Patent Application
20230242628
Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 979,248 Byte ASCII (Text) file named “Sequence_Listing_ST25.txt,” created on Nov. 28, 2022.
The field of the invention generally relates to medicine, infectious disease and in particular anti-HIV monoclonal antibodies which have enhanced therapeutic neutralizing activity and potency for treating or preventing HIV infection in a mammalian subject.
HIV is an integrating retrovirus that rapidly establishes chronic infection in CD+4 T cells with subsequent depletion of the T cell arm of immunity. This fundamental characteristic means that prevention of HIV infection largely depends on humoral responses and associated effector mechanisms directed against HIV envelope proteins (gp120 and gp41) that drive viral attachment and entry. Humoral anti-envelope responses in a minority of HIV-infected persons comprise neutralizing activity against diverse viral variants. It is widely held that these broadly neutralizing responses can be used to guide the development of effective HIV vaccines and/or other immune-based prevention measures.
We have previously found that multiple HIV-infected subjects harbor broad and potent neutralizing activities with highly shared biochemical determinants. These broadly neutralizing antibodies were isolated from two test subjects, N60 and N49. The broadly neutralizing antibodies in N49 plasma fell into two lineages distinguished by different light chain gene usage. The N49 mAbs all exhibited basic pIs and VH1-2 gene usage. However, all of the N49 mAbs used gamma light chain genes, while also containing deletions in CDRL1 and CDRL3. The binding characteristics of this N49 lineage also matched N60 neutralizing antibodies, reflecting anti-CD4BS specificity. Members of the N49P series of broadly neutralizing antibodies are the broadest and most potent naturally occurring anti-gp120 antibodies described to date capable of neutralizing viruses that are missed by other broadly neutralizing mAbs (including N6, DH411-2 and 10E8).
This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.
It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary, and thus do not restrict the scope of the embodiments.
In one aspect, the invention provides an anti-HIV antibody that is derived from a N49P series antibody, wherein the N49P series antibody is modified whereby a part or all of the framework 3 region of the heavy chain is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542). In some embodiments, the framework 3 region in the N49P series antibody is fully deleted or missing, and in those cases either SEQ ID NO: 541 or 542 is inserted therein.
In some embodiments, the N49P series of antibodies to be modified are selected from the natural antibody sequences 1-38 as shown in Table 1 below. In some embodiments, the N49P series of antibodies to be modified comprises variants of these natural antibodies. In some embodiments, the variants that can be further modified are selected from antibodies N49P6, N49P6.2, N49P7, N49P7.1, N49P7A, N49P7S, N49P7F, N49P7Y, N49P7.54TY, N49P7-LS1, N49P7-LS2, N49P7/6L, N49P7/11L, R49P7,N49P7.2, N49P11, N49P18, N49P18.2, N49P18.1, N49P19, N49P37, N49P38, N49P38.1, N49P55, N49P56, N49P57, N49P58, N49P59, N49P73, N49P74, N49P75, N49P9, N49P9.1, N49P9.2, N49P9i7, N49P22, N49P23, N49P9.3, N49P9.4, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P51, N49P52, N49P53, N49P54, N49P60, N49P61, N49P62, N49P63, N49P64, N49P65, N49P66, N49P67, N49P68, N49P69, N49P70, N49P71, and N49P72. These variants are described in Table 3, below.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody comprises the VH and VL regions of antibody N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P7-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P7-FR is SEQ ID NO:501 and the nucleotide sequence is SEQ ID NO:502. The amino acid sequence of the light chain of N49P7-FR is SEQ ID NO:503 and the nucleotide sequence is SEQ ID NO:504.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9-FR is SEQ ID NO:505 and the nucleotide sequence is SEQ ID NO:506. The amino acid sequence of the light chain of N49P9-FR is SEQ ID NO:295 and the nucleotide sequence is SEQ ID NO:296.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.3-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.3-FR is SEQ ID NO:507 and the nucleotide sequence is SEQ ID NO:508. The amino acid sequence of the light chain of N49P9.3-FR is SEQ ID NO:327 and the nucleotide sequence is SEQ ID NO:328.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR is SEQ ID NO:509 and the nucleotide sequence is SEQ ID NO:510. The amino acid sequence of the light chain of N49P9.6-FR is SEQ ID NO:511 and the nucleotide sequence is SEQ ID NO:512.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR-54W or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-54W is SEQ ID NO:513 and the nucleotide sequence is SEQ ID NO:514. The amino acid sequence of the light chain of N49P9.6-FR-54W is SEQ ID NO:515 and the nucleotide sequence is SEQ ID NO:516.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR-54F or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-54F is SEQ ID NO:517 and the nucleotide sequence is SEQ ID NO:518. The amino acid sequence of the light chain of N49P9.6-FR-54F is SEQ ID NO:519 and the nucleotide sequence is SEQ ID NO:520.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR3-06 or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR3-06 is SEQ ID NO:521 and the nucleotide sequence is SEQ ID NO:522. The amino acid sequence of the light chain of N49P9.6-FR3-06 is SEQ ID NO:523 and the nucleotide sequence is SEQ ID NO:524.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR1-D or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR1-D is SEQ ID NO:525 and the nucleotide sequence is SEQ ID NO:526. The amino acid sequence of the light chain of N49P9.6-FR1-D is SEQ ID NO:527 and the nucleotide sequence is SEQ ID NO:528.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR1-D-I or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR1-D-I is SEQ ID NO:529 and the nucleotide sequence is SEQ ID NO:530. The amino acid sequence of the light chain of N49P9.6-FR1-D-I is SEQ ID NO:531 and the nucleotide sequence is SEQ ID NO:532.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR-LS or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-LS is SEQ ID NO:533 and the nucleotide sequence is SEQ ID NO:534. The amino acid sequence of the light chain of N49P9.6-FR-LS is SEQ ID NO:535 and the nucleotide sequence is SEQ ID NO:536.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody is N49P9.6-FR-YTE or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-YTE is SEQ ID NO:537 and the nucleotide sequence is SEQ ID NO:538. The amino acid sequence of the light chain of N49P9.6-FR-YTE is SEQ ID NO:539 and the nucleotide sequence is SEQ ID NO:540.
In another aspect, the invention provides an anti-HIV antibody, wherein the antibody comprises the heavy chain CDR and light chain CDR sequences of the antibodies N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE, wherein the antibody comprises a framework 3 region of the heavy chain comprising an amino acid sequence selected from QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDIRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody neutralizes at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the HIV pseudoviruses listed Table 4 (see also
In some embodiments, the anti-HIV antibody neutralizes at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the HIV pseudoviruses listed in Table 4 with an IC50 value of less than about 1 μg/ml, between about 1-5 μg/ml or greater than about 5 μg/ml.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
From the N49P series of mAbs there is a need to develop the most broadly neutralizing and potent antibodies for clinical/therapeutic applications. Further modifications to certain of the N49P series of mAbs have been made and are herein described in detail and form the basis of this invention and the next generation of broadly neutralizing anti-HIV monoclonal antibodies.
Reference will now be made in detail to the presently preferred embodiments of the invention which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments describe in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols in Molecular Biology (F. M. Ausubel et al. eds. (1987)); the series Methods in Enzymology (Academic Press, Inc.); PCR: A Practical Approach (M. MacPherson et al. IRL Press at Oxford University Press (1991)); PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Antibodies, A Laboratory Manual (Harlow and Lane eds. (1988)); Using Antibodies, A Laboratory Manual (Harlow and Lane eds. (1999)); and Animal Cell Culture (R. I. Freshney ed. (1987)). Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).
For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of “or” means “and/or” unless stated otherwise. As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.”
Abbreviations for amino acids are used throughout this disclosure and follow the standard nomenclature known in the art. For example, as would be understood by those of ordinary skill in the art, Alanine is Ala or A; Arginine is Arg or R; Asparagine is Asn or N; Aspartic Acid is Asp or D; Cysteine is Cys or C; Glutamic acid is Glu or E; Glutamine is Gln or Q; Glycine is Gly or G; Histidine is His or H; Isoleucine is Ile or I; Leucine is Leu or L; Lysine is Lys or K; Methionine is Met or M; Phenylalanine is Phe or F; Proline is Pro or P; Serine is Ser or S; Threonine is Thr or T; Tryptophan is Trp or W; Tyrosine is Tyr or Y; and Valine is Val or V.
As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used.
The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments, dual affinity retargeting antibodies (DART)), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific and trispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
In some embodiments, an antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ, and ε isotypes. Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.
The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ) respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
The terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
The term “antigen binding fragment” or antibody fragment refers to a portion of an intact antibody and comprises the antigenic determining variable regions of an intact antibody. Examples of antigen binding fragment include, but are not limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.
A “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
The term “humanized antibody” refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539 or 5,639,641.
A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)).
The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., The Immunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique numbering is used in 2D graphical representations, designated as IMGT Colliers de Perles (Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)), and in 3D structures in IMGT/3Dstructure-DB (Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)).
In some embodiments, CDRs are determined based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)). In some embodiments, CDRs are determined based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches can be used to determine CDRs. In some embodiments, the CDRs are determined based on AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). In some embodiments, CDRs are determined based on the IMGT system.
The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
A “neutralizing antibody” may inhibit the entry of HIV-1 virus for example SF162 and/or JR-CSF with a neutralization index >1.5 or >2.0. (Kostrikis L G et al. J Virol. 1996; 70(1): 445-458.). By “broad and potent neutralizing antibodies” are meant antibodies that neutralize more than one HIV-1 virus species (from diverse clades and different strains within a clade) in a neutralization assay. A broad neutralizing antibody may neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains of HIV-1, the strains belonging to the same or different clades. A broad neutralizing antibody may neutralize multiple HIV-1 species belonging to at least 2, 3, 4, 5, or 6 different clades. In some embodiments, the \concentration of the monoclonal antibody able to neutralize at 50% of the input virus in the neutralization assay can be less than about 50 μg/ml.
An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
The antibodies herein also include antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
In some embodiments, the antibody comprises variable region antigen-binding sequences derived from human antibodies (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In some embodiments, the antibody includes those comprising a human variable region antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass.
In some embodiments, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In some embodiments, modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term “epitope” or “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
“Binding affinity” generally refers to the strength of the sum total of noncovalent 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). Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method well known in the art, e.g. flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE™analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and methods described herein. The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or Kd, Kon, Koff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art and such as the buffer described herein.
The phrase “substantially similar,” or “substantially the same”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristics measured by said values (e.g., Kd values). The difference between said two values is less than about 500%, less than about 40%, less than about 300%, less than about 200%, or less than about 10% as a function of the value for the reference/comparator antibody.
A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
An “isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present.
A “native sequence” polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature. A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from any species). Such native sequence polynucleotides and polypeptides can be isolated from nature. A polynucleotide “variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. or can be produced by recombinant or synthetic means.
As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
The term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.
An “effective amount” of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
The term “therapeutically effective amount” refers to an amount of an antibody or other drug effective to “treat” or prevent a disease or disorder in a subject or mammal.
Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.
The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity “X” of a first amino acid sequence to a second sequence amino acid is calculated as 100.times.(Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.
As a non-limiting example, whether any particular polynucleotide has a certain percentage sequence identity (e.g., is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman. Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In certain embodiments, identity exists over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value therebetween, or over a longer region than 60-80 residues, at least about 90-100 residues, or the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.
A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the gp120 to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
HIV-1 is among the most genetically diverse viral pathogens. Of the three main branches of the HIV-1 phylogenetic tree, the M (main), N (new), and O (outlier) groups, group M viruses are the most widespread, accounting for over 99% of global infections. This group is presently divided into nine distinct genetic subtypes, or clades (A through K), based on full-length sequences. Env is the most variable HIV-1 gene, with up to 35% sequence diversity between clades, 20% sequence diversity within clades, and up to 10% sequence diversity in a single infected person (Shankarappa, R. et al. 1999. J. Virol. 73:10489-10502). Clade B is dominant in Europe, the Americas, and Australia. Clade C is common in southern Africa, China, and India and presently infects more people worldwide than any other clade (McCutchan, F E. 2000. Understanding the genetic diversity of HIV-1. AIDS 14(Suppl. 3):531-S44). Clades A and D are prominent in central and eastern Africa.
In some embodiments, the invention provides antibodies that are broadly neutralizing and potent antibodies against HIV. The antibodies are modified from the N49P series of antibodies. The N49P series of antibodies are detailed and described in WO 2018/237357, filed on Jun. 22, 2018, which is hereby incorporated by reference in its entirety. The N49P series of antibodies comprises natural antibodies as well as engineered variants of the natural antibodies.
In some embodiments, the antibody is derived from a N49P series antibody, wherein the N49P series antibody is modified whereby a part or all of the framework 3 region of the heavy chain is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542). In some embodiments, the framework 3 region in the N49P series antibody is already deleted or missing, and in those cases either SEQ ID NO: 541 or 542 is inserted therein in the framework 3 region.
In some embodiments, the N49P series of antibodies to be modified are selected from the natural antibody sequences 1-38 as shown in Table 1 below. In some embodiments, the N49P series of antibodies to be modified comprises variants of these natural antibodies. In some embodiments, the variants that can be further modified are selected from antibodies N49P6, N49P6.2, N49P7, N49P7.1, N49P7A, N49P7S, N49P7F, N49P7Y, N49P7.54TY, N49P7-LS1, N49P7-LS2, N49P7/6L, N49P7/11L, R49P7,N49P7.2, N49P11, N49P18, N49P18.2, N49P18.1, N49P19, N49P37, N49P38, N49P38.1, N49P55, N49P56, N49P57, N49P58, N49P59, N49P73, N49P74, N49P75, N49P9, N49P9.1, N49P9.2, N49P9i7, N49P22, N49P23, N49P9.3, N49P9.4, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P51, N49P52, N49P53, N49P54, N49P60, N49P61, N49P62, N49P63, N49P64, N49P65, N49P66, N49P67, N49P68, N49P69, N49P70, N49P71, and N49P72. These variants are described in Table 3, below.
Without being bound by theory, modification of the framework 3 region of the heavy chain in the N49P antibodies to encode amino acid sequence motifs selected from QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542) enable the modified N49P antibodies to make additional contacts to improve binding to the CD4-binding site of HIV, resulting in increased potency of neutralization. The binding of the N49P antibodies with the CD4-binding site of HIV is described in WO 2018/237357, which is incorporated by reference herein.
In some embodiments, the modified N49P series antibody is antibody N49P7-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P7-FR is SEQ ID NO:501 and the nucleotide sequence is SEQ ID NO:502. The amino acid sequence of the light chain of N49P7-FR is SEQ ID NO:503 and the nucleotide sequence is SEQ ID NO:504.
In some embodiments, the modified N49P series antibody is antibody N49P9-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9-FR is SEQ ID NO:505 and the nucleotide sequence is SEQ ID NO:506. The amino acid sequence of the light chain of N49P9-FR is SEQ ID NO:295 and the nucleotide sequence is SEQ ID NO:296.
In some embodiments, the modified N49P series antibody is antibody N49P9.3-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.3-FR is SEQ ID NO:507 and the nucleotide sequence is SEQ ID NO:508. The amino acid sequence of the light chain of N49P9.3-FR is SEQ ID NO:327 and the nucleotide sequence is SEQ ID NO:328.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR is SEQ ID NO:509 and the nucleotide sequence is SEQ ID NO:510. The amino acid sequence of the light chain of N49P9.6-FR is SEQ ID NO:511 and the nucleotide sequence is SEQ ID NO:512.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR-54W or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-54W is SEQ ID NO:513 and the nucleotide sequence is SEQ ID NO:514. The amino acid sequence of the light chain of N49P9.6-FR-54W is SEQ ID NO:515 and the nucleotide sequence is SEQ ID NO:516.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR-54F or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-54F is SEQ ID NO:517 and the nucleotide sequence is SEQ ID NO:518. The amino acid sequence of the light chain of N49P9.6-FR-54F is SEQ ID NO:519 and the nucleotide sequence is SEQ ID NO:520.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR3-06 or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR3-06 is SEQ ID NO:521 and the nucleotide sequence is SEQ ID NO:522. The amino acid sequence of the light chain of N49P9.6-FR3-06 is SEQ ID NO:523 and the nucleotide sequence is SEQ ID NO:524.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR1-D or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR1-D is SEQ ID NO:525 and the nucleotide sequence is SEQ ID NO:526. The amino acid sequence of the light chain of N49P9.6-FR1-D is SEQ ID NO:527 and the nucleotide sequence is SEQ ID NO:528.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR1-D-I or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR1-D-I is SEQ ID NO:529 and the nucleotide sequence is SEQ ID NO:530. The amino acid sequence of the light chain of N49P9.6-FR1-D-I is SEQ ID NO:531 and the nucleotide sequence is SEQ ID NO:532.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR-LS or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-LS is SEQ ID NO:533 and the nucleotide sequence is SEQ ID NO:534. The amino acid sequence of the light chain of N49P9.6-FR-LS is SEQ ID NO:535 and the nucleotide sequence is SEQ ID NO:536.
In some embodiments, the modified N49P series antibody is antibody N49P9.6-FR-YTE or an antigen binding fragment thereof. The amino acid sequence of the heavy chain of N49P9.6-FR-YTE is SEQ ID NO:537 and the nucleotide sequence is SEQ ID NO:538. The amino acid sequence of the light chain of N49P9.6-FR-YTE is SEQ ID NO:539 and the nucleotide sequence is SEQ ID NO:540.
In some embodiments, the antibody comprises the VH and VL regions of antibody N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE as shown below.
In some embodiments, the antibody comprises the heavy chain CDR and light chain CDR sequences of the antibodies N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE as shown below, wherein the antibody comprises a framework 3 region of the heavy chain comprising an amino acid sequence selected from QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
The naturally occurring N49P series antibodies are shown below in Table 1 (antibody sequences 1-38), and the related variants are indicated.
VRDRSNGWGKRFESSNWFLDLWGRGTVVTVHSPSTKGPSVFPLAPSS
VRDRSNGWGKRFESSNWFLDLWGRGTVVTVHSPSTKGPSVFPLAPSS
RDKSNGSGKRFDSSNWFLDLWGRGTRVSISSASTKGPSVFPLAPSSKS
VRDRSHGSGRLFESSNWFLDLWGRGTVVTVQSPSTKGPSVFPLAPSSK
ARGIDGKSYPFHFWGHGTRVTVFSASTKGPSVFPLAPSSKSTSGGTAA
ARGIDGKSYPFHFWGHGTRVTVFSASTKGPSVFPLAPSSKSTSGGTAA
DNKRPSGVSDRFSASRLGSTSSLTISNVQAADDAHYVCASFEFFGGGTK
DNKRPSGVSNRFSASRLGSTSSLTISNVQAADDAHYVCASFEFFGGGTK
AAAPEEAFPLQYWGQGTQLIVSSASTKGPSVFPLAPSSKSTSGGTAALG
CKAAAPDEAFPLEYWGQGTQLIVSPASTKGPSVFPLAPSSKSTSGGTAA
SKAAAPDEAFPLEFWGQGTQVIVSSASTKGPSVFPLAPSSKSTSGGTAA
CKAAAPDEAFPLEYWGQGTQLIVSPASTKGPSVFPLAPSSKSTSGGTAA
ARGAFEADLYGPTYPFHHWGQGTQVTVSAASTKGPSVFPLAPSSKSTS
DNKRPSGVSDRFSASRLGSTSSLTISNVQAADDAHYVCASFEFFGGGTK
ARGIDGNSYPFHFWGHGTRVTVFSASTKGPSVFPLAPSSKSTSGGTAAL
In some embodiments, the above listed antibodies can be further modified.
In some embodiments, modifications can be made to improve antibody function (binding, neutralization, complement fixation, ADCC, ADCP).
In some embodiments, modifications to the heavy chain can be made: Dual substitution at positions 59 and 62 to T and Y, respectively. Substitution at position 1.4 of CH1 (1st amino acid of the constant region) to G from P or S. Substitution at position 120 of CH1 to R. Substitution at position 12 of CH3 to E, at position 14 of CH3 to M.
In some embodiments, light chain modifications can be made: Substitution at position 1.5 of the light chain constant region (1″ amino acid of the constant region) from R to S or G for those that use LC2, or from S to R or G for those that use LC7.
In some embodiments, modifications to improve antibody half-life can be made. These include the well-recognized “LS” (107L, 114S in CH3) and “YTE” (15.1Y, 16T,18E in CH2) mutations in the Fc. These also include other modifications in the Heavy chain such as: Substitution at position 120 of CH1 to R; Substitution at position 12 of CH3 to E, at position 14 of CH3 to M; Substitution at position 107 of CH3 to L, position 113 of CH3 to S, position 115 to R.
In some embodiments, modifications can be made that involve constant region swapping: Swapping of light chain constant region: light chain constant region swapped to another lambda constant region (LC1-7) for improved stability and function. Another method of swapping that can be used to make monoclonals that are “rhesus-ized,” that is, retain the variable regions in the heavy and light chains, but use constant regions from rhesus lambda chain (such as LC3), as well as rhesus IgG1. These mAbs can also have the half-life extending mutations noted above in the corresponding amino acids of the rhesus IgG1 constant region.
In some embodiments, the swapping of heavy and light chain variable regions can be made: These include taking a native or modified antibody listed in this application and mixing and matching the heavy and light chains from another in his application or N49P series to improve antibody stability or function.
In some embodiments, CDR replacement mutants can be made: These include taking the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 from one of the parental or modified N49P series antibodies or those in this application and inserting into the corresponding CDR another natural or modified antibody.
In some embodiments, bispecific and trispecific antibodies can be made, For example, an antibody listed in this application would constitute one arm of an IgG molecule, while the other arm(s) would be another anti-HIV monoclonal (or a monoclonal that binds to a cell surface receptor). In some embodiments, the other anti-HIV monoclonal is another antibody listed in this application.
In some embodiments, antibody-drug conjugates can be made: The natural and modified antibodies listed here can be conjugated (covalently or non-covalently) to markers (florescent dye or radionuclides), therapeutic agents, or toxins such as auristatin (a microtubule toxin).
In some embodiments, pharmaceutical compositions can be made: These can include antibodies (pre-made in a variety of vehicles) delivered via injection, delivered in a slow-release depot, or genes encoding antibodies delivered via a viral or other vector.
In some embodiments, antibody-bead conjugates can be made. The modified antibodies listed here can be conjugated to agarose or other beads by a variety of chemical reactions (such as but not limited to Cyanogen Bromide-Activated and NHS esters) for the purpose of creating affinity purification columns that can bind (and purify) gp120 and its mutants, gp160 and its mutants, HIV trimers, or HIV-1 virus.
In some embodiments, the invention provides an expression vector comprising a polynucleotide encoding the VH region and/or the VL region of the anti-HIV antibodies above; or a host cell that comprises an expression vector of the anti-HIV antibodies above; or a host cell comprising a polynucleotide that encodes the VH region and/or the VL region of the anti-HIV antibodies above.
All of the antibodies herein, their modifications, and fragments, can be used for the purpose of HIV prevention, treatment, or cure, and can be done individually or in combination with any number of anti-HIV treatments, including latency reversing agents.
Amino acid and nucleotide sequences of the anti-HIV variant and modified antibodies are shown below. Variable regions within the heavy and light chain in the amino acid sequence are italicized and changes to the amino acid sequence relative to the natural or parental antibody sequence are underlined. CDR residues are in bold.
AGLMQSGAVMKNSGASVRVSCQAD
IHWFRQRRGEGLEWLGW
NYPRPFQGKVT
MTRDTSTETAYLDVRGLTYDDTAVYYC
WGRGTQITVVS
ASTKGPSNPP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQQKPGQAPKLLIY
KRPSGVSDRFSGSTSGNTASLTISGL
QADDEGHYFC
GGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWK
AGLMQSGAVMKNSGASVRVSCQAD
IHWFRQRRGEGLEWLGW
NYPRPFQGKVT
MTRDTSTETAYLDVRGLTYDDTAVYYC
WGRGTQITVVS
ASTKGPSNPP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQQKPGQAPKLLIY
KRPSGVSDRFSGSTSGNTASLTISGL
QADDEGHYFC
GGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQSSSTKGPSVFPLAPS
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLQSGAVVKKPGDSVRISCEAQ
IHWIRRAVPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSW
A
QHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLQSGAVVKKPGDSVRISCEAQ
IHWIRRAVPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSW
F
QHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLQSGAVVKKPGDSVRISCEAQ
IHWIRRAVPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYCV
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSW
S
QHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLQSGAVVKKPGDSVRISCEAQ
IHWIRRAVPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSW
Y
QHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSMT
RDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAPS
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQQKPGQAPKLLIY
KRPSGVSDRFSGSTSGNTASLTISGL
QADDEGHYFC
IGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
QCVLTQPRSVSGSPGQSVTISCTGT
VSWCQHHPGNAPKLLLY
KRPSGISDRFSGSRSGNTASLTISG
LOPEDEADYFC
FGGGTKVLVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPAVFPLAPS
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAPS
QSALTQPRSVSATPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDEADYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTKGPSVFPLAP
QKTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSD
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVEVAWKA
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPWQFQGRVSMT
RDTSIETAFLDLRGLKSDDTALYYC
WGRGTAVTVHSPSTKGPSVFPLAPS
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
SAELVQSGAWKKPGTSVKVSCQAY
IHWLRQAPGQGLEWMGW
NYAQNFQGRVS
MTRDIYRETAFLEVRDLKTDDTGTYYC
WGRGTWIRVAPASTKGPSVFPL
QCVLTQPRSVSGSPGQSVTISCTGT
VSWCQHHPGNAPKLLLY
KRPSGISDRFSGSRSGNTASLTISG
LQPEDEADYFC
FGGGTKVLVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVMKKPGDSVRISCEAR
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSMT
RDTSIETAFLDLRGLKSDDTGLYYC
WGRGTVVTVHSPSTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTISG
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVMKKPGDSVRISCEAR
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSMT
RDTSIETAFLDLRGLKSDDTGLYYC
WGRGTVVTVHSPSTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAW
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSMT
RDTSIETAFLDLRGLKSDDTGLYYC
WGRGTVVTVHSPSYKGPSVFPLAP
QSALTQPRSMSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRPSGVPDRFSGSGSGGTASLTISG
LQDDDDAEYIC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAWKNAGASVRVSCEAY
IHWVRQAPGQGFEWMGY
NIARKFQGRLSLS
RDRSSETSFLDLSGLRSDDSAVYYC
WGRGTRVSIFSASTKGPSVFPLAPSS
QSALTQPRSVSATPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDEADYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
ADLVQSGAVVKKPGDSVRVSCEAY
IHWIRRAPGRGLEWMGW
NIPWNFQGRVSM
TRDTSIETAFLDLRGLRSDDTAVYYC
WGRGTAVTISS
ASTKGPSVFPLAPS
QSALTQPRSVSAAPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDEAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKTPGASVRVSCEAY
IHWVRQAPGQGFEWLGY
NIARKFQGRLSLSRD
TSIETSFLDLSGLRSDDSAVYYC
WGRGTRVSISSASTKGPSVFPLAPSSKS
QSALTQPRSVSAAPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITR
LQDDDDADYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
ADLVQSGAVVKTPGASVRVSCEAY
IHWVRQAPGQGFEWLGY
NIARKFQGRLSLSRD
TSIETSFLDLSGLRSDDSAVYYC
WGRGTRVSISSASTKGPSVFPLAPSSKS
QSALTQPRSVSAAPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITR
LQDDDDADYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
ADLVQSGAVVKKPGASVRVSCEAY
IHWIRQAPGQGLEWMGW
NIPWKFQGRVSM
TRDTSIETAFLDLSGLTSDDTAVYYC
WGRGTPVTISSPSTKGPSVFPLAPS
QSALTQPRSVSAAPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLSITG
LQDDDEAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGASVRVSCEAY
IHWVRQAPGQGFEWMGY
NIARKFQGRLSLS
RDTSIETSFLDLSGLRSDDSAVYYC
WGRGTRVSISSASTKGPSVFPLAPSS
QSALTQPRSVSAAPGQSVTISCTGT
VSWCQQHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDDADYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSMT
RDTSIETAFLELRGLKSDDTGLYYC
WGRGTVITVHSPSTKGPSVFPLAPS
QSALTQPRSMSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDDAEYIC
FGGGTKLTILRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPRNFQGRVSMT
RDTFRETAYLELRGLQSDDKGLYYC
WGRGTVVNVQSPSTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRASGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSLRISCEAQ
IHWIRRAPGQGLEWMGW
NIPRNFQGRVSM
TRDMYIETAFLDLRGLKSDDTGLYYC
WGRGTVVTVQSPSTKGPSVFPLA
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRASGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGL
NIPRKFQGRVSMT
RDTSMETAFLDFRGLNFDDTGLYYC
WGRGTVVTVQSPSTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRASGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGL
NIPRKFQGRVSMT
RDTSIETAFLDLRGLKSDDTGLYYC
WGRGTVVTVQSPSTKGPSVFPLAPS
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRPPKLLIY
KRASGVPDRFSGSGSGGTASLTISG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIHSPSTKGPSVFPLAPS
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDDAEYFC
FGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGLEWMGW
NIPWNFQGRVSM
TRDTSIETAFLDLRGLKSDDTAVYYC
WGRGTAVTIHS
ASTKGPSVFPLAP
QSALTQPRSVSASPGQSVTISCTGT
VSWCQHHPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITG
LQDDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPLAPSSKSTS
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPLAPSSKSTS
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFPLAPSSKSTS
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPLAPSSK
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFP
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTRDMDTETAFMELRGLRVDDTAVYYC
WGRGTAVTIQSSSTKGPSVFPL
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVL
RQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HIQLLQSGPQVKKSGDTVRISCETS
IHWVRQTPEKRLRWMGW
NYAPEFQGRIRMT
RDTFIDTVYVDLSGLTPADTAYYYC
WGHGTRVTVFSASTKGPSVFPLAPSSKSTSGGTA
RFALTQPASVSGSPGQTITITCAGG
VSWFHFPPGKTPRLIIY
KRPSGVSPRFSGSQSGSTASLIISGLQSDD
EGTYFC
FGRGTLVTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
QVRLVQSGAGARKTGASMKLSCSTS
INWVRQARGQGLEWMGW
NIEGKFQ
GRVTLTRDIYSDTAYMEMTRLTTGDTGTYYC
WGQGSLVIVSSASTKGPSVFPLAPSSK
LSALTQPASVSGSPGQSVTISCSGT
VSWYQQHPDKAPKLIIY
KRPSGISDRFSASRPDDTASLTISGL
QTGDEATYWC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTRDMYTDTAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPLAPSSKSTS
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
RQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTRDMYTDTAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPLAPSSKSTS
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HIQLLQSGPQVKKSGDTVRISCETS
IHWVRQTPEKRLRWMGW
NYAPEFQGRIRMT
RDTFIDTVYVDLSGLTPADTAYYYC
WGHGTRVTVFSASTKGPSVFPLAPSSKSTSGGTA
RFALTQPASVSGSPGQTITITCAGG
VSWFHFPPGKTPRLIIY
KRPSGVSPRFSGSQSGSTASLIISGLQSDD
EGTYFC
FGRGTLVTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
RVTLQQSGAIVRQPGASVTVSCETS
IYWVRQAPGQGLEWLGR
KYAPRFQGRLSMTR
DWSLDTAYLGLTGLTLGDTALYFC
WGQGTLVTVSAASTKGPSVFPLAPSSKST
SWALTQPASVSASPGQSVTMSCTGFG
DSWYQQYPGKAPKLIIY
KRPSGVSDRFSASRLGSTSSLTIS
NVQAADDAHYVC
FGGGTKLTVLSQPKAAPSNYLFPPSSEELQANKAYLNCLNSDFYPGANYNAW
RVTLQQSGATVKQPGASVTVSCETS
IHWVRQAPGQGLQWVGR
KYAPIFQGKVSMS
RDLSRDTAYLGLTRLTLADTALFFC
WGQGTLVIVSAASTKGPSVFPLAPSSKS
SWALTQPASVSASPGQSVTMSCTGF
DSWYQQYPGKAPKLIIY
KRPSGVSNRFSASRLGSTSSLTIS
NVQAADDAHYVC
FGGGTKLIVLSQPKAAPSNYLFPPSSEELQANKAYLNCLNSDFYPGANYNAW
NVQLMQSGTEVKKSGASVTISCETA
IHWLRQAPGGGFQWMGW
NYPQFLQGRVSM
TRDLSTDTVYMVLNGLTPDDTGLYYC
WGQGTLLTVSPASTKGPSVFPLAPSSKSTSGG
QSALSQPVSVSGSPGESITISCTGATTWYQQLPGRPPKLIIY
NRPSGISSRFSGSTSGHTASLTISGLQVDDE
GLYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
QVRLVQSGPQVKKTGASVRVSCETS
IHWLRLGPGEGLQWMGW
NYENKFRGRVTIT
RDTSTDTVYLDMSRLTPDDTAVYFC
WGQGTQVTVSPASTKGPSVFPLAPSSKSTSGG
SWALTQPASVSGSPGQSVAISCAGG
VSWYQVLPGRAPKLIIY
KRPSGVSARFSGSQSGNTAYLTISDLQT
EDEGIYFC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADG
QVRLQQSGVVVRKPGASVRISCETS
VHWVRRAPGRGFEWMGW
DYAPQLRGRISLT
RDIYSETVFIDVSRLTSGDTAIYFC
WGQGTQLIVSSASTKGPSVFPLAPSSKSTSGGTA
QAALTQPASVSGSPGQSVTISCLYA
ICWYQLHPGRAPKLLIV
KRPSGVSPRFSGSKSGTTASLTISGL
QADDEAEYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
QVRLQQSGWVRKPGASVRLSCETS
VNWVRRAPGRGFEWMGW
DYAPQHRGRISLT
RDIYTETVFIDLSRLTSGDTAIYFC
WGQGTQLIVSPASTKGPSVFPLAPSSKSTSGGTA
QAALTQPASVSGSPGQSVTISCLYA
ICWYQIQPGRLPKLLIV
RRPSGVSPRFSGSKSGTTASLTISGLQ
ADDEAEYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKAD
QVRLVQSGPVMRKPGASVRISCETS
IVHWVRRAPGRGFEWMGW
DYAPHLRGRISVT
RDVFSETVFLDLSRLTSGDTAMYFC
WGQGTQVIVSSASYKGPSVFPLAPSSKSTSGG
QAALTQPASVSGSPGQSVHSCLYA
ICWYQLHPGRAPKLLIL
KRPSGVSSRFSGSKSGITASLTISDL
QADDEAEYHC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
QVRLVQSGPWRKPGTSVRISCETS
IVHWVRRAPGRGFEWMGW
DYAPHLRGRISVT
RDVFSEIVFMELSRLTSGDTAMYFC
WGQGTQVIVSSASTKGPSVFPLAPSSKSTSGGT
QAALTQPASVSGSPGQSVTISCLYA
ICWYQLHPGRAPKLLIV
KRPSGVSSRFSGSKSGTTASLTISDL
QADDEAEYHC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
QVQLVQSGAGVKKPGASVRVSCETS
IHFLRQAPGQGLEWMGW
NYPRKFQGRVTL
TRDIYTTTVYMQLNGLTPDDTAVYYC
WGQGSLVTVSS
ASTKGPSVFPLAPSSKSTSGG
SWAQTQPASVSGSPGQSITISCAGI
DAWYQQYPGRPPRLILY
KRPSGVSPRFSASRAGKTASLTISGLQA
DDEAYYHC
GGVTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADG
QVRLQQSGWVRKPGASVRLSCETS
VNWVRRAPGRGFEWMGW
DYAPQHRGRISLT
RDIYTETVFIDLSRLTSGDTAIYFC
WGQGTQLIVSPASTKGPSVFPLAPSSKSTSGGTA
QAALTQPASVSGSPGQSVTISCLYA
ICWYQIQPGRLPKLLIV
RRPSGVSPRFSGSKSGTTASLTISGLQ
ADDEAEYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKAD
QVRLVQSGPVMRKPGASVRISCETS
VHWVRRAPGRGFEWMGW
DYAPHLRGRISVT
RDVFSETVFLDLSRLTSGDTAMYFC
WGQGTQVIVSSASTKGPSVFPLAPSSKSTSGG
QAALTQPASVSGSPGQSVTISCLYA
ICWYQLHPGRAPKLLIL
KRPSGVSSRFSGSKSGTTASLTISDL
QADDEAEYHC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLRQSGTEAKKSGASVTISCETA
IHWLRQAPGGGFQWMGW
NYPHYLQGRISMT
RDLSSDTVYMVLNRLTPADTGLYYC
WGQGTLLTVSPASTKGPSVFPLAPSSKSTSGGT
QSALSQPVSVSGSPGESITISCTEATTWYQQLPGKPPKLIIY
NRPSGISSRFSGSMSGRTASLTISGLQVDDE
GLYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
HVQLMQSGTQAKKSGASVTISCETA
IHWLRQAPGGGFQWMGW
NYPPYLQGRISLT
RDLSTDTIYMVLNGLTPADTGFYYC
WGQGTLLTVSPASTKGPSVFPLAPSSKSTSGGT
QSALSQPVSVSGSPGDSITISCFGATTWYQQLPGRPPKLIIY
NRPSGISGRFSGSMSGQKASLTISGLQVDD
EGLYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSP
HVQLRQSGTEAKKSGASVTISCETA
IHWLRQAPGGGFQWMGW
NYPHYLQGRISMT
RDLSSDTVYMVLNRLTPDDTGLYYC
WGQGTLLTVSPASTKGPSVFPLAPSSKSTSGGT
QSALSQPVSVSGSPGESITISCTEATTWYQQLPGRSPKLIIY
NRPSGISSRFSGSMSGRTASLTISGLQVDDE
GLYHC
FGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
RVTLQQSGATVRQPGASVTVSCETS
IHWVRQAPGQGLQWVGR
KFAPIFQGKFSMS
RDLSRDTAYLGLTRLTLADTALFFC
WGQGTQVTVSAASTKGPSVFPLAPSSKS
SWALTQPASVSASPGQSVTMSCTGF
DSWYQQYPGKAPKLIIY
KRPSGVSDRFSASRLGSTSSLTIS
NVQAADDAHYVC
FGGGTKLTVLSQPKAAPSNYLFPPSSEELQANKAYLNCLNSDFYPGANYNAW
HIQLLQSGPQVKKSGDTVRISCETS
IHWVRQTPEKRLRWMGW
NYAPEFQGRIRMT
RDTFIDTVYVDLSGLTPADTAYYYC
WGHGTRVTVFSASTKGPSVFPEAPSSKSTSGGT
RFALTQPASVSGSPGQTITITCAGG
VSWFHFPPGKTPRLIIY
KRPSGVSPRFSGSQSGSTASLIISGLQSDD
EGTYFC
FGRGTLLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV
ADLVQSGAVVKKPGDSVRISCEAQ
IHWIRRAPGQGPEWMGW
NIPWKFQGRVSM
TR
QLSQDPDDPDWG
TAFLDLRGLKSDDTAVYYC
WGRGTAVTIQS
ASTK
ALTQPRSVSASPGQSVTISCTGT
VSWCQHQPGRAPKLLIY
KRPSGVPDRFSGSGSGGTASLTITGLQ
DDDDAEYFC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVT
INWVRQAPGQGLEWMGW
NYSWRFEGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFPL
ASALTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNV
QPEDEATYIC
FGGGTRLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKA
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSSPSTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
RQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRWVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRV
TMTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVF
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
LFSQDLYYPDRG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASYKGPSVFPLA
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
YSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
HVQLVQSGGGVKKIGAAVRISCEVS
INWVRQAPGQGLEWMGW
NYSWRFQGRVT
MTR
QLSQDPDDPDWG
TAFMELRGLRVDDTAVYYC
WGQGVRVVVSS
ASTKGPSVFP
LTQPASMSASPGQSVTISCSGT
SAWFQQYPGKPPKLIIF
KRPSGVPSRFSASRPGDTASLTISNVQPE
DEATYIC
FGGGTKLTVL
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
In some embodiments, the antibodies of the invention have a particularly high potency in neutralizing HIV infection in vitro across multiple clades as shown in Table 4, below (see also
In some embodiments, the anti-HIV antibody neutralizes at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the HIV pseudoviruses listed Table 4 (see also
In some embodiments, the anti-HIV antibody neutralizes at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the HIV pseudoviruses listed in Table 4 with an IC50 value of less than about 1 μg/ml, between about 1-5 μg/ml or greater than about 5 μg/ml.
The neutralization can be performed using a luciferase-based assay in TZM.bl cells as described by M. M. Sajadi et al., J Acquir Immune Defic Syndr 57, 9-15 (2011) and M. Li et al., J Virol 79, 10108-10125 (2005)). This assay measures the reduction in luciferase expression following a single round of virus infection.
Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains.
Standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibodies or fragments of the antibodies of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab′)2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. Eukaryotic, e.g. mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6, myeloma or hybridoma cells.
In some embodiments, antibodies according to the invention may be produced by i) expressing a nucleic acid sequence according to the invention in a cell, and ii) isolating the expressed antibody product. Additionally, the method may include iii) purifying the antibody.
For the antibodies of the present invention to be expressed, the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The “nucleic acid control sequence” can be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. The term “promoter” will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein. The expression of the antibodies of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals. The promoter can also be specific to a particular cell-type, tissue or organ. Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the antibodies of the invention. For example, suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions. Specific expression and purification conditions will vary depending upon the expression system employed.
Following expression, the antibodies and/or antigens of the invention can be isolated and/or purified or concentrated using any suitable technique known in the art. For example, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immuno-affinity chromatography, hydroxyapatite chromatography, lectin chromatography, molecular sieve chromatography, isoelectric focusing, gel electrophoresis, or any other suitable method or combination of methods can be used.
In some embodiments, the antibodies can be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotides encoding a monoclonal antibody can be isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells.
The anti-HIV antibodies can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues. It should be understood that the antibodies of the invention may differ from the exact sequences illustrated and described herein. Thus, the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, leucine can be replaced with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid can be made.
The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of HIV such as gp120.
In some embodiments, the variable regions or domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, in some embodiments the CDRs will be derived from an antibody of different class.
Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention can comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased localization, increased serum half-life or reduced serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, modified constant regions wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (e.g. 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region.
Besides their configuration, it is known in the art that the constant region mediates several effector functions. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
In certain embodiments, the anti-HIV antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications, consistent with this invention, moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with this invention can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.
In some embodiments, the anti-HIV antibody is an antibody that binds to the same epitope as an antibody selected from the group consisting of N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, and N49P9.6-FR-YTE.
In some embodiments, the anti-HIV antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537.
In some embodiments, the anti-HIV antibody comprises an antigen binding fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537.
In some embodiments, the anti-HIV antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:503, 295, 327, 511, 515, 519, 523, 527, 531, 535, and 539.
In some embodiments, the anti-HIV antibody comprises an antigen binding fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:503, 295, 327, 511, 515, 519, 523, 527, 531, 535, and 539.
In some embodiments, the anti-HIV antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 and 397, except that the heavy chain amino acid sequence is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises an antigen binding fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 and 397, except that the heavy chain amino acid sequence is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 503, 511, 515, 519, 523, 527, 531, 535, and 539.
In some embodiments, the anti-HIV antibody comprises an antigen binding fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 503, 511, 515, 519, 523, 527, 531, 535, and 539.
For any of the light chain sequences described herein, and for any of the antibodies described herein, the first 1, 2, or 3 amino acids in the light chain [by IMGT numbering system] can be deleted, or can be substituted with another amino acid, e.g., a conservative amino acid substitution. In some embodiments there is a deletion of the first 2 or 3 amino acids. In some embodiments, the antibodies can comprise the variable region of such antibodies. For example, the variable region of SEQ ID NO:36 comprises amino acids 1-100 of SEQ ID NO:36. If an antibody has a light chain that has a 2 amino acid deletion in SEQ ID NO:36, the variable region will comprise amino acids 3-100 of SEQ ID NO:36.
In some embodiments, the anti-HIV antibody is selected from the group consisting of:
In some embodiments, the anti-HIV antibody is isolated and/or substantially pure.
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VL region comprises one or more VL complementary determining regions (CDRs) and wherein the VH region comprises one or more VH complementary determining regions (CDRs), wherein the VL CDRs correspond to the CDRs found within any of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 503, 511, 515, 519, 523, 527, 531, 535, and 539, wherein the heavy chain in the framework 3 region comprises an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VL region comprises one or more VL complementary determining regions (CDRs) and wherein the VH region comprises one or more VH complementary determining regions (CDRs), wherein the VH CDRs correspond to the CDRs found within any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537, wherein the heavy chain in the framework 3 region comprises an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VL region comprises one or more VL complementary determining regions (CDRs) and wherein the VH region comprises one or more VH complementary determining regions (CDRs), wherein the VL CDRs correspond to the CDRs found within any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327,331, 335, 339, 343, 347, 351, 355, 359, 363, 367,371, 375, 379, 383, 387, 391, 395, 399, 503, 511, 515, 519, 523, 527, 531, 535, and 539, or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, and wherein the VH CDRs correspond to the CDRs found within any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537 or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the heavy chain in the framework 3 region comprises an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VL region comprises an amino acid sequence selected from the group consisting of: amino acids 1-99 of SEQ ID NO:2; amino acids 1-97 of SEQ ID NO:4; amino acids 1-99 of SEQ ID NO:6; amino acids 1-99 of SEQ ID NO:8; amino acids 1-99 of SEQ ID NO:10; amino acids 1-99 of SEQ ID NO:12; amino acids 1-99 of SEQ ID NO:14; amino acids 1-99 of SEQ ID NO:16; amino acids 1-99 of SEQ ID NO:18; amino acids 1-99 of SEQ ID NO:20; amino acids 1-99 of SEQ ID NO:22; amino acids 1-99 of SEQ ID NO:24; amino acids 1-99 of SEQ ID NO:26; amino acids 1-99 of SEQ ID NO:28; amino acids 1-99 of SEQ ID NO:30; amino acids 1-99 of SEQ ID NO:32; amino acids 1-99 of SEQ ID NO:34; amino acids 1-100 of SEQ ID NO:36; amino acids 1-97 of SEQ ID NO:38; amino acids 1-100 of SEQ ID NO:40; amino acids 1-97 of SEQ ID NO:42; amino acids 1-97 of SEQ ID NO:44; amino acids 1-101 of SEQ ID NO:46; amino acids 1-101 of SEQ ID NO:48; amino acids 1-96 of SEQ ID NO:50; amino acids 1-97 of SEQ ID NO:52; amino acids 1-99 of SEQ ID NO:54; amino acids 1-99 of SEQ ID NO:56; amino acids 1-99 of SEQ ID NO:58; amino acids 1-99 of SEQ ID NO:60; amino acids 1-98 of SEQ ID NO:62; amino acids 1-99 of SEQ ID NO:64; amino acids 1-99 of SEQ ID NO:66; amino acids 1-96 of SEQ ID NO:68; amino acids 1-96 of SEQ ID NO:70; amino acids 1-96 of SEQ ID NO:72; amino acids 1-101 of SEQ ID NO:74; amino acids 1-97 of SEQ ID NO:76; amino acids 1-99 of SEQ ID NO:155; amino acids 1-99 of SEQ ID NO:159; amino acids 1-97 of SEQ ID NO:163; amino acids 1-97 of SEQ ID NO:167; amino acids 1-97 of SEQ ID NO:171; amino acids 1-97 of SEQ ID NO:175; amino acids 1-97 of SEQ ID NO:179; amino acids 1-97 of SEQ ID NO:183; amino acids 1-97 of SEQ ID NO:187; amino acids 1-97 of SEQ ID NO:191; amino acids 1-97 of SEQ ID NO:195; amino acids 1-97 of SEQ ID NO:199; amino acids 1-99 of SEQ ID NO:203; amino acids 1-99 of SEQ ID NO:207; amino acids 1-100 of SEQ ID NO:211; amino acids 1-99 of SEQ ID NO:215; amino acids 1-97 of SEQ ID NO:219; amino acids 1-99 of SEQ ID NO:223; amino acids 1-99 of SEQ ID NO:227; amino acids 1-99 of SEQ ID NO:231; amino acids 1-99 of SEQ ID NO:235; amino acids 1-99 of SEQ ID NO:239; amino acids 1-99 of SEQ ID NO:243; amino acids 1-99 of SEQ ID NO:247; amino acids 1-99 of SEQ ID NO:251; amino acids 1-99 of SEQ ID NO:255; amino acids 1-99 of SEQ ID NO:259; amino acids 1-99 of SEQ ID NO:263; amino acids 1-99 of SEQ ID NO:267; amino acids 1-99 of SEQ ID NO:271; amino acids 1-99 of SEQ ID NO:275; amino acids 1-99 of SEQ ID NO:279; amino acids 1-99 of SEQ ID NO:283; amino acids 1-99 of SEQ ID NO:287; amino acids 1-99 of SEQ ID NO:291; amino acids 1-100 of SEQ ID NO:295; amino acids 1-100 of SEQ ID NO:299; amino acids 1-100 of SEQ ID NO:303; amino acids 1-100 of SEQ ID NO:307; amino acids 1-100 of SEQ ID NO:311; amino acids 1-100 of SEQ ID NO:315; amino acids 1-97 of SEQ ID NO:319; amino acids 1-100 of SEQ ID NO:323; amino acids 1-97 of SEQ ID NO:327; amino acids 1-97 of SEQ ID NO:331; amino acids 1-97 of SEQ ID NO:335; amino acids 1-101 of SEQ ID NO:339; amino acids 1-101 of SEQ ID NO:343; amino acids 1-96 of SEQ ID NO:347; amino acids 1-97 of SEQ ID NO:351; amino acids 1-99 of SEQ ID NO:355; amino acids 1-99 of SEQ ID NO:359; amino acids 1-99 of SEQ ID NO:363; amino acids 1-99 of SEQ ID NO:367; amino acids 1-98 of SEQ ID NO:371; amino acids 1-99 of SEQ ID NO:375; amino acids 1-99 of SEQ ID NO:379; amino acids 1-96 of SEQ ID NO:383; amino acids 1-96 of SEQ ID NO:387; amino acids 1-96 of SEQ ID NO:391; amino acids 1-101 of SEQ ID NO:395; amino acids 1-97 of SEQ ID NO:399; amino acids 1-97 of SEQ ID NO:503; amino acids 1-97 of SEQ ID NO: 511; amino acids 1-97 of SEQ ID NO:515; amino acids 1-97 of SEQ ID NO:519; amino acids 1-97 of SEQ ID NO:523; amino acids 1-97 of SEQ ID NO:527; amino acids 1-97 of SEQ ID NO:531; amino acids 1-97 of SEQ ID NO:535; and amino acids 1-97 of SEQ ID NO:539, or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the heavy chain in the framework 3 region comprises an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VH region comprises an amino acid sequence selected from the group consisting of: amino acids 1-128 of SEQ ID NO:1; amino acids 1-127 of SEQ ID NO:3; amino acids 1-127 of SEQ ID NO:5; amino acids 1-128 of SEQ ID NO:7; amino acids 1-127 of SEQ ID NO:9; amino acids 1-127 of SEQ ID NO:11; amino acids 1-127 of SEQ ID NO:13; amino acids 1-127 of SEQ ID NO:15; amino acids 1-127 of SEQ ID NO:17; amino acids 1-127 of SEQ ID NO:19; amino acids 1-127 of SEQ ID NO:21; amino acids 1-127 of SEQ ID NO:23; amino acids 1-127 of SEQ ID NO:25; amino acids 1-127 of SEQ ID NO:27; amino acids 1-127 of SEQ ID NO:29; amino acids 1-127 of SEQ ID NO:31; amino acids 1-127 of SEQ ID NO:33; amino acids 1-120 of SEQ ID NO:35; amino acids 1-120 of SEQ ID NO:37; amino acids 1-123 of SEQ ID NO:39; amino acids 1-120 of SEQ ID NO:41; amino acids 1-120 of SEQ ID NO:43; amino acids 1-125 of SEQ ID NO:45; amino acids 1-125 of SEQ ID NO:47; amino acids 1-120 of SEQ ID NO:49; amino acids 1-120 of SEQ ID NO:51; amino acids 1-121 of SEQ ID NO:53; amino acids 1-121 of SEQ ID NO:55; amino acids 1-121 of SEQ ID NO:57; amino acids 1-121 of SEQ ID NO:59; amino acids 1-120 of SEQ ID NO:61; amino acids 1-121 of SEQ ID NO:63; amino acids 1-121 of SEQ ID NO:65; amino acids 1-120 of SEQ ID NO:67; amino acids 1-120 of SEQ ID NO:69; amino acids 1-120 of SEQ ID NO:71; amino acids 1-125 of SEQ ID NO:73; amino acids 1-120 of SEQ ID NO:75; amino acids 1-128 of SEQ ID NO:153; amino acids 1-128 of SEQ ID NO:157; amino acids 1-127 of SEQ ID NO:161; amino acids 1-127 of SEQ ID NO:165; amino acids 1-127 of SEQ ID NO:169; amino acids 1-127 of SEQ ID NO:173; amino acids 1-127 of SEQ ID NO:177; amino acids 1-127 of SEQ ID NO:181; amino acids 1-127 of SEQ ID NO:185; amino acids 1-127 of SEQ ID NO:189; amino acids 1-127 of SEQ ID NO:193; amino acids 1-127 of SEQ ID NO:197; amino acids 1-127 of SEQ ID NO:201; amino acids 1-127 of SEQ ID NO:205; amino acids 1-127 of SEQ ID NO:209; amino acids 1-127 of SEQ ID NO:213; amino acids 1-127 of SEQ ID NO:217; amino acids 1-127 of SEQ ID NO:221; amino acids 1-128 of SEQ ID NO:225; amino acids 1-127 of SEQ ID NO:229; amino acids 1-127 of SEQ ID NO:233; amino acids 1-127 of SEQ ID NO:237; amino acids 1-127 of SEQ ID NO:241; amino acids 1-127 of SEQ ID NO:245; amino acids 1-127 of SEQ ID NO:249; amino acids 1-127 of SEQ ID NO:253; amino acids 1-127 of SEQ ID NO:257; amino acids 1-127 of SEQ ID NO:261; amino acids 1-127 of SEQ ID NO:265; amino acids 1-127 of SEQ ID NO:269; amino acids 1-127 of SEQ ID NO:273; amino acids 1-127 of SEQ ID NO:277; amino acids 1-127 of SEQ ID NO:281; amino acids 1-127 of SEQ ID NO:285; amino acids 1-127 of SEQ ID NO:289; amino acids 1-120 of SEQ ID NO:293; amino acids 1-120 of SEQ ID NO:297; amino acids 1-120 of SEQ ID NO:301; amino acids 1-123 of SEQ ID NO:305; amino acids 1-128 of SEQ ID NO:309; amino acids 1-128 of SEQ ID NO:313; amino acids 1-120 of SEQ ID NO:317; amino acids 1-123 of SEQ ID NO:321; amino acids 1-120 of SEQ ID NO:325; amino acids 1-120 of SEQ ID NO:329; amino acids 1-120 of SEQ ID NO:333; amino acids 1-125 of SEQ ID NO:337; amino acids 1-125 of SEQ ID NO:341; amino acids 1-120 of SEQ ID NO:345; amino acids 1-120 of SEQ ID NO:349; amino acids 1-121 of SEQ ID NO:353; amino acids 1-121 of SEQ ID NO:357; amino acids 1-121 of SEQ ID NO:361; amino acids 1-121 of SEQ ID NO:365; amino acids 1-120 of SEQ ID NO:369; amino acids 1-121 of SEQ ID NO:373; amino acids 1-121 of SEQ ID NO:377; amino acids 1-120 of SEQ ID NO:381; amino acids 1-120 of SEQ ID NO:385; amino acids 1-120 of SEQ ID NO:389; amino acids 1-125 of SEQ ID NO:393; and amino acids 1-120 of SEQ ID NO:397, or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, except that the heavy chain amino acid sequence is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain comprises a heavy chain variable (VH) region and the light chain comprises a light chain variable (VL) region; wherein the VH region comprises an amino acid sequence selected from the group consisting of: amino acids 1-134 of SEQ ID NO:501; amino acids 1-127 of SEQ ID NO: 505; amino acids 1-127 of SEQ ID NO:507; amino acids 1-127 of SEQ ID NO:509; amino acids 1-127 of SEQ ID NO:513; amino acids 1-127 of SEQ ID NO:517; amino acids 1-127 of SEQ ID NO:521; amino acids 1-127 of SEQ ID NO:525; amino acids 1-127 of SEQ ID NO:529; amino acids 1-127 of SEQ ID NO:533; and amino acids 1-127 of SEQ ID NO:537, or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions,
In some embodiments, the anti-HIV antibody comprises a heavy chain or an antigen binding fragment thereof and a light chain or an antigen binding fragment thereof, wherein the heavy chain or antigen binding fragment thereof comprises a heavy chain variable (VH) region and the light chain or antigen binding fragment thereof comprises a light chain variable (VL) region; wherein the anti-HIV antibody is selected from the group consisting of:
In some embodiments, the anti-HIV antibody is selected from the group consisting of:
In some embodiments, the anti-HIV antibody is selected from the group consisting of:
In some embodiments, the anti-HIV antibody is selected from the group consisting of:
In some embodiments, the anti-HIV antibody is selected from the group consisting of
In some embodiments, the anti-HIV antibody is a non-naturally occurring antibody. In some embodiments, the anti-HIV antibody is selected from the group consisting of: N49P6; N49P6.2; N49P7; N49P7.1; N49P7A; N49P7S; N49P7F; N49P7Y; N49P7-54TY; N49P7LS-1; N49P7LS-2; N49P7YTE; N49P7L6; N49P7L11; N49P7.1L9; N49P7.1L19 R49P7; N49P7.2; N49P11; N49P18; N49P18.2; N49P18.1; N49P19; N49P37; N49P38; N49P38.1; N49P55; N49P56; N49P57; N49P58; N49P59; N49P73; N49P74; N49P75; N49P75.1; N49P9; N49P9.1; N49P9.2; N49P9i7; N49P9i7H1; N49P9i7H2; N49P22; N49P23; N49P9.3; N49P9.4; N49P51; N49P52; N49P53; N49P54; N49P60; N49P61; N49P62; N49P63; N49P64; N49P65; N49P66; N49P67; N49P68; N49P69; N49P70; N49P71; and N49P72, except that the heavy chain amino acid sequence of the above mentioned antibodies is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the invention provides isolated polypeptides comprising an individual light chain or heavy chain described herein as well as antigen binding fragments thereof. Polypeptides (e.g., intact antibodies) comprising both a light chain and a heavy chain are also provided.
Also provided are polypeptides that comprise: a polypeptide comprising SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, or 537 or an antigen binding fragment thereof.
Also provided are polypeptides that comprise: a polypeptide having at least about 90% sequence identity to SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, or 537 or an antigen binding fragment thereof.
In some embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, or 537 or an antigen binding fragment thereof.
Also provided are polypeptides that comprise: a polypeptide comprising SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397 or an antigen binding fragment thereof, with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
Also provided are polypeptides that comprise: a polypeptide having at least about 90% sequence identity to SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397 with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397, with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide as described herein, such as a heavy chain or light chain sequence of an HIV antibody or a fragment of such a polypeptide. For example, the invention provides a polynucleotide comprising a nucleic acid sequence that encodes an antibody to gp120 or encodes a fragment of such an antibody. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
In some embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the heavy chain variable region found within a sequence selected from the group consisting of SEQ ID NOS:501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537.
Also provided is a polynucleotide encoding a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOS: 501, 505, 507, 509, 513, 517, 521, 525, 529, 533, and 537.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397, with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the heavy chain variable region found within a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397, with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
Also provided is a polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393 or 397, with the exception that the heavy chain amino acid sequence of the above mentioned polypeptides is modified whereby a part or all of the framework 3 region is replaced with an amino acid sequence selected from the group consisting of QLSQDPDDPDWG (SEQ ID NO:541) and LFSQDLYYPDRG (SEQ ID NO:542).
The invention further provides a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS:502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, and 540.
Also provided is a polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOS: 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, and 540.
In some embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g. a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a proprotein which is the mature protein plus additional 5′ amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
In certain embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells) is used.
The present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and derivatives.
The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
Vectors and cells comprising the polynucleotides described herein are also provided. The term “vector” means a construct, which is capable of delivering, and expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. “Vector” also includes shuttle and expression vectors. In some embodiments, the vector is a plasmid construct and also includes an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively. An “expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragments of the invention, in bacterial or eukaryotic cells.
The anti-HIV antibodies of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment, cure, functional cure, or prevention of HIV infection. The methods of use may be in vitro, ex vivo, or in vivo methods.
In some embodiments, the antibodies disclosed herein may be used as neutralizing antibodies, passively administered or given via gene therapies.
In one aspect, the anti-HIV antibodies are useful for detecting the presence of HIV in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue.
Certain other methods can be used to detect binding of anti-HIV antibodies to antigens such as gp120. Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).
In certain embodiments, the antibodies are labeled. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
In certain embodiments, the antibodies are immobilized on an insoluble matrix. Immobilization entails separating the antibody from any antigen that remains free in solution. This conventionally is accomplished by either insolubilizing the antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760), or by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the antibody after formation of a complex between the antibody and antigen, e.g., by immunoprecipitation.
The present invention provides for methods of treating or preventing HIV infection comprising administering a therapeutically effective amount of an antibody as described herein to a subject (e.g., a subject in need of treatment). In some embodiments, the subject is a human.
Subjects at risk for HIV-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to HIV-1 in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of HIV-1-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
In some embodiments of the present invention, the subject is administered effective amounts of more than one anti-HIV antibody of the invention. In some embodiments, the subject is administered a pharmaceutical composition comprising a combination of antibodies of the invention, in order to treat or prevent HIV infection. In some embodiments, a combination of antibodies are administered, which can include a combination comprising any one or more of N49P7-FR or an antigen binding fragment thereof, N49P9-FR or an antigen binding fragment thereof, N49P9.3-FR or an antigen binding fragment thereof, N49P9.6-FR or an antigen binding fragment thereof, N49P9.6-FR-54W or an antigen binding fragment thereof, N49P9.6-FR-54F or an antigen binding fragment thereof, N49P9.6-FR3-06 or an antigen binding fragment thereof, N49P9.6-FR1-D or an antigen binding fragment thereof, N49P9.6-FR1-D-I or an antigen binding fragment thereof, N49P9.6 or an antigen binding fragment thereof, N49P9.6-54W or an antigen binding fragment thereof, N49P9.6-54F or an antigen binding fragment thereof, N49P9.6-LS or an antigen binding fragment thereof, N49P9.6-YTE or an antigen binding fragment thereof, N49P9.6-FR-LS or an antigen binding fragment thereof, or N49P9.6-FR-YTE or an antigen binding fragment thereof. In some embodiments, the antibody comprises the VH and VL regions of N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE as described herein. In some embodiments, the antibody comprises the CDRs of the VH and VL regions of N49P7-FR, N49P9-FR, N49P9.3-FR, N49P9.6-FR, N49P9.6-FR-54W, N49P9.6-FR-54F, N49P9.6-FR3-06, N49P9.6-FR1-D, N49P9.6-FR1-D-I, N49P9.6, N49P9.6-54W, N49P9.6-54F, N49P9.6-LS, N49P9.6-YTE, N49P9.6-FR-LS, or N49P9.6-FR-YTE as described herein. Such combinations can be selected according to the desired immunity. The composition can further include one or more other broadly neutralizing antibodies.
Methods for preventing an increase in HIV-1 virus titer, virus replication, virus proliferation or an amount of an HIV-1 viral protein in a subject are further provided. In one embodiment, a method includes administering to the subject an amount of an anti-HIV antibody effective to prevent an increase in HIV-1 titer, virus replication or an amount of an HIV-1 protein of one or more HIV strains or isolates in the subject.
For in vivo treatment of human patients, the patient is usually administered or provided a pharmaceutical formulation including an anti-HIV antibody of the invention. When used for in vivo therapy, the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's viral burden). The antibodies can be administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibodies may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous or subcutaneous administration of the antibody is preferred in certain embodiments. Therapeutic compositions of the invention are administered to a patient or subject systemically, parenterally, or locally.
For parenteral administration, the antibodies can be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate are also used. Liposomes are used as carriers. The vehicle contains minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies are typically formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection and the characteristics of the particular cytotoxic agent or growth inhibitory agent conjugated to the antibody (when used), e.g., its therapeutic index, the patient, and the patient's history. Generally, a therapeutically effective amount of an antibody is administered to a patient. In particular embodiments, the amount of antibody administered is in the range of about 0.1 mg/kg to about 20 mg/kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg/kg to about 20 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
Antibodies of the invention can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cells of interest, such as cells infected with HIV. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels. Labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an antibody of the invention and an epitope of interest (an HIV epitope) can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, τ3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 35S, or .sup.3H. Such labeled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like.
The antibodies can be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bid-diazotized benzadine and the like are used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. An enzyme is typically combined with an antibody using bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like. Various labeling techniques are described in Morrison, Methods in Enzymology 32b, 103 (1974), Syvanen et al., J. Biol. Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J. 133, 529 (1973).
In one embodiment, the antibodies can be administered as immunoconjugates, conjugated to a second molecule. For example, the second molecule can be a toxin, a label, a radioisotope, a drug, or a chemical compound.
An antibody according to the invention may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. Such antibody conjugates can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, TLR agonists (such as TLR7 agonist), or monomethylauristatin E.
Other therapeutic regimens can be combined with the administration of the anti-HIV antibody of the present invention. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.
For any application, the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment can be combined with anti-retroviral therapy. Antiretroviral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The disclosed antibodies can be administered in conjunction with nucleoside analog reverse-transcriptase inhibitors (such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine), nucleotide reverse transcriptase inhibitors (such as tenofovir and adefovir), non-nucleoside reverse transcriptase inhibitors (such as efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine), protease inhibitors (such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir), entry or fusion inhibitors (such as maraviroc and enfuvirtide), maturation inhibitors, (such as bevirimat and vivecon), or a broad spectrum inhibitors, such as natural antivirals. In some examples, a disclosed antibody or active fragment thereof or nucleic acids encoding such is administered in conjunction with IL-15, or conjugated to IL-15.
Single or multiple administrations of the compositions including the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment, that are disclosed herein, are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient. The dosage can be administered once, but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antibody binding fragments thereof, can be placed under the control of a promoter to increase expression. Another approach is to administer the nucleic acids in the form of mRNA.
In some embodiments, the subject is administered cells that are engineered to express the anti-HIV antibody. In some embodiments, the cells are engineered immune cells, such as B cells. In some embodiments, the cells are engineered, autologous cells.
In another approach to using nucleic acids, an anti-HIV antibody, or antibody binding fragment thereof can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the antibody. For example, vaccinia vectors and methods useful protocols are described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed antibodies (see Stover, Nature 351:456-460, 1991).
The present invention also encompasses compositions comprising one or more antibodies of the invention. In certain embodiments, the compositions are pharmaceutical compositions. In some embodiments, formulations are prepared for storage and use by combining an antibody with a pharmaceutically acceptable vehicle (e.g. carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g. less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosacchandes, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).
For the treatment or prevention of HIV, the appropriate dosage of an antibody or combination of antibodies of the present invention can depend on a variety of factors, such as the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. In certain embodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain embodiments, the antibody or combination of antibodies is given once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
Effective dosages and schedules for administering embodiments of the present invention can be determined empirically. In some embodiments, and effective amount of one or more antibodies are administered to neutralize, treat, prevent or eradicate HIV infection. In some embodiments, compositions comprising one or more nucleic acid molecules of the invention are administered to the subject. In some embodiments, genetic constructs capable of inducing production of antibodies of the present invention may be administered to a patient in need thereof.
Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 .mu.m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, (1992).
Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).
In some embodiments, the compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used. To prepare such a composition, a nucleic acid or vector of the invention, having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients. The carriers and excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™ PLURONICS™ or polyethylene glycol (PEG).
The compositions can be designed to introduce the antibodies, nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate. Methods of preparing controlled-release formulations are known in the art. For example, controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition. A controlled-release formulations can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile. Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these active ingredients into polymeric particles, it is possible to entrap these materials into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md., 1978 and Remington's Pharmaceutical Sciences, 16th edition.
The compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA into animal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960; Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994) Virology 199: 132-140; Webster et al., (1994) Vaccine 12: 1495-1498; Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et al., (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using “gene gun” technology (Johnston et al., (1994) Meth. Cell Biol. 43:353-365). Alternatively, delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
Dosing schedules (or regimens) can be readily determined for the particular subject and composition. Hence, the composition can be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the composition. While this interval varies for every subject, typically it can range from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. In some embodiments, the interval can be typically from 2 to 6 weeks.
The compositions of the invention can be administered alone, or can be co-administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with “other” immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or “cocktail” or combination compositions of the invention and methods of employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
The present invention also includes kits useful in performing diagnostic and prognostic assays using the antibodies of the present invention. Kits of the invention include a suitable container comprising an HIV-1 antibody of the invention in either labeled or unlabeled form. In addition, when the antibody is supplied in a labeled form suitable for an indirect binding assay, the kit further includes reagents for performing the appropriate indirect assay. For example, the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included.
Application of the teachings of the present invention to a specific problem is within the capabilities of one having ordinary skill in the art in light of the teaching contained herein. Examples of the compositions and methods of the invention appear in the following non-limiting Examples.
Through our efforts to deconvolute ongoing plasma bNAb responses in HIV-infected subjects, we have been isolating lineages of circulating bNAbs in HIV Elite Neutralizers. Plasma polyclonal anti-Env responses in one subject (donor N49) were particularly impressive, demonstrating near pan-neutralizing activity (Sajadi et al., Cell. 2018; 173(7):1783-95 e14. doi: 10.1016/j.cell.2018.03.061). Neutralization breadth was determined using standardized panels of pseudoviruses (representing multiple Tier 1-3 or Clade envelopes) (for detailed data see Sajadi et al., Cell. 2018; 173(7):1783-95 e14. doi: 10.1016/j.cell.2018.03.061). As previously predicted for Elite Neutralizers in our cohort, the N49 plasma bNAbs exhibited basic isoelectric points (Sajadi et al., J Virol. 2012; 86(9):5014-25) and utilized 2\., light chain genes (Sajadi et al., J Infect Dis. 2016; 213(1):156-64). Two related lineages of bNAbs (termed the N49 P series), distinguished by Lambda 2-11 or Lambda 2-23 use, were recovered from NVS 49. All of them recognize the CD4bs. Notably, a number of them have greater breadth than other CD4bs antibodies currently in clinical trials (e.g., 3BNC117 and VRC01). As we already published, one lineage of the bNAbs, exemplified by bNAb N49P7, exhibit near pan-neutralizing activity (Table 4 above). The more recently characterized (unpublished) second lineage of N49 bNAbs includes N49P9, N49P9.3, and N49P9.6. N49P9.6 has breadth comparable to the best CD4bs bNAbs (97%), combined with an overall potency that rivals the best of the PGT series of mAbs (Table 4). N49P9.3 is a clonal variant of N49P9.6 that has one Amino Acid difference in the Heavy Chain sequence.
To define the molecular basis for such breadth and potency, we solved the crystal structures of N49P9.3 Fab in complex with HIV-1 93TH057 gp120 core (
Accordingly, this series of N49 bNAbs was assessed for in vivo antiviral activity in a variety of pilot studies using humanized mouse formats we employ in our laboratories. One assay format examined bNAb efficacy in NOD scid gamma (NSG) mice (NOD.Cg-PrkcdscidIL2rgtmlWij/SzJ (NOD-scid IL2rg−/−)) reconstituted with HIV-infected human PBLs (Hu-PBL). This model can test the ability of a bNAb to suppress HIV-1 replication (which is ongoing in the infected donor cells). Groups of animals (n=5) were treated IP with 10 mg/kg of bNAb N49P9.3 or another anti-CD4bs bNAb, b12 (62). Control animals were treated with PBS. Six hours later the mice were given IP injection with 7.5×106 PBMCs from a heterologous Clade B HIV-1-infected patient not on ARVs (donor LT9). Plasma viral loads were measured periodically by our in-house TaqMan RT-qPCR assay with a 40 HIV-1 RNA copy/ml lower limit of detection (Satheesan et al., J Virol. 2018; 92(7)). As shown in
In another assay format, we tested the efficacy of bNAb P9.6 in NSG mice reconstituted with uninfected Hu-PBL (10×106 donor PBMCs/animal). Three weeks later, groups of mice were given 10 mg/kg of either bNAb P9.6 (n=7) or Synagis negative control (n=8) by IP route. Six hours later they were challenged IP with 100 TCID50 of cell-free CCR5-tropic HIV-1 BaL. Plasma viral loads were measured periodically by our in-house TaqMan RT-qPCR assay with a 40 HIV-1 RNA copy/ml lower limit of detection (Satheesan et al., J Virol. 2018; 92(7)). As shown in
To probe therapeutic effects, HIV-1 infection was established in NSG mice reconstituted with human CD34+ stem cells (Hu-CD34). There is substantially less graft versus host disease (GVHD) in this model versus Hu-PBL mice, allowing for studies of experimental therapeutic interventions. The reconstituted mice were infected by injecting 8000 TCID50 infection of HIV-1 Bal virus IP (based on our titration studies in these mice, this dose consistently results in 100% infection of the mice). On Day 0, 2 weeks after injection of HIV-1, after which all mice were infected, cART was started with 2 drugs (tenofovir and emtricitabine) to induce partial viral suppression in this robust infection model. After 2 weeks of cART, one dose (10 mg/kg) of a test mAb (N49P9.6, VRC01, or Synagis) was administered IP as cART was continued for about 10 days longer (predicting that antibody and cART would “wash out” concurrently). The ability of the mAbs to maintain viral suppression of HIV-1 in conjunction with cART up to Day 30 was then compared. bNAb VRC01 was unable to alter viral rebound in any animal compared to the Synagis control group. However, treatment with N49P9.6 caused a continuous viral load drop to baseline in all but one animal. The difference in Day 30 viral loads between the N49P9.6 and control groups was clearly evident (p=0.0014 by 2-tailed Fisher's exact test) (
Overall, these experiments demonstrate that the impressive in vitro characteristics of N49 P series bNAbs translates to potent in vivo efficacy. Accordingly, we expect that engineered improvements in potency/breadth evident in vitro should provide a superior antiviral effect in vivo.
Given the above information, we initiated engineering efforts to improve antigen binding and neutralization potency. CD4 (or CD4bs antibodies) not only bind to the CD4 binding pocket of one gp120, but have additional contacts on the opposing gp120 protomer (Liu et al., Nat Commun. 2019; 10(1):721; Liu et al., Nat Struct Mol Biol. 2017; 24(4):370-8). For CD4bs antibodies FR3 and CDR1 contacts have been described (Liu et al., Nat Commun. 2019; 10(1):721; Liu et al., Nat Struct Mol Biol. 2017; 24(4):370-8). Thus, we inserted a heavy chain framework 3 (FR3) loop from a CD4bs antibody (VRC03) into N49P9.6, which itself has a FR3 deletion, in order to enable the bNAbs to bind to the adjacent protomer in the envelope trimer (Liu et al., Nat Commun. 2019; 10(1):721; Liu et al., Nat Struct Mol Biol. 2017; 24(4):370-8). The constructs were tested for neutralization breadth and potency in the 117 multiclade, pseudovirus panels discussed above. As shown in
Two engineering strategies were launched. The first was based on observations that CDR1 contacts may naturally occur between N49 P series bNAbs and the adjacent protomer in the envelope trimer(Liu et al., Nat Commun. 2019; 10(1):721; Liu et al., Nat Struct Mol Biol. 2017; 24(4):370-8). It is expected that a bNAb with such binding characteristics will deliver a more potent effect, a concept already supported by N49P9.6-FR. Accordingly, we analyzed all available CD4bs antibodies isolated from donor N49, as well as the antibody database made from the donor's memory B cell and bone marrow. We identified one antibody N49P6 (not to be confused with the similarly named N49P9.6 from which the FR3 variants were made), that contained an aspartate residue in the CDR1 at position 29 (IMGT numbering system) mimicking the contact of CD4 the opposing gp120 protomer. Two variants of N49P9.6 made containing this aspartate, with improvement in potency seen similar to N49P9.6-FR in the 20 pseudovirus panel. These variants are currently undergoing testing with the full pseudovirus panel. In addition, more CDR1 variants have been made and are undergoing testing.
The second engineering strategy involved optimizing the FR3 contacts of N49P9.6-FR. The engineering efforts have been aided by structure based study of resistant variants.
Comparative Assessments of Frontline and Next Generation bNAbs
Comparative assessments of front-line bNAb potency and breadth were recently conducted under the aegis of the CA-VIMC, in part based on their highly accurate computational modeling algorithms developed to estimate potencies of bNAb combinations (67). Using various bNAb and pseudovirus panels, they compared IC99 and IC80 geometric means, IC99 and IC80% breadth at <10 ug/ml; median instantaneous inhibition potential (IIP) and % viruses exhibiting IIP >5 at total antibody concentrations of 30 ug/ml. An IIP value reflects the log number reduction of single-round infection events mediated by a drug or a drug combination according to additivity or independence models (Jilek et al., Nat Med. 2012; 18(3):446-51). In the context of cART therapy, it was shown that an IIP >5 for a given drug dose equated with >50% chance of clinical success (Jilek et al., Nat Med. 2012; 18(3):446-51). Results from a global cross-clade, cross-Tier panel of 96 pseudoviruses are shown as radar plots in
Finally, N49P9.6-FR has been compared with other next generation mAbs, namely 1-18 and LN02 ML85. In side-by-side comparisons, N49P9.6-FR was better alone and in combinations compared to these other next generation mAbs. Given such evidence, further efforts to test and engineer N49P9.6 series bNAbs is clearly warranted.
N49 bNAbs were assessed for in vivo antiviral activity in a variety of pilot studies using humanized mouse formats we employ in our laboratories. One assay format examined bNAb efficacy in NOD scid gamma (NSG) mice (NOD.Cg-PrkcdscidIL2rgtmlWij/SzJ (NOD-scid IL2rg−/−)) reconstituted with HIV-infected human PBLs (Hu-PBL). This model can test the ability of a bNAb to suppress HIV-1 replication (which is ongoing in the infected donor cells). Groups of animals (n=5) were treated IP with 10 mg/kg of bNAb N49P9.3 or another anti-CD4bs bNAb, b12. Control animals were treated with PBS. Six hours later the mice were given IP injection with 7.5×106 PBMCs from a heterologous Clade B HIV-1-infected patient not on ARVs (donor LT9). Plasma viral loads were measured periodically by our in-house TaqMan RT-qPCR assay with a 40 HIV-1 RNA copy/ml lower limit of detection. As shown in
In another assay format, we tested the efficacy of bNAb P9.6 in NSG mice reconstituted with uninfected Hu-PBL (10×106 donor PBMCs/animal). Three weeks later, groups of mice were given 10 mg/kg of either bNAb P9.6 (n=7) or Synagis negative control (n=8) by IP route. Six hours later they were challenged IP with 100 TCID50 of cell-free CCR5-tropic HIV-1 BaL. Plasma viral loads were measured periodically by our in-house TaqMan RT-qPCR assay with a 40 HIV-1 RNA copy/ml lower limit of detection. As shown in
To probe therapeutic effects, HIV-1 infection was established in NSG mice reconstituted with human CD34+ stem cells (Hu-CD34). There is substantially less graft versus host disease (GVHD) in this model versus Hu-PBL mice, allowing for studies of experimental therapeutic interventions. The reconstituted mice were infected by injecting 8000 TCID50 infection of HIV-1 Bal virus IP (based on our titration studies in these mice, this dose consistently results in 100% infection of the mice). On Day 0, 2 weeks after injection of HIV-1, after which all mice were infected, cART was started with 2 drugs (tenofovir and emtricitabine) to induce partial viral suppression in this robust infection model. After 2 weeks of cART, one dose (10 mg/kg) of a test mAb (N49P9.6, VRC01, or Synagis) was administered IP as cART was continued for about 10 days longer (predicting that antibody and cART would “wash out” concurrently). The ability of the mAbs to maintain viral suppression of HIV-1 in conjunction with cART up to Day 30 was then compared. bNAb VRC01 was unable to alter viral rebound in any animal compared to the Synagis control group. However, treatment with N49P9.6 caused a continuous viral load drop to baseline in all but one animal. The difference in Day 30 viral loads between the N49P9.6 and control groups was clearly evident (p=0.0014 by 2-tailed Fisher's exact test) (
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
This application claims the benefit of U.S. Provisional Appl. No. 62/940,572, filed Nov. 26, 2019, the contents of which are hereby incorporated by reference in their entirety.
This invention was made with government support under Grant No. AI110259 awarded by the National Institutes of Health and Grant No. BX002358 awarded by the US Department of Veterans Affairs. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/062493 | 11/27/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62940572 | Nov 2019 | US |
