HLA antibody products and methods

Abstract

The disclosure relates to antibody products specific for human leukocyte antigens and related methods. The products can be used in diagnosis and methods of treatment of disease, such as in methods of diagnosing and treating Graft Versus Host Disease (GVHD).

Description

HLA ANTIBODY PRODUCTS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Provisional Application No. 63 / 705,824, filed October 10, 2024, which is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (Filename: 70613P_SeqListing.xml; 21,025 bytes - XML file created October 10, 2024) which is incorporated by reference herein in its entirety.FIELD

[0003] The disclosure relates to antibody products specific for human leukocyte antigens and related methods. The products can be used in diagnosis and methods of treatment of disease, such as in methods of diagnosing and treating Graft Versus Host Disease (GVHD).BACKGROUND

[0004] Graft-versus-host disease (GVHD) is a severe complication of allogenic transplantation. GVHD occurs when immunocompetent T lymphocytes from a donor graft recognize the recipient's tissues as foreign due to histocompatibility differences and initiate an immune response against the recipient's tissues. This attack typically occurs within the first 100 days post-transplant, leading to tissue damage in various organs, including the skin, gastrointestinal tract, liver, and lungs.

[0005] GVHD arises because the cells of the immune system are trained to differentiate between self and non-self cells. The ability to recognize non-self cells depends on the histocompatibility genes, which provide instructions for making a group of related proteins known as major histocompatibility complex (MHC) proteins or human leukocyte antigens (HLAs). During transplantation, the donor graft, usually obtained from a genetically different individual (an allograft), prompts immune cells in the graft to recognize the recipient's MHC proteins as non-self, triggering GVHD.

[0006] GVHD has traditionally been categorized based on the timing of presentation into acute and chronic, with a cutoff of 100 days post-transplant. These classifications have been further refined based on clinical manifestations accepted by the National Institutes of Health (NIH): acute classic GVHD presents within 100 days of transplantation with typical clinical features of acute GVHD; persistent, recurrent, or late-onset acute GVHD manifests with clinical features of classic acute GVHD but after 100 days of transplantation; classic chronicGVHD presents after 100 days of transplant with classic clinical features of chronic GVHD; and overlap syndrome may occur at any time post-transplant with features of both acute and chronic GVHD.

[0007] GVHD commonly arises after hematopoietic cell transplantation (HCT) or allogenic bone marrow transplantation but also arises, for example, after transplantation of solid organs that are rich in lymphoid cells such as a liver. HCT is used to treat many diseases including cancer and immune-related disorders.

[0008] Acute GVHD can occur in up to 50% of patients receiving hematopoietic stem cell transplantation from an HLA-matched sibling. The occurrence is typically higher in unmatched donors. The incidence of chronic GVHD ranges from 6% to 80%. GVHD is considered one of the main causes of morbidity and mortality after hematopoietic stem cell transplantation; more than 10% of patients will die from this complication.

[0009] Examples of risk factors for acute GVHD include higher degrees of HLA mismatch, prior acute GVHD, sex disparity, older age of the donor or recipient, peripheral stem cell recipients, alloimmunization of the donor, and cytomegalovirus and Epstein Barr virus seropositivity. These factors collectively contribute to the increased risk of developing acute GVHD following hematopoietic stem cell transplantation.

[0010] Treatment for GVHD depends on the severity of symptoms and the organs involved. Most treatment options focus on immunosuppression of donor T-cells. Corticosteroids remain the most used treatment.

[0011] In prior work of the laboratory of inventors herein, various allele-specific anti-HLA monoclonal antibodies (ASHmAbs) were generated and given at standard doses to effectively treat severe GVHD by selectively killing donor-derived cells in a xenogeneic GVHD animal model (Nakauchi et al., Experimental Hematology 2015; 43: 79-88). That work was the first evidence in an animal model that GVHD could be treated by targeting only donor-derived cells. Subsequently, the efficacy of anti-HLA donor-specific polyclonal antibodies in serum against severe GVHD after renal transplantation was confirmed by another group (Zuber et al., Am J Transplant. 2020;20(8):2243-2253).

[0012] There are about 8,000-10,000 patients with severe GVHD each year in the United States. Severe GVHD is a fatal disease, and a cure is long-awaited.

[0013] There remains a need in the art for products and methods for addressing GVHD.SUMMARY

[0014] To develop new anti-HLA Abs to treat GVHD in human patients, an allogeneic GVHD model was developed that closely mimics the clinical situation. Bone marrow from HLA-A2 transgenic mice derived from C57BL / 6 was transplanted into irradiated Balb / c mice, creating a Major Histocompatibility Complex (MHC)-complete mismatched severe GVHD model. A standard dose of HLA-A2 antibody was administered after HCT when GVHD symptoms developed. The results were significant, with GVHD being cured in the antibody- treated group, demonstrating a clear difference compared to controls (p<0.05, n=5-14). Notably, the engraftment of HLA-A2 -expressing donor cells in surviving mice was also confirmed, indicating that the donor-specific antibody treatment effectively cured GVHD by targeting only mature functional donor cells with little effect on engraftment. Surprisingly, the mice could be treated with ultra-low-dose antibody administration (1- lOOng / mouse / dose).

[0015] The HLA-A2 antibody also accurately stained donor- and recipient-derived mismatched HLA in patient blood after HLA mismatch transplantation.

[0016] The disclosure therefore provides antibody products that specifically bind human leukocyte antigen-A2 (HLA-A2). The antibody can be an AN7 antibody product.

[0017] The antibody products can comprise a variable region comprising: a first variable domain comprising an AN7 CDR-H1 set forth in SEQ ID NO: 1, an AN7 CDR-H2 set forth in SEQ ID NO: 2, and an AN7 CDR-H3 set forth in SEQ ID NO: 3, and a second variable domain comprising an AN7 CDR-L1 set forth in SEQ ID NO: 4, an AN7 CDR-L2 set forth in SEQ ID NO: 5, and an AN7 CDR-L3 set forth in SEQ ID NO: 6, as specified using NCBI IGBLAST (ncbi.nlm.nih.gov / igblast / ).

[0018] The variable region can comprise a heavy chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID NO: 7.

[0019] The variable region can comprise a light chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID NO: 8.

[0020] The variable region can comprise a heavy chain variable domain comprising SEQ ID NO: 7 and a light chain variable domain SEQ ID NO: 8.

[0021] An antibody product provided herein can comprise a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain constant domain (CH).

[0022] An antibody product provided herein can comprise a light chain comprising a light chain variable domain (VL) and a light chain constant domain (CL).

[0023] An antibody product provided herein can comprise a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain constant domain (CH) and a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL).

[0024] An antibody product provided herein can comprise a heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH).

[0025] An antibody product provided herein can comprise a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL).

[0026] An antibody product provided herein can comprise a heavy chain comprising a heavy chain variable domain (VH) and a human heavy chain constant domain (CH) and a light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL).

[0027] A heavy chain constant domain of an antibody product provided herein can comprise an IgA, IgD, IgE, IgG, or IgM heavy chain constant domain. The heavy chain constant domain can be an IgGl constant domain, an IgG2 constant domain, or an IgG4 constant domain. The heavy chain constant domain can be an IgG2 constant domain.

[0028] A light chain constant region (CL) can comprise a kappa domain or a fragment thereof.

[0029] The disclosure provides an antibody product that binds human HLA-A2, comprising an IgG2 heavy chain.

[0030] The disclosure provides an antibody product that binds human HLA-A2, comprising an IgGl heavy chain.

[0031] An antibody product provided herein can be a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single chain antibody.

[0032] An antibody product provided herein can be a monospecific, bispecific, trispecific, or multispecific antibody.

[0033] Binding of the antibody product to the donor cell may or may not be cytotoxic to the cell as needed.

[0034] The disclosure provides a method of treating GVHD in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody product provided herein.

[0035] In GVHD treatment methods provided herein, the methods can further comprise administering to the subject an effective amount of an immunosuppressive agent (sometimes referred to as an immunosuppressant herein). The immunosuppressive agent can be a corticosteroid.

[0036] The disclosure provides an isolated nucleic acid, comprising a nucleotide sequence encoding a heavy chain variable domain provided herein.

[0037] The disclosure provides an isolated nucleic acid, comprising a nucleotide sequence encoding a light chain variable domain provided herein.

[0038] The disclosure provides an expression vector comprising a nucleic acid provided herein.

[0039] The disclosure provides a host cell comprising a nucleic acid or expression vector provided herein.

[0040] The disclosure provides a method of producing an antibody product comprising an immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain. Such a method can comprise growing a host cell provided herein encoding the antibody product under conditions such that the host cell expresses the antibody product comprising the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain; and purifying the antibody product comprising the immunoglobulin heavy chain variable domain or the immunoglobulin light chain variable domain.

[0041] The disclosure provides a method of producing an antibody product comprising immunoglobulin heavy and light chains. Such a method can comprise growing a host cell comprising an expression vector provided herein encoding the antibody product under conditions such that the host cell expresses the antibody product comprising the immunoglobulin heavy chain and the immunoglobulin light chain, thereby producing the antibody product; and purifying the antibody product.

[0042] The disclosure provides a composition comprising an antibody product provided herein and an excipient.

[0043] The disclosure provides an article of manufacture, comprising the composition provided herein and a container.

[0044] The disclosure provides a use of the antibody product, or a composition provided herein, for manufacture of a medicament for treatment of GVHD in a subject in need thereof.

[0045] The disclosure provides a pharmaceutical composition, comprising an antibody product provided herein and a pharmaceutically acceptable excipient.

[0046] The disclosure provides an antibody product or pharmaceutical composition provided herein, for use in treating a subject at risk for or having GVHD.

[0047] The disclosure provides a use of an antibody product in the manufacture of a medicament for treating a subject at risk for or having GVHD.

[0048] The disclosure provides a method of detecting HLA-A2 in a sample using an antibody product provided herein, comprising contacting the sample with the antibody product and detecting the antibody product or detecting binding of the antibody product to HLA-A2.

[0049] An antibody product provided herein can be detectably labeled or can comprise a therapeutic or cytotoxic moiety.

[0050] A pharmaceutical composition provided herein can comprise an antibody product provided herein that reduces or prevents a symptom caused by donor-derived cells including, but not limited to, one or more GVHD symptoms. A pharmaceutical composition provided herein can comprise an antibody product provided herein that reduces or prevents one more acute GVHD symptoms such as skin rash, skin redness, abdominal pain, abdominal cramps, nausea, vomiting, diarrhea, and liver dysfunction e.g., elevated liver enzymes and / or jaundice). A pharmaceutical composition provided herein can comprise an antibody product provided herein that reduces or prevent one or more chronic GVHD symptoms such dry itchy skin, skin thickening, skin tightening, dry eyes, dry mouth, shortness of breath, joint stiffness, and joint pain).

[0051] The disclosure provides a method of reducing a biological activity of HLA-A2 in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein.

[0052] The disclosure provides method of depleting cells expressing HLA-A2 in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antibody product or a pharmaceutical composition provided herein. The depletion of cellscan occur by antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0053] The disclosure provides a method of treating a subject at risk for or suffering from GVHD, the method comprising administering a therapeutically effective amount of an antibody product or a pharmaceutical composition provided herein.

[0054] The methods may comprise administering at least one additional therapeutic agent to the patient.

[0055] Antibody products provided herein can be administered at an ultra-low dose in treatment methods provided herein.

[0056] The disclosure provides kits including antibody products provided herein.

[0057] The following Drawings and Detailed Description (including the Examples) illustrate various non-limiting aspects of the subject matter contemplated herein.DESCRIPTION OF THE DRAWINGS

[0058] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0059] Aspects of the disclosure are illustrated by the following figures.

[0060] Fig. 1: Characteristics of allele-specific anti-HLA-A2 antibody (AN7). (A) Screening of allele-specific anti-HLA monoclonal antibodies (ASHmAb). Hybridoma clones were generated following the protocol from Nakauchi et al. (PMID: 25448490). HLA-A2 positive PBMCs from healthy donors (100,000 cells / wel I, in triplicate) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and 50-100 pL of hybridoma supernatant (each containing a single clone-derived antibody) with or without 10% baby rabbit complement. After 24 hours, dead cells were stained with propidium iodide (PI) and analyzed by flow cytometry. (B-D) Characterization of purified AN7 from ascites and recombinant AN7 using FlowPRA kits (OneLambda). HLA antigens that bind to AN7 were determined following the manufacturer's instructions (Figures IB, ID). The isotype of AN7 was identified as IgG2a kappa using Rodent Monoclonal Isotyping Strips (Figure 1C). Representative flow cytometry data are shown for FlowPRA beads coated with specific HLA antigens (Group 1: A01:01, A02:01, A03:01, B49:01, A25:01, A29:02, A30:01, control, or A26:01; Group 2: A68:01, All:01, A34:02, A24:02, A32:01, A33:01, A31:01, control, or A23:01). (E) Complement-dependent cytotoxicity (CDC) assay of AN7 against HLA-A2positive and negative cells with or without baby rabbit complement. The left panel shows AN7 effects on an HLA-A33-positive (A2-negative) B cell line (LCC), and the right panel shows effects on PBMC-derived HLA-A2-positive cells. Cells were cultured with isotype control, ascites AN7 (aAN7), recombinant AN7 (rAN7), and commercial anti-A24 or anti-A33 antibodies. Live cell percentages were determined by PI staining and flow cytometry. The red arrow indicates the cytotoxic effects of both aAN7 and rAN7. N=3. (F) Antibodydependent cellular cytotoxicity (ADCC) assay of purified mouse-AN7 (mAN7) and chimerized- AN7 (cAN7) against HLA-A2 positive NALM6 target cells. Rituximab against Raji cells served as the positive control. NK cells, isolated from HLA-A2 negative healthy donors, were used as effector cells at an effector-to-target (E) ratio of 5: 1. Data points represent the mean ± standard deviation (SD) of triplicate wells. The experiment was repeated twice with NK cells from different donors. Error bars represent ± SD. N=2. (G) Antibody-dependent cellular phagocytosis (ADCP) assay of purified mouse-AN7 (mAN7) and chimerized-AN7 (cAN7) against HLA-A2 and GFP positive NALM-6 cell line (target cells). Human monocyte-derived macrophages isolated from healthy donors (HLA-A2-negative) were used as effector cells at an effector-to-target (E:T) ratio of 5: 1. Data points represent the mean ± SD of triplicate wells. The experiment was repeated three times with different donors. Statistical significance is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001. Error bars represent ± SD. N=3. (H) Mixed Lymphocyte Reaction (MLR) assessing T cell proliferation in response to allogeneic stimulation. Responder spleen-derived T cells isolated from HLA-A2 transgenic mice (B6) were co-cultured with irradiated stimulator spleen-derived cells from Balb / c at a 1: 1 ratio for three days. T cell proliferation was measured by flow cytometry using CountBright beads. Data points represent the mean ± standard error of the mean from three independent experiments, each performed in triplicate. Isotype control at the same dose was a negative control (1 pg, 100 pg, and 10 ng). Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001. Error bars represent ± SD. (I) AN7 inhibits HLA-A2 binding to LILRB1. The left figure shows FlowPRA beads incubated with 1 pg of LILRBl-Fc, demonstrating LILRB1 binding to multiple HLA antigens. The right figure shows that 2pg of mAN7 can block LILRBl-Fc binding. A red circle highlights the binding of AN7 to the HLA-A*02:01 antigen.

[0061] Fig. 2: Mixed chimerism analysis using AN7 in HLA-A2 mismatched hematopoietic stem cell transplantation. The figure demonstrates the use of AN7 for accurately analyzing the ratio of donor to recipient cells at various cell fraction levels in patient peripheral blood mononuclear cells (PBMCs) 32 days after HLA-A2 mismatched hematopoietic stem cell transplantation. The donor cells were HLA-A2-positive, while the recipient (patient) cellswere HLA-A2-negative. Flow cytometry was performed using AN7 and commercial antibodies to distinguish between donor and recipient cells. The upper flow plots show the overall staining of the samples with AN7. The middle-left flow plots highlight CD14-positive cells within the CD45-positive blood cell population. The middle-right flow plots depict the presence of HLA-A2-positive (donor) or HLA-A2-negative (recipient) cells within the CD14+ cell population. The lower flow plots (right, middle, and left, respectively) display the proportion of HLA-A2-negative and HLA-A2-positive cells across various cell fractions, including CD3 (T cells), CD19 (B cells), CD56 (NK cells), Alpha-Beta T cells, and GammaDelta T cells, providing a comprehensive assessment of chimerism at multiple immune cell levels.

[0062] Fig. 3: AN7 depletes HLA-A2 -expressing cells in vivo. (A) Peripheral blood mononuclear cells (PBMCs) from a healthy HLA-A2-positive donor were intravenously transplanted into NSG mice (Day 0). Peripheral blood from each mouse was analyzed on Days 1, 3, and 14 before and after administering AN7 or PBS (negative control) on Day 2. The flow cytometry plots display the results of the in vivo experiments for each group: mAN7, rAN7, and PBS (negative control). Flow plots are shown for two mice per group. The X-axis represents mouse CD45 expression, and the Y-axis represents human CD45 expression. N=2. (B) A summary of the data analysis from (A). N=2.

[0063] Fig. 4: AN7 successfully cured severe GVHD in allogeneic GVHD mouse models while preserving donor cells, including hematopoietic stem cells, without fatal damage. (A) The left panel illustrates a schematic of the xenograft GVHD model based on our lab's previous study (PMID: 25448490). In this model, HLA-A2-positive healthy donor PBMCs were transplanted into NSG mice on Day 0. Mice were treated with mAN7, rAN7, or PBS (negative control) on Day 3. The right panel shows Kaplan-Meier survival curves for the mice, with N=6 for each group (mAN7, rAN7, and PBS negative control). (B) The left panel depicts a schematic of the allograft GVHD model. In this model, irradiated Balb / c mice received bone marrow cells from HLA-A2 transgenic B6 mice (HLA-A2-positive) on Day 0. Mice were treated with mAN7 or isotype control (negative control) on Day 7 after showing GVHD symptoms. The right panel presents mouse survival data as Kaplan-Meier survival curves. N=14 for each control and AN7 group, except for the AN7 60 pg group. Statistical significance is indicated as *p < 0.05 and **p < 0.01 using one-way ANOVA. Error bars represent ± SD. (C) Follow-up PBMC analysis (Day 35) from the mice in panel (B). The data represent mice treated with either isotype control or 10 pg AN7. The X-axis shows the H- 2Db expression, and the Y-axis shows H-2Kd expression. N=1 per treated mouse.

[0064] Fig 5: Schematic and survival analysis of a fully allogeneic GVHD model. (A) Lethally irradiated BALB / c mice received 5 x 106bone marrow cells from HLA-A2 transgenic C57BL / 6 donors. On day 7 post-transplantation, mice displaying GVHD symptoms were treated with a single dose of AN7 (0.1 ng, 1 ng, 10 ng, or 100 ng,) or mouse IgG isotype control (10 ng). (B) Survival was monitored and plotted as Kaplan-Meier curves. Statistical comparisons were performed as indicated. N=10 (each dose) , ns = not significant; *P < 0.05; **P < 0.01. (C) Representative flow cytometry data using peripheral blood cells from a BALB / c recipient that received a bone marrow transplant from HLA-A2 transgenic C57BL / 6 donors, followed by treatment with 10 ng of AN7. Analysis was performed 35 days after antibody administration. Anti-mH-2Kd and anti-mH-2Dd antibodies were used to detect recipient and donor chimerism, respectively. (D) Mixed lymphocyte reaction (MLR) assay. Splenocytes from HLA-A2 transgenic C57BL / 6 mice (responders) and mitomycin C-treated BALB / c splenocytes (stimulators) were co-cultured at a 1: 1 ratio in the presence of AN7 (l pg, 100 pg, 10 ng) or isotype control antibody. After 72 hours, T cell counts were determined by flow cytometry using counting beads. Data represent mean ± SD from triplicate wells (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 (paired t-test).DETAILED DESCRIPTION

[0065] Antibodies provided herein can be used for diagnostic and / or therapeutic purposes. As a diagnostic agent, ASHmAbs can be used for flow cytometry analysis of the chimeric state, a state in which donor and recipient cells are mixed after HLA mismatch transplantation (HCT / Solid Organ Transplantation (SOT)). In clinical practice, chimerism analysis, which compares the ratio of donor and recipient cells by DNA-based analysis or chromosome-based analysis, is standard, but performing chimerism analysis with ASHmAbs determines the ratio at a more detailed level, such as B cells or T cells, rather than simply the ratio of donor and recipient cells This allows a deeper understanding of the pathogenesis of post-transplantation pathologies. As a therapeutic agent, we are considering the use of this antibody for the treatment of severe GVHD after HLA mismatch transplantation (HCT / SOT); we believe that an antibody with an unprecedented mechanism of action that attacks only the donor-derived cells that cause GVHD will be a specific treatment for severe GVHD. In our studies using a mouse model of severe GVHD, we treated GVHD by temporarily disrupting the cells that cause GVHD, but the loss of donor cells, which was a concern, was easily prevented by controlling the dosage. The effect is maintained in thechimeric antibodies we have produced, and we are ready to produce humanized antibodies at any time for clinical application.Antibody products

[0066] The disclosure provides antibody products that specifically bind to HLA-A2.

[0067] "HLA-A2 antibody product" means an antibody or fragment thereof that binds to HLA-A2, but specifically relative to binding to other HLA family members. In some embodiments, HLA-A2 antibody products bind with affinities that allow the antibody product to block or substantially impair binding of HLA-A2 to natural ligands, or to modulate activities mediated by HLA-A2. HLA-A2 antibodies can be capable of HLA-A2 antagonism. As such, they may be referred to as "HLA-A2 antagonists."

[0068] "HLA-A2 antagonist" means any chemical compound or biological molecule that blocks or substantially impairs binding of HLA-A2 to any of its natural ligands, including, for example, LILRB1. HLA-A2 antagonists may block or impair interaction with ligands on another cell, thereby blocking or inhibiting functional activity associated with such "trans" interactions. HLA-A2 antagonists may block or impair interaction with ligands on the same cell, thereby blocking or inhibiting functional activity associated with such "cis" interactions. An antibody, such as an AN7 antibody provided herein, that specifically binds HLA-A2 can be an HLA-A2 antagonist. A "HLA-A2 antibody" or "HLA-A2 antibody product" binds HLA-A2 specifically relative to binding to other HLA family members.

[0069] Detailed and reviewed information for HLA-A2 is readily available, e.g., at www.ncbi.nlm.nih.gov / protein / AAA76608-2.

[0070] The terms "polypeptide" and "protein" are used interchangeably herein in the conventional way to refer to a molecule formed of amino acids. The polypeptides are not limited to a specific length. Peptides are included within polypeptides, unless specifically indicated otherwise. The terms neither specify nor exclude post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. Polypeptides of interest in the context of the antibodies of this disclosure include, but are not limited to, polypeptide fragments comprising CDRs which are capable of binding HLA-A2 proteins expressed by myeloid cells, lymphoid cells, or cancer cells.

[0071] The term "polypeptide fragment" refers to a polypeptide that has an aminoterminal deletion, a carboxyl -terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids ascompared with the native protein. Fragments are about 5 to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of HLA-A2 antibodies disclosed herein, useful fragments include, but are not limited, to a CDR region, a variable domain of a heavy or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0072] The term "isolated protein" referred to herein means that a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Genomic DNA, cDNA, mRNA, or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.

[0073] A "variant" of a polypeptide e.g., an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0074] A "derivative" of a polypeptide is a polypeptide e.g., an antibody) that has been chemically modified in some manner distinct from insertion, deletion, or substitution variants, e.g., via conjugation to another chemical moiety.

[0075] The term "antibody" generally includes an immunoglobulin protein that comprises one or more polypeptide chains and that is immunologically functional, including specifically binding to an antigen. In humans, antibodies typically comprise four linked polypeptide chains, a "tetramer" including two identical "heavy" chains and two identical smaller "light" chains. The two heavy chains are each linked to one light chain, are also linked to one another in parallel. The linkages impart a roughly Y-shaped structure to the antibody, such that the linked portions of the heavy chains form the leg of the "Y", and each light chain (with the portion of the heavy chain to which it is linked) forms an arm of the "Y". Each arm of the antibody contains an antigen binding site, so the typical antibody can bind two ofantigens. In humans there exist five basic types or classes of antibodies, differentiated by the structure of the heavy regions and by their functional purpose: IgG, IgA, IgE, IgD, IgM. Intact antibodies in some classes in humans can differ from the typical tetrameric "Y" structural unit, such as circulating IgM antibodies that contain five such units linked at their bases in a roughly circular array. More detail on antibody structure and function is provided elsewhere herein.

[0076] In a typical antibody, each pair or couplet in the tetrameric unit includes one full- length "light" chain (about 25 kDa) and one full-length "heavy" chain (about 50-70 kDa). Each individual immunoglobulin chain is composed of several "immunoglobulin domains," each consisting of roughly 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units of which antibody polypeptide chains are composed. The amino-terminal portion of each chain typically includes a variable domain that is responsible for antigen recognition. The carboxy-terminal portion is more conserved evolutionarily than the amino-terminal end of the chain and is referred to as the "constant region" or "C region."

[0077] The term "heavy chain" includes a full-length immunoglobulin heavy chain and fragments thereof having sufficient variable domain sequence to confer binding specificity, either alone or together with a light chain variable domain. Heavy chains are typically classified as mu (p), delta (3), gamma (y), alpha (a), or epsilon (E) chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgM, and IgM2. IgA subtypes include IgAl and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. Full-length IgG heavy chains, for example, each contain three C region domains known as CHI, CH2 and CH3, with the CH3 being closest to the carboxy terminus. The antibody products that are provided can have any of these isotypes and subtypes. For example, an HLA-A2 antibody product can be an intact antibody of the IgGl or IgG4 subtype.

[0078] The term "light chain" includes a full-length immunoglobulin light chain and fragments thereof having sufficient variable domain sequence to confer binding specificity, either alone or together with a heavy chain variable domain. Human light chains generallyare classified as kappa (K) or lambda (A) light chains. A full-length light chain includes an amino-terminal variable domain (VL) and a carboxy terminal constant domain (CL).

[0079] In the light and heavy chains, the variable and constant domains are naturally joined by a "J" region of about twelve or more amino acids, with the heavy chain further including a "D" region of about ten more amino acids. See, e.g., Fundamental Immunology, 2nded., Ch. 7 (Paul, ed.) 1989, New York: Raven Press.

[0080] Variable domains of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called "complementarity determining regions" or CDRs. The CDRs from the two chains of each heavy chain / light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a particular epitope on the target protein (HLA-A2). From N-terminal to C-terminal, naturally occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Numbering systems have been devised for assigning numbers to amino acids that occupy positions in each of these domains.

[0081] The current art utilizes various numbering schemes with different definitions of CDR lengths and positions. For example, the Kabat numbering scheme is based on sequence alignment and uses "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most occurring amino acid at that position) to predict CDRs [Kabat etal. in Sequences of Proteins of Immunological Interest, 5thEd., US Dept, of Health and Human Services, PHS, NIH, NIH Pub. No. 91-3242 (1991)]. The Chothia numbering scheme, on the other hand, is a structure-based numbering scheme where antibody crystal structures are aligned as define the loop structures as CDRs [Chothia and Lesk, J Moi Biol 1987 196:901-17; Chothia etal, Nature. 1989 342:878-83]. The Martin numbering scheme focuses on the structure alignment of different framework regions of unconventional lengths [Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," in: Kontermann and Dubel, eds. Antibody Engineering. Springer; Berlin, Germany: 2014. Pp. 33-51]. The ImMunoGeneTics (IMGT) numbering scheme is a standardized numbering system based on alignments of sequences from a complete reference gene database including the whole immunoglobulin superfamily [Lefranc etal., Dev Comp Immunol. 2003 27(l):55-77;(www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html)]. The Honneger numbering scheme (Aho) is based on structural alignments of the 3D structure ofthe variable regions and uses structurally conserved Co positions to deduce framework and CDR lengths [Honegger eta / ., J Mo! Biol. 2001 309(3):657-70]. One of ordinary skill in the art understands that the definition of a CDR will vary based on the method used.

[0082] Some of the antibody products that are provided have the structure typically associated with naturally occurring antibodies. Thus, the term "antibody product" includes an intact antibody of any class or subclass, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies, as well as other forms. As noted, an intact antibody generally will comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids, which can comprise only heavy chains, and V NAR domains from sharks. Antibody products can be derived solely from a single source, or can be "chimeric," that is, different portions of the antibody can be derived from two different antibodies. For example, the complementarity determining regions, which impart the binding specificity of an antibody, can be derived from a rat or murine source, while the framework portion of the variable regions is derived from a different species source, such as a human. In other chimeric forms, the light and heavy variable domains (optionally with a constant domain) can be derived from one species and one or more constant domains from another species. See, e.g., US Patent No. 11352444. The antibody products provided can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody product" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains (such as IgG antibodies), antibodies of other isotypes, derivatives, variants, and fragments thereof. The antibody products provided include, but are not limited to, monoclonal antibodies, human antibodies, chimeric antibodies, and humanized antibodies. Immunologically functional antibody fragments provided include, but are not limited to, scFv, Fab, Fab', F(ab')2, and domain antibody products.

[0083] An "immunologically functional fragment" (or simply "fragment") of an immunoglobulin, as used herein, refers to a portion of an antibody, comprising a light chain or a heavy chain (or both) and is capable of binding specifically to an antigen, but the light chain or heavy chain (or both) lacks at least some of the amino acids present in a full-length chain. Such fragments are biologically active in that they bind specifically to the target antigen and can compete with intact antibodies for specific binding to a given epitope. Such a fragment will retain at least one CDR present in the full-length light or heavy chain andcan comprise a single heavy chain and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is contemplated further that a functional portion of the inventive antibodies, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life.

[0084] A "Fab fragment" comprises one light chain (VL+CL) and a portion of a heavy chain that includes the variable domain and the CHI domain (VH+CH1). The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0085] An "Fc" region contains two heavy chain fragments each comprising the CH2 and CH3 domains of an antibody and in some cases the lower hinge region. The two heavy chain fragments are held together by two or more disulfide bonds (typically in the hinge region) and by hydrophobic interactions of the CH3 domains.

[0086] A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0087] A "F(ab')2 fragment" contains two light chains and two heavy chains each containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0088] The "Fv region" comprises the variable domains from both the heavy and light chains, but lacks the constant domains.

[0089] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable domains have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail, for example, in PCT Pub. No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0090] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable domain of a heavy chain or the variable domain of a light chain. In some instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0091] A "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies can be bi specific (see below).

[0092] A "multispecific antibody" is one that targets more than one antigen or epitope.

[0093] A "bispecific," "dual-specific" or "bifunctional" antibody is a hybrid antibody having two different antigen binding sites. Bispecific antibodies are a species of multispecific antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol. 1990 79:315-21; Kostelny etai., J Immunol. 1992 148: 1547-53. The two binding sites of a bispecific antibody will bind to two different epitopes, which can reside on the same or different protein targets. A "trispecific" antibody has three different antigen binding sites. See also, Merchant eta / , Nat BiotechnoL 1998 16:677-81.

[0094] The term "neutralizing antibody" refers to an antibody that binds to a ligand, prevents binding of the ligand to its binding partner and interrupts the biological response that otherwise would result from the ligand binding to its binding partner. In assessing the binding and specificity of an antibody or immunologically functional fragment thereof, an antibody or fragment will substantially inhibit binding of a ligand to its binding partner when an excess of antibody reduces the quantity of binding partner bound to the ligand by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in an in vitro competitive binding assay). In the case of antibody products that bind to HLA-A2 proteins, a neutralizing antibody product will diminish the ability of a HLA-A2 protein to bind to one or more of its (their) ligands thereby inhibiting HLA-A2 -mediated activity e.g., as shown in the Examples herein).

[0095] The term "competition" when used in the context of antibody products that compete for the same epitope means competition between antibodies is determined by an assay in which the antibody product under test prevents or inhibits specific binding of a reference antibody product to a common antigen (HLA-A2 or a fragment thereof). Numerous types of competitive binding assays can be used, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay [e.g., Stahli eta / ., Methods Enzymoi. 1983 9:242-53]; solid phase direct biotin-avidin EIA [e.g., Kirkland eta / ., J Immunol. 1986 137:3614-9]; solid phase direct labeled assay, solid phase direct labeled sandwich assay [e.g, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)]; solid phase direct label RIA using 1-125 label [e.g, Morel etai, Molec Immunol. 1988 25:7-15]; solid phase direct biotin-avidin EIA [e.g., Cheung etai., Virology. 1990 176:546-52]; and direct labeled RIA [Moldenhauer etai., Scand J Immunol. 1990 32:77-82]. Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually, the test antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that can interact with different antibodies.

[0096] The term "epitope" includes any determinant capable of specifically binding to an antibody or to a T-cell receptor. An epitope is a region of an antigen that is bound by an antibody that specifically targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antibody. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0097] An antibody product "specifically binds" its target antigen when the dissociation constant (Kd) is less than 100 nM. The antibody specifically binds antigen with "high affinity" when the Kd is less than 10 nM and with "very high affinity" when the Kd is less than 0.5 nM.The antibody product can have a Kd in the range from about 0.5 nM to about 500 nM. The antibody product can have a Kd in the range from about 100 to about 500 nM. One of skill in the art will recognize that specifically binding does not mean exclusive binding, rather it allows for some degree of non-specific binding as is typical in biological reactions between groups with affinity to one another.

[0098] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and can be expressed as the equilibrium dissociation constant, KD, a calculated ratio of the dissociation constant and the association constant (Koff / Kon), between the antibody and its antigen. Affinity can also be expressed as the association constant, KA, which is the reciprocal of KD. The antibody products disclosed herein exhibit binding affinity as measured by KDfor HLA-A2 in the range of 10'4M or less, or ranging down to 10'16M or lower, e.g., about 10'4, 10'5, 10'6, 10'7, 10'8, 10'9, 10'10, 10’11, 10'12, 10'13, 10'14, 10'15, 10'16M or less). Antibodies described herein can specifically bind to a HLA-A2 polypeptide with a KDof less than or equal to 10’4M, less than or equal to about 10’5M, less than or equal to about 10’6M, less than or equal to 10’7M, or less than or equal to 10'8M. Methods for determining the affinity of two molecules are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (SPR), bio-layer interferometry (BLI), and the like.

[0099] As used herein, an antibody product is said to be "immunospecific" or "specific" for, or to "specifically bind" when that an antibody product forms a complex with an antigen that is relatively stable under physiologic conditions. The terms "preferentially binds" or "specifically binds" mean that the antibodies or fragments thereof bind to an epitope with greater affinity than it binds unrelated amino acid sequences, and, if cross-reactive to other polypeptides containing the epitope, are not toxic at the levels at which they are formulated for use in administration to human subjects. The terms are also applicable where for example, an antibody product is specific for a particular epitope that is carried by more than one antigen, in which case the antibody or antigen-binding fragment thereof carrying the antigen-binding domain will be able to specifically bind to the epitope found in the different antigens. The measured relative affinity of antibodies for one epitope compared to another may differ between comparators, or between variants of the same comparator, or across experimental conditions. The HLA-A2 antibodies disclosed herein preferentially bind HLA-A2 as compared to other HLA family members. Such relative affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-foldgreater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 200-fold greater, at least 250-fold greater, at least 500-fold greater, or at least 1000-fold greater than the affinity of the antibody product for amino acid sequences derived from other HLA family members.

[0100] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (Ze., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, Ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Parti, (Griffin and Griffin, eds.), 1994, New Jersey: Humana Press; Sequence Analysis in Molecular Biology, (von Heinje), 1987, New York: Academic Press; Sequence Analysis Primer, (Gribskov and Devereux, eds.), 1991, New York: M. Stockton Press; and Carillo etal, SIAM J Applied Math. 1988 48(5): 1073-82.

[0101] In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences. An exemplary computer program used to determine percent identity is the GCG program package, which includes GAP (Devereux et al, Nuci Acid Res. 1984 12:387-95; Genetics Computer Group, University of Wisconsin, Madison, Wise.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3 times (3X) the average diagonal, in which the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. A standard comparison matrix \e.g., Dayhoff etal, Atlas of Protein Sequence andStructure, 5:345-352 (1978) for the PAM 250 comparison matrix; Henikoff eta!., Proc Nat! Acad Sci USA. 1992 89: 10915-9 for the BLOSUM 62 comparison matrix] can also be used by the algorithm.

[0102] Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following: Algorithm: Needleman et a!., J Mol Biol. 1970 48:443-53; Comparison matrix: BLOSUM 62 from Henikoff eta!, 1992, supra; Gap Penalty: 12 (but with no penalty for end gaps); Gap Length Penalty: 4; Threshold of Similarity: 0.

[0103] Certain alignment schemes for aligning two amino acid sequences can result in matching of only a short region of the two sequences, and this small aligned region can have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

[0104] Other exemplary programs that compare and align pairs of sequences include, but are not limited to, ALIGN (Myers and Miller, Comput Appt Biosci. 1988 4(1): 11-7); FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 85(8): 2444-8; Pearson, Methods Enzymoi. 1990 183:63-98); and gapped BLAST (Altschul etal, Nucleic Acids Res 1997 25(17):3389-402), BLASTP, BLASTN, or GCG (Devereux etal, Nucleic Acids Res. 1984 12(1 Pt 1): 387-95)).

[0105] "Amino acid" includes its normal meaning in the art. The twenty naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology— A Synthesis, 2nded. (Golub and Gren, Eds.), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-di substituted amino acids, N-alkyl amino acids, and other unconventional amino acids can be suitable components. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamate, £-N,N,N-trimethyllysine, s-N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, o-N-methylarginine, and other similar amino acids and imino acids e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxyl -terminal direction, in accordance with standard usage and convention.

[0106] Table 1 below sets out heavy (SEQ ID NO: 7) and light chain (SEQ ID NO: 8) variable domains for an HLA-A2 murine antibody designated "AN7." The heavy (H) and light (L) chain CDRs are shown separately in Table 2.Table 1 - Murine Variable Domains and CDRsTable 2 — Complementarity Determining Regions

[0107] Chimeric variants of the AN7 antibody were prepared as described in the Examples. Variable domains of representative chimeric IgGl heavy (SEQ ID NO: 11) and kappa light (SEQ ID NO: 12) chains are presented in Table 3.Table 3 - Chimeric Variable Domains

[0108] Antibody products can comprise a light chain variable domain comprising a sequence of amino acids that differs from the sequence of a light chain variable domain provided herein at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, in which each such sequence difference is independently either a deletion, insertion or substitution of one amino acid. The light chain variable region in some antibodies comprises a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of a light chain variable region provided herein.

[0109] Antibody products can comprise a heavy chain variable domain comprising a sequence of amino acids that differs from the sequence of a heavy chain variable domain provided herein at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, in which each such sequence difference is independently either a deletion, insertion or substitution of one amino acid. The heavy chain variable region in some antibodies comprises a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of a heavy chain variable region provided herein.

[0110] Still other antibody products include variant forms of a variant light chain and a variant heavy chain as just described.

[0111] The antibody products that are provided can include one, two, three, four, five or all six CDRs. Some antibody products include both the light chain CDR3 and the heavy chain CDR3. Certain antibody products have variant forms of the CDRs, with one or more (Ze., 2, 3, 4, 5 or 6) of the CDRs each having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a CDR sequence. For example, the antibody product can include both a light chain CDR3 and a heavy chain CDR3 that each have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the light chain CDR3 sequence and theheavy chain CDR3, respectively. The CDR sequences of the antibody products that are provided can also differ from the CDR sequences provided herein such that the amino acid sequence for any given CDR differs from the sequence provided herein by no more than one, two, three, four or five amino acid residues. Differences from the listed sequences are conservative substitutions.

[0112] When an antibody product is said to bind an epitope within HLA-A2, what is meant is that the antibody product specifically binds to a polypeptide consisting of the specified residues e.g., a specified segment of HLA-A2). Such an antibody does not necessarily contact every residue within HLA-A2. Nor does every single amino acid substitution or deletion within HLA-A2 necessarily significantly affect binding affinity. Exact epitope specificity of an antibody can be determined in variety of ways. One approach, for example, involves testing a collection of overlapping peptides of about fifteen amino acids spanning the sequence of HLA-A2 and differing in increments of a small number of amino acids (e.g., three amino acids). The peptides are immobilized within the wells of a microtiter dish. Immobilization can be achieved by biotinylating one terminus of the peptides. Optionally, different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for purposes of comparison. This is useful for identifying endspecific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments of HLA-A2. An antibody product is screened for specific binding to each of the various peptides. The epitope is defined as occurring within a segment of amino acids that is common to all peptides to which the antibody shows specific binding.

[0113] Antibody products that compete with one of the exemplified antibodies for specific binding to HLA-A2 are also provided. Such antibody products can also bind to the same epitope as one of the exemplified antibodies. Antibody products that compete with or bind to the same epitope as the exemplified antibody or fragment are expected to show similar functional properties. The exemplified antibody products include those with the heavy and light chains, variable domains and CDRs provided herein. Competing antibody products can include those that bind to the epitope described in the section on antibodies and epitopes above.

[0114] The antibody products provided include monoclonal antibodies that bind to HLA- A2. Monoclonal antibodies can be produced using any technique known in the art, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells can be immortalized using any technique known inthe art, e.g., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63- Ag8.653, NSl / l.Ag 4 1, Sp210-Agl4, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7. and S194 / 5XXO Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR- LON-hMy2, and UC729-6.

[0115] In some instances, a hybridoma cell line is produced by immunizing an animal e.g., a transgenic animal having human immunoglobulin sequences) with a HLA-A2 immunogen; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds a HLA-A2 polypeptide. Hybridoma cell lines, and HLA-A2 monoclonal antibodies produced by them, are provided herein.

[0116] Monoclonal antibodies secreted by a hybridoma cell line can be purified using any useful technique known in the antibody arts. Hybridomas or monoclonal antibodies can be further screened to identify monoclonal antibodies with particular properties. Examples of such screens are provided in the Examples below.

[0117] Humanized antibodies based upon the foregoing sequences are also provided. Generally, a humanized antibody is based on a monoclonal antibody raised initially in a nonhuman animal. Certain amino acid residues in the monoclonal antibody, typically from nonantigen recognizing portions of the antibody, are modified to be homologous to corresponding residues in a human antibody of corresponding isotype. Humanization can be performed, for example, using various methods by substituting at least a portion of a rodent variable region with the corresponding regions of a human antibody.

[0118] One example is a “chimeric” antibody, which is an antibody composed of protein segments from different antibodies that are covalently joined to produce functional immunoglobulin light or heavy chains or immunologically functional portions thereof. Generally, at least a portion of the heavy chain and / or light chain variable region is identical or homologous to a corresponding sequence in antibodies derived from one species or belonging to a particular antibody class or subclass, while the remainder of the chain(s)is / are identical or homologous to a corresponding constant region in antibodies derived from another species or belonging to another antibody class or subclass.

[0119] Generally, the goal of making a humanized antibody is to create a chimera in which the number of amino acids from the intended patient species is maximized. One such example is the "CDR-grafted" antibody, in which the antibody comprises one or more complementarity determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain(s) is / are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For use in humans, the CDRs from a rodent antibody often are grafted into a human antibody, replacing the naturally occurring CDRs of the human antibody.

[0120] Fully human antibodies are also provided. Methods are known for making fully human antibodies specific for a given antigen without exposing human beings to the antigen ("fully human antibodies"). One means for implementing the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in mouse, an animal that can be immunized with any desirable antigen. Using fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by administering mouse or mouse-derivatized monoclonal antibodies to humans as therapeutic agents.

[0121] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and optionally are conjugated to a carrier, such as a hapten. See, for example, Jakobovits eta / ., Proc Natl Acad Sci USA. 1993 90:2551-5; Jakobovits et a / ., Nature. 1993 362:255-8; and Bruggemann eta / ., Year Immunol. 1993 7:33-40. In one example of such a method, transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci encoding the mouse heavy and light immunoglobulin chains therein, and inserting into the mouse genome large fragments of human genome DNA containing loci that encode human heavy and light chain proteins. Partially modified animals, which have less than the full complement of human immunoglobulin loci, are then cross-bred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of such methods, see, for example, WO96 / 33735 and W094 / 02602. Additional methods relating to transgenic mice for making human antibodies are described in U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in PCT Pub. Nos. WO 91 / 10741, WO 90 / 04036, and in EPO Pub. No. EP 546073B1. The transgenic mice, referred to herein as "HuMab" mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (g and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous gamma and kappa chain loci (Lonberg eta / ., Nature. 1994 368:856-9). Accordingly, the aforementioned mice exhibit reduced expression of mouse IgM or kappa and in response to immunization the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG kappa monoclonal antibodies. An exemplary mouse with the entire human immunoglobulin locus in its germline is the XenoMouse (Abgenix). Another is the Velocilmmune mouse (Regeneron Pharmaceuticals). Others are the RenMab mouse and the RenLite mouse (Biocytogen), and, more recently the AlivaMab mouse (Ablexis) and ATX-GX mouse (Alloy Therapeutics).

[0122] Using hybridoma technology, antigen-specific human monoclonal antibodies with the desired specificity can be produced and selected from the transgenic mice such as those described above. Such antibodies can be cloned and expressed using a suitable vector and host cell, or the antibodies can be harvested from cultured hybridoma cells.

[0123] Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom and Winter, J Mo! Biol. 1992 227(2):381-8; and Marks eta / ., J Mo! Biol. 1991 222:581-97. Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT Pub. No. WO 99 / 10494, which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk-receptors using such an approach.

[0124] Single chain antibodies are provided. Single chain antibodies are formed by linking heavy and light chain variable domain (Fv region) fragments (such as those shown in Table 1 or in Table 3) via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) can be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH).The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains. Techniques developed for the production of single chain antibodies include those described in Bird eta / ., Science. 1988 24 2:423-6; Huston etal, Proc Natl Acad Sci USA. 1988 8 5:5879-83; Ward eta / ., Nature. 1989 334:544-6; and de Graaf etai., Methods Mol Biol. 2002 178:379-87. A"diabody" is a dimer of scFV.

[0125] Antibodies provided herein that are of one subclass can be changed to antibodies from a different subclass using subclass switching methods. For example, the variable domains provided herein can be attached to constant domains of any desired Ig subtype. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding particular antibody polypeptides can be employed in such procedures, e.g., DNA encoding the constant domain of an antibody of the desired isotype. See, e.g., Lantto eta / ., Methods Mol Biol. 2002 178:303-16. Accordingly, the antibodies that are provided include a desired isotype (for example, IgA, IgGl, IgG2, IgG3, IgG4, IgE, and IgD).

[0126] Antibody products provided can include one or more of the CDRs of any of the heavy chain variable domains exemplified herein, where such CDRs are determined according to IMGT, Kabat or other method: (i) a CDR-H1 with at least 80% sequence identity to the CDR-H1 of SEQ ID NO: 1; (ii) a CDR-H2 with at least 80% sequence identity to the CDR-H2 of SEQ ID NO: 2; and (iii) a CDR-H3 with at least 80% sequence identity to the CDR-H3 of SEQ ID NO: 3. Antibody products provided can include one or more of the CDRs of any of the light chain variable domains exemplified herein, where such CDRs are determined according to IMGT, Kabat or other method: (i) a CDR-L1 with at least 80% sequence identity to the CDR-L1 of SEQ ID NO: 4; (ii) a CDR-L2 with at least 80% sequence identity to the CDR-L2 of SEQ ID NO: 5; and (iii) a CDR-L3 with at least 80% sequence identity to the CDR-L3 of SEQ ID NO: 6. In some embodiments, the CDRs will have at least 85%, at least 90%, at least 95%, or least 99% identity to the determined CDR sequences. Antibody products can include one, two, three, four, five or all six of the foregoing CDRs, as long as they specifically bind HLA-A2.

[0127] Antibody products provided can include (a) a heavy chain variable region having 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQID NO: 7; (b) a light chain variable region having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NOs: 8; or (c) a heavy chain variable region of (a) and a light chain variable region of (b).

[0128] Other antibody products provided compete with an antibody such as those described above for specific binding to an HLA-A2 polypeptide. For example, antibody products are provided that compete with an antibody that consists of two identical heavy chains and two identical light chains, in which the heavy chains comprise SEQ ID NO: 7, and the light chains comprise SEQ ID NO: 8.

[0129] HLA-A2 antibody products are provided that have a half-life of at least one day in vitro or in vivo e.g., when administered to a human subject). The antibody product can have a half-life of at least three days. The antibody product can have a half-life of four days or longer. The antibody product can have a half-life of eight days or longer.Variants

[0130] Variant forms of HLA-A2 antibody products disclosed herein e.g., variant forms of antibody products having sequences provided herein) are provided. For example, antibody products can have one or more conservative amino acid substitutions in one or more of the heavy or light chain variable regions, or CDRs.

[0131] Naturally occurring amino acids can be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions can involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions can encompass non- naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0132] Non-conservative substitutions can involve the exchange of a member of one of the above classes for a member from another class. Such substituted residues can be introduced into regions of the antibody product that are homologous with human antibodies, or into the non-homologous regions of the molecule.

[0133] In making such changes, the hydropathic index of amino acids can be considered. The hydropathic profile of a protein is calculated by assigning each amino acid a numerical value ("hydropathy index") and then repetitively averaging these values along the peptidechain. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0134] The importance of the hydropathic profile in conferring interactive biological function on a protein is understood in the art \e.g., Kyte and Doolittle, J Mol Biol, 157(1): 105-132 (1982)]. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±0.2 can be made. The substitution of amino acids whose hydropathic indices are within ±0.1 can also be made. The substitution of amino acids whose hydropathic indices are within ±0.5 can also be made.

[0135] It is also understood in the art that the substitution of like residues in amino acid sequences can be made effectively on the basis of relative hydrophilicity or hydrophobicity of the residues, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological molecules, as in the present case. The greatest local average hydropathic character of a protein, as governed by the hydrophilicity of its adjacent amino acids, can correlate with its immunogenicity and antigen-binding or immunogenicity, that is, with a biological property of the protein.

[0136] Various methods are known for estimating the hydrophilicity or hydrophobicity of amino acid residues in proteins. A comparative survey of such methods is given at Biswas eta!., J Chromatogr A. 1000(l-2):637-55. Hopp and Woods Mol Immuno! 1983 20(4):483- 9) assigned the hydrophilicity values amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±0.1); glutamate (+3.0 ±0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ±0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In this ranking system, more hydrophilic residues are assigned positive values, and less hydrophilic residues negative. In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±0.2 is included, otherwise those which are within ±0.1 are included or those within ±0.5 are included. In some instances, one can also identifyepitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitopic core regions."

[0137] A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan also will be able to identify residues and portions of the molecules that are conserved among similar polypeptides. Even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0138] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0139] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for HLA-A2 binding activity (see Examples below), thus yielding information regarding which amino acids can be changed and which must not be changed. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations.

[0140] Substantial modifications in the functional and / or biochemical characteristics of the antibody products described herein can be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulkiness of the side chain. A "conservative amino acid substitution" can involve a substitution of a native amino acid residue with a normative residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide can also be substituted with alanine, as has been previously described for alanine scanning mutagenesis.

[0141] Amino acid substitutions (whether conservative or non-conservative) of the subject antibodies can be implemented by those skilled in the art by applying routine techniques. Amino acid substitutions include, but are not limited to, substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (4) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts). Conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the parent sequence e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the parent or native antibody). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed.), 1984, New York: W. H. Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton etal, Nature. 1991 354(6349): 105, which are each incorporated herein by reference.

[0142] Glycosylation variants of the antibody products are provided in which the number and / or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Antibody product variants can comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, in which the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. For example, one or more new N-linked sites are created. Antibodies typically have a N-linked glycosylation site in the Fc region.

[0143] Additional antibody product variants include cysteine variants in which one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia when antibodies must be refolded into a biologically active conformation. Cysteine variants can have fewer cysteine residues than the native antibody, and typically have an even number to minimize interactions resulting from unpaired cysteines.Effector Functions

[0144] Antibody structure affects the role that the antibody plays in the immune system and the effects that the antibody can induce or influence. See, e.g., Vidarsson eta / ., Front Immunol. 2014 5(Art. 5): 1-17. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors e.g., B cell receptor); and B cell activation. Typically, the Fc-mediated functions involve binding of the Fc portion of an antibody by specialized receptor molecules, "Fc receptors" or "FcR," expressed by the cell whose function is to be affected.

[0145] IgG is considered the most versatile immunoglobulin because it carries out all of the functions of immunoglobulin molecules in some embodiments. IgG is the major Ig in serum, and the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not. Macrophages, monocytes, polymorphonuclear leukocytes (PMNs), and some lymphocytes have receptors for the Fc region of IgG. Not all subclasses bind equally well: IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMNs, monocytes, and macrophages is that the cell now internalizes the antigen better in some cases. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.

[0146] In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma ("y") receptor) and includes receptors of the FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16) subclasses, including allelic variants and alternatively spliced forms of these receptors FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine- based inhibition motif (ITIM) in its cytoplasmic domain.

[0147] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells \_e.g., Natural Killer (NK) cells, neutrophils, and macrophages] enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay is performed in some embodiments. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0148] Alternatively, or additionally, in some embodiments, ADCC activity of the molecule of interest is assessed in vivo, e.g., in an animal model.

[0149] For certain therapeutic applications, the internalization process is employed for purposes of killing or decreasing the activity or proliferation of a target cell that expresses an HLA-A2 protein. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth.

[0150] "Antibody-Dependent Cellular Phagocytosis" or "ADCP" refers to a mechanism by which antibody-opsonized target cells activate the FcyRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification.

[0151] The HLA-A2 antibody or antigen-binding fragment provided herein can be conjugated or linked to a therapeutic moiety, an imaging or detectable moiety, or an affinity tag. Methods for conjugating or linking polypeptides are well known in the art. Associations (binding) between compounds and labels include any means known in the art including, but not limited to, covalent and non-covalent interactions, chemical conjugation, as well as recombinant techniques. An antibody or antigen-binding fragment thereof is conjugated to, or recombinantly engineered with, an affinity tag {e.g., a purification tag), in some embodiments. Affinity tags such as, for example, poly-histidine e.g., His6) tags are conventional in the art.

[0152] The HLA-A2 antibody or antigen-binding fragment can be conjugated or linked to a detectable moiety. Detection can be accomplished, for example, in vitro, in vivo, or ex vivo. In vitro assays for the detection and / or determination (quantification, qualification, etc.) thereof include, but are not limited to, ELISAs, RIAs, and western blots. In some embodiments, in vitro detection, diagnosis, or monitoring of the antigen of the antibodiesoccurs by obtaining a sample (e.g., a blood sample) from a subject and testing the sample in, for example, a standard ELISA assay.Derivatives

[0153] Derivatives of the HLA-A2 antibody products described herein are also provided. The derivatized antibody product can comprise any molecule or substance that imparts a desired property to the antibody product, such as increased half-life in a particular use. The derivatized antibody product can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), or a molecule that binds to another molecule e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo ov in vitro uses). Examples of molecules that can be used to derivatize an antibody product include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antibody products can be prepared using techniques well known in the art. The antibody can be conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

[0154] Other derivatives include covalent or aggregative conjugates of HLA-A2 antibody products, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an HLA-A2 antibody product. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag. HLA-A2 antibody product-containing fusion proteins can comprise peptides added to facilitate purification or identification of the HLA-A2 antibody product (e.g., poly-His). An HLA-A2 antibody product also can be linked to the FLAG peptide as described in Hopp etai., Bio / Technoiogy 1988 6: 1204-10, and U.S. Pat. No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), enabling rapid assay and facile purification of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo.).

[0155] Oligomers that contain one or more HLA-A2 antibody products can be employed as HLA-A2 antagonists. Oligomers can be in the form of covalently linked or non-covalently linked dimers, trimers, or higher. Oligomers comprising two or more HLA-A2 antibody products are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0156] Oligomers can comprise multiple HLA-A2 antibody products joined via covalent or non-covalent interactions between peptide moieties fused to the HLA-A2 antibody polypeptides. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of HLA-A2 antibody products attached thereto, as described in more detail below.

[0157] Oligomers can comprise from two to four HLA-A2 antibody products. The HLA-A2 product moieties of the oligomer can be in any of the forms described above, e.g., variants or fragments. The oligomers comprise HLA-A2 antibody products that have HLA-A2 binding activity.

[0158] Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi eta / ., Proc Natl Acad Sci USA. 1991 88(23): 10535-9; Byrn et a / ., Nature. 1990 344(6267): 667-70; and Hollenbaugh and Aruffo, Curr Protoc Immuno! 2002 48(1): 4: 10.19.1-10.19.11.

[0159] Dimers are provided comprising two fusion proteins created by fusing a HLA-A2 binding fragment of an HLA-A2 antibody to the Fc region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield the dimer.

[0160] The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

[0161] One exemplary Fc polypeptide, described in PCT Pub. No. WO 93 / 10151 and U.S. Patent Nos. 5,426,048 and 5,262,522 (each of which is hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C- terminus of the Fc region of a human IgGl antibody. Another exemplary Fc polypeptide is the Fc mutein described in U.S. Patent No. 5,457,035 and in Baum eta / ., EMBOJ. 1994 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in PCT Pub. No. WO 93 / 10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

[0162] Alternatively, the oligomer is a fusion protein comprising multiple HLA-A2 antibody polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Patent Nos. 4,751,180 and 4,935,233.

[0163] Another method for preparing oligomeric HLA-A2 antibody product derivatives involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT Pub. No. WO 94 / 10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe eta / ., FEBSLett. 1994 344: 191-5. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow eta / ., Semin Immunol. 1994 6:267-78. Generally, recombinant fusion proteins comprising an HLA- A2 antibody fragment fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomeric HLA-A2 antibody products that form are recovered from the culture supernatant.

[0164] HLA-A2 antibody products described herein can also be derivatized or modified such that the products have a longer half-life as compared to the underivatized or unmodified antibody. For example, the antibody product can contain point mutations to increase serum half-life, such as described in PCT Pub. No. WO 00 / 09560.Nucleic Acids and Cells

[0165] Nucleic acids are provided that encode one or more chains of an antibody product herein, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing are also provided.

[0166] Nucleic acids provided encode antibody products disclosed herein, such as a light chain variable region provided herein, and / or a heavy chain variable region provided herein. Due to the degeneracy of the genetic code, each of the polypeptide sequences provided herein is also encoded other nucleic acid sequences besides those provided herein. The present disclosure provides each degenerate nucleotide sequence encoding each antibody product.

[0167] Illustrative nucleic acids are provided herein.Table 4 - Nucleic Acids Encoding Variable Domains

[0168] The term "polynucleotide" or "nucleic acid" means single-stranded or doublestranded polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, and phosphoroamidate. The term includes both single and double stranded forms.

[0169] An "isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that "a nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules"comprising" specified nucleic acid sequences can include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty other proteins or portions thereof, or can include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and / or can include vector sequences.

[0170] Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[0171] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. For example, control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence. "Control sequences" can include leader sequences and / or fusion partner sequences.

[0172] The term "vector" means any molecule or entity e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information into a host cell.

[0173] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.

[0174] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0175] The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0176] DNA encoding antibody polypeptides e.g., heavy or light chain, variable domain only, or full length) can be isolated from B-cells of mice that have been immunized with HLA-A2 or an immunogenic fragment thereof. The DNA can be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique whereby nucleotide sequences encoding antibody polypeptides can be selected.

[0177] Nucleic acids are provided that hybridize to other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well-known in the art. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5X sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6X SSC, and a hybridization temperature of 55 °C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42 °C.), and washing conditions of 60 °C, in 0.5X SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6X SSC at 45 °C, followed by one or more washes in 0.1X SSC, 0.2% SDS at 68 °C. Furthermore, one of skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to each other typically remain hybridized to each other.

[0178] The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel eta!, eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and / or base composition of the DNA.

[0179] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide e.g., an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in theart. One or more particular amino acid residues can be changed using, for example, a site- directed mutagenesis protocol. One or more randomly selected residue can be changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0180] Polypeptides that are components of an antibody product of interest are expressed in any suitable recombinant expression system.

[0181] Expression vectors are provided comprising nucleic acid encoding an HLA-A2 antibody product. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.

[0182] Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0183] Optionally, the vector can contain a "tag "-encoding sequence, that is, an oligonucleotide molecule located at the 5' or 3' end of the coding sequence, the oligonucleotide sequence encoding polyHis (such as hexaHis), or another "tag" for which commercially available antibodies exist, such as FLAG, HA (hemagglutinin from influenza virus), or myc. The tag is typically fused to the antibody protein upon expression, and can serve as a means for affinity purification of the antibody from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified antibody polypeptide by various means such as using certain peptidases for cleavage.

[0184] Flanking sequences in the expression vector can be homologous (Ze., from the same species and / or strain as the host cell), heterologous (Ze., from a species other than the host cell species or strain), hybrid (Ze., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence can be anyprokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.

[0185] Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably linked to nucleic acid encoding the HLA-A2 antibody product. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, initiate continuous gene product production; that is, there is little or no experimental control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the HLA-A2 antibody product by removing the promoter from the source DNA by restriction enzyme digestion or amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.

[0186] Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

[0187] Exemplary promoters useful in recombinant expression vectors include, but are not limited to: the SV40 early promoter region (Bemoist and Chambon, 1981, Nature, 290:304- 10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, eta / ., Cell. 1980 22:787-97); the herpes thymidine kinase promoter (Wagner eta / ., Proc Natl Acad Sci USA. 1981 78: 1444-5); the regulatory sequences of the metallothionine gene (Brinster eta!, Nature. 1982 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Komaroff eta!, Proc Natl Acad Sci USA197 75:3727-31); or the tac promoter (DeBoer eta!., Proc Nat! Acad Sci USA. 1983 80:21-5). Also available for use are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift etal, Cell. 1984 38:639- 46; Ornitz etal, Cold Spring Harb Symp Quant Bioi. 1986 50:399-409; MacDonald, Hepatology. 1987 7:425-515); the insulin gene control region that is active in pancreaticbeta cells (Hanahan, Nature. 1985 315: 115-22); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder eta / ., Cell. 1986 45:485-95); the albumin gene control region that is active in liver (Pinkert eta!, Genes Dev 1987 1:268-76); the alpha-feto-protein gene control region that is active in liver (Krumlauf eta!., Mol Cell Bio! 1985 5: 1639-48; Hammer eta!., Science. 1987 235:53-8); the alpha 1- antitrypsin gene control region that is active in the liver (Kelsey eta / ., Genes Dev. 1987 1: 161-71); the beta-globin gene control region that is active in myeloid cells (Mogram eta / ., Nature. 1985 315:338-40; Kollias eta / ., Cell. 1986 46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead eta / ., Cell. 1987 48:703-12); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, Nature. 1985 314:283-6); the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason eta / ., Science. 1986 234: 1372-8); and most particularly the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl eta!., Cell. 1984 38:647-58; Adams eta!., Nature. 1985 318:533-8; Alexander et al, Moi Cell Biol 1987 7: 1436-44).

[0188] An enhancer sequence can be inserted into the vector to increase the transcription in higher eukaryotes of a nucleic acid encoding an HLA-A2 antibody product described herein. Various enhancer sequences available from mammalian genes are known e.g., globin, elastase, albumin, alpha-feto-protein and insulin). An enhancer sequence from a virus also can be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer can be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically placed at a site 5' to the promoter.

[0189] In expression vectors, a transcription termination sequence is typically located 3' of the end of a polypeptide-coding region and serves to terminate transcription. A transcription termination sequence used for expression in prokaryotic cells typically is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.

[0190] Selection genes can be used to amplify the gene that will be expressed. Amplification is a process whereby genes that cannot in single copy be expressed at high enough levels to permit survival and growth of cells under certain selection conditions are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable amplifiable selectable markers for mammalian cells includedihydrofolate reductase (DHFR) and promoterless thymidine kinase. In the use of these markers mammalian cell transformants are placed under selection pressure in which only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby permitting survival of only those cells in which the selection gene has been amplified. Under these circumstances, DNA adjacent to the selection gene, such as DNA encoding an antibody, is co-amplified with the selection gene. As a result, increased quantities of HLA-A2 polypeptide are synthesized from the amplified DNA.

[0191] A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[0192] In some cases, for example where glycosylation is desired in a eukaryotic host cell expression system, various presequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or pro-sequences added, which also can affect glycosylation. The final protein product can have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product can have one or two amino acid residues found in the peptidase cleavage site, attached to the amino terminus. Alternatively, use of some enzyme cleavage sites can result in a slightly truncated yet active form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.

[0193] Where a commercially available expression vector lacks some of the desired flanking sequences as described above, the vector can be modified by individually ligating these sequences into the vector. After the vector has been chosen and modified as desired, a nucleic acid molecule encoding an HLA-A2 antibody product is inserted into the proper site of the vector.

[0194] The completed vector containing sequences encoding the antibody product is inserted into a suitable host cell for amplification and / or polypeptide expression. The transformation of an expression vector for an HLA-A2 antibody product into a selected host cell can be accomplished by well-known methods including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran method, or other known techniques. The method selected will in part be a function of thetype of host cell to be used. These methods and other suitable methods are well known to the skilled artisan.

[0195] Antibodies can be expressed in hybridoma cell lines or in cell lines other than hybridomas. Expression constructs encoding the antibodies can be used to transform a mammalian, insect or microbial host cell. Transformation can be performed using any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus or bacteriophage and transducing a host cell with the construct by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The optimal transformation procedure used will depend upon which type of host cell is being transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively charged lipids, and direct microinjection of the DNA into nuclei.

[0196] The transformed host cell, when cultured under appropriate conditions, synthesizes an HLA-A2 antibody product that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

[0197] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells e.g., Hep G2), human embryonic kidney cells (HEK) e.g., HEK-293) and a number of other cell lines. The best cell line for expressing a particular DNA construct can be selected by testing various cell lines to determine which ones have the highest levels of expression levels and produce antibody products with the desired HLA-A2 binding properties.

[0198] Nucleic acid molecules that are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences are also provided. A nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, forexample, a fragment that can be used as a probe or primer or a fragment encoding an active portion e.g., a HLA-A2 binding portion) of a polypeptide.Compositions

[0199] Compositions that include HLA-A2 antibody products are also provided. Pharmaceutical compositions typically include one or more of a buffer, a pharmaceutically acceptable diluent, a carrier, a solubilizer, an emulsifier and a preservative. The use of the foregoing antibody products in the preparation of a pharmaceutical composition or medicament is also provided.

[0200] Acceptable formulation components for pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the antibody products that are provided herein, compositions can contain components for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants, (see Remington's Pharmaceutical Sciences, 23rded., (Adejare, ed.), 2020, Elsevier Academic Press).

[0201] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. Suitable vehicles or carriers for such compositions include water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Compositions comprising HLA-A2 antibody product can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the HLA-A2 antibody product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0202] Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 4.0 to about 8.5, or alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefor.

[0203] Additional pharmaceutical compositions are in the form of sustained- or controlled- delivery formulations. Techniques for formulating a variety of other sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections can be used (see, e.g., PCT Pub. No. WO 93 / 15722 Al, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions). Sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules, polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman eta / ., Biopolymers. 1983 22:547-56), poly (2- hydroxyethyl-methacrylate) (Langer eta / ., J Biomed Mater Res. 1981 15: 167-277) and Langer, Chem Tech. 1982 12:98-105), ethylene vinyl acetate (Langer etai., ibid.) or poly- D(-)-3-hydroxybutyric acid (EP 133,988). Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein etai., Proc Natl Acad Sci USA. 1985 82:3688-92; EPO Pub. Nos. EP 036676; EP 088046, and EP 143949.

[0204] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilizedpowder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0205] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0206] Kits are provided for multi-dose or single-dose administration units. For example, kits can each contain both a first container having a dried protein and a second container having an aqueous diluent, including for example single and multi-chambered pre-filled syringes e.g., liquid syringes, lyosyringes or needle-free syringes).

[0207] The pharmaceutical compositions can be delivered parenterally, typically by injection. Injections can be intraocular, intraperitoneal, intraportal, intramuscular, intravenous, intrathecal, intracerebral (intra-parenchymal), intracerebroventricular, intraarterial, intralesional, perilesional, or subcutaneous. Eye drops can be used for intraocular administration. In some instances, injections can be localized to the vicinity of a particular bone or bones to which the treatment is targeted. For parenteral administration, the antibodies can be administered in a pyrogen-free, parenterally acceptable aqueous solution comprising the desired HLA-A2 antibody product in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the HLA-A2 antibody product are formulated as a sterile, isotonic solution, properly preserved.

[0208] Pharmaceutical compositions comprising the subject HLA-A2 antibody products can be administered by bolus injection or continuously by infusion, by implantation device, sustained release systems or other means for accomplishing prolonged release. The pharmaceutical composition also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous release. The preparation can be formulated with agents, such as injectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid; polyglycolic acid; or copoly (lactic / glycolic) acid (PLGA), beads or liposomes, that can provide controlled or sustained release of the product which can then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation.

[0209] Subject compositions comprising an HLA-A2 antibody product also can be used ex vivo. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to or cultured with the HLA-A2 antibody product. The cultured cells can then be implanted back into the patient or a different patient or used for other purposes.

[0210] An HLA-A2 antibody product can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. Such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic, or can be immortalized. In order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. Encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

[0211] As used herein, "substantially pure" means that the described species of molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. A substantially pure molecule can be a composition in which the object species comprises at least 50% (on a molar basis) of all macromolecular species present. A substantially pure composition can comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. The object species can also be purified to essential homogeneity in which contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.Dosages

[0212] The pharmaceutical compositions that are provided can be administered for prophylactic and / or therapeutic treatment.

[0213] As used herein, the terms "treatment," "treating," and the like, refer to administering an agent or carrying out a procedure, for the purposes of obtaining an effect. The effect is prophylactic in terms of completely or partially preventing disease or symptom thereof in a subject at risk for GVHD and / or is therapeutic in terms of effecting a partial orcomplete cure for a disease and / or symptoms of the disease in a subject having (suffering from) GVHD. "Treatment," as used herein, includes treatment of a disease or disorder e.g., GVHD) in a mammal, particularly in a human, and includes: (a) prophylactic treatment, that is preventing the disease or a symptom of a disease from occurring in a subject which is predisposed to the disease but has not yet been diagnosed as having it e.g., including diseases that are associated with or caused by a primary disease); (b) therapeutic treatment inhibiting the disease, i.e., arresting its development; and (c) therapeutic treatment relieving the disease, i.e., causing regression of the disease. Treating refers to any clinical indicia of success in the treatment or amelioration or prevention, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases e.g., GVHD). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. For example, a subject is "treated" for a disease or disorder if, after receiving a therapeutic amount of a combination of an HLA-A2 antibody product provided herein, the patient shows one or more observable and / or measurable changes in a symptom of the disease condition. Treatment can reduce or prevent one more acute GVHD symptoms such as skin rash, skin redness, abdominal pain, abdominal cramps, nausea, vomiting, diarrhea, and liver dysfunction e.g., elevated liver enzymes and / or jaundice). Treatment can reduce or prevent one or more chronic GVHD symptoms such dry itchy skin, skin thickening, skin tightening, dry eyes, dry mouth, shortness of breath, joint stiffness, and joint pain).

[0214] Two systems for GVHD staging are the International Cone Marrow Transplant Registry (IBMTR) system (A to D) (Rowlings etai., Br J Haematol. 1997; 97(4): 855-864) and Glucksberg grade (1 to 4) (Glucksberg etai., Transplantation 1974; 18(4): 295- 304). Staging is based on clinical manifestations and severity of organ involvement (Jamil, Int J. Hematol. 2015; 101(5): 452-466).SkinStage 1: Maculopapular rash <25% of the bodyStage 2: Maculopapular rash 25% to 50% of the bodyStage 3: Generalized erythrodermaStage 4: Generalized erythroderma with bullaeLiver• Stage 1: Bilirubin 2 to 3, AST 150 to 750• Stage 2: Bilirubin 3 to 6• Stage 3: Bilirubin 6 to 15• Stage 4: Bilirubin >15 Gastrointestinal System• Stage 1: Diarrhea >500 cc / day• Stage 2: Diarrhea >1000 cc / day• Stage 3: Diarrhea >1500 cc / day• Stage 4: Diarrhea >2000 cc / day or severe abdominal pain Glucksberg Grade:1. Mild: No liver or GI involvement, stage 1 to 2 skin involvement2. Moderate: Stage 1 liver or GI involvement, stage 1 to 3 skin involvement3. Severe: Stage 2 to 3 skin, liver, or GI involvement4. Life-threatening: stage 2 to 4 liver or GI involvement, stage 1 to 4 skin involvement International Cone Marrow Transplant Registry Severity Index:1. Mild: No liver or GI involvement, stage 1 skin involvement2. Moderate: Stage 1 to 2 liver or GI involvement, stage 2 skin involvement3. Severe: Stage 3 skin, liver, or GI involvement4. Life-threatening: Stage 4 skin, liver, or GI involvement

[0215] An "effective response" in accordance with the present disclosure is achieved when the subject experiences partial or total alleviation or reduction of signs or symptoms of illness, retarding progression, cure, prolongation of survival, or other objective responses. The expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Prolonging survival includes without limitation times of at least 1 month (mo.), about at least 2 mos., about at least 3 mos., about at least 4 mos., about at least 6 mos., about at least 1 year, about at least 2 years, about at least 3 years, etc. Overall survival is also measured, for example, in months to years. Alternatively, an effective response can be that a subject's symptoms remain static.

[0216] Administration of a therapeutic agent in a prophylactic method occurs prior to the manifestation of symptoms of an undesired disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. Thus, when used in conjunction with prophylactic methods, the term "therapeutically effective" means that, aftertreatment, a smaller number of subjects (on average) develop the undesired disease or disorder or progress in severity of symptoms.

[0217] The terms "recipient," "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired (Ze., "in need thereof"), particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. The mammal can be a human.

[0218] In general, toxicity and therapeutic efficacy of the antibody product can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD5o (the dose lethal to 50% of the population) and the ED5o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred.

[0219] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0220] The effective amount of a pharmaceutical composition comprising an HLA-A2 antibody product to be employed therapeutically or prophylactically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, will thus vary depending, in part, upon the molecule delivered, the indication for which the HLA-A2 antibody is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. A clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 1 pg / kg to up to about 1600 mg / kg or more, depending on the factors mentioned above. The dosage can range, for example, from 1 ng / kg up to about 200 mg / kg; 1 mg / kg up to about 200 mg / kg; or 1 mg / kg up to about 1200 mg / kg; or 1 pg / kg up to about 1600 mg / kg.

[0221] The dosing frequency will depend upon the pharmacokinetic parameters of the HLA-A2 antibody product in the formulation. For example, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, or as two or more doses (which can contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Treatment can be continuous over time or intermittent. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data. An exemplary dosing schedule is every 2 to 3 weeks.

[0222] To treat a disease condition by targeting HLA-A2, a composition comprising the HLA-A2 antibody product is administered to the patient in an amount and for a time sufficient to induce a sustained improvement in at least one indicator that reflects the severity of the disorder. An improvement is considered "sustained" if the patient exhibits the improvement on at least two occasions separated by at least one to seven days, or in some instances one to six weeks. The appropriate interval will depend to some extent on what disease condition is being treated; it is within the purview of the skilled physician to determine the appropriate interval for determining whether the improvement is sustained. The degree of improvement is determined based on signs or symptoms and can also employ questionnaires that are administered to the patient, such as quality-of-life questionnaires.

[0223] Various indicators that reflect the extent of the patient's illness can be assessed for determining whether the amount and time of the treatment is sufficient. The baseline value for the chosen indicator or indicators is established by examination of the patient prior to administration of the first dose of antibody. Preferably, the baseline examination is done within about 100 days of administering the first dose.

[0224] Improvement is induced by administering the HLA-A2 antibody product until the patient manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e.g., for one, two, or three months or longer, or indefinitely. A period of one to six weeks, or even a single dose, often is sufficient for treating acute conditions.

[0225] Although the extent of the patient's illness after treatment may appear improved according to one or more indicators, treatment can be continued indefinitely at the samelevel or at a reduced dose or frequency. Once treatment has been reduced or discontinued, it later can be resumed at the original level if symptoms should reappear.

[0226] Antibody products provided herein may be administered at an ultra-low dose to subjects to treat GVHD.Methods of Use

[0227] The HLA-A2 antibody products disclosed herein have a variety of uses. The antibodies and fragments, for instance, are useful for specific binding assays, affinity purification of HLA-A2 or its ligands, and screening assays to identify other antagonists of HLA-A2 activity. The antibody products can be used to treat various diseases that are associated with the activity of HLA-A2.

[0228] HLA-A2 antibody products can be used to detect HLA-A2 in biological samples. Such uses allow the identification of cells or tissues that express HLA-A2 or serve as a diagnostic for diagnosing pathological conditions involving HLA-A2, for example, GVHD associated with a donor graft.

[0229] Accordingly, methods are provided of detecting HLA-A2 in a biological sample, such as a sample of an in vitro medium or a tissue sample from a subject, or in vivo in a subject, in which the method comprises contacting a cell expressing HLA-A2 with an HLA-A2 antibody product provided herein. The HLA-A2 antibody product can be conjugated to a detectable moiety, and the method comprises detecting the moiety directly. The method can comprise detecting binding of the HLA-A2 antibody product to the cell indirectly by way of a detectably moiety that binds to the antibody. For example, an IgG antibody conjugated to a detectable moiety can be used to bind to the HLA-A2 antibody presented as an IgG isotype.

[0230] The disclosure provides diagnostic kits comprising an HLA-A2 antibody product provided herein. The diagnostic kits can be kits, for example, for flow cytometry analysis of the chimeric state of a subject in which donor and recipient cells are mixed after HLA mismatch transplantation (HCT / SOT). Illustrative kits can comprise an HLA-A2 antibody product as well as an antibody panel such as the following.Panel 1:AF488_Open for HLA 1PE_Open for HLA 2PE_Dazzle CD16PE-Cy5_CD235a / PIPerCP-Cy5.5_CD8PE-Cy7_CD19APC_CD34 SPKR_718_CD56 APC_F750_CD3 SPKVL_423_CD45 SPKVL_500_CD4BV711 CD14Panel 2:AF488_Open for HLA 1 PE_Open for HLA 2 PE-Cy5_CD235a / PI PE-Cy7_CD19 SPKR_718_CD56 APC_F750_CD3 SPKVL_423_CD45BV711_CD14Panel 3:AF488_Open for HLA 1 PE_Open for HLA 2 PE-Cy5_CD235a / PI APC_CD34 SPKVL_423_CD45Panel 4:AF488_Open for HLA 1 PE_Open for HLA 2 PE_Dazzle_CD16 PE-Cy5_TCR a / b PerCP-Cy5.5_CD8 PE-Cy7_CD19 APC TCR g / d SPKR_718_CD56 APC_F750_CD3 SPKVL_423_CD45SPKVL_500_CD4BV711 CD14

[0231] The antibody products provided can also be used in methods to screen for a molecule that binds to HLA-A2. A variety of competitive screening methods, for example, can be used. In some methods, an HLA-A2 molecule or fragment thereof to which an HLA- A2 antibody product binds, is contacted with an antibody product disclosed herein together with another molecule (Ze., a candidate molecule). A reduction in binding between the antibody product and HLA-A2 is an indication that the candidate molecule binds HLA-A2. Binding of the antibody product can be detected using a variety of methods, e.g., an ELISA. Detection of binding between the HLA-A2 antibody product and HLA-A2 can be simplified by detectably labeling the antibody. In some methods, a molecule that exhibits binding in the initial screen is further analyzed to determine whether it inhibits or modulates aN HLA-A2 activity.

[0232] The disclosure provides methods of reducing a biological activity of HLA-A2 in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein. Biological activities contemplated herein include, but are not limited to, one or more of the following.• Antigen presentation: HLA-A2 is involved in presenting peptide antigens to CD8+ T cells. Blocking this function can reduce immune responses mediated by these T cells.• T cell recognition: the antibody product can interfere with T cell receptor (TCR) recognition of HLA-A2-peptide complexes, potentially inhibiting T cell activation.• CD8+ T cell-mediated cytotoxicity: the antibody product can inhibit MHC-restricted cellular cytotoxicity.• Immune cell interactions: Blocking HLA-A2 can disrupt interactions between antigen- presenting cells and T cells.• Allogeneic responses: in transplantation settings, reducing HLA-A2 activity can decrease allogeneic immune responses against donor tissues.• Viral antigen presentation: HLA-A2 is involved in presenting viral antigens, so blocking it can affect antiviral immune responses.• Tumor antigen presentation: in cancer immunotherapy contexts, reducing HLA-A2 activity can modulate tumor antigen presentation.• Cell surface expression: the antibody product can induce internalization of HLA-A2, reducing its surface expression on cells.• Peptide binding: blocking the peptide-binding groove of HLA-A2 can interfere with its ability to bind and present antigens.• Immune surveillance: reducing HLA-A2 activity can affect the ability of the immune system to detect and respond to altered self-antigens.

[0233] The disclosure provides methods of depleting cells expressing HLA-A2 in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein. HLA-A2-expressing cells to be depleted include, but are not limited to, one or more of the following.• Donor cytotoxic CD8+ T cells: these cells express HLA-A2 and are crucial for immune responses, including graft-versus-host disease (GVHD) and graft-versus-tumor effects.• Allogeneic hematopoietic stem cells: HLA-A2 positive donor stem cells used in transplantation can be targeted.• Antigen-presenting cells (APCs): dendritic cells, macrophages, and B cells that express HLA-A2 and present antigens to T cells.• Natural killer (NK) cells: some NK cell subsets express HLA-A2 and can be affected.• Regulatory T cells (Tregs): HLA-A2 positive Tregs involved in immune regulation and tolerance.• Memory T cells: both CD4+ and CD8+ memory T cells expressing HLA-A2.• Effector T cells: activated T cells involved in immune responses against pathogens or tumors.• Tissue-resident cells: various cell types in organs and tissues that express HLA-A2, including epithelial and endothelial cells.• Tumor cells: malignant cells expressing HLA-A2 can be targeted in cancer immunotherapy approaches.• Virus-specific T cells respond to viral infections in HLA-A2-positive individuals.

[0234] The HLA-A2 antibody products provided herein are useful for the treatment of human disease, including treatment of GVHD. The disclosure provides methods of treating a subject in need thereof (Ze., at risk for or having GVHD), comprising administering to the patient a therapeutically effective amount of an antibody product or pharmaceutical composition provided herein.

[0235] A way by which an HLA-A2 antibody product can mediate killing of HLA-A2- expressing cells is through antibody-dependent cellular toxicity ("ADCC"). ADCC is theprocess by which antibodies coat a target cell and recruit effector cells to induce target cell death via non-phagocytic mechanisms.

[0236] As noted above, in treatment methods the HLA-A2 antibody products provided herein can be used as a monotherapy or in a combination therapy. A "combination" therapy refers to administration of one treatment agent before, during, or after administration of the other treatment agent to the subject.

[0237] Provided herein are methods of treating a patient at risk for or having GVHD, comprising administering to the patient an HLA-A2 antibody product as described herein e.g., as a pharmaceutical composition thereof).

[0238] Provided herein are methods of treating a patient at risk for or having GVHD, comprising administering to the patient an HLA-A2 antibody product as described herein e.g., as a pharmaceutical composition thereof) in combination with at least one other therapeutic agent. Other therapeutic agents contemplated herein include, but are not limited to, corticosteroids e.g., Prednisolone, Methylprednisolone and Budesonide), calcineurin inhibitors e.g., Tacrolimus and Cyclosporine), mTOR inhibitors e.g., Sirolimus and Everolimus), JAK inhibitors e.g., Ruxolitinib), tyrosine kinase inhibitors (TKIs) e.g., Ibrutinib and Imatinib), ROCK2 inhibitors e.g., Belumosudil), anti-inflammatory agents (Infliximab and Etanercept), monoclonal antibodies (Rituximab and IL-2 receptor antibodies), antimetabolites (Mycophenolate mofetil and Methotrexate), chemotherapy drugs e.g., Pentostatin), immunomodulators e.g., Thalidomide), proteasome inhibitors e.g., Bortezomib), ECP (Extracorporeal photopheresis), and mesenchymal stem cells. Such combination use can comprise administering an effective amount of the HLA-A2 antibody product and an effective amount of the at least one other therapeutic agent. The HLA-A2 antibody product and the at least one other therapeutic agent can complement each other therapeutically and that one or both may be administered in subtherapeutic amounts, yet the combined use may yet be therapeutically effective. Such combination use may provide opportunities for clinical use in situations where a therapeutic window for one or another component of the combination is narrow, so to limit or mitigate adverse effects in the patient.Other Terminology and Disclosure

[0239] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0240]

[0138] As used herein and in the appended claims, the singular forms "a," "and," and "the" mean "one or more" unless the context unambiguously requires a more restricted meaning. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this sentence is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0241] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having." When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the subject matter of a claim.

[0242] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold includes 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, or 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, or 2.5-fold, etc., and so forth.

[0243] "About" a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. "About" a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0244] As used herein, "contemplated," "can," "can be," "may" and "may be" all indicate something contemplated by the inventors that is functional and available as part of the subject matter provided.

[0245] This entire document is intended to be read as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the embodiments specifically mentioned. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure.

[0246] All documents mentioned in this application are hereby incorporated herein by reference in their entirety to disclose and describe the methods and / or materials for the purpose for which the documents are cited. To the extent the material incorporated by a document contradicts or is inconsistent with this specification, the specification will supersede any such material.EXAMPLES

[0247] While the following examples describe specific embodiments, variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0248] Statistics

[0249] Survival curves of experimental groups were analyzed using the log-rank test, performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Differences between group means were assessed using Student's t-test, also performed with GraphPad Prism. Statistical significance was defined as p < 0.05.Example 1

[0250] Screening of killing allele-specific anti-HLA monoclonal antibody (ASHmAb).

[0251] Allele-specific anti-HLA monoclonal antibody (ASHmAb) hybridoma clones were generated using the standard protocol from Nakauchi et al. (PMID: 25448490). HLA-A2- positive peripheral blood mononuclear cells (PBMCs) from healthy donors (100,000 cells per well, in triplicate) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50-100 pL of hybridoma supernatant, each containing a single clone-derived antibody. Cells were cultured with or without 10% baby rabbit complement. After 24 hours of incubation at 37°C with 5% CO2, propidium iodide (PI) was added to stain dead cells, and the samples were analyzed by flow cytometry. See Fig.1A. By this screening process, the killing ASHmAb (kASHmAb) designated "AN7" was identified.Example 2

[0252] Characterization of AN7 (Isotype and FlowPRA)

[0253] The isotype of AN7 was determined using Rodent Monoclonal Isotyping Strips (AbD Serotec, Kidlington, UK), identifying it as IgG2a kappa (Fig. 1C). To assess the antigen specificity of AN7, FlowPRA screening (OneLambda) was performed by the manufacturer's instructions. Flow cytometry data are presented for FlowPRA beads coated with various HLA antigens. Group 1 included HLA-A01:01, A02:01, A03:01, B49:01, A25:01, A29:02, A30:01, and a control antigen or A26:01. Group 2 included HLA-A68:01, All:01, A34:02, A24:02, A32:01, A33:01, A31:01, and a control antigen or A23:01. (Fig. IB, ID)

[0254] LILRB1 is an inhibitory receptor expressed on NK cells and macrophages, binding to HLA class I antigens to inhibit ADCC and ADCP. An experiment was performed to demonstrate AN7 could block the interaction between LILRB1 and HLA-A antigens, confirming that recombinant LILRB1 binds to several HLA types, including HLA-A02:01.

[0255] Co-incubation with AN7 reduced LILRB1 binding to HLA-A02:01 (Fig. II), indicating AN7 functions as a blocking antibody contemplated herein as useful to enhance ADCC and ADCP.Example 3

[0256] AN7 binding to peripheral blood samples from healthy human donors

[0257] Peripheral blood samples from healthy donors were freshly obtained from the Stanford Blood Center. Mononuclear cells were isolated using Ficoll (GE Healthcare) density gradient centrifugation. Patient samples were analyzed with approval from the Stanford Institutional Review Board (IRB #46969). All sample analyses were performed using AN7 conjugated to PE (phycoerythrin) (AAT Bioquest, #1312).

[0258] Cells were washed with FACS buffer (PBS, 2% FBS, 2 mmol / L EDTA) and stained with antibodies for 20 minutes on ice in a total volume of 50 pL. Following staining, cells were washed and then stained with either propidium iodide (PI) at a final concentration of 1 pg / mL or DAPI at 0.1 pg / mL immediately before analysis. Flow cytometric analysis was performed using FACSCanto II (BD), FACSAria II (BD), or FACSymphony (BD). Data acquisition and analysis were conducted using FlowJo software (Treestar, Ashland, OR, USA), according to flow cytometry standard protocols.

[0259] Flow cytometry analysis with AN7 was performed on PBMCs from healthy individuals and post-transplant patients to test its ability to distinguish donor- and recipient- derived cells, specifically those expressing HLA-A2. The results (Fig. 2) confirmed that AN7 can clearly differentiate between these cells in both post-HSCT and post-kidney transplant patients, even when only one sample expressed HLA-A2. This indicates that AN7 can be a valuable diagnostic tool and that it can selectively target donor cells without harming recipient cells in GVHD therapy.Example 4

[0260] Complement-dependent cytotoxicity (CDC) assay

[0261] A complement-dependent cytotoxicity (CDC) assay was performed to evaluate the effect of AN7 on HLA-A2 positive and negative cells (1 x 10e5 cells / well) with and without baby rabbit complement. Cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) with 0.6 pg / well of isotype control, ascites-derived AN7 (aAN7), recombinant AN7 (rAN7), or commercial anti-A24 and anti-A33 antibodies in a total volume of 200 pL per well. The cells were incubated for 2 hours at 37°C and 5% CO2. The percentages of live cells were determined by propidium iodide (PI) staining and analyzed via flow cytometry (Fig. IE).

[0262] This experiment showed that AN7 exhibits strong CDC activity via baby rabbit complement, specifically targeting cells expressing HLA-A2. Additionally, it confirmed that not only AN7 purified from mouse ascites but also recombinant AN7, produced based on its sequence, has similar CDC activity specific to HLA-A2.

[0263] These findings highlight the consistent and potent CDC activity of AN7 against HLA-A2-expressing cells.Example 5

[0264] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0265] Natural killer (NK) cells (HLA-A2 negative) were isolated from human PBMCs using the EasySep Human CD56 Positive Selection Kit (Stemcell Technologies, #17855). Isolated NK cells were cultured overnight in RPMI 1640 medium supplemented with 10% FBS and 100 U / mL recombinant human IL-2 (PeproTech, #200-02). Target cells, HLA-A2 positive NALM6 and Rituximab control Raji cells, were labeled with 5 pM Calcein-AM (Thermo Fisher Scientific, #C3100MP) for 30 minutes at 37°C.

[0266] AN7 or isotype control antibodies were serially diluted 10-fold, ranging from 0.0003 to 30 pg / mL. For each condition, mAN7, cAN7, and Rituximab (positive control) were used at a concentration of 0.5 pg / well. Labeled HLA-A2 positive NALM6 target cells were added to the assay plate, followed by the addition of activated NK cells at a 1:5 target-to- effector ratio. The mixed cells were incubated for 2 hours at 37°C.

[0267] Total lysis was induced using Promega's lysis solution (IX), and 100 pL of supernatant was collected and transferred to a clean, flat-bottom black plate for analysis. The plate was read using a SpectraMax M3 fluorescence plate reader with 490 nm excitation, 520 nm emission, and a cutoff of 515 nm, using SoftMax Pro 7.0 software (top read, no shake) (Fig. IF).

[0268] In this study, the ADCC activity of AN7 mediated by HLA-A2 negative NK cells targeting HLA-A2 positive cells was examined. As shown in Fig. 1 F, both purified AN7 from mouse ascites and chimerized-AN7 exhibited more robust ADCC activity than Rituximab, which was used as a positive control. These results demonstrate AN7’s potent ADCC activity.Example 6

[0269] Antibody-dependent cellular phagocytosis (ADCP) assay

[0270] HLA-A2 negative monocyte-derived macrophages (50,000 cells / well) were cocultured with HLA-A2 positive and GFP positive NALM6 cell line (target cells, 100,000 cells / well) in 200 ul serum-free IMDM 200 ul for 2 hours. 0.5 ug antibody (isotype as negative control) was added to the culture medium (effector-to-target (E:T) ratio of 5: 1). CDllb positive I GFP positive cells were analyzed using flow cytometry. Here, we used 96- well ultra-low attachment plates for culturing the cells.

[0271] HLA-A2 negative monocyte-derived macrophages (50,000 cells / well) were cocultured with HLA-A2 positive and GFP-positive NALM6 target cells (100,000 cells / well) in 200 pL of serum-free IMDM for 2 hours. Antibodies (0.5 pg), including mouse-AN7 (mAN7), chimerized-AN7 (cAN7), and isotype control (negative control) were added to the culture medium at an effector-to-target ratio of 5: 1. Flow cytometry was used to analyze CDllb- positive / GFP-positive cells (Fig. 1G). The experiment was conducted in a 96-well ultra-low attachment plate to prevent cell adherence during culture.

[0272] The ADCP activity of AN7 through HLA-A2-negative macrophages was investigated. As shown in Fig. 1G, purified AN7 from mouse ascites and chimeric AN7demonstrated more robust ADCP activity than the isotype control. These results confirm the potent ADCP activity of AN7.Example 7

[0273] Mixed Lymphocyte Reaction (MLR) assay

[0274] Responder cells were isolated from HLA-A2 transgenic mouse spleens, while stimulator cells were collected from Balb / c mouse spleens. The stimulator cells were inactivated by treatment with 25 pg / mL mitomycin C for 30 minutes at 37°C. Both responder and stimulator cells were mixed at a 1: 1 ratio, using 5 x 10e5 cells of each type, and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). The assay was performed in triplicate wells for each condition. Three control conditions were included: isotype control, whole mouse IgG, and the test sample (AN7), with each condition tested at three different concentrations: 0.001 ng / mL, 0.1 ng / mL, and 10.0 ng / mL. The cells and antibody mixtures were cultured at 37°C with 5% CO2 for 72 hours. T cell proliferation was measured using flow cytometry with CountBright beads, following the manufacturer's instructions (Invitrogen, #036950) (Fig. 1H).

[0275] The extent to which HLA-A2-positive cells were damaged by adding AN7 to a mixture of HLA-A2-positive responder cells and HLA-A2-negative stimulator cells was analyzed using AN7 doses ranging from as low as 1 pg to 10 ng.

[0276] The results showed a statistically significant reduction in T cell numbers in the AN7-treated groups compared to the control under all conditions.

[0277] Donor-derived T cells, which cause GVHD, were damaged by a minimal amount of AN7, thus it is contemplated herein that T cells could be targeted explicitly with minimal antibody doses in vivo.Example 8

[0278] Mixed chimerism analysis using AN7

[0279] AN7 was used to accurately analyze the ratio of donor to recipient cells at various cell fraction levels in patient peripheral blood mononuclear cells (PBMCs) 32 days after HLA- A2 mismatched hematopoietic stem cell transplantation. The donor cells were HLA-A2- positive, while the recipient (patient) cells were HLA-A2-negative. Flow cytometry was performed using AN7 and commercial antibodies to distinguish between donor and recipient cells (Fig. 2).

[0280] The results are precise: AN7 can distinctly differentiate between HLA-A2-positive and negative cells. Furthermore, in combination with commercially available antibodies, it was also able to distinguish donor-derived and recipient-derived cells, including more specific T cells, B cells, and monocytes.

[0281] These findings indicates that AN7 possesses significant potential, such as in predicting the onset of GVHD.Example 9

[0282] Xenogeneic model of GVHD

[0283] Non-irradiated female NSG mice, aged 6 to 10 weeks, were intravenously injected with 1.0 x 10e7 human PBMCs on Day 0. The mice were treated with ASHmAb (3 mg / g / day) on Day 3, with a single dose of 60 mg / mouse. A survival curve was generated following antibody administration using a systemic assessment of GVHD. Objective measurements were used to monitor GVHD progression, with weight loss as the key criterion. Mice were euthanized if they exhibited a 20% or more significant reduction in body weight compared to their pre-transplant weight. (Fig. 3A-B)

[0284] Fig. 3A-B demonstrates that both AN7 derived from mouse ascites and recombinant AN7 can damage HLA-A2-positive cells in mice.Example 10

[0285] Allogeneic model of GVHD using HLA-A2 transgenic mouse

[0286] HLA-A2 positive PBMCs (10 million cells) were intravenously injected into HLA-A2 transgenic mice on Day 0. Mice were treated with either PBS or wide range of AN7 (Ing, 10 ng, 100 ng, and 60ug / mouse) administered intraperitoneally on Days 3 and 4. This GVHD mouse model that closely mimics the clinical setting. It allowed the study of GVHD within the same species, making it more reflective of human clinical conditions. Survival curves were generated using various doses of AN7.

[0287] The results indicated that AN7 dosages between 1 and 100 ng significantly improved survival rates (Fig. 4A, B).Example 11

[0288] Immunocompetent allo-GVHD model

[0289] Additionally, a new immunocompetent allo-GVHD model was used to test AN7. The model comprised lethally irradiated BALB / c mice transplanted with 5 million bone marrowcells from HLA-A2 transgenic C57BL / 6 donors (Le eta!., J Z / 77 / 7747 / 70 / 1989; 142(4): 1366- 1371). The fully MHC-mismatched combination induces robust and rapidly fatal GVHD. In addition, because most HSCs in the host BM are of donor origin, it was possible to examine the effect of AN7 on donor HSCs.

[0290] To determine the therapeutic window of AN7, a single dose of AN7 was administered at varying concentrations (0.1, 1, 10, 100 ng) on day 7 post-transplant (Fig. 5A), alongside a 10 ng isotype control. The rationale for evaluating such ultra-low doses was based on clinical evidence suggesting that donor-specific antibodies can mediate therapeutic effects at concentrations estimated <6 pg / kg8. Strikingly, a single administration of AN7 at 1, 10, or 100 ng conferred long-term survival in approximately half of the mice, whereas all animals in the isotype control and 0.1 ng groups succumbed within two weeks (Fig 5B).These findings define an effective and well -tolerated dose range of 1-100 ng for AN7 in the new GVHD model.

[0291] Importantly, all survivors exhibited successful donor hematopoietic reconstitution, indicating that AN7 selectively eliminates alloreactive immune cells while sparing donor hematopoietic stem cells (HSCs) (Fig. 5C). This advantageous specific AN7 activity is consistent with known HSC biology; HSCs residing in the BM niche are reported to exhibit resistance to apoptosis and immune-mediated attacks (Essers eta / ., Nature 2009;458(7240): 904-908) (Tasian eta!., Biomedicines 2018:6(1): 22).Example 12

[0292] To further validate the donor-specific immunosuppressive activity of AN7, an in vitro mixed lymphocyte reaction (MLR) was conducted using mitomycin C-treated BALB / c splenocytes as stimulators and splenocytes from HLA-A2 transgenic C57BL / 6 mice as responders. Treatment with AN7 at picogram-range concentrations (1 pg to 10 ng) significantly reduced the expansion of T cells (Fig. 5D), reinforcing the specificity and potency of AN7-mediated immunosuppressionSummary of the Examples

[0293] The present disclosure contemplates AN7 is a donor-targeted therapeutic with unprecedented efficacy at nanogram-level doses in the GVHD models. In contrast to conventional strategies that broadly suppress immune function, the strategy herein selectively targets a mismatched donor allele, enabling precise depletion of alloreactive cells while sparing stem cells. Robust in vivo activity was achieved with as little as 1-100 ng per mouse (0.05-5 pg / kg), a 1,000- to 100,000-fold reduction compared to typical therapeuticantibody doses used in clinical settings. This ultra-low dose requirement allows not only a favorable safety margin but also offers clear advantages in terms of manufacturing efficiency, cost-effectiveness, and clinical scalability.

[0294] HLA-A2 is among the most frequently mismatched alleles in allogeneic transplantation, owing to its high prevalence across global populations. AN7 exemplifies a novel class of allele-specific antibodies capable of selectively eliminating donor alloreactive immune cells. The use of a human chimeric antibody format enhances translational relevance and supports the clinical applicability of this approach. Supported by data from a clinically relevant GVHD model, the results herein lay the foundation for next-generation immunotherapies with improved specificity and reduced off-target toxicity.

Claims

CLAIMSWhat is claimed is:

1. An antibody product that specifically binds human leukocyte antigen A2 ( HLA-A2), the antibody product comprising a variable region comprising a heavy chain variable domain comprising a CDR-H1 set forth in SEQ ID NO: 1, a CDR-H2 set forth in SEQ ID NO: 2, and a CDR-H3 set forth in SEQ ID NO: 3, and a light chain variable domain comprising a CDR-L1 set forth in SEQ ID NO: 4, a CDR-L2 with the sequence set forth in SEQ ID NO: 5, and a CDR-L3 set forth in SEQ ID NO: 6.

2. The antibody product of claim 1, wherein the variable region comprises a heavy chain variable domain comprising: a) an amino acid sequence at least 80% identical to SEQ ID NO: 7; or b) an amino acid sequence set forth in SEQ ID NO: 7.

3. The antibody product of claim 1, wherein the variable region comprises a light chain variable domain comprising: a) an amino acid sequence at least 80% identical to SEQ ID NO: 8; or b) an amino acid sequence set forth in SEQ ID NO: 8.

4. The antibody product of any one of claims 1-3, wherein the variable region comprises a heavy chain variable domain comprising SEQ ID NO: 7 and a light chain variable domain SEQ ID NO: 8.

5. The antibody product of any one of claims 1-4, further comprising a heavy chain constant domain.

6. The antibody product of any one of claims 1-4, further comprising a light chain constant domain.

7. The antibody product of any one of claims 1-4, further comprising a heavy chain constant domain and a light chain constant domain.

8. The antibody product of claim 1-4, further comprising a murine heavy chain constant domain and a murine light chain constant domain.

9. The antibody product of any one of claims 1-4, further comprising a human heavy chain constant domain.

10. The antibody product of any one of claims 1-4, further comprising a human light chain constant domain.

11. The antibody product of any one of claims 1-4, further comprising a human heavy chain constant domain and a human light chain constant domain.

12. The antibody product of claim 9 or 11, wherein the human heavy chain constant domain is an IgA, IgD, IgE, IgG, or IgM human heavy chain constant domain.

13. The antibody product of claim 12, wherein the human heavy chain constant domain is an IgGl constant domain, an IgG2 constant domain, or an IgG4 constant domain.

14. The antibody product of claim 13, wherein the heavy chain constant domain is an IgGl constant domain.

15. The antibody product of claim 10 or 11, in which the human light chain constant region comprises a kappa domain or a fragment thereof.

16. The antibody product of any one of claims 1-4, in which the antibody product is a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single chain antibody.

17. The antibody product of any one of claims 1-4, in which the antibody product is a monospecific, bispecific, trispecific, or multispecific antibody.

18. An isolated nucleic acid, comprising: a) a nucleotide sequence encoding a heavy chain variable domain of SEQ ID NO: 7, b) a nucleotide sequence encoding a light chain variable domain of SEQ ID NO: 8, c) a nucleotide sequence encoding a heavy chain variable domain of SEQ ID NO: 9, or d) a nucleotide sequence encoding a light chain variable domain of SEQ ID NO: 10.

19. An expression vector comprising a nucleic acid comprising: a) a nucleotide sequence encoding a heavy chain variable domain of SEQ ID NO: 7, b) a nucleotide sequence encoding a light chain variable domain of SEQ ID NO: 8, or c) both a) and b).

20. An expression vector comprising a nucleic acid comprising:a) a nucleotide sequence encoding a heavy chain variable domain of SEQ ID NO: 9, b) a nucleotide sequence encoding a light chain variable domain of SEQ ID NO: 10, or c) both a) and b).

21. A host cell comprising the expression vector of claim 18, 19 or 20.

22. A method of producing an antibody product comprising an immunoglobulin variable domain, the method comprising: a) growing the host cell comprising an expression vector of claim 18 under conditions such that the host cell expresses the antibody product comprising the immunoglobulin variable domain; and b) purifying the protein comprising the immunoglobulin variable domain.

23. A method of producing an antibody product comprising an immunoglobulin heavy chain and an immunoglobulin light chain, comprising: a) growing a host cell comprising an expression vector of claim 19 or 20 under conditions such that the host cell expresses the antibody product; and b) purifying the antibody product.

24. A composition, comprising the antibody product of any one of claims 1-17 and an excipient.

25. An article of manufacture, comprising the composition of claim 24 and a container.

26. Use of the antibody product of any one of claims 1-17 or the composition of claim 24, for manufacture of a medicament for treatment of GVHD in a subject in need thereof.

27. A pharmaceutical composition, comprising an antibody product of any one of claims 1- 17 and a pharmaceutically acceptable excipient.

28. The antibody product of any one of claims 1-17, or the pharmaceutical composition of claim 27, for use in treating a subject at risk for or having GVHD.

29. Use of an antibody product of any one of claims 1-17 in the manufacture of a medicament for treating a subject at risk for or having GVHD.

30. A method of detecting HLA-A2 in a sample comprising: a) contacting the sample with an antibody product of any one of claims 1-17 andb) detecting the antibody product or detecting binding of the antibody product to HLA-A2.

31. A method of reducing a biological activity of HLA-A2 in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antibody product of any one of claims 1-17 or the pharmaceutical composition of claim 27.

32. A method of depleting cells expressing HLA-A2 in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antibody product of any one of claims 1-17 or the pharmaceutical composition of claim 27.

33. The method of claim 32 wherein the depletion of cells occurs by ADCC and / or ADCP.

34. A method of treating a subject at risk for or suffering from GVHD, the method comprising administering a therapeutically effective amount of the antibody product of any one of claims 1-17 or the pharmaceutical composition of claim 27.

35. The method of claim 34, further comprising administering an immunosuppressive agent to the patient.

36. The method of any one of claim 31-35, wherein the antibody product is administered at an ultra-low dose.

37. The antibody product of any one of claims 1-17, wherein the antibody product is detectably labeled, or comprises a therapeutic or cytotoxic moiety.

38. A kit comprising an antibody product of any one of claims 1-17 and instructions for using the antibody product for treatment of a subject in need thereof.

39. A kit comprising an antibody product of any one of claims 1-17 and instructions for using the antibody product for detection of HLA-A2.

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