Patent Grant
10975150
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “Sequence Listing.txt,” created May 22, 2017, and is 192 kilobytes in size. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.
Provided herein are antibodies with binding specificity for CD74 and compositions comprising the antibodies, including pharmaceutical compositions, diagnostic compositions and kits. Also provided are methods of using anti-CD74 antibodies for therapeutic and diagnostic purposes.
Human leukocyte antigen (HLA) class II histocompatibility antigen gamma chain (also known as HLA-DR antigens-associated invariant chain or CD74 (Cluster of Differentiation 74)) is a protein that is involved in the formation and transport of major histocompatibility complex (MHC) class II protein. See Claesson et al., Proc. Natl. Acad. Sci. U.S.A., 1983, 80:7395-7399; Kudo et al., Nucleic Acids Res., 1985, 13:8827-8841; and Cresswell, Ann. Rev. Immunol., 1994, 12:259-291.
One function of CD74 is to regulate peptide loading onto MHC class II heterodimers in intracellular compartments, to prevent MHC class II from binding cellular peptides. The full range of functionality of cell surface-expressed CD74 is not yet known. However, studies have demonstrated that CD74 is a receptor for the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). Binding of MIF to CD74 activates downstream signaling through the MAPK and Akt pathways and promotes cell proliferation and survival. See Gore et al., J. Biol. Chem., 2008, 283:2784-2792; and Starlets et al., Blood, 2006, 107:4807-4816.
Upregulation of CD74 expression has been observed in cancers and autoimmune disease (Borghese et al., Exp. Op. Ther. Targets, 2011, 15:237-251), as well as in infection (Hofman et al., Modern Pathology, 2007, 20:974-989) and inflammatory conditions (Vera et al., Exp. Biol. & Med., 2008, 233:620-626). CD74 is known to be expressed at moderate to high levels in multiple myeloma. Burton et al., Clin. Cancer Res., 2004, 10:6606-6611. CD74 expression is also known to be a key factor associated with the progression of pancreatic cancer. Zhang et al., Hepatobiliary Pancreat. Dis. Int., 2014, 13:81-86.
In view of the role of CD74 in multiple disease processes, there is a need for improved methods of modulating the interaction of CD74 with its ligands and the downstream signaling processes activated by CD74. Moreover, given the upregulation of CD74 in several diseases, there is also a need for therapeutics that specifically target cells and tissues overexpressing CD74.
Provided herein are antibodies that selectively bind CD74. In some embodiments, the antibodies bind to more than one isoform of CD74. In some embodiments, the antibodies bind human CD74. In some embodiments, the antibodies also bind homologs of human CD74. In some aspects, the homolog is a cynomolgus monkey homolog.
In some embodiments, the antibodies have higher melting temperatures than other anti-CD74 antibodies. In some aspects, the Tm2 of the antibodies is higher than other anti-CD74 antibodies. The Tm2 represents the melting temperature of the Fab domain of an IgG. A higher Tm2 therefore promotes stability of the antibody binding site. Such improved stability can lead to better stability of the antibody during storage, as well as improved yield during manufacturing.
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, VH, or VL sequence provided in this disclosure, or a variant thereof. In some aspects, the variant is a variant with a conservative amino acid substitution.
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-CD74 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 CD74.
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, an autoimmune disease, an infectious disease, and an inflammatory condition. In some aspects, the cancer is pancreatic cancer or multiple myeloma.
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/or parameters 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 “CD74” and “CD74 antigen” are used interchangeably herein. Unless specified otherwise, the terms include any variants, isoforms and species homologs of human CD74 that are naturally expressed by cells, or that are expressed by cells transfected with a CD74 gene.
At least four human isoforms of CD74 are known to exist, including p43, p41, p35 and p33. Borghese et al., Expert Opin. Ther. Targets, 2011, 15:237-251, incorporated by reference in its entirety. These isoforms result from alternative transcript splicing and two translation start sites.
p43 (also known as CD74 isoform 1, isoform a, or “long”; see UniProt entry P04233-1 and NCBI Reference Sequence NP 001020330, each incorporated by reference in its entirety) contains 296 amino acids, with residues 73-296 forming the extracellular portion. Protein constructs of CD74 having the extracellular part of isoform 1 are herein referred to as “variant 1” or “CD74v1.”
p35 (also known as CD74 isoform 2, isoform b or “short”; see UniProt entry P04233-2 and NCBI Reference Sequence NP 004346, each incorporated by reference in its entirety) lacks residues 209-272 from the extracellular domain due to alternative splicing. Protein constructs of CD74 having the extracellular part of isoform 2 are herein referred to as “variant 2” or “CD74v2.”
p41 and p33 arise from an alternative translation start site (48 nucleotides/16 amino acids downstream) leading to variants lacking the endoplasmic reticulum (ER) retention signal that is present within the eliminated 16 amino acids, but having extracellular domain that is identical to p43 and p35, respectively.
The sequence of another isoform (known as isoform 3 and isoform c), in which residues 148-160 are replaced and residues 161-296 are lacking, is provided in NP 001020329.
The sequences of cynomolgus CD74 homologs are provided in, e.g., NCBI Reference Sequence: XP-001099491.2 and NCBI Reference Sequence: XP-002804624.1.
The term “immunoglobulin” refers to a class of structurally related proteins generally consisting of 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 (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, CH1, CH2, and CH3. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.
The term “antibody” describes a type of immunoglobulin molecule and is used herein in its broadest sense. An antibody specifically includes monoclonal antibodies, polyclonal antibodies, intact antibodies, 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 VH-VL dimer. A “CD74 antibody,” “anti-CD74 antibody,” “CD74 Ab,” “CD74-specific antibody” or “anti-CD74 Ab” is an antibody, as described herein, which binds specifically to the antigen CD74.
The VH and VL 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 VH and VL 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 confer antigen specificity and binding affinity to 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 these two numbering schemes diverge, the numbering scheme is specified as either Kabat or Chothia.
The “EU numbering scheme” or “EU index” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., the EU index 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 (CH1) of the heavy chain. Fab fragments may be generated, for example, 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 pepsin digestion of an intact antibody. The F(ab′) fragments can be dissociated, for example, by treatment with β-mercaptoethanol.
“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). 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-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain is an IgG1 Fc domain (e.g., SEQ ID NO: 289).
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 provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of an antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent.
“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 generally be represented by the dissociation constant (KD). 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.
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 is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.
The term “kd” (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 koff value.
The term “ka” (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 kon value.
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD=kd/ka.
The term “KA” (M−1), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction. KA=ka/kd.
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 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 VH and VL 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 (e.g., CD74). In one exemplary assay, CD74 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. 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 CD74 variants with different point-mutations.
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), or CLUSTALW 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 of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. By way of example, the following groups of amino acids are considered conservative substitutions for one another.
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 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 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 prevent or ameliorate a disease or the progression of the disease, or result in amelioration of symptoms.
As used herein, the term “inhibits growth” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in cell growth when contacted with a CD74 antibody, as compared to the growth of the same cells not in contact with a CD74 antibody. In some embodiments, growth may be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. The decrease in cell growth can occur by a variety of mechanisms, including but not limited to antibody internalization, apoptosis, necrosis, and/or effector function-mediated activity.
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, goats and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an inflammatory disease or condition, or an autoimmune disease or condition, that can be treated with an antibody provided herein. In some embodiments, the subject is a human that has or is suspected to have cancer, an inflammatory disease or condition, or an autoimmune disease or condition.
Provided herein are antibodies that selectively bind human CD74. In some aspects, the antibody selectively binds to human CD74 isoform 1. In some aspects, the antibody selectively binds to human CD74 isoform 2. In some aspects, the antibody may selectively bind to more than one CD74 isoform, for example, both human CD74 isoforms 1 and 2. In some aspects, the antibody may selectively bind to one or more CD74 isoforms with the same extracellular domain as isoforms 1 and 2, such as p41 and p33, respectively.
In some embodiments, the antibody binds to homologs of human CD74. In some aspects, the antibody binds to a homolog of human CD74 from a species selected from monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep. In some aspects, the homolog is a cynomolgus monkey homolog.
In some embodiments, the antibody has one or more CDRs having particular lengths, in terms of the number of amino acid residues. In some aspects, the Chothia CDR-H1 of the antibody is seven residues in length. In some embodiments, the Kabat CDR-H1 of the antibody is five residues in length. In some aspects, the Chothia CDR-H2 of the antibody is six residues in length. In some embodiments, the Kabat CDR-H2 of the antibody is seventeen residues in length. In some aspects, the Chothia/Kabat CDR-H3 of the antibody is eleven residues in length. In some aspects, the Chothia/Kabat CDR-H3 of the antibody is twelve residues in length. In some aspects, the Chothia/Kabat CDR-H3 of the antibody is thirteen residues in length. In some aspects, the Chothia/Kabat CDR-H3 of the antibody is fourteen residues in length. In some aspects, the Chothia/Kabat CDR-H3 of the antibody is not fifteen residues in length.
In some aspects, the Kabat/Chothia CDR-L1 of the antibody is eleven residues in length. In some aspects, the Kabat/Chothia CDR-L1 of the antibody is twelve residues in length. In some aspects, the Kabat/Chothia CDR-L2 of the antibody is seven residues in length. In some aspects, the Kabat/Chothia CDR-L3 of the antibody is nine residues in length.
In some embodiments, the antibody comprises a light chain. In some aspects, the light chain is selected from a kappa light chain and a lambda light chain.
In some embodiments, the antibody comprises a heavy chain. In some aspects, the heavy chain is selected from IgA, IgD, IgE, IgG, and IgM. In some aspects, the heavy chain is selected from IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is selected from an Fv fragment, a Fab fragment, a F(ab′)2 fragment, a Fab′ fragment, an scFv (sFv) fragment, and an scFv-Fc fragment.
In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is a chimeric, humanized, or human antibody.
In some embodiments, the 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.
In some embodiments, the antibody is internalized by a cell after binding.
In some embodiments, the antibody inhibits the binding of CD74 to its ligands. In some aspects, the antibody inhibits the binding of CD74 to macrophage migration inhibitory factor (MIF).
The antibodies provided herein may be useful for the treatment of a variety of diseases and conditions, including cancers, autoimmune diseases, infection, and inflammation.
2.1. CDR-H3 Sequences
In some embodiments, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156. 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 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 aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 132. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 133. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 134. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 135. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 136. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 137. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 138. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 139. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 140. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 141. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 142. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 143. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 144. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 145. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 146. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 147. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 148. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 149. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 150. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 151. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 152. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 153. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 154. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 155. In some aspects, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 156.
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-H3 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 157-160. In some aspects, the CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 157. In some aspects, the CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 158. In some aspects, the CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 159. In some aspects, the CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 160.
2.2. VH Sequences Comprising Illustrative CDRs
In some embodiments, the antibody comprises a VH 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.
2.2.1. VH Sequences Comprising Illustrative Kabat CDRs
In some embodiments, the antibody comprises a VH 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 antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 129. In some aspects, the antibody comprises a VH 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 VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 131. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 132. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 133. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 134. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 135. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 136. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 137. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 138. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 139. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 140. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 141. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 142. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 143. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 144. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 145. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 146. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 147. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 148. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 149. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 150. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 151. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 152. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 153. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 154. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 155. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 156.
2.2.1.2. Kabat CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 97-124. In some aspects, the antibody comprises a VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 107. In some aspects, the antibody comprises a VH 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 VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 109. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 110. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 111. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 112. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 113. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 114. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 115. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 116. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 117. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 119. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 120. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 121. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 122. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 123. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 124.
2.2.1.3. Kabat CDR-H1
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 33-60. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 33. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 34. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 35. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 36 In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 37. In some aspects, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 38. In some aspects, the antibody comprises a VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 58. In some aspects, the antibody comprises a VH 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 VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 60.
2.2.1.4. Kabat CDR-H3+Kabat CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156 and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 97-124. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H2 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.1.5. Kabat CDR-H3+Kabat CDR-H1
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156 and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 33-60. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H1 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H1 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.1.6. Kabat CDR-H1+Kabat CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 33-60 and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 97-124. In some aspects, the Kabat CDR-H1 sequence and the Kabat CDR-H2 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1 and Kabat CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.1.7. Kabat CDR-H1+Kabat CDR-H2+Kabat CDR-H3
In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 33-60, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 97-124, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156. In some aspects, the Kabat CDR-H1 sequence, Kabat CDR-H2 sequence, and Kabat CDR-H3 sequence are all from a single illustrative VH 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 VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.1.8. Variants of VH Sequences Comprising Illustrative Kabat CDRs
In some embodiments, the VH 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.
2.2.1.9. Excluded VH Sequences Comprising Kabat CDRs
In some embodiments, the VH sequences provided herein do not comprise certain Kabat CDR-H3, CDR-H2, and/or CDR-H1 sequences.
In some aspects, the Kabat CDR-H3 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 157-160. In some aspects, the Kabat CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 157. In some aspects, the Kabat CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 158. In some aspects, the Kabat CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 159. In some aspects, the Kabat CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 160.
In some aspects, the Kabat CDR-H2 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 125-128. In some aspects, the Kabat CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 125. In some aspects, the Kabat CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 126. In some aspects, the Kabat CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 127. In some aspects, the Kabat CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 128.
In some aspects, the Kabat CDR-H1 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 61-64. In some aspects, the Kabat CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 61. In some aspects, the Kabat CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 62. In some aspects, the Kabat CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 63. In some aspects, the Kabat CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 64.
2.2.2. VH Sequences Comprising Illustrative Chothia CDRs
In some embodiments, the antibody comprises a VH 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 antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 129. In some aspects, the antibody comprises a VH 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 VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 131. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 132. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 133. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 134. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 135. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 136. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 137. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 138. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 139. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 140. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 141. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 142. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 143. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 144. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 145. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 146. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 147. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 148. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 149. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 150. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 151. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 152. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 153. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 154. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 155. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 156.
2.2.2.2. Chothia CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 65-92. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 65. In some aspects, the antibody comprises a VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 85. In some aspects, the antibody comprises a VH 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 VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 87. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 88. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 89. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 90. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 91. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 92.
2.2.2.3. Chothia CDR-H1
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 1-28. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2. In some aspects, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 3. In some aspects, the antibody comprises a VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH 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 VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 23. In some aspects, the antibody comprises a VH 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 VH 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 VH 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 VH 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 VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 28.
2.2.2.4. Chothia CDR-H3+Chothia CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 65-92. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H2 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.2.5. Chothia CDR-H3+Chothia CDR-H1
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156 and a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 1-28. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H1 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H1 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.2.6. Chothia CDR-H1+Chothia CDR-H2
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 1-28 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 65-92. In some aspects, the Chothia CDR-H1 sequence and the Chothia CDR-H2 sequence are both from a single illustrative VH sequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1 and Chothia CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.2.7. Chothia CDR-H1+Chothia CDR-H2+Chothia CDR-H3
In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 1-28, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 65-92, and a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 129-156. In some aspects, the Chothia CDR-H1 sequence, Chothia CDR-H2 sequence, and Chothia CDR-H3 sequence are all from a single illustrative VH 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 VH sequence selected from SEQ ID NOs: 230-251 and 273-280.
2.2.2.8. Variants of VH Sequences Comprising Illustrative Chothia CDRs
In some embodiments, the VH 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.
2.2.2.9. Excluded VH Sequences Comprising Chothia CDRs
In some embodiments, the VH sequences provided herein do not comprise certain Chothia CDR-H3, CDR-H2, and/or CDR-H1 sequences.
In some aspects, the Chothia CDR-H3 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 157-160. In some aspects, the Chothia CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 157. In some aspects, the Chothia CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 158. In some aspects, the Chothia CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 159. In some aspects, the Chothia CDR-H3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 160.
In some aspects, the Chothia CDR-H2 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 93-96. In some aspects, the Chothia CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 93. In some aspects, the Chothia CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 94. In some aspects, the Chothia CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 95. In some aspects, the Chothia CDR-H2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 96.
In some aspects, the Chothia CDR-H1 sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 29-32. In some aspects, the Chothia CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 29. In some aspects, the Chothia CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 30. In some aspects, the Chothia CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 31. In some aspects, the Chothia CDR-H1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 32.
2.3. VH Sequences
In some embodiments, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 230-251 and 273-280. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 230. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 231. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 232. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 233. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 234. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 235. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 236. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 237. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 238. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 239. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 240. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 241. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 242. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 243. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 244. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 245. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 246. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 247. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 248. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 249. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 250. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 251. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 273. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 274. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 275. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 276. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 277. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 278. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 279. In some aspects, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 280.
2.3.1. Variants of VH Sequences
In some embodiments, the VH sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative VH sequence provided in this disclosure.
In some aspects, the VH sequence comprises, consists of, or consists essentially of a variant of an illustrative VH sequence provided in this disclosure. In some aspects, the VH 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 VH sequences provided in this disclosure. In some aspects, the VH sequence comprises, consists of, or consists essentially of any of the illustrative VH sequences provided in this disclosure, with 1-25 amino acid substitutions, 1-20 amino acid substitutions, 1-15 amino acid substitutions, 1-10 amino acid substitutions, 1-5 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution. In some aspects, the amino acid substitutions are conservative amino acid substitutions.
2.3.2. Excluded VH Sequences
In some embodiments, the VH sequences provided herein do not comprise certain VH sequences.
In some aspects, the VH sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 252-255. In some aspects, the VH sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 252. In some aspects, the VH sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 253. In some aspects, the VH sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 254. In some aspects, the VH sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 255.
2.4. CDR-L3 Sequences
In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 201-219. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 201. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 202. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 203. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 204. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 205. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 206. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 207. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 208. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 209. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 210. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 211. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 212. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 213. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 214. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 215. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 216. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 217. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 218. In some aspects, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 219.
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-L3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 220.
2.5. VL Sequences Comprising Illustrative CDRs
In some embodiments, the antibody comprises a VL 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 antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 201-219. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 201. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 202. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 203. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 204. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 205. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 206. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 207. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 208. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 209. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 210. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 211. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 212. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 213. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 214. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 215. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 216. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 217. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 218. In some aspects, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 219.
2.5.2. CDR-L2
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 181-199. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 181. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 182. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 183. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 184. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 185. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 186. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 187. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 188. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 189. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 190. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 191. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 192. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 193. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 194. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 195. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 196. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 197. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 198. In some aspects, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 199.
2.5.3. CDR-L1
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 161-179. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 161. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 162. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 163. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 164. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 165. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 166. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 167. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 168. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 169. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 170. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 171. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 172. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 173. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 174. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 175. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 176. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 177. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 178. In some aspects, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 179.
2.5.4. CDR-L3+CDR-L2
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 201-219 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 181-199. In some aspects, the CDR-L3 sequence and the CDR-L2 sequence are both from a single illustrative VL sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L2 are both from a single illustrative VL sequence selected from SEQ ID NOs: 256-270 and 281-288.
2.5.5. CDR-L3+CDR-L1
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 201-219 and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 161-179. In some aspects, the CDR-L3 sequence and the CDR-L1 sequence are both from a single illustrative VL sequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L1 are both from a single illustrative VL sequence selected from SEQ ID NOs: 256-270 and 281-288.
2.5.6. CDR-L1+CDR-L2
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 161-179 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 181-199. In some aspects, the CDR-L1 sequence and the CDR-L2 sequence are both from a single illustrative VL sequence provided in this disclosure. For example, in some aspects, the CDR-L1 and CDR-L2 are both from a single illustrative VL sequence selected from SEQ ID NOs: 256-270 and 281-288.
2.5.7. CDR-L1+CDR-L2+CDR-L3
In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 161-179, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 181-199, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative VL sequence provided in this disclosure. For example, in some aspects, the CDR-L1, CDR-L2, and CDR-L3 are all from a single illustrative VL sequence selected from SEQ ID NOs: 256-270 and 281-288.
2.5.8. Variants of VL Sequences Comprising Illustrative CDR-Ls
In some embodiments, the VL 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.
2.5.9. Excluded VL Sequences Comprising CDR-Ls
In some embodiments, the VL sequences provided herein do not comprise certain CDR-L3, CDR-L2, and/or CDR-L1 sequences.
In some aspects, the CDR-L3 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 220.
In some aspects, the CDR-L2 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 200.
In some aspects, the CDR-L1 sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 180.
2.6. VL Sequences
In some embodiments, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 256. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 257. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 258. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 259. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 260. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 261. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 262. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 263. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 264. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 265. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 266. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 267. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 268. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 269. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 270. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 281. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 282. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 283. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 284. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 285. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 286. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 287. In some aspects, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 288.
2.6.1. Variants of VL Sequences
In some embodiments, the VL sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative VL sequence provided in this disclosure.
In some aspects, the VL sequence comprises, consists of, or consists essentially of a variant of an illustrative VL sequence provided in this disclosure. In some aspects, the VL sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.05% identity with any of the illustrative VL sequences provided in this disclosure. In some aspects, the VL sequence comprises, consists of, or consists essentially of any of the illustrative VL sequences provided in this disclosure, with 1-25 amino acid substitutions, 1-20 amino acid substitutions, 1-15 amino acid substitutions, 1-10 amino acid substitutions, 1-5 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution. In some aspects, the amino acid substitutions are conservative amino acid substitutions.
2.6.2. Excluded VL Sequences
In some embodiments, the VL sequences provided herein do not comprise certain VL sequences.
In some aspects, the VL sequence does not comprise, consist of, or consist essentially of a sequence selected from SEQ ID NOs: 271-272. In some aspects, the VL sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 271. In some aspects, the VL sequence does not comprise, consist of, or consist essentially of SEQ ID NO: 272.
2.7. Pairs
2.7.1. CDR-H3-CDR-L3 Pairs
In some embodiments, the antibody comprises a CDR-H3 sequence and a CDR-L3 sequence. In some aspects, the CDR-H3 sequence is part of a VH and the CDR-L3 sequence is part of a VL.
In some aspects, the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 129-156 and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 201-219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 129, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 130, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 131, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 132, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 133, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 134, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 135, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 136, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 137, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 138, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 139, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 140, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 141, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 142, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 143, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 144, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 145, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 146, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 147, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 148, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 149, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 150, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 151, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 152, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 153, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
In some aspects, the CDR-H3 sequence is SEQ ID NO: 154, and the CDR-L3 sequence is selected from SEQ ID NOs: 201-219. In some aspects, the CDR-L3 sequence is SEQ ID NO: 201. In some aspects, the CDR-L3 sequence is SEQ ID NO: 202. In some aspects, the CDR-L3 sequence is SEQ ID NO: 203. In some aspects, the CDR-L3 sequence is SEQ ID NO: 204. In some aspects, the CDR-L3 sequence is SEQ ID NO: 205. In some aspects, the CDR-L3 sequence is SEQ ID NO: 206. In some aspects, the CDR-L3 sequence is SEQ ID NO: 207. In some aspects, the CDR-L3 sequence is SEQ ID NO: 208. In some aspects, the CDR-L3 sequence is SEQ ID NO: 209. In some aspects, the CDR-L3 sequence is SEQ ID NO: 210. In some aspects, the CDR-L3 sequence is SEQ ID NO: 211. In some aspects, the CDR-L3 sequence is SEQ ID NO: 212. In some aspects, the CDR-L3 sequence is SEQ ID NO: 213. In some aspects, the CDR-L3 sequence is SEQ ID NO: 214. In some aspects, the CDR-L3 sequence is SEQ ID NO: 215. In some aspects, the CDR-L3 sequence is SEQ ID NO: 216. In some aspects, the CDR-L3 sequence is SEQ ID NO: 217. In some aspects, the CDR-L3 sequence is SEQ ID NO: 218. In some aspects, the CDR-L3 sequence is SEQ ID NO: 219.
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.
2.7.1.2. Excluded CDR-H3-CDR-L3 Pairs
In some embodiments, the CDR-H3-CDR-L3 pairs provided herein do not comprise certain CDR-H3-CDR-L3 pairs.
In some aspects, the CDR-H3 sequence is not SEQ ID NO: 157, and the CDR-L3 sequence not SEQ ID NO: 220. In some aspects, the CDR-H3 sequence is not SEQ ID NO: 158, and the CDR-L3 sequence not SEQ ID NO: 220. In some aspects, the CDR-H3 sequence is not SEQ ID NO: 159, and the CDR-L3 sequence not SEQ ID NO: 220. In some aspects, the CDR-H3 sequence is not SEQ ID NO: 160, and the CDR-L3 sequence not SEQ ID NO: 220.
2.7.2. VH-VL Pairs
In some embodiments, the antibody comprises a VH sequence and a VL sequence.
In some aspects, the VH sequence is a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 230-251 and 273-280, and the VL sequence is a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 256-270 and 281-288.
In some aspects, the VH sequence is SEQ ID NO: 230, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 231, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 232, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 233, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 234, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 235, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 236, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 237, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 238, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 239, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 240, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 241, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 242, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 243, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 244, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 245, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 246, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 247, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 248, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 249, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 250, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 251, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 273, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 274, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 275, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 276, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 277, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 278, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 279, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
In some aspects, the VH sequence is SEQ ID NO: 280, and the VL sequence is selected from SEQ ID NOs: 256-270 and 281-288. In some aspects, the VL sequence is SEQ ID NO: 256. In some aspects, the VL sequence is SEQ ID NO: 257. In some aspects, the VL sequence is SEQ ID NO: 258. In some aspects, the VL sequence is SEQ ID NO: 259. In some aspects, the VL sequence is SEQ ID NO: 260. In some aspects, the VL sequence is SEQ ID NO: 261. In some aspects, the VL sequence is SEQ ID NO: 262. In some aspects, the VL sequence is SEQ ID NO: 263. In some aspects, the VL sequence is SEQ ID NO: 264. In some aspects, the VL sequence is SEQ ID NO: 265. In some aspects, the VL sequence is SEQ ID NO: 266. In some aspects, the VL sequence is SEQ ID NO: 267. In some aspects, the VL sequence is SEQ ID NO: 268. In some aspects, the VL sequence is SEQ ID NO: 269. In some aspects, the VL sequence is SEQ ID NO: 270. In some aspects, the VL sequence is SEQ ID NO: 281. In some aspects, the VL sequence is SEQ ID NO: 282. In some aspects, the VL sequence is SEQ ID NO: 283. In some aspects, the VL sequence is SEQ ID NO: 284. In some aspects, the VL sequence is SEQ ID NO: 285. In some aspects, the VL sequence is SEQ ID NO: 286. In some aspects, the VL sequence is SEQ ID NO: 287. In some aspects, the VL sequence is SEQ ID NO: 288.
2.7.2.1. Variants of VH-VL Pairs
In some embodiments, the VH-VL pairs provided herein comprise a variant of an illustrative VH and/or VL sequence provided in this disclosure.
In some aspects, the VH sequence comprises, consists of, or consists essentially of a variant of an illustrative VH sequence provided in this disclosure. In some aspects, the VH 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 VH sequences provided in this disclosure. In some aspects, the VH sequence comprises, consists of, or consists essentially of any of the illustrative VH sequences provided in this disclosure, with 1-25 amino acid substitutions, 1-20 amino acid substitutions, 1-15 amino acid substitutions, 1-10 amino acid substitutions, 1-5 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution. In some aspects, the amino acid substitutions are conservative amino acid substitutions.
In some aspects, the VL sequence comprises, consists of, or consists essentially of a variant of an illustrative VL sequence provided in this disclosure. In some aspects, the VL sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.05% identity with any of the illustrative VL sequences provided in this disclosure. In some aspects, the VL sequence comprises, consists of, or consists essentially of any of the illustrative VL sequences provided in this disclosure, with 1-25 amino acid substitutions, 1-20 amino acid substitutions, 1-15 amino acid substitutions, 1-10 amino acid substitutions, 1-5 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution. In some aspects, the amino acid substitutions are conservative amino acid substitutions.
2.7.2.2. Excluded VH-VL Pairs
In some embodiments, the VH-VL pairs provided herein do not comprise certain VH-VL pairs.
In some aspects, the VH sequence is not selected from SEQ ID NOs: 252-255, and the VL sequence is not selected from SEQ ID NOs: 271-272.
In some aspects, the VH sequence is not SEQ ID NO: 252, and the VL sequence is not selected from SEQ ID NO: 271-272. In some aspects, the VL sequence is not SEQ ID NO: 271. In some aspects, the VL sequence is not SEQ ID NO: 272.
In some aspects, the VH sequence is not SEQ ID NO: 253, and the VL sequence is not selected from SEQ ID NO: 271-272. In some aspects, the VL sequence is not SEQ ID NO: 271. In some aspects, the VL sequence is not SEQ ID NO: 272.
In some aspects, the VH sequence is not SEQ ID NO: 254, and the VL sequence is not selected from SEQ ID NO: 271-272. In some aspects, the VL sequence is not SEQ ID NO: 271. In some aspects, the VL sequence is not SEQ ID NO: 272.
In some aspects, the VH sequence is not SEQ ID NO: 255, and the VL sequence is not selected from SEQ ID NO: 271-272. In some aspects, the VL sequence is not SEQ ID NO: 271. In some aspects, the VL sequence is not SEQ ID NO: 272.
2.8. Consensus Sequences
In particular embodiments, provided herein are anti-CD74 antibodies comprising one or more consensus sequences. Each consensus sequences is based, at least in part, on one or more alignments of two or more useful anti-CD74 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-CD74 CDR sequences.
2.8.1. CDR-H3 Consensus Sequences
In some embodiments, the antibody comprises a CDR-H3 sequence defined by the consensus sequence G-G-α3-α4-α5-α6-α7-α8-α9-α10-G-α12-D-V, where: α3 is T, S, Q, M, or A; α4 is R, L, or V; α5 is V, E, A, G, I, D, or M; α6 is R, L, H, G, Q, or T; α7 is G or R; α8 is A, L, E, or G; α9 is V, I, M, F, R, or L; α10 is Y, H, F, or S; and α12 is T, L, H, or N.
In some aspects, if α9 is M, then either α3 is not T, α4 is not L, α5 is not V, α6 is not R, α7 is not G, α8 is not A, α10 is not Y, α12 is not T, or combinations thereof.
In some aspects, α9 is V, I, F, R, or L.
In some aspects, α6 is L, H, G, Q, or T.
In some aspects, α3 is not T. In some aspects, α4 is not L. In some aspects, α5 is not V. In some aspects, α6 is not R. In some aspects, α7 is not G. In some aspects, α8 is not A. In some aspects, α9 is not M. In some aspects, α10 is not Y. In some aspects, α12 is not T.
2.8.2. Chothia CDR-H2 Consensus Sequences
In some embodiments, the antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence β1-β2-D-β4-S-β6, where: β1 is W or S; β2 is Y, D, or H; β4 is G or A; and β6 is N, I, D, H, K, or R.
In some aspects, β1 is W.
In some aspects, β6 is I.
In some aspects, β1 is not S. In some aspects, β2 is not Y. In some aspects, β4 is not G. In some aspects, β6 is not N or I.
2.8.3. Chothia CDR-H1 Consensus Sequences
In some embodiments, the antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-F-δ3-F-δ5-δ6-δ7, where: δ3 is T, N, S, A, or D; δ5 is S, G, D, or A; δ6 is S or D; and δ7 is Y, H, or F.
In some aspects, δ3 is N, S, A, or D.
In some aspects, δ3 is not T. In some aspects, δ5 is not S. In some aspects, δ6 is not S. In some aspects, δ7 is not Y.
2.8.4. Kabat CDR-H2 Consensus Sequences
In some embodiments, the antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence V-γ2-γ3-γ4-D-γ6-S-γ8-γ9-γ10-Y-A-γ13-S-V-K-G, where: γ2 is I, T, or V; γ3 is W or S; γ4 is Y, D, or H; γ6 is G or A; γ8 is N, I, D, H, K, or R; γ9 is K, E, R, S, T, or D; γ10 is Y, I, V, K, or N; and γ13 is D or G.
In some aspects, γ9 is E, R, S, T, or D.
In some aspects, γ2 is not I. In some aspects, γ3 is not S or W. In some aspects, γ4 is not Y. In some aspects, γ6 is not G. In some aspects, γ8 is not N or I. In some aspects, γ9 is not K. In some aspects, γ10 is not Y. In some aspects, γ13 is not D.
In some embodiments, the antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence V-σ2-W-σ4-D-σ6-S-σ8-σ9-σ10-Y-A-σ13-S-V-K-G, where: σ2 is I, T, or V; σ4 is Y, D, or H; σ6 is G or A; σ8 is N, I, D, H, K, or R; σ9 is K, E, R, S, T, or D; σ10 is Y, I, V, K, or N; and σ13 is D or G.
In some aspects, if σ2 is I, then either σ4 is not Y, σ6 is not G, a is not N, σ9 is not K, σ10 is not Y, σ13 is not D, or combinations thereof.
In some aspects, σ2 is not I. In some aspects, σ3 is not S or W. In some aspects, σ4 is not Y. In some aspects, σ6 is not G. In some aspects, σ8 is not N or I. In some aspects, σ9 is not K. In some aspects, σm is not Y. In some aspects, σ13 is not D.
2.8.5. Kabat CDR-H1 Consensus Sequences
In some embodiments, the antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence ε1-ε2-ε3-M-H, where: ε1 is S or D; ε2 is Y, H, or F; and ε3 is G or A.
In some aspects, ε1 is D.
In some aspects, ε1 is not S. In some aspects, ε2 is not Y. In some aspects, ε3 is not A or G.
2.8.6. CDR-L3 Consensus Sequences
In some embodiments, the antibody comprises a CDR-L3 sequence defined by the consensus sequence Q-Θ2-Θ3-Θ4-Θ5-Θ6-P-Θ8-T, where: Θ2 is Q or H; Θ3 is Y, H, Q, or N; Θ4 is N, Y, Q, H, or C; Θ5 is T, S, I, Y, P, L, or A; Θ6 is Y, T, W, or A; and Θ8 is L or P.
In some aspects, Θ5 is T, I, Y, P, L, or A.
In some aspects, Θ2 is not Q. In some aspects, Θ3 is not Y. In some aspects, Θ4 is not N. In some aspects, Θ5 is not S. In some aspects, Θ6 is not Y. In some aspects, Θ8 is not L.
2.8.7. CDR-L2 Consensus Sequences
In some embodiments, the antibody comprises a CDR-L2 sequence defined by the consensus sequence π1-π2-π3-π4-π5-π6-π7, where: π1 is G, A, L, S, or N; π2 is A, S, G, or R; π3 is S, D, T, N, or R; π4 is S, R, Y, Q, or L; π5 is L or R; π6 is Q or A; and π7 is S, T, or I.
In some aspects, π7 is S.
In some aspects, π1 is not A. In some aspects, π2 is not A. In some aspects, π3 is not S. In some aspects, π4 is not S. In some aspects, π5 is not L. In some aspects, π6 is not Q. In some aspects, π7 is not S.
2.8.8. CDR-L1 Consensus Sequences
In some embodiments, the antibody comprises a CDR-L1 sequence defined by the consensus sequence R-A-μ3-Q-μ5-μ6-μ7-μ8-μ9-μ10-μ11-μ12, where: μ3 is S or G; μ5 is G, S, D, or R; μ6 is V, I, or L; μ7 is S, G, F, A, or Y; μ8 is S, R, or G; μ9 is S, I, N, R, or nothing (i.e., not present); μ10 is W, Y, F, E, or D; μ11 is L or V; and μ12 is A, S, or G.
In some aspects, μ7 is G, F, A, or Y.
In some aspects, μ3 is not S. In some aspects, μ5 is not G. In some aspects, μ6 is not I. In some aspects, μ7 is not S. In some aspects, μ8 is not S. In some aspects, μ9 is present. In some aspects, μ10 is not W. In some aspects, μ11 is not L. In some aspects, μ12 is not A.
In some embodiments, the antibody is characterized by particular thermostability parameters. As described in Example 16, the thermostability of an antibody may be characterized by measuring its melting temperatures. The melting temperatures include Tm1 and Tm2. Tm1 represents the melting of the Fc domain of an IgG, while Tm2 represents the melting of the Fab domain of an IgG.
In some embodiments, the Tm2 of the antibody is at least 75° C., 75.5° C., 76° C., 76.5° C., 77° C., 77.5° C., 78° C., 78.5° C., or 79° C. In some embodiments, the Tm2 of the antibody is between about 75° C. and about 80° C. In some embodiments, the Tm2 of the antibody is between about 76° C. and about 79° C. In some embodiments, the Tm2 of the antibody is between about 77° C. and about 78° C. In some aspects, the Tm2s described above are for aglycosylated versions of the antibody.
In some embodiments, the Tm1 of the antibody is between about 59° C. and about 62.2° C. In some embodiments, the Tm1 of the antibody is less than 62.2° C. In some embodiments, the Tm1 of the antibody is less than 61° C. In some embodiments, the Tm1 of the antibody is less than 60° C. In some aspects, the Tm1s described above are for aglycosylated versions of the antibody.
In some embodiments, the affinity of the antibody for CD74, as indicated by KD, 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 is between about 10−7 M and 10−11 M. In some embodiments, the affinity of the antibody is between about 10−7 M and 10−10 M. In some embodiments, the affinity of the antibody is between about 10−7 M and 10−9 M. In some embodiments, the affinity of the antibody is between about 10−7 M and 10−8 M. In some embodiments, the affinity of the antibody is between about 10−8 M and 10−11 M. In some embodiments, the affinity of the antibody is between about 10−9 M and 10−11 M. In some embodiments, the affinity of the antibody is between about 10−10 M and 10−11 M. In some embodiments, the affinity of the antibody is between about 1.08×10−7 M and 9.57×10−10 M. In some embodiments, the affinity of the antibody is 2.52×10−10 M, or less. In some embodiments, the affinity of the antibody is about 2.52×10−10 M. In some embodiments, the affinity of the antibody is about 3.54×10−10 M. In some embodiments, the affinity of the antibody is between about 2.52×10−10 M and about 3.54×10−10 M. In some aspects, the KD is determined at 25° C.
In some embodiments the antibody has a ka of at least about 105 M−1×sec−1. In some embodiments the antibody has a ka of at least about 106 M−1×sec−1. In some embodiments the antibody has a ka of between about 105 M−1×sec−1 and about 106 M−1×sec−1. In some embodiments the antibody has a ka of between about 1.66×105 M−1×sec−1 and about 1.07×106 M−1×sec−1. In some embodiments the antibody has a ka of about 3.09×105 M−1×sec−1, or more. In some embodiments the antibody has a ka of about 3.09×105 M−1×sec−1. In some embodiments the antibody has a ka of about 3.38×105 M−1×sec−1. In some embodiments the antibody has a ka between about 3.09×105 M−1×sec−1 and about 3.38×105 M−1×sec−1. In some aspects, the ka is determined at 25° C.
In some embodiments the antibody has a kd of about 10−4 sec−1 or less. In some embodiments the antibody has a kd of about 10−5 sec−1 or less. In some embodiments the antibody has a kd of between about 10−4 sec−1 and about 10−5 sec−1. In some embodiments the antibody has a kd of between about 2.35×10−4 sec−1 and about 7.10×10−5 sec−1. In some embodiments the antibody has a kd of about 7.77×10−5 sec−1, or less. In some embodiments the antibody has a kd of about 7.77×10−5 sec−1. In some embodiments the antibody has a kd of about 1.20×10−4 sec−1. In some embodiments the antibody has a kd between about 1.20×10−4 sec−1 and about 7.77×10−5 sec−1. In some aspects, the kd is determined at 25° C.
In some embodiments, the antibody has an IC50 in a secondary antibody-drug conjugate (ADC) cell killing assay, as described in Example 5. In some embodiments, the IC50 is from about 0.001 to about 1 nM. In some aspects, the IC50 is from about 0.001 to about 0.5 nM. In some aspects, the IC50 is from about 0.001 to about 0.25 nM. In some aspects, the IC50 is from about 0.001 to about 0.1 nM. In some aspects, the IC50 is from about 0.001 to about 0.05 nM. In some aspects, the IC50 is from about 0.001 to about 0.025 nM. In some aspects, the IC50 is from about 0.001 to about 0.009 nM. In some aspects, the IC50 is from about 0.001 to about 0.005 nM.
In certain embodiments, an antibody 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 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 certain embodiments, amino acid modifications may be introduced into the Fc region of an 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 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 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.
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. 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.
C1q binding assays may also be carried out to confirm that the 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.
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.
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.
8.1. Antigen Preparation
The CD74 antigen to be used for production of antibodies may be intact CD74 or a fragment of CD74. Other forms of CD74 useful for generating antibodies will be apparent to those skilled in the art.
8.2. Monoclonal Antibodies
Monoclonal antibodies may be obtained, for example, using the hybridoma method first described by Kohler et al., Nature, 1975, 256:495-497, and/or by recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567). 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.
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 3rd ed. (1986) Academic Press, San Diego, Calif.
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.
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.
8.3. Humanized Antibodies
Humanized antibodies may be generated by replacing most, or all, of the structural portions of a 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.
8.4. 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. 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). 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). Human antibodies may also be derived from yeast-based libraries (see e.g., U.S. Pat. No. 8,691,730).
The invention also provides isolated nucleic acids encoding anti-CD74 antibodies, 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 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.
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.
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-CD74 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).
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-CD74 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.
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). Protein G is useful for all mouse isotypes and for human γ3 (Guss et al., EMBO J., 1986, 5:1567-1575).
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 CH3 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.
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 2.5-4.5, generally performed at low salt concentrations (e.g., from about 0-0.25 M salt).
The antibodies provided herein can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the antibodies provided herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration.
The methods provided herein encompass administering pharmaceutical compositions comprising at least one antibody provided herein and one or more compatible and pharmaceutically acceptable carriers. In this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” includes a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Martin, E. W., Remington's Pharmaceutical Sciences.
In clinical practice the pharmaceutical compositions or antibodies provided herein may be administered by any route known in the art. In certain embodiments, a pharmaceutical composition or antibody provided herein is administered parenterally.
The compositions for parenteral administration can be emulsions or sterile solutions. Parenteral compositions may include, for example, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate). These compositions can also contain wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, for example using a bacteriological filter, by radiation or by heating. Parenteral compositions can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium.
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.
Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific antibody in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate.
Further encompassed 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.
Further provided are pharmaceutical compositions and dosage forms that comprise one or more excipients that reduce the rate by which an antibody will decompose. Such antibodies, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
10.1. Parenteral Dosage Forms
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.
10.2. 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, stage of the infection and other factors specific to the subject to be treated.
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 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 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 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.25 mg to 2.5 mg, 0.5 mg to 20 mg, 0.5 to 15 mg, 0.5 to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 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 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 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 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
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 upregulation of CD74. Upregulation of CD74 expression has been observed in cancers and autoimmune disease (Borghese et al., Exp. Op. Ther. Targets, 2011, 15:237-251, incorporated by reference in its entirety), as well as in infection (Hofman et al., Modern Pathology, 2007, 20:974-989, incorporated by reference in its entirety) and inflammatory conditions (Vera et al., Exp. Biol. & Med., 2008, 233:620-626, incorporated by reference in its entirety). CD74 is known to be expressed at moderate to high levels in multiple myeloma. Burton et al., Clin. Cancer Res., 2004, 10:6606-6611, incorporated by reference in its entirety. CD74 expression is also known to be a key factor associated with the progression of pancreatic cancer. Zhang et al., Hepatobiliary Pancreat. Dis. Int., 2014, 13:81-86, incorporated by reference in its entirety.
In some embodiments, the antibodies provided herein are used in diagnostic applications. For example, an ant-CD74 antibody may be useful in assays for CD74 protein. In some aspects the antibody can be used to detect the expression of CD74 in various cells and tissues. These assays may be useful, for example, diagnosing cancer, infection and autoimmune disease.
In some diagnostic applications, the 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 of the invention, the anti-CD74 antibody need not be labeled, and the presence thereof can be detected using a labeled antibody which specifically binds to the anti-CD74 antibody.
The antibodies of the invention may be used as affinity purification agents. In this process, the antibodies may be immobilized on a solid phase such a resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the CD74 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 CD74 protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the CD74 protein from the antibody.
In some embodiments, an antibody of the present invention can be provided in 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.
Antibody Fab or scFv libraries were constructed using a standard overlap extension PCR protocol with mutagenic primers targeting CDRs. See Heckman and Pease, Nat. Protoc., 2007, 2:924-932. Selections for novel antibodies were performed using standard ribosome display protocols. See Dreir and Plückthun, Methods Mol. Biol., 2011, Clifton, N.J., 687:283-306. Specifically, scFv- or Fab-based selection formats were performed according to published protocols. See Hanes and Plückthun, Proc. Natl. Acad. Sci. U.S.A., 1997, 94:4937-4942, and Stafford et al., Protein Eng. Des. Sel. PEDS, 2014, 27:97-109. 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.
Libraries of antibody variants isolated by the selections were transformed into E. coli and grown on agar plates with antibiotic (kanamycin). Individual colonies were picked and grown in liquid broth (TB+kanamycin). Overnight cultures were used to prepare glycerol stocks, and for rolling circle amplification (RCA) to amplify the DNA encoding the variant of interest. The variants were then expressed in a cell-free protein synthesis reaction as described in Zawada et al. (Biotechnol. Bioeng., 2011, 108:1570-1578) with the modifications described below.
Cell-free extracts were treated with 50 μM iodoacetamide for 30 minutes at room temperature (RT; 20° C.) and added to a premix containing all other components except for the DNA template from the variant of interest. Cell free reactions, at a final volume of 604, were initiated by addition of 10% (v/v) amplified variant DNA template and incubated at 30° C. for 12 h on a shaker at 650 rpm in 96-well plates. A total of 88 variants (and 12 control wells, including on-plate and externally added controls) were synthesized on each plate, and about 12-14 plates were screened for each selection campaign. The reaction was incubated further at 4° C. for 6 h. The final concentration in the protein synthesis reaction was 40% cell extract which was engineered to also express disulfide bond isomerase (DsbC), 2 mM glutathione disulfide (GSSG), 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 1 mM for tyrosine and phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, Following synthesis, each reaction was diluted 1:50 into PBST (PBS at pH 7.4 with Tween-20+0.2% BSA).
Variants were analyzed using an ELISA-based assay, using 384-well plates prepared by pre-coating with 2 μg/mL hu-CD74 antigen (R&D SYSTEMS catalog #3590-CD-050; GenBank Accession No. NP_004346; GI No. GI:10835071) diluted in sodium bicarbonate buffer, and then blocked with BSA. Automated high-throughput liquid handling systems (Biomek FX® workstations) were programmed to carry out the ELISA steps, namely, addition of 30 μL of diluted cell-free reactions to assay plates, plate washing, addition of secondary antibody (anti-human Fc, HRP conjugated), addition of detection substrate (SuperSignal®, Thermo-Fisher/Pierce) and reading plate signals on a SepctraMax® M5 (Molecular Devices) plate reader. Signals obtained for all variants were graphed, and the top hits were picked on the basis of the signal:noise ratio, or by raw signal intensity, depending on the intensity of background signal. Cells containing about 300-400 of the top hits were selected from the glycerol stocks, and the sequences of the scFvs in the cells were determined using RCA sequencing. The sequences were analyzed to select variants with unique CDRs and minimal mutations in the framework regions. The selected sequences were then subjected to secondary screening, as described in Example 2.
Cells containing the top 88 hits from the primary screening were isolated from glycerol stocks and cultured overnight in liquid broth (TB+kanamycin), in 96-well plates. Plasmid DNA was isolated from the overnight cultures with a QiaPrep 96 Turbo® plasmid preparation kit (Qiagen) according to the manufacturer's instructions. Cell-free reactions were prepared to express the variants as described above, except that plasmid DNA template was added at a final concentration of 7.5 μg/mL of 6×His tagged heavy chain, and 2.5 μg/mL of light chain DNA template. Controls were also expressed in the same plate. After overnight incubation (30° C., 12 hours, followed by 6 hours at 40° C.), the mixture was centrifuged at 5000×g, 4° C. for 10 min. IMAC purification protocols were employed to capture antibody variants from the cell-free supernatant using a semi-automated high throughput batch purification method. Each milliliter of clarified supernatant was mixed in a 96-well deep well plate with 50 μL of IMAC resin (Ni Sepharose® High Performance, GE Healthcare) previously equilibrated in IMAC binding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole) by pipetting up and down for 15 minutes at slow mixing speed. The resin slurry was then transferred to a filter plate, and washed twice with IMAC binding buffer, and finally His-tagged antibody variants were eluted using 200 μL IMAC elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole). Purified variants were buffer exchanged into PBS using a 96-well ZEBA plate (7 kD MWCO, Thermo-Fisher). Because the His tag is C-terminal, this procedure allows isolation of full length protein. To quantitate the variants following purification, purified samples were analyzed using a HT Protein Express® assay on a LabChip GXII® (Perkin Elmer). Samples were prepared for analysis according to the protocol provided by manufacturer, and protein concentrations were calculated by comparison to a trastuzumab standard curve (1000−10 μg/mL) run under same conditions.
All variants that were produced as described in Example 2, and that had measurable protein concentrations, were subject to off-rate ranking using a Biacore® T200 instrument. Anti-human Fc antibody (GE Healthcare) was directly coupled to a CM5 chip using amine coupling. Antibody variants were captured on the anti-human Fc surface, and off-rate was measured by flowing 50 nM huCD74 antigen over the antibodies.
Affinity measurements (KD) were obtained, using the Biacore® T200 instrument, for variants with “slow” off-rates and good cell killing properties (as described in Example 5).
Anti-human Fc IgG (Human Antibody Capture Kit, GE Life Sciences) was immobilized onto a CM5 chip (GE Life Sciences) using amine coupling chemistry (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-human Fc IgG was injected over all 4 flow cells at a concentration of 25 μg/ml in 10 mM sodium acetate, pH 5.0, for 7 min. Ethanolamine (1 M, pH 8.5) was injected for 7 min to block any remaining activated groups. An average of 12,500 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. Kinetic data was collected by injecting test and control antibodies at concentrations of 10, 5 and 2.5 μg/mL for 12 s at a flow rate of 10 μl/min on flow cells 2, 3 and 4 respectively, followed by a buffer wash for 30 s at the same flow rate. Kinetic characterization of antibody samples was carried out with 8 concentrations of a 1:2 dilution series of rhCD74 antigen (R&D Systems) and 2 injections of 0 nM antigen (buffer only). After capturing ligand (antibody) on the anti-human Fc surface, the analyte (antigen) was bound at 50, 25, 12.5, 6.3, 3.1, 1.6, 0.8, 0.4 and 0 nM for 180 seconds, followed by a 900 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 3M MgCl2 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. KD (affinity, nM) was determined as a ratio of the kinetic rate constants calculated from the fits of the association and dissociation phases.
The internalization ability of selected antibodies was evaluated by a secondary antibody-drug conjugate (ADC) cell killing assay on CD74 positive SU-DHL-6 cells. SU-DHL-6 cells were obtained from ATCC (CRL-2959) and maintained in RPMI, high glucose (Cellgro®, Mediatech, Manassas, Va.) supplemented with 20% heat-inactivated fetal bovine serum (HyClone™; Thermo-Scientific, Waltham, Mass.), 2 mM GlutaMAX™ (Invitrogen, Carlsbad, Calif.) and 1× penicillin/streptomycin (Cellgro®, Mediatech, Manassas, Va.). Cells were collected and counted by the Vi-CELL™ cell viability analyzers (Beckman Coulter, Brea, Calif.). A total of 2×104 cells in a volume of 40 μl were seeded in each well of a 96-well half area flat bottom white polystyrene plate. Antibodies were formulated at 4× starting concentration in the cell culture medium and filtered through MultiScreen® HTS 96-well filter plates (Millipore; Billerica, Mass.). 20 μl of the serially diluted antibody (1:3 serial dilution starting from 200 nM) was added into treatment wells and 20 μl of anti-hFc-MMAF (MORADEC) was then added into each well at a fixed final concentration of 5 μg/mL. Assay plates were cultured at 37° C. in a CO2 incubator for 72 hrs before the cell viability assay. For cell viability measurement, 80 μl of CellTiter-Glo® reagent (Promega, Madison, Wis.) was added into each well, and plates were processed as per product instructions. Relative luminescence was measured on an EnVision® plate reader (Perkin Elmer, Waltham, Mass.). Relative luminescence readings were converted to percent viability using untreated cells as controls. Data was fitted with non-linear regression analysis, using log(inhibitor) vs. response-variable slope, four parameter fit equation using GraphPad Prism (GraphPad v 5.0 software, San Diego, Calif.). Data was expressed as relative cell viability (ATP content) percent vs. dose of antibody in nM.
CHO cells were transfected to stably express CD74 from cynomolgus monkey (cyno-CD74; GI #s 544440372, gene; 544440373, protein) on the cell surface. CHO parental and CHO-cyno-CD74 stable cells were washed twice with calcium and magnesium-free Hanks Balanced Salt Solution (HBSS), harvested with HyQTase® (Hyclone, Thermo Scientific, Waltham, Mass.) and suspended in FACS buffer (DPBS buffer supplemented with 1% bovine serum albumin). A total of 200,000 cells per sample were incubated on ice for 60 mins with 100 nM of anti-CD74 antibodies. Cells were washed twice with ice-cold FACS buffer and incubated with 5 μg/ml Alexa Fluor®-647 labeled goat anti-human Fcγ antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) on ice for 60 mins. Unstained cells, human IgG1 (isotype control) and secondary antibody were used as controls. Samples were washed twice using FACS buffer and analyzed using a BD FACSCalibur™ system (BD BioSciences, San Jose, Calif.). Mean fluorescence intensities were plotted using GraphPad Prism (GraphPad v 5.00 software, San Diego, Calif.).
The following eight antibodies exhibited cross-reactivity to cynomolgus monkey CD74:
1. HC 1251_A06/LC 1275_C10
2. HC 1251_B08/LC 1275_C10
3. HC 1251_B09/LC 1275_C10
Table 2 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for four scFv clones selected from a library of scFvs with randomized VH CDRs. The scFvs are based on the VH3-23 heavy chain germline and the Vk3-A27 light chain germline.
Table 3 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for four Fab clones selected from a library of Fabs with randomized heavy chain CDRs. All heavy chains were paired with the light chain from trastuzumab (SEQ ID NO: 290).
Table 4 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for sequences of thirteen heavy chains selected from a library of Fabs with soft randomized CDRs, paired with a light chain having a VL sequence provided in SEQ ID NO: 272. Soft randomization was performed by utilizing a mixture of 70% wild type (i.e., parent) nucleotide and 10% each of the remaining three nucleotides for each position within a codon. The antibodies are based on the VH3-33 heavy chain germline and the Vk1-12 light chain germline.
Table 5 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for four light chains selected from a library of Fabs with soft randomized CDRs, paired with one of two heavy chains (HC 1251-A06 (SEQ ID NO: 310); or HC 1251-F07 (SEQ ID NO: 311)) in Fab format. The antibodies are based on the VH3-33 heavy chain germline and the Vk1-12 light chain germline.
Table 6 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for seven light chains selected from a library of Fabs with randomized light chain CDRs, paired with VH 1251-A06 (SEQ ID NO: 236) in Fab format. The antibodies are based on the VH3-33 heavy chain germline and the Vk1-12 light chain germline.
Table 7 provides secondary ADC data for two heavy chains selected from a library of Fabs generated by trinucleotide-directed mutagenesis (TRIM). See U.S. Pat. No. 5,869,644. The heavy chains were paired with the trastuzumab light chain (SEQ ID NO: 290).
Table 8 provides secondary ADC data for four scFv-Fc clones generated from the soft randomization of 1193-E06b scFvs. See Wang et al. (Cancer Res., 2011, 71:7410-7422) for an example of scFv-Fc constructs.
Table 9 provides a matrix of heavy chain and light chain pairs that were evaluated. In Table 9, “(wt)” indicates that the heavy chain or light chain was used as isolated from the respective library (i.e., without reverting mutated residues to the residues found in the canonical germline gene). In contrast, “(g)” indicates that the heavy chain or light chain sequence has been altered to restore mutated framework residues to the residues most commonly found at the respective position for a particular germline. Mutated framework residues may arise, for example, from errors introduced during amplification of the variable regions during cloning.
The sequence identification numbers in Table 9 refer to the molecules as actually tested—i.e., with an N-terminal methionine (M) residue and, in some cases, a C-terminal tag with the sequence GGSHHHHHH (SEQ ID NO: 303). These sequences are optional. The sequences of the heavy chain constant regions and light chain constant regions used to produce the heavy and light chains are provided in SEQ ID NOs: 304 (HC constant) and 305 (LC constant).
Other heavy chains produced and tested in combination with 1275-C10, 1337-A04, 1337-A-7, 1337-A09, and 1337-A10 light chains include 1251-A03 (SEQ ID NO: 306), 1251-B09 (SEQ ID NO: 307), and 1251-B10 (SEQ ID NO: 308).
Table 10 provides the ka, kd, KD, Rmax, Chi2, and secondary ADC data for the 32 heavy chain-light chain pairs provided in Table 9.
The heavy chain 1251-B08-(g) and light chain 1337-A09-(g) sequences are formatted into an scFv-Fc format. The amino acid sequence of the scFv-Fc is provided in SEQ ID NO: 229. The amino acid sequence of the Fc constant region is provided in SEQ ID NO: 309.
Purified ScFv-Fc is captured on an anti-human Fc surface at a concentration of 10 μg/mL, and different concentrations of antigen (20 nM, 10 nM, 5 nM and 0 nM of human CD74, R&D Systems) are passed over the chip surface to which the ScFv-Fc is bound. Data is fit to a 1:1 binding model using BIACORE evaluation software. Table 11 shows the ka, kd, KD, Rmax, and Chi2 for this scFv-Fc.
This example describes experiments designed to measure the thermal stability (Tm) of aglycosylated anti-CD74 parent antibody and variants. The thermal shift assay was carried out by mixing the protein to be assayed (anti-CD74 parent antibody and variants) 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. The final concentration of the protein in the assay mixture was between 100-250 μg/mL, and the dye was diluted 1:1000 from the original stock (stock dye is 5000× in DMSO). After dispensing 5 μL aliquots of the protein-dye mixture in a 384-well microplate (Bio-Rad Cat #MSP-3852), 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 melting temperatures (Tm1 and Tm2) were determined using the Bio-Rad CFX manager software. Tm1 represents the melting of the Fc domain. Tm2 represents the melting of the Fab domain. The Tm2 for certain illustrative antibodies provided in this disclosure is higher than the Tm2 for the parent antibody.
For protein samples with complex thermal transition profiles, the melting temperatures are calculated from the negative first-order derivative plot of fluorescence intensity (Y-axis) against temperature (X-axis), or by fitting the data to the Boltzmann sigmoidal model. The difference in melting temperatures of anti-CD74 variants compared to the wild-type protein is a measure of the thermal shift for the protein being assayed. The results for these studies are provided in Table 12.
Table 13 provides sequences referred to herein.
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.
This application is a U.S. national phase entry of International Patent Application No. PCT/US2015/041192, filed Jul. 20, 2015, which in turn claims priority to U.S. Provisional Patent Application No. 62/027,637, filed Jul. 22, 2014. Each of the foregoing applications is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/041192 | 7/20/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/014434 | 1/28/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4816567 | Cabilly et al. | Mar 1989 | A |
| 5204244 | Fell et al. | Apr 1993 | A |
| 5229275 | Goroff | Jul 1993 | A |
| 5500362 | Robinson et al. | Mar 1996 | A |
| 5534615 | Baker et al. | Jul 1996 | A |
| 5545807 | Surani et al. | Aug 1996 | A |
| 5565332 | Hoogenboom et al. | Oct 1996 | A |
| 5567610 | Borrebaeck et al. | Oct 1996 | A |
| 5573905 | Lerner et al. | Nov 1996 | A |
| 5585089 | Queen et al. | Dec 1996 | A |
| 5589369 | Seidman et al. | Dec 1996 | A |
| 5591669 | Krimpenfort et al. | Jan 1997 | A |
| 5693761 | Queen et al. | Dec 1997 | A |
| 5693762 | Queen et al. | Dec 1997 | A |
| 5821337 | Carter et al. | Oct 1998 | A |
| 5869644 | Shortie et al. | Feb 1999 | A |
| 6180370 | Queen et al. | Jan 2001 | B1 |
| 8258082 | Ladner | Sep 2012 | B2 |
| 8691730 | Vasquez et al. | Apr 2014 | B2 |
| 20040219203 | Griffiths et al. | Nov 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2005100402 | Oct 2005 | WO |
| WO 2006029879 | Mar 2006 | WO |
| WO 2012104344 | Aug 2012 | WO |
| Entry |
|---|
| Paul, Fundamental Immunology, 3rd Edition, 1993, pp. 292-295. |
| Rudikoff et al, Proc. Natl. Acad. Sci. USA, 79(6):1979-1983, Mar. 1982. |
| Colman P. M., Research in Immunology, 145:33-36, 1994. |
| MacCallunn et al, J. Mol. Biol., 262, 732-745, 1996. |
| Pascalis et al, Journal of Immunology, 2002, vol. 169, pp. 3076-3084. |
| Casset et al, Biochemical and Biophysical Research Communications, 2003, vol. 307, pp. 198-205. |
| Invitation to Pay Additional Fees and, Where Applicable, Protest Fee dated Oct. 28, 2015 for Application No. PCT/US2015/041192, 8 pages. |
| International Search Report and Written Opinion dated Jan. 21, 2016 for Application No. PCT/US2015/041192, 17 pages. |
| Berkova et al., “Milatuzumab—apromising new immunotherapeutic agent”, Expert Opinion on Investigational Drugs, Informa Healthcare, United Kingdom, Jan. 1, 2010, vol. 19, No. 1, pp. 141-149. |
| Borghese et al., “CD74: An emerging opportunity as a therapeutic target in cancer and autoimmune disease”, Expert Opin. Ther. Targets, 2011, vol. 15, No. 3, pp. 237-251. |
| Bruggemann et al., “Comparison of the Effector Functions of Human Immunoglobulins Using a Matched Set of Chimeric Antibodies”, J. Exp. Med., Nov. 1987, vol. 166, pp. 1351-1361. |
| Bruggermann et al., “Designer Mice: The Production of Human Antibody Repertoires in Transgenic Animals”, Year Immunol. Basel, Karger, 1993, vol. 7, pp. 33-40. |
| Burton eta al., “CD74 is expressed by multiple myeloma and is a promising target for therapy”, Clinical Cancer Research, The American Association for Cancer Research, US, Oct. 1, 2004, vol. 10, No. 19, pp. 6606-6611. |
| Carter et al. “High Level Escherichia col/ Expression and Production of a Bivalent Humanized Antibody Fragment”, Biotechnology, Feb. 1992, vol. 10, pp. 163-167. |
| Claesson et al., “cDNA clone for the human invariant γ chain of class II histocompatibility antigens and its implications for the protein structure”, Proc. Natl. Acad. Sci. U.S.A., Dec. 1983, vol. 80, pp. 7395-7399. |
| Clynes et al. “Fc receptors are required in passive and active immunity to melanoma”, Proc. Natl. Acad. Sci. U.S.A., Jan. 1998, vol. 95, pp. 652-656. |
| Cragg et al., “Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents”, Blood, 2004, vol. 103, pp. 2738-2743. |
| Cragg et al., “Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts”, Blood, 2003, vol. 101, pp. 1045-1052. |
| Cresswell, “Assembly, Transport, and Function of MHC Class II Molecules”, Ann. Rev. Immunol., 1994, vol. 12, pp. 259-293. |
| Dreir et al., “Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries”, Methods in Molecular Biology, 2011, vol. 687, pp. 283-306. |
| Gazzano-Santoro et al., “A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody”, Journal of Immunological Methods, 1996, vol. 202, pp. 163-171. |
| Gore et al., “Macrophage Migration Inhibitory Factor Induces B Cell Survival by Activation of a CD74-CD44 Receptor Complex”, Journal of Biological Chemistry, Feb. 1, 2008, vol. 283, No. 5, pp. 2784-2792. |
| Hanes et al., “In vitro selection and evolution of functional proteins by using ribosome display”, Proc. Natl. Acad. Sci. U.S.A., May 1997, vol. 94, pp. 4937-4942. |
| Heckman et al., “Gene splicing and mutagenesis by PCR-driven overlap extension”, Nature Protocols, 2007, vol. 2, No. 4, pp. 924-932. |
| Hellstrom et al., “Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside”, Proc. Natl. Acad. Sci. U.S.A., Mar. 1985, vol. 82, pp. 1499-1502. |
| Hellstrom et al., “Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas”, Proc. Natl. Acad. Sci. U.S.A., Sep. 1986, vol. 83, pp. 7059-7063. |
| Hofman et al., “Gene expression profiling in human gastric mucosa infected with Helicobacter pylori”, Modern Pathology, 2007, vol. 20, pp. 974-989. |
| Hoogenboom et al., By-passing Immunisation Human Antibodies from Synthetic Repertoires of Germline VH Gene Segments Rearranged in Vitro, J. Mol. Biol., 1991, vol. 227, pp. 381-388. |
| Jakobovits et al., “Germ-line transmission and expression of a human-derived yeast artificial chromosome”, Nature, Mar. 18, 1993, vol. 362, pp. 255-258. |
| Jakobovits et al., “Analysis of homozygous mutant chimeric mice: Deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production”, Proc. Natl. Acad. Sci. U.S.A., Mar. 1993, vol. 90, pp. 2551-2555. |
| Kohler et al., “Continuous cultures of fused cells secreting antibody of predefined specificity”, Nature, Aug. 7, 1975, vol. 256, pp. 495-497. |
| Kozbor et al., “A Human Hybrid Myeloma for Production of Human Monoclonal Antibodies”, Journal of Immunology, Dec. 1984, vol. 133, No. 6, pp. 3001-3005. |
| Kudo et al., “Structure of the human gene encoding the Invariant 7-chain of class II histocompatibillty Antigens”, Nucleic Acids Research, 1985, vol. 13, No. 24, pp. 8827-8841. |
| Marks et al., “By-passing Immunization Human Antibodies from V-gene Libraries Displayed on Phage”, J. Mol. Biol., 1991, vol. 222, pp. 581-597. |
| Pawlak-Byczkowska et al., “Two New Monoclonal Antibodies, Epb-1 and Epb-2, Reactive with Human Lymphoma”, Cancer Research, American Association for Cancer Research, US, Aug. 15, 1989, vol. 49, No. 16, pp. 4568-4577. |
| Petkova et al., “Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: Potential application in humorally mediated autoimmune disease”, International Immunology, 2006, vol. 18, No. 12, pp. 1759-1769. |
| Queen et al., “A humanized antibody that binds to the interleukin 2 receptor”, Proc. Natl. Acad. Sci. U.S.A., Dec. 1989, vol. 86, pp. 10029-10033. |
| Rader et al., “A phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries”, Proc. Nat. Acad. Sci. U.S.A., Jul. 1998, vol. 95, pp. 8910-8915. |
| Ravetch et al., “Fc receptors”, Annual Review of Immunology, 1991, vol. 9, pp. 457-492. |
| Stafford et al., “In vitro Fab display: A cell-free system for IgG discovery”, Protein Engineering, Design & Selection, 2014, vol. 27, No. 4, pp. 97-109. |
| Starlets et al., “Cell-surface CD74 initiates a signaling cascade leading to cell proliferation and survival”, Blood, 2006, vol. 107, pp. 4807-4816. |
| Stein et al., “Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell Malignancies”, Blood, Dec. 1, 2004, vol. 104, No. 12, pp. 3705-3711. |
| Steinberger et al., “Generation and Characterization of a Recombinant Human CCR5-specific Antibody”, J. Biol. Chem., Nov. 17, 2000, vol. 275, No. 46, pp. 36073-36078. |
| Vera et al., “Upregulation of Macrophage Migration Inhibitory Factor (MIF) and CD74, Receptor for MIF, in Rat Bladder During Persistent Cyclophosphamide-Induced Inflammation”, Experimental Biology and Medicine, 2008, vol. 233, pp. 620-626. |
| Wang et al. “Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4”, Cancer Res., Dec. 15, 2011, vol. 71, No. 24, pp. 7410-7422. |
| Winter et al., “Man-made antibodies”, Nature, 1991, vol. 349, pp. 293-299. |
| Yin et al., “Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system”, mAbs, Mar./Apr. 2012, vol. 4, No. 2, pp. 217-225. |
| Zawada et al., “Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production A New Approach for Shortening Protein Production Development Timelines”, Biotechnology and Bioengineering, Jul. 2011, vol. 108, No. 7, pp. 1570-1578. |
| Zhang et al., “Effect of CD74 on the prognosis of patients with resectable pancreatic cancer”, Hepatobiliary Pancreat. Dis. Int., Feb. 15, 2014, vol. 13, No. 1, pp. 81-86. |
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|---|---|---|---|
| 20170253656 A1 | Sep 2017 | US |
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| 62027637 | Jul 2014 | US |
