, LAG3 proteins include cynomolgus monkey LAG3 (GI: 544483249; SEQ ID NO: 2). In some embodiments, LAG3 proteins include murine LAG3 (GI: 112293275; SEQ ID NO: 3). However, as discussed in detail elsewhere in this disclosure, in some embodiments the antibodies provided herein do not bind murine LAG3 proteins. The antibodies provided herein bind to an extracellular domain of LAG3.

The terms “PD-1” and “PD-1 antigen” are used interchangeably herein. Unless specified otherwise, the terms include any variants, isoforms and species homologs of human PD-1 that are naturally expressed by cells, or that are expressed by cells transfected with a PD-1 gene. PD-1 proteins include full-length PD-1 (e.g., human PD-1; GI: 167857792; SEQ ID NO: 318; extracellular domain: Pro21-Gln167), as well as alternative splice variants of PD-1, such as PD-1Δex2, PD-1Δex3, PD-1Δex2,3, and PD-1Δex2,3,4. See Nielsen et al., Cellular Immunology, 2005, 235:109-116, incorporated by reference in its entirety. In some embodiments, PD-1 proteins include murine PD-1 (e.g., SEQ ID NO: 319; extracellular domain: Leu25-Gln167). In some embodiments, PD-1 proteins include cynomolgus PD-1 (e.g., SEQ ID NO: 320; extracellular domain: Pro21-Gln167).

The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, Pa. Briefly, each heavy chain typically comprises a heavy chain variable region (V_H) and a heavy chain constant region (C_H). The heavy chain constant region typically comprises three domains, abbreviated C_H1, C_H2, and C_H3. Each light chain typically comprises a light chain variable region (V_L) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated C_L.

The term “antibody” describes a type of immunoglobulin molecule and is used herein in its broadest sense. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), and antibody fragments. Antibodies comprise at least one antigen-binding domain. One example of an antigen-binding domain is an antigen binding domain formed by a V_H-V_L dimer.

A “LAG3 antibody,” “anti-LAG3 antibody,” “LAG3 Ab,” “LAG3-specific antibody” or “anti-LAG3 Ab” is an antibody, as described herein, which binds specifically to the antigen LAG3. In some embodiments, the antibody binds the extracellular domain of LAG3.

A “PD-1 antibody,” “anti-PD-1 antibody,” “PD-1 Ab,” “PD-1-specific antibody” or “anti-PD-1 Ab” is an antibody, as described herein, which binds specifically to the antigen PD-1. In some embodiments, the antibody binds the extracellular domain of PD-1.

The V_H and V_L regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each V_H and V_L generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, Md., incorporated by reference in its entirety.

The light chain from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.

The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme), each of which is incorporated by reference in its entirety.

Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

Unless otherwise specified, the numbering scheme used for identification of a particular CDR herein is the Kabat/Chothia numbering scheme. Where the residues encompassed by these two numbering schemes diverge (e.g., CDR-H1 and/or CDR-H2), the numbering scheme is specified as either Kabat or Chothia. For convenience, CDR-H3 is sometimes referred to herein as either Kabat or Chothia. However, this is not intended to imply differences in sequence where they do not exist, and one of skill in the art can readily confirm whether the sequences are the same or different by examining the sequences.

CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at http://www.bioinf.org.uk/abs/anum/, and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.

TABLE 1
Residues in CDRs according to Kabat
and Chothia numbering schemes.
	CDR	Kabat	Chothia
	L1	L24-L34	L24-L34
	L2	L50-L56	L50-L56
	L3	L89-L97	L89-L97
	H1 (Kabat Numbering)	H31-H35B	H26-H32 or H34*
	H1 (Chothia Numbering)	H31-H35	H26-H32
	H2	H50-H65	H52-H56
	H3	H95-H102	H95-H102
	*The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR, as illustrated in FIG. 1.

The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

An “antibody fragment” comprises a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv (sFv) fragments, and scFv-Fc fragments.

“Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

“Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (C_H1) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.

“F(ab′)₂” fragments contain two Fab′ fragments joined, near the hinge region, by disulfide bonds. F(ab′)₂ fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab′) fragments can be dissociated, for example, by treatment with ß-mercaptoethanol.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a V_H domain and a V_L domain in a single polypeptide chain. The V_H and V_L are generally linked by a peptide linker. See Plückthun A. (1994). In some embodiments, the linker is SEQ ID NO: 168. Antibodies from Escherichia coli. In Rosenberg M. & Moore G. P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.

“scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminus of the scFv. The Fc domain may follow the V_H or V_L, depending on the orientation of the variable domains in the scFv (i.e., V_H-V_L or V_L-V_H). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG1 Fc domain. In some embodiments, the IgG1 Fc domain comprises SEQ ID NO: 300, or a portion thereof, or SEQ ID NO: 306. SEQ ID NO: 300 provides the sequence of C_H1, C_H2, and C_H3 of the human IgG1 constant region. SEQ ID NO: 306 provides the sequence of the constant region used in the illustrative scFv-Fc antibodies provided herein.

The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.

The term “chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

“Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated antibody is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator. In some embodiments, an isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain. An isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment is not present. In some aspects, an isolated antibody is prepared by at least one purification step.

In some embodiments, an isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight. In some embodiments, an isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by volume.

“Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be represented by the dissociation constant (K_D). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology, such as a Biacore® instrument. In some embodiments, the affinity is determined at 25° C.

With regard to the binding of an antibody to a target molecule, the terms “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that mimics the antibody binding site on the target. In that case, specific binding is indicated if the binding of the antibody to the target is competitively inhibited by the control molecule.

The term “k_d” (sec⁻¹), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the k_off value.

The term “k_a” (M⁻¹×sec⁻¹), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the k_on value.

The term “K_D” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. K_D=k_d/k_a.

The term “K_A” (M⁻¹), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction. K_A=k_a/k_d.

An “affinity matured” antibody is one with one or more alterations in one or more CDRs or FRs that result in an improvement in the affinity, or other properties (e.g. biophysical), of the antibody for its antigen, compared to a parent antibody which does not possess the alteration(s). In one embodiment, an affinity matured antibody has nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies may be produced using a variety of methods known in the art. For example, Marks et al. (Bio/Technology, 1992, 10:779-783, incorporated by reference in its entirety) describes affinity maturation by V_H and V_L domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example, Barbas et al. (Proc. Nat. Acad. Sci. U.S.A., 1994, 91:3809-3813); Schier et al., Gene, 1995, 169:147-155; Yelton et al., J. Immunol., 1995, 155:1994-2004; Jackson et al., J. Immunol., 1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226:889-896, each of which is incorporated by reference in its entirety.

When used herein in the context of two or more antibodies, the term “competes with” or “cross-competes with” indicates that the two or more antibodies compete for binding to an antigen. In one exemplary assay, an antigen is coated on a plate and allowed to bind a first antibody, after which a second, labeled antibody is added. If the presence of the first antibody reduces binding of the second antibody, then the antibodies compete. In another exemplary assay, a first antibody is coated on a plate and allowed to bind an antigen, and then the second antibody is added. The term “competes with” also includes combinations of antibodies where one antibody reduces binding of another antibody, but where no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one antibody reduces binding of another antibody to its antigen by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

The term “epitope” means a portion of an antigen capable of specific binding to an antibody. Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and may have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination such as, for example, testing for antibody binding to LAG3 and/or PD-1 variants with different point-mutations, or to chimeric LAG3 and/or PD-1 variants as described further in the Examples provided herein.

Percent “identity” between a polypeptide sequence and a reference sequence, is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” By way of example, the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for one another.

TABLE 2
Selected groups of amino acids that are considered conservative
substitutions for one another, in certain embodiments.
Acidic Residues                D and E
Basic Residues                 K, R, and H
Hydrophilic Uncharged Residues S, T, N, and Q
Aliphatic Uncharged Residues   G, A, V, L, and I
Non-polar Uncharged Residues   C, M, and P
Aromatic Residues              F, Y, and W

TABLE 3
Additional selected groups of amino acids that
are considered conservative substitutions for
one another, in certain embodiments.
Group 1 A, S, and T
Group 2 D and E
Group 3 N and Q
Group 4 R and K
Group 5 I, L, and M
Group 6 F, Y, and W

TABLE 4
Further selected groups of amino acids that are considered conservative
substitutions for one another, in certain embodiments.
Group A A and G
Group B D and E
Group C N and Q
Group D R, K, and H
Group E I, L, M, V
Group F F, Y, and W
Group G S and T
Group H C and M

Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, N.Y. An antibody generated by making one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a “conservatively modified variant.”

The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

“Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder.

As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or composition that when administered to a subject is effective to treat a disease or disorder.

As used herein, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, avians, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a cancer that can be treated or diagnosed with an antibody provided herein. In some embodiments, the cancer is a cancer of epithelial origin.

2. LAG3 Antibodies

Provided herein are antibodies that selectively bind human LAG3. In some aspects, the antibody selectively binds to the extracellular domain of human LAG3.

In some embodiments, the antibody binds to a homolog of human LAG3. In some aspects, the antibody binds to a homolog of human LAG3 from a species selected from monkeys, mice, dogs, cats, rats, cows, horses, goats and sheep. In some aspects, the homolog is a cynomolgus monkey homolog.

In some embodiments, the LAG3 antibody has one or more CDRs having particular lengths, in terms of the number of amino acid residues. In some embodiments, the Chothia CDR-H1 of the antibody is 6, 7, or 8 residues in length. In some embodiments, the Kabat CDR-H1 of the antibody is 4, 5, or 6 residues in length. In some embodiments, the Chothia CDR-H2 of the antibody is 5, 6, or 7 residues in length. In some embodiments, the Kabat CDR-H2 of the antibody is 16, 17, or 18 residues in length. In some embodiments, the Kabat/Chothia CDR-H3 of the antibody is 9, 10, 11, 12, or 13 residues in length.

In some aspects, the Kabat/Chothia CDR-L1 of the antibody is 11, 12, 13, 14, 15, 16, 17, or 18 residues in length. In some aspects, the Kabat/Chothia CDR-L2 of the antibody is 6, 7, or 8 residues in length. In some aspects, the Kabat/Chothia CDR-L3 of the antibody is 8, 9, or 10 residues in length.

In some embodiments, the LAG3 antibody comprises a light chain. In some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain.

In some embodiments, the LAG3 antibody comprises a heavy chain. In some aspects, the heavy chain is an IgA. In some aspects, the heavy chain is an IgD. In some aspects, the heavy chain is an IgE. In some aspects, the heavy chain is an IgG. In some aspects, the heavy chain is an IgM. In some aspects, the heavy chain is an IgG1. In some aspects, the heavy chain is an IgG2. In some aspects, the heavy chain is an IgG3. In some aspects, the heavy chain is an IgG4. In some aspects, the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.

In some embodiments, the LAG3 antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab′)2 fragment. In some aspects, the antibody fragment is a Fab′ fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

In some embodiments, the scFv-Fc fragment comprises a constant region wherein the constant region comprises SEQ ID NO: 306. The constant region in SEQ ID NO: 306 differs from the human IgG1 constant region of SEQ ID NO: 300 in several respects. First, the sequence in SEQ ID NO: 306 comprises the linker AAGSDQEPKSS (SEQ ID NO: 312). SEQ ID NO: 306 also does not comprise the CH1 domain of the IgG1 constant region. SEQ ID NO: 306 further comprises a C220S (EU numbering system) mutation, which removes an unpaired cysteine reside that is not needed when the light chain constant region is not present (e.g., in an scFv-Fc format). SEQ ID NO: 306 further comprises two, optional, P to S mutations (P230S and P238S by the EU numbering system). Either or both of these serine residues can be reverted to the naturally occurring proline residues. Finally, SEQ ID NO: 306 comprises an aspartic acid (D) residue at EU position 356 and a leucine (L) residue at EU position 358. In contrast, SEQ ID NO: 300 comprises glutamic acid (E) in EU position 356 and methionine (M) in EU position 358. In some embodiments, the antibodies provided herein comprise constant regions comprising D356/L358, E356/M358, D356/M358, or E356/L358 (EU numbering). However, a skilled person will recognize that the antibodies provide herein may comprise any suitable constant region and that the constant region sequences provided herein are for illustrative purposes.

In some embodiments, the LAG3 antibody is a monoclonal antibody. In some embodiments, the LAG3 antibody is a polyclonal antibody.

In some embodiments, the LAG3 antibody is a chimeric antibody. In some embodiments, the LAG3 antibody is a humanized antibody. In some embodiments, the LAG3 antibody is a human antibody.

In some embodiments, the LAG3 antibody is an affinity matured antibody. In some aspects, the LAG3 antibody is an affinity matured antibody derived from an illustrative sequence provided in this disclosure.

In some embodiments, the LAG3 antibody inhibits the binding of LAG3 to one or more of its ligands. In some aspects, the LAG3 antibody inhibits the binding of LAG3 to a ligand such as MHC class II.

In some embodiments, the LAG3 antibody is provided as a single arm binder. For example, the LAG3 antibody can be provided as part of a bi-specific antibody or bi-specific antibody construct as disclosed here.

The LAG3 antibodies provided herein may be useful for the treatment of a variety of diseases and conditions including cancers. In particular, the LAG3 antibodies provided herein may be useful for the treatment of cancers of epithelial origin.

2.1. LAG3 CDR-H3 Sequences

In some embodiments, the LAG3 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the CDR-H3 sequence is a CDR-H3 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the CDR-H3 sequence is a CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 110. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 111. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 112. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 113. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 114. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 115. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 116. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 117. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 119. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 120. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 121. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 122. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 123. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 124. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 125. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 126. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 127. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 128. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 129.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.2. LAG3 V_H Sequences Comprising Illustrative CDRs

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising one or more CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-H sequences provided in this disclosure, and variants thereof. In some embodiments, the CDR-H sequences comprise, consist of, or consist essentially of one or more CDR-H sequences provided in a V_H sequence selected from SEQ ID NOs: 191-210.

2.2.1. V_H Sequences Comprising Illustrative Kabat CDRs

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising one or more Kabat CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Kabat CDR-H sequences provided in this disclosure, and variants thereof.

2.2.1.1. Kabat CDR-H3

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Kabat CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H3 sequence is a Kabat CDR-H3 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Kabat CDR-H3 sequence is a Kabat CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 110. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 111. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 112. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 113. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 114. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 115. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 116. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 117. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 119. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 120. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 121. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 122. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 123. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 124. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 125. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 126. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 127. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 128. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 129.

2.2.1.2. Kabat CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Kabat CDR-H2 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H2 sequence is a Kabat CDR-H2 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Kabat CDR-H2 sequence is a Kabat CDR-H2 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 88-107. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 88. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 89. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 90. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 91. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 92. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 93. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 94. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 95. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 96. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 97. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 98. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 99. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 100. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 101. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 102. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 103. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 104. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 105. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 106. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 107.

2.2.1.3. Kabat CDR-H1

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Kabat CDR-H1 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H1 sequence is a Kabat CDR-H1 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Kabat CDR-H1 sequence is a Kabat CDR-H1 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 39-58. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 39. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 40. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 41. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 42. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 43. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 44. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 45. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 46. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 47. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 48. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 49. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 50. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 51. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 52. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 53. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 54. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 55. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 56. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 57. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 58.

2.2.1.4. Kabat CDR-H3+Kabat CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 88-107. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.1.5. Kabat CDR-H3+Kabat CDR-H1

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129, and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 39-58. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H1 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H1 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.1.6. Kabat CDR-H1+Kabat CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 39-58 and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 88-107. In some aspects, the Kabat CDR-H1 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1 and Kabat CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.1.7. Kabat CDR-H1+Kabat CDR-H2+Kabat CDR-H3

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 39-58, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 88-107, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129. In some aspects, the Kabat CDR-H1 sequence, Kabat CDR-H2 sequence, and Kabat CDR-H3 sequence are all from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1, Kabat CDR-H2, and Kabat CDR-H3 are all from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.1.8. Variants of V_H Sequences Comprising Illustrative Kabat CDRs

In some embodiments, the V_H sequences provided herein comprise a variant of an illustrative Kabat CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure.

In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H3 sequence provided in this disclosure. In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H3 sequences provided in this disclosure. In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H2 sequence provided in this disclosure. In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H2 sequences provided in this disclosure. In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H1 sequence provided in this disclosure. In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H1 sequences provided in this disclosure. In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.2.2. V_H Sequences Comprising Illustrative Chothia CDRs

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising one or more Chothia CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Chothia CDR-H sequences provided in this disclosure, and variants thereof.

2.2.2.1. Chothia CDR-H3

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Chothia CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H3 sequence is a Chothia CDR-H3 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Chothia CDR-H3 sequence is a Chothia CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 110. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 111. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 112. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 113. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 114. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 115. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 116. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 117. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 119. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 120. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 121. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 122. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 123. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 124. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 125. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 126. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 127. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 128. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 129.

2.2.2.2. Chothia CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Chothia CDR-H2 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H2 sequence is a Chothia CDR-H2 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Chothia CDR-H2 sequence is a Chothia CDR-H2 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 66-85. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 66. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 67. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 68. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 69. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 70. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 71. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 72. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 73. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 74. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 75. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 76. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 77. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 78. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 79. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 80. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 81. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 82. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 83. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 84. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 85.

2.2.2.3. Chothia CDR-H1

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Chothia CDR-H1 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H1 sequence is a Chothia CDR-H1 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the Chothia CDR-H1 sequence is a Chothia CDR-H1 sequence of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 4-23. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 4. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 5. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 6. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 7. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 8. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 9. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 10. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 11. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 12. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 13. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 14. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 15. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 16. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 17. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 18. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 19. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 20. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 21. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 22. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 23.

2.2.2.4. Chothia CDR-H3+Chothia CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 66-85. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.2.5. Chothia CDR-H3+Chothia CDR-H1

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129, and a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 4-23. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H1 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H1 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.2.6. Chothia CDR-H1+Chothia CDR-H2

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 4-23 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 66-85. In some aspects, the Chothia CDR-H1 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1 and Chothia CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.2.7. Chothia CDR-H1+Chothia CDR-H2+Chothia CDR-H3

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 4-23, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 66-85, and a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 110-129. In some aspects, the Chothia CDR-H1 sequence, Chothia CDR-H2 sequence, and Chothia CDR-H3 sequence are all from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1, Chothia CDR-H2, and Chothia CDR-H3 are all from a single illustrative V_H sequence selected from SEQ ID NOs: 191-210.

2.2.2.8. Variants of V_H Sequences Comprising Illustrative Chothia CDRs

In some embodiments, the V_H sequences provided herein comprise a variant of an illustrative Chothia CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure.

In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H3 sequence provided in this disclosure. In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H3 sequences provided in this disclosure. In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H2 sequence provided in this disclosure. In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H2 sequences provided in this disclosure. In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H1 sequence provided in this disclosure. In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H1 sequences provided in this disclosure. In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.3. LAG3 V_H Sequences

In some embodiments, the LAG3 antibody comprises, consists of, or consists essentially of a V_H sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some embodiments, the antibody comprises, consists of, or consists essentially of a V_H sequence provided in SEQ ID NOs.: 191-210.

In some embodiments, the LAG3 antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 191-210. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 191. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 192. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 193. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 194. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 195. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 196. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 197. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 198. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 199. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 200. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 201. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 202. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 203. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 204. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 205. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 206. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 207. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 208. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 209. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 210.

2.3.1. Variants of V_H Sequences

In some embodiments, the V_H sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_H sequence provided in this disclosure.

In some aspects, the V_H sequence comprises, consists of, or consists essentially of a variant of an illustrative V_H sequence provided in this disclosure. In some aspects, the V_H sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_H sequences provided in this disclosure.

In some embodiments, the V_H sequence comprises, consists of, or consists essentially of any of the illustrative V_H sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.4. LAG3 CDR-L3 Sequences

In some embodiments, the LAG3 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of a V_L sequence provided in SEQ ID NOs.: 251-266.

In some embodiments, the LAG3 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 173-188. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 173. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 174. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 175. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 176. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 177. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 178. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 179. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 180. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 181. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 182. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 183. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 184. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 185. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 186. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 187. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 188.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.5. LAG3 V_L Sequences Comprising Illustrative CDRs

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising one or more CDR-L sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-L sequences provided in this disclosure, and variants thereof.

2.5.1. CDR-L3

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L3 sequence, wherein the CDR-L3 sequence comprises, consists of, or consists essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of a V_L sequence provided in SEQ ID NOs.: 251-266.

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 173-188. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 173. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 174. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 175. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 176. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 177. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 178. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 179. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 180. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 181. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 182. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 183. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 184. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 185. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 186. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 187. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 188.

2.5.2. CDR-L2

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L2 sequence, wherein the CDR-L2 sequence comprises, consists of, or consists essentially of a CDR-L2 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L2 sequence is a CDR-L2 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the CDR-L2 sequence is a CDR-L2 sequence of a V_L sequence provided in SEQ ID NOs.: 251-266.

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 150-165. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 150. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 151. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 152. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 153. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 154. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 155. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 156. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 157. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 158. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 159. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 160. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 161. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 162. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 163. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 164. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 165.

2.5.3. CDR-L1

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L1 sequence, wherein the CDR-L1 sequence comprises, consists of, or consists essentially of a CDR-L1 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L1 sequence is a CDR-L1 sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some aspects, the CDR-L1 sequence is a CDR-L1 sequence of a V_L sequence provided in SEQ ID NOs.: 251-266.

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 132-147. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 132. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 133. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 134. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 135. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 136. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 137. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 138. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 139. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 140. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 141. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 142. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 143. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 144. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 145. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 146. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 147.

2.5.4. CDR-L3+CDR-L2

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 173-188 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 150-165. In some aspects, the CDR-L3 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L2 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 251-266.

2.5.5. CDR-L3+CDR-L1

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 173-188 and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 132-147. In some aspects, the CDR-L3 sequence and the CDR-L1 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L1 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 251-266.

2.5.6. CDR-L1+CDR-L2

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 132-147 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 150-165. In some aspects, the CDR-L1 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L1 and CDR-L2 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 251-266.

2.5.7. CDR-L1+CDR-L2+CDR-L3

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 132-147, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 150-165, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 173-188. In some aspects, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L1, CDR-L2, and CDR-L3 are all from a single illustrative V_L sequence selected from SEQ ID NOs: 251-266.

2.5.8. Variants of V_L Sequences Comprising Illustrative CDR-Ls

In some embodiments, the V_L sequences provided herein comprise a variant of an illustrative CDR-L3, CDR-L2, and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.6. LAG3 V_L Sequences

In some embodiments, the LAG3 antibody comprises, consists of, or consists essentially of a V_L sequence of an scFv-Fc sequence provided in SEQ ID NO: 379. In some embodiments, the antibody comprises, consists of, or consists essentially of a V_L sequence provided in SEQ ID NOs.: 251-266.

In some embodiments, the LAG3 antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 251-266. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 251. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 252. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 253. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 254. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 255. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 256. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 257. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 258. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 259. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 260. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 261. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 262. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 263. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 264. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 265. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 266.

2.6.1. Variants of V_L Sequences

In some embodiments, the V_L sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_L sequence provided in this disclosure.

In some aspects, the V_L sequence comprises, consists of, or consists essentially of a variant of an illustrative V_L sequence provided in this disclosure. In some aspects, the V_L sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_L sequences provided in this disclosure.

In some embodiments, the V_L sequence comprises, consists of, or consists essentially of any of the illustrative V_L sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.7. LAG3 Pairs

2.7.1. CDR-H3-CDR-L3 Pairs

In some embodiments, the LAG3 antibody comprises a CDR-H3 sequence and a CDR-L3 sequence. In some aspects, the CDR-H3 sequence is part of a V_H and the CDR-L3 sequence is part of a V_L.

In some aspects, the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 110-129, and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 173-188.

2.7.1.1. Variants of CDR-H3-CDR-L3 Pairs

In some embodiments, the CDR-H3-CDR-L3 pairs provided herein comprise a variant of an illustrative CDR-H3 and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.7.2. CDR-H1-CDR-L1 Pairs

In some embodiments, the LAG3 antibody comprises a CDR-H1 sequence and a CDR-L1 sequence. In some aspects, the CDR-H1 sequence is part of a V_H and the CDR-L1 sequence is part of a V_L.

In some aspects, the CDR-H1 sequence is a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 4-23, and the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 132-147.

In some aspects, the CDR-H1 sequence is a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 39-58, and the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 132-147.

2.7.2.1. Variants of CDR-H1-CDR-L1 Pairs

In some embodiments, the CDR-H1-CDR-L1 pairs provided herein comprise a variant of an illustrative CDR-H1 and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H1 sequence provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H1 sequences provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.7.3. CDR-H2-CDR-L2 Pairs

In some embodiments, the LAG3 antibody comprises a CDR-H2 sequence and a CDR-L2 sequence. In some aspects, the CDR-H2 sequence is part of a V_H and the CDR-L2 sequence is part of a V_L.

In some aspects, the CDR-H2 sequence is a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 66-85, and the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 150-165.

In some aspects, the CDR-H1 sequence is a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 88-107, and the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 150-165.

2.7.3.1. Variants of CDR-H2-CDR-L2 Pairs

In some embodiments, the CDR-H2-CDR-L2 pairs provided herein comprise a variant of an illustrative CDR-H2 and/or CDR-L2 sequence provided in this disclosure.

In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H2 sequence provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H2 sequences provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.7.4. V_H-V_L Pairs

In some embodiments, the LAG3 antibody comprises a V_H sequence and a V_L sequence.

In some aspects, the V_H sequence is a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210, and the V_L sequence is a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 191 and SEQ ID NO: 251; SEQ ID NO: 191 and SEQ ID NO: 252; SEQ ID NO: 191 and SEQ ID NO: 253; SEQ ID NO: 191 and SEQ ID NO: 254; SEQ ID NO: 191 and SEQ ID NO: 255; SEQ ID NO: 191 and SEQ ID NO: 256; SEQ ID NO: 191 and SEQ ID NO: 257; SEQ ID NO: 191 and SEQ ID NO: 258; SEQ ID NO: 191 and SEQ ID NO: 259; SEQ ID NO: 191 and SEQ ID NO: 260; SEQ ID NO: 191 and SEQ ID NO: 261; SEQ ID NO: 191 and SEQ ID NO: 262; SEQ ID NO: 191 and SEQ ID NO: 263; SEQ ID NO: 191 and SEQ ID NO: 264; SEQ ID NO: 191 and SEQ ID NO: 265; and SEQ ID NO: 191 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 192 and SEQ ID NO: 251; SEQ ID NO: 192 and SEQ ID NO: 252; SEQ ID NO: 192 and SEQ ID NO: 253; SEQ ID NO: 192 and SEQ ID NO: 254; SEQ ID NO: 192 and SEQ ID NO: 255; SEQ ID NO: 192 and SEQ ID NO: 256; SEQ ID NO: 192 and SEQ ID NO: 257; SEQ ID NO: 192 and SEQ ID NO: 258; SEQ ID NO: 192 and SEQ ID NO: 259; SEQ ID NO: 192 and SEQ ID NO: 260; SEQ ID NO: 192 and SEQ ID NO: 261; SEQ ID NO: 192 and SEQ ID NO: 262; SEQ ID NO: 192 and SEQ ID NO: 263; SEQ ID NO: 192 and SEQ ID NO: 264; SEQ ID NO: 192 and SEQ ID NO: 265; and SEQ ID NO: 192 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 193 and SEQ ID NO: 251; SEQ ID NO: 193 and SEQ ID NO: 252; SEQ ID NO: 193 and SEQ ID NO: 253; SEQ ID NO: 193 and SEQ ID NO: 254; SEQ ID NO: 193 and SEQ ID NO: 255; SEQ ID NO: 193 and SEQ ID NO: 256; SEQ ID NO: 193 and SEQ ID NO: 257; SEQ ID NO: 193 and SEQ ID NO: 258; SEQ ID NO: 193 and SEQ ID NO: 259; SEQ ID NO: 193 and SEQ ID NO: 260; SEQ ID NO: 193 and SEQ ID NO: 261; SEQ ID NO: 193 and SEQ ID NO: 262; SEQ ID NO: 193 and SEQ ID NO: 263; SEQ ID NO: 193 and SEQ ID NO: 264; SEQ ID NO: 193 and SEQ ID NO: 265; and SEQ ID NO: 193 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 194 and SEQ ID NO: 251; SEQ ID NO: 194 and SEQ ID NO: 252; SEQ ID NO: 194 and SEQ ID NO: 253; SEQ ID NO: 194 and SEQ ID NO: 254; SEQ ID NO: 194 and SEQ ID NO: 255; SEQ ID NO: 194 and SEQ ID NO: 256; SEQ ID NO: 194 and SEQ ID NO: 257; SEQ ID NO: 194 and SEQ ID NO: 258; SEQ ID NO: 194 and SEQ ID NO: 259; SEQ ID NO: 194 and SEQ ID NO: 260; SEQ ID NO: 194 and SEQ ID NO: 261; SEQ ID NO: 194 and SEQ ID NO: 262; SEQ ID NO: 194 and SEQ ID NO: 263; SEQ ID NO: 194 and SEQ ID NO: 264; SEQ ID NO: 194 and SEQ ID NO: 265; and SEQ ID NO: 194 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 195 and SEQ ID NO: 251; SEQ ID NO: 195 and SEQ ID NO: 252; SEQ ID NO: 195 and SEQ ID NO: 253; SEQ ID NO: 195 and SEQ ID NO: 254; SEQ ID NO: 195 and SEQ ID NO: 255; SEQ ID NO: 195 and SEQ ID NO: 256; SEQ ID NO: 195 and SEQ ID NO: 257; SEQ ID NO: 195 and SEQ ID NO: 258; SEQ ID NO: 195 and SEQ ID NO: 259; SEQ ID NO: 195 and SEQ ID NO: 260; SEQ ID NO: 195 and SEQ ID NO: 261; SEQ ID NO: 195 and SEQ ID NO: 262; SEQ ID NO: 195 and SEQ ID NO: 263; SEQ ID NO: 195 and SEQ ID NO: 264; SEQ ID NO: 195 and SEQ ID NO: 265; and SEQ ID NO: 195 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 196 and SEQ ID NO: 251; SEQ ID NO: 196 and SEQ ID NO: 252; SEQ ID NO: 196 and SEQ ID NO: 253; SEQ ID NO: 196 and SEQ ID NO: 254; SEQ ID NO: 196 and SEQ ID NO: 255; SEQ ID NO: 196 and SEQ ID NO: 256; SEQ ID NO: 196 and SEQ ID NO: 257; SEQ ID NO: 196 and SEQ ID NO: 258; SEQ ID NO: 196 and SEQ ID NO: 259; SEQ ID NO: 196 and SEQ ID NO: 260; SEQ ID NO: 196 and SEQ ID NO: 261; SEQ ID NO: 196 and SEQ ID NO: 262; SEQ ID NO: 196 and SEQ ID NO: 263; SEQ ID NO: 196 and SEQ ID NO: 264; SEQ ID NO: 196 and SEQ ID NO: 265; and SEQ ID NO: 196 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 197 and SEQ ID NO: 251; SEQ ID NO: 197 and SEQ ID NO: 252; SEQ ID NO: 197 and SEQ ID NO: 253; SEQ ID NO: 197 and SEQ ID NO: 254; SEQ ID NO: 197 and SEQ ID NO: 255; SEQ ID NO: 197 and SEQ ID NO: 256; SEQ ID NO: 197 and SEQ ID NO: 257; SEQ ID NO: 197 and SEQ ID NO: 258; SEQ ID NO: 197 and SEQ ID NO: 259; SEQ ID NO: 197 and SEQ ID NO: 260; SEQ ID NO: 197 and SEQ ID NO: 261; SEQ ID NO: 197 and SEQ ID NO: 262; SEQ ID NO: 197 and SEQ ID NO: 263; SEQ ID NO: 197 and SEQ ID NO: 264; SEQ ID NO: 197 and SEQ ID NO: 265; and SEQ ID NO: 197 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 198 and SEQ ID NO: 251; SEQ ID NO: 198 and SEQ ID NO: 252; SEQ ID NO: 198 and SEQ ID NO: 253; SEQ ID NO: 198 and SEQ ID NO: 254; SEQ ID NO: 198 and SEQ ID NO: 255; SEQ ID NO: 198 and SEQ ID NO: 256; SEQ ID NO: 198 and SEQ ID NO: 257; SEQ ID NO: 198 and SEQ ID NO: 258; SEQ ID NO: 198 and SEQ ID NO: 259; SEQ ID NO: 198 and SEQ ID NO: 260; SEQ ID NO: 198 and SEQ ID NO: 261; SEQ ID NO: 198 and SEQ ID NO: 262; SEQ ID NO: 198 and SEQ ID NO: 263; SEQ ID NO: 198 and SEQ ID NO: 264; SEQ ID NO: 198 and SEQ ID NO: 265; and SEQ ID NO: 198 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 199 and SEQ ID NO: 251; SEQ ID NO: 199 and SEQ ID NO: 252; SEQ ID NO: 199 and SEQ ID NO: 253; SEQ ID NO: 199 and SEQ ID NO: 254; SEQ ID NO: 199 and SEQ ID NO: 255; SEQ ID NO: 199 and SEQ ID NO: 256; SEQ ID NO: 199 and SEQ ID NO: 257; SEQ ID NO: 199 and SEQ ID NO: 258; SEQ ID NO: 199 and SEQ ID NO: 259; SEQ ID NO: 199 and SEQ ID NO: 260; SEQ ID NO: 199 and SEQ ID NO: 261; SEQ ID NO: 199 and SEQ ID NO: 262; SEQ ID NO: 199 and SEQ ID NO: 263; SEQ ID NO: 199 and SEQ ID NO: 264; SEQ ID NO: 199 and SEQ ID NO: 265; and SEQ ID NO: 199 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 200 and SEQ ID NO: 251; SEQ ID NO: 200 and SEQ ID NO: 252; SEQ ID NO: 200 and SEQ ID NO: 253; SEQ ID NO: 200 and SEQ ID NO: 254; SEQ ID NO: 200 and SEQ ID NO: 255; SEQ ID NO: 200 and SEQ ID NO: 256; SEQ ID NO: 200 and SEQ ID NO: 257; SEQ ID NO: 200 and SEQ ID NO: 258; SEQ ID NO: 200 and SEQ ID NO: 259; SEQ ID NO: 200 and SEQ ID NO: 260; SEQ ID NO: 200 and SEQ ID NO: 261; SEQ ID NO: 200 and SEQ ID NO: 262; SEQ ID NO: 200 and SEQ ID NO: 263; SEQ ID NO: 200 and SEQ ID NO: 264; SEQ ID NO: 200 and SEQ ID NO: 265; and SEQ ID NO: 200 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 201 and SEQ ID NO: 251; SEQ ID NO: 201 and SEQ ID NO: 252; SEQ ID NO: 201 and SEQ ID NO: 253; SEQ ID NO: 201 and SEQ ID NO: 254; SEQ ID NO: 201 and SEQ ID NO: 255; SEQ ID NO: 201 and SEQ ID NO: 256; SEQ ID NO: 201 and SEQ ID NO: 257; SEQ ID NO: 201 and SEQ ID NO: 258; SEQ ID NO: 201 and SEQ ID NO: 259; SEQ ID NO: 201 and SEQ ID NO: 260; SEQ ID NO: 201 and SEQ ID NO: 261; SEQ ID NO: 201 and SEQ ID NO: 262; SEQ ID NO: 201 and SEQ ID NO: 263; SEQ ID NO: 201 and SEQ ID NO: 264; SEQ ID NO: 201 and SEQ ID NO: 265; and SEQ ID NO: 201 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 202 and SEQ ID NO: 251; SEQ ID NO: 202 and SEQ ID NO: 252; SEQ ID NO: 202 and SEQ ID NO: 253; SEQ ID NO: 202 and SEQ ID NO: 254; SEQ ID NO: 202 and SEQ ID NO: 255; SEQ ID NO: 202 and SEQ ID NO: 256; SEQ ID NO: 202 and SEQ ID NO: 257; SEQ ID NO: 202 and SEQ ID NO: 258; SEQ ID NO: 202 and SEQ ID NO: 259; SEQ ID NO: 202 and SEQ ID NO: 260; SEQ ID NO: 202 and SEQ ID NO: 261; SEQ ID NO: 202 and SEQ ID NO: 262; SEQ ID NO: 202 and SEQ ID NO: 263; SEQ ID NO: 202 and SEQ ID NO: 264; SEQ ID NO: 202 and SEQ ID NO: 265; and SEQ ID NO: 202 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 203 and SEQ ID NO: 251; SEQ ID NO: 203 and SEQ ID NO: 252; SEQ ID NO: 203 and SEQ ID NO: 253; SEQ ID NO: 203 and SEQ ID NO: 254; SEQ ID NO: 203 and SEQ ID NO: 255; SEQ ID NO: 203 and SEQ ID NO: 256; SEQ ID NO: 203 and SEQ ID NO: 257; SEQ ID NO: 203 and SEQ ID NO: 258; SEQ ID NO: 203 and SEQ ID NO: 259; SEQ ID NO: 203 and SEQ ID NO: 260; SEQ ID NO: 203 and SEQ ID NO: 261; SEQ ID NO: 203 and SEQ ID NO: 262; SEQ ID NO: 203 and SEQ ID NO: 263; SEQ ID NO: 203 and SEQ ID NO: 264; SEQ ID NO: 203 and SEQ ID NO: 265; and SEQ ID NO: 203 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 204 and SEQ ID NO: 251; SEQ ID NO: 204 and SEQ ID NO: 252; SEQ ID NO: 204 and SEQ ID NO: 253; SEQ ID NO: 204 and SEQ ID NO: 254; SEQ ID NO: 204 and SEQ ID NO: 255; SEQ ID NO: 204 and SEQ ID NO: 256; SEQ ID NO: 204 and SEQ ID NO: 257; SEQ ID NO: 204 and SEQ ID NO: 258; SEQ ID NO: 204 and SEQ ID NO: 259; SEQ ID NO: 204 and SEQ ID NO: 260; SEQ ID NO: 204 and SEQ ID NO: 261; SEQ ID NO: 204 and SEQ ID NO: 262; SEQ ID NO: 204 and SEQ ID NO: 263; SEQ ID NO: 204 and SEQ ID NO: 264; SEQ ID NO: 204 and SEQ ID NO: 265; and SEQ ID NO: 204 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 205 and SEQ ID NO: 251; SEQ ID NO: 205 and SEQ ID NO: 252; SEQ ID NO: 205 and SEQ ID NO: 253; SEQ ID NO: 205 and SEQ ID NO: 254; SEQ ID NO: 205 and SEQ ID NO: 255; SEQ ID NO: 205 and SEQ ID NO: 256; SEQ ID NO: 205 and SEQ ID NO: 257; SEQ ID NO: 205 and SEQ ID NO: 258; SEQ ID NO: 205 and SEQ ID NO: 259; SEQ ID NO: 205 and SEQ ID NO: 260; SEQ ID NO: 205 and SEQ ID NO: 261; SEQ ID NO: 205 and SEQ ID NO: 262; SEQ ID NO: 205 and SEQ ID NO: 263; SEQ ID NO: 205 and SEQ ID NO: 264; SEQ ID NO: 205 and SEQ ID NO: 265; and SEQ ID NO: 205 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 206 and SEQ ID NO: 251; SEQ ID NO: 206 and SEQ ID NO: 252; SEQ ID NO: 206 and SEQ ID NO: 253; SEQ ID NO: 206 and SEQ ID NO: 254; SEQ ID NO: 206 and SEQ ID NO: 255; SEQ ID NO: 206 and SEQ ID NO: 256; SEQ ID NO: 206 and SEQ ID NO: 257; SEQ ID NO: 206 and SEQ ID NO: 258; SEQ ID NO: 206 and SEQ ID NO: 259; SEQ ID NO: 206 and SEQ ID NO: 260; SEQ ID NO: 206 and SEQ ID NO: 261; SEQ ID NO: 206 and SEQ ID NO: 262; SEQ ID NO: 206 and SEQ ID NO: 263; SEQ ID NO: 206 and SEQ ID NO: 264; SEQ ID NO: 206 and SEQ ID NO: 265; and SEQ ID NO: 206 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 207 and SEQ ID NO: 251; SEQ ID NO: 207 and SEQ ID NO: 252; SEQ ID NO: 207 and SEQ ID NO: 253; SEQ ID NO: 207 and SEQ ID NO: 254; SEQ ID NO: 207 and SEQ ID NO: 255; SEQ ID NO: 207 and SEQ ID NO: 256; SEQ ID NO: 207 and SEQ ID NO: 257; SEQ ID NO: 207 and SEQ ID NO: 258; SEQ ID NO: 207 and SEQ ID NO: 259; SEQ ID NO: 207 and SEQ ID NO: 260; SEQ ID NO: 207 and SEQ ID NO: 261; SEQ ID NO: 207 and SEQ ID NO: 262; SEQ ID NO: 207 and SEQ ID NO: 263; SEQ ID NO: 207 and SEQ ID NO: 264; SEQ ID NO: 207 and SEQ ID NO: 265; and SEQ ID NO: 207 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 208 and SEQ ID NO: 251; SEQ ID NO: 208 and SEQ ID NO: 252; SEQ ID NO: 208 and SEQ ID NO: 253; SEQ ID NO: 208 and SEQ ID NO: 254; SEQ ID NO: 208 and SEQ ID NO: 255; SEQ ID NO: 208 and SEQ ID NO: 256; SEQ ID NO: 208 and SEQ ID NO: 257; SEQ ID NO: 208 and SEQ ID NO: 258; SEQ ID NO: 208 and SEQ ID NO: 259; SEQ ID NO: 208 and SEQ ID NO: 260; SEQ ID NO: 208 and SEQ ID NO: 261; SEQ ID NO: 208 and SEQ ID NO: 262; SEQ ID NO: 208 and SEQ ID NO: 263; SEQ ID NO: 208 and SEQ ID NO: 264; SEQ ID NO: 208 and SEQ ID NO: 265; and SEQ ID NO: 208 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 209 and SEQ ID NO: 251; SEQ ID NO: 209 and SEQ ID NO: 252; SEQ ID NO: 209 and SEQ ID NO: 253; SEQ ID NO: 209 and SEQ ID NO: 254; SEQ ID NO: 209 and SEQ ID NO: 255; SEQ ID NO: 209 and SEQ ID NO: 256; SEQ ID NO: 209 and SEQ ID NO: 257; SEQ ID NO: 209 and SEQ ID NO: 258; SEQ ID NO: 209 and SEQ ID NO: 259; SEQ ID NO: 209 and SEQ ID NO: 260; SEQ ID NO: 209 and SEQ ID NO: 261; SEQ ID NO: 209 and SEQ ID NO: 262; SEQ ID NO: 209 and SEQ ID NO: 263; SEQ ID NO: 209 and SEQ ID NO: 264; SEQ ID NO: 209 and SEQ ID NO: 265; and SEQ ID NO: 209 and SEQ ID NO: 266.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 210 and SEQ ID NO: 251; SEQ ID NO: 210 and SEQ ID NO: 252; SEQ ID NO: 210 and SEQ ID NO: 253; SEQ ID NO: 210 and SEQ ID NO: 254; SEQ ID NO: 210 and SEQ ID NO: 255; SEQ ID NO: 210 and SEQ ID NO: 256; SEQ ID NO: 210 and SEQ ID NO: 257; SEQ ID NO: 210 and SEQ ID NO: 258; SEQ ID NO: 210 and SEQ ID NO: 259; SEQ ID NO: 210 and SEQ ID NO: 260; SEQ ID NO: 210 and SEQ ID NO: 261; SEQ ID NO: 210 and SEQ ID NO: 262; SEQ ID NO: 210 and SEQ ID NO: 263; SEQ ID NO: 210 and SEQ ID NO: 264; SEQ ID NO: 210 and SEQ ID NO: 265; and SEQ ID NO: 210 and SEQ ID NO: 266.

2.7.4.1. Variants of V_H-V_L Pairs

In some embodiments, the V_H-V_L pairs provided herein comprise a variant of an illustrative V_H and/or V_L sequence provided in this disclosure.

In some aspects, the V_H sequence comprises, consists of, or consists essentially of a variant of an illustrative V_H sequence provided in this disclosure. In some aspects, the V_H sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.1% identity with any of the illustrative V_H sequences provided in this disclosure.

In some embodiments, the V_H sequence comprises, consists of, or consists essentially of any of the illustrative V_H sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the V_L sequence comprises, consists of, or consists essentially of a variant of an illustrative V_L sequence provided in this disclosure. In some aspects, the V_L sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_L sequences provided in this disclosure.

In some embodiments, the V_L sequence comprises, consists of, or consists essentially of any of the illustrative V_L sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.8. LAG3 Antibodies Comprising all Six CDRs

In some embodiments, the LAG3 antibody comprises a CDR-H1 sequence, a CDR-H2 sequence, a CDR-H3 sequence, a CDR-L1 sequence, and a CDR-L3 sequence. In some aspects, the CDR sequences are part of a V_H (for CDR-H) or V_L (for CDR-L).

In some aspects, the CDR-H1 sequence is a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 4-23; the CDR-H2 sequence is a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 66-85; the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 110-129; the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 132-147; the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 150-165; and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 173-188.

In some aspects, the CDR-H1 sequence is a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 39-58; the CDR-H2 sequence is a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 88-107; the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 110-129; the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 132-147; the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 150-165; and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 173-188.

2.8.1. Variants of Antibodies Comprising All Six CDRs

In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided herein comprise a variant of an illustrative CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequence provided in this disclosure.

In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H1 sequence provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H2 sequence provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

2.9. LAG3 Consensus Sequences

In some embodiments, provided herein are anti-LAG3 antibodies comprising one or more sequences defined by consensus sequences. Each consensus sequence is based, at least in part, on one or more alignments of two or more useful anti-LAG3 CDR sequences provided in this disclosure. Based on such alignments, a person of skill in the art would recognize that different amino acid residues may useful in certain positions of the CDRs. Accordingly, each consensus sequence encompasses two or more useful anti-LAG3 CDR sequences.

In some embodiments, the LAG3 antibodies comprise one to six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise two to six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise three to six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise four to six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise five to six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise six of the consensus CDR sequences provided herein. In some embodiments, the antibodies comprise a V_L comprising the CDR-L consensus sequence(s). In some embodiments, the antibodies comprise a V_H comprising the CDR-H consensus sequence(s). In some embodiments, the antibodies comprise a V_H comprising the CDR-H consensus sequence(s) and a V_L comprising the CDR-L consensus sequence(s).

2.9.1. CDR-H3 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a CDR-H3 sequence defined by the consensus sequence α₁-α₂-α₃-α₄-α₅-α₆-α₇-α₈-α₉-α₁₀-α₁₁-D-α₁₃, where α₁ is absent, E, or V; α₂ is absent I, S, W, E, Y, D, or F; α₃ is absent, F, L, I, E A, A, or N; α₄ is absent, G, V, P, or D; α₅ is absent, A, S, E, V, or G; α₆ is F, S, N, or V; α₇ is absent Y, W, or R; α₈ is W, L, D, P, or S; α₉ is N, Y, A, D, or F; α₁₀ is P, A, G, S, or M; an is absent, F, L, M, or V; and α₁₃ is Y or V. In certain embodiments, each of α₁, α₂, α₃, α₄, α₅, α₆, and α₇ is absent. In certain embodiments, none of α₁, α₂, α₃, α₄, α₅, α₆, and α₇ is absent. In certain embodiments, only α₅ of α₁, α₂, α₃, α₄, as, α₆, and α₇ is absent. In certain embodiments, only as is absent. In certain embodiments, only all is absent. In certain embodiments, when α₂ is W, α₄ is V, α₅ is A, α₆ is S, and α₁₀ is G, then α₁₁ is F, L, or V. In certain embodiments, α₂ is E or D, α₄ is P, α₅ is E, α₆ is N, α₁₀ is A or G, and α₁₁ is F, L, or V. In certain embodiments, the antibody comprises a CDR-H3 sequence defined by the consensus sequence E-α₂-α₃-α₄-α₅-α₆-W-D-α₉-α₁₀-α₁₁-D-V where α₂ is S, W, or E; α₃ is A or E; α₄ is V, P, or D; α₅ is A, S, E, or V; α₆ is S or N; α₉ is Y or A; α₁₀ is A or G; and α₁₁ is L or M wherein when α₂ is W, α₄ is V, α₅ is A, α₆ is S, and α₁₀ is G, then α₁₁ is L.

In some embodiments, the LAG3 antibody comprises a CDR-H3 sequence defined by the consensus sequence V-β₂-β₃-G-G-V-R-P-β₉-S-β₁₁-D-Y, where β₂ is F, Y, or D; β₃ is E or N; β₉ is Y or F; and β₁₁ is absent.

2.9.2. Chothia CDR-H1 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-F-γ₃-γ₄-γ₅-γ₆-γ₇, where γ₃ is N or T; γ₄ is I or F; γ₅ is K, N, A, S, R, P, or T; γ₆ is D, S, or E; and γ₇ is T, N, Y, F, S, or L.

In some embodiments, the LAG3 antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-F-T-F-δ₅-δ₆-δ₇, where δ₅ is S, R, P, T, or N; δ₆ is S, D, or E; and δ₇ is F, S, or Y.

2.9.3. Chothia CDR-H2 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence ε₁-ε₂-ε₃-ε₄-ε₅-ε₆, where ε₁ is D, W, or T; ε₂ is P, Y, D, G, or S; ε₃ is Y, D, N, W, or, E; ε₄ is D, A, G, S, T, or N; ε₅ is G or S; and ε₆ is A, D, F, Y, V, N, T, or S. In certain embodiments, ε₁ is W; ε₂ is Y; ε₃ is D; ε₄ is A or G; ε₅ is S; and ε₆ is Y, N, or V. In certain embodiments, ε₁ is T or S; ε₂ is D or S; ε₃ is N or D; ε₄ is S or T; ε₅ is G; and ε₆ is N, T, or S.

2.9.4. Kabat CDR-H1 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence ξ₁-ξ₂-ξ₃-ξ₄-ξ₅, where ξ₁ is D, S, or E; ξ₂ is T, N, Y, F, S, or L; ξ₃ is Y, F, G, S, or T; ξ₄ is I or M; and ξ₅ is H or S.

In some embodiments, the LAG3 antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence S-η₂-G-M-H, where η₂ is Y or F. In some embodiments, the antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence η₁-S-η₃-M-H, where η₁ is D, E, or S; and η₃ is S or T.

2.9.5. Kabat CDR-H2 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence θ₁-I-θ₃-θ₄-θ₅-θ₆-θ₇-θ₈-θ₉-θ₁₀-Y-A-θ₁₃-θ₁₄-θ₁₅-θ₁₆-G, where θ₁ is I, A, V, R, or W; θ₃ is D, W, T, or S; θ₄ is P, Y, D, G, or S; θ₅ is Y, D, N, W, or E; θ₆ is D, A, G, S, T, or N; θ₇ is G or S; θ₈ is A, D, F, Y, N, V, T, or S; θ₉ is T or K; θ₁₀ is D, A, Y or E; θ₁₃ is D, or P; θ₁₄ is S or K; θ₁₅ is V or F; and θ₁₆ is K or Q. In some embodiments, the antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence θ₁-I-θ₃-Y-D-G-S-θ₈-K-Y-Y-A-D-S-V-K-G, where θ₁ is V or A; θ₃ is W or T; and θ₈ is Y, N, or V. In some embodiments, the antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence θ₁-I-θ₃-θ₄-θ₅-θ₆-G-θ₈-T-D-Y-A-D-S—V-K-G, where θ₁ is F or V; θ3 is T or S; θ₄ is S, D, or G; θ₅ is D or N; θ6 is S or T; and θ₈ is T, S, or N.

2.9.6. CDR-L3 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a CDR-L3 sequence defined by the consensus sequence Q-Q-ι₃-ι₄-ι₅-ι₆-P-ι₈-ι₉, where ι₃ is Y or D; ι₄ is G, D, S, M, or T; ι₅ is R, S, A, or L; ι₆ is S, T, A, or G; ι₈ is F, L or P; and ι₉ is S, T, or K. In certain embodiments, when ι₅ is S, then ι₅ is S.

In some embodiments, the LAG3 antibody comprises a CDR-L3 sequence defined by the consensus sequence ι₁-ι₂-ι₃-ι₄-ι₅-ι₆-P-Q-T where ι₁ is S or W; ι₂ is H, T, or Q; ι₃ is G or Y; ι₄ is N, I, or S; and ι₅ is V or F.

2.9.7. CDR-L2 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a CDR-L2 sequence defined by the consensus sequence GASSRAT (SEQ ID NO:150) and LVSKLDS (SEQ ID NO:161)

2.9.8. CDR-L1 Consensus Sequences

In some embodiments, the LAG3 antibody comprises a CDR-L1 sequence defined by the consensus sequence R-A-S-Q-μ₅-μ₆-μ₇-μ₈-S—V-S-S-μ₁₃-μ₁₄-μ₁₅-A, where μ₅ is absent; μ₆ is absent; μ₇ is absent; μ₈ is absent; μ₁₃ is S, N, or G; μ₁₄ is Y, P or N; and μ₁₅ is L or P. In some embodiments, the antibody comprises a CDR-L1 sequence defined by the consensus sequence KSSQSLLDSDGKTYLN (SEQ ID NO:143).

3. PD-1 Antibodies

Provided herein are PD-1 antibodies that selectively bind human PD-1. In some aspects, the antibody selectively binds to the extracellular domain of human PD-1. In some aspects, the antibody selectively binds to one or more of full-length human PD-1, PD-1Δex2, PD-1Δex3, PD-1Δex2,3, and PD-1Δex2,3,4. See Nielsen et al., Cellular Immunology, 2005, 235:109-116, incorporated by reference in its entirety.

In some embodiments, the PD-1 antibody binds to a homolog of human PD-1. In some aspects, the antibody binds to a homolog of human PD-1 from a species selected from monkeys, mice, dogs, cats, rats, cows, horses, goats and sheep. In some aspects, the homolog is a cynomolgus monkey homolog. In some aspects, the homolog is a murine homolog.

In some embodiments, the PD-1 antibody comprises a light chain. In some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain.

In some embodiments, the PD-1 antibody comprises a heavy chain. In some aspects, the heavy chain is an IgA. In some aspects, the heavy chain is an IgD. In some aspects, the heavy chain is an IgE. In some aspects, the heavy chain is an IgG. In some aspects, the heavy chain is an IgM. In some aspects, the heavy chain is an IgG1. In some aspects, the heavy chain is an IgG2. In some aspects, the heavy chain is an IgG3. In some aspects, the heavy chain is an IgG4. In some aspects, the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.

In some embodiments, the PD-1 antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab′)₂ fragment. In some aspects, the antibody fragment is a Fab′ fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

In some embodiments, the PD-1 antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

In some embodiments, the PD-1 antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

In some embodiments, the PD-1 antibody is an affinity matured antibody. In some aspects, the antibody is an affinity matured antibody derived from an illustrative sequence provided in this disclosure or in, e.g., WO 2016/077397, which is incorporated herein by reference in its entirety.

In some embodiments, the PD-1 antibody inhibits the binding of PD-1 to its ligands. In some aspects, the antibody inhibits the binding of PD-1 to PD-L1. In some aspects, the antibody inhibits the binding of PD-1 to PD-L2. In some aspects, the antibody inhibits the binding of PD-1 to PD-L1 and PD-L2.

In some embodiments, the PD-1 antibody is provided as a single arm binder. For example, the PD-1 antibody can be provided as part of a bi-specific antibody or bi-specific antibody construct as disclosed here.

The PD-1 antibodies provided herein may be useful for the treatment of a variety of diseases and conditions, including cancers, autoimmune diseases, and infections.

3.1 PD-1 CDR-H3 Sequences

In some embodiments, the PD-1 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the CDR-H3 sequence is a CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 130. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 131. In some embodiments, the PD-1 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of a V_H sequence of SEQ ID NO: 250. In some embodiments, the PD-1 antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a CDR-H3 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.2 PD-1 V_H Sequences Comprising Illustrative CDRs

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising one or more CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-H sequences provided in this disclosure, and variants thereof. In some embodiments, the CDR-H sequences comprise, consist of, or consist essentially of one or more CDR-H sequences provided in a V_H sequence selected from SEQ ID NOs: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising one or more CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-H sequences provided in WO 2016/077397.

3.3.1 V_H Sequences Comprising Illustrative Kabat CDRs

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising one or more Kabat CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Kabat CDR-H sequences provided in this disclosure, and variants thereof. In some embodiments, the CDR-H sequences comprise, consist of, or consist essentially of one or more CDR-H sequences provided in a V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.1 Kabat CDR-H3

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Kabat CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H3 sequence is a Kabat CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Kabat CDR-H3 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 130. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 131.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H3 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.2 Kabat CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Kabat CDR-H2 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H2 sequence is a Kabat CDR-H2 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Kabat CDR-H2 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 108-109. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 108. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 109.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H2 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.3 Kabat CDR-H1

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Kabat CDR-H1 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Kabat CDR-H1 sequence is a Kabat CDR-H1 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Kabat CDR-H1 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 59-65. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 59. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 60. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 61. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 62. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 63. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 64. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 65.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a Kabat CDR-H1 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.4 Kabat CDR-H3+Kabat CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 108-109. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.5 Kabat CDR-H3+Kabat CDR-H1

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131, and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 59-65. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H3 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250, and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H1 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H1 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H1 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.6 Kabat CDR-H1+Kabat CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 59-65, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 108-109. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H1 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H2 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250. In some aspects, the Kabat CDR-H1 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1 and Kabat CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.7 Kabat CDR-H1+Kabat CDR-H2+Kabat CDR-H3

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 59-65, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 108-109, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H1 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Kabat CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250. In some aspects, the Kabat CDR-H1 sequence, Kabat CDR-H2 sequence, and Kabat CDR-H3 are all from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1, Kabat CDR-H2, and Kabat CDR-H3 are all from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.8 Variants of V_H Sequences Comprising Illustrative Kabat CDRs

In some embodiments, the V_H sequences provided herein comprise a variant of an illustrative Kabat CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure. In some embodiments, the V_H sequences provided herein comprise a variant of an illustrative Kabat CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in WO 2016/077397.

In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H3 sequence provided in this disclosure. In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H3 sequences provided in this disclosure. In some aspects, the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H2 sequence provided in this disclosure. In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H2 sequences provided in this disclosure. In some aspects, the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H1 sequence provided in this disclosure. In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H1 sequences provided in this disclosure. In some aspects, the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.3.2 V_H Sequences Comprising Illustrative Chothia CDRs

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising one or more Chothia CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Chothia CDR-H sequences provided in this disclosure, and variants thereof. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising one or more Chothia CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Chothia CDR-H sequences provided in WO 2016/077397.

3.2.2.1 Chothia CDR-H3

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Chothia CDR-H3 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H3 sequence is a Chothia CDR-H3 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a Chothia CDR-H3 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 130. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 131.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H3 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.2 Chothia CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Chothia CDR-H2 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H2 sequence is a Chothia CDR-H2 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a Chothia CDR-H2 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 86-87. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 86. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 87.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 245.

In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H2 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.3 Chothia CDR-H1

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Chothia CDR-H1 sequence of an illustrative antibody or V_H sequence provided herein. In some aspects, the Chothia CDR-H1 sequence is a Chothia CDR-H1 sequence of a V_H sequence provided in SEQ ID NOs.: 211-250. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a Chothia CDR-H1 sequence of an illustrative antibody or V_H sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 24-38. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 24. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 25. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 26. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 27. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 28. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 29. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 30. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 31. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 32. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 33. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 34. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 35. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 36. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 37. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 38.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a Chothia CDR-H1 sequence of a V_H sequence of SEQ ID NO: 250.

3.2.2.4 Chothia CDR-H3+Chothia CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 86-87. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 211-250. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.5 Chothia CDR-H3+Chothia CDR-H1

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131, and a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 24-38. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H3 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250, and a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H1 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H1 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H1 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.6 Chothia CDR-H1+Chothia CDR-H2

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 24-38 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 86-87. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H1 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H2 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250. In some aspects, the Chothia CDR-H1 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1 and Chothia CDR-H2 are both from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.7 Chothia CDR-H1+Chothia CDR-H2+Chothia CDR-H3

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 24-38, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 86-87, and a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131. In some embodiments, the PD-1 antibody comprises a V_H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H1 sequence of a Vii sequence provided in any one of SEQ ID NOs.: 211-250, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H2 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250, and a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a Chothia CDR-H3 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 211-250. In some aspects, the Chothia CDR-H1 sequence, Chothia CDR-H2 sequence, and Chothia CDR-H3 sequence are all from a single illustrative V_H sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1, Chothia CDR-H2, and Chothia CDR-H3 are all from a single illustrative V_H sequence selected from SEQ ID NOs: 211-250.

3.2.2.8 Variants of V_H Sequences Comprising Illustrative Chothia CDRs

In some embodiments, the V_H sequences provided herein comprise a variant of an illustrative Chothia CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure.

In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H3 sequence provided in this disclosure. In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H3 sequences provided in this disclosure. In some aspects, the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H2 sequence provided in this disclosure. In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H2 sequences provided in this disclosure. In some aspects, the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H1 sequence provided in this disclosure. In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H1 sequences provided in this disclosure. In some aspects, the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.3 PD-1 V_H Sequences

In some embodiments, the PD-1 antibody comprises, consists of, or consists essentially of a V_H sequence provided in SEQ ID NOs.: 211-250.

In some embodiments, the PD-1 antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 211-250. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 211. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 212. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 213. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 214. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 215. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 216. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 217. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 218. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 219. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 220. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 221. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 222. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 223. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 224. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 225.

In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 226. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 227. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 228. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 229. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 230. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 231. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 232. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 233. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 234. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 235. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 236. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 237. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 238. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 239. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 240. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 241. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 242. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 243. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 244. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 245. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 246. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 247. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 248. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 249. In some aspects, the antibody comprises a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 250.

3.3.1 Variants of V_H Sequences

In some embodiments, the V_H sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_H sequence provided in this disclosure. In some embodiments, the V_H sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_H sequence provided in WO 2016/077397.

In some aspects, the V_H sequence comprises, consists of, or consists essentially of a variant of an illustrative V_H sequence provided in this disclosure. In some aspects, the V_H sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_H sequences provided in this disclosure.

In some embodiments, the V_H sequence comprises, consists of, or consists essentially of any of the illustrative V_H sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.4 PD-1 CDR-L3 Sequences

In some embodiments, the PD-1 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of a V_L sequence provided in SEQ ID NOs.: 267-299. In some embodiments, the PD-1 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 189. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 190.

In some embodiments, the PD-1 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 277. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 278. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 279. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 280. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 281. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 282. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 283. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 284. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 285. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 286. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 287. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 288. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 289. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 290. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 291. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 292. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 293. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 294. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 295. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 296. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 297. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 298. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 299.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.5 PD-1 V_L Sequences Comprising Illustrative CDRs

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising one or more CDR-L sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-L sequences provided in this disclosure, and variants thereof.

3.5.1 CDR-L3

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence, wherein the CDR-L3 sequence comprises, consists of, or consists essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L3 sequence is a CDR-L3 sequence of a V_L sequence provided in SEQ ID NOs.: 267-299. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence, wherein the CDR-L3 sequence comprises, consists of, or consists essentially of a CDR-L3 sequence of an illustrative antibody or V_L sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 189. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 190.

In some embodiments, the PD-1 antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 277. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 278. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 279. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 280. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 281. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 282. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 283. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 284. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 285. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 286. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 287. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 288. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 289. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 290. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 291. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 292. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 293. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 294. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 295. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 296. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 297. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 298. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a CDR-L3 sequence of a V_L sequence of SEQ ID NO: 299.

3.5.2 CDR-L2

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L2 sequence, wherein the CDR-L2 sequence comprises, consists of, or consists essentially of a CDR-L2 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L2 sequence is a CDR-L2 sequence of a V_L sequence provided in SEQ ID NOs.: 267-299. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L2 sequence, wherein the CDR-L2 sequence comprises, consists of, or consists essentially of a CDR-L2 sequence of an illustrative antibody or V_L sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 166. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 167. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 168. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 169. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 170. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 171. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 172.

In some embodiments, the PD-1 antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 277. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 278. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 279. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 280. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 281. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 282. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 283. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 284. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 285. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 286. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 287. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 288. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 289. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 290. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 291. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 292. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 293. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 294. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 295. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 296. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 297. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 298. In some aspects, the antibody comprises a CDR-L2 sequence comprising, consisting of, or consisting essentially of a CDR-L2 sequence of a V_L sequence of SEQ ID NO: 299.

3.5.3 CDR-L1

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence, wherein the CDR-L1 sequence comprises, consists of, or consists essentially of a CDR-L1 sequence of an illustrative antibody or V_L sequence provided herein. In some aspects, the CDR-L1 sequence is a CDR-L1 sequence of a V_L sequence provided in SEQ ID NOs.: 267-299. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence, wherein the CDR-L1 sequence comprises, consists of, or consists essentially of a CDR-L1 sequence of an illustrative antibody or V_L sequence provided in WO 2016/077397.

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 148. In some aspects, the antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 149.

In some embodiments, the PD-1 antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 277. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 278. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 279. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 280. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 281. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 282. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 283. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 284. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 285. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 286. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 287. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 288. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 289. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 290. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 291. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 292. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 293. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 294. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 295. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 296. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 297. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 298. In some aspects, the antibody comprises a CDR-L1 sequence comprising, consisting of, or consisting essentially of a CDR-L1 sequence of a V_L sequence of SEQ ID NO: 299.

3.5.4 CDR-L3+CDR-L2

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299, and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299. In some aspects, the CDR-L3 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L2 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 267-299. In some aspects, the CDR-L3 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in WO 2016/077397.

3.5.5 CDR-L3+CDR-L1

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190 and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299, and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299. In some aspects, the CDR-L3 sequence and the CDR-L1 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L1 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 267-299. In some aspects, the CDR-L3 sequence and the CDR-L1 sequence are both from a single illustrative V_L sequence provided in WO 2016/077397.

3.5.6 CDR-L1+CDR-L2

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299, and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299. In some aspects, the CDR-L1 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L1 and CDR-L2 are both from a single illustrative V_L sequence selected from SEQ ID NOs: 267-299. In some aspects, the CDR-L1 sequence and the CDR-L2 sequence are both from a single illustrative V_L sequence provided in WO 2016/077397.

3.5.7 CDR-L1+CDR-L2+CDR-L3

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190. In some embodiments, the PD-1 antibody comprises a V_L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 267-299. In some aspects, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative V_L sequence provided in this disclosure. For example, in some aspects, the CDR-L1, CDR-L2, and CDR-L3 are all from a single illustrative V_L sequence selected from SEQ ID NOs: 267-299. In some aspects, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative V_L sequence provided in WO 2016/077397.

3.5.8 Variants of V_L Sequences Comprising Illustrative CDR-Ls

In some embodiments, the V_L sequences provided herein comprise a variant of an illustrative CDR-L3, CDR-L2, and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.6 PD-1 V_L Sequences

In some embodiments, the PD-1 antibody comprises, consists of, or consists essentially of a V_L sequence provided in SEQ ID NOs.: 267-299.

In some embodiments, the PD-1 antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 267-299. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 267. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 268. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 269. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 270. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 271. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 272. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 273. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 274.

In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 275. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 276. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 277. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 278. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 279. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 280. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 281. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 282. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 283. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 284. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 285. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 286. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 287. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 288. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 289. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 290. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 291. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 292. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 293. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 294. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 295. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 296. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 297. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 298. In some aspects, the antibody comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 299.

3.6.1 Variants of V_L Sequences

In some embodiments, the V_L sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_L sequence provided in this disclosure. In some embodiments, the V_L sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative V_L sequence provided in WO 2016/077397.

In some aspects, the V_L sequence comprises, consists of, or consists essentially of a variant of an illustrative V_L sequence provided in this disclosure. In some aspects, the V_L sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_L sequences provided in this disclosure.

In some embodiments, the V_L sequence comprises, consists of, or consists essentially of any of the illustrative V_L sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.7 PD-1 Pairs

3.7.1 CDR-H3-CDR-L3 Pairs

In some embodiments, the PD-1 antibody comprises a CDR-H3 sequence and a CDR-L3 sequence. In some aspects, the CDR-H3 sequence is part of a V_H and the CDR-L3 sequence is part of a V_L.

In some aspects, the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 130-131 and a CDR-H3 sequence of a a V_H sequence selected from SEQ ID NOs.: 228-250, and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 189-190 and a CDR-L3 sequence of a a V_L sequence selected from SEQ ID NOs.: 277-299.

3.7.1.1 Variants of CDR-H3-CDR-L3 Pairs

In some embodiments, the CDR-H3-CDR-L3 pairs provided herein comprise a variant of an illustrative CDR-H3 and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.7.2 CDR-H1-CDR-L1 Pairs

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence and a CDR-L1 sequence. In some aspects, the CDR-H1 sequence is part of a V_H and the CDR-L1 sequence is part of a V_L.

In some aspects, the CDR-H1 sequence is a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 24-38 and a Chothia CDR-H1 sequence of a a V_H sequence selected from SEQ ID NOs.: 228-250, and the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149 and a CDR-L1 sequence of a a V_L sequence selected from SEQ ID NOs.: 277-299.

In some aspects, the CDR-H1 sequence is a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 59-65 and a Kabat CDR-H1 sequence of a a V_H sequence selected from SEQ ID NOs.: 228-250, and the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 148-149 and a CDR-L1 sequence of a a V_L sequence selected from SEQ ID NOs.: 277-299.

3.7.2.1 Variants of CDR-H1-CDR-L1 Pairs

In some embodiments, the CDR-H1-CDR-L1 pairs provided herein comprise a variant of an illustrative CDR-H1 and/or CDR-L1 sequence provided in this disclosure.

In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H1 sequence provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H1 sequences provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.7.3 CDR-H2-CDR-L2 Pairs

In some embodiments, the PD-1 antibody comprises a CDR-H2 sequence and a CDR-L2 sequence. In some aspects, the CDR-H2 sequence is part of a V_H and the CDR-L2 sequence is part of a V_L.

In some aspects, the CDR-H2 sequence is a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 86-87 and a Chothia CDR-H2 sequence of a a V_H sequence selected from SEQ ID NOs.: 228-250, and the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172 and a CDR-L2 sequence of a a V_L sequence selected from SEQ ID NOs.: 277-299.

In some aspects, the CDR-H2 sequence is a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 108-109 and a Kabat CDR-H2 sequence of a a V_H sequence selected from SEQ ID NOs.: 228-250, and the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 166-172 and a CDR-L2 sequence of a a V_L sequence selected from SEQ ID NOs.: 277-299.

3.7.3.1 Variants of CDR-H2-CDR-L2 Pairs

In some embodiments, the CDR-H2-CDR-L2 pairs provided herein comprise a variant of an illustrative CDR-H2 and/or CDR-L2 sequence provided in this disclosure.

In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H2 sequence provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H2 sequences provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.7.4 V_H-V_L Pairs

In some embodiments, the PD-1 antibody comprises a V_H sequence and a V_L sequence.

In some aspects, the V_H sequence is a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 211-250, and the V_L sequence is a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 267-299.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 267 and SEQ ID NO: 211; SEQ ID NO: 267 and SEQ ID NO: 212; SEQ ID NO: 267 and SEQ ID NO: 213; SEQ ID NO: 267 and SEQ ID NO: 214; SEQ ID NO: 267 and SEQ ID NO: 215; SEQ ID NO: 267 and SEQ ID NO: 216; SEQ ID NO: 267 and SEQ ID NO: 217; SEQ ID NO: 267 and SEQ ID NO: 218; SEQ ID NO: 267 and SEQ ID NO: 219; SEQ ID NO: 267 and SEQ ID NO: 220; SEQ ID NO: 267 and SEQ ID NO: 221; SEQ ID NO: 267 and SEQ ID NO: 222; SEQ ID NO: 267 and SEQ ID NO: 223; SEQ ID NO: 267 and SEQ ID NO: 224; SEQ ID NO: 267 and SEQ ID NO: 225; SEQ ID NO: 267 and SEQ ID NO: 226; SEQ ID NO: 267 and SEQ ID NO: 227; SEQ ID NO: 267 and SEQ ID NO: 228; SEQ ID NO: 267 and SEQ ID NO: 229; SEQ ID NO: 267 and SEQ ID NO: 230; SEQ ID NO: 267 and SEQ ID NO: 231; SEQ ID NO: 267 and SEQ ID NO: 232; SEQ ID NO: 267 and SEQ ID NO: 233; SEQ ID NO: 267 and SEQ ID NO: 234; SEQ ID NO: 267 and SEQ ID NO: 235; SEQ ID NO: 267 and SEQ ID NO: 236; SEQ ID NO: 267 and SEQ ID NO: 237; SEQ ID NO: 267 and SEQ ID NO: 238; SEQ ID NO: 267 and SEQ ID NO: 239; SEQ ID NO: 267 and SEQ ID NO: 240; SEQ ID NO: 267 and SEQ ID NO: 241; SEQ ID NO: 267 and SEQ ID NO: 242; SEQ ID NO: 267 and SEQ ID NO: 243; SEQ ID NO: 267 and SEQ ID NO: 244; SEQ ID NO: 267 and SEQ ID NO: 245; SEQ ID NO: 267 and SEQ ID NO: 246; SEQ ID NO: 267 and SEQ ID NO: 247; SEQ ID NO: 267 and SEQ ID NO: 248; SEQ ID NO: 267 and SEQ ID NO: 249; and SEQ ID NO: 267 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 268 and SEQ ID NO: 211; SEQ ID NO: 268 and SEQ ID NO: 212; SEQ ID NO: 268 and SEQ ID NO: 213; SEQ ID NO: 268 and SEQ ID NO: 214; SEQ ID NO: 268 and SEQ ID NO: 215; SEQ ID NO: 268 and SEQ ID NO: 216; SEQ ID NO: 268 and SEQ ID NO: 217; SEQ ID NO: 268 and SEQ ID NO: 218; SEQ ID NO: 268 and SEQ ID NO: 219; SEQ ID NO: 268 and SEQ ID NO: 220; SEQ ID NO: 268 and SEQ ID NO: 221; SEQ ID NO: 268 and SEQ ID NO: 222; SEQ ID NO: 268 and SEQ ID NO: 223; SEQ ID NO: 268 and SEQ ID NO: 224; SEQ ID NO: 268 and SEQ ID NO: 225; SEQ ID NO: 268 and SEQ ID NO: 226; SEQ ID NO: 268 and SEQ ID NO: 227; SEQ ID NO: 268 and SEQ ID NO: 228; SEQ ID NO: 268 and SEQ ID NO: 229; SEQ ID NO: 268 and SEQ ID NO: 230; SEQ ID NO: 268 and SEQ ID NO: 231; SEQ ID NO: 268 and SEQ ID NO: 232; SEQ ID NO: 268 and SEQ ID NO: 233; SEQ ID NO: 268 and SEQ ID NO: 234; SEQ ID NO: 268 and SEQ ID NO: 235; SEQ ID NO: 268 and SEQ ID NO: 236; SEQ ID NO: 268 and SEQ ID NO: 237; SEQ ID NO: 268 and SEQ ID NO: 238; SEQ ID NO: 268 and SEQ ID NO: 239; SEQ ID NO: 268 and SEQ ID NO: 240; SEQ ID NO: 268 and SEQ ID NO: 241; SEQ ID NO: 268 and SEQ ID NO: 242; SEQ ID NO: 268 and SEQ ID NO: 243; SEQ ID NO: 268 and SEQ ID NO: 244; SEQ ID NO: 268 and SEQ ID NO: 245; SEQ ID NO: 268 and SEQ ID NO: 246; SEQ ID NO: 268 and SEQ ID NO: 247; SEQ ID NO: 268 and SEQ ID NO: 248; SEQ ID NO: 268 and SEQ ID NO: 249; and SEQ ID NO: 268 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 269 and SEQ ID NO: 211; SEQ ID NO: 269 and SEQ ID NO: 212; SEQ ID NO: 269 and SEQ ID NO: 213; SEQ ID NO: 269 and SEQ ID NO: 214; SEQ ID NO: 269 and SEQ ID NO: 215; SEQ ID NO: 269 and SEQ ID NO: 216; SEQ ID NO: 269 and SEQ ID NO: 217; SEQ ID NO: 269 and SEQ ID NO: 218; SEQ ID NO: 269 and SEQ ID NO: 219; SEQ ID NO: 269 and SEQ ID NO: 220; SEQ ID NO: 269 and SEQ ID NO: 221; SEQ ID NO: 269 and SEQ ID NO: 222; SEQ ID NO: 269 and SEQ ID NO: 223; SEQ ID NO: 269 and SEQ ID NO: 224; SEQ ID NO: 269 and SEQ ID NO: 225; SEQ ID NO: 269 and SEQ ID NO: 226; SEQ ID NO: 269 and SEQ ID NO: 227; SEQ ID NO: 269 and SEQ ID NO: 228; SEQ ID NO: 269 and SEQ ID NO: 229; SEQ ID NO: 269 and SEQ ID NO: 230; SEQ ID NO: 269 and SEQ ID NO: 231; SEQ ID NO: 269 and SEQ ID NO: 232; SEQ ID NO: 269 and SEQ ID NO: 233; SEQ ID NO: 269 and SEQ ID NO: 234; SEQ ID NO: 269 and SEQ ID NO: 235; SEQ ID NO: 269 and SEQ ID NO: 236; SEQ ID NO: 269 and SEQ ID NO: 237; SEQ ID NO: 269 and SEQ ID NO: 238; SEQ ID NO: 269 and SEQ ID NO: 239; SEQ ID NO: 269 and SEQ ID NO: 240; SEQ ID NO: 269 and SEQ ID NO: 241; SEQ ID NO: 269 and SEQ ID NO: 242; SEQ ID NO: 269 and SEQ ID NO: 243; SEQ ID NO: 269 and SEQ ID NO: 244; SEQ ID NO: 269 and SEQ ID NO: 245; SEQ ID NO: 269 and SEQ ID NO: 246; SEQ ID NO: 269 and SEQ ID NO: 247; SEQ ID NO: 269 and SEQ ID NO: 248; SEQ ID NO: 269 and SEQ ID NO: 249; and SEQ ID NO: 269 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 270 and SEQ ID NO: 211; SEQ ID NO: 270 and SEQ ID NO: 212; SEQ ID NO: 270 and SEQ ID NO: 213; SEQ ID NO: 270 and SEQ ID NO: 214; SEQ ID NO: 270 and SEQ ID NO: 215; SEQ ID NO: 270 and SEQ ID NO: 216; SEQ ID NO: 270 and SEQ ID NO: 217; SEQ ID NO: 270 and SEQ ID NO: 218; SEQ ID NO: 270 and SEQ ID NO: 219; SEQ ID NO: 270 and SEQ ID NO: 220; SEQ ID NO: 270 and SEQ ID NO: 221; SEQ ID NO: 270 and SEQ ID NO: 222; SEQ ID NO: 270 and SEQ ID NO: 223; SEQ ID NO: 270 and SEQ ID NO: 224; SEQ ID NO: 270 and SEQ ID NO: 225; SEQ ID NO: 270 and SEQ ID NO: 226; SEQ ID NO: 270 and SEQ ID NO: 227; SEQ ID NO: 270 and SEQ ID NO: 228; SEQ ID NO: 270 and SEQ ID NO: 229; SEQ ID NO: 270 and SEQ ID NO: 230; SEQ ID NO: 270 and SEQ ID NO: 231; SEQ ID NO: 270 and SEQ ID NO: 232; SEQ ID NO: 270 and SEQ ID NO: 233; SEQ ID NO: 270 and SEQ ID NO: 234; SEQ ID NO: 270 and SEQ ID NO: 235; SEQ ID NO: 270 and SEQ ID NO: 236; SEQ ID NO: 270 and SEQ ID NO: 237; SEQ ID NO: 270 and SEQ ID NO: 238; SEQ ID NO: 270 and SEQ ID NO: 239; SEQ ID NO: 270 and SEQ ID NO: 240; SEQ ID NO: 270 and SEQ ID NO: 241; SEQ ID NO: 270 and SEQ ID NO: 242; SEQ ID NO: 270 and SEQ ID NO: 243; SEQ ID NO: 270 and SEQ ID NO: 244; SEQ ID NO: 270 and SEQ ID NO: 245; SEQ ID NO: 270 and SEQ ID NO: 246; SEQ ID NO: 270 and SEQ ID NO: 247; SEQ ID NO: 270 and SEQ ID NO: 248; SEQ ID NO: 270 and SEQ ID NO: 249; and SEQ ID NO: 270 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 271 and SEQ ID NO: 211; SEQ ID NO: 271 and SEQ ID NO: 212; SEQ ID NO: 271 and SEQ ID NO: 213; SEQ ID NO: 271 and SEQ ID NO: 214; SEQ ID NO: 271 and SEQ ID NO: 215; SEQ ID NO: 271 and SEQ ID NO: 216; SEQ ID NO: 271 and SEQ ID NO: 217; SEQ ID NO: 271 and SEQ ID NO: 218; SEQ ID NO: 271 and SEQ ID NO: 219; SEQ ID NO: 271 and SEQ ID NO: 220; SEQ ID NO: 271 and SEQ ID NO: 221; SEQ ID NO: 271 and SEQ ID NO: 222; SEQ ID NO: 271 and SEQ ID NO: 223; SEQ ID NO: 271 and SEQ ID NO: 224; SEQ ID NO: 271 and SEQ ID NO: 225; SEQ ID NO: 271 and SEQ ID NO: 226; SEQ ID NO: 271 and SEQ ID NO: 227; SEQ ID NO: 271 and SEQ ID NO: 228; SEQ ID NO: 271 and SEQ ID NO: 229; SEQ ID NO: 271 and SEQ ID NO: 230; SEQ ID NO: 271 and SEQ ID NO: 231; SEQ ID NO: 271 and SEQ ID NO: 232; SEQ ID NO: 271 and SEQ ID NO: 233; SEQ ID NO: 271 and SEQ ID NO: 234; SEQ ID NO: 271 and SEQ ID NO: 235; SEQ ID NO: 271 and SEQ ID NO: 236; SEQ ID NO: 271 and SEQ ID NO: 237; SEQ ID NO: 271 and SEQ ID NO: 238; SEQ ID NO: 271 and SEQ ID NO: 239; SEQ ID NO: 271 and SEQ ID NO: 240; SEQ ID NO: 271 and SEQ ID NO: 241; SEQ ID NO: 271 and SEQ ID NO: 242; SEQ ID NO: 271 and SEQ ID NO: 243; SEQ ID NO: 271 and SEQ ID NO: 244; SEQ ID NO: 271 and SEQ ID NO: 245; SEQ ID NO: 271 and SEQ ID NO: 246; SEQ ID NO: 271 and SEQ ID NO: 247; SEQ ID NO: 271 and SEQ ID NO: 248; SEQ ID NO: 271 and SEQ ID NO: 249; and SEQ ID NO: 271 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 272 and SEQ ID NO: 211; SEQ ID NO: 272 and SEQ ID NO: 212; SEQ ID NO: 272 and SEQ ID NO: 213; SEQ ID NO: 272 and SEQ ID NO: 214; SEQ ID NO: 272 and SEQ ID NO: 215; SEQ ID NO: 272 and SEQ ID NO: 216; SEQ ID NO: 272 and SEQ ID NO: 217; SEQ ID NO: 272 and SEQ ID NO: 218; SEQ ID NO: 272 and SEQ ID NO: 219; SEQ ID NO: 272 and SEQ ID NO: 220; SEQ ID NO: 272 and SEQ ID NO: 221; SEQ ID NO: 272 and SEQ ID NO: 222; SEQ ID NO: 272 and SEQ ID NO: 223; SEQ ID NO: 272 and SEQ ID NO: 224; SEQ ID NO: 272 and SEQ ID NO: 225; SEQ ID NO: 272 and SEQ ID NO: 226; SEQ ID NO: 272 and SEQ ID NO: 227; SEQ ID NO: 272 and SEQ ID NO: 228; SEQ ID NO: 272 and SEQ ID NO: 229; SEQ ID NO: 272 and SEQ ID NO: 230; SEQ ID NO: 272 and SEQ ID NO: 231; SEQ ID NO: 272 and SEQ ID NO: 232; SEQ ID NO: 272 and SEQ ID NO: 233; SEQ ID NO: 272 and SEQ ID NO: 234; SEQ ID NO: 272 and SEQ ID NO: 235; SEQ ID NO: 272 and SEQ ID NO: 236; SEQ ID NO: 272 and SEQ ID NO: 237; SEQ ID NO: 272 and SEQ ID NO: 238; SEQ ID NO: 272 and SEQ ID NO: 239; SEQ ID NO: 272 and SEQ ID NO: 240; SEQ ID NO: 272 and SEQ ID NO: 241; SEQ ID NO: 272 and SEQ ID NO: 242; SEQ ID NO: 272 and SEQ ID NO: 243; SEQ ID NO: 272 and SEQ ID NO: 244; SEQ ID NO: 272 and SEQ ID NO: 245; SEQ ID NO: 272 and SEQ ID NO: 246; SEQ ID NO: 272 and SEQ ID NO: 247; SEQ ID NO: 272 and SEQ ID NO: 248; SEQ ID NO: 272 and SEQ ID NO: 249; and SEQ ID NO: 272 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 273 and SEQ ID NO: 211; SEQ ID NO: 273 and SEQ ID NO: 212; SEQ ID NO: 273 and SEQ ID NO: 213; SEQ ID NO: 273 and SEQ ID NO: 214; SEQ ID NO: 273 and SEQ ID NO: 215; SEQ ID NO: 273 and SEQ ID NO: 216; SEQ ID NO: 273 and SEQ ID NO: 217; SEQ ID NO: 273 and SEQ ID NO: 218; SEQ ID NO: 273 and SEQ ID NO: 219; SEQ ID NO: 273 and SEQ ID NO: 220; SEQ ID NO: 273 and SEQ ID NO: 221; SEQ ID NO: 273 and SEQ ID NO: 222; SEQ ID NO: 273 and SEQ ID NO: 223; SEQ ID NO: 273 and SEQ ID NO: 224; SEQ ID NO: 273 and SEQ ID NO: 225; SEQ ID NO: 273 and SEQ ID NO: 226; SEQ ID NO: 273 and SEQ ID NO: 227; SEQ ID NO: 273 and SEQ ID NO: 228; SEQ ID NO: 273 and SEQ ID NO: 229; SEQ ID NO: 273 and SEQ ID NO: 230; SEQ ID NO: 273 and SEQ ID NO: 231; SEQ ID NO: 273 and SEQ ID NO: 232; SEQ ID NO: 273 and SEQ ID NO: 233; SEQ ID NO: 273 and SEQ ID NO: 234; SEQ ID NO: 273 and SEQ ID NO: 235; SEQ ID NO: 273 and SEQ ID NO: 236; SEQ ID NO: 273 and SEQ ID NO: 237; SEQ ID NO: 273 and SEQ ID NO: 238; SEQ ID NO: 273 and SEQ ID NO: 239; SEQ ID NO: 273 and SEQ ID NO: 240; SEQ ID NO: 273 and SEQ ID NO: 241; SEQ ID NO: 273 and SEQ ID NO: 242; SEQ ID NO: 273 and SEQ ID NO: 243; SEQ ID NO: 273 and SEQ ID NO: 244; SEQ ID NO: 273 and SEQ ID NO: 245; SEQ ID NO: 273 and SEQ ID NO: 246; SEQ ID NO: 273 and SEQ ID NO: 247; SEQ ID NO: 273 and SEQ ID NO: 248; SEQ ID NO: 273 and SEQ ID NO: 249; and SEQ ID NO: 273 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 274 and SEQ ID NO: 211; SEQ ID NO: 274 and SEQ ID NO: 212; SEQ ID NO: 274 and SEQ ID NO: 213; SEQ ID NO: 274 and SEQ ID NO: 214; SEQ ID NO: 274 and SEQ ID NO: 215; SEQ ID NO: 274 and SEQ ID NO: 216; SEQ ID NO: 274 and SEQ ID NO: 217; SEQ ID NO: 274 and SEQ ID NO: 218; SEQ ID NO: 274 and SEQ ID NO: 219; SEQ ID NO: 274 and SEQ ID NO: 220; SEQ ID NO: 274 and SEQ ID NO: 221; SEQ ID NO: 274 and SEQ ID NO: 222; SEQ ID NO: 274 and SEQ ID NO: 223; SEQ ID NO: 274 and SEQ ID NO: 224; SEQ ID NO: 274 and SEQ ID NO: 225; SEQ ID NO: 274 and SEQ ID NO: 226; SEQ ID NO: 274 and SEQ ID NO: 227; SEQ ID NO: 274 and SEQ ID NO: 228; SEQ ID NO: 274 and SEQ ID NO: 229; SEQ ID NO: 274 and SEQ ID NO: 230; SEQ ID NO: 274 and SEQ ID NO: 231; SEQ ID NO: 274 and SEQ ID NO: 232; SEQ ID NO: 274 and SEQ ID NO: 233; SEQ ID NO: 274 and SEQ ID NO: 234; SEQ ID NO: 274 and SEQ ID NO: 235; SEQ ID NO: 274 and SEQ ID NO: 236; SEQ ID NO: 274 and SEQ ID NO: 237; SEQ ID NO: 274 and SEQ ID NO: 238; SEQ ID NO: 274 and SEQ ID NO: 239; SEQ ID NO: 274 and SEQ ID NO: 240; SEQ ID NO: 274 and SEQ ID NO: 241; SEQ ID NO: 274 and SEQ ID NO: 242; SEQ ID NO: 274 and SEQ ID NO: 243; SEQ ID NO: 274 and SEQ ID NO: 244; SEQ ID NO: 274 and SEQ ID NO: 245; SEQ ID NO: 274 and SEQ ID NO: 246; SEQ ID NO: 274 and SEQ ID NO: 247; SEQ ID NO: 274 and SEQ ID NO: 248; SEQ ID NO: 274 and SEQ ID NO: 249; and SEQ ID NO: 274 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 275 and SEQ ID NO: 211; SEQ ID NO: 275 and SEQ ID NO: 212; SEQ ID NO: 275 and SEQ ID NO: 213; SEQ ID NO: 275 and SEQ ID NO: 214; SEQ ID NO: 275 and SEQ ID NO: 215; SEQ ID NO: 275 and SEQ ID NO: 216; SEQ ID NO: 275 and SEQ ID NO: 217; SEQ ID NO: 275 and SEQ ID NO: 218; SEQ ID NO: 275 and SEQ ID NO: 219; SEQ ID NO: 275 and SEQ ID NO: 220; SEQ ID NO: 275 and SEQ ID NO: 221; SEQ ID NO: 275 and SEQ ID NO: 222; SEQ ID NO: 275 and SEQ ID NO: 223; SEQ ID NO: 275 and SEQ ID NO: 224; SEQ ID NO: 275 and SEQ ID NO: 225; SEQ ID NO: 275 and SEQ ID NO: 226; SEQ ID NO: 275 and SEQ ID NO: 227; SEQ ID NO: 275 and SEQ ID NO: 228; SEQ ID NO: 275 and SEQ ID NO: 229; SEQ ID NO: 275 and SEQ ID NO: 230; SEQ ID NO: 275 and SEQ ID NO: 231; SEQ ID NO: 275 and SEQ ID NO: 232; SEQ ID NO: 275 and SEQ ID NO: 233; SEQ ID NO: 275 and SEQ ID NO: 234; SEQ ID NO: 275 and SEQ ID NO: 235; SEQ ID NO: 275 and SEQ ID NO: 236; SEQ ID NO: 275 and SEQ ID NO: 237; SEQ ID NO: 275 and SEQ ID NO: 238; SEQ ID NO: 275 and SEQ ID NO: 239; SEQ ID NO: 275 and SEQ ID NO: 240; SEQ ID NO: 275 and SEQ ID NO: 241; SEQ ID NO: 275 and SEQ ID NO: 242; SEQ ID NO: 275 and SEQ ID NO: 243; SEQ ID NO: 275 and SEQ ID NO: 244; SEQ ID NO: 275 and SEQ ID NO: 245; SEQ ID NO: 275 and SEQ ID NO: 246; SEQ ID NO: 275 and SEQ ID NO: 247; SEQ ID NO: 275 and SEQ ID NO: 248; SEQ ID NO: 275 and SEQ ID NO: 249; and SEQ ID NO: 275 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 276 and SEQ ID NO: 211; SEQ ID NO: 276 and SEQ ID NO: 212; SEQ ID NO: 276 and SEQ ID NO: 213; SEQ ID NO: 276 and SEQ ID NO: 214; SEQ ID NO: 276 and SEQ ID NO: 215; SEQ ID NO: 276 and SEQ ID NO: 216; SEQ ID NO: 276 and SEQ ID NO: 217; SEQ ID NO: 276 and SEQ ID NO: 218; SEQ ID NO: 276 and SEQ ID NO: 219; SEQ ID NO: 276 and SEQ ID NO: 220; SEQ ID NO: 276 and SEQ ID NO: 221; SEQ ID NO: 276 and SEQ ID NO: 222; SEQ ID NO: 276 and SEQ ID NO: 223; SEQ ID NO: 276 and SEQ ID NO: 224; SEQ ID NO: 276 and SEQ ID NO: 225; SEQ ID NO: 276 and SEQ ID NO: 226; SEQ ID NO: 276 and SEQ ID NO: 227; SEQ ID NO: 276 and SEQ ID NO: 228; SEQ ID NO: 276 and SEQ ID NO: 229; SEQ ID NO: 276 and SEQ ID NO: 230; SEQ ID NO: 276 and SEQ ID NO: 231; SEQ ID NO: 276 and SEQ ID NO: 232; SEQ ID NO: 276 and SEQ ID NO: 233; SEQ ID NO: 276 and SEQ ID NO: 234; SEQ ID NO: 276 and SEQ ID NO: 235; SEQ ID NO: 276 and SEQ ID NO: 236; SEQ ID NO: 276 and SEQ ID NO: 237; SEQ ID NO: 276 and SEQ ID NO: 238; SEQ ID NO: 276 and SEQ ID NO: 239; SEQ ID NO: 276 and SEQ ID NO: 240; SEQ ID NO: 276 and SEQ ID NO: 241; SEQ ID NO: 276 and SEQ ID NO: 242; SEQ ID NO: 276 and SEQ ID NO: 243; SEQ ID NO: 276 and SEQ ID NO: 244; SEQ ID NO: 276 and SEQ ID NO: 245; SEQ ID NO: 276 and SEQ ID NO: 246; SEQ ID NO: 276 and SEQ ID NO: 247; SEQ ID NO: 276 and SEQ ID NO: 248; SEQ ID NO: 276 and SEQ ID NO: 249; and SEQ ID NO: 276 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 277 and SEQ ID NO: 211; SEQ ID NO: 277 and SEQ ID NO: 212; SEQ ID NO: 277 and SEQ ID NO: 213; SEQ ID NO: 277 and SEQ ID NO: 214; SEQ ID NO: 277 and SEQ ID NO: 215; SEQ ID NO: 277 and SEQ ID NO: 216; SEQ ID NO: 277 and SEQ ID NO: 217; SEQ ID NO: 277 and SEQ ID NO: 218; SEQ ID NO: 277 and SEQ ID NO: 219; SEQ ID NO: 277 and SEQ ID NO: 220; SEQ ID NO: 277 and SEQ ID NO: 221; SEQ ID NO: 277 and SEQ ID NO: 222; SEQ ID NO: 277 and SEQ ID NO: 223; SEQ ID NO: 277 and SEQ ID NO: 224; SEQ ID NO: 277 and SEQ ID NO: 225; SEQ ID NO: 277 and SEQ ID NO: 226; SEQ ID NO: 277 and SEQ ID NO: 227; SEQ ID NO: 277 and SEQ ID NO: 228; SEQ ID NO: 277 and SEQ ID NO: 229; SEQ ID NO: 277 and SEQ ID NO: 230; SEQ ID NO: 277 and SEQ ID NO: 231; SEQ ID NO: 277 and SEQ ID NO: 232; SEQ ID NO: 277 and SEQ ID NO: 233; SEQ ID NO: 277 and SEQ ID NO: 234; SEQ ID NO: 277 and SEQ ID NO: 235; SEQ ID NO: 277 and SEQ ID NO: 236; SEQ ID NO: 277 and SEQ ID NO: 237; SEQ ID NO: 277 and SEQ ID NO: 238; SEQ ID NO: 277 and SEQ ID NO: 239; SEQ ID NO: 277 and SEQ ID NO: 240; SEQ ID NO: 277 and SEQ ID NO: 241; SEQ ID NO: 277 and SEQ ID NO: 242; SEQ ID NO: 277 and SEQ ID NO: 243; SEQ ID NO: 277 and SEQ ID NO: 244; SEQ ID NO: 277 and SEQ ID NO: 245; SEQ ID NO: 277 and SEQ ID NO: 246; SEQ ID NO: 277 and SEQ ID NO: 247; SEQ ID NO: 277 and SEQ ID NO: 248; SEQ ID NO: 277 and SEQ ID NO: 249; and SEQ ID NO: 277 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 278 and SEQ ID NO: 211; SEQ ID NO: 278 and SEQ ID NO: 212; SEQ ID NO: 278 and SEQ ID NO: 213; SEQ ID NO: 278 and SEQ ID NO: 214; SEQ ID NO: 278 and SEQ ID NO: 215; SEQ ID NO: 278 and SEQ ID NO: 216; SEQ ID NO: 278 and SEQ ID NO: 217; SEQ ID NO: 278 and SEQ ID NO: 218; SEQ ID NO: 278 and SEQ ID NO: 219; SEQ ID NO: 278 and SEQ ID NO: 220; SEQ ID NO: 278 and SEQ ID NO: 221; SEQ ID NO: 278 and SEQ ID NO: 222; SEQ ID NO: 278 and SEQ ID NO: 223; SEQ ID NO: 278 and SEQ ID NO: 224; SEQ ID NO: 278 and SEQ ID NO: 225; SEQ ID NO: 278 and SEQ ID NO: 226; SEQ ID NO: 278 and SEQ ID NO: 227; SEQ ID NO: 278 and SEQ ID NO: 228; SEQ ID NO: 278 and SEQ ID NO: 229; SEQ ID NO: 278 and SEQ ID NO: 230; SEQ ID NO: 278 and SEQ ID NO: 231; SEQ ID NO: 278 and SEQ ID NO: 232; SEQ ID NO: 278 and SEQ ID NO: 233; SEQ ID NO: 278 and SEQ ID NO: 234; SEQ ID NO: 278 and SEQ ID NO: 235; SEQ ID NO: 278 and SEQ ID NO: 236; SEQ ID NO: 278 and SEQ ID NO: 237; SEQ ID NO: 278 and SEQ ID NO: 238; SEQ ID NO: 278 and SEQ ID NO: 239; SEQ ID NO: 278 and SEQ ID NO: 240; SEQ ID NO: 278 and SEQ ID NO: 241; SEQ ID NO: 278 and SEQ ID NO: 242; SEQ ID NO: 278 and SEQ ID NO: 243; SEQ ID NO: 278 and SEQ ID NO: 244; SEQ ID NO: 278 and SEQ ID NO: 245; SEQ ID NO: 278 and SEQ ID NO: 246; SEQ ID NO: 278 and SEQ ID NO: 247; SEQ ID NO: 278 and SEQ ID NO: 248; SEQ ID NO: 278 and SEQ ID NO: 249; and SEQ ID NO: 278 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 279 and SEQ ID NO: 211; SEQ ID NO: 279 and SEQ ID NO: 212; SEQ ID NO: 279 and SEQ ID NO: 213; SEQ ID NO: 279 and SEQ ID NO: 214; SEQ ID NO: 279 and SEQ ID NO: 215; SEQ ID NO: 279 and SEQ ID NO: 216; SEQ ID NO: 279 and SEQ ID NO: 217; SEQ ID NO: 279 and SEQ ID NO: 218; SEQ ID NO: 279 and SEQ ID NO: 219; SEQ ID NO: 279 and SEQ ID NO: 220; SEQ ID NO: 279 and SEQ ID NO: 221; SEQ ID NO: 279 and SEQ ID NO: 222; SEQ ID NO: 279 and SEQ ID NO: 223; SEQ ID NO: 279 and SEQ ID NO: 224; SEQ ID NO: 279 and SEQ ID NO: 225; SEQ ID NO: 279 and SEQ ID NO: 226; SEQ ID NO: 279 and SEQ ID NO: 227; SEQ ID NO: 279 and SEQ ID NO: 228; SEQ ID NO: 279 and SEQ ID NO: 229; SEQ ID NO: 279 and SEQ ID NO: 230; SEQ ID NO: 279 and SEQ ID NO: 231; SEQ ID NO: 279 and SEQ ID NO: 232; SEQ ID NO: 279 and SEQ ID NO: 233; SEQ ID NO: 279 and SEQ ID NO: 234; SEQ ID NO: 279 and SEQ ID NO: 235; SEQ ID NO: 279 and SEQ ID NO: 236; SEQ ID NO: 279 and SEQ ID NO: 237; SEQ ID NO: 279 and SEQ ID NO: 238; SEQ ID NO: 279 and SEQ ID NO: 239; SEQ ID NO: 279 and SEQ ID NO: 240; SEQ ID NO: 279 and SEQ ID NO: 241; SEQ ID NO: 279 and SEQ ID NO: 242; SEQ ID NO: 279 and SEQ ID NO: 243; SEQ ID NO: 279 and SEQ ID NO: 244; SEQ ID NO: 279 and SEQ ID NO: 245; SEQ ID NO: 279 and SEQ ID NO: 246; SEQ ID NO: 279 and SEQ ID NO: 247; SEQ ID NO: 279 and SEQ ID NO: 248; SEQ ID NO: 279 and SEQ ID NO: 249; and SEQ ID NO: 279 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 280 and SEQ ID NO: 211; SEQ ID NO: 280 and SEQ ID NO: 212; SEQ ID NO: 280 and SEQ ID NO: 213; SEQ ID NO: 280 and SEQ ID NO: 214; SEQ ID NO: 280 and SEQ ID NO: 215; SEQ ID NO: 280 and SEQ ID NO: 216; SEQ ID NO: 280 and SEQ ID NO: 217; SEQ ID NO: 280 and SEQ ID NO: 218; SEQ ID NO: 280 and SEQ ID NO: 219; SEQ ID NO: 280 and SEQ ID NO: 220; SEQ ID NO: 280 and SEQ ID NO: 221; SEQ ID NO: 280 and SEQ ID NO: 222; SEQ ID NO: 280 and SEQ ID NO: 223; SEQ ID NO: 280 and SEQ ID NO: 224; SEQ ID NO: 280 and SEQ ID NO: 225; SEQ ID NO: 280 and SEQ ID NO: 226; SEQ ID NO: 280 and SEQ ID NO: 227; SEQ ID NO: 280 and SEQ ID NO: 228; SEQ ID NO: 280 and SEQ ID NO: 229; SEQ ID NO: 280 and SEQ ID NO: 230; SEQ ID NO: 280 and SEQ ID NO: 231; SEQ ID NO: 280 and SEQ ID NO: 232; SEQ ID NO: 280 and SEQ ID NO: 233; SEQ ID NO: 280 and SEQ ID NO: 234; SEQ ID NO: 280 and SEQ ID NO: 235; SEQ ID NO: 280 and SEQ ID NO: 236; SEQ ID NO: 280 and SEQ ID NO: 237; SEQ ID NO: 280 and SEQ ID NO: 238; SEQ ID NO: 280 and SEQ ID NO: 239; SEQ ID NO: 280 and SEQ ID NO: 240; SEQ ID NO: 280 and SEQ ID NO: 241; SEQ ID NO: 280 and SEQ ID NO: 242; SEQ ID NO: 280 and SEQ ID NO: 243; SEQ ID NO: 280 and SEQ ID NO: 244; SEQ ID NO: 280 and SEQ ID NO: 245; SEQ ID NO: 280 and SEQ ID NO: 246; SEQ ID NO: 280 and SEQ ID NO: 247; SEQ ID NO: 280 and SEQ ID NO: 248; SEQ ID NO: 280 and SEQ ID NO: 249; and SEQ ID NO: 280 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 281 and SEQ ID NO: 211; SEQ ID NO: 281 and SEQ ID NO: 212; SEQ ID NO: 281 and SEQ ID NO: 213; SEQ ID NO: 281 and SEQ ID NO: 214; SEQ ID NO: 281 and SEQ ID NO: 215; SEQ ID NO: 281 and SEQ ID NO: 216; SEQ ID NO: 281 and SEQ ID NO: 217; SEQ ID NO: 281 and SEQ ID NO: 218; SEQ ID NO: 281 and SEQ ID NO: 219; SEQ ID NO: 281 and SEQ ID NO: 220; SEQ ID NO: 281 and SEQ ID NO: 221; SEQ ID NO: 281 and SEQ ID NO: 222; SEQ ID NO: 281 and SEQ ID NO: 223; SEQ ID NO: 281 and SEQ ID NO: 224; SEQ ID NO: 281 and SEQ ID NO: 225; SEQ ID NO: 281 and SEQ ID NO: 226; SEQ ID NO: 281 and SEQ ID NO: 227; SEQ ID NO: 281 and SEQ ID NO: 228; SEQ ID NO: 281 and SEQ ID NO: 229; SEQ ID NO: 281 and SEQ ID NO: 230; SEQ ID NO: 281 and SEQ ID NO: 231; SEQ ID NO: 281 and SEQ ID NO: 232; SEQ ID NO: 281 and SEQ ID NO: 233; SEQ ID NO: 281 and SEQ ID NO: 234; SEQ ID NO: 281 and SEQ ID NO: 235; SEQ ID NO: 281 and SEQ ID NO: 236; SEQ ID NO: 281 and SEQ ID NO: 237; SEQ ID NO: 281 and SEQ ID NO: 238; SEQ ID NO: 281 and SEQ ID NO: 239; SEQ ID NO: 281 and SEQ ID NO: 240; SEQ ID NO: 281 and SEQ ID NO: 241; SEQ ID NO: 281 and SEQ ID NO: 242; SEQ ID NO: 281 and SEQ ID NO: 243; SEQ ID NO: 281 and SEQ ID NO: 244; SEQ ID NO: 281 and SEQ ID NO: 245; SEQ ID NO: 281 and SEQ ID NO: 246; SEQ ID NO: 281 and SEQ ID NO: 247; SEQ ID NO: 281 and SEQ ID NO: 248; SEQ ID NO: 281 and SEQ ID NO: 249; and SEQ ID NO: 281 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 282 and SEQ ID NO: 211; SEQ ID NO: 282 and SEQ ID NO: 212; SEQ ID NO: 282 and SEQ ID NO: 213; SEQ ID NO: 282 and SEQ ID NO: 214; SEQ ID NO: 282 and SEQ ID NO: 215; SEQ ID NO: 282 and SEQ ID NO: 216; SEQ ID NO: 282 and SEQ ID NO: 217; SEQ ID NO: 282 and SEQ ID NO: 218; SEQ ID NO: 282 and SEQ ID NO: 219; SEQ ID NO: 282 and SEQ ID NO: 220; SEQ ID NO: 282 and SEQ ID NO: 221; SEQ ID NO: 282 and SEQ ID NO: 222; SEQ ID NO: 282 and SEQ ID NO: 223; SEQ ID NO: 282 and SEQ ID NO: 224; SEQ ID NO: 282 and SEQ ID NO: 225; SEQ ID NO: 282 and SEQ ID NO: 226; SEQ ID NO: 282 and SEQ ID NO: 227; SEQ ID NO: 282 and SEQ ID NO: 228; SEQ ID NO: 282 and SEQ ID NO: 229; SEQ ID NO: 282 and SEQ ID NO: 230; SEQ ID NO: 282 and SEQ ID NO: 231; SEQ ID NO: 282 and SEQ ID NO: 232; SEQ ID NO: 282 and SEQ ID NO: 233; SEQ ID NO: 282 and SEQ ID NO: 234; SEQ ID NO: 282 and SEQ ID NO: 235; SEQ ID NO: 282 and SEQ ID NO: 236; SEQ ID NO: 282 and SEQ ID NO: 237; SEQ ID NO: 282 and SEQ ID NO: 238; SEQ ID NO: 282 and SEQ ID NO: 239; SEQ ID NO: 282 and SEQ ID NO: 240; SEQ ID NO: 282 and SEQ ID NO: 241; SEQ ID NO: 282 and SEQ ID NO: 242; SEQ ID NO: 282 and SEQ ID NO: 243; SEQ ID NO: 282 and SEQ ID NO: 244; SEQ ID NO: 282 and SEQ ID NO: 245; SEQ ID NO: 282 and SEQ ID NO: 246; SEQ ID NO: 282 and SEQ ID NO: 247; SEQ ID NO: 282 and SEQ ID NO: 248; SEQ ID NO: 282 and SEQ ID NO: 249; and SEQ ID NO: 282 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 283 and SEQ ID NO: 211; SEQ ID NO: 283 and SEQ ID NO: 212; SEQ ID NO: 283 and SEQ ID NO: 213; SEQ ID NO: 283 and SEQ ID NO: 214; SEQ ID NO: 283 and SEQ ID NO: 215; SEQ ID NO: 283 and SEQ ID NO: 216; SEQ ID NO: 283 and SEQ ID NO: 217; SEQ ID NO: 283 and SEQ ID NO: 218; SEQ ID NO: 283 and SEQ ID NO: 219; SEQ ID NO: 283 and SEQ ID NO: 220; SEQ ID NO: 283 and SEQ ID NO: 221; SEQ ID NO: 283 and SEQ ID NO: 222; SEQ ID NO: 283 and SEQ ID NO: 223; SEQ ID NO: 283 and SEQ ID NO: 224; SEQ ID NO: 283 and SEQ ID NO: 225; SEQ ID NO: 283 and SEQ ID NO: 226; SEQ ID NO: 283 and SEQ ID NO: 227; SEQ ID NO: 283 and SEQ ID NO: 228; SEQ ID NO: 283 and SEQ ID NO: 229; SEQ ID NO: 283 and SEQ ID NO: 230; SEQ ID NO: 283 and SEQ ID NO: 231; SEQ ID NO: 283 and SEQ ID NO: 232; SEQ ID NO: 283 and SEQ ID NO: 233; SEQ ID NO: 283 and SEQ ID NO: 234; SEQ ID NO: 283 and SEQ ID NO: 235; SEQ ID NO: 283 and SEQ ID NO: 236; SEQ ID NO: 283 and SEQ ID NO: 237; SEQ ID NO: 283 and SEQ ID NO: 238; SEQ ID NO: 283 and SEQ ID NO: 239; SEQ ID NO: 283 and SEQ ID NO: 240; SEQ ID NO: 283 and SEQ ID NO: 241; SEQ ID NO: 283 and SEQ ID NO: 242; SEQ ID NO: 283 and SEQ ID NO: 243; SEQ ID NO: 283 and SEQ ID NO: 244; SEQ ID NO: 283 and SEQ ID NO: 245; SEQ ID NO: 283 and SEQ ID NO: 246; SEQ ID NO: 283 and SEQ ID NO: 247; SEQ ID NO: 283 and SEQ ID NO: 248; SEQ ID NO: 283 and SEQ ID NO: 249; and SEQ ID NO: 283 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 284 and SEQ ID NO: 211; SEQ ID NO: 284 and SEQ ID NO: 212; SEQ ID NO: 284 and SEQ ID NO: 213; SEQ ID NO: 284 and SEQ ID NO: 214; SEQ ID NO: 284 and SEQ ID NO: 215; SEQ ID NO: 284 and SEQ ID NO: 216; SEQ ID NO: 284 and SEQ ID NO: 217; SEQ ID NO: 284 and SEQ ID NO: 218; SEQ ID NO: 284 and SEQ ID NO: 219; SEQ ID NO: 284 and SEQ ID NO: 220; SEQ ID NO: 284 and SEQ ID NO: 221; SEQ ID NO: 284 and SEQ ID NO: 222; SEQ ID NO: 284 and SEQ ID NO: 223; SEQ ID NO: 284 and SEQ ID NO: 224; SEQ ID NO: 284 and SEQ ID NO: 225; SEQ ID NO: 284 and SEQ ID NO: 226; SEQ ID NO: 284 and SEQ ID NO: 227; SEQ ID NO: 284 and SEQ ID NO: 228; SEQ ID NO: 284 and SEQ ID NO: 229; SEQ ID NO: 284 and SEQ ID NO: 230; SEQ ID NO: 284 and SEQ ID NO: 231; SEQ ID NO: 284 and SEQ ID NO: 232; SEQ ID NO: 284 and SEQ ID NO: 233; SEQ ID NO: 284 and SEQ ID NO: 234; SEQ ID NO: 284 and SEQ ID NO: 235; SEQ ID NO: 284 and SEQ ID NO: 236; SEQ ID NO: 284 and SEQ ID NO: 237; SEQ ID NO: 284 and SEQ ID NO: 238; SEQ ID NO: 284 and SEQ ID NO: 239; SEQ ID NO: 284 and SEQ ID NO: 240; SEQ ID NO: 284 and SEQ ID NO: 241; SEQ ID NO: 284 and SEQ ID NO: 242; SEQ ID NO: 284 and SEQ ID NO: 243; SEQ ID NO: 284 and SEQ ID NO: 244; SEQ ID NO: 284 and SEQ ID NO: 245; SEQ ID NO: 284 and SEQ ID NO: 246; SEQ ID NO: 284 and SEQ ID NO: 247; SEQ ID NO: 284 and SEQ ID NO: 248; SEQ ID NO: 284 and SEQ ID NO: 249; and SEQ ID NO: 284 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 285 and SEQ ID NO: 211; SEQ ID NO: 285 and SEQ ID NO: 212; SEQ ID NO: 285 and SEQ ID NO: 213; SEQ ID NO: 285 and SEQ ID NO: 214; SEQ ID NO: 285 and SEQ ID NO: 215; SEQ ID NO: 285 and SEQ ID NO: 216; SEQ ID NO: 285 and SEQ ID NO: 217; SEQ ID NO: 285 and SEQ ID NO: 218; SEQ ID NO: 285 and SEQ ID NO: 219; SEQ ID NO: 285 and SEQ ID NO: 220; SEQ ID NO: 285 and SEQ ID NO: 221; SEQ ID NO: 285 and SEQ ID NO: 222; SEQ ID NO: 285 and SEQ ID NO: 223; SEQ ID NO: 285 and SEQ ID NO: 224; SEQ ID NO: 285 and SEQ ID NO: 225; SEQ ID NO: 285 and SEQ ID NO: 226; SEQ ID NO: 285 and SEQ ID NO: 227; SEQ ID NO: 285 and SEQ ID NO: 228; SEQ ID NO: 285 and SEQ ID NO: 229; SEQ ID NO: 285 and SEQ ID NO: 230; SEQ ID NO: 285 and SEQ ID NO: 231; SEQ ID NO: 285 and SEQ ID NO: 232; SEQ ID NO: 285 and SEQ ID NO: 233; SEQ ID NO: 285 and SEQ ID NO: 234; SEQ ID NO: 285 and SEQ ID NO: 235; SEQ ID NO: 285 and SEQ ID NO: 236; SEQ ID NO: 285 and SEQ ID NO: 237; SEQ ID NO: 285 and SEQ ID NO: 238; SEQ ID NO: 285 and SEQ ID NO: 239; SEQ ID NO: 285 and SEQ ID NO: 240; SEQ ID NO: 285 and SEQ ID NO: 241; SEQ ID NO: 285 and SEQ ID NO: 242; SEQ ID NO: 285 and SEQ ID NO: 243; SEQ ID NO: 285 and SEQ ID NO: 244; SEQ ID NO: 285 and SEQ ID NO: 245; SEQ ID NO: 285 and SEQ ID NO: 246; SEQ ID NO: 285 and SEQ ID NO: 247; SEQ ID NO: 285 and SEQ ID NO: 248; SEQ ID NO: 285 and SEQ ID NO: 249; and SEQ ID NO: 285 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 286 and SEQ ID NO: 211; SEQ ID NO: 286 and SEQ ID NO: 212; SEQ ID NO: 286 and SEQ ID NO: 213; SEQ ID NO: 286 and SEQ ID NO: 214; SEQ ID NO: 286 and SEQ ID NO: 215; SEQ ID NO: 286 and SEQ ID NO: 216; SEQ ID NO: 286 and SEQ ID NO: 217; SEQ ID NO: 286 and SEQ ID NO: 218; SEQ ID NO: 286 and SEQ ID NO: 219; SEQ ID NO: 286 and SEQ ID NO: 220; SEQ ID NO: 286 and SEQ ID NO: 221; SEQ ID NO: 286 and SEQ ID NO: 222; SEQ ID NO: 286 and SEQ ID NO: 223; SEQ ID NO: 286 and SEQ ID NO: 224; SEQ ID NO: 286 and SEQ ID NO: 225; SEQ ID NO: 286 and SEQ ID NO: 226; SEQ ID NO: 286 and SEQ ID NO: 227; SEQ ID NO: 286 and SEQ ID NO: 228; SEQ ID NO: 286 and SEQ ID NO: 229; SEQ ID NO: 286 and SEQ ID NO: 230; SEQ ID NO: 286 and SEQ ID NO: 231; SEQ ID NO: 286 and SEQ ID NO: 232; SEQ ID NO: 286 and SEQ ID NO: 233; SEQ ID NO: 286 and SEQ ID NO: 234; SEQ ID NO: 286 and SEQ ID NO: 235; SEQ ID NO: 286 and SEQ ID NO: 236; SEQ ID NO: 286 and SEQ ID NO: 237; SEQ ID NO: 286 and SEQ ID NO: 238; SEQ ID NO: 286 and SEQ ID NO: 239; SEQ ID NO: 286 and SEQ ID NO: 240; SEQ ID NO: 286 and SEQ ID NO: 241; SEQ ID NO: 286 and SEQ ID NO: 242; SEQ ID NO: 286 and SEQ ID NO: 243; SEQ ID NO: 286 and SEQ ID NO: 244; SEQ ID NO: 286 and SEQ ID NO: 245; SEQ ID NO: 286 and SEQ ID NO: 246; SEQ ID NO: 286 and SEQ ID NO: 247; SEQ ID NO: 286 and SEQ ID NO: 248; SEQ ID NO: 286 and SEQ ID NO: 249; and SEQ ID NO: 286 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 287 and SEQ ID NO: 211; SEQ ID NO: 287 and SEQ ID NO: 212; SEQ ID NO: 287 and SEQ ID NO: 213; SEQ ID NO: 287 and SEQ ID NO: 214; SEQ ID NO: 287 and SEQ ID NO: 215; SEQ ID NO: 287 and SEQ ID NO: 216; SEQ ID NO: 287 and SEQ ID NO: 217; SEQ ID NO: 287 and SEQ ID NO: 218; SEQ ID NO: 287 and SEQ ID NO: 219; SEQ ID NO: 287 and SEQ ID NO: 220; SEQ ID NO: 287 and SEQ ID NO: 221; SEQ ID NO: 287 and SEQ ID NO: 222; SEQ ID NO: 287 and SEQ ID NO: 223; SEQ ID NO: 287 and SEQ ID NO: 224; SEQ ID NO: 287 and SEQ ID NO: 225; SEQ ID NO: 287 and SEQ ID NO: 226; SEQ ID NO: 287 and SEQ ID NO: 227; SEQ ID NO: 287 and SEQ ID NO: 228; SEQ ID NO: 287 and SEQ ID NO: 229; SEQ ID NO: 287 and SEQ ID NO: 230; SEQ ID NO: 287 and SEQ ID NO: 231; SEQ ID NO: 287 and SEQ ID NO: 232; SEQ ID NO: 287 and SEQ ID NO: 233; SEQ ID NO: 287 and SEQ ID NO: 234; SEQ ID NO: 287 and SEQ ID NO: 235; SEQ ID NO: 287 and SEQ ID NO: 236; SEQ ID NO: 287 and SEQ ID NO: 237; SEQ ID NO: 287 and SEQ ID NO: 238; SEQ ID NO: 287 and SEQ ID NO: 239; SEQ ID NO: 287 and SEQ ID NO: 240; SEQ ID NO: 287 and SEQ ID NO: 241; SEQ ID NO: 287 and SEQ ID NO: 242; SEQ ID NO: 287 and SEQ ID NO: 243; SEQ ID NO: 287 and SEQ ID NO: 244; SEQ ID NO: 287 and SEQ ID NO: 245; SEQ ID NO: 287 and SEQ ID NO: 246; SEQ ID NO: 287 and SEQ ID NO: 247; SEQ ID NO: 287 and SEQ ID NO: 248; SEQ ID NO: 287 and SEQ ID NO: 249; and SEQ ID NO: 287 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 288 and SEQ ID NO: 211; SEQ ID NO: 288 and SEQ ID NO: 212; SEQ ID NO: 288 and SEQ ID NO: 213; SEQ ID NO: 288 and SEQ ID NO: 214; SEQ ID NO: 288 and SEQ ID NO: 215; SEQ ID NO: 288 and SEQ ID NO: 216; SEQ ID NO: 288 and SEQ ID NO: 217; SEQ ID NO: 288 and SEQ ID NO: 218; SEQ ID NO: 288 and SEQ ID NO: 219; SEQ ID NO: 288 and SEQ ID NO: 220; SEQ ID NO: 288 and SEQ ID NO: 221; SEQ ID NO: 288 and SEQ ID NO: 222; SEQ ID NO: 288 and SEQ ID NO: 223; SEQ ID NO: 288 and SEQ ID NO: 224; SEQ ID NO: 288 and SEQ ID NO: 225; SEQ ID NO: 288 and SEQ ID NO: 226; SEQ ID NO: 288 and SEQ ID NO: 227; SEQ ID NO: 288 and SEQ ID NO: 228; SEQ ID NO: 288 and SEQ ID NO: 229; SEQ ID NO: 288 and SEQ ID NO: 230; SEQ ID NO: 288 and SEQ ID NO: 231; SEQ ID NO: 288 and SEQ ID NO: 232; SEQ ID NO: 288 and SEQ ID NO: 233; SEQ ID NO: 288 and SEQ ID NO: 234; SEQ ID NO: 288 and SEQ ID NO: 235; SEQ ID NO: 288 and SEQ ID NO: 236; SEQ ID NO: 288 and SEQ ID NO: 237; SEQ ID NO: 288 and SEQ ID NO: 238; SEQ ID NO: 288 and SEQ ID NO: 239; SEQ ID NO: 288 and SEQ ID NO: 240; SEQ ID NO: 288 and SEQ ID NO: 241; SEQ ID NO: 288 and SEQ ID NO: 242; SEQ ID NO: 288 and SEQ ID NO: 243; SEQ ID NO: 288 and SEQ ID NO: 244; SEQ ID NO: 288 and SEQ ID NO: 245; SEQ ID NO: 288 and SEQ ID NO: 246; SEQ ID NO: 288 and SEQ ID NO: 247; SEQ ID NO: 288 and SEQ ID NO: 248; SEQ ID NO: 288 and SEQ ID NO: 249; and SEQ ID NO: 288 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 289 and SEQ ID NO: 211; SEQ ID NO: 289 and SEQ ID NO: 212; SEQ ID NO: 289 and SEQ ID NO: 213; SEQ ID NO: 289 and SEQ ID NO: 214; SEQ ID NO: 289 and SEQ ID NO: 215; SEQ ID NO: 289 and SEQ ID NO: 216; SEQ ID NO: 289 and SEQ ID NO: 217; SEQ ID NO: 289 and SEQ ID NO: 218; SEQ ID NO: 289 and SEQ ID NO: 219; SEQ ID NO: 289 and SEQ ID NO: 220; SEQ ID NO: 289 and SEQ ID NO: 221; SEQ ID NO: 289 and SEQ ID NO: 222; SEQ ID NO: 289 and SEQ ID NO: 223; SEQ ID NO: 289 and SEQ ID NO: 224; SEQ ID NO: 289 and SEQ ID NO: 225; SEQ ID NO: 289 and SEQ ID NO: 226; SEQ ID NO: 289 and SEQ ID NO: 227; SEQ ID NO: 289 and SEQ ID NO: 228; SEQ ID NO: 289 and SEQ ID NO: 229; SEQ ID NO: 289 and SEQ ID NO: 230; SEQ ID NO: 289 and SEQ ID NO: 231; SEQ ID NO: 289 and SEQ ID NO: 232; SEQ ID NO: 289 and SEQ ID NO: 233; SEQ ID NO: 289 and SEQ ID NO: 234; SEQ ID NO: 289 and SEQ ID NO: 235; SEQ ID NO: 289 and SEQ ID NO: 236; SEQ ID NO: 289 and SEQ ID NO: 237; SEQ ID NO: 289 and SEQ ID NO: 238; SEQ ID NO: 289 and SEQ ID NO: 239; SEQ ID NO: 289 and SEQ ID NO: 240; SEQ ID NO: 289 and SEQ ID NO: 241; SEQ ID NO: 289 and SEQ ID NO: 242; SEQ ID NO: 289 and SEQ ID NO: 243; SEQ ID NO: 289 and SEQ ID NO: 244; SEQ ID NO: 289 and SEQ ID NO: 245; SEQ ID NO: 289 and SEQ ID NO: 246; SEQ ID NO: 289 and SEQ ID NO: 247; SEQ ID NO: 289 and SEQ ID NO: 248; SEQ ID NO: 289 and SEQ ID NO: 249; and SEQ ID NO: 289 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 290 and SEQ ID NO: 211; SEQ ID NO: 290 and SEQ ID NO: 212; SEQ ID NO: 290 and SEQ ID NO: 213; SEQ ID NO: 290 and SEQ ID NO: 214; SEQ ID NO: 290 and SEQ ID NO: 215; SEQ ID NO: 290 and SEQ ID NO: 216; SEQ ID NO: 290 and SEQ ID NO: 217; SEQ ID NO: 290 and SEQ ID NO: 218; SEQ ID NO: 290 and SEQ ID NO: 219; SEQ ID NO: 290 and SEQ ID NO: 220; SEQ ID NO: 290 and SEQ ID NO: 221; SEQ ID NO: 290 and SEQ ID NO: 222; SEQ ID NO: 290 and SEQ ID NO: 223; SEQ ID NO: 290 and SEQ ID NO: 224; SEQ ID NO: 290 and SEQ ID NO: 225; SEQ ID NO: 290 and SEQ ID NO: 226; SEQ ID NO: 290 and SEQ ID NO: 227; SEQ ID NO: 290 and SEQ ID NO: 228; SEQ ID NO: 290 and SEQ ID NO: 229; SEQ ID NO: 290 and SEQ ID NO: 230; SEQ ID NO: 290 and SEQ ID NO: 231; SEQ ID NO: 290 and SEQ ID NO: 232; SEQ ID NO: 290 and SEQ ID NO: 233; SEQ ID NO: 290 and SEQ ID NO: 234; SEQ ID NO: 290 and SEQ ID NO: 235; SEQ ID NO: 290 and SEQ ID NO: 236; SEQ ID NO: 290 and SEQ ID NO: 237; SEQ ID NO: 290 and SEQ ID NO: 238; SEQ ID NO: 290 and SEQ ID NO: 239; SEQ ID NO: 290 and SEQ ID NO: 240; SEQ ID NO: 290 and SEQ ID NO: 241; SEQ ID NO: 290 and SEQ ID NO: 242; SEQ ID NO: 290 and SEQ ID NO: 243; SEQ ID NO: 290 and SEQ ID NO: 244; SEQ ID NO: 290 and SEQ ID NO: 245; SEQ ID NO: 290 and SEQ ID NO: 246; SEQ ID NO: 290 and SEQ ID NO: 247; SEQ ID NO: 290 and SEQ ID NO: 248; SEQ ID NO: 290 and SEQ ID NO: 249; and SEQ ID NO: 290 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 291 and SEQ ID NO: 211; SEQ ID NO: 291 and SEQ ID NO: 212; SEQ ID NO: 291 and SEQ ID NO: 213; SEQ ID NO: 291 and SEQ ID NO: 214; SEQ ID NO: 291 and SEQ ID NO: 215; SEQ ID NO: 291 and SEQ ID NO: 216; SEQ ID NO: 291 and SEQ ID NO: 217; SEQ ID NO: 291 and SEQ ID NO: 218; SEQ ID NO: 291 and SEQ ID NO: 219; SEQ ID NO: 291 and SEQ ID NO: 220; SEQ ID NO: 291 and SEQ ID NO: 221; SEQ ID NO: 291 and SEQ ID NO: 222; SEQ ID NO: 291 and SEQ ID NO: 223; SEQ ID NO: 291 and SEQ ID NO: 224; SEQ ID NO: 291 and SEQ ID NO: 225; SEQ ID NO: 291 and SEQ ID NO: 226; SEQ ID NO: 291 and SEQ ID NO: 227; SEQ ID NO: 291 and SEQ ID NO: 228; SEQ ID NO: 291 and SEQ ID NO: 229; SEQ ID NO: 291 and SEQ ID NO: 230; SEQ ID NO: 291 and SEQ ID NO: 231; SEQ ID NO: 291 and SEQ ID NO: 232; SEQ ID NO: 291 and SEQ ID NO: 233; SEQ ID NO: 291 and SEQ ID NO: 234; SEQ ID NO: 291 and SEQ ID NO: 235; SEQ ID NO: 291 and SEQ ID NO: 236; SEQ ID NO: 291 and SEQ ID NO: 237; SEQ ID NO: 291 and SEQ ID NO: 238; SEQ ID NO: 291 and SEQ ID NO: 239; SEQ ID NO: 291 and SEQ ID NO: 240; SEQ ID NO: 291 and SEQ ID NO: 241; SEQ ID NO: 291 and SEQ ID NO: 242; SEQ ID NO: 291 and SEQ ID NO: 243; SEQ ID NO: 291 and SEQ ID NO: 244; SEQ ID NO: 291 and SEQ ID NO: 245; SEQ ID NO: 291 and SEQ ID NO: 246; SEQ ID NO: 291 and SEQ ID NO: 247; SEQ ID NO: 291 and SEQ ID NO: 248; SEQ ID NO: 291 and SEQ ID NO: 249; and SEQ ID NO: 291 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 292 and SEQ ID NO: 211; SEQ ID NO: 292 and SEQ ID NO: 212; SEQ ID NO: 292 and SEQ ID NO: 213; SEQ ID NO: 292 and SEQ ID NO: 214; SEQ ID NO: 292 and SEQ ID NO: 215; SEQ ID NO: 292 and SEQ ID NO: 216; SEQ ID NO: 292 and SEQ ID NO: 217; SEQ ID NO: 292 and SEQ ID NO: 218; SEQ ID NO: 292 and SEQ ID NO: 219; SEQ ID NO: 292 and SEQ ID NO: 220; SEQ ID NO: 292 and SEQ ID NO: 221; SEQ ID NO: 292 and SEQ ID NO: 222; SEQ ID NO: 292 and SEQ ID NO: 223; SEQ ID NO: 292 and SEQ ID NO: 224; SEQ ID NO: 292 and SEQ ID NO: 225; SEQ ID NO: 292 and SEQ ID NO: 226; SEQ ID NO: 292 and SEQ ID NO: 227; SEQ ID NO: 292 and SEQ ID NO: 228; SEQ ID NO: 292 and SEQ ID NO: 229; SEQ ID NO: 292 and SEQ ID NO: 230; SEQ ID NO: 292 and SEQ ID NO: 231; SEQ ID NO: 292 and SEQ ID NO: 232; SEQ ID NO: 292 and SEQ ID NO: 233; SEQ ID NO: 292 and SEQ ID NO: 234; SEQ ID NO: 292 and SEQ ID NO: 235; SEQ ID NO: 292 and SEQ ID NO: 236; SEQ ID NO: 292 and SEQ ID NO: 237; SEQ ID NO: 292 and SEQ ID NO: 238; SEQ ID NO: 292 and SEQ ID NO: 239; SEQ ID NO: 292 and SEQ ID NO: 240; SEQ ID NO: 292 and SEQ ID NO: 241; SEQ ID NO: 292 and SEQ ID NO: 242; SEQ ID NO: 292 and SEQ ID NO: 243; SEQ ID NO: 292 and SEQ ID NO: 244; SEQ ID NO: 292 and SEQ ID NO: 245; SEQ ID NO: 292 and SEQ ID NO: 246; SEQ ID NO: 292 and SEQ ID NO: 247; SEQ ID NO: 292 and SEQ ID NO: 248; SEQ ID NO: 292 and SEQ ID NO: 249; and SEQ ID NO: 292 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 293 and SEQ ID NO: 211; SEQ ID NO: 293 and SEQ ID NO: 212; SEQ ID NO: 293 and SEQ ID NO: 213; SEQ ID NO: 293 and SEQ ID NO: 214; SEQ ID NO: 293 and SEQ ID NO: 215; SEQ ID NO: 293 and SEQ ID NO: 216; SEQ ID NO: 293 and SEQ ID NO: 217; SEQ ID NO: 293 and SEQ ID NO: 218; SEQ ID NO: 293 and SEQ ID NO: 219; SEQ ID NO: 293 and SEQ ID NO: 220; SEQ ID NO: 293 and SEQ ID NO: 221; SEQ ID NO: 293 and SEQ ID NO: 222; SEQ ID NO: 293 and SEQ ID NO: 223; SEQ ID NO: 293 and SEQ ID NO: 224; SEQ ID NO: 293 and SEQ ID NO: 225; SEQ ID NO: 293 and SEQ ID NO: 226; SEQ ID NO: 293 and SEQ ID NO: 227; SEQ ID NO: 293 and SEQ ID NO: 228; SEQ ID NO: 293 and SEQ ID NO: 229; SEQ ID NO: 293 and SEQ ID NO: 230; SEQ ID NO: 293 and SEQ ID NO: 231; SEQ ID NO: 293 and SEQ ID NO: 232; SEQ ID NO: 293 and SEQ ID NO: 233; SEQ ID NO: 293 and SEQ ID NO: 234; SEQ ID NO: 293 and SEQ ID NO: 235; SEQ ID NO: 293 and SEQ ID NO: 236; SEQ ID NO: 293 and SEQ ID NO: 237; SEQ ID NO: 293 and SEQ ID NO: 238; SEQ ID NO: 293 and SEQ ID NO: 239; SEQ ID NO: 293 and SEQ ID NO: 240; SEQ ID NO: 293 and SEQ ID NO: 241; SEQ ID NO: 293 and SEQ ID NO: 242; SEQ ID NO: 293 and SEQ ID NO: 243; SEQ ID NO: 293 and SEQ ID NO: 244; SEQ ID NO: 293 and SEQ ID NO: 245; SEQ ID NO: 293 and SEQ ID NO: 246; SEQ ID NO: 293 and SEQ ID NO: 247; SEQ ID NO: 293 and SEQ ID NO: 248; SEQ ID NO: 293 and SEQ ID NO: 249; and SEQ ID NO: 293 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 294 and SEQ ID NO: 211; SEQ ID NO: 294 and SEQ ID NO: 212; SEQ ID NO: 294 and SEQ ID NO: 213; SEQ ID NO: 294 and SEQ ID NO: 214; SEQ ID NO: 294 and SEQ ID NO: 215; SEQ ID NO: 294 and SEQ ID NO: 216; SEQ ID NO: 294 and SEQ ID NO: 217; SEQ ID NO: 294 and SEQ ID NO: 218; SEQ ID NO: 294 and SEQ ID NO: 219; SEQ ID NO: 294 and SEQ ID NO: 220; SEQ ID NO: 294 and SEQ ID NO: 221; SEQ ID NO: 294 and SEQ ID NO: 222; SEQ ID NO: 294 and SEQ ID NO: 223; SEQ ID NO: 294 and SEQ ID NO: 224; SEQ ID NO: 294 and SEQ ID NO: 225; SEQ ID NO: 294 and SEQ ID NO: 226; SEQ ID NO: 294 and SEQ ID NO: 227; SEQ ID NO: 294 and SEQ ID NO: 228; SEQ ID NO: 294 and SEQ ID NO: 229; SEQ ID NO: 294 and SEQ ID NO: 230; SEQ ID NO: 294 and SEQ ID NO: 231; SEQ ID NO: 294 and SEQ ID NO: 232; SEQ ID NO: 294 and SEQ ID NO: 233; SEQ ID NO: 294 and SEQ ID NO: 234; SEQ ID NO: 294 and SEQ ID NO: 235; SEQ ID NO: 294 and SEQ ID NO: 236; SEQ ID NO: 294 and SEQ ID NO: 237; SEQ ID NO: 294 and SEQ ID NO: 238; SEQ ID NO: 294 and SEQ ID NO: 239; SEQ ID NO: 294 and SEQ ID NO: 240; SEQ ID NO: 294 and SEQ ID NO: 241; SEQ ID NO: 294 and SEQ ID NO: 242; SEQ ID NO: 294 and SEQ ID NO: 243; SEQ ID NO: 294 and SEQ ID NO: 244; SEQ ID NO: 294 and SEQ ID NO: 245; SEQ ID NO: 294 and SEQ ID NO: 246; SEQ ID NO: 294 and SEQ ID NO: 247; SEQ ID NO: 294 and SEQ ID NO: 248; SEQ ID NO: 294 and SEQ ID NO: 249; and SEQ ID NO: 294 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 295 and SEQ ID NO: 211; SEQ ID NO: 295 and SEQ ID NO: 212; SEQ ID NO: 295 and SEQ ID NO: 213; SEQ ID NO: 295 and SEQ ID NO: 214; SEQ ID NO: 295 and SEQ ID NO: 215; SEQ ID NO: 295 and SEQ ID NO: 216; SEQ ID NO: 295 and SEQ ID NO: 217; SEQ ID NO: 295 and SEQ ID NO: 218; SEQ ID NO: 295 and SEQ ID NO: 219; SEQ ID NO: 295 and SEQ ID NO: 220; SEQ ID NO: 295 and SEQ ID NO: 221; SEQ ID NO: 295 and SEQ ID NO: 222; SEQ ID NO: 295 and SEQ ID NO: 223; SEQ ID NO: 295 and SEQ ID NO: 224; SEQ ID NO: 295 and SEQ ID NO: 225; SEQ ID NO: 295 and SEQ ID NO: 226; SEQ ID NO: 295 and SEQ ID NO: 227; SEQ ID NO: 295 and SEQ ID NO: 228; SEQ ID NO: 295 and SEQ ID NO: 229; SEQ ID NO: 295 and SEQ ID NO: 230; SEQ ID NO: 295 and SEQ ID NO: 231; SEQ ID NO: 295 and SEQ ID NO: 232; SEQ ID NO: 295 and SEQ ID NO: 233; SEQ ID NO: 295 and SEQ ID NO: 234; SEQ ID NO: 295 and SEQ ID NO: 235; SEQ ID NO: 295 and SEQ ID NO: 236; SEQ ID NO: 295 and SEQ ID NO: 237; SEQ ID NO: 295 and SEQ ID NO: 238; SEQ ID NO: 295 and SEQ ID NO: 239; SEQ ID NO: 295 and SEQ ID NO: 240; SEQ ID NO: 295 and SEQ ID NO: 241; SEQ ID NO: 295 and SEQ ID NO: 242; SEQ ID NO: 295 and SEQ ID NO: 243; SEQ ID NO: 295 and SEQ ID NO: 244; SEQ ID NO: 295 and SEQ ID NO: 245; SEQ ID NO: 295 and SEQ ID NO: 246; SEQ ID NO: 295 and SEQ ID NO: 247; SEQ ID NO: 295 and SEQ ID NO: 248; SEQ ID NO: 295 and SEQ ID NO: 249; and SEQ ID NO: 295 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 296 and SEQ ID NO: 211; SEQ ID NO: 296 and SEQ ID NO: 212; SEQ ID NO: 296 and SEQ ID NO: 213; SEQ ID NO: 296 and SEQ ID NO: 214; SEQ ID NO: 296 and SEQ ID NO: 215; SEQ ID NO: 296 and SEQ ID NO: 216; SEQ ID NO: 296 and SEQ ID NO: 217; SEQ ID NO: 296 and SEQ ID NO: 218; SEQ ID NO: 296 and SEQ ID NO: 219; SEQ ID NO: 296 and SEQ ID NO: 220; SEQ ID NO: 296 and SEQ ID NO: 221; SEQ ID NO: 296 and SEQ ID NO: 222; SEQ ID NO: 296 and SEQ ID NO: 223; SEQ ID NO: 296 and SEQ ID NO: 224; SEQ ID NO: 296 and SEQ ID NO: 225; SEQ ID NO: 296 and SEQ ID NO: 226; SEQ ID NO: 296 and SEQ ID NO: 227; SEQ ID NO: 296 and SEQ ID NO: 228; SEQ ID NO: 296 and SEQ ID NO: 229; SEQ ID NO: 296 and SEQ ID NO: 230; SEQ ID NO: 296 and SEQ ID NO: 231; SEQ ID NO: 296 and SEQ ID NO: 232; SEQ ID NO: 296 and SEQ ID NO: 233; SEQ ID NO: 296 and SEQ ID NO: 234; SEQ ID NO: 296 and SEQ ID NO: 235; SEQ ID NO: 296 and SEQ ID NO: 236; SEQ ID NO: 296 and SEQ ID NO: 237; SEQ ID NO: 296 and SEQ ID NO: 238; SEQ ID NO: 296 and SEQ ID NO: 239; SEQ ID NO: 296 and SEQ ID NO: 240; SEQ ID NO: 296 and SEQ ID NO: 241; SEQ ID NO: 296 and SEQ ID NO: 242; SEQ ID NO: 296 and SEQ ID NO: 243; SEQ ID NO: 296 and SEQ ID NO: 244; SEQ ID NO: 296 and SEQ ID NO: 245; SEQ ID NO: 296 and SEQ ID NO: 246; SEQ ID NO: 296 and SEQ ID NO: 247; SEQ ID NO: 296 and SEQ ID NO: 248; SEQ ID NO: 296 and SEQ ID NO: 249; and SEQ ID NO: 296 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 297 and SEQ ID NO: 211; SEQ ID NO: 297 and SEQ ID NO: 212; SEQ ID NO: 297 and SEQ ID NO: 213; SEQ ID NO: 297 and SEQ ID NO: 214; SEQ ID NO: 297 and SEQ ID NO: 215; SEQ ID NO: 297 and SEQ ID NO: 216; SEQ ID NO: 297 and SEQ ID NO: 217; SEQ ID NO: 297 and SEQ ID NO: 218; SEQ ID NO: 297 and SEQ ID NO: 219; SEQ ID NO: 297 and SEQ ID NO: 220; SEQ ID NO: 297 and SEQ ID NO: 221; SEQ ID NO: 297 and SEQ ID NO: 222; SEQ ID NO: 297 and SEQ ID NO: 223; SEQ ID NO: 297 and SEQ ID NO: 224; SEQ ID NO: 297 and SEQ ID NO: 225; SEQ ID NO: 297 and SEQ ID NO: 226; SEQ ID NO: 297 and SEQ ID NO: 227; SEQ ID NO: 297 and SEQ ID NO: 228; SEQ ID NO: 297 and SEQ ID NO: 229; SEQ ID NO: 297 and SEQ ID NO: 230; SEQ ID NO: 297 and SEQ ID NO: 231; SEQ ID NO: 297 and SEQ ID NO: 232; SEQ ID NO: 297 and SEQ ID NO: 233; SEQ ID NO: 297 and SEQ ID NO: 234; SEQ ID NO: 297 and SEQ ID NO: 235; SEQ ID NO: 297 and SEQ ID NO: 236; SEQ ID NO: 297 and SEQ ID NO: 237; SEQ ID NO: 297 and SEQ ID NO: 238; SEQ ID NO: 297 and SEQ ID NO: 239; SEQ ID NO: 297 and SEQ ID NO: 240; SEQ ID NO: 297 and SEQ ID NO: 241; SEQ ID NO: 297 and SEQ ID NO: 242; SEQ ID NO: 297 and SEQ ID NO: 243; SEQ ID NO: 297 and SEQ ID NO: 244; SEQ ID NO: 297 and SEQ ID NO: 245; SEQ ID NO: 297 and SEQ ID NO: 246; SEQ ID NO: 297 and SEQ ID NO: 247; SEQ ID NO: 297 and SEQ ID NO: 248; SEQ ID NO: 297 and SEQ ID NO: 249; and SEQ ID NO: 297 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 298 and SEQ ID NO: 211; SEQ ID NO: 298 and SEQ ID NO: 212; SEQ ID NO: 298 and SEQ ID NO: 213; SEQ ID NO: 298 and SEQ ID NO: 214; SEQ ID NO: 298 and SEQ ID NO: 215; SEQ ID NO: 298 and SEQ ID NO: 216; SEQ ID NO: 298 and SEQ ID NO: 217; SEQ ID NO: 298 and SEQ ID NO: 218; SEQ ID NO: 298 and SEQ ID NO: 219; SEQ ID NO: 298 and SEQ ID NO: 220; SEQ ID NO: 298 and SEQ ID NO: 221; SEQ ID NO: 298 and SEQ ID NO: 222; SEQ ID NO: 298 and SEQ ID NO: 223; SEQ ID NO: 298 and SEQ ID NO: 224; SEQ ID NO: 298 and SEQ ID NO: 225; SEQ ID NO: 298 and SEQ ID NO: 226; SEQ ID NO: 298 and SEQ ID NO: 227; SEQ ID NO: 298 and SEQ ID NO: 228; SEQ ID NO: 298 and SEQ ID NO: 229; SEQ ID NO: 298 and SEQ ID NO: 230; SEQ ID NO: 298 and SEQ ID NO: 231; SEQ ID NO: 298 and SEQ ID NO: 232; SEQ ID NO: 298 and SEQ ID NO: 233; SEQ ID NO: 298 and SEQ ID NO: 234; SEQ ID NO: 298 and SEQ ID NO: 235; SEQ ID NO: 298 and SEQ ID NO: 236; SEQ ID NO: 298 and SEQ ID NO: 237; SEQ ID NO: 298 and SEQ ID NO: 238; SEQ ID NO: 298 and SEQ ID NO: 239; SEQ ID NO: 298 and SEQ ID NO: 240; SEQ ID NO: 298 and SEQ ID NO: 241; SEQ ID NO: 298 and SEQ ID NO: 242; SEQ ID NO: 298 and SEQ ID NO: 243; SEQ ID NO: 298 and SEQ ID NO: 244; SEQ ID NO: 298 and SEQ ID NO: 245; SEQ ID NO: 298 and SEQ ID NO: 246; SEQ ID NO: 298 and SEQ ID NO: 247; SEQ ID NO: 298 and SEQ ID NO: 248; SEQ ID NO: 298 and SEQ ID NO: 249; and SEQ ID NO: 298 and SEQ ID NO: 250.

In some aspects, the V_H-V_L pairs are selected from SEQ ID NO: 299 and SEQ ID NO: 211; SEQ ID NO: 299 and SEQ ID NO: 212; SEQ ID NO: 299 and SEQ ID NO: 213; SEQ ID NO: 299 and SEQ ID NO: 214; SEQ ID NO: 299 and SEQ ID NO: 215; SEQ ID NO: 299 and SEQ ID NO: 216; SEQ ID NO: 299 and SEQ ID NO: 217; SEQ ID NO: 299 and SEQ ID NO: 218; SEQ ID NO: 299 and SEQ ID NO: 219; SEQ ID NO: 299 and SEQ ID NO: 220; SEQ ID NO: 299 and SEQ ID NO: 221; SEQ ID NO: 299 and SEQ ID NO: 222; SEQ ID NO: 299 and SEQ ID NO: 223; SEQ ID NO: 299 and SEQ ID NO: 224; SEQ ID NO: 299 and SEQ ID NO: 225; SEQ ID NO: 299 and SEQ ID NO: 226; SEQ ID NO: 299 and SEQ ID NO: 227; SEQ ID NO: 299 and SEQ ID NO: 228; SEQ ID NO: 299 and SEQ ID NO: 229; SEQ ID NO: 299 and SEQ ID NO: 230; SEQ ID NO: 299 and SEQ ID NO: 231; SEQ ID NO: 299 and SEQ ID NO: 232; SEQ ID NO: 299 and SEQ ID NO: 233; SEQ ID NO: 299 and SEQ ID NO: 234; SEQ ID NO: 299 and SEQ ID NO: 235; SEQ ID NO: 299 and SEQ ID NO: 236; SEQ ID NO: 299 and SEQ ID NO: 237; SEQ ID NO: 299 and SEQ ID NO: 238; SEQ ID NO: 299 and SEQ ID NO: 239; SEQ ID NO: 299 and SEQ ID NO: 240; SEQ ID NO: 299 and SEQ ID NO: 241; SEQ ID NO: 299 and SEQ ID NO: 242; SEQ ID NO: 299 and SEQ ID NO: 243; SEQ ID NO: 299 and SEQ ID NO: 244; SEQ ID NO: 299 and SEQ ID NO: 245; SEQ ID NO: 299 and SEQ ID NO: 246; SEQ ID NO: 299 and SEQ ID NO: 247; SEQ ID NO: 299 and SEQ ID NO: 248; SEQ ID NO: 299 and SEQ ID NO: 249; and SEQ ID NO: 299 and SEQ ID NO: 250.

3.7.4.1 Variants of V_H-V_L Pairs

In some embodiments, the V_H-V_L pairs provided herein comprise a variant of an illustrative V_H and/or V_L sequence provided in this disclosure.

In some aspects, the V_H sequence comprises, consists of, or consists essentially of a variant of an illustrative V_H sequence provided in this disclosure. In some aspects, the V_H sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.1% identity with any of the illustrative V_H sequences provided in this disclosure.

In some embodiments, the V_H sequence comprises, consists of, or consists essentially of any of the illustrative V_H sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the V_L sequence comprises, consists of, or consists essentially of a variant of an illustrative V_L sequence provided in this disclosure. In some aspects, the V_L sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative V_L sequences provided in this disclosure.

In some embodiments, the V_L sequence comprises, consists of, or consists essentially of any of the illustrative V_L sequences provided in this disclosure having 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

3.8 PD-1 Antibodies Comprising All Six CDRs

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence, a CDR-H2 sequence, a CDR-H3 sequence, a CDR-L1 sequence, and a CDR-L3 sequence. In some aspects, the CDR sequences are part of a V_H (for CDR-H) or V_L (for CDR-L).

In some aspects, the CDR-H1 sequence is a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 24-38 or a Chothia CDR-H1 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 228-250; the CDR-H2 sequence is a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 86-87 or a Chothia CDR-H2 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 228-250; the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 130-131 or a CDR-H3 sequence of a V_H sequence provided in any one of SEQ ID NOs.: 228-250; the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 148-149 or a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299; the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 166-172 or a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299; and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 189-190 or a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299.

In some aspects, the CDR-H1 sequence is a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 59-65 or a Kabat CDR-H1 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250; the CDR-H2 sequence is a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 108-109 or a Kabat CDR-H2 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250; the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 130-131 or a Kabat CDR-H3 sequence of a V_H sequence selected from SEQ ID NOs.: 228-250; the CDR-L1 sequence is a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 148-149 or a CDR-L1 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299; the CDR-L2 sequence is a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 166-172 or a CDR-L2 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299; and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 189-190 or or a CDR-L3 sequence of a V_L sequence selected from SEQ ID NOs.: 277-299.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 24, a CDR-H2 sequence of SEQ ID NO: 86, a CDR-H3 sequence of SEQ ID NO: 130, a CDR-L1 sequence of SEQ ID NO: 148, a CDR-L2 sequence of SEQ ID NO: 166, and a CDR-L3 sequence of SEQ ID NO: 189.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 24, a CDR-H2 sequence of SEQ ID NO: 86, a CDR-H3 sequence of SEQ ID NO: 130, a CDR-L1 sequence of SEQ ID NO: 149, a CDR-L2 sequence of SEQ ID NO: 172, and a CDR-L3 sequence of SEQ ID NO: 190.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 38, a CDR-H2 sequence of SEQ ID NO: 87, a CDR-H3 sequence of SEQ ID NO: 131, a CDR-L1 sequence of SEQ ID NO: 148, a CDR-L2 sequence of SEQ ID NO: 166, and a CDR-L3 sequence of SEQ ID NO: 189.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 38, a CDR-H2 sequence of SEQ ID NO: 87, a CDR-H3 sequence of SEQ ID NO: 131, a CDR-L1 sequence of SEQ ID NO: 149, a CDR-L2 sequence of SEQ ID NO: 172, and a CDR-L3 sequence of SEQ ID NO: 190.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 59, a CDR-H2 sequence of SEQ ID NO: 108, a CDR-H3 sequence of SEQ ID NO: 130, a CDR-L1 sequence of SEQ ID NO: 148, a CDR-L2 sequence of SEQ ID NO: 166, and a CDR-L3 sequence of SEQ ID NO: 189.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 59, a CDR-H2 sequence of SEQ ID NO: 108, a CDR-H3 sequence of SEQ ID NO: 130, a CDR-L1 sequence of SEQ ID NO: 149, a CDR-L2 sequence of SEQ ID NO: 172, and a CDR-L3 sequence of SEQ ID NO: 190.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 65, a CDR-H2 sequence of SEQ ID NO: 109, a CDR-H3 sequence of SEQ ID NO: 131, a CDR-L1 sequence of SEQ ID NO: 148, a CDR-L2 sequence of SEQ ID NO: 166, and a CDR-L3 sequence of SEQ ID NO: 189.

In some embodiments, the PD-1 antibody comprises a CDR-H1 sequence of SEQ ID NO: 65, a CDR-H2 sequence of SEQ ID NO: 109, a CDR-H3 sequence of SEQ ID NO: 131, a CDR-L1 sequence of SEQ ID NO: 149, a CDR-L2 sequence of SEQ ID NO: 172, and a CDR-L3 sequence of SEQ ID NO: 190.

3.8.1 Variants of Antibodies Comprising All Six CDRs

In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided herein comprise a variant of an illustrative CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequence provided in this disclosure.

In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H1 sequence provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H2 sequence provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

4. Bi-Specific Antibodies and Antigen-Binding Constructs

Provided herein are bi-specific antigen-binding constructs, e.g., antibodies, that bind LAG3 and PD-1. The bi-specific antigen-binding construct includes two antigen-binding polypeptide constructs, e.g., antigen binding domains, wherein at least one polypeptide construct specifically binds to LAG3 and at least one polypeptide construct specifically binds to PD-1. In some embodiments, the antigen-binding construct is derived from known antibodies or antigen-binding constructs. In some embodiments, the antigen-binding polypeptide constructs comprise two antigen binding domains that comprise antibody fragments. In some embodiments, the first antigen binding domain and second antigen binding domain each independently comprises an antibody fragment selected from the group of: an scFv, a Fab, and an Fc domain. The antibody fragments may be the same format or different formats from each other. For example, in some embodiments, the antigen-binding polypeptide constructs comprise a first antigen binding domain comprising an scFv and a second antigen binding domain comprising a Fab. In some embodiments, the antigen-binding polypeptide constructs comprise a first antigen binding domain and a second antigen binding domain, wherein both antigen binding domains comprise an scFv. In some embodiments, the first and second antigen binding domains each comprise a Fab. In some embodiments, the first and second antigen binding domains each comprise an Fc domain. Any combination of antibody formats is suitable for the bi-specific antibody constructs disclosed herein.

In some embodiments, in the bi-specific antibodies disclosed herein, the first and second antigen-binding polypeptide constructs independently comprise different light chains. For example, in some embodiments, the first antigen-binding polypeptide construct comprises a V_L sequence selected from any one of SEQ ID NOs: 251-266, and the second antigen-binding polypeptide construct comprises a V_L sequence selected from any one of SEQ ID NOs: 267-276. In some embodiments, the first antigen-binding polypeptide construct comprises a V_L sequence selected from any one of SEQ ID NOs: 267-276, and the second antigen-binding polypeptide construct comprises a V_L sequence selected from any one of SEQ ID NOs: 251-266. In some embodiments, the first and second antigen-binding polypeptide constructs comprise the same light chain. For example, in some embodiments, the first and second antigen-binding polypeptide constructs comprise a same V_L sequence selected from any one of SEQ ID NOs: 251-276. In some embodiments, the first and second antigen-binding polypeptide constructs further comprise a C_L sequence selected from any one of SEQ ID NOs: 353-363 and 386-387. In some embodiments, the first and second antigen-binding polypeptide constructs comprise the same C_L sequence. In some embodiments, the first and second antigen-binding polypeptide constructs comprise different C_L sequences.

The term “antigen-binding construct” refers to any agent, e.g., polypeptide or polypeptide complex capable of binding to an antigen. In some aspects an antigen-binding construct is a polypeptide that specifically binds to an antigen of interest. An antigen-binding construct can be a monomer, dimer, multimer, a protein, a peptide, or a protein or peptide complex; an antibody, an antibody fragment, or an antigen-binding fragment thereof; an scFv and the like. An antigen-binding construct can be a polypeptide construct that is monospecific, bi-specific, or multispecific. In some aspects, an antigen-binding construct can include, e.g., one or more antigen-binding components (e.g., Fabs or scFvs) linked to one or more Fc. Further examples of antigen-binding constructs are described below and provided in the Examples.

The term “bi-specific” includes any agent, e.g., an antigen-binding construct, which has two antigen-binding moieties (e.g. antigen-binding polypeptide constructs), each with a unique binding specificity. For example, a first antigen-binding moiety binds to an epitope on a first antigen, and a second antigen-binding moiety binds to an epitope on a second antigen, where the first antigen is different from the second antigen.

For example, in some embodiments, a bi-specific agent can bind to, or interact with, (a) a cell surface target molecule and (b) an Fc receptor on the surface of an effector cell. In some embodiments, the agent can bind to, or interact with (a) a first cell surface target molecule and (b) a second cell surface target molecule that is different from the first cell surface target molecule. In some embodiments, the agent can bind to and bridge two cells, i.e. interact with (a) a first cell surface target molecule on a first call and (b) a second cell surface target molecule on a second cell that is different from the first cells surface target molecule on the first cell.

In contrast, a monospecific antigen-binding construct refers to an antigen-binding construct with a single binding specificity. In other words, both antigen-binding moieties bind to the same epitope on the same antigen. Examples of monospecific antigen-binding constructs include the anti-CD19 antibody HD37 and the anti-CD3 antibody OKT3 for example.

An antigen-binding construct can be an antibody or antigen-binding portion thereof as disclosed herein.

Methods of generating bi-specific antibodies and bi-specific antigen-binding constructs are described, for example, in Briukmann, U., and R. E. Kontermann. 2017. mAbs 9(2):182-212; and in Yang, et al. 2017. Int. J. Mol. Sci. 18(1):48 (21 pages), each of which is incorporated herein by reference in its entirety.

4.1 Antigen-Binding Polypeptide Construct—Format

The bi-specific antigen-binding construct comprises at least two antigen-binding polypeptide constructs, e.g., antigen binding domains. The format of the antigen-binding polypeptide construct determines certain functional characteristics of the bi-specific antigen-binding construct. In some embodiments, the bi-specific antigen-binding construct has an scFv-scFv format, i.e. both antigen-binding polypeptide constructs are scFvs. In some embodiments, the bi-specific antigen-binding construct has a Fab-Fab format, i.e. both antigen-binding polypeptide constructs are Fabs. In some embodiments, the bi-specific antigen-binding construct has an scFv-Fab format, i.e. a first antigen-binding polypeptide construct is an scFv, and a second antigen-binding polypeptide construct is an Fab. The bi-specific antibody or antigen-binding construct can have any form suitable for the antibody or antigen-binding construct, so long as it comprises a first antigen binding domain and a second antigen binding domain that bind to distinct targets.

In some embodiments, the bi-specific antibody or antigen-binding construct is provided, comprising a first antigen binding domain that specifically binds LAG3 and a second antigen binding domain that specifically binds PD-1. In some embodiments, a bi-specific antigen construct is provided, comprising a first scFv that specifically binds LAG3 and a second scFv that specifically binds PD-1. In some embodiments, a bi-specific antigen construct is provided, comprising a first Fab that specifically binds LAG3 and a second Fab that specifically binds PD-1. In some embodiments, a bi-specific antigen construct is provided, comprising an scFv that specifically binds LAG3 and a Fab that specifically binds PD-1. In some embodiments, a bi-specific antigen construct is provided, comprising a Fab that specifically binds LAG3 and an scFv that specifically binds PD-1.

In some embodiments, the bi-specific antibody or bi-specific antigen-binding construct can be generated as a dual-variable domain antibody. A “dual-variable domain antibody” (also referred to as a DVD-Ig) refers to fusion of an additional V_H domain and V_L domain of a second specificity to a given IgG heavy chain and light chain. Generation of dual-variable domain antibody formats are described, for example, in Wu et al. 2007. Nature Biotechnology 25:1290-1297 and U.S. 2007/0071675, each of which is incorporated herein by reference in its entirety.

In some embodiments, the bi-specific antibody or bi-specific antigen-binding construct is generated as a cross-over dual-variable domain antibody. A “cross-over dual-variable domain antibody” (also referred to as a CODV-Ig) refers to a format related to the dual-variable domain antibody format wherein the two V_H domains and two V_L domains are linked to allow cross-over pairing of the variable V_H-V_L domains. Generation of cross-over dual-variable domain antibody formats are described, for example, in Steinmetz et al. 2016. mAbs 8:867-878, which is incorporated herein by reference in its entirety.

Other formats for synthesis and use in bi-specific antibodies or bi-specific antigen-binding constructs are contemplated and described below.

The format “Single-chain Fv” or “scFv” includes the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains. See, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The “Fab fragment” (also referred to as fragment antigen-binding) contains the constant domain (C_L) of the light chain and the first constant domain (C_H1) of the heavy chain along with the variable domains V_L and V_H on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen-binding. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

The “Single domain antibodies” or “sdAb” format is an individual immunoglobulin domain. SdAbs are fairly stable and easy to express as fusion partner with the Fc chain of an antibody (see Harmsen M M, De Haard H J (2007). “Properties, production, and applications of camelid single-domain antibody fragments.” Appl. Microbiol Biotechnol. 77(1): 13-22).

4.1.1 Format scFv

Embodiments are directed to bi-specific antigen-binding constructs comprising two antigen-binding polypeptide constructs that are each capable of specific binding to a distinct antigen. In some embodiments, each antigen-binding polypeptide construct is in an scFv format. (i.e., antigen-binding domains composed of a heavy chain variable domain and a light chain variable domain, connected with a polypeptide linker). In some embodiments, the scFv molecules are human. In some embodiments, the scFv molecules are humanized. The scFvs can be optimized for protein expression and yield by the modifications disclosed herein.

In some embodiments, the scFv is optimized by changing the order of the variable domains V_L and V_H in the scFv. In some embodiments, in the scFv, the C-terminus of the light chain variable region can be linked to the N-terminus of the heavy chain variable region. In some embodiments, in the scFv, the C-terminus of the heavy chain variable region can be linked to the N-terminus of the light chain variable region.

The variable regions of the scFv can be connected via a linker peptide, or scFv linker, that allows the formation of a functional antigen-binding moiety. In some embodiments, the scFv can be optimized for protein expression and yield by changing composition and/or length of the scFv linker polypeptide. Typical peptide linkers comprise about 2-20 amino acids, and are described herein or known in the art. Suitable, non-immunogenic linker peptides include, for example, (G₄S)_n, (SG₄)_n, (G₄S)_n, G₄(SG₄)_n or G₂(SG₂)_n linker peptides, wherein n is generally a number between 1 and 10. In some embodiments, n is a number between 4 and 8. In some embodiments, n is a number between 3 and 6. In some embodiments, n is a number between 2 and 4. Other linkers are described, for example, in Bird et al. 1988. Science 242:423-426; Huston et al. 1988. PNAS 85:5879-5883; and McCafferty et al. 1990. Nature 348:552-554.

The scFv molecule can be optimized for protein expression and yield by including stabilizing disulfide bridges between the heavy and light chain variable domains, for example as described in Reiter et al. (Nat Biotechnol 14, 1239-1245 (1996)). Accordingly, in some embodiments, the bi-specific antigen-binding molecule disclosed herein can comprise an scFv molecule wherein an amino acid in the heavy chain variable domain and an amino acid in the light chain variable domain have been replaced by cysteine so that a disulfide bridge can be formed between the heavy and light chain variable domain.

ScFvs can also be stabilized by mutation of CDR sequences, as described in the art (Miller et al., Protein Eng Des Sel. 2010 July; 23(7):549-57; Igawa et al., MAbs. 2011 May-June; 3(3):243-5; Perchiacca & Tessier, Annu Rev Chem Biomol Eng. 2012; 3:263-286, each of which is incorporated herein by reference in its entirety.) and as disclosed herein in exemplary embodiments.

4.1.2 Fc Domains of Antigen-Binding Constructs

In some embodiments, the antigen-binding constructs described herein comprise an Fc domain, e.g., a dimeric Fc. The Fc domain is a heterodimeric Fc comprising first and second Fc polypeptides each comprising a modified C_H3 sequence, wherein each modified C_H3 sequence comprises asymmetric amino acid modifications that promote the formation of a heterodimeric Fc and the dimerized C_H3 domains have a melting temperature (Tm) of about 68° C. or higher, and wherein the first Fc polypeptide is linked to the first antigen-binding polypeptide construct, with a first hinge linker, and the second Fc polypeptide is linked to the second antigen-binding polypeptide construct with a second hinge linker.

The term “Fc domain” or “Fc region” herein refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions and is used interchangeably with “Fc.” Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. An “Fc polypeptide” of a dimeric Fc as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, an Fc polypeptide of a dimeric IgG Fc comprises an IgG C_H2 and an IgG C_H3 constant domain sequence.

An Fc domain comprises either a C_H3 domain or a C_H3 and a C_H2 domain. The C_H3 domain comprises two C_H3 sequences, one from each of the two Fc polypeptides of the dimeric Fc. The C_H2 domain comprises two C_H2 sequences, one from each of the two Fc polypeptides of the dimeric Fc.

In some embodiments, the Fc comprises at least one or two C_H3 sequences. In some aspects, the Fc is coupled, with or without one or more linkers, to a first antigen-binding construct and/or a second antigen-binding construct. In some embodiments, the Fc is a human Fc. In some embodiments, the Fc is a human IgG or IgG1 Fc. In some embodiments, the Fc is a heterodimeric Fc. In some embodiments, the Fc comprises at least one or two C_H2 sequences.

In some embodiments, the Fc comprises one or more modifications in at least one of the C_H3 sequences. In some embodiments, the Fc comprises one or more modifications in at least one of the C_H2 sequences. For example, the Fc can include one or modifications selected from the group consisting of: V262E, V262D, V262K, V262R, V262S, V264S, V303R, and V305R. In some embodiments, an Fc is a single polypeptide. In some embodiments, an Fc is multiple peptides, e.g., two polypeptides.

4.1.3 Modified C_H3 Domains

In some embodiments, the antigen-binding construct described herein comprises a heterodimeric Fc comprising a modified C_H3 domain that has been asymmetrically modified. The heterodimeric Fc can comprise two heavy chain constant domain polypeptides: a first Fc polypeptide and a second Fc polypeptide, which can be used interchangeably provided that Fc comprises one first Fc polypeptide and one second Fc polypeptide. Generally, the first Fc polypeptide comprises a first C_H3 sequence and the second Fc polypeptide comprises a second C_H3 sequence.

Two C_H3 sequences that comprise one or more amino acid modifications introduced in an asymmetric fashion generally results in a heterodimeric Fc, rather than a homodimer, when the two C_H3 sequences dimerize. As used herein, “asymmetric amino acid modifications” refers to any modification where an amino acid at a specific position on a first C_H3 sequence is different from the amino acid on a second C_H3 sequence at the same position, and the first and second C_H3 sequence preferentially pair to form a heterodimer, rather than a homodimer. This heterodimerization can be a result of modification of one of the two amino acids at the same respective amino acid position on each sequence; or modification of both amino acids on each sequence at the same respective position on each of the first and second C_H3 sequences. The first and second C_H3 sequence of a heterodimeric Fc can comprise one or more than one asymmetric amino acid modification.

Typically an Fc can include two contiguous heavy chain sequences (A and B) that are capable of dimerizing. With respect to the antigen binding constructs described herein, in some embodiments the first scFv is linked to chain A of the heterodimeric Fc and the second scFv is linked to chain B of the heterodimeric Fc. In some embodiments the second scFv is linked to chain A of the heterodimeric Fc and the first scFv is linked to chain B of the heterodimeric Fc.

In some embodiments, one or both sequences of an Fc include one or more mutations or modifications at the following locations: L351, L368, F405, Y407, T366, K392, T394, T350, 5400, and/or N390, using EU numbering. In some embodiments, an Fc includes the mutations as disclosed in the art (see Von Kreudenstein et al. 2013. mAbs 5(5):646-654; Von Kreudenstein et al. 2014. Methods 65:77-94; U.S. 2013/0195849; Ridgway et al. 1996. Protein Eng 9(7):617-621; and Brinkmann, U., and R. E. Kontermann. 2017. mAbs 9(2):182-212, each of which is incorporated herein by reference in its entirety.)

The first and second C_H3 sequences can comprise amino acid mutations as described herein. In some embodiments, the heterodimeric Fc comprises a modified C_H3 domain with a first C_H3 sequence having one or more amino acid modifications selected from L351Y, F405A, and Y407V, and the second C_H3 sequence having one or more amino acid modifications selected from T366L, T366I, K392L, K392M, and T394W. In some embodiments, the heterodimeric Fc comprises a modified C_H3 domain with a first C_H3 sequence having amino acid modifications T350V/T366L/K392L/T394W, and a second C_H3 sequence having amino acid modifications T350V/L351Y/F405A/Y407V.

Additional modifications within the first and second C_H3, or within the amino acid sequence of human IgG1 Fc, can be found, for example, at U.S. 2016/0326249, which is incorporated herein by reference in its entirety.

4.1.4 Modified V_H/V_L and/or C_H1/C_L1 Interactions

In some embodiments, the bi-specific antibodies or bi-specific antigen-binding constructs disclosed herein comprise one or more light chains. In some embodiments, the bi-specific antibody or bi-specific antigen-binding construct comprises two light chains. In some embodiments, the two light chains are different. In some embodiments, the two light chains are the same.

In embodiments wherein the two light chains are different, one or more modifications can be introduced into one or both light chains to allow for pairing of a cognate heavy chain and light chain. Accordingly, in some embodiments, the interaction between the variable domain a first light chain and the variable domain of a corresponding first heavy chain (i.e., V_H-V_L interaction) is modified. In some embodiments, the interaction the constant domain of a first light chain and the first constant domain of a corresponding first heavy chain (i.e., C_H1-C_L interaction) is modified. In some embodiments, the modification comprises genetically engineering or genetically modifying residues that are involved in the V_H-V_L and/or the Cal-C_L interaction. In some embodiments, the modification involves mutating residues to modify electrostatic interactions between the V_H-V_L pairs and/or the C_H1-C_L pairs. The result of modification to the V_H-V_L and/or the C_H1-C_L interactions can result in improved accuracy (or improved “steering”) in pairing of cognate heavy and light chains. Exemplary modifications in these domains are described, for example, in Lewis et al. 2014. Nature Biotechnology 32:191-198 and WO 2014/082179, each of which is incorporated herein by reference in its entirety.

4.1.5 Hinge Linkers

In some embodiments, in the bi-specific antigen-binding constructs disclosed herein, the first Fc polypeptide is linked to the first antigen-binding polypeptide construct with a first hinge linker, and the second Fc polypeptide is linked to the second antigen-binding polypeptide construct with a second hinge linker. Examples of hinge linker sequences are well-known to one of skill in the art and can be used in the antigen-binding constructs described herein. Alternatively, modified versions of known hinge linkers can be used.

The hinge linker polypeptides are selected such that they maintain or optimize the functional activity of the antigen-binding construct. Suitable linker polypeptides include IgG hinge regions such as, for example those from IgG₁, IgG₂, or IgG₄, including the upper hinge sequences and core hinge sequences. The amino acid residues corresponding to the upper and core hinge sequences vary depending on the IgG type, as is known in the art and one of skill in the art would readily be able to identify such sequences for a given IgG type. Modified versions of these exemplary linkers can also be used. For example, modifications to improve the stability of the IgG4 hinge are known in the art (see for example, Labrijn et al. (2009) Nature Biotechnology 27, 767-771). Examples of hinge linker sequences are found, for example, in U.S. 2016/0326249.

4.1.6. Bispecific Scaffold Arrangements

The bi-specific antigen-binding construct can have a variety of different arrangements. For example, the bi-specific antigen-binding construct can comprise a 2-chain scFvFc, a 3-chain Fab×scFvFc, or a 4-chain IgG-like bispecific construct as described below.

4.1.6.1. Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Two-chain scFvFc)

A general scheme for generating embodiments of scFvs for use in a PD1/LAG3 bi-specific antigen-binding construct is provided below in Tables 5 and 6. In some embodiments, the bi-specific antigen-binding construct comprises a 2-chain scFvFc HC/LC pairing maintained by genetically fusing the V_H to the V_L of both antibodies to form an scFv. In some embodiments, the order of the HC/LC pairing is V_H/V_L. In some embodiments, the order of the HC/LC pairing is V_L/V_H. In any of the foregoing embodiments, the HC/LC pairing can comprise a linker sequence. In some embodiments, the linker sequence comprises a Gly/Ser-rich linker of the sequence (GGGGS)n where n=3, 4, 5, or 6 to provide linkers with lengths of 15, 20, 25, or 30 residues, respectively.

In some embodiments, the scFv is arranged in a V_H-V_L arrangement. In some embodiments, the scFv comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the scFv comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266.

TABLE 5
Exemplary VH - VL scFv Arrangements
Design	Name	VH	Linker	VL
(a)	1353-G10	1353-G10 VH	(GGGGS)3,	1353-G10 VL
	VH -		(GGGGS)4,	(λ), or
	VL scFv		(GGGGS)5, or	1353-G10
			(GGGGS)6	VL (κ)
(b)	1449-G09.2	1449-G09.2 VH	(GGGGS)3,	1449-G09.2 VL
	VH -		(GGGGS)4,
	VL scFv		(GGGGS)5, or
			(GGGGS)6

In some embodiments, the scFv is arranged in a V_L-V_H arrangement. In some embodiments, the scFv comprises a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227. In some embodiments, the scFv comprises a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210.

TABLE 6
Exemplary VL - VH scFv Arrangements
Design	Name	VL	Linker	VH
(a)	1353-G10	1353-G10 VL	(GGGGS)3,	1353-G10
	VL -	(λ), or	(GGGGS)4,	VH
	VH scFv	1353-G10 VL (κ)	(GGGGS)5, or
			(GGGGS)6
(b)	1449-G09.2	1449-G09.2 VL	(GGGGS)3,	1449-G09.2
	VL-		(GGGGS)4,	VH
	VH scFv		(GGGGS)5, or
			(GGGGS)6

4.1.6.2. Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Two-Chain scFvFc with Knob-in-Hole Mutations)

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a two-chain scFvFc arrangement, wherein the scFvFc pairing comprises knob-in-hole mutations, is provided. In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising an anti-PD1 scFvFc knob paired with an anti-LAG3 scFvFc hole. In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising an anti-PD1 scFvFc hole paired with an anti-LAG3 scFvFc knob. The scFvFcs include scFvs generated in accordance with Section 4.1.6.1.

A general scheme for scFvFcs useful in a PD1/LAG3 bi-specific antigen-binding construct comprising a two-chain scFv arrangement with knob-in-hole mutations is provided below in Table 7.

TABLE 7
Exemplary scFvFcs (with Knob-in-hole Mutations)
for Two-chain scFvFc Arrangements
Design	Name	scFv	Linker	CH2-CH3
(a)	aPD1 scFvFc	1353-G10 VH-	Linker or	Fc-Knob, Fc-
	knob	VL scFv, or	Linker-	Knob V262E,
		1353-G10 VL-	hinge	Fc-Knob-
		VH scFv		V264S, Fc-
				knob-D399C,
				Fc-knob-S354C
(b)	aLAG3 scFvFc	1449-G09.2 VH-	Linker or	Fc-Knob, Fc-
	knob	VL scFv, or	Linker-	Knob V262E,
		1449-G09.2 VL-	hinge	Fc-Knob-
		VH scFv		V264S, Fc-
				knob-D399C,
				Fc-knob-S354C
(c)	aPD1 scFvFc	1353-G10 VH-	Linker or	Fc-Hole, Fc-
	hole	VL scFv, or	Linker-	hole V262E,
		1353-G10 VL-	hinge	Fc-hole
		VH scFv		V264S, Fc-
				hole-Y349C,
				or Fc-hole-
				K392C
(d)	aLAG3 scFvFc	1449-G09.2 VH-	Linker or	Fc-Hole, Fc-
	hole	VL scFv, or	Linker-	hole V262E,
		1449-G09.2 VL-	hinge	Fc-hole
		VH scFv		V264S, or
				Fc-hole-
				Y349C, or
				Fc-hole-K392C

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a two-chain scFvFc arrangement is prepared using the following arrangement: an anti-PD1 scFvFc knob (Table 12, design (a)) paired with an anti-LAG3 scFvFc hole (Table 12, design (d)). In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a two-chain scFvFc arrangement is prepared using the following arrangement: an anti-PD1 scFvFc hole (Table 12, design (c)) paired with an anti-LAG3 scFvFc knob (Table 12, design (b)).

In some embodiments, the anti-PD1 scFvFc knob is constructed from: (1) an anti-PD1 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-PD1 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the anti-PD1 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 267-276; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 211-227. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH2-CH3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 321-325.

In some embodiments, the anti-PD1 scFvFc hole is constructed from: (1) an anti-PD1 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-PD1 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the anti-PD1 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 267-276; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 211-227. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH2-CH3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 326-330.

In some embodiments, the anti-LAG3 scFvFc knob is constructed from: (1) an anti-LAG3 scFv; (2) a linker or linker-hinge; and (3) a CH2-CH3 region. In some embodiments, the anti-LAG3 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the anti-LAG3 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH2-CH3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 321-325.

In some embodiments, the anti-LAG3 scFvFc hole is constructed from: (1) an anti-LAG3 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-LAG3 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the anti-LAG3 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH₂-CH₃ region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 326-330.

4.1.6.3. Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Three-chain Fab×scFvFc with Knob-in-Hole Mutations)

In some embodiments, the bi-specific antigen-binding construct comprises a 3-chain Fab×scFvFc scaffolds in which an scFv is replaced with a Fab domain. In these embodiments, the asymmetry of the scaffold facilitates correct HC/LC pairing as there is only one HC/LC pairing that can correctly form the Fab domain; the other arm is an scFv.

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab×scFvFc structure can be prepared using the following arrangements: (1) an anti-PD1 scFvFc knob paired with an anti-LAG3 half IgG (HC+LC) hole, (2) an anti-PD1 scFvFc hole paired with an anti-LAG3 half IgG (HC+LC) knob, (3) an anti-PD1 half IgG (HC+LC) knob paired with an anti-LAG3 scFvFc hole, and (4) an anti-PD1 half IgG (HC+LC) hole paired with an anti-LAG3 scFvFc knob. The scFvs included within such scFvFc arrangements can be generated in accordance with Section 4.1.6.1.

A general scheme for anti-PD1 half IgGs (HC+LC, knob or hole) and anti-LAG3 half IgGs (HC+LC, knob or hole) for use in a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab×scFv arrangement with knob-in-hole mutations is provided below in Tables 8 and 9.

TABLE 8
Exemplary HCs of anti-PD1 and anti-LAG3 Half IgGs
Design	Name	VH	CH1	Linker	CH2-CH3
(a)	aPD1	1353-G10 VH	CH1-wt	Hinge-wt	Fc-Knob, Fc-
	HC				Knob V262E,
	knob				Fc-Knob-
					V264S, Fc-
					knob-D399C,
					Fc-knob-
					S354C
(b)	aLAG3	1449-G09.2 VH	CH1-wt	Hinge-wt	Fc-Knob, Fc-
	HC				Knob V262E,
	knob				Fc-Knob-
					V264S, Fc-
					knob-D399C,
					Fc-knob-
					S354C
(c)	aPD1	1353-G10 VH	CH1-wt	Hinge-wt	Fc-Hole, Fc-
	HC				hole V262E,
	hole				Fc-hole
					V264S, or
					Fc-hole-
					Y349C, or
					Fc-hole-
					K392C
(d)	aLAG3	1449-G09.2 VH	CH1-wt	Hinge-wt	Fc-Hole, Fc-
	HC				hole V262E,
	hole				Fc-hole
					V264S, or
					Fc-hole-
					Y349C, or
					Fc-hole-
					K392C

TABLE 9
Exemplary LCs of anti-PD1 and anti-LAG3 Half IgGs
	Design	Name	VL	CL
	(a)	aPD1 LC (λ)	1353-G10 VL (λ)	Cλ-wt
	(b)	aPD1 LC (κ)	1353-G10 VL (κ)	Cκ-wt
	(c)	aLAG3 LC	1449-G09.2 VL	Cκ-wt

In some embodiments, the anti-PD1 half IgG knob comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 321-325, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276 and a C_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 386-387.

In some embodiments, the anti-PD1 half IgG hole comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 326-330, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276 and a C_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs:386-387.

In some embodiments, the anti-LAG3 half IgG knob comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a C_H2-C_H3 region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 321-325, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266 and a C_L comprising, consisting of, or consisting essentially of SEQ ID NO:386.

In some embodiments, the anti-LAG3 half IgG hole comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 326-330, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266 and a C_L comprising, consisting of, or consisting essentially of SEQ ID NO:386.

In some embodiments, the anti-LAG3 scFvFc hole is constructed from: (1) an anti-LAG3 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-LAG3 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the anti-LAG3 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH₂-CH₃ region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 326-330.

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab×scFvFc arrangement is prepared using the following arrangement: an anti-PD1 scFvFc knob (Table 7, design (a)) paired with an anti-LAG3 half IgG hole (HC=Table 8, design (d), LC=Table 9, design (c)).

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab×scFvFc arrangement is prepared using the following arrangement: an anti-PD1 scFvFc hole (Table 7, design (c)) paired with an anti-LAG3 half IgG knob (HC=Table 8, design (b), LC=Table 9, design (c)).

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab× scFvFc arrangement is prepared using the following arrangement: an anti-PD1 half IgG knob (HC=Table 8, design (a), LC=Table 9, design (a) or (b)) paired with an anti-LAG3 scFvFc hole (Table 7, design (d)).

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab× scFvFc arrangement is prepared using the following arrangement: an anti-PD1 half IgG hole (HC=Table 8, design (c), LC=Table 9, design (a) or (b)) hole paired with an anti-LAG3 scFvFc knob (Table 7, design (b)).

4.1.6.4. Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Three-Chain Fab× scFvFc with zw Mutations)

In some embodiments, the bi-specific antigen-binding construct comprises a three-chain Fab× scFvFc scaffold, wherein the Fab and scFvFc structures comprise knob-in-hole mutations. In these embodiments, the asymmetry of the scaffold facilitates correct HC/LC pairing as there is only one HC/LC pairing that can correctly form the Fab domain; the other arm is an scFv.

In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising a three-chain Fab× scFvFc structure comprising an anti-PD1 scFvFc with zwA mutations paired with an anti-LAG3 half IgG (HC+LC) with zwB mutations. In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising a three-chain Fab× scFvFc structure comprising an anti-PD1 scFvFc with zwB mutations paired with an anti-LAG3 half IgG (HC+LC) with zwA mutations. In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising a three-chain Fab× scFvFc structure comprising an anti-PD1 half IgG (HC+LC) with zwA mutations paired with an anti-LAG3 scFvFc with zwB mutations. In some embodiments, a PD1/LAG3 bi-specific antigen-binding construct is provided, comprising a three-chain Fab× scFvFc structure comprising an anti-PD1 half IgG (HC+LC) with zwB mutations paired with an anti-LAG3 scFvFc with zwA mutations. The mutations encompassed by “zwA” mutations include T350V/L351Y/F405A/Y407V in the C_H3 domain. The mutations encompassed by “zwB” mutations include T350V/T366L/K392L/T394W in the C_H3 domain.

A general scheme for scFvFcs with zw mutations and for anti-PD1 half IgGs (HC+LC) and anti-LAG3 half IgGs (HC+LC) with zw mutations for use in a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab× scFv arrangement is provided below in Tables 10 and 11. Exemplary LC embodiments for the half IgGs correspond to those in Table 9 above.

TABLE 10
Exemplary scFvFcs (with zw Mutations) for
Three-chain Fab × scFvFc Arrangement
Design	Name	scFv	Linker	CH2-CH3
(a)	aPD1 scFvFc	1353-G10 VH-	Linker or	Fc-zwA, Fc-
	zwA	VL scFv, or	Linker-	zwA V262E, or
		1353-G10 VL-	hinge	Fc-zwA V264S
		VH scFv
(b)	aLAG3 scFvFc	1449-G09.2 VH-	Linker or	Fc-zwA, Fc-
	zwA	VL scFv, or	Linker-	zwA V262E, or
		1449-G09.2 VL-	hinge	Fc-zwA V264S
		VH scFv
(c)	aPD1 scFvFc	1353-G10 VH-	Linker or	Fc-zwB, Fc-
	zwB	VL scFv, or	Linker-	zwB V262E, or
		1353-G10 VL-	hinge	Fc-zwB V264S
		VH scFv
(d)	aLAG3 scFvFc	1449-G09.2 VH-	Linker or	Fc-zwB, Fc-
	zwB	VL scFv, or	Linker-	zwB V262E, or
		1449-G09.2 VL-	hinge	Fc-zwB V264S
		VH scFv

In some embodiments, the anti-PD1 scFvFc zwA is constructed from: (1) an anti-PD1 scFv; (2) a linker or linker-hinge; and (3) a CH₂—CH₃ region. In some embodiments, the anti-PD1 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the anti-PD1 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 267-276; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 211-227. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH₂-CH₃ region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 331-333.

In some embodiments, the anti-PD1 scFvFc zwB is constructed from: (1) an anti-PD1 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-PD1 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the anti-PD1 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 267-276; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 211-227. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the C_H2-C_H3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 334-336.

In some embodiments, the anti-LAG3 scFvFc zwA is constructed from: (1) an anti-LAG3 scFv; (2) a linker or linker-hinge; and (3) a CH₂-CH₃ region. In some embodiments, the anti-LAG3 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the anti-LAG3 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH₂-CH₃ region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 331-333.

In some embodiments, the anti-LAG3 scFvFc zwB is constructed from: (1) an anti-LAG3 scFv; (2) a linker or linker-hinge; and (3) a CH₂—CH₃ region. In some embodiments, the anti-LAG3 scFv comprises a V_H-V_L arrangement and comprises a V_H sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a linker selected from SEQ ID NOs: 308-314; and a V_L sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the anti-LAG3 scFv comprises a V_L-V_H arrangement and comprises a V_L sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 251-266; a linker selected from SEQ ID NOs: 308-314; and a V_H sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 191-210. In some embodiments, the linker-hinge comprises, consists of, or consists essentially of SEQ ID NO: 315. In some embodiments, the CH₂-CH₃ region comprises, consists of, or consists essentially of any one of SEQ ID NOs.: 334-336.

TABLE 11
Exemplary HCs of anti-PD1 and anti-
LAG3 Half IgGs with zw Mutations
Design	Name	VH	CH1	Linker	CH2-CH3
(a)	aPD1	1353-G10 VH	CH1-wt	Hinge-wt	Fc-zwA,
	HC				Fc-zwA
	zwA				V262E, or
					Fc-zwA
					V264S
(b)	aLAG3	1449-G09.2 VH	CH1-wt	Hinge-wt	Fc-zwA,
	HC				Fc-zwA
	zwA				V262E, or
					Fc-zwA
					V264S
(c)	aPD1	1353-G10 VH	CH1-wt	Hinge-wt	Fc-zwB,
	HC				Fc-zwB
	zwB				V262E, or
					Fc-zwB
					V264S
(d)	aLAG3	1449-G09.2 VH	CH1-wt	Hinge-wt	Fc-zwB,
	HC				Fc-zwB
	zwB				V262E, or
					Fc-zwB
					V264S

In some embodiments, the anti-PD1 half IgG with zwA mutations comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 331-333, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276 and a C_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 386-387.

In some embodiments, the anti-PD1 half IgG with zwB mutations comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 211-227; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 334-336, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 267-276 and a C_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 386-387.

In some embodiments, the anti-LAG3 half IgG with zwA mutations comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 331-333, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266 and a C_L comprising, consisting of, or consisting essentially of SEQ ID NO: 386.

In some embodiments, the anti-LAG3 half IgG with zwB mutations comprises a heavy chain and a light chain, wherein the heavy chain comprises: a V_H comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 191-210; a CH₁ region comprising, consisting of, or consisting essentially of SEQ ID NO: 385; a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316; and a CH₂-CH₃ region comprising, consisting of, or consisting essentially of any one of SEQ ID NOs.: 334-336, and wherein the light chain comprises: a V_L comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 251-266 and a C_L comprising, consisting of, or consisting essentially of SEQ ID NO: 386.

4.1.6.5. Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Four-chain Fab-Fc×Fab-Fc (IgG-like) with zw Mutations)

In some embodiments, the bi-specific antigen-binding construct comprises a four-chain IgG-like scaffold comprising a Fab domain fused to the N-termini of a heterodimeric Fc. In such embodiments, this bispecific format comprises four chains: heavy chain 1 (HC1), light chain 1 (LC1), heavy chain 2 (HC2), and light chain 2 (LC2). In some embodiments, the HC and LC sequences are mutated as described herein in sections 4.1.2-4.1.4 to facilitate correct pairing between HCs and LCs. For example, the HC/LC pairing designs listed in Tables 12-14 can be incorporated into the construct to facilitate correct HC/LC pairing. HC1 is designed such that it pairs specifically with LC1 rather than LC2. HC2 is designed such this it pairs specifically with LC2 rather than LC1. Six designs are shown below in Tables 12 and 13 that enforce correct light-chain pairing: (a), (b) (c), (d), (e), and (f). Designs (a) and (b) in Table 12 can be incorporated for an embodiment in which one light chain is a kappa chain (LC1) and the second light chain is a lambda chain (LC2). Designs (c), (d), (e) and (f) in Table 13 can be incorporated for embodiments in which the first and second light chains (LC1, LC2) are kappa chains.

TABLE 12
Exemplary embodiments of a four-chain bi-specific
antibody arrangement (kappa × lambda)
Bispecific	LAG3 HC1	LAG3 LC1	PD-1 HC2	PD-1 LC2
(a)	HC1(a)	LC1(a)	HC2(a)	LC2(a)
(b)	HC1(b)	LC1(b)	HC2(b)	LC2(b)

TABLE 13
Exemplary embodiments of a four-chain bi-specific
antibody arrangement (kappa × kappa)
Bispecific	LAG3 HC1	LAG3 LC1	PD-1 HC2	PD-1 LC2
(c)	HC1(c)	LC1(c)	HC2(c)	LC2(c)
(d)	HC1(d)	LC1(d)	HC2(d)	LC2(d)
(e)	HC1(e)(f)	LC1(e)(f)	HC2(e)(f)	LC2(e)
(f)	HC1(e)(f)	LC1(e)(f)	HC2(e)(f)	LC2(f)

In some embodiments, the HCs and LCs for a kappa×kappa bi-specific construct can be switched around. For example, in some embodiments, the (c), (d), (e), and (f) designs of Table 13 can be swapped such that the LAG3 HC1+LC1 and PD-1 HC2+LC2 use the opposite light-chain pairing mutations, as illustrated in Table 14.

TABLE 14
Exemplary embodiments of a four-chain bi-specific
antibody arrangement (kappa × kappa)
Bispecific	PD-1 HC1	PD-1 LC1	LAG3 HC2	LAG3 LC2
(c)	HC1(c)	LC1(c)	HC2(c)	LC2(c)
(d)	HC1(d)	LC1(d)	HC2(d)	LC2(d)
(e)	HC1(e)(f)	LC1(e)(f)	HC2(e)(f)	LC2(e)
(f)	HC1(e)(f)	LC1(e)(f)	HC2(e)(f)	LC2(f)

In some embodiments, HC1 and HC2 incorporate complementary Zymeworks mutations A (T350V/L351Y/F405A/Y407V; “zwA”) and B (T350V/T366L/K392L/T394W; “zwB”) to enforce heterodimerization of HC1 with HC2 (see, e.g., Von Kreudenstein et al. 2013. mAbs 5(5):646-654; Von Kreudenstein et al. 2014. Methods 65:77-94; U.S. 2013/0195849, each of which is incorporated herein by reference in its entirety). Accordingly, if HC1 has zwA then HC2 must have zwB; if HC1 has zwB then HC2 must have zwA. Many alternatives to these C_H3 pairing mutations, e.g., knobs-in-holes mutations, can be used instead. Some of these alternatives are described, for example, in Brinkmann, U., and R. E. Kontermann. 2017. mAbs 9(2):182-212; and Yang, et al. 2017. Int. J. Mol. Sci. 18(1):48 (21 pages), each of which is incorporated herein by reference in its entirety. The C_H2 domains can either be wild-type or have stability/solubility/assembly/yield enhancing mutations V262E or V264S.

4.1.6.5.1 LAG3 Heavy Chains (HC1 or HC2)

A full-length antibody LAG3 heavy chain typically includes a V_H domain, a C_H1 domain, a linker, and a C_H2-C_H3 region. In some embodiments, the V_H domain comprises, consists, or consists essentially of any one of SEQ ID NOs: 191-210. In some embodiments, the C_H1 domain is selected from SEQ ID NOs: 343-352. In some embodiments, the linker comprises, consists of, or consists essentially of SEQ ID NO: 316. In some embodiments, the C_H2-C_H3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs: 331-336. For example, in an embodiment where the anti-LAG3 arm is antibody “1” in the bi-specific antibody, an anti-LAG3 heavy chain with design (a) (“HC1(a)”) can be constructed from: (1) a V_H sequence of 1449.G09.2 (SEQ ID NO: 200); (2) C_H1-(a)1; (3) a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316, and (4) a C_H2-C_H3 region comprising, consisting of, or consisting essentially of Fc-zwA. Table 15 provides various exemplary components for the V_H domain, C_H1 domain, linker, and C_H2-C_H3 region that are contemplated for generation of a LAG-3 heavy chain sequence.

TABLE 15
Exemplary Combinations of Components
for LAG-3 Heavy Chain (HC1 or HC2)
Design	VH	CH1	Linker	CH2-CH3
(a)	1449-G09.2	CH1-(a)1	Hinge-wt	Fc-zwA;
		CH1-(a)2		Fc-zwA V262E;
				Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(b)	1449-G09.2	CH1-(b)1	Hinge-wt	Fc-zwA;
		CH1-(b)2		Fc-zwA V262E;
				Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(c)	1449-G09.2	CH1-(c)1	Hinge-wt	Fc-zwA;
		CH1-(c)2		Fc-zwA V262E;
				Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(d)	1449-G09.2	CH1-(d)1	Hinge-wt	Fc-zwA;
		CH1-(d)2		Fc-zwA V262E;
				Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(e)(f)	1449-G09.2	CH1-(e)(f)1	Hinge-wt	Fc-zwA;
		CH1-(e)(f)2		Fc-zwA V262E;
				Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449.G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(01 (SEQ ID NO: 347, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-LAG-3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(02 (SEQ ID NO: 352, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(02 (SEQ ID NO: 352, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(02 (SEQ ID NO: 352, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(02 (SEQ ID NO: 352, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(02 (SEQ ID NO: 352, a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-LAG3 heavy chain comprises a V_H sequence comprising the V_H sequence of 1449-G09.2 (SEQ ID NO: 200), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

4.1.6.5.2 LAG3 Light Chains (LC1 or LC2)

A full-length anti-LAG3 light chain typically includes a V_L domain and a C_L domain. In some embodiments, the V_L domain comprises, consists of, or consists essentially of any one of SEQ ID NOs: 251-266. In some embodiments, the C_L domain comprises, consists of, or consists essentially of any one of SEQ ID NOs: 353-364. For example, in an embodiment where the anti-LAG3 arm is antibody “1” in the bi-specific antibody, an anti-LAG3 light chain with design (a) (“LC1(a)”) can be constructed from: (1) V_L sequence 1449-G09.2 (SEQ ID NO: 254); and (2) Ck-(a)1. Table 16 provides various exemplary components for the V_L domain and C_L domain for generation of a LAG3 light chain sequence.

TABLE 16
Exemplary Combinations of Components
for LAG-3 Light Chain (LC1 or LC2)
	Design	VL	CL
	(a)	1449-G09.2	Ck-(a)1;
			Ck-(a)2
	(b)	1449-G09.2	Ck-(b)1;
			Ck-(b)2
	(c)	1449-G09.2	Ck-(c)1;
			Ck-(c)2
	(d)	1449-G09.2	Ck-(d)1;
			Ck-(d)2
	(e)	1449-G09.2	Ck-(e)(f)1;
			Ck-(e)2
	(f)	1449-G09.2	Ck-(e)(f)1;
			Ck-(f)2

In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(a)1 (SEQ ID NO: 353).

In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(b)1 (SEQ ID NO: 354).

In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(c)1 (SEQ ID NO: 355). In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(c)2 (SEQ ID NO: 360).

In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(d)1 (SEQ ID NO: 356). In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(d)2 (SEQ ID NO: 361).

In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(e)(f)1 (SEQ ID NO: 357). In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(e)2 (SEQ ID NO: 362). In some embodiments, the anti-LAG3 light chain comprises a V_L sequence comprising the V_L sequence of 1449-G09.2 (SEQ ID NO: 254) and a C_L sequence comprising the sequence of Ck-(f)2 (SEQ ID NO: 363).

4.1.6.5.3 PD-1 Heavy Chains (HC1 or HC2)

A full-length anti-PD-1 heavy chain typically includes a V_H domain, a C_H1 domain, a linker, and a C_H2-C_H3 region. In some embodiments, the V_H domain comprises, consists, or consists essentially of any one of SEQ ID NOs: 211-227. In some embodiments, the C_H1 domain is selected from SEQ ID NOs: 343-352. In some embodiments, the linker comprises, consists of, or consists essentially of SEQ ID NO: 316. In some embodiments, the C_H2-C_H3 region comprises, consists of, or consists essentially of any one of SEQ ID NOs: 331-336. For example, in an embodiment where the anti-PD-1 arm is antibody “2” in the bi-specific antibody, an anti-PD-1 hearby chain with design (a) (“HC2a”) can be constructed from: (1) V_H sequence 1353-G10 R28T/P30D/H31S (SEQ ID NO: 227); (2) C_H1(a)2; and (3) a linker comprising, consisting of, or consisting essentially of SEQ ID NO: 316, and (4) a C_H2-C_H3 region comprising, consisting of, or consisting essentially of Fc-zwA. Table 17 provides various exemplary components for the V_H domain, C_H1 domain, linker, and C_H2-C_H3 region that are contemplated for generation of a PD-1 heavy chain sequence.

TABLE 17
Exemplary Combinations of Components
for PD-1 Heavy Chain (HC1 or HC2)
Design	VH	CH1	Linker	CH2-CH3
(a)	1353-G10-wt;	CH1-(a)1;	Hinge-wt	Fc-zwA;
	1353-G10-28T/	CH1-(a)2		Fc-zwA V262E;
	P30D/H31S			Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(b)	1353-G10-wt;	CH1-(b)1;	Hinge-wt	Fc-zwA;
	1353-G10-28T/	CH1-(b)2		Fc-zwA V262E;
	P30D/H31S			Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(c)	1353-G10-wt;	CH1-(c)1;	Hinge-wt	Fc-zwA;
	1353-G10-28T/	CH1-(c)2		Fc-zwA V262E;
	P30D/H31S			Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(d)	1353-G10-wt;	CH1-(d)1;	Hinge-wt	Fc-zwA;
	1353-G10-28T/	CH1-(d)2		Fc-zwA V262E;
	P30D/H31S			Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S
(e)(f)	1353-G10-wt;	CH1-(e)(f)1;	Hinge-wt	Fc-zwA;
	1353-G10-28T/	CH1-(e)(f)2		Fc-zwA V262E;
	P30D/H31S			Fc-zwA V264S;
				Fc-zwB;
				Fc-zwB V262E;
				Fc-zwB V264S

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)1 (SEQ ID NO: 343), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(a)2 (SEQ ID NO: 348), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)1 (SEQ ID NO: 344), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(b)2 (SEQ ID NO: 349), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)1 (SEQ ID NO: 345), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(c)2 (SEQ ID NO: 350), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)1 (SEQ ID NO: 346), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(d)2 (SEQ ID NO: 351), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA (SEQ ID NO: 331).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V262E (SEQ ID NO: 332).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwA V264S (SEQ ID NO: 333).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB (SEQ ID NO: 334).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V262E (SEQ ID NO: 335).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)1 (SEQ ID NO: 347), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-wt (SEQ ID NO: 211), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336). In some embodiments, the anti-PD-1 heavy chain comprises a V_H sequence comprising the V_H sequence of 1353-G10-R28T/P30D/H31S (SEQ ID NO: 227), a C_H1 sequence comprising the sequence of C_H1-(e)(f)2 (SEQ ID NO: 352), a linker of hinge-wt (SEQ ID NO: 316), and a C_H2-C_H3 region comprising the sequence of Fc-zwB V264S (SEQ ID NO: 336).

4.1.6.5.4 PD-1 Light Chains (LC1 or LC2)

A full-length anti-PD-1 light chain typically includes a V_L domain and a C_L domain. In some embodiments, the V_L domain comprises, consists of, or consists essentially of any one of SEQ ID NOs: 267-276. In some embodiments, the C_L domain comprises, consists of, or consists essentially of any one of SEQ ID NOs: 353-364. For example, in an embodiment where the anti-PD-1 arm is antibody “2” in the bi-specific antibody, an anti-PD-1 light chain with design (a) (“LC2a”) can be constructed from: (1) V_L sequence 1353-G10 wt (SEQ ID NO: 267); and (2) Cl-(a)2. Table 18 provides various exemplary components for the V_L domain and C_L domain for generation of a PD-1 light chain sequence.

TABLE 18
Exemplary Combinations of Components
for PD-1 Light Chain (LC1 or LC2)
	Design	VL	CL
	(a)	1353-G10 wt (λ)	Cl-(a)2
	(b)	1353-G10 wt (λ)	Cl-(b)2
	(c)	1353-G10 Vk1-39 (κ)	Ck-(c)1;
			Ck-(c)2
	(d)	1353-G10 Vk1-39 (κ)	Ck-(d)1;
			Ck-(d)2
	(e)	1353-G10 Vk1-39 (κ)	Ck-(e)2;
			Ck-(e)(f)2
	(f)	1353-G10 Vk1-39 (κ)	Ck-(f)2;
			Ck-(e)(f)2

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 wt (λ) (SEQ ID NO: 267) and a C_L sequence comprising the sequence of Cl-(a)2 (SEQ ID NO: 358).

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 wt (λ) (SEQ ID NO: 267) and a C_L sequence comprising the sequence of Cl-(b)2 (SEQ ID NO: 359).

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(c)1 (SEQ ID NO: 355). In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (K) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(c)2 (SEQ ID NO: 360).

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(d)1 (SEQ ID NO: 356). In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(d)2 (SEQ ID NO: 361).

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(e)2 (SEQ ID NO: 362). In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(e)(f)1 (SEQ ID NO: 357).

In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (κ) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(f)2 (SEQ ID NO: 363). In some embodiments, the anti-PD-1 light chain comprises a V_L sequence comprising the V_L sequence of 1353-G10 Vk1-39 (K) (SEQ ID NO: 275) and a C_L sequence comprising the sequence of Ck-(e)(f)1 (SEQ ID NO: 357).

4.1.6.5.5 Hybrid Light Chains (LC1 or LC2=Vλ+Cκ)

In some embodiments, the anti-LAG3 or anti-PD-1 light chain comprises a V_L lambda sequence (“Vλ”) and a C_L kappa sequence (“Cκ”). In such embodiments, the Vλ, sequence comprises one or more mutations selected from the group consisting of: E38F, D85T, T105E, V1061, and L106K. In some embodiments, the light chain is an anti-PD-1 light chain comprising, consisting of, or consisting essentially of SEQ ID NOs: 394 or 395.

In some embodiments, a PD-1/LAG3 bi-specific antigen-binding construct is provided, comprising a hybrid light chain comprising a Vλ, and a Cκ sequence, wherein HC1, LC1, HC2, and LC2 comprise one or more mutations selected from the Table 19:

TABLE 19
Table of mutations for a bi-specific
construct with a hybrid light chain
	Bispecific	HC1	LC1	HC2	LC2
	(c)	A139W;	F116A;	Q179K	Q124E;
		L143E;	Q124R;		L135W;
		K145T;	L135V;		Q160E;
		Q179E	T178R		T180E
	(d)	Q39E;	Q38R;	Q39R;	Q38E;
		L143E;	Q124R;	H172R;	Q124E;
		K145T;	Q160K;	Q179K	Q160E;
		Q179E	T178R		T180E

In some embodiments, HC1 and LC1 indicate PD-1 heavy chain and light chain sequences, respectively, and HC2 and LC2 indicate LAG3 heavy chain and light chain sequences, respectively. In some embodiments, HC1 and LC1 indicate LAG3 heavy chain and light chain sequences, respectively, and HC2 and LC2 indicate PD-1 heavy chain and light chain sequences, respectively.

In some embodiments, the V_H and V_L sequences of HC1, HC2, LC1 and LC2 comprise the following mutations from Table 20:

TABLE 20
Exemplary mutations for a bi-specific
construct with a hybrid light chain
	VH1	VL1	VH2	VL2
	Q39R	Q38E	Q39E	Q38R

In some embodiments, HC1 comprises, consists of, or consists essentially of SEQ ID NO: 397; LC1 comprises, consists of, or consists essentially of SEQ ID NO: 395; HC2 comprises, consists of, or consists essentially of SEQ ID NO: 398; and LC2 comprises, consists of, or consists essentially of SEQ ID NO: 396.

The sequences of the various components contemplated for V_H, C_H1, linker, C_H2-C_H3, V_L, and C_L in Section 4.1.6 can be found in Table 35.

4.1.7. Additional Mutations

In some embodiments, an bi-specific antibody or antigen-binding construct as disclosed herein can include additional mutations. In some embodiments, the bi-specific antibody or antigen-binding construct can include a mutation to remove a methionine start residue. In some embodiments, the bi-specific antibody or antigen-binding construct can include a mutation to remove glycosylation (e.g. N297A). In some embodiments, the bi-specific antibody or antigen-binding construct can include a mutation to remove effector function (e.g., AAS mutation, as described in U.S. Patent Publication No. 2016/0075792, which is incorporated herein by reference in its entirety). For example, in some embodiments, one or more of the additional mutations disclosed herein can be used to improve production of bi-specific antibody constructs or bi-specific antibody components in a host cell.

5. Germline

In some embodiments, an antibody or bi-specific antibody as disclosed herein that specifically binds LAG3 is an antibody comprising a variable region that is encoded by a particular germline gene, or a variant thereof. The illustrative antibodies provided herein comprise variable regions that are encoded by the heavy chain variable region germline genes VH3-23 and VH5-51, or variants thereof; and the light chain variable region germline genes Vκ3-20 and Vκ4-1, or variants thereof.

One of skill in the art would recognize that the CDR sequences provided herein may also be useful when combined with variable regions encoded by other variable region germline genes, or variants thereof. In particular, the CDR sequences provided herein may be useful when combined with variable regions encoded by variable region germline genes, or variants thereof, that are structurally similar to the variable region germline genes recited above. For example, in some embodiments, a CDR-H sequence provided herein may be combined with a variable region encoded by a variable region germline gene selected from the V_H 3 or V_H 5 families, or a variant thereof. In some embodiments, a CDR-L sequence provided herein may be combined with a variable region encoded by a variable region germline gene selected from the Vκ3 or Vκ4 families, or a variant thereof.

6. Affinity

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for LAG3 as indicated by K_D, is less than about 10⁻⁵ M, less than about 10⁻⁶ M, less than about 10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, less than about 10⁻¹⁰ M, less than about 10⁻¹¹ M, or less than about 10⁻¹² M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻¹⁰ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻⁹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻⁸ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁸ M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁸ M and 10⁻¹⁰ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁹ M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻¹⁰ M and 10⁻¹¹M.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for human LAG3, as determined by surface plasmon resonance at 25° C., and as indicated by K_D, is between about 1.3×10⁻⁸ M and about 1.93×10⁻¹⁰ M. In some embodiments, the affinity of the antibody or bi-specific antibody for human LAG3 is about 8.63×10⁻⁷ M, about 4.33×10⁻⁸ M, about 3.90×10⁻⁸ M, about 3.10×10⁻⁸ M, about 2.40×10⁻⁸ M, about 2.13×10⁻⁸ M, about 1.89×10⁻⁸ M, about 1.52×10⁻⁸ M, about 1.47×10⁻⁸M, about 1.35×10⁻⁸ M, about 1.30×10⁻⁸ M, about 1.03×10⁻⁸ M, about 3.10×10⁻⁹ M, about 2.46×10⁻⁹ M, about 2.27×10⁻⁹ M, about 1.36×10⁻⁹ M, about 6.76×10⁻¹⁰ M, about 6.40×10⁻¹⁰ M, or about 4.12×10⁻¹¹ M.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_a of at least about 10⁴ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of at least about 10⁵ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of at least about 10⁶ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of between about 10⁴ M⁻¹×sec⁻¹ and about 10⁵ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of between about 10⁵ M⁻¹×sec⁻¹ and about 10⁶ M⁻¹×sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_a when associating with human LAG3, as determined by surface plasmon resonance at 25° C., of between about 5.02×10⁴ M⁻¹×sec⁻¹ and about 5.31×10⁷ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a when associating with human LAG3 of about 2.67×10³ M⁻¹×sec⁻¹, about 5.02×10⁴ M⁻¹×sec⁻¹, about 1.61×10⁵ M⁻¹×sec⁻¹, about 2.61×10⁵ M⁻¹×sec⁻¹, about 3.12×10⁵ M⁻¹×sec⁻¹, about 4.35×10⁵ M⁻¹×sec⁻¹, about 4.60×10⁵ M⁻¹×sec⁻¹, about 4.72×10⁵ M⁻¹×sec⁻¹, about 5.60×10⁵ M⁻¹×sec⁻¹, about 7.90×10⁵ M⁻¹×sec⁻¹, about 7.94×10⁵ M⁻¹×sec⁻¹, about 1.06×10⁶ M⁻¹×sec⁻¹, about 1.24×10⁶ M⁻¹×sec⁻¹, about 1.29×10⁶ M⁻¹×sec⁻¹, about 1.31×10⁶ M⁻¹×sec⁻¹, about 1.64×10⁶M⁻¹×sec⁻¹, about 1.65×10⁶M⁻¹×sec⁻¹, about 1.12×10⁷ M⁻¹×sec⁻¹, or about 5.35×10⁷M⁻¹×sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_d of about 10⁻⁵ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of about 10⁻⁴ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of about 10⁻³ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of between about 10⁻² sec⁻¹ and about 10⁻⁵ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_d of between about 10⁻² sec⁻¹ and about 10⁻⁴ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_d of between about 10⁻³ sec⁻¹ and about 10⁻⁵ sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_d when dissociating from human LAG3, as determined by surface plasmon resonance at 25° C., of between about 2.79×10⁻² sec⁻¹ and about 6.78×10⁻⁵ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_d when dissociating from human LAG3 of about 1.22×10⁻¹ sec⁻¹, about 7.10×10⁻² sec⁻¹, about 2.79×10⁻² sec⁻¹, about 2.75×10⁻² sec⁻¹, about 2.34×10⁻² sec⁻¹, about 1.96×10⁻² sec⁻¹, about 1.70×10⁻² sec⁻¹, about 1.52×10⁻² sec⁻¹, about 1.10×10⁻² sec⁻¹, about 9.90×10⁻³ sec⁻¹, about 6.20×10⁻³ sec⁻¹, about 4.22×10⁻³ sec⁻¹, about 2.30×10⁻³ sec⁻¹, about 8.07×10⁻⁴ sec⁻¹, about 6.27×10⁻⁴ sec⁻¹, about 5.36×10⁻⁴ sec⁻¹, about 5.15×10⁻⁴ sec⁻¹, about 3.02×10⁻⁴ sec⁻¹, or about 6.78×10⁻⁵ sec⁻¹.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for human LAG3 expressed on the surface of a cell, as indicated by K_D, is between about 78.0 and about 0.19 nM. In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for human LAG3 expressed on the surface of a cell is about 78.0 nM, about 40.6 nM, about 39.4 nM, about 35.0 nM, about 3.37 nM, about 1.92 nM, about 1.54 nM, about 1.06 nM, about 0.97 nM, about 0.74 nM, about 0.50 nM, about 0.40 nM, about 0.32 nM, about 0.30 nM, and about 0.19 nM. In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a 293T cell.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for cynomolgus LAG3, as determined by surface plasmon resonance at 25° C., and as indicated by K_D, is between about 4.5×10⁻⁹ M and about 0.3×10⁻⁹ M. In some embodiments, the affinity of the antibody or bi-specific antibody for cynomolgus LAG3 is about 4.5×10⁻⁹ M, about 1.6×10⁻⁹ M, about 1.0×10⁻⁹ M, about 0.7×10⁻⁹ M, or about 0.3×10⁻⁹ M.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for PD-1, as indicated by K_D, is less than about 10⁻⁵ M, less than about 10⁻⁶ M, less than about 10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, less than about 10⁻¹⁰ M, less than about 10⁻¹¹ M, or less than about 10⁻¹² M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻¹⁰ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻⁹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁷ M and 10⁻⁸ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁸ M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁸ M and 10⁻¹⁰ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻⁹M and 10⁻¹¹ M. In some embodiments, the affinity of the antibody or bi-specific antibody is between about 10⁻¹⁰ M and 10⁻¹¹M.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for human PD-1 is between about 3.85×10⁻⁸ M and 2.52×10⁻¹° M. In some embodiment, the affinity of the antibody or bi-specific antibody for human PD-1 is about 2.55×10⁻⁸ M, about 1.52×10⁻⁸ M, about 9.52×10⁻⁹ M, about 1.09×10⁻⁸ M, about 4.50×10⁻⁹ M, about 1.90×10⁻⁹ M, about 4.76×10⁻⁹ M, about 4.5×10⁻⁹ M, about 1.04×10⁻⁸ M, about 9.90×10⁻⁹ M, about 9.13×10⁻¹⁰ M, about 2.52×10⁻¹° M, about 2.58×10⁻⁹ M, about 3.85×10⁻⁸ M, about 3.66×10⁻⁹ M, about 3.15×10⁻⁹ M, about 5.14×10⁻⁹ M, about 2.47×10⁻⁹ M, about 2.79×10⁻⁹ M, about 1.20×10⁻⁹ M, or about 1.28×10⁻⁸ M

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for human PD-1 expressed on the surface of a cell is between about 3.2 and about 0.2 nM. In some embodiment, the affinity of the antibody or bi-specific antibody for human PD-1 expressed on the surface of a cell is about 0.2 nM, about 0.4 nM, about 0.9 nM, about 1 nM, about 0.3 nM, about 0.7 nM, about 0.2 nM, about 0.8 nM, about 3.2 nM, about 2.9 nM, about 1.39 nM, or about 1.34 nM.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for murine PD-1 is between about 6.09×10⁻⁸ M and 9.08×10⁻⁹ M. In some embodiment, the affinity of the antibody or bi-specific antibody for murine PD-1 is about 6.09×10⁻⁸ M, about 6.22×10⁻⁸M, or about 9.08×10⁻⁹ M.

In some embodiments, the affinity of an antibody or bi-specific antibody as disclosed herein for cynomolgus PD-1 is between about 2.43×10⁻⁸ M and 1.95×10⁻¹° M. In some embodiment, the affinity of the antibody or bi-specific antibody for cynomolgus PD-1 is about 2.43×10⁻⁸ M, about 1.55×10⁻⁸ M, about 2.22×10⁻⁸ M, about 2.56×10⁻⁹ M, about 2.54×10⁻⁹M, about 5.61×10⁻¹⁰ M, or about 1.95×10⁻¹⁰ M

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_a of at least about 10⁴ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of at least about 10⁵ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of at least about 10⁶ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of between about 10⁴ M⁻¹×sec⁻¹ and about 10⁵ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of between about 10⁵ M⁻¹×sec⁻¹ and about 10⁶ M⁻¹×sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_a when associating with human PD-1 of between about 4.74×10⁴ M⁻¹×sec⁻¹ and about 1.23×10⁶ M⁻¹×sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a when associating with human PD-1 of about 4.88×10⁵ M⁻¹×sec⁻¹, about 1.23×10⁶ M⁻¹×sec⁻¹, about 7.37×10⁵ M⁻¹×sec⁻¹, about 6.87×10⁵ M⁻¹×sec⁻¹, about 5.63×10⁵ M⁻¹×sec⁻¹, about 5.16×10⁵ M⁻¹×sec⁻¹, about 2.48×10⁵ M⁻¹×sec⁻¹, about 7.98×10⁵ M⁻¹×sec⁻¹, about 1.82×10⁵ M⁻¹×sec⁻¹, about 4.74×10⁴ M⁻¹×sec⁻¹, about 1.85×10⁵ M⁻¹×sec⁻¹, about 2.00×10⁵ M⁻¹×sec⁻¹, about 8.12×10⁴ M⁻¹×sec⁻¹, about 1.21×10⁶ M⁻¹×sec⁻¹, about 1.16×10⁶M⁻¹×sec⁻¹, about 5.13×10⁵ M⁻¹×sec⁻¹, or about 1.86×10⁵ M⁻¹×sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_d of about 10⁻⁵ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of about 10⁻⁴ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of about 10⁻³ sec⁻¹ or less. In some embodiments the antibody or bi-specific antibody has a k_d of between about 10⁻² sec⁻¹ and about 10⁻⁵ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_d of between about 10⁻² sec⁻¹ and about 10⁻⁴ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_a of between about 10⁻³ sec⁻¹ and about 10⁻⁵ sec⁻¹.

In some embodiments an antibody or bi-specific antibody as disclosed herein has a k_d when dissociating from human PD-1 of between about 1.87×10⁻² sec⁻¹ and about 4.17×10⁻⁴ sec⁻¹. In some embodiments the antibody or bi-specific antibody has a k_d when dissociating from human PD-1 of about 1.24×10⁻² sec⁻¹, about 1.87×10⁻² sec⁻¹, about 7.01×10⁻³ sec⁻¹, about 7.74×10⁻³ sec⁻¹, about 2.54×10⁻³ sec⁻¹, about 9.80×10⁻⁴ sec⁻¹, about 1.18×10⁻³ sec⁻¹, about 3.59×10⁻³ sec⁻¹, about 4.68×10⁻⁴ sec⁻¹, about 1.82×10⁻³ sec⁻¹, about 6.79×10⁻⁴ sec⁻¹, about 6.28×10⁻⁴ sec⁻¹, about 4.17×10⁻⁴ sec⁻¹, about 2.99×10⁻³ sec⁻¹, about 3.24×10⁻³ sec⁻¹, about 6.17×10⁻⁴ sec⁻¹, or about 2.39×10⁻³ sec⁻¹.

In some aspects, the K_D, k_a, and k_d are determined at 25° C. In some embodiments, the KID, k_a, and k_d are determined by surface plasmon resonance. In some embodiments, the K_D, k_a, and k_d are determined according to the methods described in the Examples provided herein.

7. Inhibition of PD-L1 and PD-L2 Binding

In some embodiments, an antibody or bi-specific antibody as disclosed herein inhibits binding of one or more of PD-L1 and PD-L2 to PD-1.

In some embodiments, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 1 to about 7 nM. In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 1.99, about 2.53, about 5.86, or about 5.96 nM.

In some embodiments, the antibody or bi-specific antibody inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.01 to about 1 nM. In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.01, about 0.18, about 0.56, or about 0.58 nM.

In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 5.96 nM, and inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.56 nM. In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 5.86 nM, and inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.58 nM. In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 1.99 nM, and inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.01 nM. In some aspects, the antibody or bi-specific antibody inhibits binding of PD-L1 to PD-1 with an IC₅₀ of about 2.53 nM, and inhibits binding of PD-L2 to PD-1 with an IC₅₀ of about 0.18 nM.

8. Epitope Bins

In some embodiments, an antibody or bi-specific antibody as disclosed herein binds the same epitope as the scFv antibody provided in SEQ ID NO: 379. In some embodiments, the antibody or bi-specific antibody binds to a different epitope from the scFv antibody provided in SEQ ID NO: 379. In some embodiments, the antibody or bi-specific antibody binds the same epitope as antibody encompassing any of SEQ ID NOs: 191-210. In some embodiments, the antibody or bi-specific antibody binds the same epitope as an antibody comprising any of the V_H-V_L pairs, above. In some embodiments, the antibody or bi-specific antibody binds to part of the epitope bound by the scFv antibody provided in SEQ ID NO: 379. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with the scFv antibody provided in SEQ ID NO: 379. In some embodiments, the antibody or bi-specific antibody does not compete for epitope binding with scFv antibody provided in SEQ ID NO: 379. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with an antibody encompassing any of SEQ ID NOs: 191-210. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with an antibody comprising any of the V_H-V_L pairs, above.

In some embodiments, an antibody or bi-specific antibody as disclosed herein binds the same epitope as the scFv antibody provided in SEQ ID NO: 367. In some embodiments, the antibody or bi-specific antibody binds to a different epitope from the scFv antibody provided in SEQ ID NO: 367. In some embodiments, the antibody or bi-specific antibody binds the same epitope as antibody encompassing any of SEQ ID NOs: 211-250. In some embodiments, the antibody or bi-specific antibody binds the same epitope as an antibody comprising any of the V_H-V_L pairs, above. In some embodiments, the antibody or bi-specific antibody binds to part of the epitope bound by the scFv antibody provided in SEQ ID NO: 367. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with the scFv antibody provided in SEQ ID NO: 367. In some embodiments, the antibody or bi-specific antibody does not compete for epitope binding with scFv antibody provided in SEQ ID NO: 367. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with an antibody encompassing any of SEQ ID NOs: 211-250. In some embodiments, the antibody or bi-specific antibody competes for epitope binding with an antibody comprising any of the V_H-V_L pairs, above.

9. Glycosylation Variants

In certain embodiments, an antibody or bi-specific antibody as disclosed herein may be altered to increase, decrease or eliminate the extent to which it is glycosylated. Glycosylation of polypeptides is typically either “N-linked” or “O-linked.”

“N-linked” glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.

“O-linked” glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition or deletion of N-linked glycosylation sites to the antibody or bi-specific antibody may be accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences is created or removed. Addition or deletion of O-linked glycosylation sites may be accomplished by addition, deletion, or substitution of one or more serine or threonine residues in or to (as the case may be) the sequence of an antibody.

In some embodiments, an antibody or bi-specific antibody as disclosed herein may be aglycosylated.

In some embodiments, an antibody or bi-specific antibody as disclosed herein may be deglycosylated.

10. Fc Variants

In certain embodiments, amino acid modifications may be introduced into the Fc region of an antibody or bi-specific antibody provided herein to generate an Fc region variant. In certain embodiments, the Fc region variant possesses some, but not all, effector functions. Such antibodies may be useful, for example, in applications in which the half-life of the antibody or bi-specific antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-directed complement-mediated cytotoxicity (ADCC). Numerous substitutions or substitutions or deletions with altered effector function are known in the art.

An alteration in in CDC and/or ADCC activity can be confirmed using in vitro and/or in vivo assays. For example, Fc receptor (FcR) binding assays can be conducted to measure FcγR binding. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Ann. Rev. Immunol., 1991, 9:457-492, incorporated by reference in its entirety.

Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are provided in U.S. Pat. Nos. 5,500,362 and 5,821,337; Hellstrom et al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83:7059-7063; Hellstrom et al., Proc. Natl. Acad. Sci. U.S.A., 1985, 82:1499-1502; and Bruggemann et al., J. Exp. Med., 1987, 166:1351-1361; each of which is incorporated by reference in its entirety. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, using an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. U.S.A., 1998, 95:652-656, incorporated by reference in its entirety.

C1q binding assays may also be carried out to confirm that the antibody or bi-specific antibody is unable to bind C1q and hence lacks CDC activity. Examples of C1q binding assays include those described in WO 2006/029879 and WO 2005/100402, each of which is incorporated by reference in its entirety.

Complement activation assays include those described, for example, in Gazzano-Santoro et al., J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003, 101:1045-1052; and Cragg and Glennie, Blood, 2004, 103:2738-2743; each of which is incorporated by reference in its entirety.

FcRn binding and in vivo clearance (half-life determination) can also be measured, for example, using the methods described in Petkova et al., Intl. Immunol., 2006, 18:1759-1769, incorporated by reference in its entirety.

11. Preparation of Antibodies and Bi-Specific Antigen Binding Constructs

11.1. Antigen Preparation

The LAG3 antigen to be used for isolation of the antibodies and bi-specific antigen binding constructs disclosed herein may be intact LAG3 or a fragment of LAG3. The intact LAG3, or fragment of LAG3, may be in the form of an isolated protein or protein expressed by a cell. Other forms of LAG3 useful for generating antibodies will be apparent to those skilled in the art.

The PD-1 antigen to be used for production of antibodies and bi-specific antigen binding constructs disclosed herein may be intact PD-1 or a fragment of PD-1. The intact PD-1, or fragment of PD-1, may be in the form of an isolated protein or expressed by a cell. Other forms of PD-1 useful for generating antibodies will be apparent to those skilled in the art.

11.2. Monoclonal Antibodies

Monoclonal antibodies may be obtained, for example, using the hybridoma method first described by Kohler et al., Nature, 1975, 256:495-497 (incorporated by reference in its entirety), and/or by recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567, incorporated by reference in its entirety). Monoclonal antibodies may also be obtained, for example, using phage or yeast-based libraries. See e.g., U.S. Pat. Nos. 8,258,082 and 8,691,730, each of which is incorporated by reference in its entirety.

In the hybridoma method, a mouse or other appropriate host animal is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See Goding J. W., Monoclonal Antibodies: Principles and Practice 3^rd ed. (1986) Academic Press, San Diego, Calif., incorporated by reference in its entirety.

The hybridoma cells are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Useful myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive media conditions, such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, Calif.), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Md.). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. See e.g., Kozbor, J. Immunol., 1984, 133:3001, incorporated by reference in its entirety.

After the identification of hybridoma cells that produce antibodies of the desired specificity, affinity, and/or biological activity, selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

DNA encoding the monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Thus, the hybridoma cells can serve as a useful source of DNA encoding antibodies with the desired properties. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as bacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to produce the monoclonal antibodies.

11.3. Humanized Antibodies

Humanized antibodies may be generated by replacing most, or all, of the structural portions of a non-human monoclonal antibody with corresponding human antibody sequences. Consequently, a hybrid molecule is generated in which only the antigen-specific variable, or CDR, is composed of non-human sequence. Methods to obtain humanized antibodies include those described in, for example, Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. U.S.A., 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86:10029-10033; and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; each of which is incorporated by reference in its entirety.

11.4. Human Antibodies

Human antibodies can be generated by a variety of techniques known in the art, for example by using transgenic animals (e.g., humanized mice). See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807; each of which is incorporated by reference in its entirety. Human antibodies can also be derived from phage-display libraries (see e.g., Hoogenboom et al., J. Mol. Biol., 1991, 227:381-388; Marks et al., J. Mol. Biol., 1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905; each of which is incorporated by reference in its entirety). Human antibodies may also be generated by in vitro activated B cells (see e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety). Human antibodies may also be derived from yeast-based libraries (see e.g., U.S. Pat. No. 8,691,730, incorporated by reference in its entirety). Human antibodies can also be generated and screened by ribosome display (see, e.g., WO 2014/176327 and WO 2014/176439, each of which is incorporated herein by reference in its entirety).

12. Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acids encoding an antibody or bispecific antigen binding construct disclosed herein, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies.

For recombinant production of the antibody, the nucleic acid(s) encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression. In some aspects, the nucleic acid may be produced by homologous recombination, for example as described in U.S. Pat. No. 5,204,244, incorporated by reference in its entirety.

Many different vectors are known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Pat. No. 5,534,615, incorporated by reference in its entirety.

Illustrative examples of suitable host cells are provided below. These host cells are not meant to be limiting.

Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), and Streptomyces. One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-LAG3 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is a commonly used lower eukaryotic host microorganism. However, a number of other genera, species, and strains are available and useful, such as Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus K. wickeramii, K. waltii, K. drosophilarum, K thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as, for example Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

Suitable host cells can also include insect cells, such as, for example, Drosophila systems S2, SF9, and SF21 cells and High Five™ cells (ThermoFisher Scientific). Drosophila cells can be grown in a suitable medium, such as, for example, Schneider's Drosophila medium or other commercially available media,

Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertoli cells; African green monkey kidney cells (VERO-76), and the like.

The host cells used to produce the anti-LAG3 antibody of this invention may be cultured in a variety of media. Commercially available media such as, for example, Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz., 1979, 58:44; Barnes et al., Anal. Biochem., 1980, 102:255; and U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469, or WO 90/03430 and WO 87/00195 may be used. Each of the foregoing references is incorporated by reference in its entirety.

Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. For example, Carter et al. (Bio/Technology, 1992, 10:163-167) describes a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min Cell debris can be removed by centrifugation.

In some embodiments, the antibody is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs, 2012, 4:217-225, incorporated by reference in its entirety. In some aspects, the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell. In some aspects, the prokaryotic cell is E. coli. Cell-free expression of the antibody may be useful, for example, where the antibody accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.

Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore® Pellcon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth., 1983, 62:1-13, incorporated by reference in its entirety). Protein G is useful for all mouse isotypes and for human γ3 (Guss et al., EMBO J., 1986, 5:1567-1575, incorporated by reference in its entirety).

The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a C_H3 domain, the BakerBond ABX® resin is useful for purification.

Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin Sepharose®, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, and can be applied by one of skill in the art. In embodiments where the antibody is a bi-specific antibody or antigen binding construct, separation techniques suitable for such molecules are described, for example, in Xu et al. 2015. mAbs 7:231-242, which is incorporated herein by reference in its entirety.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 to about 4.5, generally performed at low salt concentrations (e.g., from about 0 to about 0.25 M salt).

13. Pharmaceutical Compositions and Methods of Administration

Any of the antibodies or bi-specific antigen binding constructs provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, the inhalation, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises an anti-foaming agent. Any suitable anti-foaming agent may be used. In some aspects, the anti-foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof. In some aspects, the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, and propylene glycol.

In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.

In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.

In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.

In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.

Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars. Specific examples of each of these agents are described, for example, in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is saline solution, such as a sterile isotonic saline solution or dextrose solution. In some aspects, the solvent is water for injection.

In some embodiments, the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles may be formed from any suitable material, such as a polymer or a lipid. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymersomes.

Further provided herein are anhydrous pharmaceutical compositions and dosage forms comprising an antibody, since water can facilitate the degradation of some antibodies.

Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided herein comprise a variant of an illustrative CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequence provided in this disclosure.

In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H1 sequence provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure. In some aspects, the CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia or Kabat CDR-H2 sequence provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure. In some aspects, the CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia or Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure. In some aspects, the CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some aspects, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some aspects, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some aspects, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In certain embodiments, provided are parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses subjects' natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Excipients that increase the solubility of one or more of the antibodies disclosed herein can also be incorporated into the parenteral dosage forms.

13.1. Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which he considers most appropriate according to a preventive or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated.

In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies.

The amount of the antibody or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the antibody is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In certain embodiments, exemplary doses of a composition include milligram or microgram amounts of the antibody per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram). In certain embodiment, the dosage of the antibody provided herein, based on weight of the antibody, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is about 0.1 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 10 mg/kg, or about 15 mg/kg or more of a subject's body weight. In another embodiment, the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is about 0.1 mg to about 200 mg, about 0.1 mg to about 100 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 25 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 7.5 mg, about 0.1 mg to about 5 mg, about 0.1 to about 2.5 mg, about 0.25 mg to about 20 mg, about 0.25 to about 15 mg, about 0.25 to about 12 mg, about 0.25 to about 10 mg, about 0.25 mg to about 7.5 mg, about 0.25 mg to about 5 mg, about 0.25 mg to about 2.5 mg, about 0.5 mg to about 20 mg, about 0.5 to about 15 mg, about 0.5 to about 12 mg, about 0.5 to about 10 mg, about 0.5 mg to about 7.5 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 2.5 mg, about 1 mg to about 20 mg, about 1 mg to about 15 mg, about 1 mg to about 12 mg, about 1 mg to about 10 mg, about 1 mg to about 7.5 mg, about 1 mg to about 5 mg, or about 1 mg to about 2.5 mg.

The dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the antibody outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.

Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the antibodies provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.

In certain embodiments, treatment or prevention can be initiated with one or more loading doses of an antibody or composition provided herein followed by one or more maintenance doses.

In certain embodiments, a dose of an antibody or composition provided herein can be administered to achieve a steady-state concentration of the antibody in blood or serum of the subject. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight and age.

In certain embodiments, administration of the same composition may be repeated and the administrations may be separated by at least about 1 day, about 2 days, about 3 days, about 5 days, about 10 days, about 15 days, about 30 days, about 45 days, about 2 months, about 75 days, about 3 months, or about 6 months. In other embodiments, administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least about 1 day, about 2 days, about 3 days, about 5 days, about 10 days, about 15 days, about 30 days, about 45 days, about 2 months, about 75 days, about 3 months, or about 6 months.

14. Therapeutic Applications

For therapeutic applications, the antibodies of the invention are administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above. For example, the antibodies of the invention may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, or intratumoral routes. The antibodies also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.

The antibodies provided herein may be useful for the treatment of any disease or condition involving LAG3 and/or PD-1. In some embodiments, the disease or condition is a disease or condition that can be diagnosed by overexpression of LAG3 and/or PD-1. In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with an anti-LAG3 antibody and/or anti-PD-1 antibody. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an infection.

Any suitable cancer may be treated with the antibodies provided herein. Illustrative suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

In particular embodiments, the cancer is a cancer of epithelial origin. In some aspects, the cancer is a carcinoma. In some aspects, the cancer is selected from an adenocarcinoma, a squamous cell carcinoma, an adenosquamos carcinoma, an anaplastic carcinoma, a large cell carcinoma, small cell carcinoma, and carcinoma of unknown primary origin.

15. Diagnostic Applications

In some embodiments, the antibodies provided herein are used in diagnostic applications.

In some embodiments, an antibody or bi-specific antibody as disclosed herein may be useful in assays for LAG3 protein. In some aspects the antibody or bi-specific antibody can be used to detect the expression of LAG3 in various cells and tissues. These assays may be useful, for example, in making a diagnosis and/or prognosis for a disease, such as a cancer.

In some embodiments, an antibody or bi-specific antibody as disclosed herein may be useful in assays for PD-1 protein. In some aspects the antibody or bi-specific antibody can be used to detect the expression of PD-1 in various cells and tissues. These assays may be useful, for example, in making a diagnosis and/or prognosis for a disease, such as a cancer.

In some diagnostic and prognostic applications, the antibody or bi-specific antibody may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to radioisotopes, fluorescent labels, and enzyme-substrate labels. In another embodiment, the antibody or bi-specific antibody need not be labeled, and the presence of the antibody or bi-specific antibody can be detected using a labeled antibody which specifically binds to the anti-LAG3 antibody.

16. Affinity Purification Reagents

The antibodies and bi-specific antigen binding constructs disclosed herein may be used as affinity purification agents. In this process, the antibodies or bi-specific antibodies may be immobilized on a solid phase such a resin or filter paper, using methods well known in the art. The immobilized antibody or bi-specific antibody is contacted with a sample containing an antigen protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the antigen protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0 or glycine buffer, pH 3 to 4, that will release the antigen protein from the antibody. In some embodiments, the antigen protein is LAG3. In some embodiments, the antigen protein is PD-1.

17. Kits

In some embodiments, an antibody or bi-specific antigen binding construct provided herein is provided in the form of a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure. In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure.

In some embodiments, the kit further comprises a solvent for the reconstitution of the antibody or bispecific antibody. In some embodiments, the antibody or bispecific antibody is provided in the form of a pharmaceutical composition.

EXAMPLES

Example 1: Generation of Mouse and Humanized LAG3 Antibodies

Balb/C mice were immunized with the extracellular domain of human LAG3 fused with human Fc (R&D Systems) using standard immunization methods. The spleens and/or lymph nodes of the mice were harvested and fused with P3X cells to generate hybridomas (Aragen Biosciences, Morgan Hill, Calif.), similar to what has been previously described (Chronopoulou et al., 2014, Methods Mol Biol. 1131:47-70; Kim, et al., 2014, Methods Mol Biol. 1131:33-45; each incorporated by reference in its entirety).

Total RNA was extracted from hybridoma cells using QIAGEN RNeasy Mini Kit (Cat No. 74104) and converted to cDNA using a Clontech SMARTer RACE cDNA Amplification Kit (Cat. No. 634923; Lake Pharma, Belmont, Calif.). Positive clones were identified by gel electrophoresis, cloned using an Invitrogen TOPO kit, and sequenced using standard Sanger methods. Mouse single-chain antibodies were constructed by using total gene synthesis using optimized E. Coli codons and cloned into a standard cell-free expression vector (Yin et al., 2012, mAbs 4:217-225, incorporated by reference in its entirety). Murine IgG 421.61.4.5G11 (5G11) was selected.

The CDRs for 5G11 were grafted onto human antibody frameworks V_H1-69 and Vk2-30 by standard methodology (Kuramochi et al., 2014, Methods Mol. Biol. 1060:123-137, incorporated by reference in its entirety) to yield humanized antibody h5G11-2.

Example 2: Generation and Primary Screening of Anti-LAG3 Antibodies

Antibody Fab and scFv libraries were constructed using a standard overlap extension PCR protocol with mutagenic primers targeting complementary determining regions (CDRs). See Heckman and Pease, Nat. Protoc., 2007, 2:924-932, incorporated by reference in its entirety. Selections for novel antibodies were performed using standard ribosome display protocols. See Dreier and Plückthun, Methods Mol. Biol., 2003, 687:283-306, Clifton, N.J., incorporated by reference in its entirety. Specifically, scFv and Fab ribosome display selections were performed according to published protocols. See Hanes and Plückthun, Proc. Natl. Acad. Sci. U.S.A., 1997, 94:4937-4942; Stafford et al., 2014, Protein Eng. Des. Sel. 27:97-109; each incorporated by reference in its entirety. After multiple rounds of selection, the DNA from RT-PCR output was cloned into an optimized vector for cell-free expression using standard molecular biology techniques. See Yin et al., mAbs, 2012, 4:217-225, incorporated by reference in its entirety. All constructs were HIS- and FLAG-tagged to streamline purification and testing during screening.

Libraries of antibody variants generated by the selection workflow were transformed into E. coli and grown on agar plates with antibiotic (kanamycin). Individual colonies were grown in liquid broth (TB+kanamycin), and used as a template for DNA amplification via rolling circle amplification (RCA). The variants were then expressed in cell-free protein synthesis reactions as described in Zawada et al., 2011, Biotechnol. Bioeng. 108:1570-1578, incorporated by reference in its entirety.

Briefly, cell-free extracts were treated with 50 μM iodoacetamide for 30 min at room temperature (20° C.) and added to a premix containing cell-free components (see Groff et al., mAbs, 2014, 6:671-678, incorporated by reference in its entirety) and 10% (v/v) RCA DNA template (approximately 10 μg/mL DNA) for variants of interest. For Fab selection, 2.5 μg/mL trastuzumab LC DNA was also added to the reactions. Sixty microliters of cell-free reactions were incubated at 30° C. for 12 hr on a shaker at 650 rpm in 96-well plates. Four hundred to one-thousand-five-hundred colonies were screened, depending on the predicted diversity of different selection campaigns.

Following synthesis, each reaction was diluted 1:50 into PBST (PBS at pH 7.4 with 0.2% Tween-20+0.2% BSA) and expressed variants were tested for functional activity via ELISA-based binding to recombinant human LAG3 extracellular domain (ECD) (Acro Biosystems; R&D Systems). Standard ELISA-based methods were employed. Specifically, 384-well plates were coated with 2 μg/mL recombinant LAG3 diluted in bicarbonate buffer, and then blocked with BSA. Antibody variants of interest were allowed to bind to the LAG3-coated plates, and detected with secondary antibodies (e.g., HRP-conjugated anti-human Fc or anti-FLAG) and then detected with chemiluminescent substrate (Pierce ELISA SuperSignal™ Substrate). Chemiluminescence was quantified on a Molecular Devices SpectraMax® M5 plate reader. Top hits were selected based on ELISA signal or signal/noise ratio and their associated DNA constructs were sequenced. Based on functional activity and sequence analysis, a subset of variants was selected for further scale-up and characterization.

Example 3: Secondary Screening of LAG3 Antibodies

The top leads from the initial round of screening were cultured and plasmid minipreps were performed using a QIAprep® 96 Turbo miniprep kit (Qiagen) according to the manufacturer's instructions. 10 μg/mL miniprepped DNA was added to 4 mL cell-free reactions and incubated overnight for 12 hr at 30° C., at 650 rpm. For Fab selection, 2.5 ug/mL trastuzumab LC DNA was also added.

Expressed variants from clarified cell-free reactions were purified via immobilized metal ion affinity chromatography (IMAC) purification using a semi-automated high throughput batch purification method. Briefly, purifications were performed in a 96-well plate format where 50 μL/well of IMAC resin (Ni Sepharose High Performance, GE Healthcare) was equilibrated in IMAC binding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole), incubated with 1 mL cell-free reaction for 15 minutes followed by two washes in IMAC binding buffer. His-tagged antibody variants were then eluted using 200 μL IMAC elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole) and buffer exchanged into PBS using a 96-well Zeba plate (7 kD MWCO, Thermo Fisher). Purified antibodies were quantified via high throughput capillary electrophoresis using the LabChip GXII (Perkin Elmer) against a Herceptin standard curve, according to the manufacturer's instructions.

Example 4: LAG3 Antibody Selection and Maturation

Primary and secondary screening with humanized antibody h5G11-2 yielded antibodies designated SRP1627 in the Examples below. Ribosome display was used to affinity mature antibody 26H10 (SEQ ID NOs: 191 and 251) yielding antibodies designated SRP1449 or 1449 in the Examples below. Antibody SRP1448-D09 was identified by screening a naive scFv antibody library against LAG3. Affinity maturation of SRP1448-D09 using ribosome display yielded antibodies designated SRP1558 in the Examples below. Anti-LAG-3 Fabs were identified by selecting from a Fab TRIM library against recombinant LAG3 protein using ribosome display (Stafford et al., Protein Eng Des Sel 2014, 27:97-109, incorporated by reference in its entirety). Primary and secondary screening yielded LAG-3 Fab antibodies designated SRP1496 in the Examples below. Affinity maturation of antibody SRP1496-A04 using ribosome display yielded antibodies designated SRP1648 in the Examples below. All ribosome display selections were screened by cloning the output into a cell free expression vector for small-scale expression followed by characterization by ELISA, biacore, cell binding, and ligand competition.

The mouse antibody 421.61.4.5G11 was constructed from the V_H and V_L variable domains in the table below and mouse constant domains. The human and humanized antibodies were constructed from the V_H and V_L variable domains in the table below and human constant domains. Additional human antibodies are constructed in either scFvFc or IgG format. The scFvFc format contains a V_H domain, followed by a linker domain (for instance, a GGGGSGGGGSGGGGS linker (SEQ ID NO: 308) or a APGPSAPSHRSLPSRAFG linker (SEQ ID NO: 314) from Tang et al., 1996, J. Biol. Chem. 271:15682-15686, incorporated by reference in its entirety), then the V_L domain, and then the human scFvFc constant domains. The mouse and human antibody sequences start with an N-terminal methionine to enable expression in cell-free. Additional variable domains can also be expressed in a mammalian system by fusing an N-terminal signal peptide instead of an N-terminal methionine. Additional antibodies can also be expressed with or without a C-terminal affinity tags (e.g., His or FlagHis, SEQ ID NO: 307).

TABLE 21
Antibody Sequences
	VH	VL
		SEQ		SEQ
Antibody		ID		ID
Name	Name	NO	Name	NO
421.61.4.5G11	5G11-VH	210	5G11-VL	266
SRP1627-A02	SRP1627-A02-VH	206	SRP1627-A02-VL	262
SRP1627-A11	SRP1627-A11-VH	207	SRP1627-A11-VL	263
SRP1627-B01	SRP1627-B01-VH	209	SRP1627-B01-VL	264
h5G11-2	h5G11-2-VH	208	h5G11-2-VL	265
SRP1449-B03	SRP1449-B03-VH	197	SRP1449-B03-VL	256
SRP1449-B07	SRP1449-B07-VH	199	SRP1449-B07-VL	255
SRP1449-D05	SRP1449-D05-VH	201	SRP1449-D05-VL	252
SRP1449-F01	SRP1449-F01-VH	198	SRP1449-F01-VL	253
SRP1449-	SRP1449-G09.2-	200	SRP1449-G09.2-	254
G09.2	VH		VL
SRP1558-A06	SRP1558-A06-VH	204	SRP1558-A06-VL	258
SRP1558-E11	SRP1558-E11-VH	205	SRP1558-E11-VL	257
SRP1558-F01	SRP1558-F01-VH	202	SRP1558-F01-VL	259
SRP1448-D09	SRP1448-D09-VH	203	SRP1448-D09-VL	260
SRP1496-A03	SRP1496-A03-VH	192	trastuzumab-VL	261
SRP1496-A04	SRP1496-A04-VH	193	trastuzumab-VL	261
SRP1496-B08	SRP1496-B08-VH	194	trastuzumab-VL	261
SRP1648-B07	SRP1648-B07-VH	195	trastuzumab-VL	261
SRP1648-E02	SRP1648-E02-VH	196	trastuzumab-VL	261

Example 5: Affinity and Kinetic Binding Analyses of LAG3 Antibodies

Anti-Flag M2 IgG (Sigma-Aldrich # F9291) was immobilized onto a CMS chip (GE Life Sciences) using amine coupling chemistry (from Amine Coupling Kit, GE Life Sciences). The immobilization steps were carried out at a flow rate of 25 μL/min in 1×HBS-EP+ buffer (GE Life Sciences; 10×Stock diluted before use). The sensor surfaces were activated for 7 min with a mixture of NHS (0.05 M) and EDC (0.2 M). The Anti-Flag M2 IgG was injected over all 4 flow cells at a concentration of 25 μg/mL in 10 mM sodium acetate, pH 4.5, for 7 min. Ethanolamine (1 M, pH 8.5) was injected for 7 min to block any remaining activated groups. An average of 12,000 response units (RU) of capture antibody was immobilized on each flow cell.

Off-rate and kinetic binding experiments were performed at 25° C. using 1×HBS-EP+buffer. Test and control antibodies were injected over the Anti-Flag surface at concentrations of 5-10 μg/mL for 12 seconds at a flow rate of 10 μL/min on flow cells 2, 3 and 4, followed by a buffer wash for 30 seconds at the same flow rate. Kinetic characterization of antibody samples was carried out with a single concentration of antigen (for off-rate ranking) or a dilution series of antigen (for kinetic characterization) and 1 injection of 0 nM antigen. After capturing ligand (antibody) on the anti-Flag surface, the analyte (human LAG3-Fc, R&D Systems #2319-L3; or cynomolgus LAG3-Fc, accession # NC 022282.1) was bound at 50, 25, 12.5, 6.25, and 0 nM for 180 seconds, followed by a 600 second dissociation phase at a flow rate of 50 μl/min Between each ligand capture and analyte binding cycle, regeneration was carried out using 2 injections of 10 mM glycine pH 2.0 for 30 seconds at 30 μL/min, followed by a 30 second buffer wash step.

The data were fit with the Biacore T200 Evaluation software, using a 1:1 Langmuir binding model. K_D (affinity, nM) was determined as a ratio of the kinetic rate constants calculated from the fits of the association and dissociation phases.

Example 6: ELISA Binding Assay of Anti-LAG3 Antibodies

Standard ELISA methods were used to compare binding to human and cynomolgus recombinant LAG-3. Specifically, 384-well plates were coated with 2 μg/mL recombinant LAG3 (human LAG3-Fc or cynomolgus LAG3-Fc) diluted in bicarbonate buffer, and then blocked with BSA. A dilution series of antibody variants were allowed to bind to the LAG3-coated plates, and detected with secondary antibodies (e.g., HRP-conjugated anti-human Fab or anti-FLAG) and then detected with chemiluminescent substrate (Pierce ELISA SuperSignal™ Substrate). Chemiluminescence was quantified on a Molecular Devices SpectraMax® M5 plate reader. ELISA EC50s were calculated.

Example 7: Cell Binding Assay of Anti-LAG3 Antibodies

LAG3 antibody variants were tested in a fluorescence-activated cell sorting (FACS) cell-binding assay. Chinese Hamster Ovary (CHO) cells or HEK293T cells stably expressing the human target molecule LAG3 on the cell surface (CHO-LAG3, 293T-LAG3) were used to screen for cell binders by flow cytometry. Parental CHO or 293T cells were used as a negative control to determine background-binding levels. Cells were cultured in RPMI with 10% FCS Penicillin/Streptomycin (or Pen/Strep) and glutamine (or Gln) and split every 3-4 days at 10⁵ cells/ml.

A mix of parental CHO cells and CHO-LAG3 cells (or 293T and 293T-LAG3 cells) was prepared as follows: Parental CHO cells were washed 2× in PBS then incubated in PBS containing 1 nM CellTrace™ Oregon Green488® (Life Technologies) at 37° C. for 30 minutes. Cells were then washed 2× with RPMI w/10% fetal calf serum (or FCS), washed 2× with FACS buffer (PBS w/2% FCS), suspended thoroughly in ice-cold FACS buffer at a final concentration of 2×106 cells/ml and kept on ice. CHO-LAG3 cells were similarly washed with FACS buffer and kept on ice at 2×106 cells/ml. Parental CHO cells and CHO-LAG3 cells were then mixed to obtain a 1:1 cell suspension and seeded at 100 μl per well on 96 well polypropylene plates. Plates were spun at 1500 rpm for 5 minutes and cell pellets were suspended in 50 μl FACS buffer containing 6-12 point dilutions of anti-LAG3 variants starting from concentrations of ˜100-200 nM antibody, dispensed using BioMekFX (Beckman Coulter). Cells were then incubated on ice for 1 hr, washed with FACS buffer and incubated for 1 hr on ice with 50 μl FACS buffer containing 2.5 μg/ml R-Phycoerythrin-conjugated Goat Anti-Human IgG (Jackson ImmunoResearch) or AF647-conjugated Goat Anti-mouse IgG (Life Technologies) dispensed using BioMekFX (Beckman Coulter). Cells were then washed 2× with FACS buffer and fixed for 10 minutes in 200 μl PBS with 2% PFA prior to fluorescence detection. Samples were acquired using a Beckton Dickinson LSRII FACS. Mean Fluorescence Intensity of LAG3 antibody binding was analyzed using Tree Star, Inc. FlowJo® software.

Example 8: Cell-Based MHCII Competition of LAG3 Antibodies

Top variants that showed cell-binding activity were tested in a fluorescence-activated cell sorting (FACS) cell-based competition assay. DAUDI cells express high levels of Major Histocompatibility Class II (MHCII) molecules, a natural ligand for LAG3, on the cell surface. DAUDI cells were used to screen for antibodies that inhibit binding of HIS-tagged (ACRO) or biotinylated recombinant human LAG3 protein (rhLAG3) to MHCII expressed on the cell surface.

DAUDI cells were cultured in RPMI w/10% FCS Pen/Strep and Gln and split every 3-4 days at 105 cells/ml. Cells were washed 2× with FACS buffer (PBS w/2% FCS), thoroughly in ice-cold FACS buffer at a final concentration of 1×10⁶ cells/ml and seeded at 100 μl per well on 96 well polypropylene plates. Plates were spun at 1500 rpm for 5 minutes and cell pellets were suspended in 50 μl FACS buffer containing 8 point 1:3 dilutions (2× concentrated) of anti-LAG3 antibody variants, starting from high concentration of −600 nM. 50 μl FACS buffer containing 10-20 μg/ml of the HIS-tagged rhLAG3 protein or 40 μg/ml of the biotinylated rhLAG3 protein were then added to the cells. Cell were then incubated in ice for 1 hr, washed with FACS buffer and incubated for 1 hr in ice with 50 μl FACS buffer containing 2 μg/ml R-Phycoerythrin-conjugated Streptavidin (eBiosciences) or 1 μg/ml R-Phycoerythrin-conjugated anti-HIS IgG (Abcam). Cell were washed 2× with FACS buffer and fixed for 10 minutes in 200 μl PBS w/2% PFA prior to acquisition.

Example 9: Effect of Anti-PD-1 in Combination with Anti-LAG3 Antibodies on IFN γ Production in a CMV Recall Assay and Dendritic Cell (DC)/CD-4+ T Cell Mixed Lymphocyte Reaction (MLR)

CMV Recall Assay

CD14⁺ monocytes and CD3⁺ T cells were obtained from peripheral blood mononuclear (PBMC) isolated from CMV⁺ human donors (AllCells, Alameda, Calif.) using MACS Cell Separation kits (Miltenyi Biotec). CD14⁺ monocytes were differentiated into immature dendritic cells (DC) by culturing cells at 1e6 cells/ml for 7 days in presence of GM-CSF and IL-4 (Peprotech) in X-Vivo 15 media (Lonza) containing 2% human AB serum (Sigma-Aldrich), penicillin-streptomycin (Corning Mediatech) and GlutaMAX (Life Technologies). Following differentiation, DCs were matured by culturing in X-Vivo 15+2% human AB serum media at 1e6 cells/ml for 2 days in the presence of GM-CSF, IL-4, TNF-a, IL-1b, IL-6 (Peprotech) and prostaglandin E2 (Sigma-Aldrich). To set-up the CMV recall assay, mature DCs were collected, washed and 10,000 DCs and 100,000 pan CD3⁺ T cells were plated per well in a 96-well U-bottom plate in a total volume of 100 ul media containing peptide pools for the CMV IE-1 and CMV pp65 protein (Miltenyi Biotec). Anti-PD-1 and/or anti-LAG-3 IgG antibodies (50 ul) were added starting at a final concentration of 133-400 nM with 5-fold serial dilutions. Cells were co-cultured with peptides and antibodies for 5-6 days. Conditioned media was collected and tested for human IFN-g levels by ELISA (BD Biosciences).

DC/CD4⁺ T Cell Mixed Lymphocyte Reaction (MLR)

Allogeneic CD14⁺ monocytes and CD4⁺ T cells were obtained from PBMC isolated from human donors using MACS Cell Separation kits. CD14⁺ monocytes were differentiated into immature DC by culturing cells at 1e6 cells/ml cell density for 7 days in presence of GM-CSF and IL-4 in RPMI media containing 10% fetal bovine serum, penicillin-streptomycin and GlutaMAX. Following differentiation, DCs were matured by culturing in RPMI+10% FBS media at 1e6 cells/ml cell density for 2 days in the presence of GM-CSF, IL-4, TNF-α, IL-1b, IL-6 and prostaglandin E2. To set-up the DC/CD4⁺ T cell MLR, mature DCs were collected, washed and 10,000 DCs and 100,000 CD4⁺ T cells were plated per well in a 96-well U-bottom plate in a total volume of 100 ul media. Anti-PD-1 and/or anti-LAG-3 IgG antibodies (50 ul, final volume of 150 ul per well) were added starting at a final concentration of 133-400 nM with 5-fold serial dilutions. Cells were co-cultured with peptides and antibodies for 5-6 days. Conditioned media was collected and tested for human IFN-g levels by ELISA.

Example 10: Characteristics of Illustrative Anti-LAG3 Antibodies

FIG. 2 (A-B) provides an alignment of the V_H sequences provided herein. FIG. 3 provides an alignment of the V_L sequences provided herein. Chothia CDR sequences are highlighted, and Kabat CDR sequences are underlined.

Tables 22-23 provide results obtained using the illustrative LAG3 antibodies described herein. Table 22 presents the results of binding assays for antibodies provided herein. Table 23 provides the results of functional assays provided herein.

TABLE 22
Binding Assays
								Human	Human	Cyno
			Human LAG3	Cyno LAG3	LAG3	LAG3	LAG3
Antibody	(Biacore)	(Biacore)	(CHO)	(293T)	(293T)
		SEQ ID	ka	kd	KD	kd	KD	KD	KD	KD
Name	Scaffold	NO(s)	(1/Ms)	(1/s)	(M)	(1/s)	(M)	(nM)	(nM)	(nM)
421.61.4.5G11	Murine IgG	210 (VH);	5.02E+04	5.15E−04	1.03E−08	6.41E−04	4.51E−09	18.0		4.5
		266 (VL)
SRP1627-A02	ScFvFc	216 (VH);	1.64E+06	7.10E−02	4.33E−08				0.97	+−−
		262 (VL)
SRP1627-A11	ScFvFc	207	3.12E+05	4.22E−03	1.35E−08				0.74	+−−
		(VH); 263
		(VL)
SRP1627-B01	ScFvFc	209 (VH);	1.04E+06	1.52E−02	1.47E−08				0.19	nd
		264 (VL)
h5G11-2	ScFvFc	208 (VH);	2.67E+03	2.30E−03	8.63E−07				78	nd
		265 (VL)
SRP1449-B03	ScFvFc	197 (VH);	4.72E+05	3.02E−04	6.4E−10			1.92
		256 (VL)
SRP1449-B07	ScFvFc	199 (VH);	7.94E+05	5.36E−04	6.76E−10			1.06
		255 (VL)
SRP1449-D05	ScFvFc	201 (VH);	4.60E+05	6.27E−04	1.36E−09			1.54
		252 (VL)
SRP1449-F01	ScFvFc	198 (VH);	2.61E+05	8.07E−04	3.1E−09			3.37
		253 (VL)
1449-G09.2	ScFvFc	200 (VH);	1.65E+06	6.78E−05	4.12E−11			0.32
		254 (VL)
SRP1558-A06	ScFvFc	204 (VH);	7.9E+05	9.9E−03	1.3E−08				0.3	0.3
		258 (VL)
SRP1558-E11	ScFvFc	205 (VH);	5.6E+05	1.7E−02	3.1E−08				0.5	1.0
		257 (VL)
SRP1558-F01	ScFvFc	202 (VH);	4.35E+05	1.1E−02	2.4E−08				0.5	0.7
		259 (VL)
SRP1448-D09	ScFvFc	203 (VH);	1.61E+05	6.2E03−	3.9E−08				0.4	1.6
		260 (VL)
SRP1496-A03	IgG	192 (VH);	1.24E+06	2.34E−02	1.89E−08				40.6
		261 (VL)
SRP1496-A04	IgG	193 (VH);	1.29E+06	1.96E−02	1.52E−08				35.0
		261 (VL)
SRP1496-B08	IgG	194 (VH);	1.31E+06	2.79E−02	2.13E−08				39.4
		261 (VL)
SRP1648-B07	IgG	197 (VH);	1.12E+07	2.75E−02	2.46E−09				positive
		261 (VL)
SRP1648-E02	IgG	196 (VH);	5.35E+07	1.22E−01	2.27E−09				positive
		261 (VL)
nd = not detected;
+++ = binding observed, KD not calculated

TABLE 23
Functional Assays.
				Cyno
		MHCII		ELISA
		Blockade		Reactivity
		IC50	Functional	EC50
	Antibody	(nM)	Activity	(nM)
	421.61.4.5G11	64.3	positive	NT
	SRP1627-A02	1.7	NT	NT
	SRP1627-A11	1.6	NT	NT
	SRP1627-B01	0.4	NT	NT
	h5G11-2	nd	NT	NT
	SRP1449-B03	1.0	NT	NT
	SRP1449-B07	1.2	NT	NT
	SRP1449-D05	2.3	NT	NT
	SRP1449-F01	1.7	NT	NT
	1449-G09.2	1.1	Positive	NT
	SRP1558-A06	Not tested	Positive	NT
	SRP1558-E11	Not tested	NT	NT
	SRP1558-F01	Not tested	NT	NT
	SRP1448-D09	1.9	NT	NT
	SRP1496-A03	41.3	NT	4.2
	SRP1496-A04	55.8	NT	1.6
	SRP1496-B08	28.6	NT	2.6
	SRP1648-B07	4.5	NT	NT
	SRP1648-E02	4.1	NT	NT
	NT = not tested

Example 11: Methods for Assessing Bi-Specific Antigen-Binding Constructs

Bi-specific antigen-binding constructs as described in Examples 12 to 17 were assessed for functional characteristics based on the following methods.

Cell Lines and Reagents

Stably transfected CHO-k and U2OS cells were cultured in RPMI or DMEM/F12, respectively, supplemented with 10% fetal calf serum, penicillin/streptomycin and glutamine. On day of assay, cells were washed with PBS, detached with Accutase™ (BD Biosciences; San Jose, Calif.), and resuspended in culture media. Daudi cells were cultured in RPMI with 10% fetal calf serum, penicillin/streptomycin and glutamine and maintained at 0.3 to 3×10⁶ cells/mL.

Cell Binding Assay

CHO-k cells stably expressing PD-1 or LAG3 (human or cynomolgus) on the cell surface were used to assess binding affinity of anti-PD-1/LAG3 bispecific antibody constructs. Cells were seeded at 100,000 cells/well in 100 μL of FACS buffer (1×PBS, 0.5% BSA, 0.1% sodium azide) in 96-well polypropylene plates. The cells were centrifuged at 1.5K rpm and resuspended with test antibodies diluted in FACS buffer and incubated on ice for 1 hour. The cells were washed twice with FACS buffer and incubated on ice for 30 mins with R-Phycoerythrin-AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG (H+L) secondary detection antibody (1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, Pa.). The cells were washed twice with FACS buffer, fixed in 4% paraformaldehyde in PBS (Santa Cruz Biotechnology; Dallas, Tex.) for 20 mins on ice in the dark, washed twice with FACS buffer, and analyzed using the BD LSR II Flow Cytometer (BD Biosciences; San Jose, Calif.). Data were analyzed using FlowJo (FlowJo, LLC; Ashland, Oreg.) to determine mean fluorescence intensities. Binding constants were calculated using the statistical software, GraphPad Prism (GraphPad Software; La Jolla, Calif.) using the nonlinear regression equation, one site—specific binding with Hill slope. The secondary antibody by itself was used as a control.

Affinity and Kinetic Binding Analyses

Anti-Fc polyclonal antibodies were immobilized onto a CMS chip (GE Life Sciences) using amine coupling chemistry (from Amine Coupling Kit, GE Life Sciences). The immobilization steps were carried out at a flow rate of 25 μl/min in 1×HBS-EP+buffer (GE Life Sciences; 10× Stock diluted before use). The sensor surfaces were activated for 7 min with a mixture of NHS (0.05 M) and EDC (0.2 M). The Anti-Fc antibodies were injected over all 4 flow cells at a concentration of 25 ug/ml in 10 mM sodium acetate, pH 4.5, for 7 min. Ethanolamine (1 M, pH 8.5) was injected for 7 min to block any remaining activated groups. An average of 12,000 response units (RU) of capture antibody was immobilized on each flow cell.

Kinetic binding experiments were performed at 25° C. using 1×HBS-EP+buffer. Test and control antibodies were injected over the anti-Fc surface at concentrations of 5-10 μg/mL for 12 seconds at a flow rate of 10 μl/min on flow cells 2, 3 and 4, followed by a buffer wash for 30 seconds at the same flow rate. Kinetic characterization of antibody samples was carried out with a dilution series of antigen and 1 injection of 0 nM antigen. After capturing ligand (antibody) on the anti-Fc surface, the analyte (hPD1 or hLAG3, Acro Biosystems, Newark, Del.) was bound at 100, 25, 6.25 and 0 nM for 180 seconds, followed by a 450 second dissociation phase at a flow rate of 50 μl/min. Between each ligand capture and analyte binding cycle, regeneration was carried out using 2 injections of 10 mM Glycine pH 2.0 for 30 seconds at 30 μL/min, followed by a 30 second buffer wash step.

The data was fit with the Biacore T200 Evaluation software, using a 1:1 Langmuir binding model. K_D (affinity, nM) was determined as a ratio of the kinetic rate constants calculated from the fits of the association and dissociation phases.

PD1 Competition Assay

CHO-k cells stably expressing human PD-L1 or PD-L2 were used to measure the ability of anti-PD1/LAG3 bi-specific antibody constructs to block rabbit Fc-tagged recombinant human PD1 (rhPD1-rabbit Fc) protein binding. Cells were seeded at 100,000 cells/well in 100 μL of FACS buffer in 96-well polypropylene plates. The cells were centrifuged and resuspended with test antibodies (2× concentration) diluted in FACS buffer, immediately followed by equal volume of rhPD1-rabbit Fc (added at 2× concentration; 0.2 ng/mL and 0.1 ng/mL final for PD-L1 and PD-L2, respectively). Cells were incubated on ice for 1 hour and washed twice. rhPD1-rabbit Fc binding on CHO-human PD-L1 or PD-L2 cells was detected with R-Phycoerythrin AffiniPure F(ab′)₂ Fragment Goat Anti-Rabbit IgG (H+L) (1:200 dilution, Jackson ImmunoResearch) for 30 minutes. The cells were washed twice with FACS buffer, fixed in 4% paraformaldehyde in PBS for 20 mins on ice in the dark, washed twice with FACS buffer, and analyzed using the BD LSR II Flow Cytometer. Data were analyzed using FlowJo (FlowJo, LLC; Ashland, Oreg.) to determine mean fluorescence intensities. Prism 6 software was used to create one site, specific binding with Hill slope curves (Log transform) to determine IC₅₀ values.

LAG3 Competition Assay

Daudi cells expressing Major Histocompatibility Class II (MHCII) molecules, the natural ligand for LAG3, were used to measure the ability of anti-PD1/LAG3 bi-specific antibody constructs to block biotinylated (ThermoFisher, Cat #21329) recombinant human LAG3 protein (rhLAG3, R&D Cat #2319-C3) binding. Cells were seeded at 100,000 cells/well in 100 μL of FACS buffer in 96-well polypropylene plates. The cells were centrifuged and resuspended with test antibodies (2× concentration) diluted in FACS buffer, immediately followed by equal volume of biotinylated rhLAG3 (added at 2× concentration; 7.5 μg/mL final). Cells were incubated on ice for 1 hour and washed twice. Biotinylated rhLAG3 binding was detected with 2 μg/mL R-Phycoerythrin-conjugated Streptavidin (eBioscience) for 30 minutes. Cells were washed twice with FACS buffer, fixed in 4% paraformaldehyde in PBS for 20 mins on ice in the dark, washed twice with FACS buffer, and analyzed using the BD LSR II Flow Cytometer. Data were analyzed using FlowJo (FlowJo, LLC; Ashland, Oreg.) to determine mean fluorescence intensities. Prism 6 software was used to create one site, specific binding with Hill slope curves (Log transform) to determine IC₅₀ values.

PD1×LAG3 Cell-Based Bridging Assay

A cell-based bridging assay was developed using the PathHunter® platform (DiscoverX, Fremont, Calif.) to detect simultaneous binding of PD1 and LAG3 on the cell surface. Briefly, a split beta-galactosidase reporter enzyme was utilized such that enzyme activity could only be detected when the enzyme acceptor (EA) and ProLink (PK) fragments can assemble. The EA-fragment was C-terminally fused to PD-1 (residues 1-199) and the PK fragment was C-terminally fused to LAG3 (residues 1-477). Both fusion constructs were co-expressed in U2OS cells, and stable cell pools were generated. To assess PD1×LAG3 bispecific bridging, 10,000 cells per well were added to opaque 96-well polystyrene plates. The bi-specific antibodies were serially diluted and incubated with the cells for 16 hours at 37° C. Binding was subsequently detected by measuring beta-galactosidase activity using the Beta-Glo® Assay System (Promega Cat. # TM239) and plates were read on an Envision luminometer (integration time of 0.5 sec/well). Prism 6 software was used to create agonist vs. response curves with variable slope to determine EC50 values.

CMV Recall Assay for Bi-Specific Antibodies

Peripheral blood mononuclear cells (PBMC) were initially isolated from CMV-positive human donors (Stanford Blood Center) by differential gradient centrifugation using NycoPrep™ 1.077 (Axis-Shield). PBMC were cryopreserved with Recovery™ Cell Culture Freezing Medium (Life Technologies) in liquid nitrogen. For the assay, PBMC (0.2×10⁶ cells/well) were cultured in serum-free media containing 2% human AB serum and CMV peptide pools for the CMV IE-1 and CMV pp65 proteins (Miltenyi Biotec) in a total volume of 100 μL per well. Anti-PD1/LAG3 bi-specific antibody candidates (50 μL/well) were added as 3× stock with 5-fold serial dilution titration. Cells were cultured for 5 days. Conditioned media was collected and tested for human IFN-γ levels by ELISA (BD Biosciences).

Example 12: Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Two-Chain scFvFc with Knob-in-Hole Mutations)

Embodiments of a PD1/LAG3 bi-specific antigen-binding construct comprising a two-chain scFvFc arrangement were prepared using the following arrangements: (1) an anti-PD1 scFvFc knob paired with an anti-LAG3 scFvFc hole, and (2) an anti-PD1 scFvFc hole paired with an anti-LAG3 scFvFc knob. The scFvFcs include scFvs generated in accordance with Section 4.1.1.1. Table 24 lists exemplary scFvFc fragments and their corresponding SEQ ID NOs that are found within such constructs.

TABLE 24
Exemplary scFvFc Fragments and Sequences
for Two-chain scFvFc Arrangements
scFvFc                           SEQ ID NO.
Anti-PD1 1353-G10 scFvFc hole    368
Anti-LAG3 1449-G09.2 scFvFc knob 371
Anti-PD1 1353-G10 scFvFc knob    369
Anti-LAG3 1449-G09.2 scFvFc hole 380

Functional characteristics for the exemplary two-chain scFvFc arrangements described above are provided in Table 25.

TABLE 25
Functional Characteristics of PD1/LAG3 Bispecific Antibodies (Two-chain scFvFc
Arrangements)
		AlphaLISA	PD1	LAG3
		bridging	ELISA	ELISA	293T-	CHO-
		(max	EC50	EC50	LAG3 Kd	PD1 Kd
Construct	Description	signal)	(nM)	(nM)	(nM)	(nM)
E	aLAG3 1449-G09.2_K	31576	1.1	0.69	8	3
	x aPD1 1353-G10_H
F	aLAG3 1449-G09.2_H	20892	1.2	0.44	15	6
	x aPD1 1353-G10_K
“K” = knob
“H” = hole

Example 13: Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Three-Chain Fab× scFvFc with Knob-in-Hole Mutations)

Embodiments of a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab× scFvFc structure were prepared using the following arrangements: (1) an anti-PD1 scFvFc knob paired with an anti-LAG3 half IgG (HC+LC) hole, (2) an anti-PD1 scFvFc hole paired with an anti-LAG3 half IgG (HC+LC) knob, (3) an anti-PD1 half IgG (HC+LC) knob paired with an anti-LAG3 scFvFc hole, and (4) an anti-PD1 half IgG (HC+LC) hole paired with an anti-LAG3 scFvFc knob. Table 26 lists exemplary scFvFc and half IgG fragments and their corresponding SEQ ID NOs that can be used in these embodiments.

TABLE 26
Exemplary scFvFc and Half IgG Fragments and Sequences for Three-
chain Fab × scFvFc Arrangements with Knob-in-Hole Mutations
scFvFc                           SEQ ID NO.
Anti-PD1 1353-G10 scFvFc knob    369
Anti-PD1 1353-G10 scFvFc hole    368
Anti-PD1 1353-G10 HC knob        381
Anti-PD1 1353-G10 HC hole        382
Anti-PD1 1353-G10 LC             383
Anti-LAG3 1449-G09.2 scFvFc knob 371
Anti-LAG3 1449-G09.2 scFvFc hole 384
Anti-LAG3 1449-G09.2 HC hole     372
Anti-LAG3 1449-G09.2 LC          366

Exemplary anti-PD1 scFvFcs and anti-LAG3 scFvFcs that can be included in the three-chain arrangement are described in Example 13.

Functional characteristics for the exemplary three-chain Fab× scFvFc arrangements described above are provided in Table 27. The half IgG fragments are labeled as “Fab_K” or “Fab_H” where “Fab” indicates the antigen-binding region and “K” or “H” indicates a knob or hole mutation in the Fc region.

TABLE 27
Functional Characteristics of PD1/LAG3 Bispecific Antibodies (Comparison of Two-
chain scFvFc × scFvFc Arrangements to Three-chain Fab × scFvFc Arrangements)
				CHO-PD1	CHO-PD1
		AlphaLISA	AlphaLISA	cell binding,	cell binding,
Construct	Description	Bmax	EC50 (nM)	Kd (nM)	Bmax
F	aPD1 1353-G10 scFvFc_K ×	50547	0.069	1.1	7201
	aLAG3 1449-G09.2
	scFvFc_H
E	aPD1 1353-G10 scFvFc_H ×	36902	0.011	1	6296
	aLAG3 1449-G09.2
	scFvFc_K
G	aPD1 1353-G10 Fab_K ×	210201	0.051	0.8	6375
	aLAG3 1449-G09.2
	scFvFc_H
H	aPD1 1353-G10 Fab_H ×	86841	0.011	0.4	5455
	aLAG3 1449-G09.2
	scFvFc_K

Example 14: Generation of a PD1/LAG3 Bi-Specific Antigen-Binding Construct (Three-Chain Fab× scFvFc with zw Mutations)

Embodiments of a PD1/LAG3 bi-specific antigen-binding construct comprising a three-chain Fab× scFvFc structure were prepared using the following arrangements: (1) an anti-PD1 scFvFc knob with zwA mutations paired with an anti-LAG3 half IgG (HC+LC) with zwB mutations, (2) an anti-PD1 scFvFc with zwB mutations paired with an anti-LAG3 half IgG (HC+LC) with zwA mutations, (3) an anti-PD1 half IgG (HC+LC) with zwA mutations paired with an anti-LAG3 scFvFc with zwB mutations, and (4) an anti-PD1 half IgG (HC+LC) with zwB mutations paired with an anti-LAG3 scFvFc with zwA mutations. The mutations encompassed by “zwA” mutations include T350V/L351Y/F405A/Y407V in the CH3 domain. The mutations encompassed by “zwB” mutations include T350V/T366L/K392L/T394W in the CH3 domain. Table 28 lists exemplary scFvFc and half IgG fragments and their corresponding SEQ ID NOs that can be used in these embodiments.

TABLE 28
Exemplary scFvFc and Half IgG Fragments and Sequences for
Three-chain Fab × scFvFc Arrangements with zw Mutations
scFvFc                          SEQ ID NO.
Anti-PD1 1353-G10 scFvFc zwA    370
Anti-PD1 1353-G10 scFvFc zwB    388
Anti-PD1 1353-G10 HC zwA        389
Anti-PD1 1353-G10 HC zwB        390
Anti-PD1 1353-G10 LC            383
Anti-LAG3 1449-G09.2 scFvFc zwA 391
Anti-LAG3 1449-G09.2 scFvFc zwB 392
Anti-LAG3 1449-G09.2 HC zwA     393
Anti-LAG3 1449-G09.2 HC zwB     373
Anti-LAG3 1449-G09.2 LC         376

Functional characteristics for the exemplary three-chain Fab× scFvFc arrangements described above are provided in Table 29. The half IgG fragments are labeled as “Fab-zwA” or “Fab-zwB” where “Fab” indicates the antigen-binding region and “zwA” or “zwB” indicates the Fc region with the mutations described above.

TABLE 29
Functional Characteristics of PD1/LAG3 Bispecific Antibodies
(Three-chain Fab × scFvFc Arrangements with zw Mutations)
	DSC Analysis
		TM Onset	TM1	TM2
Construct	Sample ID	° C.	° C.	° C.
I	1353-G10-Fab-zwB;	52.4	61.1	65.6
	1449-G09.2-scFv-zwA
A	1353-G10-scFv-zwB;	51.9	62.8	78.1
	1449-G09.2-Fab-zwA

Functional characteristics for exemplary three-chain Fab× scFvFc arrangements described above are provided in Tables 30 to 32.

TABLE 30
Functional Characteristics of PD1/LAG3 Bispecific Antibodies (Three-chain scFvFc × Fab-Fc Arrangements)
								AlphaLISA
								(LAG3-Fc
								Acceptor/PD1-Fc
	Description	Human PD1 kinetics	Human LAG3 kinetics	Donor)	DSF (° C.)
Construct	scFv × Fab	ka (1/Ms)	kd (1/s)	KD (M)	ka (1/Ms)	kd (1/s)	KD (M)	EC50	Bmax	TM1	TM2
A	aPD1 1353-G10 scFv_zwB ×	5.59E+05	8.49E−04	1.52E−09	1.21E+06	1.02E−04	8.45E−11	0.01	4688	51.3	66
	aLAG3 Fab_zwA
C	aPD1 1353-G10 Vk1-39LC	5.03E+05	6.49E−04	1.29E−09	1.34E+06	6.94E−04	5.19E−10	ND	ND	54.5	72.8
	scFvFc zwB V262E; aLAG3
	1449-G09.2 Fab zwA V262E
B	aPD1 1353-G10 scFvFc zwB	1.12E+06	4.87E−03	4.37E−09	1.46E+06	1.38E−04	9.44E−11	ND	ND	52.5	72.7
	R28T/P30D/H31S V262E;
	aLAG3 1449-G09.2 Fab zwA
	V262E
J	aPD1 1353-G10 R28T/P30D	9.22E+06	1.09E−03	1.19E−09	ND	ND	ND	ND	ND	ND	ND
	zwB × aLAG3 1449-G09.2
	Fab zwA
K	a1353-G10 R28T/P30T/H31S	1.37E+06	7.24E−03	5.3E−09	ND	ND	ND	ND	ND	ND	ND
	zwB × 1449-G09.2 Fab zwA
L	1353-G10 R28D/P30T/H31S	1.50E+06	7.77E−03	5.18E−09	ND	ND	ND	ND	ND	ND	ND
	zwB × 1449-G09.2 Fab zwA
M	1353-G10 R28T/P30T/H31D	2.07E+06	1.02E−02	4.91E−09	ND	ND	ND	ND	ND	ND	ND
	zwB × 1449-G09.2 Fab zwA
N	1353-G10 R28T/P30D/H31S	1.38E+06	55.2E−03	4.01E−09	ND	ND	ND	ND	ND	ND	ND
	zwB × 1449-G09.2 Fab zwA
O	1353-G10 R28T/H31D zwB ×	1.68E+06	7.48E−03	4.44E−09	ND	ND	ND	ND	ND	ND	ND
	1449-G09.2 Fab zwA
P	1353-G10 R28D/P30D zwB ×	9.85E+05	1.00E−03	1.02E−09	ND	ND	ND	ND	ND	ND	ND
	1449-G09.2 Fab zwA
Q	1353-G10 R28D/H31D zwB ×	1.55E+06	6.70E−03	4.32E−09	ND	ND	ND	ND	ND	ND	ND
	1449-G09.2 Fab zwA

TABLE 31
									AlphaLISA (LAG3-Fc
									Acceptor/PD1-Fc
			human PD1 kinetics	human LAG3 kinetics	Donor)
Construct	Ligand	Scaffold	ka (1/Ms)	kd (1/s)	KD (M)	ka (1/Ms)	kd (1/s)	KD (M)	EC50	Bmax
A	PD1 scFv_zwB ×	scFv × Fab	5.59E05	8.49E−04	1.52E−09	1.21E06	1.02E−04	8.45E−11	0.01	4688
	LAG3 Fab_zwA
R	LAG × PD1 (I)	fab × fab	6.30E05	6.42E−04	1.02E−09	1.46E06	1.29E−04	8.85E−11	0.015	33678
S	LAG × PD1 (II)	fab × fab	6.65E05	7.71E−04	1.16E−09	1.41E06	1.36E−04	9.65E−11	0.015	31794
T	LAG × PD1 (III)	fab × fab	7.24E05	8.06E−04	1.11E−09	1.34E06	1.52E−04	1.13E−10	0.006	25158
U	LAG × PD1 (I) +	fab × fab	5.20E05	1.15E−03	2.22E−09	1.09E06	2.63E−04	2.41E−10	ND	ND
	V262E
ND = not determined

TABLE 32
Functional Characteristics of PD1/LAG3 Bispecific Antibodies (Four-chain Fab × Fab-Fc Arrangements)
			CHO-PD1 cell
			binding	CHO-LAG3 cell	PD1/PDL1	LAG3/MHCII	PxL cell
			Kd		binding	competition	competition	bridging
Construct	Ligand	Scaffold	(nM)	Bmax	Kd (nM)	Bmax	IC50 (nM)	IC50 (nM)	EC50 (nM)
A	PD1 scFv_zwB ×	scFv × Fab	3.7	10425	3	2751	118	17	0.6
	LAG3 Fab_zwA
R	LAG × PD1 (I)	fab × fab	3.1	13671	1.8	2947	34	11	0.1
S	LAG × PD1 (II)	fab × fab	8	16739	2.4	2770	69	16	0.1
T	LAG × PD1 (III)	fab × fab	4.6	11999	2.4	2689	149	19	0.8

Example 15: Mutation Design in Anti-PD1

To improve solubility and reduce aggregation propensity, an aspartic acid mutation was also made to PD-1 antibody 1353-G10 in CDR H1 (P30D). See, e.g., WO 2016/060033, incorporated herein by reference in its entirety. The mutation was introduced using the general approach described above and in the literature (Dudgeon et al. 2012. PNAS 109(27):10879-10884). IgBLAST (Ye et al. 2013. Nucleic Acids Res 41 (Web Server issue):W34-W40, incorporated herein by reference in its entirety) was used to analyze the 1353-G10 heavy chain. The analysis showed that R28T and H31S mutations would render the sequence closer to the native VH1-18 human germline sequence, which could reduce immunogenicity in humans and improve biophysical behavior, e.g., reduction of aggregation propensity, transition melting temperature, etc. Lastly, to improve the stability of the light chain of 1353-G10, the CDRs were grafted onto a kappa framework Vk1-39 in a similar manner as described previously (Lehmann et al. 2015. mAbs 7(6):1058-1071, incorporated herein by reference in its entirety). The stabilizing mutations and kappa-grafted light chain were also made in the context of an scFv framework, both alone and in combination with each other. The sequences of these scFv are presented as SEQ ID NO: 364 (stabilizing mutations and kappa grafted light chain), SEQ ID NO: 365 (stabilizing mutations), and SEQ ID NO: 366 (kappa grafted light chain), respectively.

Example 16: Differential Scanning Fluorimetry

A protein thermal shift assay was carried out by mixing the protein to be assayed with an environmentally sensitive dye (SYPRO® Orange, Life Technologies Cat. # S-6650) in a phosphate buffered solution (PBS), and monitoring the fluorescence of the mixture in real time as it underwent controlled thermal denaturation. Protein solutions between 0.2-2 mg/mL were mixed at a 1:1 volumetric ratio with a 1:500 PBS-diluted solution of SYPRO® Orange (SYPRO® Orange stock dye is 5000× in DMSO). Aliquots of 10 μL of the protein-dye mixture were dispensed in quadruplicate in a 384-well microplate (Bio-Rad Cat # MSP-3852), and the plate was sealed with an optically clear sealing film (Bio-Rad Cat # MSB-1001) and placed in a 384-well plate real-time thermocycler (Bio-Rad CFX384 Real Time System). The protein-dye mixture was heated from 25° C. to 95° C., at increments of 0.1° C. per cycle (˜1.5° C. per minute), allowing 3 seconds of equilibration at each temperature before taking a fluorescence measurement. At the end of the experiment, the first and second transition melting temperatures (Tm1 and Tm2, respectively) were determined using the Bio-Rad CFX manager software.

Example 17: In Vivo Efficacy of Combination Therapy (Anti-PD-1 and Anti-LAG3 Antibodies) on Tumor Establishment and Growth

To determine if dual-targeting of PD-1 and LAG3 on mouse T-cells provides additive benefits over treatment with anti-PD-1 alone, MC38 colorectal murine cancer cells (National Cancer Institute, Bethesda, Md.) were subcutaneously implanted in C57BL/6 mice (2e6 cells in 100 μL PBS) in the flank region (10 mice per treatment group). Commercially available mouse surrogate antibodies were dosed in mice intraperitoneally 6 days after tumor cell implantation with a total of 5 doses at 3-4 day intervals (post-initial treatment at days 0, 3, 6, 10 and 14). Commercial antibodies administered (Bio×Cell, West Lebanon, N.H.) were 10 mg/kg anti-PD-1 (clone RMP1-14 rat IgG2A), 10 mg/kg control rat IgG2A (clone 2A3), 30 mg/kg anti-LAG3 (clone C9B7W rat IgG1) and 30 mg/kg for control rat IgG1 (clone HRPN). Treatment groups, dose levels and antibody combinations are shown in Table 33. Results are shown in FIG. 4.

TABLE 33
Treatment Groups
Treatment Groups
control rat IgG2A (10 mg/kg) +
control rat IgG1 (30 mg/kg)
anti-PD-1 RMP1-14 rat IgG2A (10 mg/kg) +
control rat IgG1 (30 mg/kg)
anti-PD-1 RMP1-14 rat IgG2A (10 mg/kg) +
anti-LAG3 C9B7W rat IgG1 (30 mg/kg)

Mean tumor volumes for each treatment groups (mean±standard error of mean (SEM)) are depicted in FIG. 4. Tumor growth is significantly inhibited with anti-PD-1 alone and anti-PD-1/anti-LAG3 combination relative to the control Ab-treated group. Furthermore, addition of anti-LAG3 with anti-PD-1 significantly suppressed tumor growth compared to anti-PD-1 treatment alone suggesting additive effects in targeting both PD-1 and LAG3 in the MC38 tumor model.

Example 18: Pharmacokinetics and ADA Response of a PD-1/LAG3 Bi-Specific Construct in Cynomolgus Monkeys

PD-1/LAG3 Bi-specific Construct A is a three-chain bi-specific antibody that includes an anti-PD-1 scFvFc with zwB mutation (T350V/T366L/K392L/T394W in C_H3 domain) and an anti-LAG3 Fab with zwA mutation (T350V/L351Y/F405A/Y407V in C_H3 domain). Bi-specific Construct A was assessed for pharmacokinetic (PK) properties and anti-drug antibody (ADA) response in cynomolgus non-human primates. The bi-specific construct was administered as IV injection to female cynomolgus monkeys (n=5 per group) on day 1 and day 15 at doses of 10, 30 and 100 mg/kg. Serum samples were collected at various timepoints for pharmacokinetic (PK) and ADA assessments. For PK assessments, a sandwich ELISA-based bridging immuno-assay using recombinant PD-1 and LAG3 was used to determine concentrations of the bi-specific construct in serum samples. This assay can detect antibody levels in serum at which the antibody is capable of binding to both PD-1 and LAG3. The assay uses human LAG3 human-Fc recombinant fusion protein as capture protein for the bi-specific construct. Following serum sample binding at appropriate dilutions and wash steps, human PD-1 mouse-Fc recombinant fusion protein was added, and after another series of wash steps, plate-bound bi-specific construct was detected using horse-radish peroxidase (HRP)-conjugated polyclonal anti-mouse-IgG Fc protein. The same dosing material was spiked into cynomolgus monkey serum to generate a standard curve to determine serum concentrations of the bi-specific construct. PK parameters were calculated using the Phoenix WinNonlin (version 6.1) program. The peak serum concentration (C_max) and half-life was determined, and the area under the serum concentration-time curve from 0.25 hour to 168 hours post dose 1 (AUC₁₆₈) was determined by the non-compartmental model using the linear/log trapezoidal rule. Serum concentrations below the limit of quantitation were not used for AUC calculations.

For immunogenicity analysis, pre-dose, day 15 and day 43 samples (last PK timepoint) were tested for ADA levels using an ELISA-based immunogenicity assay. Bi-specific Construct A dosing material was used as the capture reagent and HRP-conjugated mouse anti-non-human primate Fc antibody for detection of ADA complexes. The cut-off point for the assay is defined as the assay response (OD at 450 nm) above which, a sample is identified as ADA-positive, and below which, a sample is identified as ADA-negative. The assay cut-off point was calculated as three times the mean OD of pre-dose monkey serum samples. The cut-point for anti-construct antibody was at an OD of 0.70.

Serum levels of the bi-specific construct in all cynomolgus monkeys (n=5 animals/group) dosed at 10, 30 and 100 mg/kg (Q2W×2) at day 1 and day 15 (x-axis solid lines) are shown in FIG. 5 (left panel). Note that following day 16 (1 day post 2^nd dose at day 15), only 2 out of 5 animals per group were sampled for PK and ADA analysis up to day 43 (x-axis dotted line). Mean serum levels for Bi-specific Construct A (±SEM) per dose group are shown in FIG. 5 (right panel). Following day 16, only 1 of 2 animals in the 100 mg/kg group had measurable concentrations up to day 25 (FIG. 5). All other animals (n=2) in 10 and 30 mg/kg dose groups had no measurable concentrations after day 16 (FIG. 5). Rapid clearance and low serum half-life of the bi-specific construct (<4 days) in cynomolgus monkeys were observed at all 3 dose levels post 1^st dose up to day 8 (Table 34). Table 34 provides mean pharmacokinetic values (with standard deviation values in parentheses) for each dosing group.

TABLE 34
Pharmacokinetic Profile of Bi-specific Construct A in Cynomolgus Monkeys
							Half-
	Cmax	C,ax/D	AUC0-8	AUC0-8/Dose	Vz	Cl	life
Dose	μg/mL	μg/mL	μg * day/mL	μg * day/mL/(mg/kg)	mL/kg	mL/d/kg	days
10	283.02	28.30 (3.52)	740.74	74.07 (6.62)	49.89	10.34	3.33
mg/kg	(35.24)		(66.22)		(6.55)	(0.66)	(0.27)
30	846.79	28.23 (4.58)	2120.37	70.68 (10.57)	54.49	10.56	3.58
mg/kg	(137.27)		(317.07)		(9.80)	(1.66)	(0.37)
100	4451.29	44.51 (3.12)	9927.57	99.28 (6.85)	33.41	8.19	2.84
mg/kg	(312.19)		(684.81)		(1.98)	(0.77)	(0.27)
Cmax = the maximum observed concentration measured after dosing
Cmax/D = Cmax divided by the administered dose
AUC0-8 = the area under the concentration versus time curve from the start of dosing, including all time points up to day 8 post Dose 1, using linear/log trapezoidal method
AUC0-8/D = AUC0-8 divided by the administered dose
Vz = apparent volume of distributiuon during terminal phase
Cl = apparent clearance

The results of the immunogenicity analysis for PD-1/LAG3 Bi-specific Construct A indicated that all monkeys dosed with the bi-specific construct at 10, 30 and 100 mg/kg tested positive for ADA on day 15 (n=5) and day 43 (n=2) (data not shown). This suggests rapid PK clearance and low serum half-life of the bi-specific construct may be attributed to cynomolgus ADA response at all 3 dose levels even after the first dose.

This information from this study showed fast clearance of PD-1/LAG3 Bi-specific Construct A and strong anti-drug antibody (ADA) response. Based on this information, new bi-specific constructs were generated and evaluated to determine if a candidate bi-specific construct with lower clearance and ADA response could be obtained.

Example 19: Pharmacokinetics and ADA Response of Additional PD-1/LAG3 Bi-Specific Construct in Cynomolgus Monkeys

Given the higher clearance, lower serum half-life and strong ADA response with PD-1/LAG3 Bi-specific Construct A, two additional PD-1/LAG3 constructs (“B” and “C”) were assessed for pharmacokinetic (PK) properties and anti-drug antibody (ADA) responses in cynomolgus non-human primates. Bi-specific Construct B is a three-chain bi-specific antibody that includes an anti-PD-1 scFvFc with mutations: R28T/P30D/H31S in V_H; V262E in C_H2 domain; and T350V/T366L/K392L/T394W in C_H3; and an anti-LAG3 Fab with mutations: V262E in C_H2 and T350V/L351Y/F405A/Y407V in C_H3. Bi-specific Construct C is a three-chain bi-specific antibody that includes an anti-PD-1 scFvFc with mutations: V262E in C_H2 domain; and T350V/T366L/K392L/T394W in C_H3, in which the scFv is grafted to a kappa light chain; and an anti-LAG3 Fab with mutations: V262E in C_H2 and T350V/L351Y/F405A/Y407V in C_H3. Both bi-specific constructs were administered as IV injection to female cynomolgus monkeys (n=2 per group) on day 1 and day 15 at a dose of 10 mg/kg. Serum samples were collected at various timepoints for PK and ADA assessments (pre-dose and following post-dose after day 1: 0.25 hour to 240 hours, and pre-dose and following post-dose after day 15: 0.25 hour to 336 hours). For PK assessments, an ELISA method using mouse anti-human IgG (BioRad; clone R10Z8E9), a monoclonal Ab specific for C_H2 domain of human IgG with no non-human primate species reactivity, was used as capture Ab and HRP-conjugated polyclonal goat anti-human IgG with minimal cross-reactivity to cyno IgG (Bethyl Laboratories, A80-319P) was used as detection Ab to determine concentrations of Bi-specific Constructs B and C in serum samples. The same dosing materials were spiked into cynomolgus monkey serum to generate a standard curve to determine Bi-specific Construct B and C serum concentrations.

For immunogenicity analysis, pre-1^st dose (day 0), pre-2^nd dose (day 14), day 28 and day 42 (post-dose 2) serum samples were titrated with 2-fold serial dilutions (20- to 2560-fold, 8 point titration) and tested for ADA levels using an ELISA-based immunogenicity assay. Mouse anti-human IgG (BioRad; clone R10Z8E9), a monoclonal Ab specific for C_H2 domain of human IgG with no non-human primate species reactivity, was coated at 2 μg/ml in carbonate/bicarbonate pH 9.6 buffer (Sigma-Aldrich, C3041) overnight at 4° C. in 96-well Nunc MaxiSorp plates. All following steps were performed at room temperature. Plates were washed with PBST buffer (PBS+0.05% Tween-20) and blocked with ELISA blocking buffer (PBS+1% BSA) for 1 hour. Serum samples were serially diluted 2-fold in diluent buffer from 10- to 1280-fold in diluent buffer (PBS, 0.5% BSA, 0.05% Tween-20, 0.35 M NaCl, 0.25% CHAPS) as 2× samples. Respective dosing Abs were diluted at 5 μg/ml in diluent buffer as 2× samples. Diluted serum samples (final 20- to 2560-fold dilutions final) and diluted test Abs (2.5 μg/ml final) were mixed in a 1:1 volume ratio and incubated for 2 hours to allow formation of ADA-Ab complexes before adding to anti-human IgG coated-plates for 1 hour. Plates were washed and HRP-conjugated goat anti-monkey IgG secondary antibody (Bethyl Laboratories, A140-202P) was diluted 1:40,000-fold in diluent buffer and added to plates for 1 hour in the dark. Plates were washed and TMB substrate (SureBlue Reserve, KPL, 53-00-03) was added for 30 min in the dark. Substrate reaction was quenched with equal volume of 1M phosphoric acid and plates read at 450 nm on M5 SpectraMax plate reader (Molecular Devices). Serum samples for each timepoint per animal titrated at various dilutions were plotted as OD fold-increase at 450 nm (OD sample/OD pre-1^st dose) versus the inverse of serum dilutions. ADA response is considered positive if the OD fold-increase is greater than 3× OD pre-dose for each individual animal.

One out of 2 cynomolgus monkeys treated with PD-1/LAG3 Bi-specific Construct B (animal 2) and Bi-specific Construct C (animal 1) exhibited better PK drug profiles with 2 doses at 10 mg/kg after day 1 and day 15 (Q2W×2) (FIG. 6) compared to Bi-specific Construct A (FIG. 5). This suggests that stabilizing mutations in the 1353-G10 a-PD1 arm (e.g. R28T/P30D/H31S in V_H) in the a-PD-1/a-LAG3 bispecific improve antibody clearance and half-life in cynomolgus monkeys to potentially achieve sustained exposures for assessments in toxicological studies in non-human primates.

One out of 2 cynomolgus monkeys treated with PD-1/LAG3 Bi-specific Construct B (animal 2) and Bi-specific Construct C (animal 1) showed lower ADA response to the respective bispecific antibodies with 2 doses at 10 mg/kg after day 1 and day 15 (Q2W×2) (FIG. 7). The data suggests that stabilizing mutations and germ-lining mutations in the 1353-G10 a-PD1 arm (e.g. R28T/P30D/H31S in V_H) in the a-PD-1/a-LAG3 bispecific is less immunogenic than native a-PD1 arm in Bi-specific Construct A in cynomolgus monkey. In addition, the data indicates that using a kappa graft in the bi-specific construct (Bi-specific construct C) results in reduced immunogenicity and improved pharmacokinetics relative to Bi-specific Construct A, which includes a lambda light chain.

Example 20: Comparison of PD-1/LAG3 Bi-Specific Constructs B and C to B-Specific Construct A

The methods described in Example 12 were used to assess the characteristics of PD-1/LAG3 Bi-specific Constructs A, B, and C.

Bi-specific Constructs B and C are PD1×LAG3 bispecific antibodies with different re-engineered anti-PD1 arms relative to Bi-specific Construct A. The three constructs were compared for binding to cell-surface overexpressed human and cynomolgus PD-1 in CHO cells (FIG. 8). Both Bi-specific Constructs A and C bound to human and cynomolgus PD-1 with equivalent binding affinities. In contrast, Bi-Specific Construct B bound to human and cynomolgus PD-1 with reduced binding affinities compared to Bi-Specific Constructs A and C. All three bi-specific constructs showed similar cell binding affinities to cell-surface overexpressed human and cynomolgus LAG3 in CHO cells (FIG. 9).

Bi-specific Constructs B and C were also compared to Bi-Specific Construct A in inhibiting recombinant human PD-1 protein binding to CHO cells overexpressing either PD-L1 or PD-L2, two cell surface ligands that bind to PD-1 (FIG. 10). Similar to PD-1 cell binding results, both Constructs A and C block recombinant PD-1 protein binding to CHO-PD-L1 and CHO-PD-L2 cells with similar IC50 values, whereas Construct B showed relative weaker inhibitory cell binding activity.

Bi-specific Constructs B and C were tested relative to Bi-Specific Construct A in inhibiting recombinant human LAG3 protein binding to Daudi cell expressing endogenous MHC-class II, a cell-surface expressed ligand that binds to LAG3 (FIG. 11). All three PD1× LAG3 bispecifics inhibited human LAG3 protein binding to Daudi cells with similar IC50 values.

In addition, all three PD1×LAG3 bispecifics showed similar EC50 beta-galactosidase activation curves on U2OS cells co-expressing both PD-1 and LAG3 that are intracellularly fused to beta-galactosidase components in an enzyme-fragment complementation assay (FIG. 12), suggesting that all three bispecifics can bind to both PD-1 and LAG3 simultaneously on same cell.

Bi-specific Constructs B and C were compared to Bi-specific Construct A and to Construct D in a PBMC CMV antigen recall functional assay (FIG. 13). Whereas Bi-specific Constructs A, B, and C are PD×LAG3 bi-specific antibodies, Construct D is a monovalent version of Bi-specific Construct A with a single a-PD-1 binding arm. All three bi-specific antibodies induced similar IFN-g secretion dose-response in a CMV-peptide treated human PBMC culture over a 5-day period compared to Construct D, the monovalent a-PD1 control antibody lacking the anti-LAG3 binding arm. Increased IFN-g secretion by all three bi-specifics suggest additive or synergistic effects in targeting both PD-1 and LAG-3 on T-cells, and Bi-specific Constructs B and C are functionally active, similar to Bi-specific Construct A on human T cells.

Example 21: Sequences

Table 35 provides sequences referred to herein.

TABLE 35
Sequences
SEQ ID
NO:	Molecule	Region	Scheme	Sequence
1	Human LAG3			MWEAQFLGLLFLQPLWVAPVKPLQPGA
				EVPVVWAQEGAPAQLPCSPTIPLQDLS
				LLRRAGVTWQHQPDSGPPAAAPGHPLA
				PGPHPAAPSSWGPRPRRYTVLSVGPGG
				LRSGRLPLQPRVQLDERGRQRGDFSLW
				LRPARRADAGEYRAAVHLRDRALSCRL
				RLRLGQASMTASPPGSLRASDWVILNC
				SFSRPDRPASVHWFRNRGQGRVPVRES
				PHHHLAESFLFLPQVSPMDSGPWGCIL
				TYRDGFNVSIMYNLTVLGLEPPTPLTV
				YAGAGSRVGLPCRLPAGVGTRSFLTAK
				WTPPGGGPDLLVTGDNGDFTLRLEDVS
				QAQAGTYTCHIHLQEQQLNATVTLAII
				TVTPKSFGSPGSLGKLLCEVTPVSGQE
				RFVWSSLDTPSQRSFSGPWLEAQEAQL
				LSQPWQCQLYQGERLLGAAVYFTELSS
				PGAQRSGRAPGALPAGHLLLFLILGVL
				SLLLLVTGAFGFHLWRRQWRPRRFSAL
				EQGIHPPQAQSKIEELEQEPEPEPEPE
				PEPEPEPEPEQL
2	Cynomolgus LAG3			MWEAQFLGLLFLQPLWVAPVKPPQPGA
				EISVVWAQEGAPAQLPCSPTIPLQDLS
				LLRRAGVTWQHQPDSGPPAXAPGHPPV
				PGHRPAAPYSWGPRPRRYTVLSVGPGG
				LRSGRLPLQPRVQLDERGRQRGDFSLW
				LRPARRADAGEYRATVHLRDRALSCRL
				RLRVGQASMTASPPGSLRTSDWVILNC
				SFSRPDRPASVHWFRSRGQGRVPVQGS
				PHHHLAESFLFLPHVGPMDSGLWGCIL
				TYRDGFNVSIMYNLTVLGLEPATPLTV
				YAGAGSRVELPCRLPPAVGTQSFLTAK
				WAPPGGGPDLLVAGDNGDFTLRLEDVS
				QAQAGTYICHIRLQGQQLNATVTLAII
				TVTPKSFGSPGSLGKLLCEVTPASGQE
				HFVWSPLNTPSQRSFSGPWLEAQEAQL
				LSQPWQCQLHQGERLLGAAVYFTELSS
				PGAQRSGRAPGALRAGHLPLFLILGVL
				FLLLLVTGAFGFHLWRRQWRPRRFSAL
				EQGIHPPQAQSKIEELEQEPELEPEPE
				LERELGPEPEPGPEPEPEQL
3	Mouse LAG3			MREDLLLGFLLLGLLWEAPVVSSGPGK
				ELPVVWAQEGAPVHLPCSLKSPNLDPN
				FLRRGGVIWQHQPDSGQPTPIPALDLH
				QGMPSPRQPAPGRYTVLSVAPGGLRSG
				RQPLHPHVQLEERGLQRGDFSLWLRPA
				LRTDAGEYHATVRLPNRALSCSLRLRV
				GQASMIASPSGVLKLSDWVLLNCSFSR
				PDRPVSVHWFQGQNRVPVYNSPRHFLA
				ETFLLLPQVSPLDSGTWGCVLTYRDGF
				NVSITYNLKVLGLEPVAPLTVYAAEGS
				RVELPCHLPPGVGTPSLLIAKWTPPGG
				GPELPVAGKSGNFTLHLEAVGLAQAGT
				YTCSIHLQGQQLNATVTLAVITVTPKS
				FGLPGSRGKLLCEVTPASGKERFVWRP
				LNNLSRSCPGPVLEIQEARLLAERWQC
				QLYEGQRLLGATVYAAESSSGAHSARR
				ISGDLKGGHLVLVLILGALSLFLLVAG
				AFGFHWWRKQLLLRRFSALEHGIQPFP
				AQRKIEELERELETEMGQEPEPEPEPQ
				LEPEPRQL
4	26H10	CDR-H1	Chothia	GFTSSY
5	SRP1496-A03-VH	CDR-H1	Chothia	GFNINDT
6	SRP1496-A04-VH	CDR-H1	Chothia	GFNINDT
7	SRP1496-B08-VH	CDR-H1	Chothia	GFNINDT
8	SRP1648-B07-VH	CDR-H1	Chothia	GFNIADT
9	SRP1648-E02-VH	CDR-H1	Chothia	GFNINDN
10	SRP1449-B03-VH	CDR-H1	Chothia	GFTFSSY
11	SRP1449-F01-VH	CDR-H1	Chothia	GFTFSSY
12	SRP1449-B07-VH	CDR-H1	Chothia	GFTFSSY
13	1449-G09.2-VH	CDR-H1	Chothia	GFTFSSY
14	SRP1449-D05-VH	CDR-H1	Chothia	GFTFRSF
15	SRP1558-F01-VH	CDR-H1	Chothia	GFTFPDS
16	SRP1448-D09-VH	CDR-H1	Chothia	GFTFTDS
17	SRP1558-A06-VH	CDR-H1	Chothia	GFTFSES
18	SRP1558-E11-VH	CDR-H1	Chothia	GFTFTSS
19	SRP1627-A02-VH	CDR-H1	Chothia	GFNINDY
20	SRP1627-A11-VH	CDR-H1	Chothia	GFNINDY
21	h5G11-2-VH	CDR-H1	Chothia	GFNIKDY
22	SRP1627-B01-VH	CDR-H1	Chothia	GFNITDL
23	421.61.4.5G11-VH	CDR-H1	Chothia	GFNIKDY
24	1353-G10-wt	CDR-H1	Chothia	GYRFPHY
25	1353-G10 Y27D	CDR-H1	Chothia	GDRFPHY
26	1353-G10 R28D	CDR-H1	Chothia	GYDFPHY
27	1353-G10 F29D	CDR-H1	Chothia	GYRDPHY
28	1353-G10 P30D	CDR-H1	Chothia	GYRFDHY
29	1353-G10 H31D	CDR-H1	Chothia	GYRFPDY
30	1353-G10 Y32D	CDR-H1	Chothia	GYRFPHD
31	1353-G10	CDR-H1	Chothia	GYTFDHY
	R28T/P30D
32	1353-G10	CDR-H1	Chothia	GYDFDHY
	R28D/P30D
33	1353-G10	CDR-H1	Chothia	GYDFPDY
	R28D/H31D
34	1353-G10	CDR-H1	Chothia	GYTFPDY
	R28T/H31D
35	1353-G10	CDR-H1	Chothia	GYDFTSY
	R28D/P30T/H31S
36	1353-G10	CDR-H1	Chothia	GYTFTDY
	R28T/P30T/H31D
37	1353-G10	CDR-H1	Chothia	GYTFTSY
	R28T/P30T/H31S
38	1353-G10-	CDR-H1	Chothia	GYTFDSY
	R28T/P30D/H31S
39	26H10	CDR-H1	Kabat	SYGMH
40	SRP1496-A03-VH	CDR-H1	Kabat	DTYIH
41	SRP1496-A04-VH	CDR-H1	Kabat	DTYIH
42	SRP1496-B08-VH	CDR-H1	Kabat	DTYIH
43	SRP1648-B07-VH	CDR-H1	Kabat	DTFIH
44	SRP1648-E02-VH	CDR-H1	Kabat	DNYIH
45	SRP1449-B03-VH	CDR-H1	Kabat	SYGMH
46	SRP1449-F01-VH	CDR-H1	Kabat	SYGMH
47	SRP1449-B07-VH	CDR-H1	Kabat	SYGMH
48	1449-G09.2-VH	CDR-H1	Kabat	SYGMH
49	SRP1449-D05-VH	CDR-H1	Kabat	SFGMH
50	SRP1558-F01-VH	CDR-H1	Kabat	DSSMS
51	SRP1448-D09-VH	CDR-H1	Kabat	DSSMS
52	SRP1558-A06-VH	CDR-H1	Kabat	ESTMS
53	SRP1558-E11-VH	CDR-H1	Kabat	SSSMS
54	SRP1627-A02-VH	CDR-H1	Kabat	DYFMH
55	SRP1627-A11-VH	CDR-H1	Kabat	DYFMH
56	h5G11-2-VH	CDR-H1	Kabat	DYYMH
57	SRP1627-B01-VH	CDR-H1	Kabat	DLYMH
58	421.61.4.5G11-VH	CDR-H1	Kabat	DYYMH
59	1353-G10-wt	CDR-H1	Kabat	HYGIS
60	1353-G10 H31D	CDR-H1	Kabat	DYGIS
61	1353-G10 Y32D	CDR-H1	Kabat	HDGIS
62	1353-G10G33D	CDR-H1	Kabat	HYDIS
63	1353-G10 S35D	CDR-H1	Kabat	HYGID
64	1353-G10	CDR-H1	Kabat	SYGIS
	R28D/P30T/H31S
65	1353-G10-	CDR-H1	Kabat	SYGIS
	R28T/P30D/H31S
66	26H10	CDR-H2	Chothia	WYDGSN
67	SRP1496-A03-VH	CDR-H2	Chothia	DPYDGA
68	SRP1496-A04-VH	CDR-H2	Chothia	DPYDGA
69	SRP1496-B08-VH	CDR-H2	Chothia	DPYDGA
70	SRP1648-B07-VH	CDR-H2	Chothia	DPYDGD
71	SRP1648-E02-VH	CDR-H2	Chothia	DPYDGF
72	SRP1449-B03-VH	CDR-H2	Chothia	WYDASY
73	SRP1449-F01-VH	CDR-H2	Chothia	WYDGSY
74	SRP1449-B07-VH	CDR-H2	Chothia	WYDGSN
75	1449-G09.2-VH	CDR-H2	Chothia	WYDGSY
76	SRP1449-D05-VH	CDR-H2	Chothia	WYDGSV
77	SRP1558-F01-VH	CDR-H2	Chothia	TDNSGN
78	SRP1448-D09-VH	CDR-H2	Chothia	TGNSGT
79	SRP1558-A06-VH	CDR-H2	Chothia	TSDSGT
80	SRP1558-E11-VH	CDR-H2	Chothia	SDDTGS
81	SRP1627-A02-VH	CDR-H2	Chothia	DPWNGD
82	SRP1627-A11-VH	CDR-H2	Chothia	DPWNGD
83	h5G11-2-VH	CDR-H2	Chothia	DPENGD
84	SRP1627-B01-VH	CDR-H2	Chothia	DPWNGD
85	421.61.4.5G11-VH	CDR-H2	Chothia	DPENGD
86	1353-G10-wt	CDR-H2	Chothia	SAYNGN
87	1353-G10-	CDR-H2	Chothia	SAYNGN
	R28T/P30D/H31S
88	26H10	CDR-H2	Kabat	VIWYDGSNKYYADSVKG
89	SRP1496-A03-VH	CDR-H2	Kabat	IIDPYDGATDYADSVKG
90	SRP1496-A04-VH	CDR-H2	Kabat	IIDPYDGATDYADSVKG
91	SRP1496-B08-VH	CDR-H2	Kabat	IIDPYDGATDYADSVKG
92	SRP1648-B07-VH	CDR-H2	Kabat	IIDPYDGDTDYADSVKG
93	SRP1648-E02-VH	CDR-H2	Kabat	IIDPYDGFTAYADSVKG
94	SRP1449-B03-VH	CDR-H2	Kabat	AIWYDASYKYYADSVKG
95	SRP1449-F01-VH	CDR-H2	Kabat	VIWYDGSYKYYADSVKG
96	SRP1449-B07-VH	CDR-H2	Kabat	VIWYDGSNKYYADSVKG
97	1449-G09.2-VH	CDR-H2	Kabat	VIWYDGSYKYYADSVKG
98	SRP1449-D05-VH	CDR-H2	Kabat	VIWYDGSVKYYADSVKG
99	SRP1558-F01-VH	CDR-H2	Kabat	VITDNSGNTDYADSVKG
100	SRP1448-D09-VH	CDR-H2	Kabat	VITGNSGTTDYADSVKG
101	SRP1558-A06-VH	CDR-H2	Kabat	FITSDSGTTDYADSVKG
102	SRP1558-E11-VH	CDR-H2	Kabat	VISDDTGSTDYADSVKG
103	SRP1627-A02-VH	CDR-H2	Kabat	RIDPWNGDTEYAPKFQG
104	SRP1627-A11-VH	CDR-H2	Kabat	RIDPWNGDTEYAPKFQG
105	h5G11-2-VH	CDR-H2	Kabat	WIDPENGDTEYAPKFQG
106	SRP1627-B01-VH	CDR-H2	Kabat	RIDPWNGDTEYAPKFQG
107	421.61.4.5G11-VH	CDR-H2	Kabat	WIDPENGDTEYAPKFQG
108	1353-G10-wt	CDR-H2	Kabat	WISAYNGNTNYAQKLQG
109	1353-G10-	CDR-H2	Kabat	WISAYNGNTNYAQKLQG
	R28T/P30D/H31S
110	26H10	CDR-H3		EWAVASWDYGMDV
111	SRP1496-A03-VH	CDR-H3		EIFG-FYWNPFDY
112	SRP1496-A04-VH	CDR-H3		EIFG-FYWNPFDY
113	SRP1496-B08-VH	CDR-H3		EIFG-FYWNPFDY
114	SRP1648-B07-VH	CDR-H3		EILG-FYWNPFDY
115	SRP1648-E02-VH	CDR-H3		ESIG-FYLNPFDY
116	SRP1449-B03-VH	CDR-H3		EWAVASWDYALDV
117	SRP1449-F01-VH	CDR-H3		ESEVASWDYGLDV
118	SRP1449-B07-VH	CDR-H3		EWAVSSWDYGMDV
119	1449-G09.2-VH	CDR-H3		EEAPENWDYALDV
120	SRP1449-D05-VH	CDR-H3		EWADVSWDAGLDV
121	SRP1558-F01-VH	CDR-H3		VFEGGVRPYS-DY
122	SRP1448-D09-VH	CDR-H3		VYEGGVRPYS-DY
123	SRP1558-A06-VH	CDR-H3		VFEGGVRPFS-DY
124	SRP1558-E11-VH	CDR-H3		VDNGGVRPYS-DY
125	SRP1627-A02-VH	CDR-H3		-------SDALDY
126	SRP1627-A11-VH	CDR-H3		-------SDALDY
127	h5G11-2-VH	CDR-H3		-------PDALDY
128	SRP1627-B01-VH	CDR-H3		-------SEMVDY
129	421.61.4.5G11-VH	CDR-H3		-------PDALDY
130	1353-G10-wt	CDR-H3		DVDYG-T-GS-GY
131	1353-G10-	CDR-H3		DVDYG-T-GS-GY
	R28T/P30D/H31S
132	26H10	CDR-L1		RASQ----SVSSSYLA
133	SRP1449-D05-VL	CDR-L1		RASQ----SVSSSYLA
134	SRP1449-F01-VL	CDR-L1		RASR----SVSSSYLA
135	1449-G09.2-VL	CDR-L1		RASQ----SVSSSYLA
136	SRP1449-B07-VL	CDR-L1		RASQ----SVSSSYLA
137	SRP1449-B03-VL	CDR-L1		RASQ----SVSSSYLA
138	SRP1558-E11-VL	CDR-L1		RASQ----SVSSSYLA
139	SRP1558-A06-VL	CDR-L1		RASQ----SVSSNPLA
140	SRP1558-F01-VL	CDR-L1		RASQ----SVSSGNPA
141	SRP1448-D09-VL	CDR-L1		RASQ----SVSSSYLA
142	trastuzumab-VL	CDR-L1		RASQ----DVNTA-VA
143	SRP1627-A02-VL	CDR-L1		KSSQSLLDSDGKTYLN
144	SRP1627-A11-VL	CDR-L1		KSSQSLLDSDGKTYLN
145	SRP1627-B01-VL	CDR-L1		KSSQSLLDSDGKTYLN
146	h5G11-2-VL	CDR-L1		KSSQSLLDSDGKTYLN
147	421.61.4.5G11-VL	CDR-L1		KSSQSLLDSDGKTYLN
148	1353-G10-wt	CDR-L1		SGDALPKQYAY
149	1353-G10-kappa	CDR-L1		SGDALPKQYAY
150	26H10	CDR-L2		GASSRAT
151	SRP1449-D05-VL	CDR-L2		GASSRAT
152	SRP1449-F01-VL	CDR-L2		GASSRAT
153	1449-G09.2-VL	CDR-L2		GASSRAT
154	SRP1449-B07-VL	CDR-L2		GASSRAT
155	SRP1449-B03-VL	CDR-L2		GASSRAT
156	SRP1558-E11-VL	CDR-L2		GASSRAT
157	SRP1558-A06-VL	CDR-L2		GASSRAT
158	SRP1558-F01-VL	CDR-L2		GASSRAT
159	SRP1448-D09-VL	CDR-L2		GASSRAT
160	trastuzumab-VL	CDR-L2		SASFLYS
161	SRP1627-A02-VL	CDR-L2		LVSKLDS
162	SRP1627-A11-VL	CDR-L2		LVSKLDS
163	SRP1627-B01-VL	CDR-L2		LVSKLDS
164	h5G11-2-VL	CDR-L2		LVSKLDS
165	421.61.4.5G11-VL	CDR-L2		LVSKLDS
166	1353-G10-wt	CDR-L2		KDTERPS
167	1353-G10 K50D	CDR-L2		DDTERPS
168	1353-G10T52D	CDR-L2		KDDERPS
169	1353-G10 E53D	CDR-L2		KDTDRPS
170	1353-G10 P55D	CDR-L2		KDTERDS
171	1353-G10 S56D	CDR-L2		KDTERPD
172	1353-G10-kappa	CDR-L2		KDTERPS
173	26H10	CDR-L3		QQYGSSPFT
174	SRP1449-D05-VL	CDR-L3		QQYGSTPFK
175	SRP1449-F01-VL	CDR-L3		QQYGSSPFT
176	1449-G09.2-VL	CDR-L3		QQYGRSPFS
177	SRP1449-B07-VL	CDR-L3		QQYGASPFT
178	SRP1449-B03-VL	CDR-L3		QQYDRSPLT
179	SRP1558-E11-VL	CDR-L3		QQYSLAPPT
180	SRP1558-A06-VL	CDR-L3		QQYMAGPPT
181	SRP1558-F01-VL	CDR-L3		QQYTAGPPT
182	SRP1448-D09-VL	CDR-L3		QQDTAGPPT
183	trastuzumab-VL	CDR-L3		QQHYTTPPT
184	SRP1627-A02-VL	CDR-L3		SHGNPVPQT
185	SRP1627-A11-VL	CDR-L3		WHGINFPQT
186	SRP1627-B01-VL	CDR-L3		STYSHFPQT
187	h5G11-2-VL	CDR-L3		WQGSHFPQT
188	421.61.4.5G11-VL	CDR-L3		WQGSHFPQT
189	1353-G10-wt	CDR-L3		QSADNSITYRV
190	1353-G10-kappa	CDR-L3		QSADNSITYRV
191	26H10	VH		QVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYD
				GSNKYYADSVKGRFTISRDNSKNTLYL
				QMNSLRAEDTAVYYCAREWAVASWDYG
				MDVWGQGTTVTVSS
192	SRP1496-A03-VH	VH		EVQLVESGGGLVQPGGSLRLSCAASGF
				NINDTYIHWVRQAPGKGLEWVGIIDPY
				DGATDYADSVKGRFTISADTSKNTAYL
				QMNSLRAEDTAVYYCAREIFGFYWNPF
				DYWGQGTLVTVSS
193	SRP1496-A04-VH	VH		EVQLVESGGGLVQPGGSLRLSCAASGFN
				INDTYIHWVRQAPGKGLEWVGIIDPYDG
				ATDYADSVKGRFTISADTSKNTAYLQMN
				SLRAEDTAVYYCAREIFGFYWNPFDYWG
				QGTLVTVSS
194	SRP1496-B08-VH	VH		EVQLVESGGGLVQPGGSLRLSCAASGF
				NINDTYIHWVRQAPGKGLEWVGIIDPY
				DGATDYADSVKGRFTISADTSKNTAYL
				QMNSLRAEDTAVYYCAREIFGFYWNPF
				DYWGQGTLVTVSS
195	SRP1648-B07-VH	VH		EVQLVESGGGLVQPGGSLRLSCAASGFN
				IADTFIHWVRQAPGKGLEWVGIIDPYDG
				DTDYADSVKGRFTISADTSKNTAYLQMN
				SLRAEDTAVYYCAREILGFYWNPFDYWG
				QGTLVTVSS
196	SRP1648-E02-VH	VH		EVQLVESGGGLVQPGGSLRLSCAASGFN
				INDNYIHWVRQAPGKGLEWVGIIDPYDG
				FTAYADSVKGRFTISADTSKNTAYLQMN
				SLRAEDTAVYYCARESIGFYLNPFDYWG
				QGTLVTVSS
197	SRP1449-B03-VH	VH		QVQLVESGGGVVQPGRSLRLSCAASGFT
				FSSYGMHWVRQAPGKGLEWVAAIWYDAS
				YKYYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAREWAVASWDYALDVW
				GQGTTVTVSS
198	SRP1449-F01-VH	VH		QVQLVESGGGVVQPGRSLRLSCAASGFT
				FSSYGMHWVRQAPGKGLEWVAVIWYDGS
				YKYYADSVKGRFAISRDNSKNTLYLQMN
				SLRAEDTAVYYCARESEVASWDYGLDVW
				GQGTTVTVSS
199	SRP1449-B07-VH	VH		QVQLVESGGGVVQPGRSLRLSCAASGFT
				FSSYGMHWVRQAPGKGLEWVAVIWYDGS
				NKYYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAREWAVSSWDYGMDVW
				GQGTTVTVSS
200	1449-G09.2-VH	VH		QVQLVESGGGVVQPGRSLRLSCAASGFT
				FSSYGMHWVRQAPGKGLEWVAVIWYDGS
				YKYYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAREEAPENWDYALDVW
				GQGTTVTVSS
201	SRP1449-D05-VH	VH		QVQLVESGGGVVQPGRSLRLSCAASGFT
				FRSFGMHWVRQAPGKGLEWVAVIWYDGS
				VKYYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAREWADVSWDAGLDVW
				GQGTTVTVSS
202	SRP1558-F01-VH	VH		EVQLLESGGGLVQPGGSLRLSCAASGFT
				FPDSSMSWVRQAPGKGLEWVGVITDNSG
				NTDYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAKVFEGGVRPYSDYWG
				QGTLVTVSS
203	SRP1448-D09-VH	VH		EVQLLESGGGLVQPGGSLRLSCAASGFT
				FTDSSMSWVRQAPGKGLEWVGVITGNSG
				TTDYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAKVYEGGVRPYSDYWG
				QGTLVTVSS
204	SRP1558-A06-VH	VH		EVQLLESGGGLVQPGGSLRLSCAASGFT
				FSESTMSWVRQAPGKGLEWVGFITSDSG
				TTDYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAKVFEGGVRPFSDYWG
				QGTLVTVSS
205	SRP1558-E11-VH	VH		EVQLLESGGGLVQPGGSLRLSCAASGFT
				FTSSSMSWVRQAPGKGLEWVGVISDDTG
				STDYADSVKGRFTISRDNSKNTLYLQMN
				SLRAEDTAVYYCAKVDNGGVRPYSDYWG
				QGTLVTVSS
206	SRP1627-A02-VH	VH		QVQLVQSGAEVKKPGSSVKVSCKASGFN
				INDYFMHWVRQAPGQGLEWIARIDPWNG
				DTEYAPKFQGRVTITADESTSTAYMELS
				SLRSEDTAVYYCGMSDALDYWGQGTLVT
				VSS
207	SRP1627-A11-VH	VH		QVQLVQSGAEVKKPGSSVKVSCKASGFN
				INDYFMHWVRQAPGQGLEWIARIDPWNG
				DTEYAPKFQGRVTITADESTSTAYMELS
				SLRSEDTAVYYCGMSDALDYWGQGTLVT
				VSS
208	h5G11-2-VH	VH		QVQLVQSGAEVKKPGSSVKVSCKASGFN
				IKDYYMHWVRQAPGQGLEWIAWIDPENG
				DTEYAPKFQGRVTITADESTSTAYMELS
				SLRSEDTAVYYCNAPDALDYWGQGTLVT
				VSS
209	SRP1627-B01-VH	VH		QVQLVQSGAEVKKPGSSVKVSCKASGFN
				ITDLYMHWVRQAPGQGLEWIARIDPWNG
				DTEYAPKFQGRATITADESTSTAYMELS
				SLRSEDTAVYYCIASEMVDYWGQGTLVT
				VSS
210	421.61.4.5G11-VH	VH		EVQLQQSGAELVRSGASVKLSCTASGFN
				IKDYYMHWVKQRPEQGLEWIAWIDPENG
				DTEYAPKFQGRATLTADTSSNTAYLHLS
				SLTSEDTAVYYCNAPDALDYWGQGTSVT
				VSS
211	1353-G10-wt	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
212	1353-G10Y27D	VH		EVQLVQSGAEVKKPGASVKVSCKASGDR
				FPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
213	1353-G10R28D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYD
				FPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
214	1353-G10F29D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				DPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
215	1353-G10P30D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FDHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
216	1353-G10 H31D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPDYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
217	1353-G10 Y32D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPHDGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
218	1353-G10 G33D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPHYDISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
219	1353-G10 S35D	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPHYGIDWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
220	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYT
	R28T/P30D			FDHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
221	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYD
	R28D/P30D			FDHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
222	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYD
	R28D/H31D			FPDYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
223	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYT
	R28T/H31D			FPDYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
224	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYD
	R28D/P30T/H31S			FTSYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
225	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYT
	R28T/P30T/H31D			FTDYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
226	1353-G10	VH		EVQLVQSGAEVKKPGASVKVSCKASGYT
	R28T/P30T/H31S			FTSYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
227	1353-G10-	VH		EVQLVQSGAEVKKPGASVKVSCKASGYT
	R28T/P30D/H31S			FDSYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSS
228	10B4	VH		QVQLQQSGAELMKPGASVKMSCKTTGYI
				FSSYWIGWVKQRPGHGLEWIGKIFPGSG
				SADYNENFKGKATFTVDTSSNTAYMQLS
				SLTSEDSAVYYCARGYGNYLYFDVWGAG
				TTVTVSS
229	1353-A09	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FTWYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDSEYSSGSGYWGQG
				TLVTVSS
230	1353-C07	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FSTFGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYSSGSGYWGQG
				TLVTVSS
231	1353-E07	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FETYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDAEYSLGSGYWGQG
				TLVTVSS
232	1353-F09	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FRQYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDAEYGSGSGYWGQG
				TLVTVSS
233	1353-G08	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FTRYGISWVRQAPGQGLEWMGWVSAHNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDADYGSGSGYWGQG
				TLVTVSS
234	1353-H08	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FTRQGISWVRQAPGQGLEWMGWISAYNG
				NTKYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGSGSGYWGQG
				TLVTVSS
235	1353-H09	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDAEYGSGSGYWGQG
				TLVTVSS
236	1B10	VH		DVQLQESGPGLVKPSQSLSLTCTVTGHS
				ITSDYAWNWIRQFPGNKLEWMGYISYSG
				RTSYNPSLTSRISITRDTSKNQFFLQLN
				SVTTEDTATYYCARGYALDYWGQGTSVT
				VSS
237	1E9	VH		EVKLVESGGGLVSPGGSLKLSCAASGFT
				FSTFGMSWVRQTPEKRLEWVATISGGGS
				DTYYPDSVQGRFIISRYNAKNNLYLQMN
				SLRPEDTALYYCARQGYDVYSWFAYWGQ
				GTLVTVSA
238	4B10	VH		EVKLVESGGGLVKPGGSLKLSCAASGFT
				FSTYGMSWVRQTPEKRLQWVATISGGGS
				NTYYSDSVKGRFTISRDNAKNNLYLQMS
				SLRSEDTALYYCARQRDSAWFASWGQGT
				LVTVSA
239	h1E9-1	VH		EVQLVESGGGLVKPGGSLRLSCAASGFT
				FSTFGMSWVRQAPGKGLEWVSTISGGGS
				DTYYPDSVQGRFTISRDNAKNSLYLQMN
				SLRAEDTAVYYCARQGYDVYSWFAYWGQ
				GTLVTVSS
240	h1E9-2	VH		EVQLVESGGGLVKPGGSLRLSCAASGFT
				FSTFGMSWVRQAPGKGLEWVSTISGGGS
				DTYYPDSVQGRFTISRDNAKNSLYLQMN
				SLRAEDTAVYYCARQGYDVYSWFAYWGQ
				GTLVTVSS
241	h1E9-4	VH		EVQLVESGGGLVQPGGSLRLSCAASGFT
				FSTFGMSWVRQAPGKGLEWVATISGGGS
				DTYYPDSVQGRFTISRDNAKNSLYLQMN
				SLRAEDTAVYYCARQGYDVYSWFAYWGQ
				GTLVTVSS
242	h1E9-5	VH		EVQLVESGGGLVQPGGSLRLSCAASGFT
				FSTFGMSWVRQAPGKGLEWVATISGGGS
				DTYYPDSVQGRFTISRDNAKNSLYLQMN
				SLRAEDTAVYYCARQGYDVYSWFAYWGQ
				GTLVTVSS
243	h4B10-1	VH		EVQLVESGGGLVKPGGSLRLSCAASGFT
				FSTYGMSWVRQAPGKGLEWVATISGGGS
				NTYYSDSVKGRFTISRDDSKNTLYLQMN
				SLKTEDTAVYYCARQRDSAWFASWGQGT
				LVTVSS
244	h4B10-2	VH		EVQLVESGGGLVKPGGSLRLSCAASGFT
				FSTYGMSWVRQAPGKGLEWVATISGGGS
				NTYYSDSVKGRFTISRDDSKNTLYLQMN
				SLKTEDTAVYYCARQRDSAWFASWGQGT
				LVTVSS
245	h4B10-3	VH		EVQLVESGGGLVKPGGSLRLSCAASGFT
				FSTYGMSWVRQAPGKGLEWVATISGGGS
				NTYYSDSVKGRFTISRDDSKNTLYLQMN
				SLKTEDTAVYYCARQRDSAWFASWGQGT
				LVTVSS
246	PD1-17	VH		QVQLQESGPGVVKPSGTLSLTCAISGGS
				IGSGGSIRSTRWWSWVRQSPGKGLEWIG
				EIYHSGSTNYNPSLKSRVTISLDKSRNH
				FSLRLNSVTAADTAVYYCARQDYGDSGD
				WYFDLWGKGTMVTVSS
247	PD1-28	VH		EVQLVQSGAEVKKPGASVKVSCKASGYR
				FTSYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDADYSSGSGYWGQG
				TLVTVSS
248	PD1-33	VH		QVQLVQSGAEVKKPGASVRVSCKASGYT
				LTSYYIHWVRQAPGQGLEWMGIINPRGA
				TISYAQKFQGRVTMTRDTSTSTVYMELR
				NLKSEDTALYYCATAGIYGFDFDYWGRG
				TLVTVSS
249	PD1-35	VH		QVQLQESGPGLVKPSQTLSLTCTVSGGS
				ISSGAYYWSWIRQHPGKGLEWIGYIYYN
				GNTYYNPSLRSLVTISVDASKNQFSLKL
				SSVTAADTAVYYCARASDYVWGGYRYMD
				AFDIWGRGTLITVSS
250	PD1-F2	VH		EVQLVQSGGGVVQPGRSLRLSCAASGFT
				FSSYWCDRMSWVRQAPGKGLEWVSAISG
				SGGSTYYADSVKGRFTISRDNSKNTLYL
				QMNSLRAEDTAVYYCAKENWGSYFDLWG
				QGTTVTVSS
251	26H10	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYGSSPFTFGPGTKVDIK
252	SRP1449-D05-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYGSTPFKFGPGTKVDIK
253	SRP1449-F01-VL	VL		EIALTQSPGTLSLSPGERATLSCRASR
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYGSSPFTFGPGTKVDIK
254	1449-G09.2-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYGRSPFSFGPGTKVDIK
255	SRP1449-B07-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPNRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYGASPFTFGPGTKVDIK
256	SRP1449-B03-VL	VL		EIVLTQSPGTMSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYDRSPLTFGPGTKVDIK
257	SRP1558-E11-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYSLAPPTLGQGTKVEIK
258	SRP1558-A06-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSNPLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQYMAGPPTFGQGTKVEIK
259	SRP1558-F01-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSGNPAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				XDFAVYYCQQYTAGPPTFGQGTKVEIK
260	SRP1448-D09-VL	VL		EIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASS
				RATGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQDTAGPPTFGQGTKVEIK
261	trastuzumab-VL	VL		DIQMTQSPSSLSASVGDRVTITCRASQ
				DVNTAVAWYQQKPGKAPKLLIYSASFL
				YSGVPSRFSGSRSGTDFTLTISSLQPE
				DFATYYCQQHYTTPPTFGQGTKVEIK
262	SRP1627-A02-VL	VL		DVVMTQSPLSLPVTLGQPASISCKSSQS
				LLDSDGKTYLNWFQQRPGQSPRRLIYLV
				SKLDSGVPDRFSGSGSGTDFTLKISRVE
				AEDVGVYYCSHGNPVPQTFGQGTKVEIK
263	SRP1627-A11-VL	VL		DVVMTQSPLSLPVTLGQPASISCKSSQ
				SLLDSDGKTYLNWFQQRPGQSPRRLIY
				LVSKLDSGVPDRFSGSGSGTDFTLKIS
				RVEAEDVGVYYCWHGINFPQTFGQGTK
				VEIK
264	SRP1627-B01-VL	VL		DVVMTQSPLSLPVTLGQPASISCKSSQ
				SLLDSDGKTYLNWFQQRPGQSPRRLIY
				LVSKLDSGVPDRFSGSGSGTDFTLKIS
				RVEAEDVGVYYCSTYSHFPQTFGQGTK
				VEIK
265	h5G11-2-VL	VL		DVVMTQSPLSLPVTLGQPASISCKSSQ
				SLLDSDGKTYLNWFQQRPGQSPRRLIY
				LVSKLDSGVPDRFSGSGSGTDFTLKIS
				RVEAEDVGVYYCWQGSHFPQTFGQGTK
				VEIK
266	421.61.4.5G11-VL	VL		DVVMTQTPLTLSVTIGQIASISCKSSQ
				SLLDSDGKTYLNWLLQRPGQSPKRLIY
				LVSKLDSGVPDRFTGSGSGTDFTLKIS
				RVEAEDLGVYYCWQGSHFPQTFGGGTK
				LEIK
267	1353-G10-wt (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
268	1353-G10 K50D (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYDDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
269	1353-G10 T52D (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDDERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
270	1353-G10 E53D (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTDRPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
271	1353-G10 P55D (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERDSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
272	1353-G10 S56D (λ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPDG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
273	1353-G10 VL1c (κ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDFAT
				YYCQSADNSITYRVFGGGTKVEIK
274	1353-G10 VL1d (κ)	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQRKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDFAT
				YYCQSADNSITYRVFGGGTKVEIK
275	1353-G10 Vk1-39 (κ)	VL		DIQLTQSPSSLSASVGDRVTITCSGDAL
				PKQYAYWYQQKPGKAPKLLIYKDTERPS
				GVPSRFSGSSSGTKVTLTISSLQPEDFA
				TYYCQSADNSITYRVFGGGTKVEIK
276	1353-G10 Vk3-20 (κ)	VL		EIVLTQSPGTLSLSPGERATLSCSGDAL
				PKQYAYWYQQKPGQAPRLLIYKDTERPS
				GIPDRFSGSSSGTKVTLTISRLEPEDFA
				VYYCQSADNSITYRVFGGGTKVEIK
277	10B4	VL		NIVMTQTPKFLLVSAGDRITITCKASQS
				VSDDVAWYQQKPGQSPKLLISYAFKRYI
				GVPDRFTGSGYGTDFTFTISTVQAEDLA
				VYFCQQNYNSPYTFGGGTKLELKR
278	1353-A09	VL		SYELTQPPSVSVSPGQTARITCSGDALT
				TQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
279	1353-C07	VL		SYELTQPPSVSVSPGQTARITCSGDALS
				EQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
280	1353-E07	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
281	1353-F09	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVLYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
282	1353-G08	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				MQYGYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
283	1353-H08	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
284	1353-H09	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
285	1B10	VL		QIVLSQSPAILSASPGEKVTMTCRTSSS
				VNYMHWFQQKPGSSPKPWIYATSKLASG
				VPARFSGSGSGTSYSLTISRVEAEDAAT
				YFCQQWISDPWTFGGGTKLEIK
286	1E9	VL		DIILTQSPASLAVSLGQRAAISCRASES
				VDNSGISFMSWFQQKPGQPPKLLIYTAS
				NQGSGVPARFSGSGSGTEFSLNIHPMEE
				DDTAMYFCQQSKEVPWTFGGGTKLEIR
287	4B10	VL		DIVLTQSPASLAVSLGQRATISCRASEN
				VDDYGVSFMNWFQQKPGQPPKLLIYPAS
				NQGSGVPARFSGSGSGTDFSLNIHPMEE
				DDTAMYFCQQSKEVPWTFGGGTKLEIK
288	h1E9-1	VL		DIQLTQSPSFLSASVGDRVTITCRASES
				VDNSGISFMSWYQQKPGKAPKLLIYTAS
				NQGSGVPSRFSGSGSGTEFTLTISSLQP
				EDFATYYCQQSKEVPWTFGQGTKVEIK
289	h1E9-2	VL		EIVLTQSPATLSLSPGERATLSCRASES
				VDNSGISFMSWYQQKPGQAPRLLIYTAS
				NQGSGIPARFSGSGSGTDFTLTISSLEP
				EDFAVYYCQQSKEVPWTFGQGTKVEIK
290	h1E9-4	VL		DIQLTQSPSFLSASVGDRVTITCRASES
				VDNSGISFMSWYQQKPGKAPKLLIYTAS
				NQGSGVPSRFSGSGSGTEFTLTISSLQP
				EDFATYYCQQSKEVPWTFGQGTKVEIK
291	h1E9-5	VL		EIVLTQSPATLSLSPGERATLSCRASES
				VDNSGISFMSWYQQKPGQAPRLLIYTAS
				NQGSGIPARFSGSGSGTDFTLTISSLEP
				EDFAVYYCQQSKEVPWTFGQGTKVEIK
292	h4B10-1	VL		EIVLTQSPATLSLSPGERATLSCRASEN
				VDDYGVSFMNWYQQKPGQAPRLLIYPAS
				NQGSGIPARFSGSGSGTDFTLTISSLEP
				EDFAVYYCQQSKEVPWTFGQGTKVEIK
293	h4B10-2	VL		EIVLTQSPGTLSLSPGERATLSCRASEN
				VDDYGVSFMNWYQQKPGQAPRLLIYPAS
				NQGSGIPDRFSGSGSGTDFTLTISRLEP
				EDFAVYYCQQSKEVPWTFGQGTKVEIK
294	h4B10-3	VL		DIVMTQSPDSLAVSLGERATINCRASEN
				VDDYGVSFMNWYQQKPGQPPKLLIYPAS
				NQGSGVPDRFSGSGSGTDFTLTISSLQA
				EDVAVYYCQQSKEVPWTFGGGTKLEIK
295	PD1-17	VL		NFMLTQPHSVSESPGKTVTISCTRSSGS
				IASNSVQWYQQRPGSSPTTVIYEDNQRP
				SGVPDRFSGSIDSSSNSASLTVSGLKTE
				DEADYYCQSSDSSAVVFGSGTKLTVL
296	PD1-28	VL		SYELTQPPSVSVSPGQTARITCSGDALP
				KQYAYWYQQKPGQAPVMVIYKDTERPSG
				IPERFSGSSSGTKVTLTISGVQAEDEAD
				YYCQSADNSITYRVFGGGTKVTVL
297	PD1-33	VL		QSALTQPASVSGSPGQSITISCTGTSND
				VGGYNYVSWYQHHPGKAPKLIIYDVTNR
				PSGVSDRFSGSKSGNTASLTISGLLAED
				EGDYYCSSYTIVTNFEVLFGGGTKLTV
298	PD1-35	VL		QSVLTQPPSASGTPGQRVTISCSGSNSN
				IGSNSVNWYQQLPGTAPKLLIYGNNQRP
				SGVPDRFSGSKSGTSASLAISGLQSENE
				ADYYCAAWDDSLNGPVFGRGTKVTVL
299	PD1-F2	VL		DIVMTQSPSTLSASVGDRVTITCRASQG
				ISSWLAWYQQKPGRAPKVLIYKASTLES
				GVPSRFSGSGSGTDFTLTISSLQPEDFA
				TYYCQQSYSTPWTFGQGTKLEIK
300	Human IgG1 HC			ASTKGPSVFPLAPSSKSTSGGTAALGCL
	Constant			VKDYFPEPVTVSWNSGALTSGVHTFPAV
				LQSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSCDKTHTCPPC
				PAPELLGGPSVFLFPPKPKDTLMISRTP
				EVTCVVVDVSHEDPEVKFNWYVDGVEVH
				NAKTKPREEQYNSTYRVVSVLTVLHQDW
				LNGKEYKCKVSNKALPAPIEKTISKAKG
				QPREPQVYTLPPSREEMTKNQVSLTCLV
				KGFYPSDIAVEWESNGQPENNYKTTPPV
				LDSDGSFFLYSKLTVDKSRWQQGNVFSC
				SVMHEALHNHYTQKSLSLSPGK
301	Human IgG LC			RTVAAPSVFIFPPSDEQLKSGTASVVCL
	Constant Ckappa			LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLSSTLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
302	Mouse IgG1 HC			AKTTPPSVYPLAPGSAAQTNSMVTLGCL
	Constant			VKGYFPEPVTVTWNSGSLSSGVHTFPAV
				LQSDLYTLSSSVTVPSSTWPSETVTCNV
				AHPASSTKVDKKIVPRDCGCKPCICTVP
				EVSSVFIFPPKPKDVLTITLTPKVTCVV
				VDISKDDPEVQFSWFVDDVEVHTAQTQP
				REEQFNSTFRSVSELPIMHQDWLNGKEF
				KCRVNSAAFPAPIEKTISKTKGRPKAPQ
				VYTIPPPKEQMAKDKVSLTCMITDFFPE
				DITVEWQWNGQPAENYKNTQPIMDTDGS
				YFVYSKLNVQKSNWEAGNTFTCSVLHEG
				LHNHHTEKSLSHSPG
303	Mouse IgG LC			RADAAPTVSIFPPSSEQLTSGGASVVCF
	Constant Ckappa			LNNFYPKDINVKWKIDGSERQNGVLNSW
				TDQDSKDSTYSMSSTLTLTKDEYERHNS
				YTCEATHKTSTSPIVKSFNRNEC
304	Kappa LC			HMTVAAPSVFIFPPSDEQLKSGTASVVC
				LLNNFYPREAKVQWKVDNALQSGNSQES
				VTEQDSKDSTYSLSSTLTLSKADYEKHK
				VYACEVTHQGLSSPVTKSFNRGEC
305	Lambda LC			GQPKAAPSVTLFPPSSEELQANKATLVC
				LISDFYPGAVTVAWKADSSPVKAGVETT
				TPSKQSNNKYAASSYLSLTPEQWKSHRS
				YSCQVTHEGSTVEKTVAPTECS
306	IgG1 Fc from scFv-Fc			AAGSDQEPKSSDKTHTCPPCSAPELLGG
				SSVFLFPPKPKDTLMISRTPEVTCVVVD
				VSHEDPEVKFNWYVDGVEVHNAKTKPRE
				EQYNSTYRVVSVLTVLHQDWLNGKEYKC
				KVSNKALPAPIEKTISKAKGQPREPQVY
				TLPPSRDELTKNQVSLTCLVKGFYPSDI
				AVEWESNGQPENNYKTTPPVLDSDGSFF
				LYSKLTVDKSRWQQGNVFSCSVMHEALH
				NHYTQKSLSLSPGKGSGDYKDDDDKGSG
307	FlagHis Tag			GSGDYKDDDDKGSGHHHHHH
308	Linker (GGGGS)3			GGGGSGGGGSGGGGS
309	Linker (GGGGS)4			GGGGSGGGGSGGGGSGGGGS
310	Linker (GGGGS)5			GGGGSGGGGSGGGGSGGGGSGGGGS
311	Linker (GGGGS)6			GGGGSGGGGSGGGGSGGGGSGGGGSGGG
				GS
312	Linker			AAGSDQEPKSS
313	Linker			AAGSDQ
314	Linker			APGPSAPSHRSLPSRAFG
315	Linker-hinge			AAGSDQEPKSSDKTHTCPPCP
316	Hinge-wt			DKTHTCPPCP
317	26H10	scFv		QVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYD
				GSNKYYADSVKGRFTISRDNSKNTLYL
				QMNSLRAEDTAVYYCAREWAVASWDYG
				MDVWGQGTTVTVSSGGGGSGGGGSGGG
				GSEIVLTQSPGTLSLSPGERATLSCRA
				SQSVSSSYLAWYQQKPGQAPRLLIYGA
				SSRATGIPDRFSGSGSGTDFTLTISRL
				EPEDFAVYYCQQYGSSPFTFGPGTKVD
				IK
318	hPD-1			MQIPQAPWPVVWAVLQLGWRPGWFLDSP
				DRPWNPPTFSPALLVVTEGDNATFTCSF
				SNTSESFVLNWYRMSPSNQTDKLAAFPE
				DRSQPGQDCRFRVTQLPNGRDFHMSVVR
				ARRNDSGTYLCGAISLAPKAQIKESLRA
				ELRVTERRAEVPTAHPSPSPRPAGQFQT
				LVVGVVGGLLGSLVLLVWVLAVICSRAA
				RGTIGARRTGQPLKEDPSAVPVFSVDYG
				ELDFQWREKTPEPPVPCVPEQTEYATIV
				FPSGMGTSSPARRGSADGPRSAQPLRPE
				DGHCSWPL
319	murine PD-1			MWVRQVPWSFTWAVLQLSWQSGWLLEVP
				NGPWRSLTFYPAWLTVSEGANATFTCSL
				SNWSEDLMLNWNRLSPSNQTEKQAAFCN
				GLSQPVQDARFQIIQLPNRHDFHMNILD
				TRRNDSGIYLCGAISLHPKAKIEESPGA
				ELVVTERILETSTRYPSPSPKPEGRFQG
				MVIGIMSALVGIPVLLLLAWALAVFCST
				SMSEARGAGSKDDTLKEEPSAAPVPSVA
				YEELDFQGREKTPELPTACVHTEYATIV
				FTEGLGASAMGRRGSADGLQGPRPPRHE
				DGHCSWPL
320	cyno PD-1			MWVRQVPWSFTWAVLQLSWQSGWLLEVP
				NGPWRSLTFYPAWLTVSEGANATFTCSL
				SNWSEDLMLNWNRLSPSNQTEKQAAFCN
				GLSQPVQDARFQIIQLPNRHDFHMNILD
				TRRNDSGIYLCGAISLHPKAKIEESPGA
				ELVVTERILETSTRYPSPSPKPEGRFQG
				MVIGIMSALVGIPVLLLLAWALAVFCST
				SMSEARGAGSKDDTLKEEPSAAPVPSVA
				YEELDFQGREKTPELPTACVHTEYATIV
				FTEGLGASAMGRRGSADGLQGPRPPRHE
				DGHCSWPL
321	CH2—CH3, Fc-knob			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLWCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
322	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-knob-V262E			VTCEVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLWCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
323	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-knob-V264S			VTCVVSDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLWCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
324	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-knob-D399C			VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSRDELTKNQVSLWCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				CSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
325	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-knob-S354C			VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPCRDELTKNQVSLWCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
326	CH2—CH3, Fc-hole			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLSCAVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
327	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-hole-V262E			VTCEVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLSCAVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
328	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-hole-V264S			VTCVVSDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSREEMTKNQVSLSCAVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
329	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-hole-Y349C			VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVCTLPPSRDELTKNQVSLSCAVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFFLVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
330	CH2—CH3,			APELLGGPSVFLFPPKPKDTLMISRTPE
	Fc-hole-K392C			VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYTLPPSRDELTKNQVSLSCAVK
				GFYPSDIAVEWESNGQPENNYCTTPPVL
				DSDGSFFLVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
331	Fc-zwA			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVYPPSREEMTKNQVSLTCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFALVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
332	Fc-zwA-V262E			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCEVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVYPPSREEMTKNQVSLTCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFALVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
333	Fc-zwA-V264S			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVSDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVYPPSREEMTKNQVSLTCLVK
				GFYPSDIAVEWESNGQPENNYKTTPPVL
				DSDGSFALVSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
334	Fc-zwB			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVLPPSREEMTKNQVSLLCLVK
				GFYPSDIAVEWESNGQPENNYLTWPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
335	Fc-zwB-V262E			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCEVVDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVLPPSREEMTKNQVSLLCLVK
				GFYPSDIAVEWESNGQPENNYLTWPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
336	Fc-zwB-V264S			APELLGGPSVFLFPPKPKDTLMISRTPE
				VTCVVSDVSHEDPEVKFNWYVDGVEVHN
				AKTKPREEQYNSTYRVVSVLTVLHQDWL
				NGKEYKCKVSNKALPAPIEKTISKAKGQ
				PREPQVYVLPPSREEMTKNQVSLLCLVK
				GFYPSDIAVEWESNGQPENNYLTWPPVL
				DSDGSFFLYSKLTVDKSRWQQGNVFSCS
				VMHEALHNHYTQKSLSLSPGK
337	hinge-CH2-CH3-zwA			DKTHTCPPCPAPELLGGPSVFLFPPKPK
				DTLMISRTPEVTCVVVDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVYPPSREEMTK
				NQVSLTCLVKGFYPSDIAVEWESNGQPE
				NNYKTTPPVLDSDGSFALVSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
338	hinge-CH2-CH3-zwA			DKTHTCPPCPAPELLGGPSVFLFPPKPK
	V262E			DTLMISRTPEVTCEVVDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVYPPSREEMTK
				NQVSLTCLVKGFYPSDIAVEWESNGQPE
				NNYKTTPPVLDSDGSFALVSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
339	hinge-CH2-CH3-zwA			DKTHTCPPCPAPELLGGPSVFLFPPKPK
	V264S			DTLMISRTPEVTCVVSDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVYPPSREEMTK
				NQVSLTCLVKGFYPSDIAVEWESNGQPE
				NNYKTTPPVLDSDGSFALVSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
340	hinge-CH2-CH3-zwB			DKTHTCPPCPAPELLGGPSVFLFPPKPK
				DTLMISRTPEVTCVVVDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVLPPSREEMTK
				NQVSLLCLVKGFYPSDIAVEWESNGQPE
				NNYLTWPPVLDSDGSFFLYSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
341	hinge-CH2-CH3-zwB			DKTHTCPPCPAPELLGGPSVFLFPPKPK
	V262E			DTLMISRTPEVTCEVVDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVLPPSREEMTK
				NQVSLLCLVKGFYPSDIAVEWESNGQPE
				NNYLTWPPVLDSDGSFFLYSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
342	hinge-CH2-CH3-zwB			DKTHTCPPCPAPELLGGPSVFLFPPKPK
	V264S			DTLMISRTPEVTCVVSDVSHEDPEVKFN
				WYVDGVEVHNAKTKPREEQYNSTYRVVS
				VLTVLHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYVLPPSREEMTK
				NQVSLLCLVKGFYPSDIAVEWESNGQPE
				NNYLTWPPVLDSDGSFFLYSKLTVDKSR
				WQQGNVFSCSVMHEALHNHYTQKSLSLS
				PGK
343	CH1-(a)1			ASTKGPSVFPEAPSSKSTSGGTAALGCL
				VTDYFPEPVTVSWNSGALTSGVHTFPAV
				LESSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
344	CH1-(b)1			ASTKGPSVFPRAPSSKSTSGGTAALGCL
				VKDYFPEPVTVSWNSGALTSGVHTFPAV
				LQSSGLYKLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
345	CH1-(c)1			ASTKGPSVFPLAPSSKSTSGGTAWLGCE
				VTDYFPEPVTVSWNSGALTSGVHTFPAV
				LESSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
346	CH1-(d)1			ASTKGPSVFPLAPSSKSTSGGTAALGCE
				VTDYFPEPVTVSWNSGALTSGVHTFPAV
				LESSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
347	CH1-(e)(f)1			ASTKGPSVFPLAPSSKSTSGGTAALGCL
				VKGYFPEPVTVSWNSGALTSGVHTFPAV
				LKSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
348	CH1-(a)2			ASTKGPSVFPLAPSSKSTSGGTAALGCL
				VKDYFPEPVTVSWNSGALTSGVHTFPAV
				LQSSGLYSLKSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
349	CH1-(b)2			ASTKGPSVFPLAPSSKSTSGGTAALGCE
				VTDYFPEPVTVSWNSGALTSGVHTFPAV
				LESSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
350	CH1-(c)2			ASTKGPSVFPLAPSSKSTSGGTAALGCL
				VKDYFPEPVTVSWNSGALTSGVHTFPAV
				LKSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
351	CH1-(d)2			ASTKGPSVFPLAPSSKSTSGGTAALGCL
				VKDYFPEPVTVSWNSGALTSGVRTFPAV
				LKSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
352	CH1-(e)(f)2			ASTKGPSVFPLAPSSKSTSGGTAALGCE
				VTDYFPEPVTVSWNSGALTSGVHTFPAV
				LQSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
353	Ck-(a)1			RTVAAPSVFIFPPSDEQLKSGTARVGCL
				LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLRSTLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
354	Ck-(b)1			RTVAAPSVFIFPPSDEQLKSGTASVGCL
				LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLDSELTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
355	Ck-(c)1			RTVAAPSVAIFPPSDERLKSGTASVVCV
				LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLSSRLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
356	Ck-(d)1			RTVAAPSVFIFPPSDERLKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSKESV
				TEQDSKDSTYSLSSRLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
357	Ck-(e)(f)1			RTVAAPSVFIFPPSDEELKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSEESV
				TEQDSKDSTYSLSSTLELSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
358	Cl-(a)2			GQPKAAPSVTLFPPSSEELQANKATLVC
				LISDFYPGAVTVAWKADSSPVKAGVETT
				TPSKQSNNKYAAESELSLTPEQWKSHRS
				YSCQVTHEGSTVEKTVAPTECS
359	Cl-(b)2			GQPKAAPSVTLFPPSSEQLQANKARLVC
				LISDFYPGAVTVAWKADSSPVKAGVETT
				TPSKQSNNKYAASSYLSLTPEQWKSHRS
				YSCQVTHEGSTVEKTVAPTECS
360	Ck-(c)2			RTVAAPSVFIFPPSDEELKSGTASVVCW
				LNNFYPREAKVQWKVDNALQSGNSEESV
				TEQDSKDSTYSLSSTLELSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
361	Ck-(d)2			RTVAAPSVFIFPPSDEELKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSEESV
				TEQDSKDSTYSLSSTLELSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
362	Ck-(e)2			RTVAAPSVFIFPPSDERLKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSKESV
				TEQDSKDSTYSLSSRLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
363	Ck-(f)2			RTVAAPSVFIFPPSDERLKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLSSTLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
364	aPD-1-1353-	scFv		EVQLVQSGAEVKKPGASVKVSCKASGYT
	G10_R28T/P30D/H31S_Vk1-			FDSYGISWVRQAPGQGLEWMGWISAYNG
	39_LC			NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSSGGGGSGGGGSGGGGSDIQLTQ
				SPSSLSASVGDRVTITCSGDALPKQYAY
				WYQQKPGKAPKLLIYKDTERPSGVPSRF
				SGSSSGTKVTLTISSLQPEDFATYYCQS
				ADNSITYRVFGGGTKVEIK
365	aPD1 1353-G10	scFv		EVQLVQSGAEVKKPGASVKVSCKASGYT
	scFvFc zwB			FDSYGISWVRQAPGQGLEWMGWISAYNG
	R28T/P30D/H31S			NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSSGGGGSGGGGSGGGGSSYELTQ
				PPSVSVSPGQTARITCSGDALPKQYAYW
				YQQKPGQAPVMVIYKDTERPSGIPERFS
				GSSSGTKVTLTISGVQAEDEADYYCQSA
				DNSITYRVFGGGTKVTVL
366	aPD1 1353-G10	scFv		EVQLVQSGAEVKKPGASVKVSCKASGYR
	Vk1-39			FPHYGISWVRQAPGQGLEWMGWISAYNG
				NTNYAQKLQGRVTMTTDTSTNTAYMELR
				SLRSDDTAVYYCARDVDYGTGSGYWGQG
				TLVTVSSGGGGSGGGGSGGGGSDIQLTQ
				SPSSLSASVGDRVTITCSGDALPKQYAY
				WYQQKPGKAPKLLIYKDTERPSGVPSRF
				SGSSSGTKVTLTISSLQPEDFATYYCQS
				ADNSITYRVFGGGTKVEIK
367	1353-G10	scFv-Fc		MEVQLVQSGAEVKKPGASVKVSCKASGY
				RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSGGGGSGGGGSGGGGSSYELT
				QPPSVSVSPGQTARITCSGDALPKQYAY
				WYQQKPGQAPVMVIYKDTERPSGIPERF
				SGSSSGTKVTLTISGVQAEDEADYYCQS
				ADNSITYRVFGGGTKVTVLAAGSDQEPK
				KLAAGSDQEPKSSDKTHTCPPCSAPELL
				GGSSVFLFPPKPKDTLMISRTPEVTCVV
				VDVSHEDPEVKFNWYVDGVEVHNAKTKP
				REEQYNSTYRVVSVLTVLHQDWLNGKEY
				KCKVSNKALPAPIEKTISKAKGQPREPQ
				VYTLPPSRDELTKNQVSLTCLVKGFYPS
				DIAVEWESNGQPENNYKTTPPVLDSDGS
				FFLYSKLTVDKSRWQQGNVFSCSVMHEA
				LHNHYTQKSLSLSPGKGSGDYKDDDDKG
				SGHHHHHH
368	1353-G10 scFv-Fc	scFv-Fc		MEVQLVQSGAEVKKPGASVKVSCKASGY
	hole			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSGGGGSGGGGSGGGGSSYELT
				QPPSVSVSPGQTARITCSGDALPKQYAY
				WYQQKPGQAPVMVIYKDTERPSGIPERF
				SGSSSGTKVTLTISGVQAEDEADYYCQS
				ADNSITYRVFGGGTKVTVLAAGSDQEPK
				SSDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYTLPPSRDEL
				TKNQVSLSCAVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFFLVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
369	1353-G10 scFv-Fc	scFv-Fc		MEVQLVQSGAEVKKPGASVKVSCKASGY
	knob			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSGGGGSGGGGSGGGGSSYELT
				QPPSVSVSPGQTARITCSGDALPKQYAY
				WYQQKPGQAPVMVIYKDTERPSGIPERF
				SGSSSGTKVTLTISGVQAEDEADYYCQS
				ADNSITYRVFGGGTKVTVLAAGSDQEPK
				SSDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYTLPPSRDEL
				TKNQVSLWCLVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFFLYSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
370	1353-G10 scFv-Fc	scFv-Fc		MEVQLVQSGAEVKKPGASVKVSCKASGY
	zwA			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSGGGGSGGGGSGGGGSSYELT
				QPPSVSVSPGQTARITCSGDALPKQYAY
				WYQQKPGQAPVMVIYKDTERPSGIPERF
				SGSSSGTKVTLTISGVQAEDEADYYCQS
				ADNSITYRVFGGGTKVTVLAAGSDQEPK
				SSDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVYPPSREEM
				TKNQVSLTCLVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFALVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
371	1449-G09.2 scFv-Fc	scFv-Fc		MQVQLVESGGGVVQPGRSLRLSCAASGF
	knob			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSAPGPSAPSHRSLPSRAF
				GEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKAAG
				SDQEPKSSDKTHTCPPCPAPELLGGPSV
				FLFPPKPKDTLMISRTPEVTCVVVDVSH
				EDPEVKFNWYVDGVEVHNAKTKPREEQY
				NSTYRVVSVLTVLHQDWLNGKEYKCKVS
				NKALPAPIEKTISKAKGQPREPQVYTLP
				PSRDELTKNQVSLWCLVKGFYPSDIAVE
				WESNGQPENNYKTTPPVLDSDGSFFLYS
				KLTVDKSRWQQGNVFSCSVMHEALHNHY
				TQKSLSLSPGK
372	1449-G09.2 HC hole	HC		MQVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSASTKGPSVFPLAPSSKS
				TSGGTAALGCLVKDYFPEPVTVSWNSGA
				LTSGVHTFPAVLQSSGLYSLSSVVTVPS
				SSLGTQTYICNVNHKPSNTKVDKKVEPK
				SCDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYTLPPSREEM
				TKNQVSLSCAVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFFLVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
373	1449-G09.2 HC zwB	HC		MQVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSASTKGPSVFPLAPSSKS
				TSGGTAALGCLVKDYFPEPVTVSWNSGA
				LTSGVHTFPAVLQSSGLYSLSSVVTVPS
				SSLGTQTYICNVNHKPSNTKVDKKVEPK
				SCDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVLPPSREEM
				TKNQVSLLCLVKGFYPSDIAVEWESNGQ
				PENNYLTWPPVLDSDGSFFLYSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
374	1449-G09.2 HC1a	HC		MQVQLVESGGGVVQPGRSLRLSCAASGF
	zwA			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSASTKGPSVFPEAPSSKS
				TSGGTAALGCLVTDYFPEPVTVSWNSGA
				LTSGVHTFPAVLESSGLYSLSSVVTVPS
				SSLGTQTYICNVNHKPSNTKVDKKVEPK
				SCDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVYPPSREEM
				TKNQVSLTCLVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFALVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
375	1449-G09.2 HC2a	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
	zwB			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLKSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYVLPPSREEMTKN
				QVSLLCLVKGFYPSDIAVEWESNGQPEN
				NYLTWPPVLDSDGSFFLYSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
376	1449-G09.2 LC	LC		MEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKRTV
				AAPSVFIFPPSDEQLKSGTASVVCLLNN
				FYPREAKVQWKVDNALQSGNSQESVTEQ
				DSKDSTYSLSSTLTLSKADYEKHKVYAC
				EVTHQGLSSPVTKSFNRGEC
3771	1449-G09.2 LC1a	LC		MEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKRTV
				AAPSVFIFPPSDEQLKSGTARVGCLLNN
				FYPREAKVQWKVDNALQSGNSQESVTEQ
				DSKDSTYSLRSTLTLSKADYEKHKVYAC
				EVTHQGLSSPVTKSFNRGEC
378	1449-G09.2 LC2a	LC		MSYELTQPPSVSVSPGQTARITCSGDAL
				PKQYAYWYQQKPGQAPVMVIYKDTERPS
				GIPERFSGSSSGTKVTLTISGVQAEDEA
				DYYCQSADNSITYRVFGGGTKVTVLGQP
				KAAPSVTLFPPSSEELQANKATLVCLIS
				DFYPGAVTVAWKADSSPVKAGVETTTPS
				KQSNNKYAAESELSLTPEQWKSHRSYSC
				QVTHEGSTVEKTVAPTECS
379	LAG3 scFvFc	scFv		QVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYD
				GSYKYYADSVKGRFTISRDNSKNTLYL
				QMNSLRAEDTAVYYCAREEAPENWDYA
				LDVWGQGTTVTVSSGGGGSGGGGSGGG
				GSEIVLTQSPGTLSLSPGERATLSCRA
				SQSVSSSYLAWYQQKPGQKVDIK
380	1449-G09.2 scFv-Fc	scFv-Fc		MQVQLVESGGGVVQPGRSLRLSCAASGF
	hole			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSAPGPSAPSHRSLPSRAF
				GEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKAAG
				SDQEPKSSDKTHTCPPCSAPELLGGSSV
				FLFPPKPKDTLMISRTPEVTCVVVDVSH
				EDPEVKFNWYVDGVEVHNAKTKPREEQY
				NSTYRVVSVLTVLHQDWLNGKEYKCKVS
				NKALPAPIEKTISKAKGQPREPQVYTLP
				PSRDELTKNQVSLSCAVKGFYPSDIAVE
				WESNGQPENNYKTTPPVLDSDGSFFLVS
				KLTVDKSRWQQGNVFSCSVMHEALHNHY
				TQKSLSLSPGK
381	Anti-PD1 1353-G10	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
	HC knob			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLSSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYTLPPSREEMTKN
				QVSLWCLVKGFYPSDIAVEWESNGQPEN
				NYKTTPPVLDSDGSFFLYSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
382	Anti-PD1 1353-G10	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
	HC hole			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLSSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYTLPPSREEMTKN
				QVSLSCAVKGFYPSDIAVEWESNGQPEN
				NYKTTPPVLDSDGSFFLVSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
383	Anti-PD1 1353-G10	LC		MSYELTQPPSVSVSPGQTARITCSGDAL
	LC			PKQYAYWYQQKPGQAPVMVIYKDTERPS
				GIPERFSGSSSGTKVTLTISGVQAEDEA
				DYYCQSADNSITYRVFGGGTKVTVLGQP
				KAAPSVTLFPPSSEELQANKATLVCLIS
				DFYPGAVTVAWKADSSPVKAGVETTTPS
				KQSNNKYAASSYLSLTPEQWKSHRSYSC
				QVTHEGSTVEKTVAPTECS
384	Anti-LAG3 1449-	scFv-Fc		MQVQLVESGGGVVQPGRSLRLSCAASGF
	G09.2 scFvFc hole			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSAPGPSAPSHRSLPSRAF
				GEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKAAG
				SDQEPKSSDKTHTCPPCSAPELLGGSSV
				FLFPPKPKDTLMISRTPEVTCVVVDVSH
				EDPEVKFNWYVDGVEVHNAKTKPREEQY
				NSTYRVVSVLTVLHQDWLNGKEYKCKVS
				NKALPAPIEKTISKAKGQPREPQVYTLP
				PSRDELTKNQVSLSCAVKGFYPSDIAVE
				WESNGQPENNYKTTPPVLDSDGSFFLVS
				KLTVDKSRWQQGNVFSCSVMHEALHNHY
				TQKSLSLSPGK
385	CH1-wt			ASTKGPSVFPLAPSSKSTSGGTAALGCL
				VKDYFPEPVTVSWNSGALTSGVHTFPAV
				LQSSGLYSLSSVVTVPSSSLGTQTYICN
				VNHKPSNTKVDKKVEPKSC
386	CK-wt			RTVAAPSVFIFPPSDEQLKSGTASVVCL
				LNNFYPREAKVQWKVDNALQSGNSQESV
				TEQDSKDSTYSLSSTLTLSKADYEKHKV
				YACEVTHQGLSSPVTKSFNRGEC
387	Cλ-wt			GQPKAAPSVTLFPPSSEELQANKATLVC
				LISDFYPGAVTVAWKADSSPVKAGVETT
				TPSKQSNNKYAASSYLSLTPEQWKSHRS
				YSCQVTHEGSTVEKTVAPTECS
388	1353-G10 scFv-Fc	scFv-Fc		MEVQLVQSGAEVKKPGASVKVSCKASGY
	zwB			RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSGGGGSGGGGSGGGGSSYELT
				QPPSVSVSPGQTARITCSGDALPKQYAY
				WYQQKPGQAPVMVIYKDTERPSGIPERF
				SGSSSGTKVTLTISGVQAEDEADYYCQS
				ADNSITYRVFGGGTKVTVLAAGSDQEPK
				SSDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVLPPSREEM
				TKNQVSLLCLVKGFYPSDIAVEWESNGQ
				PENNYLTWPPVLDSDGSFFLYSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
389	1353-G10 HC zwA	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
				RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLSSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYVYPPSREEMTKN
				QVSLTCLVKGFYPSDIAVEWESNGQPEN
				NYKTTPPVLDSDGSFALVSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
390	1353-G10 HC zwB	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
				RFPHYGISWVRQAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLSSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYVLPPSREEMTKN
				QVSLLCLVKGFYPSDIAVEWESNGQPEN
				NYLTWPPVLDSDGSFFLYSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
391	1449-G09.2 scFv-Fc	scFv-Fc		MQVQLVESGGGVVQPGRSLRLSCAASGF
	zwA			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSAPGPSAPSHRSLPSRAF
				GEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKAAG
				SDQEPKSSDKTHTCPPCPAPELLGGPSV
				FLFPPKPKDTLMISRTPEVTCVVVDVSH
				EDPEVKFNWYVDGVEVHNAKTKPREEQY
				NSTYRVVSVLTVLHQDWLNGKEYKCKVS
				NKALPAPIEKTISKAKGQPREPQVYVYP
				PSREEMTKNQVSLTCLVKGFYPSDIAVE
				WESNGQPENNYKTTPPVLDSDGSFALVS
				KLTVDKSRWQQGNVFSCSVMHEALHNHY
				TQKSLSLSPGK
392	1449-G09.2 scFv-Fc	scFv-Fc		MQVQLVESGGGVVQPGRSLRLSCAASGF
	zwB			TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSAPGPSAPSHRSLPSRAF
				GEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQQKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKAAG
				SDQEPKSSDKTHTCPPCPAPELLGGPSV
				FLFPPKPKDTLMISRTPEVTCVVVDVSH
				EDPEVKFNWYVDGVEVHNAKTKPREEQY
				NSTYRVVSVLTVLHQDWLNGKEYKCKVS
				NKALPAPIEKTISKAKGQPREPQVYVLP
				PSREEMTKNQVSLLCLVKGFYPSDIAVE
				WESNGQPENNYLTWPPVLDSDGSFFLYS
				KLTVDKSRWQQGNVFSCSVMHEALHNHY
				TQKSLSLSPGK
393	1449-G09.2 HC zwA	HC		MQVQLVESGGGVVQPGRSLRLSCAASGF
				TFSSYGMHWVRQAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSASTKGPSVFPLAPSSKS
				TSGGTAALGCLVKDYFPEPVTVSWNSGA
				LTSGVHTFPAVLQSSGLYSLSSVVTVPS
				SSLGTQTYICNVNHKPSNTKVDKKVEPK
				SCDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVYPPSREEM
				TKNQVSLTCLVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFALVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK
394	1353-G10 LC1c	LC		MSYELTQPPSVSVSPGQTARITCSGDAL
				PKQYAYWYQQKPGQAPVMVIYKDTERPS
				GIPERFSGSSSGTKVTLTISGVQAEDFA
				TYYCQSADNSITYRVFGGGTKVEIKRTV
				AAPSVAIFPPSDERLKSGTASVVCVLNN
				FYPREAKVQWKVDNALQSGNSQESVTEQ
				DSKDSTYSLSSRLTLSKADYEKHKVYAC
				EVTHQGLSSPVTKSFNRGEC
395	1353-G10 LC1d	LC		MSYELTQPPSVSVSPGQTARITCSGDAL
				PKQYAYWYQRKPGQAPVMVIYKDTERPS
				GIPERFSGSSSGTKVTLTISGVQAEDFA
				TYYCQSADNSITYRVFGGGTKVEIKRTV
				AAPSVFIFPPSDERLKSGTASVVCLLNN
				FYPREAKVQWKVDNALQSGNSKESVTEQ
				DSKDSTYSLSSRLTLSKADYEKHKVYAC
				EVTHQGLSSPVTKSFNRGEC
396	1449-G09.2 LC2d	LC		MEIVLTQSPGTLSLSPGERATLSCRASQ
				SVSSSYLAWYQEKPGQAPRLLIYGASSR
				ATGIPDRFSGSGSGTDFTLTISRLEPED
				FAVYYCQQYGRSPFSFGPGTKVDIKRTV
				AAPSVFIFPPSDEELKSGTASVVCLLNN
				FYPREAKVQWKVDNALQSGNSEESVTEQ
				DSKDSTYSLSSTLELSKADYEKHKVYAC
				EVTHQGLSSPVTKSFNRGEC
397	1353-G10 HC1d zwB	HC		MEVQLVQSGAEVKKPGASVKVSCKASGY
				RFPHYGISWVREAPGQGLEWMGWISAYN
				GNTNYAQKLQGRVTMTTDTSTNTAYMEL
				RSLRSDDTAVYYCARDVDYGTGSGYWGQ
				GTLVTVSSASTKGPSVFPLAPSSKSTSG
				GTAALGCEVTDYFPEPVTVSWNSGALTS
				GVHTFPAVLESSGLYSLSSVVTVPSSSL
				GTQTYICNVNHKPSNTKVDKKVEPKSCD
				KTHTCPPCPAPELLGGPSVFLFPPKPKD
				TLMISRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYRVVSV
				LTVLHQDWLNGKEYKCKVSNKALPAPIE
				KTISKAKGQPREPQVYVLPPSREEMTKN
				QVSLLCLVKGFYPSDIAVEWESNGQPEN
				NYLTWPPVLDSDGSFFLYSKLTVDKSRW
				QQGNVFSCSVMHEALHNHYTQKSLSLSP
				GK
398	1449-G09.2 HC2d	HC		MQVQLVESGGGVVQPGRSLRLSCAASGF
	zwA			TFSSYGMHWVRRAPGKGLEWVAVIWYDG
				SYKYYADSVKGRFTISRDNSKNTLYLQM
				NSLRAEDTAVYYCAREEAPENWDYALDV
				WGQGTTVTVSSASTKGPSVFPLAPSSKS
				TSGGTAALGCLVKDYFPEPVTVSWNSGA
				LTSGVRTFPAVLKSSGLYSLSSVVTVPS
				SSLGTQTYICNVNHKPSNTKVDKKVEPK
				SCDKTHTCPPCPAPELLGGPSVFLFPPK
				PKDTLMISRTPEVTCVVVDVSHEDPEVK
				FNWYVDGVEVHNAKTKPREEQYNSTYRV
				VSVLTVLHQDWLNGKEYKCKVSNKALPA
				PIEKTISKAKGQPREPQVYVYPPSREEM
				TKNQVSLTCLVKGFYPSDIAVEWESNGQ
				PENNYKTTPPVLDSDGSFALVSKLTVDK
				SRWQQGNVFSCSVMHEALHNHYTQKSLS
				LSPGK

EQUIVALENTS

The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

Claims

1. A bi-specific antibody or antigen-binding construct, comprising a first binding domain that specifically binds to LAG3, and a second binding domain that specifically binds to PD-1, wherein: i. the first binding domain comprises a CDR-H3 sequence selected from SEQ ID NOs: 110-129; andii. the second binding domain comprises a CDR-H3 sequence selected from the group consisting of: SEQ ID NOs: 130-131 and the CDR-H3 sequences of the VH sequences SEQ ID NOs: 212-250.

2. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a Chothia CDR-H2 sequence selected from SEQ ID NOs: 66-85.

3. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a Chothia CDR-H1 sequence selected from SEQ ID NOs: 4-23.

4. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a Kabat CDR-H2 sequence selected from SEQ ID NOs: 88-107.

5. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a Kabat CDR-H1 sequence selected from SEQ ID NOs: 39-58.

6. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a CDR-L3 sequence selected from SEQ ID NOs: 173-188.

7. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a CDR-L2 sequence selected from SEQ ID NOs: 150-165.

8. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a CDR-L1 sequence selected from SEQ ID NOs: 132-147.

9. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises: i. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 7 and 42; a CDR-H2 comprising one or more of SEQ ID NOs: 69 and 91; and a CDR-H3 comprising SEQ ID NO: 113;ii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 4 and 39; a CDR-H2 comprising one or more of SEQ ID NOs: 66 and 88; and a CDR-H3 comprising SEQ ID NO: 110;iii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 5 and 40; a CDR-H2 comprising one or more of SEQ ID NOs: 67 and 89; and a CDR-H3 comprising SEQ ID NO: 111;iv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 6 and 41; a CDR-H2 comprising one or more of SEQ ID NOs: 68 and 90; and a CDR-H3 comprising SEQ ID NO: 112;v. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 8 and 43; a CDR-H2 comprising one or more of SEQ ID NOs: 70 and 92; and a CDR-H3 comprising SEQ ID NO: 114;vi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 9 and 44; a CDR-H2 comprising one or more of SEQ ID NOs: 71 and 93; and a CDR-H3 comprising SEQ ID NO: 115;vii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 10 and 45; a CDR-H2 comprising one or more of SEQ ID NOs: 72 and 94; and a CDR-H3 comprising SEQ ID NO: 116;viii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 11 and 46; a CDR-H2 comprising one or more of SEQ ID NOs: 73 and 95; and a CDR-H3 comprising SEQ ID NO: 117;ix. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 12 and 47; a CDR-H2 comprising one or more of SEQ ID NOs: 74 and 96; and a CDR-H3 comprising SEQ ID NO: 118;x. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 13 and 48; a CDR-H2 comprising one or more of SEQ ID NOs: 75 and 97; and a CDR-H3 comprising SEQ ID NO: 119;xi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 14 and 49; a CDR-H2 comprising one or more of SEQ ID NOs: 76 and 98; and a CDR-H3 comprising SEQ ID NO: 120;xii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 15 and 50; a CDR-H2 comprising one or more of SEQ ID NOs: 77 and 99; and a CDR-H3 comprising SEQ ID NO: 121;xiii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 16 and 51; a CDR-H2 comprising one or more of SEQ ID NOs: 78 and 100; and a CDR-H3 comprising SEQ ID NO: 122;xiv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 17 and 52; a CDR-H2 comprising one or more of SEQ ID NOs: 79 and 101; and a CDR-H3 comprising SEQ ID NO: 123;xv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 18 and 53; a CDR-H2 comprising one or more of SEQ ID NOs: 80 and 102; and a CDR-H3 comprising SEQ ID NO: 124;xvi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 19 and 54; a CDR-H2 comprising one or more of SEQ ID NOs: 81 and 103; and a CDR-H3 comprising SEQ ID NO: 125;xvii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 20 and 55; a CDR-H2 comprising one or more of SEQ ID NOs: 82 and 104; and a CDR-H3 comprising SEQ ID NO: 126;xviii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 21 and 56; a CDR-H2 comprising one or more of SEQ ID NOs: 83 and 105; and a CDR-H3 comprising SEQ ID NO: 127;xix. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 22 and 57; a CDR-H2 comprising one or more of SEQ ID NOs: 84 and 106; and a CDR-H3 comprising SEQ ID NO: 128; orxx. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 23 and 58; a CDR-H2 comprising one or more of SEQ ID NOs: 85 and 107; and a CDR-H3 comprising SEQ ID NO: 129.

10. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a VH selected from SEQ ID NOs: 191-210.

11. The bi-specific antibody or antigen binding construct of claim 9, wherein the first binding domain comprises: i. a VL comprising: a CDR-L1 comprising SEQ ID NO: 135; a CDR-L2 comprising SEQ ID NO: 153; and a CDR-L3 comprising SEQ ID NO: 176;ii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 132; a CDR-L2 comprising SEQ ID NO: 150; and a CDR-L3 comprising SEQ ID NO: 173;iii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 133; a CDR-L2 comprising SEQ ID NO: 151; and a CDR-L3 comprising SEQ ID NO: 174;iv. a VL comprising: a CDR-L1 comprising SEQ ID NO: 134; a CDR-L2 comprising SEQ ID NO: 152; and a CDR-L3 comprising SEQ ID NO: 175;v. a VL comprising: a CDR-L1 comprising SEQ ID NO: 136; a CDR-L2 comprising SEQ ID NO: 154; and a CDR-L3 comprising SEQ ID NO: 177;vi. a VL comprising: a CDR-L1 comprising SEQ ID NO: 137; a CDR-L2 comprising SEQ ID NO: 155; and a CDR-L3 comprising SEQ ID NO: 178;vii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 138; a CDR-L2 comprising SEQ ID NO: 156; and a CDR-L3 comprising SEQ ID NO: 179;viii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 139; a CDR-L2 comprising SEQ ID NO: 157; and a CDR-L3 comprising SEQ ID NO: 180;ix. a VL comprising: a CDR-L1 comprising SEQ ID NO: 140; a CDR-L2 comprising SEQ ID NO: 158; and a CDR-L3 comprising SEQ ID NO: 181;x. a VL comprising: a CDR-L1 comprising SEQ ID NO: 141; a CDR-L2 comprising SEQ ID NO: 159; and a CDR-L3 comprising SEQ ID NO: 182;xi. a VL comprising: a CDR-L1 comprising SEQ ID NO: 142; a CDR-L2 comprising SEQ ID NO: 160; and a CDR-L3 comprising SEQ ID NO: 183;xii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 143; a CDR-L2 comprising SEQ ID NO: 161; and a CDR-L3 comprising SEQ ID NO: 184;xiii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 144; a CDR-L2 comprising SEQ ID NO: 162; and a CDR-L3 comprising SEQ ID NO: 185;xiv. a VL comprising: a CDR-L1 comprising SEQ ID NO: 145; a CDR-L2 comprising SEQ ID NO: 163; and a CDR-L3 comprising SEQ ID NO: 186;xv. a VL comprising: a CDR-L1 comprising SEQ ID NO: 146; a CDR-L2 comprising SEQ ID NO: 164; and a CDR-L3 comprising SEQ ID NO: 187; orxvi. a VL comprising: a CDR-L1 comprising SEQ ID NO: 147; a CDR-L2 comprising SEQ ID NO: 165; and a CDR-L3 comprising SEQ ID NO: 188.

12. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises a VL sequence selected from SEQ ID NOs: 251-266.

13. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain comprises: i. the VH region SEQ ID NO: 200, or a variant thereof, and the VL region SEQ ID NO: 254, or a variant thereof;ii. the VH region is SEQ ID NO: 191, or a variant thereof, and the VL region is SEQ ID NO: 251, or a variant thereof;iii. the VH region is SEQ ID NO: 192, or a variant thereof, and the VL region is SEQ ID NO: 261, or a variant thereof;iv. the VH region is SEQ ID NO: 193, or a variant thereof, and the VL region is SEQ ID NO: 261, or a variant thereof;v. the VH region is SEQ ID NO: 194, or a variant thereof, and the VL region is SEQ ID NO: 261, or a variant thereof;vi. the VH region is SEQ ID NO: 195, or a variant thereof, and the VL region is SEQ ID NO: 261, or a variant thereof;vii. the VH region is SEQ ID NO: 196, or a variant thereof, and the VL region is SEQ ID NO: 261, or a variant thereof;viii. the VH region is SEQ ID NO: 197, or a variant thereof, and the VL region is SEQ ID NO: 256, or a variant thereof;ix. the VH region is SEQ ID NO: 198, or a variant thereof, and the VL region is SEQ ID NO: 253, or a variant thereof;x. the VH region is SEQ ID NO: 199, or a variant thereof, and the VL region is SEQ ID NO: 255, or a variant thereof;xi. the VH region is SEQ ID NO: 201, or a variant thereof, and the VL region is SEQ ID NO: 252, or a variant thereof;xii. the VH region is SEQ ID NO: 202, or a variant thereof, and the VL region is SEQ ID NO: 259, or a variant thereof;xiii. the VH region is SEQ ID NO: 203, or a variant thereof, and the VL region is SEQ ID NO: 260, or a variant thereof;xiv. the VH region is SEQ ID NO: 204, or a variant thereof, and the VL region is SEQ ID NO: 258, or a variant thereof;xv. the VH region is SEQ ID NO: 205, or a variant thereof, and the VL region is SEQ ID NO: 257, or a variant thereof;xvi. the VH region is SEQ ID NO: 206, or a variant thereof, and the VL region is SEQ ID NO: 262, or a variant thereof;xvii. the VH region is SEQ ID NO: 207, or a variant thereof, and the VL region is SEQ ID NO: 263, or a variant thereof;xviii. the VH region is SEQ ID NO: 208, or a variant thereof, and the VL region is SEQ ID NO: 265, or a variant thereof;xix. the VH region is SEQ ID NO: 209, or a variant thereof, and the VL region is SEQ ID NO: 264, or a variant thereof; orxx. the VH region is SEQ ID NO: 210, or a variant thereof, and the VL region is SEQ ID NO: 266, or a variant thereof.

14.-21. (canceled)

22. The bi-specific antibody or antigen binding construct of claim 13, wherein the second binding domain comprises: i. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 38 and 65; a CDR-H2 comprising one or more of SEQ ID NOs: 87 and 109; and a CDR-H3 comprising SEQ ID NO: 131;ii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 24 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;iii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 25 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;iv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 26 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;v. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 27 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;vi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 28 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;vii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 29 and 60; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;viii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 30 and 61; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;ix. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 24 and 62; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;x. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 24 and 63; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 31 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 32 and 59; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xiii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 33 and 60; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xiv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 34 and 60; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xv. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 35 and 64; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xvi. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 36 and 60; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xvii. a VH comprising: a CDR-H1 comprising one or more of SEQ ID NOs: 37 and 64; a CDR-H2 comprising one or more of SEQ ID NOs: 86 and 108; and a CDR-H3 comprising SEQ ID NO: 130;xviii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 228; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 228; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 228;xix. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 229; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 229; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 229;xx. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 230; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 230; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 230;xxi. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 231; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 231; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 231;xxii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 232; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 232; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 232;xxiii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 233; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 233; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 233;xxiv. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 234; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 234; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 234;xxv. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 235; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 235; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 235;xxvi. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 236; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 236; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 236;xxvii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 237; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 237; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 237;xxviii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 238; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 238; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 238;xxix. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 239; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 239; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 239;xxx. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 240; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 240; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 240;xxxi. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 241; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 241; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 241;xxxii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 242; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 242; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 242;xxxiii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 243; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 243; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 243;xxxiv. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 244; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 244; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 244;xxxv. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 245; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 245; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 245;xxxvi. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 246; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 246; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 246;xxxvii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 247; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 247; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 247;xxxviii. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 248; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 248; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 248;xxxix. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 249; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 249; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 249; orxl. a VH comprising: a CDR-H1 comprising a Chothia and/or Kabat CDR-H1 sequence of SEQ ID NO: 250; a CDR-H2 comprising a Chothia and/or Kabat CDR-H2 sequence of SEQ ID NO: 250; and a CDR-H3 comprising a CDR-H3 sequence of SEQ ID NO: 250.

23. The bi-specific antibody or antigen binding construct of claim 1, wherein the second binding domain comprises a VH selected from SEQ ID NOs: 211-250.

24. The bi-specific antibody or antigen binding construct of claim 22, wherein the second binding domain comprises: i. a VL comprising: a CDR-L1 comprising SEQ ID NO: 149; a CDR-L2 comprising SEQ ID NO: 172; and a CDR-L3 comprising SEQ ID NO: 190;ii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 166; and a CDR-L3 comprising SEQ ID NO: 189;iii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 167; and a CDR-L3 comprising SEQ ID NO: 189;iv. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 168; and a CDR-L3 comprising SEQ ID NO: 189;v. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 169; and a CDR-L3 comprising SEQ ID NO: 189;vi. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 170; and a CDR-L3 comprising SEQ ID NO: 189;vii. a VL comprising: a CDR-L1 comprising SEQ ID NO: 148; a CDR-L2 comprising SEQ ID NO: 171; and a CDR-L3 comprising SEQ ID NO: 189;ix. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 277; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 277; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 277;x. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 278; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 278; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 278;xi. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 279; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 279; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 279;xii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 280; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 280; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 280;xiii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 281; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 281; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 281;xiv. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 282; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 282; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 282;xv. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 283; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 283; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 283;xvi. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 284; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 284; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 284;xvii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 285; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 285; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 285;xviii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 286; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 286; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 286;xix. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 287; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 287; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 287;xx. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 288; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 288; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 288;xxi. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 289; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 289; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 289;xxii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 290; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 290; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 290;xxiii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 291; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 291; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 291;xxiv. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 292; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 292; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 292;xxv. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 293; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 293; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 293;xxvi. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 294; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 294; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 294;xxvii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 295; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 295; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 295;xxviii. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 296; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 296; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 296;xxix. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 297; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 297; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 297;xxx. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 298; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 298; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 298; orxxxi. a VL comprising: a CDR-L1 comprising a CDR-L1 sequence of SEQ ID NO: 299; a CDR-L2 comprising a CDR-L2 sequence of SEQ ID NO: 299; and a CDR-L3 comprising a CDR-L3 sequence of SEQ ID NO: 299.

25. The bi-specific antibody or antigen binding construct of claim 13, wherein the second binding domain comprises a VH sequence selected from SEQ ID NOs: 211-227 and a VL sequence selected from SEQ ID NOs: 267-276.

26. The bi-specific antibody or antigen binding construct of claim 25, wherein the second binding domain comprises: i. the VH region is SEQ ID NO: 227 and the VL region is SEQ ID NO: 275;ii. the VH region is SEQ ID NO: 211 and the VL region is SEQ ID NO: 275;iii. the VH region is SEQ ID NO: 227 and the VL region is SEQ ID NO: 267;iv. the VH region is SEQ ID NO: 211 and the VL region is SEQ ID NO: 267;v. the VH region is SEQ ID NO: 228 and the VL region is SEQ ID NO: 277;vi. the VH region is SEQ ID NO: 229 and the VL region is SEQ ID NO: 278;vii. the VH region is SEQ ID NO: 230 and the VL region is SEQ ID NO: 279;viii. the VH region is SEQ ID NO: 231 and the VL region is SEQ ID NO: 280;ix. the VH region is SEQ ID NO: 232 and the VL region is SEQ ID NO: 281;x. the VH region is SEQ ID NO: 233 and the VL region is SEQ ID NO: 282;xi. the VH region is SEQ ID NO: 234 and the VL region is SEQ ID NO: 283;xii. the VH region is SEQ ID NO: 235 and the VL region is SEQ ID NO: 284;xiii. the VH region is SEQ ID NO: 236 and the VL region is SEQ ID NO: 285;xiv. the VH region is SEQ ID NO: 237 and the VL region is SEQ ID NO: 286;xv. the VH region is SEQ ID NO: 238 and the VL region is SEQ ID NO: 287;xvi. the VH region is SEQ ID NO: 239 and the VL region is SEQ ID NO: 288;xvii. the VH region is SEQ ID NO: 240 and the VL region is SEQ ID NO: 289;xviii. the VH region is SEQ ID NO: 241 and the VL region is SEQ ID NO: 290;xix. the VH region is SEQ ID NO: 242 and the VL region is SEQ ID NO: 291;xx. the VH region is SEQ ID NO: 243 and the VL region is SEQ ID NO: 292;xxi. the VH region is SEQ ID NO: 244 and the VL region is SEQ ID NO: 293;xxii. the VH region is SEQ ID NO: 245 and the VL region is SEQ ID NO: 294;xxiii. the VH region is SEQ ID NO: 246 and the VL region is SEQ ID NO: 295;xxiv. the VH region is SEQ ID NO: 247 and the VL region is SEQ ID NO: 296;xxv. the VH region is SEQ ID NO: 248 and the VL region is SEQ ID NO: 297;xxvi. the VH region is SEQ ID NO: 249 and the VL region is SEQ ID NO: 298; orxxvii. the VH region is SEQ ID NO: 250 and the VL region is SEQ ID NO: 299.

27.-28. (canceled)

29. The bi-specific antibody or antigen binding construct of claim 1, wherein the antibody comprises at least one constant region domain.

30. (canceled)

31. The bi-specific antibody or antigen binding construct of claim 1, wherein the bi-specific antibody or antigen binding construct is humanized or human.

32. The bi-specific antibody or antigen binding construct of claim 1, wherein the bi-specific antibody or antigen binding construct is aglycosylated.

33.-39. (canceled)

40. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain has a ka of about 5.02×104 M−1×sec−1 to about 5.31×107 M−1×sec−1 when associating with human LAG3 at a temperature of 25° C.

41.-42. (canceled)

43. The bi-specific antibody or antigen binding construct of claim 1, wherein the first binding domain specifically binds cynomolgus LAG3.

44.-46. (canceled)

47. The bi-specific antibody or antigen binding construct of claim 1, wherein the second binding domain specifically binds one or more of murine PD-1 and cynomolgus PD-1.

48.-54. (canceled)

55. The bi-specific antibody or antigen binding construct of claim 1, comprising: i. a first antigen-binding polypeptide comprising: i. a first VH comprising a sequence selected from SEQ ID NOs: 211-227;ii. a first VL comprising a sequence selected from SEQ ID NOs: 267-276;iii. a linker-hinge sequence comprising SEQ ID NO: 315; andiv. a CH2-CH3 region comprising a sequence selected from SEQ ID NOs: 321-325; andii. a second antigen-binding polypeptide comprising: i. a second VH comprising a sequence selected from SEQ ID NOs: 191-210;ii. a linker hinge sequence comprising SEQ ID NO: 316;iii. a CH2-CH3 region comprising a sequence selected from SEQ ID NOs: 326-330;iv. a second VL comprising a sequence selected from SEQ ID NOs: 251-266; andv. a CL comprising SEQ ID NO: 386.

56.-72. (canceled)

73. An isolated antibody that specifically binds to LAG3, wherein the antibody comprises: i. a VH comprising SEQ ID NO: 200;ii. a CH1 region comprising a sequence selected from SEQ ID NOs: 343-352;iii. a linker sequence comprising SEQ ID NO: 316; andiv. a CH2-CH3 region comprising a sequence selected from SEQ ID NOs: 331-336.

74.-75. (canceled)

76. The antibody of claim 73, wherein the antibody is a monoclonal antibody.

77. The antibody of claim 73, wherein the antibody is an IgA, an IgD, an IgE, an IgG, or an IgM.

78. The antibody of claim 73, wherein the antibody is humanized or human.

79. The antibody of claim 73, wherein the antibody is aglycosylated.

80.-85. (canceled)

86. A kit comprising an antibody of claim 1, and instructions for use of the antibody.

87.-88. (canceled)

89. A polynucleotide encoding an antibody of claim 73.

90. A vector comprising the polynucleotide of claim 89.

91. A recombinant host cell comprising the vector of claim 90.

92.-94. (canceled)

95. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

96. A method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an antibody of claim 1, or a pharmaceutical composition of claim 95.

97. A method of diagnosing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an antibody of claim 1, or a pharmaceutical composition of claim 95.

98. (canceled)