HLA-a2 p53 antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antigen-binding domains that specifically bind to human p53R175H:HLA-A2 and compositions comprising such antibodies or antigen-binding domains. In further aspects, the antibodies or antigen-binding domains can be used to diagnose and treat diseases such as cancer.

Description

HLA-A2 P53 ANTIBODIES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application Nos.63 / 711,485, filed October 24, 2024, and 63 / 804,382, filed May 12, 2025, each of which is incorporated by reference herein in its entirety.1. FIELD

[0002] The present disclosure relates to molecules comprising antibody binding domains, such as antibodies, that specifically bind to human HLA-A2 presenting p53 peptides, compositions comprising such molecules, and methods of making and using the molecules that specifically bind to human HLA-A2:p53.2. DESCRIPTION OF RELATED ART

[0003] Targeted therapies, such as trastuzumab and imatinib, have revolutionized the treatment of cancer patients. All such drugs used thus far in the clinic target oncogenes rather than tumor suppressor genes. This is because drugs generally inhibit the action of proteins, and oncogenic proteins are activated by mutations or amplifications. By contrast, the proteins encoded by tumor suppressor genes are already inactivated, and reactivation is extremely difficult or impossible, depending on the nature of the alteration. This challenge is highlighted by p53, which was the first tumor suppressor gene identified and is inactivated in the great majority of human tumors. Despite extensive efforts, no drug that targets mutant p53 has been approved for treatment of the large number of patients whose tumors contain these mutations.

[0004] Mutations in p53 most commonly occur as single-nucleotide variants at positions that cluster in the DNA-binding domain. Proteins derived from mutant p53 alleles can be degraded by the proteasome, processed, and presented by the major histocompatibility complex to generate neoantigens recognizable by T cell receptors (TCRs). R175H, in which arginine at position 175 is replaced with histidine, is the most common mutation observed in p53 as well as the most frequent mutation in any tumor suppressor gene.

[0005] HLA Class I molecules are present on all nucleated cells. These molecules are responsible for presenting peptide fragments derived from intracellular proteins on thecell surface. These peptide-MHC complexes are recognized by cytotoxic T lymphocytes (CTLs), which are essential for immune surveillance and the elimination of infected or malignant cells. HLA-A*02:01 (HLA-A2) molecules are the most HLA Class I subtype present in approximately 40% of the American population, thus generating cancer immunotherapies against antigens presented by HLA-A2 would benefit a substantial population.

[0006] Peptide-HLA (pHLA) complexes are the natural ligands for T cell receptors (TCRs). Recently, TCR-mimic (TCRm) antibodies (Abs) have emerged as a class of agents that target pHLA derived from intracellular proteins in cancer cells. Compared with TCRs, TCRmAbs are of higher affinity and can be easily converted to various therapeutic formats such as full-length antibodies, antibody-drug conjugates (ADC), and bispecific antibodies.

[0007] Targeting tumor-associated pHLA antigens is often confronted by the challenge of low antigen density. Neoantigen pHLAs are present in a few to a few dozen copies per cell based on MS quantification of multiple cancer cell lines. Adoptive cell therapy has been used to target pHLA derived from not only intracellular tumor associated antigens (TAAs) and WT oncogenes but also mutant oncogenes and tumor suppressor genes. Although adoptive cell therapy has been successful in some patients, its widespread implementation is constrained by the need for patients’ autologous cells and for sophisticated manipulation of the cells in an individualized manner. Therefore, there exists a need in the art for new therapies that target specific HLA-A2 peptide complexes that are useful in diagnosis and disease treatment.3. SUMMARY

[0008] Provided herein are isolated antibodies and antigen-binding fragments thereof that specifically bind to human HLA-A2 p53 peptides and methods of use thereof. In one aspect, the peptide is the p53R175H peptide HLA-A2 (p53R175H:HLA-A2) complex. In one aspect, the disclosure provides an isolated antibody or antigen-binding domain that specifically binds to human p53R175H:HLA-A2 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of: SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively; SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively; SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively; SEQ ID NOs: 8, 9,10, and 24, 25, and 26, respectively; SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively. In another aspect, the disclosure provides an isolated antibody or antigen-binding domain that competitively inhibits binding of a reference antibody to human p53R175H:HLA-A2, wherein the reference antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of: SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively; SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively; SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively; SEQ ID NOs: 8, 9, 10, and 24, 25, and 26, respectively; SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively. In another aspect, the disclosure provides an isolated antibody or antigen-binding domain that binds to the same human p53R175H:HLA-A2 epitope as an antibody comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences of: SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively; SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively; SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively; SEQ ID NOs: 8, 9, 10, and 24, 25, and 26, respectively; SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively.

[0009] In one aspect, the disclosure provides an isolated antibody or antigen-binding domain that specifically binds to human p53R175H:HLA-A2, wherein the antibody or antigen-binding domain comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of an antibody selected from the group consisting of IM811, IM810, IM48, IM37, IM42, or IM710. In another aspect, the CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, the IMGT-defmed CDRs, or the AbM-defined CDRs. In another aspect, the antibody or antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NOs: 33-38. In another aspect, the antibody or antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NOs: 39-44. In another aspect, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of: (a) SEQ ID NOs: 33 and 39, respectively; (b) SEQ ID NOs: 34 and 40, respectively; (c) SEQ ID NOs: 35 and 41, respectively; (d) SEQ ID NOs: 36 and 42, respectively; (e) SEQ ID NOs: 37 and 43, respectively; or (f) SEQ ID NOs: 38 and 44, respectively. In another aspect, the antibody is humanized.

[0010] In another aspect, the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region. In another aspect, the heavy chain constant region is an isotype selected from the group consisting of human IgGl, IgG2, IgG3, and IgG4 isotypes. In another aspect, the antibody comprises an Fc domain that is engineered to reduce effector function. In another aspect, the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgGl heavy chain constant region, and wherein the light chain constant region is a human IgG? light chain constant region. In another aspect, the antibody or antigen-binding fragment is a monoclonal antibody. In another aspect, the antibody or antigen-binding domain is a murine, chimeric, humanized, or human antibody or antigen-binding domain. In another aspect, the antibody is a full length antibody. In another aspect, the antigen binding domain is an antigen binding fragment. In another aspect, the antigen binding fragment is a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgG?CH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0011] In one aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding domain disclosed herein. In another aspect, the nucleic acid molecule encodes the VH of SEQ ID NOs: 33-38. In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or light chain of the antibody or antigen-binding domain disclosed herein. In another aspect, the nucleic acid molecule encodes the VL of SEQ ID NOs: 39- 44. In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding domain and the light chain variable region or light chain of an antibody or antigen-binding domain disclosed herein.

[0012] In another aspect, the disclosure provides an isolated vector comprising the polynucleotides disclosed herein. In another aspect, the disclosure provides a host cell comprising the polynucleotides or vectors disclosed herein. In another aspect, the disclosure provides a method of producing an antibody or antigen-binding domain that binds to human p53R175H:HLA-A2 comprising culturing the host cell so that the nucleic acid molecule is expressed and the antibody or antigen-binding domain is produced,optionally wherein the method further comprises isolating the antibody or antigen-binding domain from the culture.

[0013] The disclosure also provides a T cell engager molecule comprising an antigenbinding domain disclosed herein.

[0014] The disclosure also provides a pharmaceutical composition comprising an antibody or antigen-binding domain, or a T cell engager and a pharmaceutically acceptable excipient.

[0015] The disclosure also provides a method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody or antigen binding domain, a T cell engager, or a pharmaceutical composition disclosed herein. In one aspect, the cancer is a solid tumor or a hematopoietic cancer.4. BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 shows phage display discovery of p53-specific antibody clones. Three consecutive Competitive panning rounds were performed on four naive antibody libraries with non-biotinylated non-target pHLA complexes in the presence of biotinylated target p53 complexes. Positive phage clones were determined by standard Phage-ELISA method against biotinylated single chain HLA-A2 / p53R175H peptide complexes. A total of 52 clones possessed unique DNA coding sequences.

[0017] Figure 2 shows 8 unique clones bind specifically to membrane-bound p53 Target. T2 cells were left unpulsed or treated with 100 pM of p53R175H or CMVpp65 peptides. After 24 hours, single cells were incubated with periplasmic extracts (PE) containing soluble Fabs or ScFvs and monitored for clone binding by flow cytometry. (A) Binding Profile of the 52 PE. (B) p53R175H-specific clones. Clone binding is expressed as the mean Fi value ± SD of independent replicates (n=3). Fi is calculated as (Sample MFI - No peptide MFI) / No peptide MFI.

[0018] Figure 3 shows that 6 out of 8 clones bind the p53R175H target in human IgGl format and do not interact with p53WT. T2 cells were treated for 24 hours with 100 pM of p53R175H or p53WT peptide followed by incubation with primary TCRmimic antibodies (TCRmAbs) at indicated concentrations (Range 0.03 to 100 nM). TCRmAb binding to single cells was monitored by Flow Cytometry with an anti-human IgG PE-labelled secondary Antibody. Three parameter nonlinear regression was used tocalculate the ECso (value indicated on graph) for each antibody (Unit Molar). Data is expressed as the mean fluorescence intensity (MFI), n=2.

[0019] Figure 4 shows a schematic of the bispecific T-Cell engager format. The Fc domain (CH2) was modified to abrograte FcR binding complement fixation & homotypic dimerization.

[0020] Figure 5 shows the affinity of four p53 specific T-Cell Engagers to the Target pHLA complex. T-Cell Engager binding to p53R175H / HLA-A*02:01 was measured with single-cycle kinetics by SPR. SA Series Sensor Chips were immobilised with Biotinylated p53 Neoantigen between 100-110 RU. TCEs were subsequently loaded at increasing concentrations. The reference-subtracted binding is shown in Red.

[0021] Figure 6 shows that p53-specific T-Cell Engagers trigger T-Cell activation against cells displaying the mutant but not WT p53 complex. 2.5xl04T2 cells pulsed with 100 pM p53WT or p53R175H were treated with indicated concentrations of TCEs and 5xl04CD3+ Donor cells for 72 hours. As a negative control for T cell activation, cells were treated with an isotype TCE. IFN-y release was measured by ELISA. Data is expressed as the mean ± SD of technical replicates and is representative of five independent experiments. Statistical significance was determined by one-way ANOVA. HIV vs treatment, ****p<0.001. Abbreviations: pHLA; peptideHLA; TCE; T Cell engager, UT; untreated.

[0022] Figure 7 shows IM810-TCE specifically triggers T cell activation & tumour cytotoxicity against cells displaying mutant but not WT p53 complex. 2.5xl04T2 cells pulsed with 100 pM p53WT or p53R175H were treated with indicated concentrations of TCEs and 5xl04CD3+ Donor cells labelled with CFSE proliferation dye for 72 hours. TransAct was included as a positive control for non-specific T-cell proliferation and tumor cell death. Cell viability was assessed by flow cytometry with FarRed viability dye and CyQuant Absolute counting beads. (A) Histogram overlap of CFSE fluorescence in single, live T Cells. (B) Effector to tumour (E:T) cell ratio after treatments. (C) % Cytotoxicity after 72 hour treatment. Data is representative of two independent experiments. Abbreviations: UT; untreated, TCE; T cell Engager. E:T;Effector: Target, MFI; Mean fluorescent intensity. Calculations: E:T Ratio= Total T Cells (CFSE+) per 1000 Bead Events / Total Tumour Cells per 1000 Bead events.. % Cytotoxicity= (Untreated control - Treatment) / Untreated control )*100)= whereby the unit is the number of viable Tumour cells per 1000 bead events.

[0023] Figure 8 shows IM810-TCE specifically triggers T cell activation against tumour cell lines that naturally express the target complex. KMS26, SKBR3, KLE, TYK-NU and AU565 tumor cells were treated with titrating amounts of of IM810-TCE or the isotype-TCE in combination with CFSE-labelled CD3+ Donor cells at an effector to target ratio of 2:1 for 72 hours. Levels of IFN-Y Release were measured by ELISA. Data is expressed as the mean +- SD of technical replicates and is representative of at least two independent experiments.

[0024] Figure 9 shows IM810-TCE specifically triggers tumour cytotoxicity against tumour cell lines that naturally express the target complex. KMS26, SKBR3, TYK- NU and AU565 tumor cells were treated with titrating amounts of of IM810-TCE or the isotype-TCE in combination with CFSE-labelled CD3+ Donor cells at an effector to target ratio of 2: 1 for 72 hours. Cell viability was assessed by flow cytometry with FarRed viability dye and CyQuant Absolute counting beads. % Cytotoxicity was calculated by normalizing to the number of viable tumour (CFSE-) cells in the untreated control. Data is expressed as the mean +- SD of technical replicates and is representative of at least two independent experiments. Abbreviations: TCE; T cell Engager.Calculations: % Cytotoxicity= (Untreated control - Treatment) / Untreated control)* 100)= whereby the unit is the number of viable Tumour cells per 1000 bead events.

[0025] Figure 10 shows IM810-TCE specifically triggers T cell proliferation against tumour cell lines that naturally express the target complex. KMS26, SKBR3, TYK- NU and AU565 tumor cells were treated with titrating amounts of of IM810-TCE or the isotype-TCE in combination with CFSE-labelled CD3+ Donor cells at an effector to target ratio of 2: 1 for 72 hours. As a positive control for T-cell activation, cocultures were treated with TransAct Dynabeads. Levels of CFSE fluorescence in single live cells was monitored by flow Cytometry. Data is representative of at least two independent experiments.

[0026] Figure 11 shows the HLA-A2 expression levels of p53R175H+ Tumour cells.Indicated cell lines were monitored for HLA-A2 expression by flow cytometry with a chimeric IgGl antibody (Clone BB7.2). BB7.2 binding to single cells was monitored by secondary staining with an anti-human IgG PE labelled antibody. As a control for nonspecific BB7.2 binding, cells were incubated with the anti-IgG PE Antibody Alone. Data is Expressed as the mean +- SD of technical Duplicates.

[0027] Figure 12 the molecular signature of IM810 to the p53 Target. Biotinylated HLA monomers presenting mutated variants of the p53R175H peptide were loaded onto Streptavidin-coated beads and monitored for IM810 binding by FACS. (A) Data is expressed as the mean +- SD of independent duplicates and normalized to the p53R175H pHLA treatment.

[0028] Figure 13 shows that IM810 weakly interacts with three healthy peptides (A) HLA-UVX monomers were co-treated for 30 minutes with UV light alone (no peptide) or UV light in the presence of 100 pM Regex peptides. The CMVpp65 and EBV EBAN peptide were used as positive & negative controls respectively for HLA-A2 binding. Peptide:HLA complex formation is expressed as absorbance intensity index (Ai) ± SD of independent replicates. Ai calculated as Sample AB450 -x(0) No peptide AB450 x(0) No peptide AB450 . Figure 13B shows (B) Recombinant peptideHLA complexes were monitored for cross-reactivity by FACS. % cross-reactivity was calculating by normalization to the p53R175H pHLA target complex. A cross-reactive hit is defined as > 20 % Antibody Binding. Data is expressed as the mean +-SD of independent duplicates..

[0029] Figure 14 shows that IM810 does not bind to p53mutant- HLA-A2+ Cell lines that express VATF, NIBAN1 & VAPA. THP-1, HEK293T and A498 cells were treated with indicated concentrations of TCEs in combination with CD3+ cells from healthy Donors at an E:T ratio of 2: 1 for 72 hours. HLA-A2 & HIV-1 specific TCEs were used as positive and negative treatment controls respectively for T Cell activation. The KMS26 cell line was used as positive control cell line. IFN-y release was measured by ELISA. Data is expressed as the mean ± SD of technical triplicates and is representative of four independent experiments. Statisitical significance was determined by one-way ANOVA. HIV vs treatment, *p<0.05,****p<0.0001. Abbreviations: UT; untreated, TCE; T cell Engager. E:T; Effector: Target, SD; standard deviation.5. DETAILED DESCRIPTION

[0030] The p53 transcription factor plays an important role in response to cellular stress and is crucial in the protection against cancer development. Through the regulation of genes involved in DNA repair, cell cycle arrest and apoptosis, p53 ensures genetic integrity by preventing the accumulation of aberrations that would otherwise lead to malignancy and oncogenesis. In the absence of stress signals, p53 is kept at low levels by the continuous ubiquitination by E3 ubiquitin ligases such as MDM2, and degradation bythe proteasome. Stress signals result in post-translational modifications of the p53 protein, leading to the increased stability of the p53 protein by disrupting the p53-MDM2 interaction, and the subsequent activation of p53.

[0031] The p53 gene is the most mutated gene found in human malignancies, with a missense mutation at position 175 to hisitidine (p53R175H) observed in 4-7% of cancer patients. This mutation increases the stability and disrupts the native conformation of the p53 protein, resulting in the inability to recognize and bind the cognate p53 response elements, while suppressing wild-type (WT) p53. Despite extensive efforts, no drug that targets mutant p53 has been approved for treatment of the large number of patients whose tumors contain these mutations.

[0032] Proteins derived from mutant TP53 alleles can be degraded by the proteasome, processed, and presented by the major histocompatibility complex to generate neoantigens recognizable by T cell receptors (TCRs). R175H, in which Arginine at position 175 is replaced with histidine, is the most commonly observed SNP in TP53. The peptide HMTEVVRHC, derived from p53R175H, binds to a human leukocyte antigen (HLA) allele (A*02:01) that is present in more than 40% of U.S. Caucasians.

[0033] Provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to HLA-A2 p53 peptides. Anti-human HLA-A2 p53 antibodies and antigen-binding fragments thereof can, for example, be used to diagnose and treat cancer. Exemplary anti-human P53R175H:HLA-A2 antibodies are provided herein that demonstrate these activities.

[0034] Also provided are isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such antibodies and antigen-binding fragments thereof. Further provided are vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding such antibodies and antigen-binding fragments thereof. Also provided are methods of making such antibodies and antigen-binding fragments thereof.5.1 Terminology

[0035] HLA class I molecules consist of two non-covalently linked polypeptide chains, an HLA-encoded a chain or heavy chain, and a non-HLA encoded subunit called P2 microglobulin. The a chain has three regions including a cytoplasmic region containing a peptide-binding groove made from the al and a2 domains, a transmembrane regioncontaining hydrophobic amino acids by which the molecule is anchored in the cell membrane, and a highly conserved a3 immunoglobulin-like domain to which CD8 binds. A peptide-binding groove is formed between the al and a2 helices with a P-pleated sheet as its floor. Usually, the groove will accommodate peptides of approximately 8-10 amino acids in length. As used herein, the term “HLA-A2” refers to the human leukocyte antigen having the antibody recognition serotype of the a2 domain of the HLA-A a-chain. “HLA-A2 p53 peptide complex” refers to HLA-A2 molecules containing peptides derived from the p53 protein. “p53” is a tumor suppressor protein involved in the formation of tumors.

[0036] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing (e.g., a glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked, part of a fusion protein, or conjugated to other molecules such as toxins, radioisotopes, etc.

[0037] The term “antibody fragment” refers to a portion of an antibody. An “antigenbinding fragment,” “antigen-binding domain,” or “antigen-binding region,” refers to a portion of an antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDR)). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0038] The terms "anti-p53R175H:HLA-A2 antibody," "p53R175H:HLA-A2 antibody" and "antibody that binds to p53R175H:HLA-A2" refer to an antibody that is capable of binding p53R175H:HLA-A2 with sufficient affinity such that the antibody is useful as a diagnostic, or a therapeutic. The extent of binding of an anti-p53R175H:HLA-A2 antibody to an unrelated, non-p53R175H:HLA-A2 protein can be less than about 10% of the binding of the antibody to p53R175H:HLA-A2 as measured, e.g., by a radioimmunoassay (RIA) or Enzyme-linked immunosorbent assay (ELISA).

[0039] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigenbinding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, a “monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0040] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or murine CDRs andhuman framework regions (FRs). In some aspects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0041] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0042] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0043] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35 A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0044] Chothia refers instead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.Loop Kabat AbM ChothiaLI L24-L34 1.24-1.34 1.24-L34L.2 L50-L.56 1.50-156 L50-L56L3 1.89-1.97 L89-L97 L89-L97Hl H31-H35B H26-H35B H26-H32..34(Kabat Numbering)Hl H31-H35 1126-1135 H26-H32(Chothia Numbering)H2 H50-H65 H50-H58 H52-H56H3 H95-H102 H95-H102 H95-H102

[0045] As used herein, the term “constant region” or “constant domain” are interchangeable and have the meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigenbinding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0046] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG2, IgGs, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0047] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0048] The term "chimeric" antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragmentsthereof derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0049] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capability of the antibody or antigen-binding fragment thereof. In general, the humanized antibody or antigen-binding fragment thereof will comprise VH and VL that comprise substantially all of at least one, and typically two or three, of the CDR regions that correspond to the non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some aspects, a "humanized antibody" is a resurfaced antibody.

[0050] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0051] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigenbinding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigenbinding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of kOff / kOn, whereas KA is calculated from the quotient of kon / koff. kOn refers to the association rate constant of, e.g., an antibody or antigenbinding fragment thereof to an antigen, and kOff refers to the dissociation rate constant of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The kon and kOff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.

[0052] A antibody that is “blocking” or that “blocks” or that is “inhibitory” of that “inhibits” is an antibody that reduces or inhibits (partially or completely) binding of its target protein to one or more ligands when the antibody is bound to the target protein, and / or that reduces or inhibits (partially or completely) one or more activities or functions of the target protein when the antibody is bound to the target protein.

[0053] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more noncontiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., alanine scanning or other site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr DBiol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23;ChayenNE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300- 6303). Crystals of an antibody or antigen-binding fragment thereof and its antigen can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.;, U.S.2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323).Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0054] A p53R175H:HLA-A2 antibody that “binds to the same epitope” as a reference p53R175H:HLA-A2 antibody refers to an antibody that contacts the same p53R175H:HLA-A2 amino acid residues as the reference p53R175H:HLA-A2 antibody. The ability of a p53R175H:HLA-A2 antibody to bind to the same epitope as a reference p53R175H:HLA-A2 antibody is determined using peptide scanning mutagenesis or high throughput alanine scanning mutagenesis (see Davidson and Doranz, 2014 Immunology 143, 13-20). In the latter methodology, a comprehensive mutation library of p53R175H:HLA-A2, or a portion thereof (e.g., the extracellular domain), can be generated by mutating each individual amino acid residue to alanine (or if the amino acid residue is alanine, then to another residue such as serine) and testing each mutant for binding to an anti-p53R175H:HLA-A2 antibody or antigen binding fragment thereof. Amino acids that are required for binding, and therefore are epitope residues, are identified by loss of interaction.

[0055] As used herein, the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding entails complementarity between the antigen binding domain and the epitope. Accordingly, an antibody that “specifically binds” to human p53R175H:HLA-A2 (e.g., SEQ ID NO:1) may also bind to p53R175H:HLA-A2 fromother species (e.g., cynomolgus monkey p53R175H:HLA-A2) and / or p53R175H:HLA- A2 proteins produced from other human alleles, but the extent of binding to an un-related, non-p53R175H:HLA-A2 protein (e.g., other immunomodulatory proteins containing ITIM domains) is less than about 10% of the binding of the antibody to p53R175H:HLA- A2 as measured, e.g., by a radioimmunoassay (RIA).

[0056] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope such that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0057] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0058] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure are based upon antibodies, in some aspects, the polypeptides can occur as single chains or associated chains.

[0059] As used herein, the term “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In some aspects, the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0060] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0061] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that may be used to deliver a drug, e.g., an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof, to the desired site of biological action. Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0062] As used herein, the terms “subject” and “patient” are used interchangeably. The subject can be a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a human.

[0063] The term "therapeutically effective amount" refers to an amount of a drug, e.g., an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof, effective to treat a disease or condition in a subject. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size or burden; inhibit (i.e., slow to some extent and in some aspects, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in some aspects, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and / or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression(TTP), or any combination thereof. To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0064] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In some aspects, a subject is successfully "treated" for cancer according to the methods provided herein if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.

[0065] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Such cancers can include solid tumors or hematopoietic cancers.

[0066] As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0067] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can mean "includes," "including," and the like; "consisting essentially of' or "consists essentially of are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0068] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0069] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range are also provided.

[0070] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.5.2 Antibodies and antigen binding domains

[0071] In one aspect, provided herein are antibodies (e.g., monoclonal antibodies, such as chimeric, humanized, or human antibodies) and antigen-binding fragments thereof which specifically bind to an HLA-A2 p53 peptide complex. The amino acid sequences of human p53 is known in the art and also provided herein as represented by SEQ ID NO: 1.Human p53:MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGP DEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAK SVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPH HERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYM CNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHH ELPPGSTKRALPNNTS S SPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKD AQ AGK EPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO:1)

[0072] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2. In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises thesix CDRs of an antibody listed in Tables 1 and 2 (i.e., the three VH CDRs of the antibody listed in Table 1 and the three VL CDRs of the same antibody listed in Table 2).Table 1. VH CDR Amino Acid Sequences1AntiVH CDR1 VH CDR2 VH CDR3body (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)NYGIS (SEQ ID WISVYNGKTKYAQNLQ DQHYSYYGLDV (SEQ IM811 NO:2) D (SEQ ID NO: 3) ID NO:4)NYGIS (SEQ ID WISVYNGKTKYAQNLQ DQHYSYYGLDV (SEQ IM810 NO:2) D (SEQ ID NO: 3) ID NO:4)FHGMH (SEQ ID YISSSGSTIYYADSVKG SGYDFWSGYYTGGYY IM48 NO: 5) (SEQ ID NO: 6) YYGMDV (SEQ ID NO: 7)TNYMS (SEQ ID IIYSGGSTYYADSVKG GEKASWHGFDI (SEQ IM37NO: 8) (SEQ ID NO: 9) ID NO: 10)SYAMS (SEQ ID SISGTDGSTYYADSVKG YRWLSPPNWFDT (SEQ IM42NO: 11) (SEQ ID NO: 12) ID NO:13)TNWIG (SEQ ID IIYPGDSDTRYSPSFEG PQTYSDFWGGSGTYYF IM710NO: 14) (SEQ ID NO: 15) DN (SEQ ID NO: 16)xThe VH CDRs in Table 1 are determined according to Kabat.Table 2. VL CDR Amino Acid Sequences2VL CDR1 VL CDR2 VL CDR3 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) RASQGISSSLA GASTLQS (SEQ ID QHLSGYPQS (SEQ ID IM811(SEQ ID NO: 17) NO: 18) NO: 19) RASQGISTSLA GASTLQS (SEQ ID QHLSGYPQS (SEQ ID IM810(SEQ ID NO:20) NO: 18) NO: 19) TRSSGNIASNFVQ EDNQRAS (SEQ ID QSYDSSNWA (SEQ ID IM48(SEQ ID NO:21) NO:22) NO:23) RASQRIDTYLN GASNLQS (SEQ ID QQGYSTIYT (SEQ ID IM37(SEQ ID NO:24) NO:25) NO:26) TGSSGSIASNYVQ EDNQRPS (SEQ ID QSYDSSNHAL (SEQ ID IM42(SEQ ID NO:27) NO:28) NO:29) SGSSSNIGSNTVN SNNQRPS (SEQ ID ASWDDSLTWV (SEQ IM710(SEQ ID NO: 30) NO:31) IDNO:32)2The VL CDRs in Table 2 are determiner according to Kabat.

[0073] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises the VH of an antibody listed in Table 3.Table 3: Variable Heavy Chain (VH) Amino Acid SequencesAntibody VH Amino Acid Sequence (SEQ ID NO)EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGISWVRQAPGQGLE IM811 WMGWISVYNGKTKYAQNLQDRVTMTTDTSTSTAYMELRSLRSDDT AVYYCARDQHYSYYGLDVWGQGTTVTVSS (SEQ ID NO:33) EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGISWVRQAPGQGLE IM810 WMGWISVYNGKTKYAQNLQDRVTMTTDTSTSTAYMELRSLRSDDT AVYYCARDQHYSYYGLDVWGQGTTVTVSS (SEQ ID NO:34) EVQLVQSGGGLVKPGGSLRLSCAASGFTFSFHGMHWVRQAPGKGLE IM48 WVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARSGYDFWSGYYTGGYYYYGMDVWGQGTTVTVSS (SEQ ID NO:35) EVKLLESGGGLIQPGGSLRLSCAASGFTVSTNYMSWVRQAPGKGLE IM37 WVSIIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCASGEKASWHGFDIWGQGTMVTVSS (SEQ ID NO:36) EVKLLESGGGL VQPGGSLRLSCT ASGFTF S S YAMSWVRQAPGKGLE IM42 WVSSISGTDGSTYYADSVKGRFTISRDNSKNTLNLQMNNLRVEDTAV YYCARYRWLSPPNWFDTWGQGTLVTVSS (SEQ ID NO:37) QVQLVQSGPEVKKPGESLKISCKGTGYTFSTNWIGWVRQMPGKGLE IM710 WMGIIYPGDSDTRYSPSFEGHVTISVDKSINTAYLQWSSLKASDSAIYYCARPQTYSDFWGGSGTYYFDNWGQGTLVTVSS (SEQ ID NO:38)

[0074] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises the VL of an antibody listed in Table 4.Table 4: Variable Light Chain (VL) Amino Acid SequencesAntibody VL Amino Acid Sequence (SEQ ID NO)DIVLTQ SPPFLS AS VGDKVTISCRASQGIS S SLAWYQQKPGRAPKVLIY IM811 GASTLQSGVPSRFSGSGSGTEFTLTISRLQPEDFATYYCQHLSGYPQSF GQGTKLDIK (SEQ ID NO: 39) AIRMTQSPSTLSASVGDTVTISCRASQGISTSLAWYQQKPGRVPKVLI IM810 YGASTLQSGVPSRFSGSGSGTEFTLTISRLQPEDFATYYCQHLSGYPQS FGQGTKLEIK (SEQ ID NO:40) NFMLTQPHSVSESPEKTVTISCTRSSGNIASNFVQWYQQRPGSAPTLLI IM48 FEDNQRASGVPDRF SGSIDS S SNS ASLTISGLKTEDEAD YYCQS YDS S NWAFGGGTKLTVL (SEQ ID NO:41)IM37 DIVMTQSPSSLSASVGDRVTITCRASQRIDTYLNWYQQRPGKAPKLLIYGASNLQSGVPSRFSGSGSGTEFALTISSLQPEDFATYYCQQGYSTIYT FGQGTKLEIN (SEQ ID NO:42) NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPTTV IM42 IYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSS NHALFGGGTKLTVL (SEQ ID NO:43) QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLI IM710 YSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCASWDDSLTWVFGGGTQLTVL (SEQ ID NO:44)

[0075] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises the VH and the VL of an antibody listed in Tables 3 and 4 (i.e., the VH of the antibody listed in Table 3 and the VL of the same antibody listed in Table 4).

[0076] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises one, two, three or all of the VH framework regions of an antibody listed in Table 5.Table 5. VH FR Amino Acid Sequences3VHFR1 VHFR2 VHFR3 VHFR4 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)EVQLVQSGAEVK WVRQAPGQGL RVTMTTDTSTSTAY WGQGTTVT IM811 KPGASVKVSCKAS EWMG (SEQ ID MELRSLRSDDTAVY VSS (SEQ ID GYTFK (SEQ ID NO:46) YCAR (SEQ ID NO:48) NO:45) NO:47)EVQLVQSGAEVK WVRQAPGQGL RVTMTTDTSTSTAY WGQGTTVT IM810 KPGASVKVSCKAS EWMG (SEQ ID MELRSLRSDDTAVY VSS (SEQ ID GYTFK (SEQ ID NO:46) YCAR (SEQ ID NO:48) NO:45) NO:47)EVQLVQSGGGLV WVRQAPGKGL RFTISRDNAKNSLYL WGQGTTVT IM48 KPGGSLRLSCAAS EWVS (SEQ ID QMNSLRAEDTAVY VSS (SEQ ID GFTFS (SEQ ID NO:50) YCAR (SEQ ID NO:48) NO:49) NO:51)EVKLLESGGGLIQP WVRQAPGKGL RFTISRDNSKNTLYL WGQGTMVT IM37 GGSLRLSCAASGF EWVS (SEQ ID QMNSLRAEDTAVY VSS (SEQ ID TVS (SEQIDNO:52) NO:53) YCAS (SEQ IDNO:54) NO:55) EVKLLESGGGLVQ WVRQAPGKGL RFTISRDNSKNTLNL WGQGTLVT IM42 PGGSLRLSCTASGF EWVS (SEQ ID QMNNLRVEDTAVY VSS (SEQ ID TFS (SEQ ID NO:56) NO:53) YCAR (SEQ ID NO:58) NO:57)QVQLVQSGPEVKK WVRQMPGKGL HVTISVDKSINTAYL WGQGTLVT IM710 PGESLKISCKGTGY EWMG (SEQ ID QWSSLKASDSAIYY VSS (SEQ IDTFS (SEQ ID NO:59) NO: 60) CAR (SEQ ID NO:61) NO:58)3The VH framework regions described in Table 5 are determined based upon the boundaries of the Kabat numbering system for CDRs. In other words, the VH CDRs are determined by Kabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0077] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises one, two, three or all of the VL framework regions of an antibody listed in Table 6.Table 6. VL FR Amino Acid Sequences4VLFR1 VLFR2 VLFR3 VLFR4 Anti-body(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) DIVLTQSPPFLSAS WYQQKPGRAP GVPSRFSGSGSGT FGQGTKLDI VGDKVTISC (SEQ KVLIY (SEQ ID EFTLTISRLQPEDF K (SEQ ID IM811 ID NO: 62) NO:63) ATYYC (SEQ ID NO:65)NO: 64)AIRMTQSPSTLSAS WYQQKPGRVP GVPSRFSGSGSGT FGQGTKLEI VGDTVTISC (SEQ KVLIY (SEQ ID EFTLTISRLQPEDF K (SEQ ID IM810 ID NO: 66) NO: 67) ATYYC (SEQ ID NO:68)NO: 64)NFMLTQPHSVSES WYQQRPGSAP GVPDRFSGSIDSS FGGGTKLTV PEKTVTISC (SEQ TLLIF (SEQ ID SNSASLTISGLKT L (SEQ ID IM48ID NO: 69) NO: 70) EDEADYYC (SEQ NO: 72)IDNO:71) DIVMTQSPSSLSAS WYQQRPGKAP GVPSRFSGSGSGT FGQGTKLEI VGDRVTITC (SEQ KLLIY (SEQ ID EFALTISSLQPEDF N (SEQ ID IM37 ID NO:73) NO: 74) ATYYC (SEQ ID NO: 76)NO:75)NFMLTQPHSVSES WYQQRPGSAP GVPDRFSGSIDSS FGGGTKLTV PGKTVTISC (SEQ TTVIY (SEQ ID SNSASLTISGLKT L (SEQ ID IM42 ID NO: 77) NO:78) EDEADYYC (SEQ NO: 80)IDNO:79) QAVLTQPPSASGT WYQQLPGTAP GVPDRFSGSKSGT FGGGTQLTV PGQRVTISC (SEQ KLLIY (SEQ ID SASLAISGLQSED L (SEQ ID IM710 ID N0:81) NO: 82) EADYYC (SEQ ID NO: 84)NO:83)4The VL framework regions described in Table 6 are determined based upon the boundaries of the Kabat numbering system for CDRs. In other words, the VL CDRs are determined by Kabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0078] In some aspects, an antibody or antigen-binding fragment thereof described herein binds to human p53R175H:HLA-A2 and comprises the four VH framework regions and the four VL framework regions of an antibody listed in Tables 5 and 6 (i.e., the four VH framework regions of the antibody listed in Table 5 and the four VL framework regions of the same antibody listed in Table 6.)

[0079] In certain aspects, an antibody or antigen-binding fragment thereof described herein may be described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also Clackson T et al., (1991) Nature 352: 624-628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementary variable domains. The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VH domain and screening a library (e.g., human VL library) for complementary VL domains; the selected VL domains in turn could be used to guide selection of additional complementary (e.g., human) VH domains.

[0080] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop ispresent at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0081] In certain aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 and comprise the Chothia VH and VL CDRs of an antibody listed in Tables 3 and 4. In some aspects, antibodies or antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 comprise one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 and comprise combinations of Kabat CDRs and Chothia CDRs.

[0082] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M- P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL- CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 and comprise the IMGT VH and VL CDRs of an antibody listed in Tables 3 and 4, for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et al., (1999) supra).

[0083] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422- 439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 and comprise VH and VL CDRs of an antibody listed in Tables 3 and 4 as determined by the method in MacCallum RM et al.

[0084] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human p53R175H:HLA-A2 and comprise VH and VL CDRs of an antibody listed in Tables 3 and 4 as determined by the AbM numbering scheme.

[0085] In some aspects, provided herein are antibodies that comprise a heavy chain and a light chain. With respect to the heavy chain, in some aspects, the heavy chain of an antibody described herein can be an alpha (a), delta (5), epsilon (a), gamma (y) or mu (p) heavy chain. In some aspects, the heavy chain of an antibody described can comprise a human alpha (a), delta (5), epsilon (a), gamma (y) or mu (p) heavy chain. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a heavy chain wherein the amino acid sequence of the VH domain comprises an amino acid sequence set forth in Table 3 and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (y) heavy chain constant region. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a heavy chain wherein the amino acid sequence of the VH domain comprises an amino acid sequence set forth in Table 3 and wherein the constant region of the heavy chain comprises the amino acid sequence of an IgGl heavy chain constant region. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA- A2, comprises a heavy chain wherein the amino acid sequence of the VH domain comprises an amino acid sequence set forth in Table 3 and wherein the constant region of the heavy chain comprises the amino acid sequence of an IgG (e.g., IgGl, IgG2a, IgG2b, IgG3, or IgG4) heavy chain constant region. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a heavy chain wherein the amino acid sequence of the VH domain comprises a sequence set forth in Table 3, and wherein the constant region of the heavy chain comprises the amino acid of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) supra.

[0086] With respect to the light chain, in some aspects, the light chain of an antibody described herein is a kappa light chain. In some aspects, the light chain of an antibody described herein is a lambda light chain. In some aspects, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain.

[0087] In some aspects, an antibody described herein, which immunospecifically binds to a human p53R175H:HLA-A2, comprises a light chain wherein the amino acid sequence of the VL domain comprises a sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a light chain wherein the amino acid sequence of the VL domain comprises a sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a light chain wherein the amino acid sequence of the VL domain comprises a sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat EA et al., (1991) supra.

[0088] In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA-A2 comprises a VH domain and a VL domain comprising the amino acid sequence of any of the anti-human p53R175H:HLA-A2 antibodies described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, an antibody described herein, which immunospecifically binds to human p53R175H:HLA- A2 comprises a VH domain and a VL domain comprising the amino acid sequences of any of the anti-human p53R175H:HLA-A2 antibodies described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In some aspects, the constant regions comprise the amino acid sequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulinmolecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0089] Non-limiting examples of human constant regions are described in the art, e.g., see Kabat EA et al. , (1991) supra.Exemplary Fc Domains

[0090] In some aspects, an anti-p53R175H:HLA-A2 antibody, and in particular, an antihuman p53R175H:HLA-A2 antibody as provided herein, may comprise a complete or partial Fc domain. In some aspects, the Fc domain is a human IgGl, IgG2, IgG3, and / or IgG4 isotype.

[0091] In certain aspects, the Fc domain has an IgGl isotype. In some aspects, an anti- p53R175H:HLA-A2 antibody contains a murine IgGl Fc domain. In some aspects, an anti-human p53R175H:HLA-A2 antibody contains a human IgGl Fc domain (hlgGl), e.g., as provided in SEQ ID NO:85.EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO:85)

[0092] In some aspects, the human IgGl Fc domain of an anti-human p53R175H:HLA- A2 antibody binds an activating Fc receptor. In certain aspects, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb.

[0093] In some aspects, the human IgGl Fc domain of an anti-human p53R175H:HLA- A2 antibody does not bind or has reduced binding to FcyRIII(CD16) and / or Clq. In some aspects, the human IgGl Fc domain of an anti-human p53R175H:HLA-A2 antibody has reduced antibody-dependent cellular cytotoxicity (ADCC) and / or complement binding activity, respectively, which in each case may reduce undesired killing of cells, e.g., myeloid cells, to which the anti-p53R175H:HLA-A2 antibody binds. The above effects may be achieved by certain amino acid modifications, e.g., the “NSLF” mutations, in which a human IgGl Fc domain contains the mutations N325S and L328F (by EU numbering of the IgGl Fc domain), as shown, e.g., in SEQ ID NO:86. In another aspect, the human IgGl Fc domain comprises a mutation corresponding to K322A (EU numbering), e.g., as provided in SEQ ID NO:87.EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO:86) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO:87)

[0094] Exemplary modifications to the human IgGl Fc domain are listed below in Table 7.Table 7: Exemplary modifications to the human IgGl Fc domainMutation (EU numbering scheme)N325S and L328F (“NSLF”)S267E and L328F (“SELF”)P331S (“PS”)P331 S and E430G (“PSEG”)K322AL234A, L235A, and P331S (“LALAPS”) (Substantiallyabolishes Fc binding to FcR)

[0095] In certain aspects of an anti-p53R175H:HLA-A2 antibody provided herein, the Fc domain has an IgG2 isotype. In some aspects, an anti-p53R175H:HLA-A2 antibody contains a murine IgG2 Fc domain, e.g., murine IgG2a (mIgG2a). In some aspects, an anti-human p53R175H:HLA-A2 antibody contains a human IgG2 Fc domain (hIgG2). In some aspects, the human IgG2 Fc domain of an anti-human p53R175H:HLA-A2 antibody binds an activating Fc receptor. In certain aspects, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb.

[0096] In certain aspects of an anti-p53R175H:HLA-A2 antibody provided herein, the Fc domain has an IgG4 isotype. In some aspects, an anti-human p53R175H:HLA-A2antibody contains a human IgG4 Fc domain (h!gG4), e.g., as provided in SEQ ID NO:88. In some aspects, the human IgG4 Fc region of the anti-human p53R175H:HLA-A2 antibody binds an activating Fc receptor. In certain aspects, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb. In certain aspects, the human IgG4 Fc region comprises a mutation corresponding to S228P (by EU numbering), e.g., as provided in SEQ ID NO:89.ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAI<TI<PREEQFNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO:88) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAI<TI<PREEQFNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO:89)

[0097] In some aspects, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein having two heavy chain constant regions.

[0098] In some aspects, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences of an antibody listed in Table 1 (e.g., SEQ ID N0s:xx-xx); (ii) the light chain comprises a VL domain comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences of the same antibody listed in Table 2 (e.g., SEQ ID NOs:xx-xx); (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgGl heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.

[0099] In some aspects, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises aheavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequence of an antibody listed in Table 3 (e.g., SEQ ID NO:33); (ii) the light chain comprises a VL domain comprising the amino acid sequence of the same antibody listed in Table 4 (e.g., SEQ ID NO:39); (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgGl heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.

[0100] In some aspects, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to human p53R175H:HLA-A2, comprises framework regions (e.g., framework regions of the VH domain and / or VL domain) that are human framework regions or derived from human framework regions. Non-limiting examples of human framework regions are described in the art, e.g., see Kabat EA et al., (1991) supra). In some aspects, an antibody or antigen-binding fragment thereof described herein comprises framework regions (e.g., framework regions of the VH domain and / or VL domain) that are primate (e.g., non-human primate) framework regions or derived from primate (e.g., non-human primate) framework regions.Antibodies That Bind Same Epitope / Competitively Inhibit

[0101] In another aspect, provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope of human p53R175H:HLA-A2 as an antibody or antigen-binding fragment thereof described herein (e.g., IM811, IM810, IM48, IM37, IM42, IM710).

[0102] Competitive binding assays can be used to determine whether two antibodies bind to overlapping epitopes. Competitive binding can be determined in an assay in which an immunoglobulin to be tested inhibits specific binding of a reference antibody to a common antigen, such as p53R175H:HLA-A2. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solid phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); solid phase direct biotin-avidin EIA (Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled RIA. (Moldenhauer G et al., (1990) Scand I Immunol 32: 77-82).Typically, such an assay involves the use of purified antigen (e.g., p53R175H:HLA-A2 such as human p53R175H:HLA-A2) bound to a solid surface or cells bearing such antigen, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. 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 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition binding assay can be configured in a large number of different formats using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96- well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels. For further details see, for example, Wagener C et al., (1983) I Immunol 130: 2308-2315; Wagener C et al., (1984) I Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872- 4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11 : 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp. 386-389.

[0103] In some aspects, a competitive binding assay is performed using surface plasmon resonance (BIAcore®), e.g., by an ‘in tandem approach’ such as that described by Abdiche YN et al., (2009) Analytical Biochem 386: 172-180, whereby p53R175H:HLA- A2 antigen is immobilized on the chip surface, for example, a CM5 sensor chip and the anti-p53R175H:HLA-A2 antibodies are then run over the chip. To determine if an antibody or antigen-binding fragment thereof competitively inhibits binding of an anti- p53R175H:HLA-A2 antibody described herein, the anti-p53R175H:HLA-A2 antibody is first run over the chip surface to achieve saturation and then the potential, competing antibody is added. Binding of the competing antibody or antigen-binding fragment thereof can then be determined and quantified relative to a non-competing control.

[0104] In some aspects, a Fortebio Octet competitive binding is used to determine that a p53R175H:HLA-A2 antibody or antigen-binding fragment thereof competitively inhibitsthe binding of another p53R175H:HLA-A2 antibody or antigen-binding fragment thereof to p53R175H:HLA-A2.

[0105] In another aspect, provided herein are antibodies that competitively inhibit (e.g., in a dose dependent manner) an antibody or antigen-binding fragment thereof described herein (e.g., IM811, IM810, IM48, IM37, IM42, IM710) from binding to human p53R175H:HLA-A2, as determined using assays known to one of skill in the art or described herein (e.g., ELISA competitive assays, or suspension array or surface plasmon resonance assay). An antibody that “competitively inhibits” may also be referred to as an antibody that “competes for binding” to a reference antibody.

[0106] In specific aspects, provided herein is an antibody or antigen-binding fragment which competitively inhibits (e.g., in a dose dependent manner) binding of an antibody to human p53R175H:HLA-A2, wherein the antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO:33, and a VL domain having the amino acid sequence set for the in SEQ ID NO:39.

[0107] In specific aspects, provided herein is an antibody or antigen-binding fragment which competitively inhibits (e.g., in a dose dependent manner) binding of an antibody to human p53R175H:HLA-A2, wherein the antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO:34, and a VL domain having the amino acid sequence set for the in SEQ ID NO:40.

[0108] In specific aspects, provided herein is an antibody or antigen-binding fragment which competitively inhibits (e.g., in a dose dependent manner) binding of an antibody to human p53R175H:HLA-A2, wherein the antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO:35, and a VL domain having the amino acid sequence set for the in SEQ ID NO:41.

[0109] In specific aspects, provided herein is an antibody or antigen-binding fragment which competitively inhibits (e.g., in a dose dependent manner) binding of an antibody to human p53R175H:HLA-A2, wherein the antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain having the amino acid sequence set for the in SEQ ID NO:42.Antigen Binding Fragments

[0110] In some aspects, an antigen-binding fragment or an antigen binding domain of an anti-p53R175H:HLA-A2 antibody described herein, such as an anti-humanp53R175H:HLA-A2 antibody, is provided. Exemplary antigen-binding fragments include but are not limited to Fab, Fab', F(ab')2, and scFv, wherein the Fab, Fab', F(ab')2, or scFv comprises a heavy chain variable region sequence and a light chain variable region sequence of an anti-human p53R175H:HLA-A2 antibody as described herein. A Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those of skill in the art, including, but not limited to, those discussed in Section 5.3, infra. In some aspects, an antigen-binding fragment, such as a Fab, Fab', F(ab')2, or scFv, further comprises a moiety that extends the half-life of the antibody in vivo. The moiety is also termed a "half-life extending moiety." Any moiety known to those of skill in the art for extending the half-life of a an antigen-binding fragment, such as a Fab, Fab', F(ab')2, or scFv, in vivo can be used. For example, the half-life extending moiety can include an Fc region, a polymer, an albumin, or an albumin binding protein or compound. The polymer can include a natural or synthetic, optionally substituted straight or branched chain polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivative thereof. Substituents can include one or more hydroxy, methyl, or methoxy groups. In some aspects, an antigen-binding fragment, such as an Fab, Fab', F(ab')2, or scFv, can be modified by the addition of one or more C-terminal amino acids for attachment of the half-life extending moiety. In some aspects, the halflife extending moiety is polyethylene glycol or human serum albumin. In some aspects, an antigen-binding fragment, such as a Fab, Fab', F(ab')2, or scFv, is fused to a Fc region.

[0111] An anti-p53R175H:HLA-A2 antibody (such as an anti-human p53R175H:HLA- A2 antibody) or antigen-binding fragment thereof can be fused or conjugated (e.g., covalently or noncovalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (12 lln), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labeled antibodies or antigen-binding fragments thereof can be used to detect p53R175H:HLA- A2 (e.g., human p53R175H:HLA-A2) protein. See, e.g., Sections 5.5 and 5.6, infra.5.3 Antibody Production

[0112] Antibodies and antigen-binding fragments thereof that immunospecifically bind to human p53R175H:HLA-A2 can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g., Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0113] In a certain aspect, provided herein is a method of making an antibody or antigenbinding fragment which immunospecifically binds to human p53R175H:HLA-A2 comprising culturing a cell or host cell described herein (e.g., a cell or a host cell comprising polynucleotides encoding an antibody or antigen-binding fragment thereof described herein). In a certain aspect, provided herein is a method of making an antibody or antigen-binding fragment thereof which immunospecifically binds to human p53R175H:HLA-A2 comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or a host cell comprising polynucleotides encoding an antibody or antigen-binding fragment thereof described herein). In some aspects, the cell is an isolated cell. In some aspects, the encoding polynucleotides have been introduced into the cell. In some aspects, the method further comprises the step of purifying the antibody or antigenbinding fragment obtained from the cell or host cell.

[0114] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0115] Monoclonal antibodies or antigen-binding fragments thereof can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, yeast-based presentation technologies, or a combination thereof. For example, monoclonal antibodies or antigen-binding fragments thereof can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), or as described in Kohler G & Milstein C (1975) Nature 256: 495. Examples of yeast-based presentation methods that can be employed to select and generate the antibodies described herein include those disclosed in, for example, W02009 / 036379A2; W02010 / 105256; and W02012 / 009568, each of which is herein incorporated by reference in its entirety.

[0116] In some aspects, a monoclonal antibody or antigen-binding fragment is an antibody or antigen-binding fragment produced by a clonal cell (e.g., hybridoma or host cell producing a recombinant antibody or antigen-binding fragment), wherein the antibody or antigen-binding fragment immunospecifically binds to human p53R175H:HLA-A2 as determined, e.g., by ELISA or other antigen-binding assays known in the art or in the Examples provided herein. In some aspects, a monoclonal antibody or antigen-binding fragment thereof can be a chimeric or a humanized antibody or antigen-binding fragment thereof. In some aspects, a monoclonal antibody or antigenbinding fragment thereof can be a Fab fragment or a F(ab’)2 fragment. Monoclonal antibodies or antigen-binding fragments thereof described herein can, for example, be made by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phage libraries using the techniques as described herein, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies and antigen-binding fragments thereof expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0117] Antigen-binding fragments of antibodies described herein can be generated by any technique known to those of skill in the art. For example, Fab and F(ab’)2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments). A Fab fragment corresponds to one of the two identical arms of a tetramericantibody molecule and contains the complete light chain paired with the VH and CHI domains of the heavy chain. A F(ab’)2 fragment contains the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0118] Further, the antibodies or antigen-binding fragments thereof described herein can also be generated using various phage display and / or yeast-based presentation methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues). The DNA encoding the VH and VL domains are recombined together with a scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antibody or antigen-binding fragment thereof that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies or fragments described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41- 50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application No.PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 1 1236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0119] A humanized antibody or antigen-binding fragment thereof can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.5.3.1 Polynucleotides

[0120] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described herein or adomain thereof e.g., a variable light chain region and / or variable heavy chain region) that immunospecifically binds to human p53R175H:HLA-A2, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0121] In particular aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof, which immunospecifically bind to human p53R175H:HLA-A2 and comprise an amino acid sequence as described herein, as well as antibodies or antigen-binding fragments that compete with such antibodies or antigen-binding fragments for binding to a human p53R175H:HLA-A2 (e.g., in a dose-dependent manner), or which bind to the same epitope as that of such antibodies or antigen-binding fragments.

[0122] Also provided herein is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs :xx. In some aspects, an antibody or antigen-binding fragment thereof comprising the polypeptide immunospecifically binds to human p53R175H:HLA-A2.

[0123] Also provided herein are kits, vectors, or host cells comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:33 and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:39. Also provided herein are kits, vectors, or host cells comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:34 and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:40. Also provided herein are kits, vectors, or host cells comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:35 and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:41. Also provided herein are kits, vectors, or host cells comprising (i) a first polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:36 and (ii) a second polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:42. In a kit comprising such first and second polynucleotides, the first and second polynucleotides can be in the same vector or can be in different vectors. In a host cell comprising such first and second polynucleotides, the first and second polynucleotides can in the same vector or can be in different vectors.

[0124] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding three VH domain CDRs, e.g., a polypeptide containing VH CDR1,VH CDR2, and VH CDR3 of any one of antibodies described herein (e.g., see Table 1), e.g., wherein the three VH domain CDRs are in the context of a VH. In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding three VL domain CDRs, e.g., a polypeptide containing VL CDR1, VL CDR2, and VL CDR3 of any one of antibodies described herein (e.g., see Table 2) ), e.g., wherein the three VL domain CDRs are in the context of a VL. In some aspects, provided herein are polynucleotides (or combinations of polynucleotides) comprising a nucleotide sequence encoding an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof comprising (i) three VH domain CDRs, e.g., a polypeptide containing VH CDR1, VH CDR2, and VH CDR3 of any one of antibodies described herein (e.g., see Table 1) e.g., wherein the three VH domain CDRs are in the context of a VH and (ii) three VL domain CDRs, e.g., a polypeptide containing VL CDR1, VL CDR2, and VL CDR3 of any one of antibodies described herein (e.g., see Table 2) e.g., wherein the three VL domain CDRs are in the context of a VL.

[0125] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-human p53R175H:HLA-A2 antibody or an antigen-binding fragment thereof or a fragment thereof comprising a VH domain, e.g., containing FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequence described herein (e.g., see Tables 1 and 5, e.g., the VH CDRs and VH FRs of a particular antibody identified by name in the tables). In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof or a fragment thereof comprising a VL domain, e.g., containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, comprising an amino acid sequence described herein (e.g., see Tables 2 and 6, e.g., the VL CDRs and VL FRs of a particular antibody identified by name in the Tables).

[0126] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding a heavy chain variable region (e.g., a VH comprising the amino acid sequence of SEQ ID NO :xx) and a heavy chain constant region, e.g., a human gamma (y) heavy chain constant region.

[0127] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding a light chain variable region (e.g., a VL comprising the amino acid sequence of SEQ ID NO :xx) and a light chain constant region, e.g., a human lambda or kappa light chain constant region.

[0128] Also provided herein are polynucleotides encoding an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein or a domain thereof that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof or a domain thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726;6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.

[0129] A polynucleotide encoding an antibody or antigen-binding fragment thereof described herein or a domain thereof can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and / or heavy chain of an antibody or antigen-binding fragment thereof. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and / or the variable heavy chain region of an antibody or antigen-binding fragment thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies or antigen-binding fragments thereof.

[0130] Polynucleotides provided herein can be, e.g., in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be double-stranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In some aspects, the polynucleotide is a cDNA or a DNA lacking one more endogenous introns. In some aspects, a polynucleotide is a non- naturally occurring polynucleotide. In some aspects, a polynucleotide is recombinantly produced. In some aspects, the polynucleotides are isolated. In some aspects, thepolynucleotides are substantially pure. In some aspects, a polynucleotide is purified from natural components.5.3.2 Cells and Vectors

[0131] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-human p53R175H:HLA-A2 antibodies and antigen-binding fragments thereof or a domain thereof for recombinant expression in host cells, preferably in mammalian cells. Also provided herein are cells, e.g. host cells, comprising such vectors for recombinantly expressing anti-human p53R175H:HLA-A2 antibodies or antigen-binding fragments thereof described herein (e.g., human or humanized antibodies or antigen-binding fragments thereof). In some aspects, provided herein are methods for producing an antibody or antigen-binding fragments thereof described herein, comprising expressing such antibody or antigen-binding fragment thereof in a host cell.

[0132] In some aspects, recombinant expression of an antibody or antigen-binding fragment thereof or domain thereof described herein (e.g., a heavy or light chain described herein) that specifically binds to human p53R175H:HLA-A2 involves construction of an expression vector containing a polynucleotide that encodes the antibody or antigen-binding fragment thereof or domain thereof. Once a polynucleotide encoding an antibody or antigen-binding fragment thereof or domain thereof (e.g., heavy or light chain variable domain) described herein has been obtained, the vector for the production of the antibody or antigen-binding fragment thereof can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody or antigenbinding fragment thereof or domain thereof (e.g., light chain or heavy chain) encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antigenbinding fragment thereof or domain thereof (e.g., light chain or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described herein, a heavy or light chain, a heavy or light chain variable domain, ora heavy or light chain CDR, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and variable domains of the antibody or antigen-binding fragment thereof can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

[0133] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs, the VH, the VL, the VH and the VL, the heavy chain, the light chain, or the heavy and the light chain of IM811, IM810, IM48, IM37, IM42, IM710) or a domain thereof (e.g. , the VH, the VL, the VH and the VL, the heavy chain, or the light chain of IM811, IM810, IM48, IM37, IM42, IM710). Thus, provided herein are host cells containing a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs, the VH, the VL, the VH and the VL, the heavy chain, the light chain, or the heavy and the light chain of IM811, IM810, IM48, IM37, IM42, IM710 or a domain thereof (e.g, the VH, the VL, the VH and the VL, the heavy chain, or the light chain of IM811, IM810, IM48, IM37, IM42, IM710, operably linked to a promoter for expression of such sequences in the host cell. In some aspects, for the expression of double-chained antibodies or antigen-binding fragments thereof, vectors encoding both the heavy and light chains, individually, can be coexpressed in the host cell for expression of the entire immunoglobulin, as detailed below. In some aspects, a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody described herein (e.g., the heavy and the light chain of IM811, IM810, IM48, IM37, IM42, IM710), or a domain thereof (e.g, the VH and the VL of IM811, IM810, IM48, IM37, IM42, IM710). In some aspects, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein (e.g., an antibody comprising the six CDRs of IM811, IM810, IM48, IM37, IM42, IM710), or a domain thereof. In some aspects, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof described herein , and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of IM811, IM810, IM48, IM37, IM42, IM710). In some aspects, a heavy chain / heavy chain variable region expressed by a first cell associated with a light chain / light chain variable region of a second cell to form an human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein (e.g., antibody or antigen-binding fragment thereof comprising the six CDRs of IM811, IM810, IM48, IM37, IM42, IM710). In some aspects, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0134] In some aspects, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein, and a second vector comprising a polynucleotide encoding a heavy chain / heavy chain variable region of an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein (e.g., antibody or antigen-binding fragment thereof comprising the CDRs of IM811, IM810, IM48, IM37, IM42, IM710). Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides.

[0135] A variety of host-expression vector systems can be utilized to express antibodies and antigen-binding fragments thereof described herein (e.g., an antibody or antigenbinding fragment thereof comprising the CDRs of IM811, IM810, IM48, IM37, IM42, IM710) (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or antigen-binding fragment thereof described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insectcell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., C0S1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB / 20 andBMTIO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, cells for expressing antibodies and antigen-binding fragments thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of IM811, IM810, IM48, IM37, IM42, IM710) are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In some aspects, cells for expressing antibodies described herein are human cells, e.g., human cell lines. In some aspects, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, antibodies or antigen-binding fragments thereof described herein are produced by CHO cells or NSO cells.

[0136] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cellline that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., C0S1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B- W, L-M, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some aspects, antihuman p53R175H:HLA-A2 antibodies or antigen-binding fragments thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of IM811, IM810, IM48, IM37, IM42, IM710) are produced in mammalian cells, such as CHO cells.

[0137] Once an antibody or antigen-binding fragment thereof described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies or antigenbinding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0138] In some aspects, an antibody or antigen-binding fragment thereof described herein is isolated or purified. Generally, an isolated antibody or antigen-binding fragment thereof is one that is substantially free of other antibodies or antigen-binding fragments thereof with different antigenic specificities than the isolated antibody or antigen-binding fragment thereof. For example, in some aspects, a preparation of an antibody or antigenbinding fragment thereof described herein is substantially free of cellular material and / or chemical precursors.5.4 Chimeric antigen receptors and T cell engagers

[0139] The present disclosure provides chimeric antigen receptors (CAR) comprising an extracellular and intracellular domain. The extracellular domain comprises a targetspecific binding element otherwise referred to as an antigen binding moiety. In one aspect, the CAR is engineered to target HLA-A2:p53. In another aspect, the antigen binding moiety is an antigen binding domain described herein. The intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen. Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasniic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain, As used herein, the term "spacer domain" generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain, A spacer domain may comprise tip to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

[0140] The cytoplasmnic domain or otherwise the intracellular signaling domain of the CAR is responsible for activation ofat least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolyti cacti vity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent thata truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0141] The present disclosure encompasses a DNA construct comprising sequences of a CAR, wherein the sequence comprises the nucleic acid sequence of an antigen binding moiety operably linked to the nucleic acid sequence of an intracellular domain.

[0142] The disclosure also provides the use of a CAR to redirect the specificity of a primary T cell to a tumor antigen, for example p53. Thus, the present disclosure also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal comprising the step of administering to the mammal a T cell that expresses a CAR, wherein the CAR comprises a binding moiety that specifically interacts with HLA-A2:p53, a zeta chain portion, and a costimulatory signaling region, In one aspect, the present invention includes a type of cellular therapy where T cells are genetically modified to express a CAR and the CAR T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR T cells are able to replicate in vivo resulting in long-term persistence thatcan lead to sustained tumor control, In one aspect, the CAR T cells can undergo robust in vivo T cell expansion and can persist for an extended amount of time. In some aspects, the anti-tumor immunity response elicited by the CAR-modified T cells may be an active or a passive immune response. In addition, the CAR mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified T cells induce an immune response specific to the antigen binding moiety in the CAR.

[0143] The antigen binding domains described herein can also be engineered into T cell engagers (TCEs), which are also able to redirect the immune system to attack tumor cells. In one aspect, the TCE comprises an antigen binding domain described herein and an antigen binding domain that binds to the CD3 receptor on T cells.5.5 Pharmaceutical Compositions

[0144] Provided herein are compositions comprising an anti-p53R175H:HLA-A2 antibody (such as an anti-human p53R175H:HLA-A2 antibody) or antigen-binding fragment thereof, as described herein. In some aspects, the antibody or antigen-binding fragment thereof having the desired degree of purity is present in a formulation comprising, e.g., a physiologically acceptable carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can comprise antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0145] In some aspects, a pharmaceutical composition comprises an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Pharmaceutical compositions described herein are, in some aspects, for use as a medicament. The compositions to be used for invivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.

[0146] In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as cancer. Examples of cancers that can be treated as provided herein include solid tumors. In some aspects, a cancer is a hematopoietic cancer, such as a leukemia, lymphoma, or myeloma. In some aspects, a cancer may be an early stage cancer or a late stage cancer. In some aspects, a cancer is a primary tumor. In some aspects, a cancer is a metastatic tumor at a second site derived from any of the above types of cancer. In some aspects, a cancer is a p53R175H:HLA-A2-positive cancer.Administration and Dosing

[0147] An anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof as provided herein, or a pharmaceutical composition thereof as provided herein, can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intratumoral, intralesional administration, intracerobrospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous administration as a bolus or by continuous infusion over a period of time, intraarterial, intra-articular, intraperitoneal, or subcutaneous administration. In some aspects, the administration is intravenous administration. In some aspects, the administration is subcutaneous.

[0148] The appropriate dosage and dosing regimen of an anti-human p53R175H:HLA- A2 antibody or antigen-binding fragment thereof as provided herein, or a pharmaceutical composition thereof as provided herein, when used alone or in combination with one or more other additional therapeutic agents, will depend on the disease to be treated, the severity and course of the disease, the route of administration and other factors.

[0149] In some aspects, provided herein is an antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein for use as a medicament.

[0150] In some aspects, provided herein is an antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein, for use in a method for the treatment of cancer. In some aspects, provided herein is an antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein, for use in a method for the treatment of cancer in a subject, comprising administering to the subject an effectiveamount of an antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein.5.5.2 Detection and Diagnostic Uses

[0151] An anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein (see, e.g., Section 5.2) can be used to assay p53R175H:HLA-A2 complex levels in a biological sample using classical methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium ("Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label an antibody or antigenbinding fragment thereof described herein. Alternatively, a second antibody or antigenbinding fragment thereof that recognizes an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof described herein can be labeled and used in combination with an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof to detect p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) levels.

[0152] Assaying for the expression level of p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) is intended to include qualitatively or quantitatively measuring or estimating the level of a p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the disease associated protein level in a second biological sample). p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) expression level in the first biological sample can be measured or estimated and compared to a standard p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once the “standard” p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) level is known, it can be used repeatedly as a standard for comparison.

[0153] As used herein, the term “biological sample” refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are well known in the art.

[0154] An anti-human p53R175H:HLA-A2 antibody described herein can be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response may be utilized to predict, diagnose and monitor to evaluate patient samples including those known to have or suspected of having an immune system-dysfunction, or cancer.

[0155] Anti-human p53R175H:HLA-A2 antibodies and antigen-binding fragments thereof described herein can carry a detectable or functional label. When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or combination of both methods procedures known in the art may be utilized to identify and to quantitate the specific binding members. Anti-human p53R175H:HLA-A2 antibodies or antigen-binding fragments thereof described herein can carry a fluorescence label. Exemplary fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-human p53R175H:HLA-A2 antibody can carry a radioactive label, such as the isotopes3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,67Cu,90Y, "TC,niIn,117LU,121I,124I,125I,131I,198AU,211At,213Bi,225Ac and186Re. When radioactive labels are used, currently available counting procedures known in the art may be utilized to identify and quantitate the specific binding of anti-human p53R175H:HLA- A2 antibody or antigen-binding fragment to p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein). In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with an anti-human p53R175H:HLA-A2 antibody or antigen-binding fragment thereof under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein). Anycomplexes formed between the antibody or antigen-binding fragment thereof and p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) are detected and compared in the sample and the control. In light of the specific binding of the antibodies or antigen-binding fragments thereof described herein to human p53R175H:HLA-A2, the antibodies or antigen-binding fragments thereof can be used to specifically detect p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) expression on the surface of cells. The antibodies or antigen-binding fragments thereof described herein can also be used to purify p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) via immunoaffinity purification.

[0156] Also included herein is an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein). The system or test kit may comprise a labeled component, e.g., a labeled antibody or antigen-binding fragment, and one or more additional immunochemical reagents. See, e.g., Section 5.6 below for more on kits.

[0157] In some aspects, methods for in vitro detection of p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) in a sample, comprising contacting said sample with an antibody or antigen-binding fragment thereof, are provided herein. In some aspects, provided herein is the use of an antibody or antigen-binding fragment thereof provided herein, for in vitro detection of p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) in a sample. In some aspects, provided herein is an antibody or antigen-binding fragment thereof or composition provided herein for use in the detection of p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) in a subject or a sample obtained from a subject. In some aspects, provided herein is an antibody or antigen-binding fragment thereof provided herein for use as a diagnostic. In some aspects, the antibody comprises a detectable label.5.6 Kits

[0158] Provided herein are kits comprising one or more antibodies or antigen-binding fragments thereof described herein. In some aspects, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulatingthe manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0159] Also provided herein are kits that can be used in detection methods. In some aspects, a kit comprises an antibody or antigen-binding fragment thereof described herein, preferably a purified antibody or antigen-binding fragment thereof, in one or more containers. In some aspects, kits described herein contain a substantially isolated p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) that can be used as a control. In some aspects, the kits described herein further comprise a control antibody or antigen-binding fragment thereof which does not react with p53R175H:HLA- A2 protein (e.g., human p53R175H:HLA-A2 protein). In some aspects, kits described herein contain one or more elements for detecting the binding of an antibody or antigenbinding fragment thereof to p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA- A2 protein) (e.g., the antibody or antigen-binding fragment thereof can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody or antigenbinding fragment thereof which recognizes the first antibody or antigen-binding fragment thereof can be conjugated to a detectable substrate). In some aspects, a kit provided herein can include a recombinantly produced or chemically synthesized p53R175H:HLA- A2 protein (e.g., human p53R175H:HLA-A2 protein). The p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) provided in the kit can also be attached to a solid support. In some aspects, the detecting means of the above described kit includes a solid support to which a p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) is attached. Such a kit can also include a non-attached reporter-labeled antihuman antibody or antigen-binding fragment thereof or anti-mouse / rat antibody or antigen-binding fragment thereof. In this aspect, binding of the antibody or antigenbinding fragment thereof to the p53R175H:HLA-A2 protein (e.g., human p53R175H:HLA-A2 protein) can be detected by binding of the said reporter-labeled antibody or antigen-binding fragment thereof.6. EXAMPLES

[0160] The examples in this Section (z.e., Section 6) are offered by way of illustration, and not by way of limitation.Example 1: Phage Display discovery of p53-specific antibody clones.

[0161] Antibody discovery against the p53 pHLA target was performed by phage display on four distinct naive libraries with greater than IxlO8diversity (Figure 1 A). Enrichment of p53R175H binders was achieved by positive selection with the p53R175H pHLA complex in the presence of excess amounts of recombinant p53WT or CMVpp65 pHLA complexes. All libraries and panning outputs demonstrating enrichment were screened for p53R175H affinity by Phage ELISA (Figure IB). Clones that demonstrated selective binding to the p53R175H were sent for sequencing, revealing 52 unique p53 -targetting clones in total. All libraries revealed unique multiple p53-specific clones (Figure 1C).

[0162] The 52 clones identified by phage ELISA were further evaluated for p53 affinity using a cell-based flow cytometry assay. T2 cells left untreated or treated with 100 pM of p53R175H or CMVpp65 peptides were incubated with periplasmic extracts (PE) containing c-myc tagged soluble Fabs or ScFvs, and clone binding was assessed by flow cytometry. A reference clone, with known specificity for the p53R175H complex, was expressed in P.E. format and included as a reference control. In total, 8 clones were identified that exhibited no interaction with CMVpp65-pulsed cells and demonstrated binding to the p53R175H complex that was either similar to or stronger than the reference control (Figure 2A-B). The remaining P.E.s screened were categorized into three groups: inert clones (no interaction with any T2 treatments), non-specific clones (binding to untreated T2 cells), and HLA-A2 specific clones (strong affinity for both CMVpp65 and p53R175H-pulsed cells) (Figure 1A).Example 2: Clones display a range of affinities to the p53R175H complex and no affinity to the p53WT.

[0163] Eight clones were assessed for cell-based affinity to the p53R175H and p53WT pHLA using TAP-deficient T2 cells. For antibody production, the variable domains of the p53R175H:HLA-A2 specific were cloned into the human IgGl backbone and transiently expressed in CHO cells. None of the antibodies bound to p53WT+ T2 cells at any antibody concentration tested. In contrast, six out of eight antibodies displayed antibody binding to the p53R175H+ T2 cells at multiple antibody concentrations tested (Figure 3). Four clones- IM810, IM810, IM48 & IM42- displayed higher affinity to the p53R175H complex than the reference positive control, with EC50 values of 0.97 nM, 0.96 nM, 1.34nM, and 3,30 nM respectively, compared to reference value of 3.47 nM. The remaining two binders- IM710 & IM42- displayed EC50 values of 8.29 nM & 5.98 nM (Table 8).Table 8Clone Antibody EC50 Against T2 Antibody EC50 Against T2 Pulsed with p53R175H Pulsed with p53WTIM811 0.96 nM Not determined-No BindingIM810 0.97 nM Not determined-No BindingIM48 1.34 nM Not determined-No Binding IM37 3.30 nM Not determined-No Binding Reference Clone 3.47 nM Not determined-No Binding IM42 5.98 nM Not determined-No Binding IM710 8.29 nM Not determined-No Binding IM610 Not determined-No Binding Not determined-No BindingExample 3: IM810-TCE Binding Characteristics to p53R175H:HLA-A2

[0164] Four p53-specific clones - IM810, IM48, IM42 & the reference clone (ref) were synthesized as bispecific T-Cell Engagers composed of anti-p53R175H Fab fused to an anti-CD3 ScFv with a partial FC domain (Figure 4). In addition, an isotype control TCE was engineered to account for non-specific CD3 activation. The variable domains of the p53R175H:HLA-A2 specific clones or isotype control and the anti-CD3 clone were cloned into two vectors & transiently expressed in CHO. The CH2 domain of human Igyl (CH2Mut) was modified as previously described (US 2018 / 0057608 Al) to abrogate FcR binding and complement fixation and prevent glycosylation & disulfide mediated homotypic dimerization.

[0165] The equilibrium binding constant (Kd) of P53-specific T-cell Engagers for the p53R175H:HLA-A2 complex were assessed with surface plasmon resonance (SPR) using BiaCore 8K+. The Kd values, calculated by Langmuir 1 : 1 binding model, identified IM810 as the highest affinity TCE for the p53R175H:HLA-A2 complex, with a Kd value of 2.9 nM (Figure 5a). IM48 displayed a higher affinity for the p53R175H complexcompared to the Reference clone, with Kds of 72.7 nM & 119.7 nM respectively (Figure 5B-C). A kd value was not determined for IM42.Table 9pHLA 2Antibody Immobilization Chi Ka(l / Ms) Kd(l / s) Kd (nM)(RU)H2-TCE 136 26.1 1.66E+05 1.98E-02 119.73IM811-TCE 129 7.4 1.92E+05 5.53E-04 2.87IM48 120 10.5 1.58E+06 1.15E-01 72.77IM42 117 ND ND ND NDExample 4: IM810-TCE triggers potent & specific T-Cell activation against the p53R175H mutation

[0166] To assess the in vitro activity & specificity of synthesized T-Cell Engagers, T2 wells were pulsed with p53R175H or P53WT peptide and treated with T-Cell Engagers in combination with CD3+ cells isolated from healthy donors. As a negative control, cocultures were left untreated or incubated with the isotype T-Cell Engager. As expected, IM810-TCE triggered the highest IFN-Y production against T2 cells pulsed with p53R175H. IM42-TCE and the reference-TCE triggered comparable T-Cell activation across all concentrations tested. In line with the weak affinity defined by SPR, IM42 failed to trigger p53R175H-specific T-cell activation at the concentrations tested. IM810, IM42 & ref-TCE were p53R175H-specific, displaying no interaction with the p53WT pulsed cells. Furthermore, no activity was observed in the isotype TCE control across all concentrations tested (Figure 6).

[0167] To assess whether IM810-TCE could trigger p53R175H-specific T-cell proliferation, CD3+ effector cells were labeled with the CFSE proliferation dye. This enabled (a) differentiation between tumor cells (CFSE-) and effector cells (CFSE+) and (b) quantification of T-cell proliferation. As seen in the IFN-y data, IM810-TCE induced T-cell proliferation, demonstrated by the appearance of multiple distinct CFSE peaks, which were absent in the T2 p53WT culture. These peaks reflect the progressive dilutionof CFSE fluorescence as T-cells divide, indicating a response specifically triggered by the p53R175H peptide (Figure 7A).

[0168] T cell proliferation & Tumour Cytoxicity data was further supported by the effector-to-tumor (E:T) ratio, calculated using absolute counting beads. IM810 triggered robust increases in the E:T ratio when cocultured with p53R175H T2 cells, while no impact on the E:T ratio was observed in the presence of the p53WT cell line (Figure 7B). This further confirms the specificity and potency of IM810-TCE in promoting tumorspecific T-cell responses.Example 5. IM810 is cytotoxic against Tumour cells that endogenously express the p53R175H mutated gene and HLA-A*02:01 allele.

[0169] The activity of IM810-TCE was assessed in various tumour cell lines that naturally express the p53R175H mutation & the HLA-A*02:01 allele to various degrees (Figure 11). KMS26, SKBR3, KLE, TYK-NY & AU565 cocultures treated with titrating amounts of IM810-TCE revealed potent T cell activation (Figure 8) T-Cell proliferation (Figure 10) & Tumour cytotoxicity (Figure 9).. IM810’s anti -tumour activity was target (surface pHLA) & drug concentration dependent (Figure 11), with the KMS26 cell line that expresses high levels of p53 and HLA-A2displaying the lowest IFNy production & tumour cell death EC50 values (Figure 8 & 9).Example 6: Peptide Scanning Assays reveal that IM810 interacts with the target peptide at multiple sites & has no affinity for healthy peptideHLA complexes.

[0170] The specificity of IM810 for the p53R175H target was assessed by site-directed mutagenesis by systemically substituting amino acids at each position of the target p53R175H peptide (HMTEVVRHC) with each of the remaining 19 common amino acids. The synthetic variants of the target pHLA complex were coated onto beads and monitored for interation with the IM810 IgGl antibody by flow cytometry. Any change In P8, where the mutant histidine lies, completely abolished recognition of the peptide. All exposed non-anchor positions, except Pl, favored the parental peptide (Figure 12). This is particularly true for P7, P4, P3 & p5. Searching the X-Scan motif in the UniProtKB human protein database via Scan Prosite identified 48 homologous peptides (Figure 13 A), of which 3 (VATF, NIBAN1, and VAPA) displayed weak binding to the IM810 antibody in a recombinant peptide-HLA assay (Figure 13B).

[0171] The recombinant pHLA binding assay has a Signal to blank assay window of >6000, indicating high sensitivity that is outside the clinical relevant range (data not shown). To investigate the clinical relevance of IM810’s weak interaction with VATF, NIBAN1 & VAPA, three p53R175H- cell lines with varying expression of vatf, nibanl. and vapa were monitored for interaction with the IM810-TCE (Figure 14A). As a positive control for T-Cell responses, all cocultures were treated with a T-Cell Engager specific to the HLA-A2 main chain (HLA-TCE). The HLA- TCE drove T Cell activation in all cell lines tested. As previously observed, the IM810-TCE triggered IFNy against the KMS26 target+ cell line. In contrast, IM180 drove no significant response against the HEK293T, THP1 or A498 cell lines (Figure 14B).

[0172] The aim of the study was to identify a highly specific Scfv or Fab to the HLA- A*02:01 restricted p53R175H neoantigen for eventual therapeutic treatment in an antibody format for HLA-A*02:01+ cancer patients bearing the p53R175H mutation. Periplasmic extracts (p.e) containing soluble Scfv or Fab with reported affinity for p53R175H: HLA-A*02:01 by phage ELISA were be monitored for binding to target and p53R175H X-scan peptides utilizing T2 cells and flow cytometry.

[0173] X-scan peptides are peptides which vary from the parent peptide by one amino acid. They enable the characterization of binding motifs. It is known that peptide selectivity, i.e the degree of off-target pHLA cross-reactivity, is associated with an energetic signature characterized by broad interactions with several amino acid side chains as well as the peptide backbone. Thus, the function of the X-scan peptides in this study will be to determine the specificity of the clone and provide insight to the binding motif on individual clones.

[0174] The H2 Fab IS used as positive control antibody fragment for binding to p53R175H:HLA neoantigen. The major contacts positions of the H2 Ab for p53R175H:HLA were determined by X-scan mutagenesis and are, in order of importance, the mutant histidine residue Hisl75 (position 8), the adjacent residue Argl74 (position 7) and positions 3 to 6 (Han-Chung Hsiue et al., Targeting a neoantigen derived from a common TP53 mutation. Science. 2021 March 05; 371. doi:10.1126 / science.abc8697).* * *

[0175] The invention is not to be limited in scope by the specific aspects described herein. Indeed, various modifications of the invention in addition to those described willbecome apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0176] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0177] Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED:

1. An isolated antibody or antigen-binding domain that specifically binds to human p53R175H:HLA-A2 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of:(a) SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively;(b) SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively;(c) SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively;(d) SEQ ID NOs: 8, 9, 10, and 24, 25, and 26, respectively;(e) SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or (f) SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively.An isolated antibody or antigen-binding domain that competitively inhibits binding of a reference antibody to human p53R175H:HLA-A2, wherein the reference antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:(a) SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively;(b) SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively;(c) SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively;(d) SEQ ID NOs: 8, 9, 10, and 24, 25, and 26, respectively;(e) SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or (f) SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively.3 An isolated antibody or antigen-binding domain that binds to the same human p53R175H:HLA-A2 epitope as an antibody comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:(a) SEQ ID NOs: 2, 3, 4, and 17, 18, and 19, respectively;(b) SEQ ID NOs: 2, 3, 4, and 20, 18, and 19, respectively;(c) SEQ ID NOs: 5, 6, 7, and 21, 22, and 23, respectively;(d) SEQ ID NOs: 8, 9, 10, and 24, 25, and 26, respectively;(e) SEQ ID NOs: 11, 12, 13, and 27, 28, and 29, respectively; or(f) SEQ ID NOs: 14, 15, 16, and 30, 31, and 32, respectively.

4. An isolated antibody or antigen-binding domain that specifically binds to human p53R175H:HLA-A2, wherein the antibody or antigen-binding domain comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of an antibody selected from the group consisting of IM811, IM810, IM48, IM37, IM42, or IM710.5 The antibody or antigen-binding domain of claim 4, wherein the CDRs are the Kabat- defined CDRs, the Chothia-defined CDRs, the IMGT-defined CDRs, or the AbM-defined CDRs.6 The antibody or antigen-binding domain of any one of claims 1-5, wherein the antibody or antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NOs: 33-38.7 The antibody or antigen-binding domain of any one of claims 1-6, wherein the antibody or antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NOs: 39-44.8 The antibody or antigen-binding domain of any one of claims 1-7, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:(a) SEQ ID NOs: 33 and 39, respectively;(b) SEQ ID NOs: 34 and 40, respectively;(c) SEQ ID NOs: 35 and 41, respectively;(d) SEQ ID NOs: 36 and 42, respectively;(e) SEQ ID NOs: 37 and 43, respectively; or(f) SEQ ID NOs: 38 and 44, respectively.9 A humanized form of the antibody of claim 8.

10. The antibody or antigen-binding domain of any one of claims 1-9, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region.

11. The antibody or antigen-binding domain of claim 10, wherein the heavy chain constant region is an isotype selected from the group consisting of human IgGi, IgG2, IgGs, and IgG4isotypes.

12. The antibody or antigen-binding domain of claim 11, which comprises an Fc domain that is engineered to reduce effector function.

13. The antibody or antigen-binding domain of any one of claims 1-12, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgGi heavy chain constant region, and wherein the light chain constant region is a human IgGK light chain constant region.

14. The antibody or antigen-binding domain of any one of claims 1-13, wherein the antibody or antigen-binding fragment is a monoclonal antibody.

15. The antibody or antigen-binding domain of any one of claims 1-14 wherein the antibody or antigen-binding domain is a murine, chimeric, humanized, or human antibody or antigen-binding domain.

16. The antibody or antigen binding domain of any one of claims 1-15, which is a full length antibody.

17. The antibody or antigen binding domain of any one of claims 1-16, which is an antigen binding fragment.

18. The antigen binding fragment of claim 17, wherein the antigen binding fragment is a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

19. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding domain of any one of claims 1-18.

20. The isolated polynucleotide of claim 19, wherein the nucleic acid molecule encodes the VH of SEQ ID NOs: 33-38.

21. An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or light chain of the antibody or antigen-binding domain of any one of claims 1-18.

22. The isolated polynucleotide of claim 38, wherein the nucleic acid molecule encodes the VL of SEQ ID NOs: 39-44.

23. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding domain of any one of claims 1-20 and the light chain variable region or light chain of the antibody or antigenbinding domain of any one of claims 1-18.

24. An isolated vector comprising the polynucleotide of any one of claims 19-23.

25. A host cell comprising (a) the polynucleotide of any one of claims 19-23, or (b) the vector of claim 24.

26. A method of producing an antibody or antigen-binding domain that binds to human p53R175H:HLA-A2 comprising culturing the host cell of claim 25 so that the nucleicacid molecule is expressed and the antibody or antigen-binding domain is produced, optionally wherein the method further comprises isolating the antibody or antigen-binding domain from the culture.

27. A T cell engager molecule comprising the antigen-binding domain of any one of claims 1-9.

28. A pharmaceutical composition comprising the antibody or antigen-binding domain of any one of claims 1-18, or the T cell engager of claim 27 and a pharmaceutically acceptable excipient.

29. A method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of the antibody or antigen binding domain of any one of claims 1-18, the T cell engager of claim 27, or the pharmaceutical composition of claim 28.

30. The method of claim 29, wherein the cancer is a solid tumor or a hematopoietic cancer.

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