US20210340259A1

Movatterモバイル変換

Info

Publication number: US20210340259A1
Application number: US17/280,755
Authority: US
Inventors: Courtney BEERS; John Corbin; Doug Hodges; Achim Moesta; Vanessa Soros; Paul Fredrick Widboom; Joseph Robert Warfield
Original assignee: Tizona Therapeutics
Current assignee: Tizona Therapeutics
Priority date: 2018-09-27
Filing date: 2019-09-26
Publication date: 2021-11-04
Also published as: ECSP21026339A; DOP2021000049A; JP2022502052A; EP3856780A1; CL2021000761A1; CO2021004719A2; CN113166243B; MX2021003555A; CA3114407A1; US11208487B2; TWI828767B; WO2020069133A1; UY38391A; SG11202103104RA; US20220135689A1; ZA202102034B; KR20210098956A; AR116526A1; CN115057932A; UA129052C2

Abstract

Provided herein are antibodies that selectively bind to HLA-G and and compositions comprising the antibodies. Also provided are methods of using the antibodies, such as therapeutic and diagnostic methods.

Description

RELATED APPLICATION

This application claims priority to U.S. provisional application No. 62/737,666, filed filed Sep. 27, 2018, which is incorporated by reference herein in its entirety.

FIELD

Provided herein are antibodies with binding specificity for HLA-G and compositions comprising the antibodies, including pharmaceutical compositions, diagnostic compositions, and kits. Also provided are methods of using anti-HLA-G antibodies for therapeutic and diagnostic purposes.

BACKGROUND

HLA-G histocompatibility antigen, class I, G, also known as human leukocyte antigen G (HLA-G), is a protein that in humans is encoded by the HLA-G gene. HLA-G belongs to the HLA nonclassical class I heavy chain paralogues. HLA-G is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). There are membrane bound and soluble forms of HLA-G.

HLA-G is normally expressed at the maternal-fetal interface and other immune-privileged sites. HLA-G may play a role in immune tolerance in pregnancy, being expressed in the placenta by extravillous trophoblast cells, while the classical MHC class I genes (HLA-A and HLA-B) are not. As HLA-G was first identified in placenta samples, many studies have evaluated its role in pregnancy disorders, such as preeclampsia and recurrent pregnancy loss. See, Michita, Rafael Tomoyaet al.,Human Immunology.2016, 77 (10): 892-897, which is incorporated by reference herein in its entirety, including any drawings.

HLA-G has been shown to be immune-suppressive. By binding receptors expressed on various myeloid and lymphoid cells, HLA-G may directly inhibit the functions of NK cells, cytotoxic T-lymphocytes, B cells, neutrophils, monocytes, macrophages and dendritic cells. HLA-G also inhibits T and NK cell proliferation and cytolytic activities. HLA-G suppresses phagocytosis and induces the generation or expansion of regulatory T cells.

HLA-G mediates immune function through at least three ITIM-containing inhibitory receptors, ILT2, ILT4, and KIR2DL4. On lymphoid and myeloid cells, for example, HLA-G mediates function through ILT2. On myeloid cells, HLA-G mediates function through ILT4. On decidual NK cells, HLA-G mediates immune function through KIR2DL4 and ILT2.

HLA-G is an immune checkpoint target. HLA-G can directly inhibit immune cell function through receptor binding and/or trogocytosis and impairment of chemotaxis. HLA-G can lend tumor cells a higher invasive and metastatic potential. HLA-G promotes evasion of tumor immune surveillance, and enhances metastasis and the progression of malignancies. During tumor progression HLA-G has other effects, such as, inhibition of immune cell cytolysis, induction of immune cell apoptosis, and/or the generation of regulatory cells through receptor binding and/or trogocytosis.

HLA-G expression is upregulated on a broad spectrum of tumors and is associated with poor prognosis and disease progression. Serum HLA-G levels are elevated in breast, lung, colorectal cancer (CRC), gastric, esophageal, neuroblastoma, cervical, and hematological cancers. HLA-G has also been found to be correlated with clinical parameters in advanced disease, such as, tumor metastasis, poor prognosis, immune escape, and tumor invasiveness.

HLA-G is an attractive target for diseases, such as, for example, cancer.

SUMMARY

Provided herein are antibodies that selectively bind HLA-G. In some embodiments, the antibodies bind human HLA-G. In some embodiments, the antibodies comprise at least one CDR sequence defined by a consensus sequence provided in this disclosure. In some embodiments, the antibodies comprise one or more of an illustrative CDR, VH, or V_Lsequence provided in this disclosure, or a variant thereof. In some aspects, the variant is a variant with one or more conservative amino acid substitutions.

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

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

In some embodiments, the method is a diagnostic method. In some embodiments, the diagnostic method comprises or consists of detecting tumor expressed HLA-G. In some embodiments, the diagnostic method comprises or consists of detecting soluble HLA-G. In some embodiments, the detection method comprises of consists of detecting HLA-G expression on immune cells.

In some embodiments, the antibodies are used to treat a disease or condition. In some aspects, the disease or condition is selected from a cancer, autoimmune disease, and infection. Some aspects provide for the use of any of the antibodies or pharmaceutical compositions provided herein to treat a disease or condition selected from a cancer, autoimmune disease, and infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a table showing avid and monomeric affinities of anti-HLA-G antibodies to recombinant HLA-G protein.

FIG. 2A andFIG. 2B provide biolayer interferometry sensorgrams showing anti-HLA-G antibodies that bind to HLA-G and can be separated into three biochemical bins based on their ability to cross-block each other when tested for binding in pairwise fashion.

FIG. 3 provide biolayer interferometry sensorgrams showing antibodies that bind and block HLA-G interaction with ILT2 and ILT4 at varying levels of effectiveness.

FIG. 4 shows evaluation of anti-HLA-G antibodies binding to naturally expressed HLA-G found on JEG-3 tumor cells.

FIG. 5A,FIG. 5B,FIG. 5C, andFIG. 5D provide data showing anti-HLA-G Fabs and antibodies that restore NKL killing activity by blocking suppression mediated through interaction of HLA-G with ILT2 or ILT4.

FIG. 6 provides evaluation of anti-HLA-G antibodies to reverse HLA-G mediated suppression of primary human cells.FIG. 6A andFIG. 6B provide evaluation of phagocytosis in a human macrophage assay.FIG. 6C provides evaluation of primary human NK cell cytotoxic activity.FIG. 6D provides evaluation of primary human CD8⁺ T cell function.

FIG. 7A andFIG. 7B shows results representing values for binding of anti-HLA-G antibodies to individual recombinant HLA class Ia antigens immobilized on beads.

FIG. 8 shows the evaluation of anti-HLA-G antibodies binding to a panel of 28 HLA-typed B-LCL lines.

FIG. 9A andFIG. 9B show the evaluation of anti-HLA-G antibodies binding to various forms of HLA-G after site-directed mutagenesis.

FIG. 10 provides evidence for tumor growth inhibition by an anti-HLA-G antibody with and Fc effector function in a mouse tumor xenograft tumor model using 721.221 cells expressing HLA-G.

DETAILED DESCRIPTION

1. Definitions

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al.,Molecular Cloning: A Laboratory Manual2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

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

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

The term “combinations thereof” includes every possible combination of elements to which the term refers.

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

The term “antibody” describes a type of immunoglobulin molecule and is used herein in its broadest sense. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), and antibody fragments and antigen binding proteins. Antibodies comprise at least one antigen-binding domain. One example of an antigen-binding domain is an antigen binding domain formed by a V_H-V_Ldimer. An “HLA-G antibody,” “anti-HLA-G antibody,” “HLA-G Ab,” “HLA-G-specific antibody,” or “anti-HLA-G Ab” is an antibody, as described herein, which binds specifically to the antigen HLA-G.

The V_Hand V_Lregions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each V_Hand V_Lgenerally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 -CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding, and confer antigen specificity and binding affinity to the antibody. See Kabat et al.,Sequences of Proteins of Immunological Interest5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, Md., incorporated by reference in its entirety.

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

The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses:IgG 1, IgG2, IgG3, IgG4, IgA1, and IgA2.

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

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

Unless otherwise specified, the numbering scheme used for identification of a particular CDR herein is the Kabat/Chothia numbering scheme. Where the residues encompassed by these two numbering schemes diverge, the numbering scheme is specified as either Kabat or Chothia.

TABLE 1

Residues in CDRs according to Kabat and Chothia
numbering schemes.

CDR	Kabat	Chothia

L1	L24-L34	L24-L34
L2	L50-L56	L50-L56
L3	L89-L97	L89-L97
H1 (Kabat Numbering)	H31-H35B	H26-H32 or H34*
H1 (Chothia Numbering)	H31-H35	H26-H32
H2	H50-H65	H52-H56
H3	H95-H102	H95-H102

*The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR.

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

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

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

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

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

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a V_Hdomain and a V_Ldomain in a single polypeptide chain. The V_Hand V_Lare generally linked by a peptide linker. See Plückthun A. (1994). Antibodies fromEscherichia coli.In Rosenberg M. & Moore G. P. (Eds.),The Pharmacology of Monoclonal Antibodiesvol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety. “scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the V_Hor V_L, depending on the orientation of the variable domains in the scFv (i.e., V_H-V_Lor V_L-V_H). Any suitable Fc domain known in the art or described herein may be used.

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

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

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

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

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

In some embodiments, an isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of an antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent.

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

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

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

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

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

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

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

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

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

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

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


	Acidic Residues	D and E
	Basic Residues	K, R, and H
	Hydrophilic Uncharged Residues	S, T, N, and Q
	Aliphatic Uncharged Residues	G, A, V, L, and I
	Non-polar Uncharged Residues	C, M, and P
	Aromatic Residues	F, Y, and W


Alcohol Group-Containing	S and T
Residues
Aliphatic Residues	I, L, V, and M
Cycloalkenyl -associated	F, H, W, and Y
Residues
Hydrophobic Residues	A, C, F, G, H, I, L, M, V, W, and Y
Negatively Charged Residues	D and E
Polar Residues	C, D, E, H, K, N, Q, R, S, and T
Positively Charged Residues	H, K, and R
Small Residues	A, C, D, G, N, P. S, T, and V
Very Small Residues	A, G, and S
Residues Involved in Turn	A, C, D, E, G, H, K, N, Q, R, S, P,
Formation	and T
Flexible Residues	Q, T, K, S, G, P, D, E, and R


	Group 1	A, S, and T
	Group 2	D and E
	Group 3	N and Q
	Group 4	Rand K
	Group 5	I, L, and M
	Group 6	F, Y, and W


	Group A	A and G
	Group B	D and E
	Group C	N and Q
	Group D	R, K, and H
	Group E	I, L, M, V
	Group F	F, Y, and W
	Group G	S and T
	Group H	C and M

Additional conservative substitutions may be found, for example, in Creighton,Proteins: Structures and Molecular Properties2nd ed. (1993) W. H. Freeman & Co., New York, N.Y. An antibody generated by making one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a “conservatively modified variant.”

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

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

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

As used herein, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, avians, goats and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an autoimmune disease or condition, and/or an infection that can be treated with an antibody provided herein. In some embodiments, the subject is a human that is suspected to have cancer, an autoimmune disease or condition, and/or an acute infection and chronic infection.

2. Antibodies

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

In some embodiments, the antibody has one or more CDRs having particular lengths, in terms of the number of amino acid residues. In some embodiments, the Chothia CDR-H1 of the antibody is 6, 7, 8, or 9 residues in length. In some embodiments, the Kabat CDR-HI of the antibody is 4, 5, 6, or 7 residues in length. In some embodiments, the Chothia CDR-H2 of the antibody is 5, 6, or 7 residues in length. In some embodiments, the Kabat CDR-H2 of the antibody is 15, 16, 17, or 18 residues in length. In some embodiments, the Kabat/Chothia CDR-H3 of the antibody is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 residues in length.

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

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

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

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

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

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

In some embodiments, the antibody blocks HLA-G interaction and/or binding to an ITIM inhibitory receptor. In some embodiments, the antibody blocks HLA-G interaction and/or binding to ILT2. In some embodiments, the antibody blocks HLA-G interaction and/or binding to ILT4. In some embodiments, the antibody blocks HLA-G interaction and/or binding to KIR2DL4. In embodiments, the antibody binds HLA-G but does not block the interaction between HLA-G and ILT2, ILT4, and/or KIRDL4. In some embodiments, the antibody disrupts HLA-G heterodimer and/or prevents dimerization of HLA-G.

In some embodiments, the antibody prevents or inhibits HLA-G mediated suppression of phagocytosis. In some embodiments, the antibody mediates HLA-G mediated induction of T regulatory cells. In some embodiments, the antibody prevents or inhibits the generation or expansion of regulatory T cells.

The antibodies provided herein may be useful for the treatment of a variety of diseases and conditions, including cancers, autoimmune diseases, and infections. In some embodiments, the antibody inhibits HLA-G function on tumor cells. In some embodiments, the antibody inhibits HLA-G function on immune cells. In some embodiments, the antibody inhibits HLA-G function on myeloid cells. In some embodiments, the antibody inhibits HLA-G function on Tcell subsets. In some embodiments, the antibody inhibits metastasis. In some embodiments, the antibody inhibits angiogenesis.

In some embodiments, the antibody competes or is capable of competing for binding to human HLA-G with another antibody. In some embodiments, the antibody comprises or consists an antibody that is capable of competing for binding to human HLA-G with a reference antibody, wherein the reference antibody binds to an epitope comprising position 195, 197, and/or 198 of SEQ ID NO: 342 on a human HLA-G polypeptide. In some embodiments, the antibody and the reference antibody cross-compete or are capable of cross-competing for binding to human HLA-G with another antibody.

In some embodiments, the antibody binds to an epitope comprising position 195, 197, and/or 198 of SEQ ID NO: 342 on a human HLA-G polypeptide. In some aspects, the epitope comprises or consists of a contiguous or non-contiguous span of amino acids including residues 195, 197, and/or 198 of the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope comprises a sequence that is identical or corresponds to residues 195, 197, and/or 198 of a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has a sequence that has a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions from a sequence that is within the sequence set forth in forth in SEQ ID NO: 342. In some aspects, the epitope has 1, 2, or 3 substitutions from residues a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the antibody makes contact with any of the residues set forth inFIG. 9.

2.1. CDR-H3 Sequences

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

2.2. V_HSequences Comprising Illustrative CDRs

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

2.2.1. V_HSequences Comprising Illustrative Kabat CDRs

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

2.2.1.1.Kabat CDR-H3

2.2.1.2.Kabat CDR-H2

2.2.1.3.Kabat CDR-H1

2.2.1.4.Kabat CDR-H3 +Kabat CDR-H2

In some embodiments, the antibody comprises a V_Hsequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101, and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 54-71. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H2 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

2.2.1.5.Kabat CDR-H3 +Kabat CDR-H1

In some embodiments, the antibody comprises a V_Hsequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101, and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 18-34. In some aspects, the Kabat CDR-H3 sequence and the Kabat CDR-H1 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H3 and Kabat CDR-H1 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

2.2.1.6.Kabat CDR-H1+Kabat CDR-H2

In some embodiments, the antibody comprises a V_Hsequence comprising a Kabat CDR-HI sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 18-34 and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 54-71. In some aspects, the Kabat CDR-H1 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1 and Kabat CDR-H2 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

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

In some embodiments, the antibody comprises a V_Hsequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 18-34, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 54-71, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101. In some aspects, the Kabat CDR-H1 sequence, Kabat CDR-H2 sequence, and Kabat CDR-H3 sequence are all from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Kabat CDR-H1, Kabat CDR-H2, and Kabat CDR-H3 are all from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

2.2.1.8.Variants of V_HSequences Comprising Illustrative Kabat CDRs

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

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

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

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

2.2.2. V_HSequences Comprising Illustrative Chothia CDRs

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

2.2.2.1.Chothia CDR-H3

2.2.2.2.Chothia CDR-H2

2.2.2.3.Chothia CDR-H1

2.2.2.4.Chothia CDR-H3+Chothia CDR-H2

In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 38-50. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H2 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

2.2.2.5.Chothia CDR-H3+Chothia CDR-H1

In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101, and a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-14. In some aspects, the Chothia CDR-H3 sequence and the Chothia CDR-H1 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H3 and Chothia CDR-H1 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

2.2.2.6.Chothia CDR-H1 +Chothia CDR-H2

In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-14 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 38-50. In some aspects, the Chothia CDR-H1 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1 and Chothia CDR-H2 are both from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

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

In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-14, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 38-50, and a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 76-101. In some aspects, the Chothia CDR-H1 sequence, Chothia CDR-H2 sequence, and Chothia CDR-H3 sequence are all from a single illustrative V_Hsequence provided in this disclosure. For example, in some aspects, the Chothia CDR-H1, Chothia CDR-H2, and Chothia CDR-H3 are all from a single illustrative V_Hsequence selected from SEQ ID NOS: 170-200.

In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 11, a Chothia CDR-H2 sequence comprising SEQ ID NO: 50, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 100. In some embodiments, the antibody comprises a V_Hsequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 6, a Chothia CDR-H2 sequence comprising SEQ ID NO: 44, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 101.

2.2.2.8.Variants of Vu Sequences Comprising Illustrative Chothia CDRs

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

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

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

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

2.3. V_HSequences

2.3.1. Variants of V_HSequences

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

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

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

2.4. CDR-L3 Sequences

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

2.5. V_LSequences Comprising Illustrative CDRs

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

2.5.1. CDR-L3

2.5.2. CDR-L2

2.5.3. CDR-L1

2.5.4. CDR-L3 +CDR-L2

In some embodiments, the antibody comprises a V_Lsequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 149-166 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 128-145. In some aspects, the CDR-L3 sequence and the CDR-L2 sequence are both from a single illustrative V_Lsequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L2 are both from a single illustrative V_Lsequence selected from SEQ ID NOS: 204-228.

2.5.5. CDR-L3+CDR-L1

In some embodiments, the antibody comprises a V_Lsequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 149-166 and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 105-124. In some aspects, the CDR-L3 sequence and the CDR-L1 sequence are both from a single illustrative V_Lsequence provided in this disclosure. For example, in some aspects, the CDR-L3 and CDR-L1 are both from a single illustrative V_Lsequence selected from SEQ ID NOS: 204-228.

2.5.6. CDR-L1 +CDR-L2

In some embodiments, the antibody comprises a V_Lsequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 105-124 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 128-145. In some aspects, the CDR-L1 sequence and the CDR-L2 sequence are both from a single illustrative V_Lsequence provided in this disclosure. For example, in some aspects, the CDR-L1 and CDR-L2 are both from a single illustrative V_Lsequence selected from SEQ ID NOS: 204-228.

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

In some embodiments, the antibody comprises a V_Lsequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 105-124, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 128-145, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 149-166. In some aspects, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative V_Lsequence provided in this disclosure. For example, in some aspects, the CDR-L1, CDR-L2, and CDR-L3 are all from a single illustrative VL sequence selected from SEQ ID NOS: 204-228.

2.5.8. Variants of V_LSequences Comprising Illustrative CDR-Ls

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

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

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

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

2.6. V_LSequences

2.6.1. Variants of V_LSequences

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

In some aspects, the V_Lsequence comprises, consists of, or consists essentially of a variant of an illustrative V_Lsequence provided in this disclosure. In some aspects, the V_Lsequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.05% identity with any of the illustrative V_Lsequences provided in this disclosure.

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

2.7. Pairs2.7.1. CDR-H3-CDR-L3 Pairs

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

In some aspects, the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 76-101, and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 149-166.

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

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

2.7.2. VH-V_LPairs

In some embodiments, the antibody comprises a V_Hsequence and a V_Lsequence.

In some aspects, the V_Hsequence is a V_Hsequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 170-200 and the V_Lsequence is a V_Lsequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 204-228.

In some aspects, the V_Lsequence is SEQ ID NO: 225. In some aspects, the V_Lsequence is SEQ ID NO: 226. In some aspects, the V_Lsequence is SEQ ID NO: 227. In some aspects, the V_Lsequence is SEQ ID NO: 228.

In some aspects, the V_Lsequence is SEQ ID NO: 220. In some aspects, the V_Lsequence is SEQ ID NO: 221. In some aspects, the V_Lsequence is SEQ ID NO: 222. In some aspects, the V_Lsequence is SEQ ID NO: 223. In some aspects, the V_Lsequence is SEQ ID NO: 224. In some aspects, the V_Lsequence is SEQ ID NO: 225. In some aspects, the V_Lsequence is SEQ ID NO: 226. In some aspects, the V_Lsequence is SEQ ID NO: 227. In some aspects, the V_Lsequence is SEQ ID NO: 228.

In some aspects, the V_Hsequence is SEQ ID NO: 189 and the V_Lsequence is selected from SEQ ID NOS: 204-228. In some aspects, the V_Lsequence is SEQ ID NO: 204. In some aspects, the V_Lsequence is SEQ ID NO: 205. In some aspects, the V_Lsequence is SEQ ID NO: 206. In some aspects, the V_Lsequence is SEQ ID NO: 207. In some aspects, the V_Lsequence is SEQ ID NO: 208. In some aspects, the V_Lsequence is SEQ ID NO: 209.

2.7.3. CDR-H1 +CDR-H2 +CDR-H3 +CDR-L1 +CDR-L2 +CDR-L3

2.7.3.1.Variants of V_H−V_LPairs

In some embodiments, the V_H−V_Lpairs provided herein comprise a variant of an illustrative V_Hand/or V_Lsequence provided in this disclosure.

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

2.7.4 HC+LC

In some aspects, the antibody comprises or consists of one or more heavy chains consisting of an HC sequence and one or more light chains consisting of an LC sequence. In some aspects, the antibody comprises or consists of two identical heavy chains consisting of an HC sequence and two identical light chains consisting of an LC sequence.

In some aspects, the HC sequence is an HC sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 232-262 and the LC sequence is an LC sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 300-330. In some embodiments, the HC sequence is an HC sequence consisting of a sequence selected from SEQ ID NOS: 232-262 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 300-330. In some embodiments, the HC sequence is an HC sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 266-296 and the LC sequence is an LC sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 300-330. In some embodiments, the HC sequence is an HC sequence consisting of a sequence selected from SEQ ID NOS: 266-296 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 300-330.

2.8. Consensus Sequences

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

2.8.1. CDR-H3 Consensus Sequences

In some embodiments, the antibody comprises a CDR-H3 sequence defined by the consensus sequence G-y₂-y₃-R-A-V-P-F-y₉-Y₁₀(SEQ ID NOS: 76-84), where y₂is I, P, Q, T, or V; y₃is A, F, K, or R; y₉is A, D, Q, or V; y₁₀is D, R, or Y.

In some aspects, when Y₂is I; y₃is A or R; y₉is F; and y₁₀is Y. In some aspects, when y₂is V; y₃is R; y₉is A, D, Q, or V; and Y₁₀is D, R, or Y. In some aspects, when Y₃is R; y₂is I, T, or V; y₉is A, D, or Q; and y₁₀is D, R, or Y. In some aspects, when y₉is D; y₂is I, P, Q, or V; y₃is A, F, K, or R; and y₁₀is Y. In some aspects, when y₁₀is Y; y₂is I, P, R, or V; y₃is A, F, K, or R; and y₉is D. In some aspects, when y₁₀is D; y₂is V; y₃is R; and y₉is A or V.

In some aspects, when y2 is V; y₃is R; y₉is D; and y₁₀is Y. In some aspects, when y₂is I; y₃is A; y₉is D; and y₁₀is Y. In some aspects, when y₂is P; y₃is K; y₉is D; and y₁₀is Y. In some aspects, when Y₂is V; y₃is R; y₉is V; and y₁₀is D. In some aspects, when y₂is V; y₃is R; y₉is Q; and y₁₀is R. In some aspects, when y₂is T; y₃is R; y₉is D; and y₁₀is Y. In some aspects, when y₂is V; y₃is R; y₉is A; and y₁₀is D. In some aspects, when y₂is I; y₃is R; y₉is D; and y₁₀is Y. In some aspects, when y₂is Q; y₃is F; y₉is D; and y₁₀is Y.

In some embodiments, the antibody comprises a CDR-H3 sequence defined by the consensus sequence G-G-Φ₃-Φ₄-Φ₅-Y-S-R-G-P-Φ₁₁-D-V (SEQ ID NOS: 85-93), where Φ₃is E, G, Q, or T; Φ₄is A, H, P, Q, or V; Os is K or T; and Φ₁₁is F, L, or M.

In some aspects, Φ₃is G; when Φ₄is A or Q; Φ₅is T; and Φ₁₁is V. In some aspects, Φ₃is T; when Φ₄is H, P, or V; Φ₅is I, T, or Y; and Φ₁₁is V. In some aspects, Φ₄is H; when Φ₃is T; Φ₅is T; and Φ₁₁is F, L, or M. In some aspects, Φ₄is V; when Φ₃is E, T, or Q; Φ₅is K or T; and Φ₁₁is L. In some aspects, Φ₅is T; when Φ₃is E, G, T, or Q; Φ₄is A, H, Q, or V; and1-1₁₁is F, L, or M. In some aspects, Φ₁₁is L; when Φ₃is E, G, T, or Q; Φ₄is A, H, P, Q, or V; and Φ₅is I, K, or T.

2.8.2. Chothia CDR-H2 Consensus Sequences

In some embodiments, the antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence Y-ε₂-S-ε₄-S (SEQ ID NOS: 38 and 44-45), where ε₂is H or Y and ε₄is A or G.

In some aspects, when ε₂is H; ε₄is A or G. In some aspects, when ε₂is G, ε₂is H or Y.

In some aspects, when ε₂is Y; ε₄is G. In some aspects, when ε₂is H; ε₄is G. In some aspects, when ε₂is H; ε₄is A.

In some embodiments, the antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence α₁-α₂-S-G-S (SEQ ID NOS: 39, 41-42, and 49), where α₁is A, H, or S; and α₂is S or Y.

In some aspects, when α₁, is S; α₂is S or Y.

In some aspects, when α₁is S; α₂is S. In some aspects, when al is S; α₂is Y. In some aspects, when al is H; α₂is Y. In some aspects, when α₁is A; α₂is Y.

In some embodiments, the antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence β₁-β₂-S-G-β₅-β₆(SEQ ID NOS: 56-60), where β₁i is A or S; β₂is G or S; β₅is I or S; and β₆is T or V.

In some aspects, when β₁is S, β₂is G or S; is I or S; and β₆is T or V. In some aspects, when β₂is S, β₁is A or S; β₅S; and β₆is T or V. In some aspects, when β₅is S, β₁is A or S; β₂is S; and β_{b 6}is T or V. In some aspects, when β₆is T, β₁is S; β₂is G or S; and β₅is I or T.

In some aspects, when β₁is A; β₂is S; β₅is S; and β₆is V. In some aspects, when β₁is S; β₂is G; β₅is I; and β₆is T. In some aspects, when β₁is S; β₂is S; β₅is S; and β₆is T.

2.8.3. Chothia CDR-H1 Consensus Sequences

In some embodiments, the antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-G-S-I-S-S-Ω₇-Ω₈-Ω₉(SEQ ID NOS: 1-4 and 13-14), where Ω₇is S or A; Ω₈is D, S, or N; and Ω₉is T, N, Y, or is absent.

In some aspects, when Ω₇is S; Ω₈is D, N, or S; and Ω₉is T, Y, or is absent.

In some aspects, when Ω₈is D; is S or A; and Ω₉is N, T, or Y.

In some aspects, when Ω₉is T; Ω₇is S; and Ω₈is D or S. In some aspects, when Ω₉is Y; Ω₇is S; and Ω₈is D or S.

In some aspects, when Ω₇is S; Ω₈is D; and Ω₉is Y. In some aspects, when Ω₇is S; Ω₈is S; and Ω₉is T. In some aspects, when Ω₇is S; Ω₈is D; and Ω₉is T. In some aspects, when Ω₇is A; Ω₈is D; and Ω₉is N. In some aspects, when Ω₇is S; Ω₈is N; and Ω₉is absent. In some aspects, when Ω₇is S; Ω₈is S; and Ω₉is Y.

In some embodiments, the antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-Y-S-I-vs-S-G-v8 (SEQ ID NOS: 6-9), where v₅is S or L and v₈is F, H, or Y.

In some aspects, when v₅is S, v₈is F, H, or Y.

In some aspects, when v₅is S, v₈is F. In some aspects, when v₈is S, v₈is H. In some aspects, when v₈is S, v₈is Y. In some aspects, when v₈is L, v₈is Y.

In some embodiments, the antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-F-T-F-κ₅-κ₆-κ₇(SEQ ID NOS: 10-12), where κ₅is D or s; κ₆is D, N, or S; and κ₇is S or Y.

In some aspects, when κ₅is S; κ₆is D or S; and κ₇is S or Y. In some aspects, when κ₇is Y; δ₅is D or S; and κ₆is N or D.

In some aspects, when κ₅is D; κ₆is N; and κ₇is Y. In some aspects, when κ₅is S; κ₆is D; and κ₇is Y. In some aspects, when κ₅is S; κ₆is S; and κ₇is S.

2.8.4. Kabat CDR-H2 Consensus Sequences

In some embodiments, the antibody comprises a Kabat CDR-H2 sequence defined by the consensus sequence β₄-β₃-β₄-β₅-β₆-β₇-T-β₉-Y-N-P-S-L-K-S (SEQ ID NOS: 54-65 and 69-70) where β₁is A, E, G, or S; β₃is A, H, S, or Y; β₄is H, S, or Y; η₅is N or S; β₆is A or G; β₇is A, L, or S; and β₉A, N, L, V, or Y.

In some aspects, when β₁is S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₅is N or S; β₆is A or G; β₇is A, L, or S; and β₉is A, N, L, V, or Y. In some aspects, when β₁is G; β₃is A or Y; β₄is H or Y; β₅is G or S; β₆is A or G; β₇is S; and β₉is A or Y. In some aspects, when β₃is Y; β₁is A, E, G, or S; β₄is H or Y; β₅is S; β₆is A or G; β₇is S; and β₉is A, N, V, or Y. In some aspects, when β₃is S; β₁is S; β₄is S or Y; β₅is N or S; β₆is A or G; β₇is L or S; and β₉is Y. In some aspects, when β₃is H; β₁is S; β₄is H or Y; β₅is S; β₆is G; β₇is A or S; and β₉is L or Y. In some aspects, when β₄is Y; β₁is S or G; β₃is A, H, S, or Y; β₅is N or S; β₆is A or G; β₇is L or S; and β₉is L or Y. In some aspects, when β₄is H; β₁is A, E, G, S; β₃is H or Y; β₅is S; β₆is A or G; β₇is A or S; and β₉is A, N, Y or V. In some aspects, when β₅is S; β₁is A, E, G, or S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₆is A or G; β₇is A or S; and β₉is A, L, N, Y, or V. In some aspects, when β₆is G; β₁is A, E, G, or S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₅is S; β₇is S; and β₉is A, N, L, V, or Y. In some aspects, when β₆is A; β₁is G or S; β₃is S or Y; β₄is H or Y; β₅is N or S; β₇is L or S; and β₉is A or Y. In some aspects, when β₇is S; β₁is A, E, G, or S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₅is S; β₆is A or G; and β₉is A, L, N, V, or Y. In some aspects, when β₉is Y; β₁is G or S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₅is N or S; β₆is A or G; and β₇is A, L, or S. In some aspects, when β₉is A; β₁is G; β₃is Y; β₄is H; β₅is S; β₆is A or G; and β₇is S.

2.8.5. Kabat CDR-H1 Consensus Sequences

In some embodiments, the antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence S-S-Δ₃-Δ₄-Y-W-Δ₇(SEQ ID NOS: 18-21, 23, and 34), where Δ₃is D or S; Δ₄is T or Y; and Δ₇is A, G, or S.

In some aspects, when Δ₃is D; Δ₄is T or Y; and Δ₇is G. In some aspects, when Δ₃is S; Δ₄is T or Y; and Δ₇is A, G, or S. In some aspects, when Δ₄is T; Δ₃is D or S; and Δ₇is A, G, or S. In some aspects, when Δ₄is Y; Δ₃is D or S; and Δ₇is G. In some aspects, when Δ₇is G; Δ₃is D or S; and Δ₄is T or Y.

In some aspects, when Δ₃is D; Δ₄is Y; and Δ₇is G. In some aspects, when Δ₃is S; Δ₄is T; and Δ₇is A. In some aspects, when Δ₃is S; Δ₄is T; and Δ₇is G. In some aspects, when Δ₃is D; Δ₄is T; and Δ₇is G. In some aspects, when Δ₃is S; Δ₄is T; and Δ₇is S. In some aspects, when Δ₃is S; Δ₄is Y; and Δ₇is G.

In some embodiments, the antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence S-G-θ₃-Y-W-θ₆(SEQ ID NOS: 24-29), where θ₃is F, H, or Y; and θ₆is F, G, I, L, or T.

In some aspects, when θ₃is H, θ₆is I or T. In some aspects, when θ₃is Y, θ₆is F, G, or L. In some aspects, when θ₆is T, θ₃is F or H.

In some aspects, when θ₃is Y, θ₆is G. In some aspects, when θ₃is Y, θ₆is F. In some aspects, when θ₃is H, θ₆is I. In some aspects, when θ₃is F, θ₆is T. In some aspects, when θ₃is Y, θ₆is L. In some aspects, when θ₃is H, θ₆is T.

2.8.6. CDR-L3 Consensus Sequences

In some embodiments, the antibody comprises a CDR-L3 sequence defined by the consensus sequence Q-π₂-λ₃-π₄-H-S-P-Y-T (SEQ ID NOS: 149-153), where π₂is Q or W; π₃is A, T, or V; and π₄is I or V.

In some aspects, when π₂is Q; π₃is A, T, or V; and π₄is I or V. In some aspects, when π₃is A; π₂is Q or W; and π₄is I or V. In some aspects, when π₄is V; π₂is Q or W; and π₃is A, T, or V.

In some aspects, when π₂is Q; π₃is A; and π₄is V. In some aspects, when π₂is W; π₃is A; and π₄is V. In some aspects, when π₂is Q; π₃is V; and π₄is V. In some aspects, when π₂is Q; π₃is T; and π₄is V. In some aspects, when π₂is Q; π₃is A; and π₄is I.

In some embodiments, the antibody comprises a CDR-L3 sequence defined by the consensus sequence Q-Q-λ₃-S-λ₅-Y-P-P-T (SEQ ID NOS: 154-158), where λ₃is F, H, or V; and λ₅is I, L, or S.

In some aspects, when λ₃is H; λ₅is I, L, or S. In some aspects, when λ₅is H; λ₃is F, H, or V.

In some aspects, when λ₃is H, λ₅is S. In some aspects, when λ₃is H, λ₅is L.

In some aspects, when λ₃is F, λ₅is S. In some aspects, when λ₃is V, λ₅is S. In some aspects, when λ₃is H, λ₅is I.

In some embodiments, the antibody comprises a CDR-L3 sequence defined by the consensus sequence Q-Q-ω₃-ω₄ω₅-ω₆-P-I-T (SEQ ID NOS: 160-161 and 163-164), where ω₃is A, L, V, or Y; ω₄is G, P, V, or Y; ω₅is S, L, or F; and ω₆is D, L, S, or Y.

In some aspects, when ω₅is S; ω₃is V or Y; ω₄is G or V; and ω₆is D or S.

In some aspects, when ω₃is A; ω₄is Y; ω₅is L; and ω₆is Y. In some aspects, when ω₃is L; ω₅is P; ω₅is F; and ω₆is L. In some aspects, when ω₃is Y; ω₄is V; ω₅is S; and ω₆is D. In some aspects, when ω₃is V; ω₄is G; ω₅is S; and ω₆is S.

2.8.7. CDR-L2 Consensus Sequences

In some embodiments, the antibody comprises a CDR-L2 sequence defined by the consensus sequence ψ₁-A-S-ψ₄-R-A-ψ₇(SEQ ID NOS: 128, 130, 132, 134-138, 143, and 145), where ψ₁is D or G; ψ₄is A, D, N, R, S, T, or Y; and ψ₇is A, N, S, or T.

In some aspects, when ψ₁is G; ψ₄is A, D, N, R, S, T, or Y; and ψ₇is A, N, or T. In some aspects, when ψ₁is D; ψ₄is S or T; and ψ₇is S or T. In some aspects, when ψ₄is S; ψ₁is G or D; and ψ₇is S or T. In some aspects, when ψ₄is N; ψ₁is G or D; and ψ₇is A or T. In some aspects, when ψ₄is T; ψ₁is G or D; and ψ₇is T. In some aspects, when ψ₇is T; ψ₁is G or D; and ψ₄is A, N, R, S, T, or Y.

2.8.8. CDR-L1 Consensus Sequences

In some embodiments, the antibody comprises a CDR-L1 sequence defined by the consensus sequence (ϕ₁-A-S-Q-ϕ₅-V-S-S-ϕ₉-ϕ₁₀-L-A (SEQ ID NOS: 105-112 and 117), where ϕ₁is E, G, K, Q, or R; ϕ₅is A or S; ϕ₉is A, D, N, S, or T; and ϕ₁₀is F or Y.

In some aspects, when ϕ₁is R; ϕ₅is Q or S; ϕ₉is A, S, or T; and ϕ₁₀is S or Y. In some aspects, when ϕ₁is G; ϕ₅is Q or S; ϕ₉is A or D; and ϕ₁₀is F or Y. In some aspects, when ϕ₁)i is Q; ϕ₅is A or S; ϕ₉is N or S; and ϕ₁₀is Y. In some aspects, when ϕ₅is S; ϕ₂is E, G, Q, or R; ϕ₉is A, D, S, or T; and ϕ₁₀is F or Y. In some aspects, when ϕ₅is A; ϕ₁is K or Q; ϕ₁₀is N or S; and ϕ₁₀is Y. In some aspects, when ϕ₉is S; ϕ₁is E, K, Q, or R;99₅is A or S; and ϕ₁₀is Y. In some aspects, when ϕ₉is A; ϕ₁is G or R; ϕ₅is S; and ϕ₁₀is F or Y. In some aspects, when ϕ₁₀is Y; ϕ₁is E, G, K, Q, or R; ϕ₅is A or S; and ϕ₉is A, D, N, S, or T.

In some embodiments, the antibody comprises a CDR-L1 sequence defined by the consensus sequence R-A-S-Q-S-
₆-
₇-S-
₉-L-
₁₁(SEQ ID NOS: 119 and 123-124), where
₆is I or V;
₇is N or S;
₉is N, W, or Y;
₁₁is A or N.
In some aspects, when
₆is I,
₇is N or S;
₉is W or Y; and
₁₁is A or N. In some aspects, when
₇is S,
₆is I or V;
₉is N or Y; and
₁₁is A or N. In some aspects, when
₁₁is A,
₆is I or V;
₇is N or S; and
₉is N or W.
In some aspects, when
₆is I;
₇is N;
₉is W; and
₁₁is A. In some aspects, when
₆is I;
₇is S;
₉is Y; and
₁is N. In some aspects, when
₆is V;
₇is S;
₉is N; and
₁₁is A.
In some embodiments, the antibody comprises a CDR-L1 sequence defined by the consensus sequence Γ₁,Γ₂-S-Q-S-V-S-Γ₈-Γ₉-Y-L-A (SEQ ID NOS: 113-116), where Γ₁is E or R; Γ₂r is A or V; Γ₈is A, D, or S; and Γ₁₀is A or S.
In some aspects, when Γ₁is E; Γ₂is A or V; Γ₈A or S; and Γ₉is A or S. In some aspects, when Γ₂is A; Γ₁is E; Γ₈is A or S; and Γ₉is A or S. In some aspects, when Γ₂is V; Γ₁is E or R; Γ₈A or D; and Γ₉is A or S. In some aspects, when Γ₈is A; Γ1 is E; Γ₂is A or V; and Γ₉is S. In some aspects, when Γ₉is S; Γ₁is E; Γ₂is A or V; and Γ₈is A. In some aspects, when Γ₉is A; ⊖₁is E or R; Γ₂is A or V; and Γ₈D or S.
In some aspects, when Γ₁is E; Γ₂is A; Γ8 is A; and Γ₉is S. In some aspects, when Γ₁, is E; Γ₂is A; Γ₈is S; and Γ₉is A. In some aspects, when Γ₁is R; Γ₂is V; Γ₈is A; and Γ₉is A. In some aspects, when Γ₁is E; Γ₂is V; Γ₈is A; and Γ₉is S.

3. Germline

In some embodiments, the antibody that specifically binds HLA-G is an antibody comprising a variable region that is encoded by a particular germline, gene, or a variant thereof. The illustrative antibodies provided herein comprise variable regions that are encoded by the heavy chain variable region germline genes VH3-23, VH4-39, VH3-11, and VH4-0B or variants thereof; and the light chain variable region germline genes VK1-33, VK3-20, VK1-12, and VK1-05 or variants thereof. One of skill in the art would recognize that the CDR sequences provided herein may also be useful when combined with variable regions encoded by other variable region germline genes or variants thereof. In particular, the CDR sequences provided herein may be useful when combined with variable regions encoded by variable region germline genes, or variants thereof, that are structurally similar to the variable region germline genes recited above. For example, in some embodiments, a CDR-H sequence provided herein may be combined with a variable region encoded by a variable region germline gene selected from the VH1 or VH3 family, or a variant thereof. In some embodiments, a CDR-L sequence provided herein may be combined with a variable region encoded by a variable region germline gene selected from the Vλ3, Vκ1, Vκ3, and Vκ4 families, or a variant thereof.

4. Affinity

In some embodiments, the affinity of the antibody for HLA-G, as indicated by K_D, is less than about 10⁻⁵M, less than about 10⁻⁶M, less than about 10⁻⁷M, less than about 10⁻⁸M, less than about 10⁻⁹M, less than about 10⁻¹⁰M, less than about 10⁻¹¹M, or less than about 10⁻¹²M. In some embodiments, the affinity of the antibody is between about 10⁻⁷M and 10⁻¹¹M. In some embodiments, the affinity of the antibody is between about 10⁻⁷M and 10¹⁰M. In some embodiments, the affinity of the antibody is between about 10⁻⁷M and 10⁻⁹M. In some embodiments, the affinity of the antibody is between about 10⁻⁷M and 10⁻⁸M. In some embodiments, the affinity of the antibody is between about 10⁻⁸M and 10¹¹M. In some embodiments, the affinity of the antibody is between about 10⁻⁸M and 10⁻¹⁰M. In some embodiments, the affinity of the antibody is between about 10⁻⁹M and 10⁻¹¹M. In some embodiments, the affinity of the antibody is between about 10¹⁰M and 10⁻¹¹M.
In some embodiments, the affinity of the antibody for human HLA-G is between about 1.00×10⁻⁸M and 9.43×10⁻¹⁰M. In some embodiment, the affinity of the antibody for human HLA-G is about 1.00×10⁻⁸M, about 1.08×10⁸M, about 1.10×10⁸M, about 1.13×10⁻⁸M, about 1.14×10⁻⁸M, about 1.16×10⁻⁸M, about 1.29×10⁻⁸M, about 1.40×10⁻⁸M, about 1.41×10⁻⁸M, about 1.46×10⁻⁸M, about 1.67×10⁻⁸M, about 1.79×10⁻⁸M, about 1.81×10⁻⁸M, about 2.04×10⁻⁸M, about 2.30×10⁻⁸M, about 2.49×10⁻⁸M, about 2.59×10⁻⁸M, about 2.94×10⁻⁸M, about 2.95×10⁻⁸M, about 3.11×10⁻⁸M, about 3.98×10⁻⁹M, about 4.11×10⁻⁹M, about 4.20×10⁻⁹M, about 4.33×10⁻⁹M, about 4.39×10⁻⁹M, about 4.42×10⁻⁹M, about 4.72×10⁻⁹M, about 5.24×10⁻⁹M, about 5.30×10⁻⁹M, about 5.35×10⁻⁹M, about 5.40×10⁻⁹M, about 5.55 ×10⁻⁹M, about 5.56×10⁻⁹M, about 5.80×10⁻⁹M, about 5.89×10⁻⁹M, about 5.92×10⁻⁹M, about 5.98×10⁻⁹M, about 5.99×10⁻⁹M, about 6.10×10⁻⁹M, about 6.34×10⁻⁹M, about 6.66×10⁻⁹M, about 6.75 ×10⁻⁹M, about 7.19×10⁻⁹M, about 7.69×10⁻⁹M, about 7.93×10⁻⁹M, about 8.23×10⁻⁹M, about 8.34×10⁻⁹M, about 8.37×10⁻⁹M, about 8.62×10⁻⁹M, about 8.82×10⁻⁹M, about 9.21×10⁻⁹M, about 9.51×10⁻⁹M, about about 1.62×10⁻¹⁰M, about 1.63×10¹⁰M, 1.64×10⁻¹⁰about 1.65×10⁻¹⁰M, about 1.66×10⁻¹⁰M, about 1.71 ×10⁻¹° M, about 1.72 ×10⁻¹° M, about 1.86×10⁻¹° M, about 1.78×10⁻¹⁰M, about 1.97×10⁻¹° M, about 1.98×10⁻¹° M, about 1.99×10⁻¹° M, about 2.29×10⁻¹⁰M, about 3.24 ×10⁻¹⁰M, about 6.47×10⁻¹° M, about 6.96×10⁻¹⁰M, about 7.84×10⁻¹⁰M, about 9.41×10¹⁰M, or about 9.43×10⁻¹⁰M.
In some embodiments the antibody has a k_onwhen associating with human HLA-G of between about 1.41×10⁵M⁻¹×sec⁻¹and about 1.07×10⁶×sec⁻¹. In some embodiments the antibody has a k_onwhen associating with human HLA-G of about about 1.41×10⁵M⁻¹×sec⁻¹, about 1.49 ×10⁵M⁻¹×sec⁻¹, about 1.66×10⁵M⁻¹×sec⁻¹, about 2.90×10⁵M⁻1 ×sec⁻¹, about 3.60×10⁵M⁻¹×sec⁻¹, about 3.74×10⁵M⁻¹×sec⁻¹, about 3.78×10⁵×sec⁻¹, about 4.03×10⁵×sec⁻¹, about 4.30×10⁵M⁻¹×sec⁻¹, about 4.32×10⁵M⁻¹×sec⁻¹, about 4.34 ×10⁵×sec⁻¹, about 4.60×10⁵M⁻¹×sec⁻¹, about 4.64×10⁵M⁻¹×sec⁻¹, about 4.80×10⁵m⁻ⁱ×sec⁻¹, about 4.84×10⁵M⁻¹×sec⁻¹, about 4.87×10⁵M⁻¹×sec⁻¹, about 4.91 ×10⁵MH ×sec⁻¹, about 4.96×10⁵×sec⁻¹, about 4.97×10⁵M⁻¹x sec⁻¹, about 4.98×10⁵M⁻¹×sec⁻¹, about 5.01 ×10⁵M⁻¹×sec⁻¹, about 5.14×10⁵×sec⁻¹, about 5.19×10⁵M⁻¹×sec⁻¹, about 5.20×10⁵m⁻ⁱ×sec⁻¹, about 5.26×10⁵M⁻¹×sec⁻¹, about 5.27×10⁵M⁻¹×sec⁻¹, about 5.30×10⁵M⁻¹×sec⁻¹, about 5.61 ×10⁵M⁻I x sec⁻¹, about 5.90×10⁵×sec⁻¹, about 6.31 ×10⁵×sec⁻¹, about 6.32×10⁵×sec⁻¹, about 6.36×10⁵M⁻¹×sec⁻¹, about 6.46×10⁵M⁻¹×sec⁻¹, about 6.53×10⁵m⁻ⁱ×sec⁻¹, about 6.66×10⁵M⁻¹×sec⁻¹, about 6.84×10⁵NV ×sec⁻¹, about 7.20×10⁵M⁻¹×sec⁻¹, about 7.24×10⁵×sec⁻¹, about 7.48×10⁵M⁻¹×sec⁻¹, about 7.36×10⁵M⁻¹×sec⁻¹, about 7.58×10⁵M⁻¹×sec⁻¹, about 7.77×10⁵M⁻¹×sec⁻¹, about 7.92×10⁵M⁻¹×sec⁻¹, about 7.94×10⁵m⁻i ×sec⁻¹, about 7.96×10⁵M⁻¹×sec⁻¹, about 8.03×10⁵M⁻¹×sec⁻¹, about 8.24×10⁵M⁻¹×sec⁻¹, about 8.26×10⁵M⁻¹×sec⁻¹, about 8.55 ×10⁵M^−I×sec⁻¹, about 8.63×10⁵M⁻¹×sec⁻¹, about 8.66×10⁵M⁻¹×sec⁻¹, about 8.74×10⁵M⁻¹×sec⁻¹,about 8.79×10⁵×sec⁻¹, about 8.92×10⁵m⁻i x sec⁻¹, about 8.96×10⁵×sec⁻¹, about 9.09×10⁵×sec⁻¹, about 9.31 ×10⁵×sec⁻¹, about 9.35 ×10⁵M⁻¹×sec⁻¹, about 9.38×10⁵M⁻¹×sec⁻¹, about 9.46×10⁵M⁻¹×sec⁻¹, about 9.54×10⁵×sec⁻¹, about 9.73 ×10⁵×sec⁻¹, about 9.83 ×10⁵×sec⁻¹, about 9.84×10⁵M¹×sec⁻¹, about 9.91×10⁵M⁻¹×sec⁻¹, about 1.05×10⁶M⁻¹×sec⁻¹, or about 1.07 ×10⁶M⁻¹×sec⁻¹.
In some embodiments the antibody has a k_offwhen associating with human HLA-G of between about 1.06×10²sec⁻¹and about 8.55×10⁵sec⁻¹. In some embodiments the antibody has a k_offof about 1.06×10⁻²sec⁻¹, about 1.13×10⁻²sec⁻¹, about 1.87×10⁻²sec⁻¹, about 2.13 ×10⁻²sec⁻¹, about 3.62×10⁻³sec⁻¹, about 3.66×10⁻³sec⁻¹, about 3.75×10⁻³sec⁻¹, about 3.78×10⁻³sec⁻¹, about 3.83 ×10⁻³sec⁻¹, about 3.96×10⁻³sec⁻¹, about 4.17×10⁻³sec⁻¹, about 4.27×10⁻³sec⁻¹, about 4.29×10⁻³sec⁻¹, about 4.41×10⁻³sec⁻¹, about 4.42 ×10⁻³sec⁻¹, about 4.46 ×10⁻³sec⁻¹, about 4.54×10⁻³sec⁻¹, about 4.65×10⁻³sec⁻¹, about 4.85 ×10⁻³sec⁻¹, about 4.88×10³sec⁻¹, about 4.89×1 0⁻³sec⁻¹, about 4.98×10⁻³sec⁻¹, about 5.26 ×10⁻³sec⁻¹, about 5.44×10⁻³sec⁻¹, about 5.47×10⁻³sec⁻¹, about 5.71×10⁻³sec⁻¹, about 5.72 ×10⁻³sec⁻¹, about 5.84×10⁻³sec⁻¹, about 5.90×10⁻³sec⁻¹, about 5.92 ×10⁻³sec⁻¹, about 5.93 ×10⁻³sec⁻¹, about 6.24×1 0⁻³sec⁻¹, about 6.25x 0⁻³sec⁻¹, about 6.28x 0⁻³sec⁻¹, about 6.49x 10⁻³sec⁻¹, about 6.50×1 0⁻³sec⁻¹, about 6.71 ×10⁻³sec⁻¹, about 6.78×10⁻³sec⁻¹, about 6.83 xl 0⁻³sec⁻¹, about 6.98×10⁻³sec⁻¹, about 7.17×10⁻³sec⁻¹, about 7.42×10⁻³sec⁻¹, about 8.12×10⁻³sec⁻¹, about 8.16×10⁻³sec⁻¹, about 8.26×10⁻³sec⁻¹, about 8.64×10⁻³sec⁻¹, about 8.76x 10⁻³sec⁻¹, about 8.91 ×10⁻³sec⁻¹, about 9.31 ×10⁻³sec⁻¹, about 9.32×10⁻³sec⁻¹, about 9.87 ×10⁻³sec⁻¹, about 1.82 ×10⁻⁴sec⁻¹, about 4.38 ×10⁻⁴sec⁻¹, about 4.59×10⁻⁴sec⁻¹, about 4.99×4.99×10⁴sec⁻¹, about 5.73×10⁻⁴sec⁻¹, about 6.03×10⁻⁴sec⁻¹, or about 8.55×10⁻⁵sec⁻¹.
In some aspects, the K_D, k_a, and k_dare determined at 25° C. In some embodiments, the K_D, k_a, and k_dare determined by surface plasmon resonance. In some embodiments, the K_D, k_a, and k_dare determined according to the methods described in the examples.

5. Inhibition of HLA-G

In some aspects, the antibody decreases the affinity of HLA-G for its ligand(s). In some aspects, the antibody disrupts the association of HLA-G with beta-2-microglobulin and/or its cognate peptide. In some aspects the antibody prevents HLA-G dimerization or oligomizeration.
In some aspects, the antibody inhibits HLA-G function on tumor cells. In some aspects, the antibody inhibits HLA-G function on immune cells. In some embodiments, the antibody blocks HLA-G interaction and/or binding to an ITIM inhibitory receptor. In some embodiments, the antibody blocks HLA-G interaction and/or binding to ILT2. In some embodiments, the antibody blocks HLA-G interaction and/or binding to ILT4. In some embodiments, the antibody blocks HLA-G interaction and/or binding to KIR2DL4.
In some embodiments, the antibody inhibits immune suppressive function. In some embodiments, the antibody inhibits HLA-G mediated suppression of NK cells. In some embodiments, the antibody inhibits HLA-G mediated suppression of cytotoxic T lymphocytes. In some embodiments, the antibody inhibits HLA-G mediated suppression of B cells. In some embodiments, the antibody inhibits HLA-G mediated suppression of neutrophils. In some embodiments, the antibody inhibits HLA-G mediated suppression of dendritic cells. In some embodiments, the antibody inhibits HLA-G mediated suppression of macrophages. In some embodiments, the antibody inhibits HLA-G mediated suppression of monocytes. In some embodiments, the antibody inhibits HLA-G mediated suppression of NK and/or T cell cytolysis and/or proliferation.
In some embodiments, the antibody prevents or inhibits inhibits HLA-G mediated suppression of phagocytosis. In some embodiments, the antibody mediates HLA-G mediated induction of T regulatory cells. In some embodiments, the antibody prevents or inhibits the generation or expansion of regulatory T cells. In some embodiments, the antibody inhibits tumor growth by antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP).
In some aspects, the decrease is about or less than a 10% decrease, about or less than a 20% decrease, about or less than a 30% decrease, about or less than a 40% decrease, about or less than a 50% decrease, about or less than a 60% decrease, about or less than a 70% decrease, about or less than an 80% decrease, about or less than a 90% decrease, or about a complete decrease. In some aspects, the increase is about or greater than a 10% increase, about or greater than a 20% increase, about or greater than a 30% increase, about or greater than a 40% increase, about or greater than a 50% increase, about or greater than a 60% increase, about or greater than a 70% increase, about or greater than an 80% increase, about or greater than a 90% increase, or a complete increase.

6. HLA-G Assays

In some embodiments, the antibody binds to an epitope of HLA-G. An epitope often consists of a number of contiguous amino acids such as, for example, without limitation, 5-6 amino acids. In some embodiments, the epitope comprises or consists of contiguous or non-contiguous amino acids. In some embodiments, the contiguous or non-contiguous amino acids are within a domain of HLA-G. In some embodiments, the domain comprises or consists of an alpha three domain.
In some aspects, HLA-G has a sequence identical to the amino acid sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has an amino acid sequence that is within the amino acid sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to a sequence that is within the sequence set forth in SEQ ID NO: SEQ ID NO: 342.
In some aspects, the epitope comprises or consists of a contiguous or non-contiguous span of amino acids including residues 195, 197, and/or 198 of the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope comprises a sequence that is identical or corresponds to residues 195, 197, and/or 198 of a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has a sequence that has a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the epitope has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions from a sequence that is within the sequence set forth in forth in SEQ ID NO: 342. In some aspects, the epitope has 1, 2 or 3 substitutions from residues a sequence that is within the sequence set forth in SEQ ID NO: 342. In some aspects, the antibody makes contact with any of the residues set forth inFIG. 9.
In some aspects, the antibody competes with any of the antibodies set forth herein. Competition could be, for example, without limitation, binding competition, inhibition competition, or any other form of competition. In some aspects, the antibody competes with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 ofantibodies 38410, 38418, 38422, 38426, 38381, 38358, 37323, 38373, 38375, 38379, 38389, 33303, or 33357. In some aspects, the antibody competes with any of the antibodies set forth inFIG. 2 orFIG. 3. In some aspects, the antibody competes with 1, 2, 3, 4, 5, 6, 7, or 8 of 38373, 38375, 38379, 38418, 38422, 38426, 38410 or 38381. In some aspects, the antibody competes with 38358. In some aspects, the antibody competes with 1, 2, 3, 4, 5, 6, 7, 8, or 9 of 38373, 38375, 38379, 38418, 38422, 38426, 38410 or 38381, or 38358. In some aspects, the antibody competes with one or both of 37323 and/or 38389. In some aspects, the antibody competes with one or both of 33303 and/or 33357.

7. Glycosylation Variants

In some embodiments, an antibody may be altered to increase, decrease or eliminate the extent to which it is glycosylated. Glycosylation of polypeptides is typically either “N-linked” or “O-linked.”
“N-linked” glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
“O-linked” glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition or deletion of N-linked glycosylation sites to the antibody may be accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences is created or removed. Addition or deletion of O-linked glycosylation sites may be accomplished by addition, deletion, or substitution of one or more serine or threonine residues in or to (as the case may be) the sequence of an antibody.
In certain embodiments, the antibody is glycosylated. In certain embodiments, the antibody is deglycosylated. Carbohydrates may be removed by standard techniques. In certain embodiments, the antibody is aglycosylated, for instance by expression in a system that does not glycosylate.

8. Fc Variants

In some embodiments, amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. In some embodiments, the Fc region variant possesses some, but not all, effector functions. Such antibodies may be useful, for example, in applications in which the half-life of the antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-directed complement-mediated cytotoxicity (ADCC). Numerous substitutions or substitutions or deletions with altered effector function are known in the art.
In some embodiments, the Fc is modified. In some embodiments, a hinge of an IgG1 or IgG4 antibody is modified. Modification of a hinge region stabilizes an antibody and prevents formation of unwanted bispecific antibodies. In some embodiments, the modification comprises an L234A, L235A, and/or G237A according to a Kabat numbering scheme or residues number 117, 118, and 120, respectively, wherein residues are numbered according to any of SEQ ID NO: 334. In some embodiments, the modification comprises an EU S228P or an S241P according to a Kabat numbering scheme or number 108 according to SEQ ID NO: 335. In some embodiments, an IgG Fc is engineered to modulate antibody effector function (See Wang et al., Protein Cell, 2018, January; 9(1): 63-73), which is incorporated by reference herein in its entirety, including any drawings).
Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are provided in U.S. Pat. Nos. 5,500,362 and 5,821,337; Hellstrom et al.,Proc. Natl. Acad. Sci. U.S.A.,1986, 83:7059-7063; Hellstrom et al.,Proc. Natl. Acad. Sci. U.S.A.,1985, 82:1499-1502; and Bruggemann et al.,J. Exp. Med.,1987, 166:1351-1361. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, using an animal model such as that disclosed in Clynes et al.Proc. Natl. Acad. Sci. US.A.,1998, 95:652-656.
C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. Examples of C1q binding assays include those described in WO 2006/029879 and WO 2005/100402.
Complement activation assays include those described, for example, in Gazzano-Santoro et al.,J Immunol. Methods,1996, 202:163-171; Cragg et al.,Blood,2003, 101:1045-1052; and Cragg and Glennie,Blood,2004, 103:2738-2743.
FcRn binding and in vivo clearance (half-life determination) can also be measured, for example, using the methods described in Petkova et al.,Intl. Immunol.,2006, 18:1759-1769.

9. Preparation of Antibodies

9.1. Antigen Preparation

The HLA-G antigen to be used for production of antibodies may be intact HLA-G or a fragment of HLA-G. The intact HLA-G, or fragment of HLA-G, may be in the form of an isolated protein or expressed by a cell. Other forms of HLA-G useful for generating antibodies will be apparent to those skilled in the art.

9.2. Monoclonal Antibodies

Monoclonal antibodies may be obtained, for example, using the hybridoma method first described by Kohler et al.,Nature,1975, 256:495-497, and/or by recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be obtained, for example, using phage or yeast-based libraries. See e.g., U.S. Pat. Nos. 8,258,082 and 8,691,730.
In the hybridoma method, a mouse or other appropriate host animal is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See Goding J. W.,Monoclonal Antibodies: Principles andPractice3^rded. (1986) Academic Press, San Diego, Calif.
The hybridoma cells are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Useful myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells; and are sensitive media conditions, such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, Calif.), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Md.). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. See e.g., Kozbor,J. Immunol.,1984, 133:3001.
After the identification of hybridoma cells that produce antibodies of the desired specificity, affinity, and/or biological activity, selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
DNA encoding the monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Thus, the hybridoma cells can serve as a useful source of DNA encoding antibodies with the desired properties. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as bacteria (e.g.,E. coli), yeast (e.g.,SaccharomycesorPichiasp.), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to produce the monoclonal antibodies.

9.3. Humanized Antibodies

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

9.4. Human Antibodies

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

10. Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acids encoding anti-HLA-G antibodies, vectors and host cells comprising the nucleic acids and recombinant techniques for the production of the antibodies.
For recombinant production of the antibody, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression. In some aspects, the nucleic acid may be produced by homologous recombination, for example as described in U.S. Pat. No. 5,204,244.
Many different vectors are known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Pat. No. 5,534,615.
Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia(E. coli),Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella(S. typhimurium),Serratia(S. marcescans),Shigella, Bacilli(B. subtilisandB. licheniformis),Pseudomonas(P. aeruginosa), andStreptomyces.One usefulE. colicloning host isE. coli294, although other strains such asE. coliB,E. coliX1776, andE. coliW3110 are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-HLA-G antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is a commonly used lower eukaryotic host microorganism. However, a number of other genera, species, and strains are available and useful, such asSchizosaccharomyces pombe, Kluyveromyces(K. lactis, K. fragilis, K. bulgaricus K wickeramii, K. waltii, K. drosophilarum, K thermotolerans,andK. marxianus),Yarrowia, Pichia pastoris, Candida(C. albicans),Trichoderma reesia, Neurospora crassa, Schwanniomyces(S. occidentalis), and filamentous fungi such as, for examplePenicillium, Tolypocladium,andAspergillus(A. nidulansandA. niger).
Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertoli cells; African green monkey kidney cells (VERO-76), and the like.
The host cells used to produce the anti-HLA-G antibody of this invention may be cultured in a variety of media. Commercially available media such as, for example, Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing the host cells. In addition, any of the media described in Ham et al.,Meth. Enz.,1979, 58:44; Barnes et al.,Anal. Biochem.,1980, 102:255; and U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469, or WO 90/03430 and WO 87/00195 may be used.
Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. For example, Carter et al. (Bio/Technology,1992, 10:163-167) describes a procedure for isolating antibodies which are secreted to the periplasmic space ofE. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation.
In some embodiments, the antibody is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs, 2012, 4:217-225, incorporated by reference in its entirety. In some aspects, the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell. In some aspects, the prokaryotic cell isE. coli.Cell-free expression of the antibody may be useful, for example, where the antibody accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.
Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore® Pellcon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al.,J. Immunol. Meth.,1983, 62:1-13). Protein G is useful for all mouse isotypes and for human γ3 (Guss et al.,EMBO J.,1986, 5:1567-1575).
The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the BakerBond ABX® resin is useful for purification.
Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin Sepharose®, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, and can be applied by one of skill in the art.
Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 to about 4.5, generally performed at low salt concentrations (e.g., from about 0 to about 0.25 M salt).

11. Pharmaceutical Compositions and Methods of Administration

Any of the antibodies provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, the inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.
The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in theHandbook of Pharmaceutical Excipients,Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.
In some embodiments, the pharmaceutical composition comprises an anti-foaming agent. Any suitable anti-foaming agent may be used. In some aspects, the anti-foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof. In some aspects, the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.
In some embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, and propylene glycol.
In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.
In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.
In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid,macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.
In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.
Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars. Specific examples of each of these agents are described, for example, in theHandbook of Pharmaceutical Excipients,Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated by reference in its entirety.
In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is saline solution, such as a sterile isotonic saline solution or dextrose solution. In some aspects, the solvent is water for injection.
In some embodiments, the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles may be formed from any suitable material, such as a polymer or a lipid. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymersomes. In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies.
Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising an antibody, since water can facilitate the degradation of some antibodies.
Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
In some embodiments, the pharmaceutical composition further comprises one or both of an antibody to an immune inhibitory receptor or ligand and/or an antibody to an immune stimulatory receptor and/or ligand. In some embodiments, the pharmaceutical composition further comprises an effective amount of at least one of the following: an anti-ILT2 antibody; an anti-ILT-4 antibody; an anti-ILT4 antibody; an anti-KIR2DL4 antibody; an anti-HLA-E antibody; an anti-NKG2A antibody; an anti-HLA-F antibody; an anti-PD-L1 antibody; an anti-PD-1 antibody; an anti-CTLA4 antibody; an anti-CD38 antibody; an anti-CD73 antibody; an anti-A2A receptor antibody; an anti-A2B receptor antibody; an anti-A2A/A2B dual receptor antibody or a combination thereof; an anti-CD39 antibody; an anti-CD73 antibody; an anti-CD47 antibody; and/or a small molecule inhibitor. In some embodiments, the pharmaceutical composition further comprises an anti-Tim-3 antibody; an anti-TIGIT antibody; an anti-Vista antibody; an anti-CD94 antibody; an anti-ILT2 antibody, an anti-ILT4 antibody, an anti-PD-Ll antibody, and/or an anti-CD47 antibody; a small molecule inhibitor; a bi-specific T cell engager, CAR-T therapy, CAR-NK therapy, CAR-Macrophage therapy, engineered cell therapy, and/or adaptive T cell therapy; an oncolytic virus; a chemotherapy; and/or an ADCC capable therapy using effector competent antibodies such as anti-CD19, anti-CD20, anti-EGFR, anti-Her2, anti-SLAMF7, anti-CD52, anti-BCMA, anti-GD2, anti-CD38, and/or anti-CCR4. In some embodiments, ADCC capable therapy is enhanced ADCC capable therapy. In some embodiments, the effector is enhanced through afucosylation, point mutations, or another method.

11.1. Parenteral Dosage Forms

In certain embodiments, provided are parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Because their administration typically bypasses subjects' natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
Excipients that increase the solubility of one or more of the antibodies disclosed herein can also be incorporated into the parenteral dosage forms.

11.2. Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which he considers most appropriate according to a preventive or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated.
The amount of the antibody or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the antibody is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
In certain embodiments, exemplary doses of a composition include milligram or microgram amounts of the antibody per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram). In certain embodiment, the dosage of the antibody provided herein, based on weight of the antibody, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. In another embodiment, the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.25 mg to 2.5 mg, 0.5 mg to 20 mg, 0.5 to 15 mg, 0.5 to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
The dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the antibody outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.
Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the antibodies provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.
In certain embodiments, treatment or prevention can be initiated with one or more loading doses of an antibody or composition provided herein followed by one or more maintenance doses.
In certain embodiments, a dose of an antibody or composition provided herein can be administered to achieve a steady-state concentration of the antibody in blood or serum of the subject. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight and age.
In certain embodiments, administration of the same composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
12. Therapeutic Applications
For therapeutic applications, the antibodies of the invention are administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above. For example, the antibodies of the invention may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, or intratumoral routes. The antibodies also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.
The antibodies provided herein may be useful for the treatment of any disease or condition involving HLA-G, such as cancer, autoimmune disease, and infection.
Any suitable cancer may be treated with the antibodies provided herein. Illustrative suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor. In some embodiments, the cancer is selected from breast, lung, CRC, gastric, esophageal, neuroblastoma, cervical, and hematological cancers.
Any suitable autoimmune disease may be treated with the antibodies provided herein. Illustrative suitable autoimmune diseases, or diseases with an autoimmune component, include, for example, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, colitis, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, inflammatory bowel disease. interstitial cystitis, juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease (chronic), Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, rimary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombotic disease, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).
Any suitable infection may be treated with the antibodies provided herein. Illustrative suitable infections include, for example, hepatitis A virus, hepatitis B virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), and other viral infections.
Some embodiments provide for treatment of a subject suffering from cancer, a chronic infection, or from an inflammatory disease, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of any of the antibodies set forth herein. Some embodiments provide for treatment of a subject suffering from cancer, a chronic infection; or from an inflammatory disease, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of any of the antibodies set forth herein in combination with an effective amount of another antibody set forth herein. In some embodiments, the cancer is a hematological cancer.
Some embodiments provide a method for modulating immune system function in a subject in need thereof, comprising the step of contacting a population of immune cells of the subject with a pharmaceutical composition comprising an effective amount of the antibody as set forth herein, under conditions such that the immune system is modulated. Some embodiments provide a method for modulating immune system function in a subject in need thereof, comprising the step of contacting a population of immune cells of the subject with an effective amount of any of the antibodies set forth herein in combination with an effective amount of another antibody set forth herein. In some embodiments, the antibody comprises a bispecific antibody or a complexing antibody.
In some embodiments, the bispecific antibody or the complexing antibody is administered in an amount sufficient to achieve 1, 2, 3, 4, 5, 6, 7, or 8 of the following in the subject:

- a) inhibition of immune suppression;
- b) reduction of levels of regulatory T cells;
- c) increase an activity of myeloid cells;
- d) increase in activity of cytotoxic T lymphocytes, NK cells, B cells, neutrophils, monocytes, macrophages, and/or dendritic cells;
- e) increase in phagocytic activity;
- f) inhibition of metastasis;
- g) inhibition of tumor growth; and/or
- h) induction of tumor regression.
  In some embodiments, the bispecific antibody binds HLA-G and HLA-E.

In some embodiments, the method for modulating immune system function in a subject in need thereof further comprises administering chemotherapy, administering radiation therapy, and/or administering one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise one or more immunostimulatory agents. In some embodiments, the one or more immunostimulatory agents comprise an antagonist to an inhibitory receptor of an immune cell. In some embodiments, the inhibitory receptor is at least one of ILT2, ILT3, ILT4, KIR2DL4, CTLA-4, PD-1, CD39, CD73, PD-L1, PD-L2, LAG-3, TIGIT, B7-H3, B7-H4, Tim3, neuritin, BTLA, CECAM-1, CECAM-5, VISTA, LAIR1, CD160, 2B4, TGF-B, NKG2A, and/or a Killer-cell immunoglobulin-like receptor (KIR).
In some embodiments, the one or more immunostimulatory agents comprise an agonist of a co-stimulatory receptor of an immune cell. In some embodiments, the co-stimulatory receptor comprises one or more of OX40, CD2, CD27, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp30, NKp46, NKp80, CD160, and/or a CD83 ligand.
In some embodiments, the one or more immunostimulatory agents comprise a cytokine. In some embodiments, the the cytokine is at least one of IL-1, IL-2, IL-5, IL-7, IL-10, IL-12, IL-15, IL-21, and/or IL-27. In some embodiments, the one or more immunostimulatory agents comprise an oncolytic virus. In some embodiments, the oncolytic virus comprises one or more of the oncolytic virus is a Herpes simplex virus, a Vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, or a maraba virus. In some embodiments, the one or more immunostimulatory agents comprise a chimeric antigen engineered T cell. In some embodiments, immunostimulatory agents comprise a bi- or multi-specific T cell directed antibody. In some embodiments, the one or more immunostimulatory agents comprises or consists of an ADCC competent antibody that may target CD19, CD20, EGFR, Her2, SLAMF7, CD52, BCMA, GD2, CD38, or CCR4. In some embodiments, the ADCC competent antibody is effector enhanced through afucosylation, point mutations, or otherwise.
In some embodiments, the one or more immunostimulatory agents comprise a bi-specific T cell engager and/or CAR-T therapy, CAR-NK therapy, CAR-macrophage therapy, adoptive T cell therapy.

13. Diagnostic Applications and Diagnostic Methods

In some embodiments, the antibodies provided herein are used in diagnostic applications and/or diagnostic methods. For example, an anti-HLA-G antibody may be useful in assays for HLA-G protein. In some aspects, the antibody can be used to detect the expression of HLA-G in various cells and tissues. In some embodiments, the antibody can be used to detect the soluble HLA-G in fluids. In some embodiments, the fluids comprise or consist of blood, serum, ascites, and/or other fluids. The assays may be useful, for example, evaluating cancer and autoimmune disease.
In some embodiments, the diagnostic method comprises or consists of detecting tumor expressed HLA-G. In some embodiments, the diagnostic method comprises or consists of detecting soluble HLA-G. In some embodiments the detection method comprises or consists of detecting HLA-G expression on immune cells.
In some diagnostic applications, the antibody may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to radioisotopes, fluorescent labels, and enzyme-substrate labels. In another embodiment of the invention, the anti-HLA-G antibody need not be labeled, and the presence thereof can be detected using a labeled antibody which specifically binds to the anti-HLA-G antibody.

14. Affinity Purification Reagents

The antibodies of the invention may be used as affinity purification agents. In this process, the antibodies may be immobilized on a solid phase such a resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the HLA-G protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the HLA-G protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the HLA-G protein from the antibody.

15. Kits

In some embodiments, an anti-HLA-G antibody provided herein is provided in the form of a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure. In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure.
In some embodiments, the kit further comprises a solvent for the reconstitution of the anti-HLA-G antibody. In some embodiments, the anti-HLA-G antibody is provided in the form of a pharmaceutical composition.

EXAMPLES

Example 1

Selection of HLA-G Antibodies

HLA-G antibodies were selected from a synthetic library of human antibodies presented on the surface of yeast cells in IgG format, as generally described, e.g., in WO2009036379; WO2010105256; WO2012009568; and Xu et al., Protein Eng. Des. Sel., 2013, 26:663-670 (each incorporated by reference in its entirety), and more specifically as provided below. The sequences and characteristics of the antibodies isolated from the recombinant library are provided in Table S.
Eight naïve human synthetic yeast libraries each of^˜10E+09 diversity were propagated as described in WO2009036379; WO2010105256; WO2012009568; and Xu et al., Protein Eng. Des. Sel., 2013, 26:663-670; each incorporated by reference in its entirety. For the first two rounds of selection, a magnetic bead sorting technique utilizing the Miltenyi MACS® system was performed, as described in Siegel et al., J. Immunol. Meth., 2004, 286:141-153. The following rounds of selection were performed using flow cytometry-based sorting. For all rounds of selection, the antigen was biotinylated human HLA-G, decreasing concentrations of antigen were used in each subsequent round of selection. In addition to selection on antigen, some rounds of selection were employed to reduce the number of non-specific binders utilizing soluble membrane proteins from CHO cells (see WO2014179363 and Xu et al., Protein Eng. Des. Sel., 2013, 26:663-670, each incorporated by reference in its entirety). In addition to the CHO cell proteins, deselections against recombinant HLA-A/B/C proteins were performed to maintain specific binding to HLA-G. After the final round of sorting, yeast were plated and individual colonies were picked for characterization and for nomination of clones for affinity maturation.
Antibody variable domains of interest were synthesized, with codon optimization to maximize transient expression in host cells. The variable regions were cloned into expression vectors containing human immunoglobulin constant domains and their sequence confirmed. Antibody heavy and light chain vector pairings were transfected into Expi293 cells using the Expifectamine system (Invitrogen). Transient cultures were harvested on day 4 and clarified cell culture supernatant IgG titer was estimated using Bio-Layer Interferometry (BLI) using Octet (ForteBio) alongside standards. Antibodies were subsequently purified on a Protein A column and eluted using low pH glycine. Purified antibody samples were then buffer-exchanged or dialyzed into downstream assay-compatible buffers.
Antibody purity was assessed by running samples on SDS-PAGE and on an analytical size exclusion chromatography column.
Light Chain Shuffling: Heavy chain plasmids were extracted from naïve outputs (described herein) and transformed into a pre-made naïve light chain library with a diversity of 10E+06. Selections were performed as described above with one round of MACS sorting and three rounds of FACS sorting using decreasing amounts of biotinylated HLA-G antigen for respective rounds.

Example 2

Affinity Maturation

Optimization of naïve clones was carried out utilizing four maturation strategies; diversification of CDR-H1 and CDR-H2; diversification of CDR-H3; diversification of CDR-L1, L2 and L3; shuffling of diversified heavy and light chains.
CDR-H1 and CDR-H2 Selection: The CDR-H3s from clones selected from light chain batch diversification, light chain diversification, and naive discovery efforts were independently recombined into premade libraries with CDR-H1 and CDR-H2 variants of a diversity of >10E+8. Selections were performed using HLA-G antigen. Affinity pressures were applied by using decreasing concentrations of antigen and HLA-G specificity was maintained with deselections against HLA-A/B/C antigens.
CDR-H3 Selection: After characterization of CDR-H1 and CDR-H2 variants, clones with binding to HLA antigens outside of HLA-G were removed. Chemical liabilities were also removed from the variable regions when applicable. The remaining clones obtained from the CDR-H1 and CDR-H2 selection procedure were subject to additional rounds of affinity maturation via walking dimer mutagenesis of the CDR-H3. Selections were performed using HLA-G as antigen generally as described above, except for employing FACS sorting for all selection rounds.
CDR-L1, L2, L3 Selection: Clones obtained from the CDR-H1 and CDR-H2 selection procedure were subject to additional rounds of affinity maturation via mutagenesis of the light chain. The CDR-L1 and CDR-L2 diversity was derived from a pre-made library while CDR-L3 diversity was derived from walking monomer mutagenesis. Selections were performed using HLA-G as antigen, starting with one round of MACS followed by three rounds of FACS in the CDR-L1, L2, L3 process described here.
Diversified Heavy Chain and Light Chain Shuffling: Outputs from CDR-H3 diversification and CDR-L1, L2, L3 diversification described above were recombined and selections were performed using HLA-G as antigen generally as described above, except for employing FACS sorting for all selection rounds.

Example 3

Binding Affinity of Anti-HLA-G Antibodies to Recombinant HLA-G

Binding kinetics were measured using the Octet Red96 system (Pall ForteBio) at 30° C. in running buffer (1× Pall ForteBio Kinetics Buffer diluted into 1× PBS pH 7.4). In brief, 0.16 μg/ml of biotinylated recombinant HLA-G/β2 m/peptide heterotrimer was loaded onto streptavidin (SA) biosensors to a binding response of approximately 0.25 nm. After a short baseline step in running buffer, the biosensors were exposed to varying concentrations (1.5, 3.0, or 30 nM) of full-length anti-HLA-G mAbs for the association step. Dissociation of the complex was monitored upon dipping the sensors to running buffer once again for up to 30 min. Data was processed using ForteBio Octet DataAnalysis software (version 10) with background subtraction of biosensors without HLA-G. A 1:1 Langmuir binding model was fit to each sensorgram to obtain association and dissociation rates via local-full or local-partial fitting. K_Dwas calculated from the ratio of k_dto k_a. Monovalent affinities were obtain using identical methods but Fabs were used instead of IgGs.
FIG. 1 provides a table showing avid and monovalent affinities of anti-HLA-G antibodies to recombinant HLA-G protein. Data shown inFIG. 1 had R²>0.98. ND=not determined using Fabs. The avid K_Dvalues ranged from 11.7 nanomolar to sub picomolar with off-rates (k_off) ranging from 0.007 sec⁻¹to the Octet off-rate limit (1.0×10⁻⁷sec⁻¹). Monovalent K_Dvalues ranged from 0.187 nM to 208 nM, but monovalent affinities for clones that had weaker avid affinities were not determined.

Example 4

Antibodies That Bind to HLA-G and Bin Separately From Other Antibodies

Epitope binning assays to recombinant HLA-G heterotrimer were determined using the Octet Red96 system (Pall ForteBio) at 30° C. in running buffer (lx Pall ForteBio Kinetics Buffer diluted into lx PBS pH 7.4). Briefly, 0.16 μg/ml of biotinylated recombinant HLA-G/132m/peptide heterotrimer was loaded onto streptavidin (SA) biosensors to a binding response of approximately 0.25 nm. After a short baseline step in running buffer, the biosensor was dipped into 20 μg/ml of each mAb and continued until the binding response to HLA-G reached saturation. For the competing association step, equimolar concentrations of each mAb to the saturating mAb was used.
Epitope binning was performed in each direction (every mAb was used as both the saturating mAb (association step 1) as well as the competing mAb (association step 2)). Self-blocking sensorgrams are shown in gray. Non-self sensograms are shown in black; black sensorgrams represent the pairwise binning mAb. Sensorgrams from only the second association step were shown inFIG. 2.
FIG. 2A provides biolayer interferometry sensorgrams showing tested antibodies that bind HLA-G can be divided into three distinct chemical bins, with one chemical bin being further divided. Gray sensorgrams indicate self-blocking; black sensorgrams indicate pairwise binning. The y-axis show saturating antibody; the x-axis shows competing antibody.
FIG. 2B provides biolayer sensorgrams showing antibodies (Fabs) that bind HLA-G and can be separated into two sub-bins from a broader bin 1. Bin 1a Fabs do not block competing bin 1b Fabs. However, bin 1b Fabs block bin 1a Fabs. As described above, gray sensorograms indicated self-blocking and black sensorograms represent pairwise binning. The y-axis represents the saturating Fab and the x-axis represents the competing Fab.

Example 5

Anti-HLA-G Antibodies That Block HLA-G Interaction/Binding to Recombinant ILT2 and ILT4

Blocking assays between recombinant HLA-G heterotrimer and recombinant extracellular domain of ILT2 or ILT4 proteins was determined using the Octet Red96 system (Pall ForteBio) at 30° C. in running buffer 1× Pall ForteBio Kinetics Buffer diluted into 1× PBS pH 7.4). Briefly, 0.16 μg/ml of biotinylated recombinant HLA-G432m/peptide heterotrimer was loaded onto streptavidin (SA) biosensors to a binding response of approximately 0.25 nm. After a short baseline step in running buffer, the biosensor was dipped into at least 200 nM of each mAb (up to 0.5 μM for weakly binding mAbs) and continued until the binding response to HLA-G reached saturation. A second association step immediately followed by dipping into wells containing dimerized ILT2 or ILT4. Dimers were obtained via pre-incubation of the His-tagged ILT2 or ILT4 with anti-His mAb (MAB050, R&D Systems). ILT2/ILT4 binding response to mAb-saturated HLA-G (black sensorgrams) was compared to HLA-G in the absence of anti-HLA-G mAb (gray sensorgrams).
Sensorgrams from the second association step are shown inFIG. 3. The provided sensorgrams show antibodies that bind and fully block HLA-G interaction with ILT2 and ILT4, for example, the binla and binlb antibodies 38381 and 38358, respectively. As indicated, gray sensorgrams indicate ILT2 or ILT4 binding in the absence of antibody; black sensorgrams represent ILT2 or ILT4 binding in the presence of antibody.
FIG. 3 also provides biolayer interferometry sensorgrams showing antibodies that bind but do not block HLA-G interaction with ILT2 or ILT4, for example, the bin 3 antibodies such as 33357. Gray sensorgrams indicate ILT2 or ILT4 binding response in the absence of antibody; black sensorgrams indicate ILT2 or ILT4 binding in the presence of antibody.
The provided sensorgrams also show antibodies that bind and block HLA-G interaction with ILT2 or ILT4 to an intermediate effect, such as the bin 2 antibody 38389.

Example 6

Anti-HLA-G Antibodies Bind to HLA-G Naturally Expressed on JEG-3 Cells

JEG-3 parentals or HLA-G-knock out (JEG-3 KO) cells were incubated with 100 nM of anti-HLA-G antibodies for 2 hours at 4° C. in wash buffer (Phosphate Buffered Saline (PBS), 2% Fetal bovine serum (FBS) and 2 mM EDTA). Cells were then washed in wash buffer and incubated with an APC conjugated secondary antibody (goat anti-human IgG Kappa) at a 1:500 dilution in wash buffer for 30 minutes at 4° C. Cells were subsequently washed and resuspended in 1% paraformaldehyde/wash buffer followed by sample acquisition using a BD Fortessa X-20 flow cytometer (Becton Dickinson). Sample data was exported as FCS files and analyzed using FlowJo software v10 (Tree Star, Inc.).
The results are shown inFIG. 4. The shaded histogram represents binding of an isotype control to JEG-3 cells at the same concentration.

EXAMPLE 7

Anti-HLA-G Antibodies Abrogate Suppression Mediated Through HLA-G-ILT2 or ILT4 Interaction in NKL Killing Assay

Inhibition of NKL cytotoxicity via the ILT2-HLA-G interaction or ILT4-HLA-G interaction is abrogated by anti-HLA-G antibodies. NKL (Effector) cells were co-incubated with 721.221 (Target) cells+/−HLA-G expression at an E:T ratio of 4:1 overnight at 37° C. 721.221s were pre-incubated with 200 nanomolar (nM) Fab fragments or full-length intact anti-HLA-G antibodies for 60 minutes at 37° C. Prior to antibody pre-treatment, 721.221 cells were labelled with 1:1000 CellTrace Violet (CTV) (Invitrogen) in 37° C. PBS.
After overnight co-incubation, cells were washed once in 4° C. wash buffer (Phosphate Buffeed Saline, 2% FBS, 2 mM EDTA) and then stained with 1:2000 Fixable Viability Dye eFluor™ 780 (Invitrogen) 30 minutes at 4° C. Cells were washed again in 4° C. wash buffer resuspended in wash buffer and analyzed for percent dead 721.221 cells by flow cytometry analysis on a BD Celesta flow cytometer. Percent of dead 721.221 cells was determined by gating on CTV positive cells, then dividing the Fixable Viability Dye positive portion of these cells by the total CTV count.
Antibody MEM-G/9 is a commercially available antibody (See, Fournel et al., Tissue Antigens 200: 55: 510-518 (1999), which is incorporated by reference in its entirety herein, including any drawings).
FIG. 5A (Fab fragments) andFIG. 5B (intact IgG) show the effect on NKL cells that endogenously express ILT2. The percent of dead target cells is shown for each antibody (indicated as a unique clone number in the figure) in addition to control values representing minimum and maximum killing in the presence and absence of HLA-G, respectively. As can be seen fromFIG. 5, Bin 1 antibodies show the best blockade of all the antibodies tested.
FIG. 5C demonstrates the dose response of a specific Bin 1 anti-HLA-G Fab fragment for blockade of HLA-G-mediated suppression of NKL killing through endogenously expressed ILT2.
FIG. 5D demonstrates the dose response of a specific Bin 1 anti-HLA-G Fab fragment for blockade of HLA-G-mediated suppression of NKL killing through engineered expression of ILT4 (engineered cell line modified by knocking down ILT2 and retrovirally transduced to express ILT4).

Example 8

Fab Fragments of Anti-HLA-G Antibodies Reverse HLA-G Mediated Suppression of Macrophage Phagocytosis, NK Cell Cytotoxicity, and CDs⁺ T Cell Activity

CD14⁺ enriched cells were differentiated into adherent macrophages by incubating at 37° C., 5% CO₂for 7 days in complete RPMI (cRPMI) with recombinant human M-CSF. Cells were harvested after 7 days, washed, and resuspended in cRPMI. Cells were plated in a 96-well round bottom plate at 50,000 cells/well in 50 μl cRPMI and incubated at 37° C., 5% CO₂, before being combined with the target cells.
The target cells, consisting of either 721.221 parental cells or 721.221-HLA-G, were stained with CTV (1:1000) at 37° C. in PBS, washed and plated in cRPMI at 25,000 cells per well. Cells were subsequently incubated with anti-CD47 antibody with or without anti-HLA-G Fabs for 1 hour at 37° C., 5% CO₂. The mixture of 721.221 and antibody was then combined with macrophages and incubated for either 2 hours or overnight at 37° C., 5% CO₂. The final ratio of macrophage to 721.221 target cell was 2:1. After either the 2 hour or overnight incubation, cells were blocked with wash buffer/BD Fc Block^TM/2% mouse serum for 20 minutes and then stained with anti-CD11b and live/dead discrimination dye for 20 minutes. Cells were then washed, resuspended in wash buffer and analyzed on the BD Fortessa flow cytometer. Sample data was exported as FCS files and analyzed using FlowJo software v10.
In the 2-hour format assay, macrophages were assayed for the presence of CTV as a readout of phagocytic uptake of CTV⁺ target cells. Live cells were gated on CD11b^±/CTV⁺ and reported as a percent of total live macrophages. For the overnight assay, macrophage killing activity was assessed by measuring absolute counts of remaining live CTV cells per well.
FIG. 6A compares results between the two hour and overnight assay formats using an anti-HLA-G antibody (Fab fragment). The overnight assay achieves similar results as the two hour assay that directly measures phagocytosis of target cells.FIG. 6B shows the results of Fab fragments of each anti-HLA-G antibody that were tested in the overnight assay to determine the level of restored phagocytic activity.
To demonstrate inhibition of primary human NK cell cytotoxicity is abrogated by an anti-HLA-G antibody, HLA-G expressing target cells (721.221-HLA-G) were pre-incubated with arepresentative Bin 1 antibody (Fab fragment) and then co-cultured with human primary NK (Effector) cells from 3 separate donors at an E:T ratio of 4:1 overnight at 37° C. Prior to antibody pre-treatment, 721.221 cells were labelled with 1:1000 CellTrace Violet (CTV) (Invitrogen) in 37° C. PBS.
After overnight co-incubation, cells were washed once in 4° C. wash buffer (Phosphate Buffered Saline, 2% FBS, 2 mM EDTA) and then stained with 1:2000 Fixable Viability Dye eFluor 780 (Invitrogen) for 30 minutes at 4° C. Cells were washed, resuspended in wash buffer, and analyzed for percent dead 721.221 cells by flow cytometry analysis on a BD Celesta flow cytometer. Percent of dead 721.221 cells was determined by gating on CTV positive cells, then dividing the Fixable Viability Dye positive portion of these cells by the total CTV count.
The results are shown inFIG. 6C. The percent of dead target cells is shown for theBin 1 antibody (Fab) in addition to control values representing co-cultures with 721.221 target cells with and without HLA-G expression (noted as HLA-G and no HLA-G in figure, respectively). An isotype control Fab (Ctrl Fab) was used as a negative control.
To demonstrate an anti-HLA-G antibody reverses HLA-G mediated suppression of cytotoxic primary CD8⁺ T cell function, human CD8⁺ T cells treated with CD3/CD28 activator (ImmunoCultTM, Stem Cell Technologies) for 1 hour were mixed with antibody pre-treated 721.221 cells (with and without expression of HLA-G) and co-cultured overnight at 37° C. After overnight co-incubation, Monensin Solution (ThermoFisher) and eFluor 450-CD107a (ThermoFisher) were added; the final amount of each reagent was 0.5 μg/mL antibody and 2 μM Monensin. The assay was incubated for 4 hours.
After the incubation, cells were washed and stained over several steps which included the following: (1) live/dead cell staining using Fixable Viability Dye eFluor 780 (ThermoFisher); (2) Fc blocking with staining buffer (Phosphate Buffered Saline, 2% FBS, 2 mM EDTA) containing 6% normal mouse serum and 5 μL Human TruStain FcX™ per sample (Biolegend) for 15 minutes at 4° C.; (3) CD8 and ILT2 staining with Alexa Fluor 700-CD8 (Biolegend, Clone SK1) and PE-CD85j/ILT2 (Biolegend, Clone HP-F1, ThermoFisher); Fixation and permeabilization using fix/perm reagent (ThermoFisher); (5) Intracellular staining with Brilliant Violet 711-IFN-γ (Biolegend, Clone 4S.B3) and APC-TNF-α (Biolegend, Clone MAb11) diluted in Permeabilization Buffer (ThermoFisher) for 20 minutes at 4° C. After the staining steps, cells were washed, centrifuged, and resuspended in staining buffer followed by sample acquisition on a BD Fortessa X-20 flow cytometer using the high throughput sampler.
FCS data was analyzed using FlowJoversion 10 analysis software (FlowJo, LLC). Gates were made to select single cells, live cells, and the cell type of interest. CD8⁺ T cell populations were defined based on CD8⁺ILT2⁺ or CD8⁺ILT2⁻. To determine the percent of cells positive for CD107a, IFN-γ, or TNF-α, gating was performed on each CD8⁺ T cell population and determined using fluorescence minus one cells and unstimulated cells.
Results depicted inFIG. 6D demonstrate that blockade of HLA-G with an anti-HLA-G antibody (representative Bin 1 clone) restored CD107a, IFN-γ, and TNF-α expression in CD8⁺ ILT2⁺ T cells in a dose-dependent manner. There was no effect from HLA-G target cell expression or anti-HLA-G antibody treatment on CD8⁺ ILT2⁻ cells (data not shown).

Example 9

Anti-HLA-G Antibodies That are Selective Binders to HLA-G and Do Not Bind to Classical HLA Class I Antigens

Binding of antibodies to HLA antigens (97 different antigens spanning HLA-A, -B, and -C haplotypes) was determined using the LABScreen single antigen class I- combi assay (One Lambda). Antibodies at concentrations greater than 300 nM were incubated with single antigen LABScreen beads in binding buffer (Phosphate Buffered Saline, 10% FBS, 2 mM EDTA) for 45 minutes in the dark at room temperature with gentle shaking. Beads were washed twice with 200 μl , of 1× LABScreen wash buffer in a 96-well V-bottom plate by centrifugation at 1500 g and removal of buffer by aspiration. Beads were then incubated with a PE-conjugated secondary antibody (polyclonal goat anti-human or anti-mouse in 1× wash buffer) for 30 minutes at room temperature in dark with gentle shaking. After incubation, beads were washed twice, resuspended in 90 μL of wash buffer, and then transferred to a 96-well flat bottom plate. Acquisition was performed on a Luminex 100 instrument and data output as a CSV file. Results for each single HLA antigen bead group were tabulated from median fluorescent intensity values.
Antibody 87G is a commercially available antibody and represents a blocking antibody (See, Odum et al., Eur. J. Immunol. 22: 2121-2131 (1991) and Lee et al., Immunity, 591-600Volume 3, Issue 5 (1995), each of which is incorporated by reference in its entirety herein, including any drawings).
The results are shown inFIG. 7. Binding values (median fluorescent signal) to each single HLA antigen are shown for each anti-HLA-G antibody.

Example 10

Anti-HLA-G Antibodies that Do Not Bind to Classical HLA Class Antigens Expressed at Physiological Levels on B-LCL Cells

Binding of antibodies to physiological levels of native HLA class Ia proteins was assessed by flow cytometry on a collection of twenty-eight B-LCL cell lines (shown in Table X). Cells were obtained from the International Histocompatibility Working Group cell line collection that holds a repository of B-LCL cell lines which have been HLA-typed. The cells were maintained in supplemented RPMI-1640 media containing 15% fetal bovine serum.
For antibody binding, anti-HLA antibodies were first conjugated to DyLight 650 (Thermo Fisher) by primary amine labeling via the NHS-ester moiety. Unconjugated dye was removed by flowing the antibody preparation through dye removal resin. Conjugation efficiency was between 1 to 3 moles of DyLight 650 per mole of antibody. B-LCL cell lines (ranging from 10,000 to 100,000 cells per well) were incubated with 300 nM of labeled antibodies in cold PBS containing 10% FBS and human Fc block (BD Biosciences). After 2 hours at 4° C., cells were washed twice in cold wash buffer (Phosphate Buffered Saline, 2% FBS, 2 mM EDTA) followed by sample acquisition on a BD FACS Celesta instrument. To assess overall HLA class I expression, a pan HLA class I antibody, clone W6/32, conjugated to FITC was incubated separately with each individual cell line. All cell lines were strongly positive for class I expression (data not shown). Data was exported as FCS files and analyzed using FlowJo software v10. Gating was performed on live singlet cells using DAPI for live-dead discrimination. Geometric MFI is shown for each B-LCL cell line.

TABLE X

	/	Workshop

IHW ID	Alternate ID	Sex	Population Group	Involvement	HLA A*	HLA B*	HLA Cw*

IHW09367	LCK		Asian	13	0203	1102	4601	380201	0702	1202
IHW09458	FH70EY	M		13	3002	6802	0801	5301	0401	07
IHW09383	FH9	M	Other	13	2402	3303	4801	44032	0801	0701
IHW09397	DUG150		Unknown	13	02	680101	5802	4501	0602	1601
IHW09057	TEM	M	Jewish	10, 12	6601		3801		1203
IHW09010	AMAI	M	Algerian	10, 12	6802		5301		0401
IHW09436	FH48	M	Caucasian	13	2402	0201	4427	070201	070201	0704
IHW01167	1413-1090	M	Caucasian	CEPH	680102	0201	0801	4402	0701	0501
IHW01075	1344-8354	F	Caucasian	CEPH	2402	0301	4001	5101	030401	0102
IHW01185	1416-1337	M	Caucasian	CEPH	0205	2501	4901	4402	0701	0501
IHW09431	FH43	M	American Black	13	3001	3301	5301	8101	04	08
IHW01124	1362-8563	M	Caucasian	CEPH	0201	2902	3501	4403	0401	1601
IHW01133	1362-8572	F	Caucasian	CEPH	2902	2601	4403	4402	1601	0501
IHW01092	1346-8603	M	Caucasian	CEPH	0201	1101	0702	3501	0702	0401
IHW09371	ISH4		Asian	13	0218	1101	1501	4601	0102	040101
IHW09433	FH45	M	Asian	13	1101	02	3802	4101	0702	17
IHW09380	FH6	M	Hispanic	13	2402	2901	2702	0705	1505	020202
IHW09411	FH29	F	Hispanic	13	0201	2902	1516	440301	1402	1601
IHW09401	TER-259		USA White/American Indian	13	3201	6802	0801	44020101	0102	07
IHW09398	FH18	F	American Black	13	3601	7401	5301	5703	0401	0701
IHW09382	FH8	F	Am. Black	13	110101	3402	8201	270502	0302	0102
IHW01026	1332-8257	F	Caucasian	CEPH	0301	3001	3501	0702	0401	0702
IHW09409	FH27	M	Native American	13	310102	3401	350101	380201	0401	0702
IHW09452	FH64		Unknown	13	02	3201	44	1801	05	07
IHW01028	1332-8259	F	Caucasian	CEPH	0301	0301	3501	2705	0401	020202
IHW01061	1341-8465	F	Caucasian	CEPH	0201	680102	0702	0702	0702	0702
IHW01099	1347-8434	M	Caucasian	CEPH	2501	0101	0801	1501	0701	0602
IHW01136	1362-8575	M	Caucasian	CEPH	0101	1101	0702	5101	0702	1502

indicates data missing or illegible when filed

The results are shown inFIG. 8. Binding levels are provided as geometric MFI. Class I-negative 721.221 cell lines with and without expression of HLA-G were used as controls.

Example 11

Critical HLA-G Amino Acid Determinants Required for HLA-G Antibody Binding

721.221 LCL cells transiently expressing various point mutants of HLA-G were established using the Neon® Transfection System (ThermoFisher Scientific). Cells were transfected with 1 μg of plasmid per point mutanin the 10 μL reaction format. Cells were cultured for 72 hours prior to staining with anti-HLA-G antibodies.
721.221 LCL cells (expressing various point mutants or a domain swap where the primary amino acid sequence encoding thealpha 3 domain was substituted with the analogous sequence from HLA-A*1101) were incubated with each antibody (100 nM) in wash buffer (Phosphate Buffered Saline, 2% FBS, 2 mM EDTA) for 60 minutes at 4° C. Cells were then washed with cold wash buffer and incubated with fluorescently labelled secondary antibody at 4° C. for 30 minutes. Numbered antibodies were incubated with 1:500 R-Phycoerythrin labelled goat-anti-human IgG (Jackson ImmunoResearch). W6/32 was incubated with 1:500 Alexa Fluor 488 goat-anti-mouse IgG (Jackson ImmunoResearch). MEMG/9 (Invitrogen) was pre-conjugated with Allophycocyanin. After incubation with secondary antibody, cells were washed and resuspended in cold wash buffer prior to analysis by flow cytometry on a BD Celesta instrument. Sample data was exported as FCS files and analyzed using FlowJo software v10. As shown inFIG. 9, mutation of residues 195, 197, and/or 198 results in loss of binding bybin 1 anti-HLA-G antibodies, revealing those residues are critical, though unlikely to be the only determinants forbin 1 antibody binding. Intriguingly, the biochemical bin defined by Octet as bin la (FIG. 2B) can be further distinguished based on either sensitivity to mutation at residue 197 (for example, 38379) or mutation at residue 198 (for example, 38410)
FIG. 9A shows the evaluation of HLA-G antibodies binding to various forms of HLA-G after site directed mutagenesis.FIG. 9B provides representative antibody binding histograms for each of the three point mutants (F195S, Y197A, Y197H, E198A).

Example 12

Tumor Growth Inhibition for an Ant-HLA-G Antibody With and Without Effector Function in a Mouse Xenograft Model Using 721.221 Cells Expressing HLA-G

NOD/SCID mice were implanted subcutaneously with 10 million HLA-G expressing 721.221 cells in Matrigel on the right flank. On day 29 post-implantation, mice were randomized into groups based on tumor volume and dosed intraperitoneally with 300 μg of either an effector competent anti-HLA-G human IgG1 antibody (hIgG1 WT), the same anti-HLA-G antibody with mutations iin the Fc domain to abrogate Fc receptor interactions (hIgGl_AAA), or left untreated. A second dose of antibody was given three days later. Data provided inFIG. 10 represents the percent change in tumor volume six days after initial treatment. These results demonstrate that an anti-HLA-G antibody can inhibit the growth of tumors expressing HLA-G by engaging Fc receptor dependent effector mechanisms, which may include antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP).

Example S

Sequences

Table S provides sequences referred to herein.

TABLE S

Sequences.

SEQ ID NO:	Region	Scheme/Clone	Sequence

1	CDR-H1	Chothia	GGSISSDY

2	CDR-H1	Chothia	GGSISSSST

3	CDR-H1	Chothia	GGSISSSDT

4	CDR-H1	Chothia	GGSISSADN

5	CDR-H1	Chothia	GGSVSSSST

6	CDR-H1	Chothia	GYSISSGY

7	CDR-H1	Chothia	GYSILSGY

8	CDR-H1	Chothia	GYSISSGH

9	CDR-H1	Chothia	GYSISSGF

10	CDR-H1	Chothia	GFTFDNY

11	CDR-H1	Chothia	GFTFSDY

12	CDR-H1	Chothia	GFTFSSS

13	CDR-H1	Chothia	GGSISSSN

14	CDR-H1	Chothia	GGSISSSSY

15

16

17

18	CDR-H1	Ka bat	SSDYYWG

19	CDR-H1	Kabat	SSSTYWA

20	CDR-H1	Ka bat	SSSTYWG

21	CDR-H1	Kabat	SSDTYWG

22	CDR-H1	Ka bat	SADNYWG

23	CDR-H1	Kabat	SSSTYWS

24	CDR-H1	Kabat	SGYYWG

25	CDR-H1	Kabat	SGYYWF

26	CDR-H1	Kabat	SGHYWI

27	CDR-H1	Kabat	SGFYWT

28	CDR-H1	Kabat	SGYYWL

29	CDR-H1	Kabat	SGHYWT

30	CDR-H1	Kabat	NYAMH

31	CDR-H1	Kabat	DYYMS

32	CDR-H1	Kabat	SSAMA

33	CDR-H1	Kabat	SSNWWS

34	CDR-H1	Kabat	SSSYYWG

35

36

37

38	CDR-H2	Chothia	YYSGS

39	CDR-H2	Chothia	SSSGS

40	CDR-H2	Chothia	HHSGA

41	CDR-H2	Chothia	HYSGS

42	CDR-H2	Chothia	AYSGS

43	CDR-H2	Chothia	SYNAL

44	CDR-H2	Chothia	YHSGS

45	CDR-H2	Chothia	YHSAS

46	CDR-H2	Chothia	SARAGI

47	CDR-H2	Chothia	ASSGSV

48	CDR-H2	Chothia	SGSGIT

49	CDR-H2	Chothia	SYSGS

50	CDR-H2	Chothia	SSSGST

51

52

53

54	CDR-H2	Kabat	SIYYSGSTYYNPSLKS

55	CDR-H2	Kabat	SISSSGSTYYNPSLKS

56	CDR-H2	Kabat	SIHHSGATYYNPSLKS

57	CDR-H2	Kabat	SIHYSGSTLYNPSLKS

58	CDR-H2	Kabat	SIHYSGSTYYNPSLKS

59	CDR-H2	Kabat	GIAYSGSTYYNPSLKS

60	CDR-H2	Kabat	SISYNALTYYNPSLKS

61	CDR-H2	Kabat	SIYHSGSTYYNPSLKS

62	CDR-H2	Kabat	GIYHSASTAYNPSLKS

63	CDR-H2	Kabat	GIYHSGSTYYNPSLKS

64	CDR-H2	Kabat	AIYHSGSTVYNPSLKS

65	CDR-H2	Kabat	GIYHSGSTAYNPSLKS

66	CDR-H2	Kabat	AISARAGITYYADSVKG

67	CDR-H2	Kabat	YIASSGSVIYYADSVKG

68	CDR-H2	Kabat	TISGSGITTWYADSVKG

69	CDR-H2	Kabat	EIYHSGSTNYNPSLKS

70	CDR-H2	Kabat	SISYSGSTYYNPSLKS

71	CDR-H2	Kabat	YISSSGSTIYYADSVKG

72

73

74

75

76	CDR-H3		GVRRAVPFDY

77	CDR-H3		GIARAVPFDY

78	CDR-H3		GPKRAVPFDY

79	CDR-H3		GVRRAVPFVD

80	CDR-H3		GVRRAVPFQR

81	CDR-H3		GTRRAVPFDY

82	CDR-H3		GVRRAVPFAD

83	CDR-H3		GIRRAVPFDY

84	CDR-H3		GQFRAVPFDY

85	CDR-H3		GGTHTYSRGPMDV

86	CDR-H3		GGTHTYSRGPFDV

87	CDR-H3		GGTPIYSRGPLDV

88	CDR-H3		GGGQTYSRGPLDV

89	CDR-H3		GGGATYSRGPLDV

90	CDR-H3		GGTHTYSRGPLDV

91	CDR-H3		GGTVKYSRGPLDV

92	CDR-H3		GGQVTYSRGPLDV

93	CDR-H3		GGEVTYSRGPLDV

94	CDR-H3		RIGYSYGTAPPFDV

95	CDR-H3		HGTPRAFDI

96	CDR-H3		GSRHLNAFNR

97	CDR-H3		GVYHYDPYGMDV

98	CDR-H3		TELGKMHFDY

99	CDR-H3		GSPRYMQD

100	CDR-H3		HSSLGTHNWFDP

101	CDR-H3		EGALSYSWLAAFDI

102

103

104

105	CDR-L1		RASQSVSSSYLA

106	CDR-L1		GASQSVSSDYLA

107	CDR-L1		QASQAVSSNYLA

108	CDR-L1		GASQSVSSAFLA

109	CDR-L1		RASQSVSSTYLA

110	CDR-L1		QASQSVSSSYLA

111	CDR-L1		KASQAVSSSYLA

112	CDR-L1		EASQSVSSSYLA

113	CDR-L1		EASQSVSASYLA

114	CDR-L1		EASQSVSSAYLA

115	CDR-L1		RVSQSVSDAYLA

116	CDR-L1		EVSQSVSASYLA

117	CDR-L1		RASQSVSSAYLA

118	CDR-L1		RASNAVSSSYLA

119	CDR-L1		RASQSINSWLA

120	CDR-L1		AASQGISSDLA

121	CDR-L1		RASQDISTYLN

122	CDR-L1		RSSQSLLHSNGYNYLD

123	CDR-L1		RASQSISSYLN

124	CDR-L1		RASQSVSSNLA

125

126

127

128	CDR-L2		GASSRAT

129	CDR-L2		GAYSLAT

130	CDR-L2		GASARAT

131	CDR-L2		GASSREA

132	CDR-L2		GASNRAA

133	CDR-L2		GASSRQD

134	CDR-L2		GASNRAT

135	CDR-L2		DASSRAS

136	CDR-L2		DASTRAT

137	CDR-L2		GASDRAN

138	CDR-L2		GASYRAT

139	CDR-L2		DASSLES

140	CDR-L2		SASSTQS

141	CDR-L2		DAFNLET

142	CDR-L2		LGSNRAS

143	CDR-L2		GASRRAT

144	CDR-L2		AASSLQS

145	CDR-L2		GASTRAT

146

147

148

149	CDR-L3		QQAVHSPYT

150	CDR-L3		QWAVHSPYT

151	CDR-L3		QQVVHSPYT

152	CDR-L3		QQTVHSPYT

153	CDR-L3		QQAIHSPYT

154	CDR-L3		QQHSSYPPT

155	CDR-L3		QQHSLYPPT

156	CDR-L3		QQFSSYPPT

157	CDR-L3		QQVSSYPPT

158	CDR-L3		QQHSIYPPT

159	CDR-L3		QQYDSHIT

160	CDR-L3		QQAYLYPIT

161	CDR-L3		QQLPFLPIT

162	CDR-L3		MQALGGPWT

163	CDR-L3		QQYVSDPIT

164	CDR-L3		QQVGSSPIT

165	CDR-L3		QQSHLVPRT

166	CDR-L3		QQANHHPPFT

167

168

169

170	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSS

171	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWAWIRQPPGKGLEWIGSISSSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIARAVPFDYWGQGTLVTVSS

172	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWGWIRQSPGKGLEWIGSIHHSGATYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GPKRAVPFDYWGQGTLVTVSS

173	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFVDWGQGTLVTVSS

174	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSAD
			NYWGWIRQPPGKGLEWIGSIHYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFQRWGQGTLVTVSS

175	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGPEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSS

176	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSS

177	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGSISYNALTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSS

178	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFADWGQGTLVTVSS

179	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSVSSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSS

180	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSS

181	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIRRAVPFDYWGQGTLVTVSS

182	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GQFRAVPFDYWGQGTLVTVSS

183	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPMDVWGQGTTVTVSS

184	VH		QLQLQESGPRLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPFDVWGQGTTVTVSS

185	VH		LVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTPIYSRGPLDVWGQGTTVTVSS

186	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKDQFSLKLSSVTAADTAVYYCARG
			GGQTYSRGPLDVWGQGTTVTVSS

187	VH		QVQLQESGPGLVKPPETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GGATYSRGPLDVWGQGTTVTVSS

188	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSS

189	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGF
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSS

190	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWLWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTVKYSRGPLDVWGQGTTVTVSS

191	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GQVTYSRGPLDVWGQGTTVTVSS

192	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GEVTYSRGPLDVWGQGTTVTVSS

193	VH		EVQLLESGGGLVQPGGSLRLSCAASGFTFDNYA
			MHWVRQAPGKGLEWVSAISARAGITYYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR
			IGYSYGTAPPFDVWGQGTTVTVSS

194	VH		QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYIASSGSVIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			GTPRAFDIWGQGTTVTVSS

195	VH		EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSA
			MAWVRQAPGKGLEWVSTISGSGITTWYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG
			SRHLNAFNRWGQGTTVTVSS

196	VH		QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSN
			WWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKS
			RVTISVDKSKNQFSLKLSSVTAADTAVYYCARG
			VYHYDPYGMDVWGQGTTVTVSS

197	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			YYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			TELGKMHFDYWGQGTLVTVSS

198	VH		QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GSPRYMQDWGQGTLVTVSS

199	VH		QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			SSLGTHNWFDPWGQGTLVTVSS

200	VH		QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
			GALSYSWLAAFDIWGQGTMVTVSS

201

202

203

204	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIK

205	VL		EIVLTQSPGTLSLSPGERATLSCGASQSVSSDY
			LAWYQQKPGQAPRLLIYGAYSLATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQWAVHSPYTF
			GGGTKVEIK

206	VL		EIVLTQSPGTLSLSPGERATLSCQASQAVSSNY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIK

207	VL		EIVLTQSPGTLSLSPGERATLSCGASQSVSSAF
			LAWYQQKPGQAPRLLIYGASARATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIK

208	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSTY
			LAWYQQKPGQAPRLLIYGASSREAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQTVHSPYTF
			GGGTKVEIK

209	VL		EIVLTQSPGTLSLSPGERATLSCQASQAVSSNY
			LAWYQQKPGQAPRLLIYGASNRAAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAIHSPYTF
			GGGTKVEIK

210	VL		EIVLTQSPGTLSLSPGERATLSCQASQSVSSSY
			LAWYQQKPGQAPRLLIYGASNRAAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIK

211	VL		EIVLTQSPGTLSLSPGERATLSCKASQAVSSSY
			LAWYQQKPGQAPRLLIYGASSRQDGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIK

212	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSSYPPTF
			GGGTKVEIK

213	VL		EIVLTQSPGTLSLSPGERATLSCEASQSVSSSY
			LAWYQQKPGQAPRLLIYGASNRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIK

214	VL		EIVLTQSPGTLSLSPGERATLSCEASQSVSASY
			LAWYQQKPGQAPRLLIYDASSRASGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQFSSYPPTF
			GGGTKVEIK

215	VL		EIVLTQSPGTLSLSPGERATLSCEASQSVSSAY
			LAWYQQKPGQAPRLLIYDASTRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVSSYPPTF
			GGGTKVEIK

216	VL		EIVLTQSPGTLSLSPGERAALSCRVSQSVSDAY
			LAWYQQKPGQAPRLLIYDASSRASGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVSSYPPTF
			GGGTKVEIK

217	VL		EIVLTQSPGTLSLSPGERATLSCEVSQSVSASY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIK

218	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSAY
			LAWYQQKPGQAPRLLIYGASDRANGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIK

219	VL		EIVLTQSPGTLSLSPGERATLSCRASNAVSSSY
			LAWYQQKPGQAPRLLIYGASDRANGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSIYPPTF
			GGGTKVEIK

220	VL		EIVLTQSPGTLSLSPGERATLSCRASNAVSSSY
			LAWYQQKPGQAPRLLIYGASYRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIK

221	VL		DIQMTQSPSTLSASVGDRVTITCRASQSINSWL
			AWYQQKPGKAPKLLISDASSLESGVPSRFSGSG
			SGTEFTLTISSLQPDDFATYYCQQYDSHITFGG
			GTKVEIK

222	VL		DIQMTQSPSSVSASVGDRVTITCAASQGISSDL
			AWYQQKPGKAPKLLIYSASSTQSGVPSRFSGSG
			SGTDFTLTISSLQPEDFATYYCQQAYLYPITFG
			GGTKVEIK

223	VL		GVQMTQSPSSLSASVGDRVTITCRASQDISTYL
			NWYQQKPGKAPKLLIYDAFNLETGVPSRFSGSG
			SGTDFTFTISSLQPEDIATYYCQQLPFLPITFG
			GGTKVEIK

224	VL		DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSN
			GYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDR
			FSGSGSGTDFTLKISRVEAEDVGVYYCMQALGG
			PWTFGGGTKVEIK

225	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASRRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQYVSDPITF
			GGGTKVEIK

226	VL		EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASRRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVGSSPITF
			GGGTKVEIK

227	VL		DIQMTQSPSSLSASVGDRVTITCRASQSISSYL
			NWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG
			SGTDFTLTISSLQPEDFATYYCQQSHLVPRTFG
			GGTKVEIK

228	VL		EIVMTQSPATLSVSPGERATLSCRASQSVSSNL
			AWYQQKPGQAPRLLIYGASTRATGIPARFSGSG
			SGTEFTLTISSLQSEDFAVYYCQQANHHPPFTF
			GGGTKVEIK

229

230

231

232	IGG1 AAA HC	33343	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

233	IGG1 AAA HC	37268	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWAWIRQPPGKGLEWIGSISSSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIARAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK
234	IGG1 AAA HC	37269	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWGWIRQSPGKGLEWIGSIHHSGATYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GPKRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

235	IGG1 AAA HC	37271	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFVDWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSNEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

236	IGG1 AAA HC	37272	QLQLQESGPGLVKPSETLSLTCTVSGGSISSAD
			NYWGWIRQPPGKGLEWIGSIHYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFQRWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

237	IGG1 AAA HC	37277	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGPEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

238	IGG1 AAA HC	38373	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

239	IGG1 AAA HC	38375	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGSISYNALTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

240	IGG1 AAA HC	38379	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFADWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

241	IGG1 AAA HC	38381	QLQLQESGPGLVKPSETLSLTCTVSGGSVSSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

242	IGG1 AAA HC	38383	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

243	IGG1 AAA HC	38386	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

244	IGG1 AAA HC	37273	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GQFRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

245	IGG1 AAA HC	33361	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPMDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

246	IGG1 AAA HC	35624	QLQLQESGPRLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPFDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

247	IGG1 AAA HC	38410	LVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTPIYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

248	IGG1 AAA HC	38418	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKDQFSLKLSSVTAADTAVYYCARG
			GGQTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

249	IGG1 AAA HC	38420	QVQLQESGPGLVKPPETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GGATYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

250	IGG1 AAA HC	38421	QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

251	IGG1 AAA HC	38422	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGF
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

252	IGG1 AAA HC	38424	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWLWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTVKYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

253	IGG1 AAA HC	38425	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GQVTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

254	IGG1 AAA HC	38426	QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GEVTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
			PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEV
			TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
			EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
			KALPAPIEKTISKAKGQPREPQVYTLPPSREEM
			TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
			KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
			CSVMHEALHNHYTQKSLSLSPGK

255	IGG1 AAA HC	37323	EVQLLESGGGLVQPGGSLRLSCAASGFTFDNYA
			MHWVRQAPGKGLEWVSAISARAGITYYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR
			IGYSYGTAPPFDVWGQGTTVTVSSASTKGPSVF
			PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
			SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
			LGTQTYKNVNHKPSNTICVDKKVEPKSCDKTHT
			CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPE
			VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
			REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
			NKALPAPIEKTISKAKGQPREPQVYTLPPSREE
			MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
			YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
			SCSVMHEALHNHYTQKSLSLSPGK

256	IGG1 AAA HC	38389	QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYIASSGSVIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			GTPRAFDIWGQGTTVTVSSASTKGPSVFPLAPS
			SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
			SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
			YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
			APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVV
			VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
			NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
			APIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
			VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
			PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
			HEALHNHYTQKSLSLSPGK

257	IGG1 AAA HC	38358	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSA
			MAWVRQAPGKGLEWVSTISGSGITTWYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG
			SRHLNAFNRWGQGTTVTVSSASTKGPSVFPLAP
			SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
			TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
			TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
			PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
			YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
			PAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

258	IGG1 AAA HC	33303	QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSN
			WWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKS
			RVTISVDKSKNQFSLKLSSVTAADTAVYYCARG
			VYHYDPYGMDVWGQGTTVTVSSASTKGPSVFPL
			APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG
			ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
			TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
			PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVT
			CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
			EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
			ALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
			KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
			TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
			SVMHEALHNHYTQKSLSLSPGK

259	IGG1 AAA HC	33342	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			YYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			TELGKMHFDYWGQGTLVTVSSASTKGPSVFPLA
			PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
			CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
			QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
			LPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

260	IGG1 AAA HC	33299	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GSPRYMQDWGQGTLVTVSSASTKGPSVFPLAPS
			SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
			SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
			YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
			APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVV
			VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
			NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
			APIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
			VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
			PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
			HEALHNHYTQKSLSLSPGK

261	IGG1 AAA HC	33351	QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			SSLGTHNWFDPWGQGTLVTVSSASTKGPSVFPL
			APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG
			ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
			TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
			PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVT
			CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
			EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
			ALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
			KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
			TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
			SVMHEALHNHYTQKSLSLSPGK

262	IGG1 AAA HC	33357	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
			GALSYSWLAAFDIWGQGTMVTVSSASTKGPSVF
			PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
			SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
			LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
			CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPE
			VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
			REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
			NKALPAPIEKTISKAKGQPREPQVYTLPPSREE
			MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
			YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
			SCSVMHEALHNHYTQKSLSLSPGK

263

264

265

266	IGG4 HC	33343	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYNGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTINDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

267	IGG4 HC	37268	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWAWIRQPPGKGLEWIGSISSSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIARAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

268	IGG4 HC	37269	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWGWIRQSPGKGLEWIGSIHHSGATYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GPKRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

269	IGG4 HC	37271	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLSLSSVTAADTAVYYCAR
			GVRRAVPFVDWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

270	IGG4 HC	37272	QLQLQESGPGLVKPSETLSLTCTVSGGSISSAD
			NYWGWIRQPPGKGLEWIGSIHYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFQRWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

271	IGG4 HC	37277	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGPEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

272	IGG4 HC	38373	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

273	IGG4 HC	38375	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGSISYNALTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

274	IGG4 HC	38379	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFADWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

275	IGG4 HC	38381	QLQLQESGPGLVKPSETLSLTCTVSGGSVSSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GVRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

276	IGG4 HC	38383	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			TYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GTRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

277	IGG4 HC	38386	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GIRRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

278	IGG4 HC	37273	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			TYWGWIRQPPGKGLEWIGSIHYSGSTLYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GQFRAVPFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

279	IGG4 HC	33361	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPMDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

280	IGG4 HC	35624	QLQLQESGPRLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPFDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

281	IGG4 HC	38410	LVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTPIYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

282	IGG4 HC	38418	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKDQFSLKLSSVTAADTAVYYCARG
			GGQTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

283	IGG4 HC	38420	QVQLQESGPGLVKPPETLSLTCAVSGYSISSGH
			YWIWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GGATYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

284	IGG4 HC	38421	QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

285	IGG4 HC	38422	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGF
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTHTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

286	IGG4 HC	38424	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWLWIRQPPGKGLEWIGGIYHSASTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GTVKYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTEN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

287	IGG4 HC	38425	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGH
			YWTWIRQPPGKGLEWIGAIYHSGSTVYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GQVTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

288	IGG4 HC	38426	QVQLQESGPGLVKPSETLSLTCAVSGYSILSGY
			YWFWIRQPPGKGLEWIGGIYHSGSTAYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
			GEVTYSRGPLDVWGQGTTVTVSSASTKGPSVFP
			LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
			GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
			GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
			PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
			VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
			FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
			PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN
			QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
			PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
			MHEALHNHYTQKSLSLSPGK

289	IGG4 HC	37323	EVQLLESGGGLVQPGGSLRLSCAASGFTFDNYA
			MHWVRQAPGKGLEWVSAISARAGITYYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR
			IGYSYGTAPPFDVWGQGTTVTVSSASTKGPSVF
			PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
			SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
			LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
			CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
			QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG
			LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

290	IGG4 HC	38389	QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYIASSGSVIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			GTPRAFDIWGQGTTVTVSSASTKGPSVFPLAPC
			SRSTSESTAALGCLVKDYFPEPVTVSWNSGALT
			SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT
			YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
			FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
			SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
			YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
			EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
			TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
			DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
			LHNHYTQKSLSLSPGK

291	IGG4 HC	38358	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSA
			MAWVRQAPGKGLEWVSTISGSGITTWYADSVKG
			RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG
			SRHLNAFNRWGQGTTVTVSSASTKGPSVFPLAP
			CSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
			TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
			TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
			EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
			VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
			TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
			IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS
			LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
			LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
			ALHNHYTQKSLSLSPGK

292	IGG4 HC	33303	QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSN
			WWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKS
			RVTISVDKSKNQFSLKLSSVTAADTAVYYCARG
			VYHYDPYGMDVWGQGTTVTVSSASTKGPSVFPL
			APCSRSTSESTAALGCLVKDYFPEPVTVSWNSG
			ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
			TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
			APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV
			VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF
			NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP
			SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
			VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
			PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
			HEALHNHYTQKSLSLSPGK

293	IGG4 HC	33342	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSS
			YYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			TELGKMHFDYWGQGTLVTVSSASTKGPSVFPLA
			PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
			LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
			KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
			PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
			DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
			STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
			SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
			SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH
			EALHNHYTQKSLSLSPGK

294	IGG4 HC	33329	QLQLQESGPGLVKPSETLSLTCTVSGGSISSSD
			YYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLK
			SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
			GSPRYMQDWGQGTLVTVSSASTKGPSVFPLAPC
			SRSTSESTAALGCLVKDYFPEPVTVSWNSGALT
			SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT
			YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
			FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
			SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
			YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
			EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
			TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
			DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
			LHNHYTQKSLSLSPGK

295	IGG4 HC	33351	QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY
			MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKG
			RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARH
			SSLGTHNWFDPWGQGTLVTVSSASTKGPSVFPL
			APCSRSTSESTAALGCLVKDYFPEPVTVSWNSG
			ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
			TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
			APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV
			VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF
			NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP
			SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
			VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
			PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
			HEALHNHYTQKSLSLSPGK

296	IGG4 HC	33357	QVQLQESGPGLVKPSETLSLTCAVSGYSISSGY
			YWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKS
			RVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
			GALSYSWLAAFDIWGQGTMVTVSSASTKGPSVF
			PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
			SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
			LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
			CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC
			VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
			QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG
			LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK
			NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
			TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
			VMHEALHNHYTQKSLSLSPGK

297

298

299

300	LC	33343	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

301	LC	37268	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

302	LC	37269	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

303	LC	37271	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

304	LC	37272	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

305	LC	37277	EIVLTQSPGTLSLSPGERATLSCGASQSVSSDY
			LAWYQQKPGQAPRLLIYGAYSLATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQWAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

306	LC	38373	EIVLTQSPGTLSLSPGERATLSCQASQAVSSNY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

307	LC	38375	EIVLTQSPGTLSLSPGERATLSCGASQSVSSAF
			LAWYQQKPGQAPRLLIYGASARATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

308	LC	38379	EIVLTQSPGTLSLSPGERATLSCRASQSVSSTY
			LAWYQQKPGQAPRLLIYGASSREAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQTVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

309	LC	38381	EIVLTQSPGTLSLSPGERATLSCQASQAVSSNY
			LAWYQQKPGQAPRLLIYGASNRAAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAIHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

310	LC	38383	EIVLTQSPGTLSLSPGERATLSCQASQSVSSSY
			LAWYQQKPGQAPRLLIYGASNRAAGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

311	LC	38386	EIVLTQSPGTLSLSPGERATLSCKASQAVSSSY
			LAWYQQKPGQAPRLLIYGASSRQDGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

312	LC	37273	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQAVHSPYTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

313	LC	33361	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSSYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

314	LC	35624	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSSYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

315	LC	38410	EIVLTQSPGTLSLSPGERATLSCEASQSVSSSY
			LAWYQQKPGQAPRLLIYGASNRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

316	LC	38418	EIVLTQSPGTLSLSPGERATLSCEASQSVSASY
			LAWYQQKPGQAPRLLIYDASSRASGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQFSSYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

317	LC	38420	EIVLTQSPGTLSLSPGERATLSCEASQSVSSAY
			LAWYQQKPGQAPRLLIYDASTRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVSSYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

318	LC	38421	EIVLTQSPGTLSLSPGERAALSCRVSQSVSDAY
			LAWYQQKPGQAPRLLIYDASSRASGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVSSYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

319	LC	38422	EIVLTQSPGTLSLSPGERATLSCEVSQSVSASY
			LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

320	LC	38424	EIVLTQSPGTLSLSPGERATLSCRASQSVSSAY
			LAWYQQKPGQAPRLLIYGASDRANGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

321	LC	38425	EIVLTQSPGTLSLSPGERATLSCRASNAVSSSY
			LAWYQQKPGQAPRLLIYGASDRANGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSIYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

322	LC	38426	EIVLTQSPGTLSLSPGERATLSCRASNAVSSSY
			LAWYQQKPGQAPRLLIYGASYRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQHSLYPPTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

323	LC	37323	DIQMTQSPSTLSASVGDRVTITCRASQSINSWL
			AWYQQKPGKAPKLLISDASSLESGVPSRFSGSG
			SGTEFTLTISSLQPDDFATYYCQQYDSHITFGG
			GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV
			CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
			DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
			GLSSPVTKSFNRGEC

324	LC	38389	DIQMTQSPSSVSASVGDRVTITCAASQGISSDL
			AWYQQKPGKAPKLLIYSASSTQSGVPSRFSGSG
			SGTDFTLTISSLQPEDFATYYCQQAYLYPITFG
			GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV
			VCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
			QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
			QGLSSPVTKSFNRGEC

325	LC	38358	GVQMTQSPSSLSASVGDRVTITCRASQDISTYL
			NWYQQKPGKAPKLLIYDAFNLETGVPSRFSGSG
			SGTDFTFTISSLQPEDIATYYCQQLPFLPITFG
			GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV
			VCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
			QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
			QGLSSPVTKSFNRGEC

326	LC	33303	DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSN
			GYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDR
			FSGSGSGTDFTLKISRVEAEDVGVYYCMQALGG
			PWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
			GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
			ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
			CEVTHQGLSSPVTKSFNRGEC

327	LC	33342	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASRRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQYVSDPITF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

328	LC	33299	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY
			LAWYQQKPGQAPRLLIYGASRRATGIPDRFSGS
			GSGTDFTLTISRLEPEDFAVYYCQQVGSSPITF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

329	LC	33351	DIQMTQSPSSLSASVGDRVTITCRASQSISSYL
			NWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG
			SGTDFTLTISSLQPEDFATYYCQQSHLVPRTFG
			GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV
			VCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
			QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
			QGLSSPVTKSFNRGEC

330	LC	33357	EIVMTQSPATLSVSPGERATLSCRASQSVSSNL
			AWYQQKPGQAPRLLIYGASTRATGIPARFSGSG
			SGTEFTLTISSLQSEDFAVYYCQQANHHPPFTF
			GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
			VVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
			EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
			HQGLSSPVTKSFNRGEC

331

332

333

334	Fc for IGG1		ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
			PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
			SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
			PKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKD
			TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
			EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
			GKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
			YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
			ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
			SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

335	Fc region for		ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
	IGG4		PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
			SVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE
			SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM
			ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
			NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
			YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
			PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN
			GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
			QEGNVFSCSVMHEALHNHYTQKSLSLSPGK

336	Kappa region		RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY
	for LC		PREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
			SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
			KSFNRGEC

337		hHLA-G1	MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFS
			AAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSAC
			PRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDR
			MNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGR
			LLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQ
			ISKRKCEAANVAEQRRAYLEGTCVEWLHRYLEN
			GKEMLQRADPPKTHVTHHPVFDYEATLRCWALG
			FYPAEIILTWQRDGEDQTQDVELVETRPAGDGT
			FQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLR
			WKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAV
			LWRKKSSD

338		hHLA-G5	MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFS
			AAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSAC
			PRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDR
			MNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGR
			LLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQ
			ISKRKCEAANVAEQRRAYLEGTCVEWLHRYLEN
			GKEMLQRADPPKTHVTHHPVFDYEATLRCWALG
			FYPAEIILTWQRDGEDQTQDVELVETRPAGDGT
			FQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLR
			WSKEGDGGIMSVRESRSLSEDL

339		Cyno HLA-AG	MAVMAPRTLLLVLSGVLALTQPRAGSHSMRYFY
			TAVSRPGRGQPRFIAVGYVDDTQFVRFDSDAES
			PRMEPRAPWVEQEGPEYWDRETQNMKTATQTYQ
			ANLRTLLRYYNQSEAGSHTFQKMYGCDLGPDGR
			LLRGYEQFAYDGRDYIILNEDLRSWTAADMAAQ
			NTQRKWEAAGAAEQHRTYLEGECLEWLRRYLEN
			GKETLQRADPPKTNVTHHPVSDYEATLRCWALG
			FYPAEITLTWQRDGEEQTEDTELVETRPTGDGT
			FQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR
			WEPSSQSTILIVGIIAGLVLLGTVVTGAVVAAV
			MWRRKS

340		Rhesus HLA-AG	MAVMAPRTLLLVLSGVLALTQTRAGSHSMRYFY
			TSMSRPGRGQPRFIAVGYVDDTQFVRFDSDAES
			PRMEPRAPWVEQEGPEYWDRETQNMKTATQTYR
			ENLRTLLRYYNQSEAGSHTIQKMYGCDLGPDGR
			LLRGYEQFAYDGRDYIALNEDLRSWTAADMAAQ
			FTQRKWEAAGAAEQHRTYLEGECLEWLRRYLEN
			GKETLQRADPPKTNVTHHPVSDYEATLRCWALG
			FYPAEITLTWQRDGEEQTEDTELVETRPTGDGT
			FQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLR
			WEPSSQSTILIVGIIAGLVLLGTVVTGAVVAAV
			MWRRKSSDR

341		hHLA-G ECD	MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFS
		with signal	AAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSAC
		peptide	PRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDR
			MNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGR
			LLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQ
			ISKRKCEAANVAEQRRAYLEGTCVEWLHRYLEN
			GKEMLQRADPPKTHVTHHPVFDYEATLRCWALG
			FYPAEIILTWQRDGEDQTQDVELVETRPAGDGT
			FQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLR
			W

342		hHLA-G ECD	GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQF
		without signal	VRFDSDSACPRMEPRAPWVEQEGPEYWEEETRN
		peptide	TKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMI
			GCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRS
			WTAADTAAQISKRKCEAANVAEQRRAYLEGTCV
			EWLHRYLENGKEMLQRADPPKTHVTHHPVFDYE
			ATLRCWALGFYPAEIILTWQRDGEDQTQDVELV
			ETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHE
			GLPEPLMLRW

Equivalents

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

Claims

What is claimed is:

1. An antibody that binds specifically to a human HLA-G (hHLA-G) and is capable of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the following:

a) inhibiting an HLA-G immune suppressive function;

b) blocking HLA-G interaction and/or binding to ILT2;

c) blocking HLA-G interaction and/or binding to ILT4;

d) blocking HLA-G interaction and/or binding to KIR2DL4;

e) inhibiting HLA-G mediated suppression of NK cells;

f) inhibiting HLA-G mediated suppression of cytotoxic T lymphocytes;

g) inhibiting HLA-G mediated suppression of B cells;

h) inhibiting HLA-G mediated suppression of neutrophils;

i) inhibiting HLA-G mediated suppression of monocytes;

j) inhibiting HLA-G mediated suppression of macrophages;

k) inhibiting HLA-G mediated suppression of dendritic cells;

l) inhibiting HLA-G mediated suppression of NK and/or T cell cytolysis and/or proliferation;

m) inhibiting HLA-G suppression of myeloid cells;

n) inhibiting HLA-G mediated suppression of phagocytosis;

o) inhibiting the HLA-G mediated generation, expansion, or function of T regulatory cells;

p) inhibiting metastasis; or

q) inhibiting tumor growth by antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP).

2. The antibody ofclaim 1, wherein the antibody has 1, 2, 3, 4, 5, 6, or 7 of the following characteristics:

a) is a monoclonal antibody;

b) is a human antibody, a humanized antibody, or a chimeric antibody;

c) is a bispecific antibody, a multi-specific antibody, a diabody, or a multivalent antibody;

d) is of the IgG1, IgG2, IgG3, IgG4, or IgM type;

e) is an antigen-binding antibody fragment;

f) is a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, or an Fv fragment; and/or g) is a single chain antibody, a single domain antibody, or a nanobody.

3. A pharmaceutical composition comprising an effective amount of an antibody which binds to hHLA-G and has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the following of the following characteristics:

a) inhibiting an HLA-G immune suppressive function;

b) blocking HLA-G interaction and/or binding to ILT2;

c) blocking HLA-G interaction and/or binding to ILT4;

d) blocking HLA-G interaction and/or binding to KIR2DL4;

e) inhibiting HLA-G mediated suppression of NK cells;

f) inhibiting HLA-G mediated suppression of cytotoxic T lymphocytes;

g) inhibiting HLA-G mediated suppression of B cells;

h) inhibiting HLA-G mediated suppression of neutrophils;

i) inhibiting HLA-G mediated suppression of monocytes;

j) inhibiting HLA-G mediated suppression of macrophages;

k) inhibiting HLA-G mediated suppression of dendritic cells;

I) inhibiting HLA-G mediated suppression of NK and/or T cell cytolysis and/or proliferation;

m) inhibiting HLA-G suppression of myeloid cells;

n) inhibiting HLA-G mediated suppression of phagocytosis;

o) inhibiting the HLA-G mediated generation or expansion of T regulatory cells;

p) inhibiting metastasis; or q) inhibiting tumor growth by antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP).

4. A pharmaceutical composition comprising the antibody ofclaim 1 orclaim 2.

5. The pharmaceutical composition ofclaim 4, further comprising an effective amount of at least one of the following

a) an anti-ILT2 antibody;

b) an anti-ILT3 antibody;

c) an anti-ILT4 antibody;

d) an anti-KIR2DL4 antibody;

e) an anti-HLA-E antibody;

f) an anti-NKG2A antibody

f) an anti-HLA-F antibody

f) an anti-PD-L1 antibody;

g) an anti-PD-1 antibody;

h) an anti-CD38 antibody;

i) an anti-CD39 antibody;

j) an anti-CD73 antibody;

k) an anti-A2A receptor antibody;

I) an anti-A2B receptor antibody;

m) an anti-A2A/A2B dual receptor antibody or a combination thereof;

n) an anti-CD47 antibody;

o) a small molecule inhibitor;

p) a bi-specific T cell engager and/or CAR-T therapy and or CAR-NK therapy, CAR-Macrophage therapy

q) an oncolytic virus;

r) a chemotherapy;

s) ADCC capable therapies using effector (enhanced or otherwise) competent antibodies such as anti-CD19, anti-CD20, anti-EGFR, anti-Her2, anti-SLAMF7, anti-CD52, anti-BCMA, anti-GD2, or anti-CCR4

6. The pharmaceutical composition ofclaim 4 orclaim 5, further comprising one or both of

a) an antibody to an immune inhibitory receptor or ligand and/or

b) an antibody to an immune stimulatory receptor or ligand.

7. The antibody ofclaim 1, wherein the antibody binds to a human HLA-G polypeptide or a variant thereof with a KD of less than about 20 nM.

8. An antibody that competes or is capable of competing for binding to human HLA-G with a reference antibody, wherein the reference antibody is the antibody set forth inclaim 1.

9. An antibody that binds to, or is capable of competing for binding to, human HLA-G with a reference antibody, wherein the reference antibody binds to an epitope at position 195, 197, and/or 198 of SEQ ID NO: 342 on a human HLA-G polypeptide.

10. The antibody ofclaim 1, comprising a human heavy chain constant region or fragment or a variant thereof and/or a light chain constant region or fragment or variant thereof, wherein the constant region or fragment of variant thereof comprises up to 20 conservatively modified amino acid substitutions from any sequence set forth SEQ ID NOS: 170-200 and/or SEQ ID NOS: 204-228.

11. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and a light chain variable region (VL), the V_Hand/or V_Lcomprising 1, 2, 3, 4, 5, or 6 of:

a) a VHCDR1 having the sequence set forth in SEQ ID NOS: 1-14 or SEQ ID NOS: 18-34,

b) a VHCDR2 having the sequence set forth in SEQ ID NOS: 38-50 or SEQ ID NOS: 54-71,

c) a VHCDR3 having the sequence set forth in SEQ ID NOS: 76-101,

d) a VLCDR1 having the sequence set forth in SEQ ID NOS: 105-124,

e) a VLCDR2 having the sequence set forth in SEQ ID NOS: 128-145, and

f) a VLCDR3 having the sequence set forth in SEQ ID NOS: 149-166.

12. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and a light chain variable region (VL), the V_Hcomprising:

a) a VHCDR1 having a sequence set forth in SEQ ID NOS: 1-14 or SEQ ID NOS: 18-34,

b) a VHCDR2 having a sequence set forth in SEQ ID NOS: 38-50 or SEQ ID NOS: 54-71, and

c) a VHCDR3 having a sequence set forth in SEQ ID NOS: 76-101; and

the V_Lcomprising:

a) a VLCDR1 having a sequence set forth in SEQ ID NO: 105-124,

b) a VLCDR2 having a sequence set forth in SEQ ID NO: 128-145, and

c) a VLCDR3 having a sequence set forth in SEQ ID NO: 149-166.

13. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), the V_Hcomprising at least one sequence set forth in any of SEQ ID NOS: 170-200 and the V_Lcomprising at least one sequence set forth in any of SEQ ID NOS: 204-228.

14. An isolated nucleic acid encoding an antibody according toclaim 1,claim 12, orclaim 13.

15. An expression vector comprising the nucleic acid according toclaim 14.

16. A prokaryotic or eukaryotic host cell comprising the vector ofclaim 15.

17. An oncolytic virus encoding the nucleic acid of any ofclaims 14-16.

18. A method for the production of a recombinant protein comprising the steps of expressing a nucleic acid according toclaim 14 orclaim 15 in a prokaryotic or eukaryotic host cell and recovering the protein from the cell or the cell culture supernatant.

19. A method for treatment of a subject suffering from cancer, a chronic infection, or from an inflammatory disease, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of the antibody ofclaim 1 or the pharmaceutical composition ofclaim 3.

20. A method for treatment of a subject suffering from cancer, a chronic infection, or from an inflammatory disease, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of the antibody ofclaim 1 or the pharmaceutical composition ofclaim 3 in combination with an antibody or a pharmaceutical composition comprising an effective amount of:

a) an anti-ILT2 antibody;

b) an anti-ILT3 antibody;

c) an anti-ILT4 antibody;

d) an anti-KIR2DL4 antibody;

e) an anti-HLA-E antibody;

an anti-NKG2A antibody

g) an anti-HLA-F antibody

h) an anti-PD-L 1 antibody;

i) an anti-PD-1 antibody;

j) an anti-CD38 antibody;

k) an anti-CD39 antibody;

1) an anti-CD73 antibody;

m) an anti-A2A receptor antibody;

n) an anti-A2B receptor antibody;

o) an anti-A2A/A2B dual receptor antibody and/or a combination;

p) an anti-CD47 antibody;

q) an anti-CTLA-4 antibody;

r) an anti-LAG3 antibody;

s) an anti-TIM-3 antibody;

t) an anti-TIGIT antibody;

u) an anti-VISTA antibody;

w) an anti-CD94 antibody;

x) a small molecule inhibitor;

y) a bi-specific T cell engager, CAR-T therapy, CAR-NK therapy, CAR-Macrophage therapy, engineered cell therapy, and/or adaptive T cell therapy

z) an oncolytic virus;

aa) a chemotherapy; and/or

ab) an ADCC capable therapy using effector competent antibodies such as anti-CD19, anti-CD20, anti-EGFR, anti-Her2, anti-SLAMF7, anti-CD52, anti-BCMA, anti-GD2, and/or anti-CCR4.

21. The method ofclaim 19 orclaim 20, wherein the cancer is a solid cancer.

22. The method ofclaim 21, wherein the cancer is a hematological cancer.

23. A method for modulating immune system function in a subject in need thereof, comprising the step of contacting a population of immune cells of the subject with a pharmaceutical composition comprising an effective amount of the antibody ofclaim 1, under conditions such that the immune system is modulated.

24. The method ofclaim 22, wherein the subject is a human subject.

25. The method ofclaim 23, wherein the antibody comprises a bispecific antibody or a complexing antibody.

26. The method ofclaim 25, wherein the antibody, the bispecific antibody, or the complexing antibody is administered in an amount sufficient to achieve 1, 2, 3, 4, 5, 6, or 7 of the following in the subject:

a) inhibition of immune suppression;

b) reduction of levels of regulatory T cells;

c) increase in activity of myeloid cells, cytotoxic T lymphocytes, NK cells, B cells, neutrophils, monocytes, macrophages, and/or dendritic cells;

d) increase in phagocytic activity;

e) inhibition of metastasis;

f) inhibition of tumor growth; and/or

g) induction of tumor regression.

27. The method ofclaim 20, wherein the method further comprises one or more of the following:

a) administering chemotherapy;

b) administering radiation therapy; and/or

c) administering one or more additional therapeutic agents.

28. The method ofclaim 27, wherein the one or more additional therapeutic agents comprise one or more immunostimulatory agents.

29. The method ofclaim 28, wherein the one or more immunostimulatory agents comprise an antagonist to an inhibitory receptor of an immune cell.

30. The method ofclaim 29, wherein the inhibitory receptor is at least one of ILT2, ILT3, ILT4, KIR2DL4, CTLA-4, PD-1, CD39, CD73, PD-L1, PD-L2, LAG-3, Tim3, TIGIT, B7-H3, B7-H4, neuritin, BTLA, CECAM-1, CECAM-5, VISTA, LAIR1, CD160, 2B4,TGF-B NKG2A, and/or a Killer-cell immunoglobulin-like receptor (KIR).

31. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise an agonist of a co-stimulatory receptor of an immune cell.

32. The method ofclaim 31, wherein the co-stimulatory receptor is at least one of OX40, CD2, CD27, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp30, NKp46, NKp80, CD160, and/or a CD83 ligand.

33. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise or consist of an ADCC competent antibody comprising or consisting of an anti-CD19, anti-CD20, anti-EGFR, anti-Her2, anti-SLAMF7, anti-CD52, anti-BCMA, anti-GD2, anti-CD38, and/or or anti-CCR4 antibody.

34. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise a cytokine.

35. The method ofclaim 34, wherein the cytokine is at least one of IL-1, IL-2, IL-5, IL-7, IL-10, IL-12, IL-15, IL-21, and/or IL-27.

36. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise an oncolytic virus.

37. The method ofclaim 36, wherein the oncolytic virus is a Herpes simplex virus, a Vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, or a maraba virus.

38. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise a chimeric antigen engineered T cell.

39. The method ofclaim 29, wherein the one or more immunostimulatory agents comprise a bi- or multi-specific T cell directed antibody.

40. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and a light chain variable region (VL), the V_Hcomprising 1, 2, or 3 of:

a) a VHCDR1 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 1-14 or 18-34,

b) a VHCDR2 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 54-71, and

c) a VHCDR3 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 76-101; and

the V_Lcomprising 1, 2, or 3 of:

a) a VLCDRI having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 105-124,

b) a VLCDR2 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 128-145, and

c) a VLCDR3 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOs 149-166.

41. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and a light chain variable region (VL),

V_Hcomprising 1, 2, or 3 of:

a) a VHCDRI having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 1-14 or SEQ ID NOS: 18-34,

b) a VHCDR2 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 38-50 or SEQ ID NOS: 54-71, and

c) a VHCDR3 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 76-101; and

VL comprising 1, 2, or 3 of:

a) a VLCDR1 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 105-124,

b) a VLCDR2 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 128-145, and

c) a VLCDR3 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 149-166.

42. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain and a light chain, the heavy chain comprising one or more molecules having a sequence consisting of one of SEQ ID NOS: 232-262 or SEQ ID NOS: 266-296 and the light chain comprising one or more molecules having a sequence consisting of one of SEQ ID NOS: 300-330.

43. An isolated antibody molecule capable of binding to human HLA-G (HLA-G), comprising a heavy chain and a light chain,

a) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 232 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 300;

b) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 233 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 301;

c) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 234 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 302;

d) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 235 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 303;

e) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 236 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 304;

f) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 237 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 305;

g) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 238 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 306;

h) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 239 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 307;

i) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 240 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 308;

j) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 241 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 309;

k) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 242 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 310;

l) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 243 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 311;

m) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 244 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 312;

n) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 245 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 313;

o) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 246 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 314;

p) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 247 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 315;

q) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 248 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 316;

r) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 249 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 317;

s) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 250 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 318;

t) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 251 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 319;

u) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 252 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 320;

v) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 253 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 321;

w) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 254 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 322;

x) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 255 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 323;

y) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 256 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 324;

z) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 257 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 325;

aa) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 258 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 326;

ab) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 259 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 327;

ac) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 260 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 328;

ad) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 261 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 329;

ae) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 262 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 330;

af) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 266 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 300;

ag) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 267 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 301;

ah) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 268 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 302;

ai) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 269 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 303;

aj) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 270 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 304;

ak) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 271 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 305;

al) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 272 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 306;

am) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 273 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 307;

an) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 274 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 308;

ao) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 275 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 309;

ap) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 276 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 310;

aq) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 277 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 311;

ar) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 278 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 312;

as) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 279 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 313;

at) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 280 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 314;

au) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 281 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 315;

av) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 282 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 316;

aw)the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 283 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 317;

ax) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 284 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 318;

ay) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 285 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 319;

az) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 286 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 320;

ba) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 287 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 321;

bb) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 288 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 322;

bc) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 289 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 323;

bd) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 290 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 324;

be) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 291 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 325;

bf) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 292 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 326;

bg) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 293 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 327;

bh) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 294 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 328;

bi) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 295 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 329; or

bj) the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 296 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 330.

44. An isolated nucleic acid encoding an antibody according to any of claimsclaim 40-43.

45. An isolated antibody molecule capable of binding to human HLA-G (hHLA-G), comprising a heavy chain variable region (VH) and a light chain variable region (VL), V_Hand/or V_Lcomprising 1, 2, 3, 4, 5, Or 6 of:

a) a VHCDR1 sequence comprising:

(i) Kabat CDR-H1 sequence defined by the consensus sequence S-S-Δ₃-Δ₄-Y-W-Δ₇(SEQ ID NOS: 18-21, 23, and 34), where Δ₃is D or S; Δ₄is T or Y; and Δ₇is A, G, or S;

(ii) a Kabat CDR-H1 sequence defined by the consensus sequence S-G-θ₃-Y-W-θ₆(SEQ ID NOS: 24-29), where θ₃is F, H, or Y; and θ₆is F, G, I, L, or T;

(iii) a Chothia CDR-H1 sequence defined by the consensus sequence G-G-S-I-S-S-θ₇Ω₈-Ω₉(SEQ ID NOS: 1-4 and 13-14), where Ω₇is S or A; Ω₈is D, S, or N; and Ω₉is T, N, Y, or is absent; or

(iv) a Chothia CDR-H1 sequence defined by the consensus sequence G-F-T-F-κ5-κ6-κ7 (SEQ ID NOS: 10-12), where κ₅is D or s; κc6 is D, N, or S; and κ₇is S or Y,

b) a VHCDR2 sequence comprising:

(i) a Kabat CDR-H2 sequence defined by the consensus sequence β₁-I-β₃-β₄-β₅-β₆-β₇-T-β₉-Y-N-P-S-L-K-S (SEQ ID NOS: 54-65 and 69-70) where β₁is A, E, G, or S; β₃is A, H, S, or Y; β₄is H, S, or Y; β₅is N or S; β₆is A or G; β₇is A, L, or S; and β₉A, N, L, V, or Y;

(ii) a Chothia CDR-H2 sequence defined by the consensus sequence Y-ε₂-S-ε₄-S (SEQ ID NOS: 38 and 44-45), where ε2 is H or Y and ε₄is A or G;

(iii) a Chothia CDR-H2 sequence defined by the consensus sequence α1-α2-S-G-S (SEQ ID NOS: 39, 41-42, and 49), where ai is A, H, or S; and az is S or Y; or

(iv) a Chothia CDR-H2 sequence defined by the consensus sequence β₁-β₂-S-G-β₅-β₆(SEQ ID NOS: 56-60), where β₁is A or S; β₂is G or S; β₅is I or S; and β₆is T or V,

c) a VHCDR3 sequence comprising:

(i) a CDR-H3 sequence defined by the consensus sequence G-y₂-y₃-R-A-V-P-F-y₉-y₁₀(SEQ ID NOS: 76-84), where y₂is I, P, Q, T, or V; y₃is A, F, K, or R; y₉is A, D, Q, or V; y₁₀is D, R, or Y; or

(ii) a CDR-H3 sequence defined by the consensus sequence G-G-Φ₃-Φ₄-Φ₅-Y-S-R-G-P-101₁₁-D-V (SEQ ID NOS: 85-93), where Φ₃is E, G, Q, or T; is A, H, P, Q, or V; Φ₅is K or T; and Φ₁₁is F, L, or M,

d) a VLCDR1 sequence comprising:

(i) a CDR-L1 sequence defined by the consensus sequence ϕ₁-A-S-Q-ϕ₅-V-S-S-ϕ₉-ϕ₁₀-L-A (SEQ ID NOS: 105-112 and 117), where ϕ₁is E, G, K, Q, or R; ϕ₅is A or S; ϕ₉is A, D, N, S, or T; and ϕ₁₀is F or Y;

(ii) a CDR-L1 sequence defined by the consensus sequence R-A-S-Q-S-

₆-

₇-S-

₉-L-

₁₁(SEQ ID NOS: 119 and 123-124), where

₆is I or V;

₇is N or S;

₉is N, W, or Y;

₁₁is A or N; or

(iii) a CDR-L1 sequence defined by the consensus sequence Γ₁-Γ₂-S-Q-S-V-S-Γ₈-Γ₉-Y-L-A (SEQ ID NOS: 113-116), where Γ₁is E or R; Γ₂is A or V; Γ₈is A, D, or S; and Γ₁₀is A or S,

e) a VLCDR2 sequence comprising:

(i) a CDR-L2 sequence defined by the consensus sequence ψ₁-A-S-ψ₄-R-A-ψ₇(SEQ ID NOS: 128, 130, 132, 134-138, 143, and 145), where ψ₁is D or G; wa is A, D, N, R, S, T, or Y; and ψ₇is A, N, S, or T, and

f) a VLCDR3 sequence comprising:

(i) a CDR-L3 sequence defined by the consensus sequence Q-π₂-π₃-π₄-H-S-P-Y-T (SEQ ID NOS: 149-153), where π₂is Q or W; π₃is A, T, or V; and π₄is I or V;

(ii) a CDR-L3 sequence defined by the consensus sequence Q-Q-λ₃-S-λ₅-Y-P-P-T (SEQ ID NOS: 154-158), where λ₃is F, H, or V; and λ₅is I, L, or S; or

(iii) a CDR-L3 sequence defined by the consensus sequence Q-Q-ω₃-ω₄-ω₅-ω₆-P-I-T (SEQ ID NOS: 160-161 and 163-164), where co3 is A, L, V, or Y; ω₄is G, P, V, or Y; ω₅is S, L, or F; and ω₆is D, L, S, or Y.