Anti-new york esophageal squamous cell carcinoma 1 (ny-ESO-1) antigen-binding proteins and methods of use thereof

Abstract

To provide anti-New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen-binding proteins and methods of using the same.SOLUTION: The present disclosure provides antigen-binding proteins that specifically bind to an HLA-displayed New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, and therapeutic and diagnostic methods of using those binding proteins. The antigen-binding proteins of the present disclosure bind with a high degree of specificity to HLA-displayed NY-ESO-1 and do not bind to HLA-displayed peptides that differ by 2, 3, 4, 5, or more amino acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 870,232, filed July 3, 2019, U.S. Provisional Patent Application No. 63 / 020,177, filed May 5, 2020, and U.S. Provisional Patent Application No. 63 / 021,826, filed May 8, 2020, the entire contents of each of the foregoing being incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on July 2, 2020, is named 118003_10520_SL.txt and is 245,227 bytes in size.

[0003] The present disclosure relates to antigen binding proteins that specifically bind to HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptides, and therapeutic and diagnostic methods using those binding proteins. [Background technology]

[0004] New York esophageal squamous cell carcinoma 1 (NY-ESO-1), also known as CTAG1B, is a cancer-testis antigen (CTA). Expression of NY-ESO-1, encoded by the CTAG1B gene, is restricted to germ cells. However, NY-ESO-1 is often aberrantly re-expressed in various tumor types. The major protein product of the CTAG1B gene is an 18-kDa protein with a glycine-rich N-terminal region and a highly hydrophobic C-terminal region. However, the function of NY-ESO-1 remains unclear.

[0005] Spontaneous CD8 against NY-ESO-1 in cancer patients + and CD4 + T cell responses are observed and analyzed (Jager, E. et al. (1998) J Exp Med 187:265-270, Jager, E. et al. (2000) J Exp Med 191:628-630). In particular, NY-ESO-1 peptides 157-165, 157-167, and 155-163 bind to tumor-reactive CD8 + Restricted by HLA-A2 in T cell lines, peptides 56-62 bind to HLA-A31 CD8 + It was found to be recognized by T cells (Jager et al. (1998) J. Exp Med 187:265-270; Wang, RF et al. (1998) J. Immunol 161:3598-3606). Several epitopes restricted by HLA-DR4 in CD4 T cell responses have also been demonstrated, and responses to peptide 157-170 were restricted by HLA-DP4, an allele found in the majority of Caucasians (Jager et al. (2000) J. Exp Med 191:625-630; Zang et al. (2001) Proc Natl Acad Sci USA 98:3964-3969).

[0006] The ability of NY-ESO-1 to elicit spontaneous humoral and cellular immune responses, along with its restricted expression pattern, makes it an excellent candidate target for cancer therapy. While the NY-ESO-1 antigen has been evaluated as a cancer vaccine candidate, few have been able to elicit complete humoral and cellular immune responses. Indeed, the use of NY-ESO-1 as a therapeutic target is challenging due to the difficulty in designing antigen-binding proteins that target HLA-presented antigens and the need to avoid off-target binding to prevent nonspecific binding, which may result in reduced therapeutic efficacy and / or increased adverse effects (e.g., nonspecific cytotoxicity, which reduces tumor cell killing activity and / or causes side effects in the subject). Therefore, there is an unmet need in the art for novel therapeutic strategies that target NY-ESO-1 with high specificity to treat NY-ESO-1-associated cancers. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jager, E. et al. (1998) J Exp Med 187:265-270 [Non-patent document 2] Jager, E. et al. (2000) J Exp Med 191:628-630 [Non-patent document 3] Wang, RF et al. (1998) J. Immunol 161:3598-3606 [Non-patent document 4] Zang et al. (2001) Proc Natl Acd Sci USA 98:3964-3969 Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides antigen binding proteins that specifically bind to a conformational epitope of the HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide. The antigen binding proteins of the present disclosure bind with high specificity to HLA-presented NY-ESO-1 and do not bind to HLA-presented peptides that differ by two, three, four, five, or more amino acids. The antigen binding proteins of the present disclosure enable specific targeting of NY-ESO-1 peptide-presenting cells (i.e., cells that present NY-ESO-1 peptide bound to an MHC molecule, e.g., HLA-A2, on their surface), such as NY-ESO-1-expressing cancer cells, and in some embodiments stimulate T cell activation, for example, to stimulate T cell-mediated killing of such cells. Furthermore, when fused to a detectable moiety, the antigen binding proteins of the present disclosure enable the diagnosis and prognosis of NY-ESO-1 positive diseases or disorders with high sensitivity to changes in the number and distribution of NY-ESO-1 peptide-presenting cells, which are a more relevant measure of disease progression than circulating NY-ESO-1 levels.

[0009] The antigen-binding proteins of the present disclosure may be antibodies, such as full-length (e.g., IgG1 or IgG4 antibodies), or may comprise only the antigen-binding portion of an antibody (e.g., a Fab, F(ab')2, or scFv fragment), and may be modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In some embodiments, the antigen-binding proteins of the present disclosure may be antibodies, or antigen-binding fragments thereof. In certain embodiments, the antigen-binding proteins may be bispecific.

[0010] In a first aspect, the present disclosure provides recombinant antigen binding proteins that specifically bind to a conformational epitope of an HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, such as an HLA-presented peptide comprising amino acid residues 157-165 of NY-ESO-1. In certain embodiments, the antigen binding protein is an antibody. In some embodiments, the antibody is fully human.

[0011] Exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins are listed herein in Tables 1 and 2. Table 1 shows the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-HLA-A2:NY-ESO-1 antibodies. Table 2 shows the nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-HLA-A2:NY-ESO-1 antibodies.

[0012] The present disclosure provides antigen binding proteins comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% sequence identity thereto. In some embodiments, antigen binding proteins having less than 100% sequence identity comprise CDR sequences from an HCVR in Table 1. For example, such antigen binding proteins can comprise those CDR sequences but have differences in the framework regions compared to the HCVRs in Table 1.

[0013] The present disclosure also provides antigen binding proteins comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% sequence identity thereto. In some embodiments, antigen binding proteins having less than 100% sequence identity comprise CDR sequences from an LCVR of Table 1. For example, such antigen binding proteins can comprise those CDR sequences but have differences in the framework regions compared to the LCVR of Table 1.

[0014] The present disclosure also provides antigen binding proteins comprising a pair of HCVR and LCVR amino acid sequences (HCVR / LCVR), including any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antigen binding proteins comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, and 230 / 238. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 2 / 10 (e.g., mAb24955N), 22 / 30 (e.g., mAb24956N), 42 / 50 (e.g., mAb24958N), and 62 / 70 (e.g., mAb24959N).

[0015] In certain embodiments, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 with five or fewer amino acid substitutions, and the LCVR comprises an amino acid sequence listed in Table 1 with five or fewer amino acid substitutions. For example, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO:2 with five or fewer amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO:10 with five or fewer amino acid substitutions. In another exemplary embodiment, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO:2 with at least one amino acid substitution, and the LCVR comprises the amino acid sequence of SEQ ID NO:10 with at least one amino acid substitution.

[0016] The present disclosure also provides antigen binding proteins comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0017] The present disclosure also provides antigen binding proteins comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0018] The present disclosure also provides antigen binding proteins comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0019] The present disclosure also provides antigen binding proteins comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0020] The present disclosure also provides antigen binding proteins comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0021] The present disclosure also provides antigen binding proteins comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0022] The present disclosure also provides antigen binding proteins comprising a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), including any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antigen binding proteins comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16 (e.g., mAb24955N), 28 / 36 (e.g., mAb24956N), 48 / 56 (e.g., mAb25958N), and 68 / 76 (e.g., mAb24959N).

[0023] The present disclosure also provides antigen binding proteins comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an HCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an HCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. In certain embodiments, the present disclosure provides antigen binding proteins comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an LCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an LCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. For example, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence that differs by one amino acid from SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs by one amino acid from SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that differs by one amino acid from SEQ ID NO:8. In another exemplary embodiment, the present disclosure provides an antigen binding protein comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:12 or an amino acid sequence that differs by one amino acid from SEQ ID NO:12, an LCDR2 comprising the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs by one amino acid from SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16 or an amino acid sequence that differs by one amino acid from SEQ ID NO:16.

[0024] The present disclosure also provides antigen binding proteins comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary antigen binding proteins listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 (e.g., mAb24955N), 24-26-28-32-34-36 (e.g., mAb24956N), 44-46-48-52-54-56 (e.g., mAb24958N), and 64-66-68-72-74-76 (e.g., mAb24959N).

[0025] In related embodiments, the present disclosure provides antigen binding proteins comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary antigen binding proteins listed in Table 1. For example, the present disclosure includes antigen binding proteins comprising a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 (e.g., mAb24955N), 22 / 30 (e.g., mAb24956N), 42 / 50 (e.g., mAb24958N), and 62 / 70 (e.g., mAb24959N).

[0026] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within antigen binding proteins.

[0027] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding patterns with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites can be useful; i.e., antibodies lacking fucose moieties present in the oligosaccharide chains increase antibody-dependent cellular cytotoxicity (ADCC) function, for example (see Shield et al. (2002) JBC 277:26733). In other applications, modifications to galactosylation can be made to alter complement-dependent cytotoxicity (CDC).

[0028] In certain embodiments, the antigen binding protein of the disclosure is a monoclonal antibody comprising a pair of an HCVR amino acid sequence and an LCVR amino acid sequence (HCVR / LCVR), comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the monoclonal antibody comprises an Fc domain of an isotype selected from the group consisting of IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM, and variants thereof.

[0029] The present disclosure provides antigen binding proteins, or antigen-binding fragments thereof, comprising a heavy chain comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, antigen binding proteins with less than 100% sequence identity comprise CDR sequences from a HC of Table 3. For example, such antigen binding proteins can comprise those CDR sequences but have differences in the framework regions compared to a HC of Table 3.

[0030] The present disclosure also provides antigen binding proteins, or antigen-binding fragments thereof, comprising a light chain comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, antigen binding proteins with less than 100% sequence identity comprise CDR sequences from a LC of Table 3. For example, such antigen binding proteins can comprise those CDR sequences but have differences in the framework regions compared to the LC of Table 3.

[0031] The present disclosure also provides antigen-binding proteins, or antigen-binding fragments thereof, comprising a pair of HC and LC amino acid sequences (HC / LC), comprising any of the HC amino acid sequences listed in Table 3 paired with any of the LC amino acid sequences listed in Table 3. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HC / LC amino acid sequence pair contained within any of the exemplary anti-NY-ESO-1 antibodies listed in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18 / 20, 38 / 40, 58 / 60, and 78 / 80. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18 / 20, 38 / 40, 58 / 60, and 78 / 80.

[0032] In one aspect, the present disclosure provides an antigen-binding protein or antigen-binding fragment thereof that binds to an HLA-peptide complex, wherein the antigen-binding protein or antigen-binding fragment thereof contacts at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the peptide contained in the HLA-peptide complex. In certain embodiments, the antigen-binding protein or antigen-binding fragment thereof "covers" or contacts all of the amino acid residues of the peptide contained in the HLA-peptide complex. In certain embodiments, the antigen-binding protein or antigen-binding fragment thereof binds to the HLA-peptide complex with high affinity and specificity, wherein the antigen-binding protein or antigen-binding fragment thereof contacts the entire length of the presented peptide. "Contact," as used herein, includes direct or water-mediated hydrogen bonding, charge-charge interactions, or hydrophobic / van der Waals interactions. In one embodiment, an antigen binding protein or antigen-binding fragment thereof binds to the HLA-A2-NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to at least three of the nine amino acid residues of peptide 157-165 (SEQ ID NO: 269) and binds to HLA-A2 such that the antigen binding protein is roughly centered with the peptide in the HLA-A2 peptide-binding groove, thereby "covering" (physically blocking) the HLA-A2 peptide complex. In another embodiment, an antigen binding protein or antigen-binding fragment thereof binds to the HLA-A2-NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to at least three of the nine amino acid residues of peptide 157-165 (SEQ ID NO: 270 or 291) and binds to HLA-A2 such that the antigen binding protein is roughly centered with the peptide in the HLA-A2 peptide-binding groove, thereby "covering" (physically blocking) the HLA-A2 peptide complex. In a specific embodiment, the antigen binding protein or antigen-binding fragment thereof comprises the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In one embodiment, the antigen binding protein is fully human.In certain embodiments, fully human antigen binding proteins are not obtained using phage display methods and techniques. In some embodiments, the antigen binding protein comprises a light chain variable region of the IGKV1-39 subtype. In some embodiments, the antigen binding protein comprises a light chain variable region of the IGKJ1 subtype.

[0033] In certain embodiments, the present disclosure provides an antigen binding protein or antigen-binding fragment thereof that binds to the HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to one or more amino acids of SEQ ID NO: 269. In one embodiment, the antigen binding protein binds to at least three amino acids of SEQ ID NO: 269.

[0034] In certain embodiments, the present disclosure provides an antigen binding protein or antigen binding fragment thereof that binds to the HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to one or more amino acids of SEQ ID NO: 270 or 291. In one embodiment, the antigen binding protein binds to at least three amino acids of SEQ ID NO: 270 or 291. In one embodiment, the antigen binding protein binds to one or more amino acids selected from the group consisting of M160, W161, and Q164 of SEQ ID NO: 270.

[0035] In certain embodiments, the present disclosure provides antigen binding proteins that specifically bind to a conformational epitope of an HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, wherein the conformational epitope comprises one or more amino acids of SEQ ID NO: 269.

[0036] In certain embodiments, the present disclosure provides antigen binding proteins that specifically bind to a conformational epitope of an HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, wherein the conformational epitope comprises one or more amino acids of SEQ ID NO: 270 or 291. In certain embodiments, the conformational epitope comprises one or more amino acids selected from the group consisting of M160, W161, and Q164 of SEQ ID NO: 270 or 291.

[0037] The present disclosure also provides an antigen binding protein that competes for specific binding to HLA-A2:NY-ESO-1 with an antigen binding protein comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0038] The present disclosure also provides antigen binding proteins that cross-compete for binding to HLA-A2:NY-ESO-1 with a reference antigen binding protein comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0039] The present disclosure also provides antigen binding proteins that bind to the same epitope as a reference antigen binding protein comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present disclosure provides antigen binding proteins that bind to the same epitope as a reference antigen binding protein comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR is selected from the group consisting of SEQ ID NOs: 2, 22, 42, and 62, and the LCVR is selected from the group consisting of SEQ ID NOs: 10, 30, 50, and 70.

[0040] In one embodiment, the present disclosure provides a recombinant antigen binding protein that specifically binds to a conformational epitope of an HLA-A2-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, wherein the antigen binding protein (a) has a binding dissociation equilibrium constant (K) of less than about 1 nM for a monomeric HLA-A2:NY-ESO-1 157-165 peptide complex (C165 or V165) as measured at 25° C. in a surface plasmon resonance assay. D ) and (b) an EC of less than about 10 nM to cells expressing the HLA-A2:NY-ESO-1 157-165 peptide complex as determined by flow cytometry assay. 50 and (c) the epitope comprises one or more amino acids of SEQ ID NO: 269, 270, or 291. As disclosed elsewhere herein, an "off-target peptide" refers to a peptide that differs from a target peptide (e.g., the NY-ESO-1 157-165 peptide (SEQ ID NO: 269 and / or SEQ ID NO: 270 or 291)) by two, three, four, five, or more amino acids.

[0041] In a second aspect, the present disclosure provides nucleic acid molecules encoding anti-HLA-A2:NY-ESO-1 antigen binding proteins. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0042] The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0043] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0044] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0045] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0046] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0047] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0048] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0049] The present disclosure also provides nucleic acid molecules encoding HCVRs, wherein the HCVRs comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1.

[0050] The present disclosure also provides nucleic acid molecules encoding LCVRs, wherein the LCVRs comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1.

[0051] The disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or substantially thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-HLA-A2:NY-ESO-1 antigen binding protein listed in Table 1.

[0052] The present disclosure provides nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in Table 3. The present disclosure also provides nucleic acid molecules encoding any of the light chain amino acid sequences listed in Table 3.

[0053] The present disclosure also provides nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), wherein the HC comprises any of the HC amino acid sequences listed in Table 3, and the LC comprises any of the LC amino acid sequences listed in Table 3.

[0054] In a related aspect, the present disclosure provides recombinant expression vectors capable of expressing polypeptides comprising the heavy and / or light chain variable regions of an anti-HLA-A2:NY-ESO-1 antigen binding protein. For example, the present disclosure includes recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 1. The present disclosure also provides recombinant expression vectors capable of expressing polypeptides comprising the heavy and / or light chains of an anti-HLA-A2:NY-ESO-1 antigen binding protein. For example, the present disclosure includes recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the heavy or light chain sequences shown in Table 2. The scope of the present disclosure also includes host cells into which such vectors have been introduced, as well as methods of producing the antigen binding protein by culturing the host cells under conditions that allow for the production of the antigen binding protein, and recovering the antigen binding protein so produced.

[0055] In a third aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a recombinant antigen binding protein that specifically binds to a conformational epitope of an HLA-A2-presented NY-ESO-1 peptide (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1) and a pharmaceutically acceptable carrier. In a related aspect, the present disclosure features a composition that is a combination of an anti-HLA-A2:NY-ESO-1 antigen binding protein and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-HLA-A2:NY-ESO-1 antigen binding protein. Exemplary agents that may be advantageously combined with the anti-HLA-A2:NY-ESO-1 antigen binding protein include, but are not limited to, other agents that modulate immune cell activation. Additional therapies that can be used in combination with the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure are disclosed elsewhere herein.

[0056] In a fourth aspect, the present disclosure provides a method of treating a subject with a NY-ESO-1-associated disease or disorder, such as NY-ESO-1-positive cancer. The method comprises administering a therapeutically effective amount of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need thereof. The disorder being treated is any disease or condition that is improved, ameliorated, inhibited, or prevented by the antigen binding proteins and compositions provided herein. In certain embodiments, the antigen binding protein (or pharmaceutical composition) of the present disclosure is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of antibodies against T-cell co-inhibitors, antibodies against tumor cell antigens, antibodies against T-cell receptors, cytotoxic agents, anti-cancer agents, anti-inflammatory agents (e.g., corticosteroids), chemotherapeutic agents, surgery, radiation therapy, immunosuppressants, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent may be an agent that serves to offset or reduce any potential side effects associated with the antigen binding protein of the present disclosure, if such side effects occur.

[0057] In certain embodiments, the present disclosure provides a method for suppressing the growth of NY-ESO-1-associated cancer. For example, the present disclosure provides a method for suppressing tumor growth from a primary tumor or a metastatic tumor in a subject. In certain embodiments, the present disclosure provides a method for improving the survival (e.g., progression-free survival or overall survival) of a subject with NY-ESO-1-associated cancer. Examples of cancer include, but are not limited to, liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer.

[0058] In certain embodiments, the present disclosure provides a method for inhibiting or suppressing the growth of an established tumor. The method comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antigen-binding protein of the present disclosure. In certain embodiments, the antigen-binding protein is administered in combination with a second therapeutic agent.

[0059] The antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, can be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight.

[0060] In a fifth aspect, the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), or a cell expressing such a CAR (e.g., a cell expressing a CAR on its surface). The CAR may comprise an extracellular binding domain that specifically binds to a conformational epitope of the HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, e.g., amino acid residues 157-165 of NY-ESO-1, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the extracellular binding domain is an anti-HLA-A2:NY-ESO-1 antigen-binding protein or an antigen-binding fragment thereof. Exemplary anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure are any of the antigen-binding proteins described herein.

[0061] For example, in certain embodiments, an antigen binding protein that is suitable for use in a CAR of the present disclosure comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised in any one of the heavy chain variable region (HCVR) sequences listed in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in any one of the light chain variable region (LCVR) sequences listed in Table 1.

[0062] In other embodiments, antigen binding proteins suitable for use in a CAR of the present disclosure comprise an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and / or an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0063] In some embodiments, an antigen binding protein that is suitable for use in a CAR of the present disclosure comprises (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and (b) an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0064] In one embodiment, an antigen binding protein that is suitable for use in a CAR of the present disclosure comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213, and 232; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 182, 199, 215, and 234; and (c) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, and 236. (d) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, and 240; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, and 242; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224, and 244.

[0065] In further embodiments, antigen binding proteins that are suitable for use in a CAR of the present disclosure comprise an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, and 230 / 238.

[0066] In some embodiments, the antigen binding protein for use in the CAR of the present disclosure is an scFv.

[0067] In other aspects, the present disclosure provides vectors comprising the isolated CAR nucleic acid molecules, and immune effector cells comprising such vectors.

[0068] In yet another embodiment of the present disclosure, methods are provided for treating a subject having a NY-ESO-1-associated disease or disorder, such as a NY-ESO-1-positive cancer, e.g., liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer. The method includes administering to the subject a population of immune effector cells comprising a CAR of the present disclosure.

[0069] In some embodiments, the present disclosure provides a method for detecting NY-ESO-1 positive cells, e.g., in a subject or a sample obtained from a subject, comprising contacting a cell, such as a cell sample obtained from a subject, with an antigen binding protein of the present disclosure comprising a detectable moiety, or administering the antigen binding protein to the subject, and detecting the presence of the detectable moiety.

[0070] Other embodiments will become apparent from review of the following detailed description. [Brief explanation of the drawings]

[0071] [Figure 1A] A diagram of three different CAR constructs is shown, including promoter and vector elements. [Figure 1B] Tumor volumes in mice treated for 0 to 21 days with T cells expressing either the non-binding control BB / z CAR (control CAR T), the anti-HLA-A2 / NY-ESO-1157~165 BB / z CAR, or the anti-HLA-A2 / NY-ESO-1157~165 28 / z CAR are shown. Left panel: average tumor volume; right panel: tumor volumes of individual mice. [Figure 1C]Tumor volumes in mice treated with T cells expressing either the non-binding control BB / z CAR (control CAR T), the anti-HLA-A2 / NY-ESO-1157~165 BB / z CAR, or the anti-HLA-A2 / NY-ESO-1157~165 28 / z CAR for days 0-38 are shown. Left panel: average tumor volume; right panel: tumor volumes of individual mice. DETAILED DESCRIPTION OF THE INVENTION

[0072] Before the present methods are described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to those described herein can be used to implement or test this disclosure, preferred method and material are described herein.All publications mentioned herein are incorporated herein by reference in their entirety.

[0074] The terms "NY-ESO-1," "NY-ESO-1," "New York esophageal squamous cell carcinoma 1," and "CTAG1B" refer to a well-known cancer-testis antigen (CTA) that is re-expressed in many cancer types and is encoded by the CTAG1B gene.

[0075] The amino acid sequence of full-length NY-ESO-1 is provided in GenBank under accession number NP_001318.1 (SEQ ID NO: 271). The term "NY-ESO-1" includes recombinant NY-ESO-1 or a fragment thereof. The term also encompasses NY-ESO-1 or a fragment thereof linked to a signal sequence, such as, for example, a histidine tag, mouse or human Fc, or ROR1. In a specific embodiment, the term includes NY-ESO-1 or a fragment thereof linked to or presented by HLA-A2 in the context of HLA-A2.

[0076] In certain embodiments, the NY-ESO-1 peptide comprises amino acids 157-165 of SEQ ID NO:271 (SLLMWITQC (SEQ ID NO:269)), and is referred to herein as the "NY-ESO-1_157-165C peptide" or "NY-ESO-1(157-165) peptide" (except where "NY-ESO-1(157-165) peptide" is further clarified as having a C or V at position 165). In other embodiments, the NY-ESO-1 peptide comprises amino acid residues 157-165 of SEQ ID NO:271 (SLMMWITQV (SEQ ID NO:270)), in which the cysteine ​​at residue 165 has been substituted with a valine, and is referred to herein as the "NY-ESO-1_157-165V peptide" or "NY-ESO-1(157-165V peptide)," or otherwise designated "C165V" or "V165." Briefly, the terms "NY-ESO-1_157-165 peptide," "NY-ESO-1(157-165)," and "a peptide comprising amino acid residues 157-165 of NY-ESO-1" are understood to encompass both the NY-ESO-1_157-165C peptide and the NY-ESO-1_157-165V peptide, unless otherwise specified.

[0077] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, a complex of genes that encodes major histocompatibility complex (MHC) proteins in humans. These cell surface proteins are involved in regulating the immune system in humans. HLA corresponding to MHC class I (A, B, and C) presents peptides from within cells.

[0078] The term "HLA-A" refers to a group of human leukocyte antigens (HLA) and is encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. The receptor is a heterodimer, consisting of a heavy α chain and a smaller β chain. The α chain is encoded by the variant HLA-A gene, and the β chain (β2-microglobulin) is the invariant β2-microglobulin molecule.

[0079] The term "HLA-A2" refers to one of a group of specific class I major histocompatibility complex (MHC) alleles at the HLA-A locus, where the alpha chain is encoded by the HLA-A*02 gene and the beta chain is encoded by the beta2-microglobulin or B2M locus.

[0080] As used herein, the terms "antigen binding protein," "binding protein," or "binding molecule" include molecules that contain at least one antigen-binding site that specifically binds to a molecule of interest, such as a conformational epitope of the HLA-A2-presented New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide), e.g., an HLA-A2-presented peptide comprising amino acid residues 157-165. The binding protein can be an antibody, including a full-length antibody, or an antigen-binding fragment of an antibody, or a chimeric antigen receptor (CAR), or any other polypeptide, such as a receptor-antibody (Rab) protein.

[0081] Terms such as "HLA-A2:NY-ESO-1 antigen-binding protein" or "HLA-A2:NY-ESO-1 antigen-binding protein" refer to an antigen-binding protein, such as an antibody or antigen-binding portion thereof, that specifically binds to a conformational epitope via presentation of a peptide fragment of NY-ESO-1, e.g., amino acid residues 157-165, by HLA-A2. In certain embodiments, the conformational epitope is generated on the surface of a cell by HLA-A2-presented NY-ESO-1 peptide. As used herein, terms such as "HLA-A2:NY-ESO-1 157-165 peptide complex" refer to a complex between HLA-A2 and the NY-ESO-1 polypeptide, where HLA-A2 presents amino acid residues 157-165 of NY-ESO-1. In this context, NY-ESO-1 can be a full-length NY-ESO-1 polypeptide or can be a truncated form, so long as amino acids 157-165 (relative to SEQ ID NO: 271) are present in the NY-ESO-1 polypeptide and are presented by HLA-A2. For the avoidance of doubt, the term "NY-ESO-1 157-165 peptide" encompasses peptides as short as 157-165 (relative to SEQ ID NO: 271) of NY-ESO-1 or up to the full-length NY-ESO-1 protein (e.g., the sequence of SEQ ID NO: 271).

[0082] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antigen-binding protein, known as the paratope. A single antigen can have two or more epitopes. Thus, different antigen-binding proteins may bind to different regions of an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen bound by an antigen-binding protein. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes, possessing those residues that directly contribute to the affinity of the interaction. Epitopes can also be "conformational," i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that group molecules with chemically active surfaces, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural and / or charge characteristics. In some embodiments, the antigen binding proteins of the present disclosure interact with a conformational epitope of the HLA-A2:NY-ESO-1 peptide complex. In some embodiments, this conformational epitope includes one or more amino acids (e.g., 1, 2, or 3 amino acids) corresponding to M160, W161, and Q164 of SEQ ID NO: 271. To determine the amino acids corresponding to one or more of M160, W161, or Q164, sequence alignments can be performed as described herein. In some embodiments, the antigen binding proteins of the present disclosure specifically bind to an HLA-A2:NY-ESO-1 peptide complex containing amino acids corresponding to amino acids 157-165 of SEQ ID NO: 271 as determined by X-ray crystallography at a resolution of 4.0 Å or better.For the avoidance of doubt, a resolution of 4.0 Å or greater includes a resolution of 3.9 Å or greater, 3.8 Å or greater, 3.7 Å or greater, 3.6 Å or greater, 3.5 Å or greater, 3.4 Å or greater, 3.3 Å or greater, 3.2 Å or greater, 3.1 Å or greater, 3.0 Å or greater, 2.9 Å or greater, 2.8 Å or greater, 2.7 Å or greater, 2.6 Å or greater, 2.5 Å or greater, 2.4 Å or greater, 2.3 Å or greater, 2.2 Å or greater, 2.1 Å or greater, 2.0 Å or greater, 1.9 Å or greater, 1.8 Å or greater, 1.7 Å or greater, 1.6 Å or greater, 1.5 Å or greater, 1.4 Å or greater, 1.3 Å or greater, 1.2 Å or greater, 1.1 Å or greater, 1.0 Å or greater, 0.9 Å or greater, 0.8 Å or greater, 0.7 Å or greater, 0.6 Å or greater, or 0.5 Å or greater.

[0083] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof, such as a full-length antibody or antigen-binding fragment thereof.

[0084] The term "antibody," as used herein, is intended to refer to an immunoglobulin consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (domain C H 1. C H 2, and C H Each light chain consists of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L consists of three CDRs and four FRs, and The CDRs are arranged in the following order from the CDR1 to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0085] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. It has been reported in the scientific literature that antigen-binding proteins, such as antibodies, can omit one or two CDRs for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0086] CDR residues that do not contact antigen can be identified based on previous studies from regions of the Kabat CDRs outside the Chothia CDRs, by molecular modeling and / or experience (e.g., residues H60-H65 in CDRH2 are often not required). When a CDR or its residues are omitted, they are typically substituted with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the substituting amino acids can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0087] The anti-HLA-A2:NY-ESO-1 antigen-binding proteins, e.g., fully human anti-HLA-A2:NY-ESO-1 monoclonal antibodies, or antigen-binding fragments thereof, or CARs disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions within the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antigen-binding proteins, e.g., antibodies, or antigen-binding fragments thereof, or CARs derived from any of the amino acid sequences disclosed herein, in which one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antigen-binding protein is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antigen binding proteins, e.g., antibodies, or antigen-binding fragments thereof, or CARs, containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are mutated back to residues found in the original germline sequence from which the antigen binding protein, e.g., antibody, was derived. In other embodiments, only specific residues are mutated back to the original germline sequence, e.g., only the mutated residues are found within the first eight amino acids of FR1, or the last eight amino acids of FR4, or only the mutated residues are found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, an antigen binding protein, e.g., an antibody, or antigen-binding fragment thereof, or CAR of the present disclosure can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antigen binding proteins, e.g., antibodies and antigen-binding fragments, containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antigen binding proteins, e.g., antibodies, or antigen-binding fragments thereof, or CARs obtained by this general means are encompassed within the present disclosure.

[0088] The present disclosure also includes antigen binding proteins, e.g., fully human anti-HLA-A2:NY-ESO-1 monoclonal antibodies, or antigen-binding fragments thereof, or CARs, that comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes HLA-A2:NY-ESO-1 antigen binding proteins that have HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0089] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies (mAbs) of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The term is not intended to include antibodies isolated from or produced in a human subject.

[0090] The term "recombinant," as used herein, refers to antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, of the present disclosure that are made, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to antigen-binding proteins, e.g., antibodies, that are expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cell (e.g., a CHO cell) expression system, or that are isolated from a recombinant combinatorial human antibody library.

[0091] As used herein, the terms "chimeric antigen receptor" or "CAR" are used interchangeably herein and refer to a recombinant fusion protein comprising an extracellular domain capable of binding to an antigen (e.g., a conformational epitope of the HLA-A2-presented NY-ESO-1 peptide, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1), a transmembrane domain, and at least one intracellular signaling domain.

[0092] As used herein, "immune effector cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC). In one embodiment, the immune effector cells used in the CARs described herein are T lymphocytes, particularly cytotoxic T cells (CTLs, CD8+ T cells) and helper T cells (HTLs, CD4+ T cells). Other populations of T cells are also useful herein, such as naive T cells and memory T cells. As will be understood by those skilled in the art, other cells can also be used as immune effector cells with the CARs described herein. In particular, immune effector cells include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include effector cell progenitors, which can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in this regard, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs), contained within CD34+ cell populations derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells upon administration in a subject, or which can be induced in vitro to differentiate into mature immune effector cells.

[0093] As disclosed herein, the term "off-target peptide" refers to a peptide that differs from a target peptide (e.g., the NY-ESO-1_157-165 peptide) by two, three, four, five, or more amino acids. In certain embodiments, the term includes peptides that differ by three or more fewer amino acids than the target peptide. For example, a 9-mer peptide is considered an "off-target" peptide if two, three, or four amino acids are not identical to the target peptide. In certain embodiments, amino acid identity is expressed in terms of "degree of similarity" (DoS). If six amino acids in a 9-mer peptide are identical, the DoS is 6. In certain embodiments, a peptide with a DoS ≤ 6 is considered an "off-target" peptide. The term "off-target" peptide also refers to a peptide that is similar to a target peptide based on sequence homology, predicted to bind to HLA-A2, and contained in a protein expressed in essential normal tissues.

[0094] Terms such as "specifically binds to" or "specifically binds to" mean that an antigen-binding protein, e.g., an antibody, or antigen-binding fragment thereof, or a CAR, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 M or less (e.g., a smaller K D (A higher number indicates stronger binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antigen binding proteins, e.g., antibodies, that specifically bind to a conformational epitope of an HLA-A2-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1, have been identified by surface plasmon resonance, e.g., BIACORE™.

[0095] The term "high affinity" antigen binding protein, e.g., antibody, refers to an antigen binding protein that binds to a conformational epitope of the HLA-A2-presented NY-ESO-1 peptide, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1, with an affinity of at least 10, as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -8 M, preferably 10 -9 M, more preferably 10 -10 M, even more preferably 10 -11 M, even more preferably 10 -12 K of M D The term "antigen binding protein" refers to these antigen-binding proteins, e.g., mAbs, that have binding affinities expressed as

[0096] By the term "slow off rate", "Koff" or "kd" is meant a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 Less than 1 × 10 -4 s -1 It means an antigen-binding protein that dissociates from HLA-A2:NY-ESO-1 with the following rate constant:

[0097] The terms "antigen-binding portion" of an antigen-binding protein (e.g., an antibody), "antigen-binding fragment" of an antigen-binding protein (e.g., an antibody), and the like, as used herein, include naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. The term "antigen-binding fragment" of an antibody, or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to a conformational epitope of the HLA-A2-presented NY-ESO-1 peptide, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1 bound to HLA-A2.

[0098] In certain embodiments, an antigen binding protein of the present disclosure, e.g., an antibody or antibody fragment, or a CAR, may be conjugated to a moiety such as a ligand, a detectable moiety, or a therapeutic moiety (an "immunoconjugate") such as a cytotoxin, a second anti-HLA-A2:NY-ESO-1 antigen binding protein, an antibody against a tumor-specific antigen, an anti-cancer agent, or any other therapeutic moiety useful for treating a disease or condition, including a NY-ESO-1-associated disease or disorder such as a NY-ESO-1-positive cancer.

[0099] An "isolated antigen binding protein," e.g., an isolated antibody, as used herein, refers to an antigen binding protein, e.g., an antibody, that is substantially free of other antigen binding proteins, e.g., antibodies (Abs), having different antigen specificities (e.g., an isolated antibody that specifically binds to HLA-A2:NY-ESO-1, or a fragment thereof, is substantially free of antigen binding proteins, e.g., antibodies, that specifically bind to antigens other than the conformational epitope of the HLA-A2-presented NY-ESO-1 peptide).

[0100] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions through the detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0101] "K D The term " ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antigen-binding protein-antigen interaction.

[0102] As used herein, the term "cross-compete" refers to an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, binding to an antigen and inhibiting or blocking the binding of another antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof. The term also includes competition between two antigen-binding proteins, e.g., antibodies, in both contexts, i.e., a first antigen-binding protein, e.g., an antibody, binding to and blocking the binding of a second antigen-binding protein, e.g., an antibody, or vice versa. In certain embodiments, a first antigen-binding protein, e.g., an antibody, and a second antigen-binding protein, e.g., an antibody, may bind to the same epitope. Alternatively, the first and second antigen-binding proteins, e.g., antibodies, may bind to different but overlapping epitopes, such that the binding of one inhibits or blocks the binding of the second, e.g., through steric hindrance. Cross-competition between antigen-binding proteins, e.g., antibodies, can be measured by methods known in the art, for example, by real-time label-free biolayer interferometry assays. Cross-competition between two antigen-binding proteins, e.g., antibodies, can be expressed as the binding of the second antigen-binding protein, e.g., antibody, below the background signal due to self-self binding (where the first and second antigen-binding proteins, e.g., antibodies, are the same antigen-binding protein, e.g., antibody). Cross-competition between two antigen-binding proteins, e.g., antibodies, can be expressed, for example, as the % binding of the second antigen-binding protein, e.g., antibody, below baseline self-self background binding (where the first and second antigen-binding proteins, e.g., antibodies, are the same antigen-binding protein, e.g., antibody).

[0103] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by well-known algorithms of sequence identity discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0104] Sequence identity can be calculated using algorithms such as the Needleman-Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment, or the Smith-Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195-197) for local alignment. Another preferred algorithm is reported by Dufresne et al. (vol. 20, pp. 1269-71) in Nature Biotechnology in 2002, and is used in the software GenePAST (GQ Life Sciences, Inc. Boston, MA).

[0105] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, when optimally aligned, such as by programs like GAP or BESTFIT using default gap weighting. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0106] Sequence similarity in polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein, using default parameters. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm, using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, in the BLOSUM62 matrix. Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0107] The phrase "therapeutically effective amount" means that amount administered to produce the desired effect. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0108] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a NY-ESO-1-associated disease or disorder, such as a NY-ESO-1-associated cancer (e.g., a NY-ESO-1-positive cancer). The term includes human subjects having or at risk of having a NY-ESO-1-associated disease or disorder, such as a NY-ESO-1-associated cancer or metastatic NY-ESO-1-associated cancer.

[0109] As used herein, "anticancer agent" means any agent that is useful for treating or ameliorating or inhibiting cancer, and includes, but is not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents. As used herein, "cytotoxin or cytotoxic agent," which also refers to chemotherapeutic agents, means any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coitisin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0110] As used herein, the term "antiviral agent" refers to any drug or therapy used to treat, prevent, or ameliorate a viral infection in a host subject. The term "antiviral agent" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids.

[0111] An immunogen comprising any one of the following can be used to generate antigen binding proteins, e.g., antibodies, against a conformational epitope of an HLA-A2-presented NY-ESO-1 peptide, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1 linked to HLA-A2. In certain embodiments, antigen binding proteins, e.g., antibodies, of the present disclosure can be obtained from mice immunized with a full-length native NY-ESO-1 protein (see GenBank Accession No. NP_001318.1) (SEQ ID NO: 271) or a recombinant NY-ESO-1 peptide (e.g., a peptide comprising amino acid residues 157-165 (SLLMWITQC, SEQ ID NO: 269) of GenBank Accession No. NP_001318.1 (SEQ ID NO: 271), or a sequence thereof with the C165V substitution (SLMMWITQV, SEQ ID NO: 270) in GenBank accession), bound to an HLA protein such as HLA-A2.

[0112] Alternatively, NY-ESO-1 protein or a fragment thereof can be produced using standard biochemical techniques, modified in the context of HLA-A2, and used as an immunogen.

[0113] In some embodiments, the immunogen may be a recombinant NY-ESO-1 polypeptide (which may be recombinant NY-ESO-1 presented by HLA) expressed in E. coli or any other eukaryotic or mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0114] In certain embodiments, antigen-binding proteins that specifically bind to conformational epitopes of HLA-A2-presented NY-ESO-1 peptides can be prepared using the above polypeptides, or fragments thereof. In some embodiments, the HLA-A2-presented NY-ESO-1 peptides can extend beyond the designated regions by about 5 to about 20 amino acid residues from either the N-terminus or C-terminus of the regions described herein, or both. In certain embodiments, any combination of the above regions or fragments thereof can be used in preparing HLA-A2:NY-ESO-1-specific antigen-binding proteins, e.g., antibodies.

[0115] Peptides can be modified to include the addition or substitution of specific residues for tagging or for conjugation with carrier molecules such as KLH. For example, cysteine ​​can be added to either the N- or C-terminus of the peptide, or a linker sequence can be added to prepare the peptide for conjugation to, for example, KLH for immunization.

[0116] Non-limiting exemplary in vitro assays for measuring binding activity are described in the Examples herein. In Example 3, the binding affinity and kinetic constants of human anti-HLA-A2:NY-ESO-1-specific antigen-binding proteins, e.g., antibodies, were determined by surface plasmon resonance, with measurements performed on a Biacore 4000 or T200 instrument. Example 4 describes antibody binding to cells overexpressing a fragment of NY-ESO-1.

[0117] Antigen-binding proteins, e.g., antibodies, specific for HLA-A2:NY-ESO-1 may contain no additional labels or moieties, or they may contain N- or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin is positioned so that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label can be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antigen-binding proteins can be used in diagnostic assays, including imaging assays.

[0118] antigen-binding proteins The present disclosure provides antigen-binding proteins, including antibodies, or antigen-binding fragments thereof, and CARs (e.g., nucleic acid molecules encoding the CARs of the present disclosure) (described below). Unless otherwise indicated, the term "antibody" as used herein should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "complete antibody molecules"), as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a conformational epitope of an HLA-A2-presented NY-ESO-1 peptide. Antigen-binding proteins, such as antibody fragments, may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding proteins, such as antigen-binding fragments of antibodies, can be derived, for example, from intact antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion techniques or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0119] Non-limiting examples of antigen-binding fragments of antibodies include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0120] An antigen-binding fragment of an antigen-binding protein (e.g., an antibody) typically comprises at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in-frame with one or more framework sequences. L V bound to the domain H In antibody-binding proteins with V domains, H Domains and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H Domain or V L It may include a domain.

[0121] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary conformations of variable and constant domains that may be found in an antigen-binding fragment of an antigen-binding protein of the present disclosure include: (i) V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Domain or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the above-listed variable domain and constant domain configurations in non-covalent association with the domains (e.g., by disulfide bonds).

[0122] Similar to intact antibody molecules, antigen-binding proteins, e.g., antigen-binding fragments of antibodies, can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0123] Preparation of antigen-binding proteins Methods for generating antigen-binding proteins, such as human antibodies, in transgenic mice are known in the art. Any such known method can be used in the context of the present disclosure to generate human antibodies that specifically bind to conformational epitopes of HLA-A2-presented New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide).

[0124] Using VELOCIMMUNE® technology (e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating antigen-binding proteins, e.g., monoclonal antibodies, high-affinity antigen-binding proteins, e.g., chimeric antibodies, directed against conformational epitopes of the HLA-A2-presented NY-ESO-1 peptide, are first isolated with human variable regions and mouse constant regions. VELOCIMMUNE® technology involves generating transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, thereby enabling the mice to produce antigen-binding proteins, e.g., antibodies, containing human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0125] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) expressing an antigen-binding protein, e.g., an antibody, are collected from the mice. The lymphocytes can be fused with a myeloma cell line to prepare an immortalized hybridoma cell line, which is then screened and selected to identify a hybridoma cell line that produces an antibody specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antigen-binding proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding an antigen-specific antigen-binding protein, e.g., a chimeric antibody, or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0126] First, a high-affinity antigen-binding protein, such as a chimeric antibody, is isolated having a human variable region and a mouse constant region. As described in the experimental section below, the antigen-binding protein is characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate an antigen-binding protein of the present disclosure, such as a fully human antibody, for example, a wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the specific use, the characteristics of high-affinity antigen binding and target specificity reside in the variable region.

[0127] biological equivalent Anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure include proteins having amino acid sequences that differ from those of the described antigen binding proteins, e.g., antibodies, but retain the ability to bind to a conformational epitope of an HLA-A2-presented NY-ESO-1 peptide. Such variant antigen binding proteins contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit substantially equivalent biological activity to the described antigen binding proteins. Similarly, antibody binding protein-encoding DNA sequences of the present disclosure include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antigen binding proteins that are essentially biologically equivalent to the antigen binding proteins of the present disclosure.

[0128] Two antigen-binding proteins or antibodies are considered bioequivalents if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antigen-binding proteins or antibodies are considered equivalents or pharmaceutical substitutes if they are equivalent in their extent of absorption but not in their rate of absorption, and can nevertheless be considered bioequivalent because such differences in absorption rate are intentional, reflected in the labeling, not essential to achieving effective body drug concentrations, e.g., with long-term use, and not considered medically significant for the particular pharmaceutical product tested.

[0129] In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if there are no clinically meaningful differences in their safety, purity, or efficacy.

[0130] In one embodiment, two antigen binding proteins (e.g., antibodies) are bioequivalent if a patient can be switched one or more times between a reference product and a biological product without a predicted increase in the risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to therapy continued without such switching.

[0131] In one embodiment, two antigen binding proteins (or antibodies) are biologically equivalent if they both operate by a common mechanism(s) of action for the condition(s) of use, to the extent that such mechanism(s) are known.

[0132] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antigen binding protein or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the antigen binding protein (or its target) are measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0133] Biologically equivalent variants of the antigen-binding proteins (or antibodies) of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antigen-binding proteins can include variants of antigen-binding proteins that contain amino acid changes that alter the glycosylation characteristics of the antigen-binding protein, for example, mutations that eliminate or remove glycosylation.

[0134] Anti-HLA-A2:NY-ESO-1 antigen-binding protein containing Fc variants Certain embodiments of the present disclosure provide anti-HLA-A2:NY-ESO-1 antigen binding proteins, e.g., antibodies, comprising an Fc domain comprising one or more mutations that enhance or decrease binding of the antigen binding protein to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides H 2 or C HThe present invention includes an anti-HLA-A2:NY-ESO-1 antigen-binding protein containing mutations in region 3 that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., within the endosome, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antigen-binding protein when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0135] For example, the present disclosure provides 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), 428L and 434S (e.g., M428L and N434S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H). , 376V and 434H (e.g., D376V and N434H), 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 376V and 434H (e.g., D376V and N434H), 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising an Fc domain comprising an S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the foregoing Fc domain mutations, and other mutations in the antigen binding protein variable domains disclosed herein, are contemplated within the scope of the present disclosure.

[0136] The present disclosure also provides chimeric heavy chain constant (C H ) region, and an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising the chimeric C H The region is composed of C H For example, the antigen binding proteins of the present disclosure may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule combined with some or all of the three domains H Chimeric C containing part or all of the 2 domains H In certain embodiments, the antigen binding proteins of the present disclosure may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. Chimeric C as described herein may be used in combination with a chimeric C as described herein. H Antigen binding proteins comprising the region may, in certain embodiments, exhibit improved Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antigen binding protein (see, e.g., U.S. Patent Publication No. 2014 / 0243504, which is incorporated herein by reference in its entirety).

[0137] Biological characteristics of antigen-binding proteins In general, the antigen binding proteins of the present disclosure function by binding to a conformational epitope of the HLA-A2-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide.

[0138] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind with high specificity to the NY-ESO-1 peptide in the context of HLA-A2. The anti-HLA-A2:NY-ESO-1 antigen binding proteins do not bind to the NY-ESO-1 peptide in the absence of HLA-A2. Furthermore, the anti-HLA-A2:NY-ESO-1 antigen binding proteins do not bind to off-target peptides in the context of HLA-A2.

[0139] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind with high affinity to the monomeric HLA-A2:NY-ESO-1(157-165) peptide (the NY-ESO-1 157-165 peptide can include either C165 or V165; i.e., the antigen binding proteins of the present disclosure can be specific for either form or non-specific with respect to either form). For example, the ... peptide (157-165) with a K of less than about 1 nM as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein. D In certain embodiments, the antigen binding protein binds to a monomeric HLA-A2:NY-ESO-1_157-165 peptide (optionally with a C165V substitution) at room temperature (e.g., 25°C or 37°C). In certain embodiments, the antigen binding protein has a K of less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.04 nM to the monomeric HLA-A2:NY-ESO-1_157-165 peptide when measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D Combine with.

[0140] The present disclosure also provides antigen binding proteins that have an EC of less than about 10 nM for cells expressing the HLA-A2:NY-ESO-1:157-165 peptide complex (the NY-ESO-1 157-165 peptide may include either C165 or V165, i.e., the antigen binding proteins of the present disclosure may be specific for either form or may be non-specific with respect to any form), as determined by a flow cytometry assay as defined in Examples 4 and 5 herein, or by a substantially similar assay. 50and does not bind to cells expressing the predicted off-target peptide. In certain embodiments, the antigen binding protein has an EC50 of less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM to cells expressing the HLA-A2:NY-ESO-1_157-165 peptide, as determined by a flow cytometry assay as defined in Examples 4 and 5 herein, or a substantially similar assay, e.g., using the assay format in Examples 4 and 5 herein, or a substantially similar assay. 50 and does not bind to cells expressing predicted off-target peptides.

[0141] In certain embodiments, the antigen binding proteins of the present disclosure are useful for inhibiting tumor growth or delaying the progression of cancer when administered prophylactically to a subject in need thereof, and may increase the survival of the subject. For example, administration of an antigen binding protein of the present disclosure may result in the shrinkage of a primary tumor and may prevent the development of metastasis or secondary tumors. In certain embodiments, the antigen binding proteins of the present disclosure are useful for inhibiting tumor growth and may increase the survival of a subject when administered therapeutically to a subject in need thereof. For example, administration of a therapeutically effective amount of an antigen binding protein of the present disclosure to a subject may result in the shrinkage and disappearance of an established tumor in the subject.

[0142] In one embodiment, the disclosure provides an isolated recombinant antigen binding protein that binds to a conformational epitope of an HLA-A2-presented NY-ESO-1 peptide, wherein the antigen binding protein comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 180, 196, 211, and 230, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 110, 130, 150, 170, 186, 203, 219, and 238, or a sequence substantially similar thereto having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity thereto; (iii) an LCVR having a substantially similar sequence thereof having at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and (iv) an LCVR selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, and 236. HCDR3 domains having amino acid sequences or substantially similar sequences thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, as well as those of SEQ ID NOs: 16, 36, 56, 76, 96,116, 136, 156, 190, 205, 224, and 244, or a substantial portion thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. and (iv) an LCDR3 domain having a sequence similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213, and 232, or at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least HCDR1 domains having a substantially similar sequence thereof with at least 97%, at least 98%, or at least 99% sequence identity, an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 215, and 234, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least an HCDR2 domain having a substantially similar sequence having 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, and 240; or an HCDR3 domain having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,an LCDR1 domain having a substantially similar sequence thereof with at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, and 242, or at least 80%, at least 81%, at least 82%, at least 83% , an LCDR2 domain having a substantially similar sequence thereof having at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and (v) a monomeric HLA-A2:NY-ESO-1 as measured at 25°C in a surface plasmon resonance assay. (vi) binding to a monomeric HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex with a binding dissociation equilibrium constant (KD) of less than about 1 nM as measured at 25°C in a surface plasmon resonance assay; and (vii) an EC2 of less than about 10 nM on HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex-expressing cells. 50 and (viii) not binding to HLA-A2-presented off-target peptides that differ from SEQ ID NO: 271 by 2, 3, 4, 5 or more amino acids.

[0143] The antigen binding proteins of the present disclosure may have one or more of the above biological characteristics, or any combination thereof. Other biological characteristics of the antigen binding proteins of the present disclosure will be apparent to those of skill in the art from a review of this disclosure, including the working examples herein.

[0144] Epitope mapping and related techniques The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that interact with one or more amino acids found within one or more domains of an HLA-A2-presented NY-ESO-1 peptide. The epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within either or both of the domains of the NY-ESO-1 molecule (e.g., a conformational epitope).

[0145] A variety of techniques known to those skilled in the art can be used to determine whether an antigen binding protein "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, those described in Antibodies, Harlow and Lane (Cold These include routine cross-blocking assays such as those described in Spring Harbor Press, Cold Spring Harbor, NY. Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallography, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (see Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antigen-binding protein interacts is hydrogen / deuterium exchange, detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the antigen-binding protein to the deuterium-labeled protein. The protein / antigen-binding protein complex is then transferred to water, and exchangeable protons in amino acids protected by the antigen-binding protein complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, the amino acid that forms part of the protein / antigen-binding protein interface can retain deuterium, and therefore exhibits a relatively high mass compared with the amino acid that is not included in the interface.After the antigen-binding protein is dissociated, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids that the antigen-binding protein interacts with.For example, see Ehring (1999) Analytical Biochemistry 267:252-259, Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0146] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed both from contiguous amino acids juxtaposed by tertiary folding of a protein or from noncontiguous amino acids. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, or 8-10 amino acids in a unique spatial configuration.

[0147] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying multiple monoclonal antigen-binding proteins, e.g., antibodies (mAbs), directed against the same antigen according to the similarity of each antibody's binding profile to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is either distinct from or partially overlaps with the epitopes represented by other categories. This technique allows for rapid filtering of genetically identical antigen-binding proteins, thereby allowing characterization to focus on genetically distinct antigen-binding proteins. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce antigen-binding proteins with desired characteristics. MAP can be used to classify the antigen-binding proteins of the present disclosure into groups of antigen-binding proteins that bind to different epitopes.

[0148] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind to the same epitope or portion of an epitope as any of the specific exemplary antigen binding proteins set forth herein in Table 1, or antigen binding proteins that have the CDR sequences of any of the exemplary antigen binding proteins set forth in Table 1. Similarly, the present disclosure also includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that compete with any of the specific exemplary antigen binding proteins set forth in Table 1, or with antigen binding proteins that have the CDR sequences of any of the exemplary antigen binding proteins set forth in Table 1, for binding to HLA-A2:NY-ESO-1 or a fragment thereof.

[0149] Whether an antigen-binding protein binds to the same epitope as a reference anti-HLA-A2:NY-ESO-1 antigen-binding protein or competes for binding with the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein can be readily determined using routine methods known in the art. For example, to determine whether a test antigen-binding protein binds to the same epitope as a reference anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure, the reference antigen-binding protein is allowed to bind to an HLA-A2:NY-ESO-1 protein or peptide under saturating conditions. The ability of the test antigen-binding protein to bind to the HLA-A2:NY-ESO-1 molecule is then evaluated. If the test antigen-binding protein is able to bind to HLA-A2:NY-ESO-1 after saturation binding of the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein, it can be concluded that the test antigen-binding protein binds to a different epitope than the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein. On the other hand, if the test antigen binding protein is unable to bind to HLA-A2:NY-ESO-1 after saturation binding with the reference anti-HLA-A2:NY-ESO-1 antigen binding protein, then the test antigen binding protein may bind to the same epitope as the epitope bound by the reference anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure.

[0150] To determine whether an antigen-binding protein competes for binding with a reference anti-HLA-A2:NY-ESO-1 antigen-binding protein, the above binding methodology is performed in two applications. In the first application, the reference antigen-binding protein is allowed to bind to the HLA-A2:NY-ESO-1 protein under saturating conditions, followed by assessing the binding of the test antigen-binding protein to the HLA-A2:NY-ESO-1 molecule. In the second application, the test antigen-binding protein is allowed to bind to the HLA-A2:NY-ESO-1 molecule under saturating conditions, followed by assessing the binding of the reference antigen-binding protein to the HLA-A2:NY-ESO-1 molecule. In both applications, if only the first (saturating) antigen-binding molecule can bind to the HLA-A2:NY-ESO-1 molecule, it is concluded that the test antigen-binding protein and the reference antigen-binding protein compete for binding to HLA-A2:NY-ESO-1. As will be appreciated by one of skill in the art, an antigen binding protein that competes for binding with a reference antigen binding protein may not necessarily bind to the same epitope as the reference antigen binding protein, but may sterically block binding of the reference antigen binding protein by binding to an overlapping or adjacent epitope.

[0151] Two antigen-binding proteins bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antigen-binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein also reduce or eliminate binding of the other. Two antigen-binding proteins have overlapping epitopes if amino acid mutations that reduce or eliminate binding of one antigen-binding protein also reduce or eliminate binding of the other.

[0152] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antigen-binding protein is indeed due to binding to the same epitope as the reference antigen-binding protein, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antigen-binding protein binding assay available in the art.

[0153] Immunoconjugates The present disclosure encompasses anti-HLA-A2:NY-ESO-1 antigen binding proteins ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxin or chemotherapeutic agent, for treating cancer. As used herein, the term "immunoconjugate" refers to an antigen binding protein that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety such as a detectable moiety, enzyme, toxin, peptide or protein, or therapeutic agent. The antigen binding protein may be conjugated to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide, or therapeutic agent at any position along the molecule, so long as the antigen binding protein is capable of binding to its target. Examples of immunoconjugates include antigen binding protein-drug conjugates and antigen binding protein-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody against NY-ESO-1 or HLA-A2:NY-ESO-1. In certain embodiments, the antigen binding protein may be conjugated to an agent specific for tumor cells. The type of therapeutic moiety that can be conjugated to the anti-HLA-A2:NY-ESO-1 antigen binding protein will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of agents suitable for forming immunoconjugates are known in the art; see, e.g., PCT Publication No. 05 / 103081.

[0154] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) redirect T cell specificity to antibody-recognized antigens expressed on the surface of cancer cells, while T cell receptors (TCRs) expand their targeting range to include intracellular tumor antigens. CARs redirecting T cells specific for the B cell differentiation antigen CD19 have shown dramatic efficacy in treating B cell malignancies, while TCR-redirected T cells have shown benefit in patients with solid tumors. Stauss et al. describe strategies for modifying therapeutic CARs and TCRs to enhance antigen-specific effector function and limit the toxicity of engineered T cells for use in cancer treatment (Current Opinion in Pharmacology 2015, 24:113-118).

[0155] One aspect of the present disclosure includes a chimeric antigen receptor (CAR) specific for a NY-ESO-1 peptide presented on the surface of tumor cells by HLA-A2, such as a peptide comprising amino acid residues 157-165 of NY-ESO-1. In one embodiment of the present disclosure, the CAR described herein comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3 zeta or FcR gamma), and / or one or more costimulatory signaling domains derived from costimulatory molecules such as, but not limited to, CD28, CD137, CD134, or CD278. In one embodiment, the CAR comprises a hinge or spacer region, such as a CD8 alpha hinge or a CD28 hinge, between the extracellular binding domain and the transmembrane domain. In another embodiment of the present disclosure, the CAR described herein comprises an extracellular target-specific binding domain and a T cell receptor constant domain (a "T body construct"). In some embodiments, the hinge / transmembrane domain comprises the amino acid sequence of SEQ ID NO: 296. In some embodiments, the hinge region comprises the CD28 sequence of SEQ ID NO: 305. In some embodiments, the transmembrane domain comprises the CD28 sequence of SEQ ID NO: 304. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 297. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 299. In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 298.

[0156] It is understood that for use in any of the CARs described herein, the extracellular target-specific binding domain can comprise a Fab, Fab', (Fab')2, Fv, or single-chain Fv (scFv) of an antigen binding protein of the disclosure.

[0157] As used herein, the binding domain or extracellular domain of a CAR provides the CAR with the ability to bind to a target antigen of interest. A binding domain can be any protein, polypeptide, oligopeptide, or peptide capable of specifically recognizing and binding to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, as further described herein, a binding domain can be an antibody light chain and heavy chain variable region, or the light chain and heavy chain variable region can be linked together in a single chain in any configuration (e.g., VL-VH or VH-VL). Various assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis), and are described herein. A target can be any antigen of clinical interest for which it is desirable to elicit an effector immune response that results in tumor killing. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is a conformational epitope of the NY-ESO-1 peptide presented by HLA-A2 on the surface of tumor cells, such as a peptide comprising amino acid residues 157 to 165 of NY-ESO-1.

[0158] Exemplary binding domains include antigen-binding proteins, including, for example, antigen-binding fragments of antibodies such as scFvs, scTCRs, extracellular domains of receptors, ligands of cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins. In certain embodiments, the antigen-binding domains included in the CARs of the present disclosure can be variable regions (Fvs), CDRs, Fabs, scFvs, VHs, VLs, domain antibody variants (dAbs), camelid antibodies (VHHs), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen-specific binding domains derived from other protein scaffolds.

[0159] In one embodiment, the binding domain of the CAR is an anti-HLA-A2:NY-ESO-1 single-chain antibody (scFv), which can be a mouse, human, or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas specific to the desired target. Techniques that can be used to clone variable heavy chains (VH) and variable light chains (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but can also be a non-antibody-derived binding domain. An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, both of which are involved in binding to an antigen.

[0160] In certain embodiments, the CAR of the present disclosure may include linkers between various domains, added for proper spacing and conformation of the molecule. For example, in some embodiments, a linker may be present between the VH and VL binding domains, which may be 1 to 10 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1 to 20, or 20 amino acids in length. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges inclusive of the numbers described herein are also included herein, such as linkers 10 to 30 amino acids in length.

[0161] In certain embodiments, a linker suitable for use in the CARs described herein is a flexible linker. Suitable linkers can be easily selected and can be any of different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0162] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (G)nS (where n is an integer of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In addition, linkers can include multiple units of the above sequences, e.g., ((G)nS)m), where n is an integer of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), and m is an integer of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, n is 4 and the linker includes the sequence GGGGS (SEQ ID NO: 295). In some embodiments, n is 4, m is 3, and the linker comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO: 303). Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has access to significantly more pi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that CAR designs can include linkers that are fully or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that confer a less flexible structure to provide the desired CAR structure.

[0163] The binding domain of a CAR may be followed by a "spacer," or "hinge," which refers to a region that separates the antigen-binding domain from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region in a CAR is generally located between the transmembrane (TM) domain and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or a mutated wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, and CD7, and may be wild-type hinge regions derived from these molecules or may be mutated. In one embodiment, the hinge region comprises a CD8 alpha hinge.

[0164] The "transmembrane" region or domain is the part of the CAR that anchors the extracellular binding moiety to the plasma membrane of immune effector cells and promotes the binding of the binding domain to the target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain, but other transmembrane domains that can be used include those obtained from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain is the transmembrane domain of CD137. In certain embodiments, the transmembrane domain is a synthetic domain, in which case it contains primarily hydrophobic residues such as leucine and valine.

[0165] The term "intracellular signaling domain" refers to the portion of a chimeric antigen receptor protein that is involved in transmitting the message of effective CAR binding to a target antigen to the interior of immune effector cells, thereby inducing effector cell functions, such as cytotoxic activity, including activation, cytokine production, proliferation, and release of cytotoxic factors in CAR-bound target cells, or other cellular responses triggered by antigen binding to the extracellular CAR domain. The term "effector function" refers to the specialized function of a cell. T cell effector functions can be, for example, cytolytic activity or aiding or activating activities, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transmits the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal. The intracellular signaling domain, also known as the "signal transduction domain," is typically derived from a portion of the human CD3 or FcRy chain.

[0166] It is known that signals generated solely through the T cell receptor are insufficient for full T cell activation; secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation via the T cell receptor (primary cytoplasmic signal sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signal sequences). Primary cytoplasmic signaling sequences control primary activation of the T cell receptor complex in either an inhibitory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0167] Examples of primary cytoplasmic signaling sequences containing ITAMs of particular use in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the intracellular signaling domain of the anti-HLA-A2:NY-ESO-1 CAR described herein is derived from CD3 zeta or FcR gamma.

[0168] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal necessary for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD3 zeta, CD28, and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells expressing a CAR receptor. The intracellular signaling domain and the costimulatory signaling domain can be tandemly linked in any order to the carboxyl terminus of the transmembrane domain.

[0169] Although scFv-based CARs engineered to contain signaling domains derived from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of concomitant costimulatory signals. Other CARs that contain signaling domains derived from CD3 zeta or FcR gamma, along with a binding domain, hinge, transmembrane, and one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278), can more efficiently direct anti-tumor activity in vitro, animal models, and cancer patients, as well as increased cytokine secretion, lytic activity, survival, and proliferation of CAR-expressing T cells (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0170] In one embodiment, an HLA-A2:NY-ESO-1 CAR of the present disclosure comprises (a) an anti-HLA-A2:NY-ESO-1 scFv (e.g., an scFv having a binding region (e.g., a CDR or variable domain) from any one or more of the HLA-A2:NY-ESO-1 antibodies listed in Table 1) as a binding domain, (b) a hinge region derived from human CD8 alpha, (c) a human CD8 alpha transmembrane domain, and (d) a human T cell receptor CD3 zeta chain (CD3) intracellular signaling domain, and optionally one or more costimulatory signaling domains derived from CD28, CD137, CD134, and CD278. In one embodiment, the different protein domains are arranged from the amino terminus to the carboxyl terminus in the following order: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are linked in tandem to the transmembrane carboxy terminus in any order to form a single-chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, a nucleic acid molecule comprising (5' to 3') the coding sequences for a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, a CD137 costimulatory domain, and a CD3 zeta intracellular signaling domain. In another embodiment, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, a nucleic acid molecule comprising (5' to 3') the coding sequences for a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8 alpha-hinge, a human CD8 alpha transmembrane domain, a CD137 costimulatory domain, and a CD3 zeta costimulatory domain.In certain embodiments, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, a nucleic acid molecule comprising (5' to 3') the coding sequences for a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8 alpha hinge, a human CD8 alpha transmembrane domain, a CD137 costimulatory domain, and a CD3 zeta costimulatory domain, wherein the anti-HLA-A2:NY-ESO-1 scFv is selected from the group consisting of SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 180, 196, 211, 230, and 250. H or the anti-HLA-A2:NY-ESO-1 scFv comprises a V selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 110, 130, 150, 170, 186, 203, 219, 238, and 258. L or the anti-HLA-A2:NY-ESO-1 scFv comprises a V selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, 230 / 238, and 250 / 258. H / V L In some embodiments, the present disclosure includes a nucleic acid molecule encoding an HLA-A2:NY-ESO-1 CAR selected from the group consisting of the sequences in Table 2.

[0171] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be covalently inserted to result in the expression of the protein and / or cloning of the polynucleotide. Such a vector may be referred to as an "expression vector." An isolated polynucleotide can be inserted into a vector using any suitable method known in the art, for example, but not limited to, a vector can be digested with an appropriate restriction enzyme and then ligated to an isolated polynucleotide with matching restriction ends. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences that encode at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription, and optionally translation, of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions and are discussed below. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification.

[0172] For efficient gene transcription and translation in their respective host cells, expression vectors may contain necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences such as the CMV, PGK, and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and 3' UTR AAUAAA transcription termination sequences. Other suitable promoters include the constitutive promoters of the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters may also be used, including, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of vectors expressing chimeric antigen receptors. This provides a molecular switch that can turn on or off expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0173] The expression vector may have additional sequences such as 6x histidine (SEQ ID NO: 292), c-Myc, and FLAG tags that are incorporated into the expressed CAR. Thus, the expression vector may be engineered to contain 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that can promote or enhance the efficient transcription of the nucleic acid of interest carried in the expression vector. The expression vector may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a specific cell type, cell location, or tissue type. The expression vector may contain a selectable marker for maintaining the vector in a host or recipient cell.

[0174] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements.Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of animal virus categories that are useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Examples of expression vectors are the Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. The coding sequence of the CAR disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0175] In certain embodiments, the nucleic acid encoding the CAR of the present disclosure is provided in a viral vector. The viral vector can be derived from a retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain the coding sequence of various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particle can be used to transfer DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0176] In certain embodiments, the viral vector that comprises the coding sequence of CAR described herein is a retroviral vector or a lentiviral vector.The term " retroviral vector " refers to the vector that comprises the structural and functional gene elements that are mainly derived from retrovirus.The term " lentiviral vector " refers to the vector that comprises the structural and functional gene elements that are mainly derived from lentivirus outside the LTR.

[0177] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retrovirus" of the present disclosure also includes lentiviruses of the Retroviridae family, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, and human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0178] As used herein, lentiviral vector refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included in this group include HIV (human immunodeficiency virus, including HIV types 1 and 2), visna-maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using the methods described by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998;72:8463-8471 and Zufferey et al., J. Virol., 1998;72:9873-9880).

[0179] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present disclosure can be generated using standard cloning techniques by combining desired DNA sequences with the instructions and adaptations described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145, Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et. al. (1993) J. Immunol 150:4104-4115, U.S. Patent No. 4,868,116, U.S. Patent No. 4,980,286, PCT Application No. 89 / 07136, PCT Application No. 89 / 02468, PCT Application No. 89 / 05345, and PCT Application No. 92 / 07573).

[0180] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in generating vectors include genomic RNA and cDNA available from commercial sources including, for example, Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0181] For the expression of HLA-A2:NY-ESO-1 CAR, a vector can be introduced into a host cell to allow expression of the polypeptide within the host cell. An expression vector can contain various elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable marker, and a signal sequence. These elements can be appropriately selected by those skilled in the art, as described above. For example, a promoter sequence can be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, a T7 promoter, a T3 promoter, an SP6 promoter, a beta-actin promoter, an EF1a promoter, a CMV promoter, and an SV40 promoter. An enhancer sequence can be selected to enhance transcription of a polynucleotide. A selectable marker can allow host cells into which a vector has been inserted to be selected from those without the vector; for example, a selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside the host cell.

[0182] In cloning a polynucleotide, a vector is introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally contain sequence components, including, but not limited to, a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, a replication origin can be selected to promote the autonomous replication of the vector in the host cell.

[0183] In certain embodiments, the present disclosure provides an isolated host cell comprising a vector provided herein. Host cells comprising the vector may be useful for expressing or cloning a polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, gram-negative or gram-positive organisms, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0184] The CAR of the present disclosure is introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which exogenous nucleic acid sequences are introduced into host cells. Nucleic acid can be integrated into host cell DNA or maintained extrachromosomally. Nucleic acid can be transiently maintained or stably introduced. Transfection can be achieved by various means known in the art, including, but not limited to, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun. Transduction refers to the delivery of genes using viral or retroviral vectors by viral infection rather than transfection. In certain embodiments, retroviral vectors are transduced by packaging the vector into virions before contacting cells. For example, anti-HLA-A2:NY-ESO-1 carried by a retroviral vector Nucleic acids encoding CARs can be transduced into cells through infection and proviral integration.

[0185] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "reintroduced cell" are used interchangeably.

[0186] In particular, the CARs of the present disclosure are introduced into and expressed in immune effector cells, thereby redirecting their specificity to a target antigen of interest, e.g., an HLA-A2-presented NY-ESO-1 peptide, e.g., amino acid residues 157-165.

[0187] The present disclosure provides a method for producing immune effector cells that express a CAR described herein. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from a subject, such as a subject with an NY-ESO-1-associated disease or disorder, so that the immune effector cells express one or more CARs described herein. In a specific embodiment, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to the same individual (for autologous therapy) or to another individual (for allogeneic therapy). In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR described herein).

[0188] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, a cell source can be obtained from a subject. In particular, the immune effector cells for use in the CAR described herein include T cells. Such recombinant T cells are referred to herein as "T cells."

[0189] In one embodiment of the present disclosure, the T body comprises a CAR of the present disclosure comprising an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3 zeta or FcR gamma), and / or a costimulatory signaling domain derived from one or more costimulatory molecules (such as, but not limited to, CD28, CD137, CD134, or CD278). In another embodiment of the present disclosure, the T body comprises a CAR of the present disclosure comprising an extracellular target-specific binding domain, a transmembrane domain, a hinge or spacer region between the extracellular binding domain and the transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3 zeta or FcR gamma), and / or one or more costimulatory signaling domains derived from costimulatory molecules. In yet another embodiment of the present disclosure, the T body comprises a T body construct CAR comprising an extracellular target-specific binding domain and a T cell receptor constant domain. An extracellular target-specific binding domain suitable for use in a T body comprising any of the CARs described herein can comprise a Fab, Fab', (Fab')2, Fv, or single-chain Fv (scFv) of an antigen binding protein of the disclosure.

[0190] T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus issue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing. In one embodiment of the present disclosure, the cells are washed with PBS. In an alternative embodiment, the wash solution can lack calcium and magnesium, or can lack many, but not all, divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solutions, with or without buffer. In certain embodiments, undesirable components of the apheresis sample can be removed directly in the medium in which the cells are resuspended.

[0191] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers unique to the negatively selected cells. Cell sorting and / or selection via flow cytometry uses a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0192] PBMCs can be directly used for genetic modification with CARs using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In embodiments, memory T cells exist in both the CD62L+ and CD62L+ subsets of CD8+ peripheral blood lymphocytes. PBMCs are stained with anti-CD8 and anti-CD62L antibodies and then sorted into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0193] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L- and CD45RO-positive. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-negative.

[0194] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified. In another embodiment, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, 6,797,514, WO 2012 / 079000, and US 2016 / 0175358. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, typically anti-CD3 and anti-CD28 antibodies bound to beads or other surfaces, in culture medium supplemented with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen-presenting cells (APCs). In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012 / 129514.

[0195] The present disclosure provides a population of engineered immune effector cells for the treatment of a NY-ESO-1 associated disease or disorder, e.g., cancer, wherein the engineered immune effector cells comprise an HLA-A2:NY-ESO-1 CAR as disclosed herein.

[0196] The CAR-expressing immune effector cells prepared as described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al., and U.S. Patent No. 4,690,915 by Rosenberg.

[0197] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0198] The number of therapeutically effective cells in the composition is at least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset), or typically at least 10 cells. 2 More than 10 6 and 10 cells 8 or 10 9 Contains 10 cells 10 The number of cells will depend on the end use for which the composition is intended and the cell types contained therein.

[0199] The cells can be autologous or heterologous to the patient receiving therapy. If desired, the treatment can also include administration of a mitogen (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) described herein to enhance the induction of an immune response.

[0200] The CAR-expressing immune effector cell population of the present disclosure can be administered alone or as a pharmaceutical composition in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present disclosure can include a CAR-expressing immune effector cell population, such as a T cell described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0201] The anti-tumor immune response induced in a subject by administering the CAR-expressing T cells described herein using the methods described herein or other methods known in the art can include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells, which can activate B cells and thus lead to antibody production, can also be induced. Various techniques can be used to analyze the type of immune response induced by the compositions of the present disclosure, and they are fully described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0202] Accordingly, the present disclosure provides a method for treating an individual diagnosed with, suspected of having, or at risk of developing an NY-ESO-1-associated disease or disorder, e.g., an NY-ESO-1-positive cancer, comprising administering to the individual a therapeutically effective amount of a CAR-expressing immune effector cell described herein.

[0203] In one embodiment, the present disclosure provides a method of treating a subject diagnosed with NY-ESO-1-positive cancer, the method comprising removing immune effector cells from the subject diagnosed with NY-ESO-1-positive cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the present disclosure, thereby producing a population of modified immune effector cells, and administering the modified immune effector cell population to the subject. In one embodiment, the immune effector cells comprise T cells.

[0204] Methods of administering the cell compositions described herein include any method effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing a CAR of the present disclosure in the subject, or expressing a CAR upon the reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells upon introduction into the subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the present disclosure and returning the transduced cells to the subject.

[0205] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising an anti-HLA-A2:NY-ESO-1 antigen-binding protein, e.g., an antibody, or antigen-binding fragment thereof, or a CAR, of the present disclosure. Therapeutic compositions according to the present disclosure are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary known to all pharmaceutical chemists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0206] The dose of an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, can vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When an antigen-binding protein of the present disclosure is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is generally advantageous to administer an antigen-binding protein of the present disclosure, e.g., an antibody or antigen-binding fragment thereof, at a single dose of about 0.1 to about 60 mg / kg body weight, more preferably about 5 to about 60, about 20 to about 50, about 10 to about 50, about 1 to about 10, or about 0.8 to about 11 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, an antigen-binding protein of the present disclosure, e.g., an antibody or antigen-binding fragment thereof, can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antigen binding protein, e.g., antibody, or antigen-binding fragment thereof, which can be about the same as or less than the initial dose, and the subsequent doses are separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0207] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0208] The use of nanoparticles to deliver the antigen-binding proteins of the present disclosure, such as antibodies or antigen-binding fragments thereof, is also contemplated herein. Antigen-binding protein-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. For antigen-binding protein-conjugated nanoparticles and methods of preparation and use, see Arruebo, M. et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can be developed to target tumor cells, autoimmune tissue cells, or virus-infected cells and conjugated to antigen-binding proteins contained in pharmaceutical compositions. Nanoparticles for drug delivery are also described, for example, in U.S. Patent No. 8,257,740 or U.S. Patent No. 8,246,995, each of which is incorporated herein in its entirety.

[0209] In certain situations, the pharmaceutical composition can be delivered in a sustained release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the sustained release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0210] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antigen-binding protein or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0211] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is held in a reservoir within the device. Once the reservoir is emptied of pharmaceutical composition, the entire device is discarded.

[0212] Numerous reusable pen delivery devices and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN IV ... Examples of disposable pen delivery devices that have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK auto-injector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA pen (Abbott Labs, Abbott Park, IL).

[0213] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared in a suitable unit dose dosage form compatible with the dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antigen-binding protein contained is generally about 5 to about 500 mg per unit dose dosage form, and particularly in the form of injection, the antigen-binding protein is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, about 10 to about 250 mg.

[0214] Therapeutic Uses of Antigen-Binding Proteins The antibodies of the present disclosure are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by NY-ESO-1. For example, the present disclosure provides methods for treating (tumor growth inhibition) an NY-ESO-1-associated disease or disorder, such as an NY-ESO-1-associated cancer (e.g., an NY-ESO-1-positive cancer), by administering to a patient in need of such treatment an anti-HLA-A2:NY-ESO-1 antigen binding protein (or a pharmaceutical composition comprising an anti-HLA-A2:NY-ESO-1 antigen binding protein) described herein, and anti-HLA-A2:NY-ESO-1 antigen binding protein (or a pharmaceutical composition comprising an anti-HLA-A2:NY-ESO-1 antigen binding protein) for use in the treatment (tumor growth inhibition) of an NY-ESO-1-associated cancer. The antigen binding proteins of the present disclosure are useful for the treatment, prevention, and / or alleviation of a disease, disorder, or condition, such as an NY-ESO-1-associated cancer, and / or for alleviating at least one symptom associated with such a disease, disorder, or condition. In the context of the therapeutic methods described herein, the anti-HLA-A2:NY-ESO-1 antigen binding protein may be administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0215] In some embodiments, the antibodies described herein are useful for treating a subject suffering from a primary or recurrent cancer, including, but not limited to, a NY-ESO-1 associated cancer, such as liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer.

[0216] The antigen binding proteins may be used to treat early or late symptoms of NY-ESO-1-associated cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure may be used to treat advanced or metastatic cancer. The antigen binding proteins are useful for reducing, inhibiting, or shrinking tumor growth. In certain embodiments, treatment with the antigen binding proteins of the present disclosure results in greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% regression of tumors in a subject. In certain embodiments, the antigen binding proteins may be used to prevent tumor recurrence. In certain embodiments, the antigen binding proteins are useful for extending progression-free survival or overall survival in subjects with NY-ESO-1-associated cancer. In some embodiments, the antibodies are useful for reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients with NY-ESO-1-associated cancer.

[0217] One or more antibodies of the disclosure may be administered to alleviate or prevent or reduce the severity of one or more of the symptoms or conditions of a disease or disorder.

[0218] Also contemplated herein is the prophylactic use of one or more antibodies of the present disclosure in patients at risk of developing a disease or disorder, such as a NY-ESO-1-associated disease or disorder, such as a NY-ESO-1-associated cancer.

[0219] In a further embodiment of the disclosure, the antibody is used for the preparation of a pharmaceutical composition for treating a patient suffering from a NY-ESO-1-associated disease or disorder, such as a NY-ESO-1-associated cancer. In another embodiment of the disclosure, the antibody is used as an adjunctive therapy with any other agent or therapy known to those skilled in the art to be useful in treating a NY-ESO-1-associated cancer.

[0220] Combination Therapies and Formulations The combination therapy can include an anti-HLA-A2:NY-ESO-1 antigen binding protein of this disclosure, such as a CAR of this disclosure (e.g., an immune effector cell comprising a CAR of this disclosure), or a pharmaceutical composition of this disclosure, and any additional therapeutic agent that can be advantageously combined with the antigen binding protein of this disclosure. The antigen binding proteins of the disclosure may be synergistically combined with one or more anti-cancer agents or therapies used to treat or inhibit an NY-ESO-1 associated disease or disorder, such as an NY-ESO-1 positive cancer, e.g., liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer.

[0221] It is contemplated herein that the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be used in combination with immunostimulatory and / or immunosupportive therapies to inhibit tumor growth and / or enhance survival in cancer patients. Immunostimulatory therapies include direct immunostimulatory therapies, which enhance immune cell activity to activate immune responses by either "taking the brakes" or "stepping on the gas" on suppressed immune cells. Examples include targeting other checkpoint receptors, vaccinations, and adjuvants. Immune supportive modalities may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, antiangiogenic agents, and surgery.

[0222] In various embodiments, one or more antigen binding proteins of the disclosure are administered in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), , TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of another T cell co-inhibitor or ligand (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3, dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists (e.g., as described in U.S. Pat. No. 7,087,411 ). "VEGF-Trap," such as aflibercept or other VEGF inhibitory fusion proteins as indicated, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., BacillusCalmette-Guérin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3×CD20 bispecific antibody or PSMA×CD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, surgery, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC), anti-inflammatory agents (e.g., corticosteroids, nonsteroidal anti-inflammatory agents), nutritional supplements such as antioxidants, or any other therapy for treating cancer. In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure may be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance anti-tumor responses. Examples of cancer vaccines that can be used in combination with the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., rindopepimut) for brain tumors (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers).

[0223] In certain embodiments, anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be administered in combination with radiation therapy in a manner that produces a long-lasting anti-tumor response and / or enhances survival in patients with cancer. In some embodiments, anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be administered before, simultaneously with, or after radiation therapy to cancer patients. For example, radiation therapy can be administered in one or more doses to the tumor lesion, followed by administration of one or more doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. In some embodiments, radiation therapy can be administered locally to the tumor lesion, followed by systemic administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure, to enhance the local immunogenicity of the patient's tumor (active radiation) and / or kill tumor cells (ablative radiation). For example, intracranial radiation may be administered to patients with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. In certain embodiments, an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0224] The additional therapeutically active agent / component may be administered prior to, simultaneously with, or following administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. For purposes of this disclosure, such administration regimens will be considered administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein "in combination" with the second therapeutically active ingredient.

[0225] An additional therapeutically active ingredient may be administered to a subject prior to administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. For example, a first ingredient may be considered to be administered "before" the second ingredient if it is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second ingredient. In other embodiments, an additional therapeutically active ingredient may be administered to a subject after administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. For example, a first component can be considered to be administered "after" administration of a second component if it is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or 1 week after administration of the second component. In still other embodiments, an additional therapeutically active component can be administered to a subject simultaneously with administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. "Concurrent" administration, for purposes of this disclosure, includes, for example, administering the anti-HLA-A2:NY-ESO-1 antigen binding protein and the additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated) or in separate dosage forms that are administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-HLA-A2:NY-ESO-1 antigen binding protein and the additional therapeutically active ingredient may be administered intravenously, subcutaneously, etc.), or each dosage form may be administered via a different route (e.g., the anti-HLA-A2:NY-ESO-1 antigen binding protein may be administered intravenously and the additional therapeutically active ingredient may be administered subcutaneously). In any event, administration of the ingredients in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "co-administration" for purposes of this disclosure.For purposes of this disclosure, administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein "before," "concurrently with," or "after" (as these terms are defined above) an additional therapeutically active ingredient will be considered administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein "in combination with" the additional therapeutically active ingredient.

[0226] The present disclosure includes pharmaceutical compositions in which the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein, using various administration combinations.

[0227] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of anti-HLA-A2:NY-ESO-1 antigen binding protein (or a pharmaceutical composition comprising a combination of an anti-HLA-A2:NY-ESO-1 antigen binding protein and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. A method according to this aspect of the present disclosure comprises sequentially administering multiple doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of the anti-HLA-A2:NY-ESO-1 antigen binding protein is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a subject a single primary dose of an anti-HLA-A2:NY-ESO-1 antigen binding protein, followed by one or more secondary doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein, and then optionally, one or more tertiary doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein. The anti-HLA-A2:NY-ESO-1 antigen binding protein may be administered at a dose of 0.1 mg / kg to 100 mg / kg of the subject's body weight.

[0228] The terms "primary dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure. Thus, a "primary dose" refers to the dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" refers to the dose administered after the primary dose, and a "tertiary dose" refers to the dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of anti-HLA-A2:NY-ESO-1 antigen-binding protein, but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of anti-HLA-A2:NY-ESO-1 antigen-binding protein contained in the primary, secondary, and / or tertiary doses differs from one another (e.g., adjusted up or down as appropriate) over the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0229] In certain embodiments, the amount of anti-HLA-A2:NY-ESO-1 antigen binding protein contained in the primary, secondary, and / or tertiary doses may be suboptimal or subtherapeutic. As used herein, the terms "subtherapeutic" or "suboptimal" refer to an antibody dose administered at a level too low to produce a therapeutic effect or below the level required to treat a disease such as cancer.

[0230] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13 , 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2, or more) weeks later). The phrase "immediately preceding dose" as used herein refers to a dose of anti-HLA-A2:NY-ESO-1 antigen binding protein administered to a patient prior to administration of the immediately next dose in the series, without an intervening dose, in a multiple dose series.

[0231] Methods according to this aspect of the disclosure can include administering any number of secondary and / or tertiary doses of anti-HLA-A2:NY-ESO-1 antigen binding protein to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0232] In embodiments comprising multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments comprising multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the individual patient's needs after clinical testing.

[0233] Diagnostic Uses of Antigen Binding Proteins The anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be used to detect and / or measure NY-ESO-1 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antigen binding proteins of the present disclosure in assays to detect diseases or disorders, such as NY-ESO-1-associated diseases or disorders, such as NY-ESO-1-positive cancer. An exemplary diagnostic assay for NY-ESO-1 can include, for example, contacting a sample obtained from a subject (e.g., a patient) with an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure, where the anti-HLA-A2:NY-ESO-1 antigen binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate NY-ESO-1 from the subject sample. Alternatively, unlabeled anti-HLA-A2:NY-ESO-1 antigen binding protein can be used in diagnostic applications in combination with a second antigen binding protein, e.g., an antibody, that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125The NY-ESO-1 signaling molecule can be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure NY-ESO-1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0234] Samples that can be used in the NY-ESO-1 diagnostic assays of the present disclosure include any tissue or body fluid sample that can be obtained from a patient under normal or pathological conditions and contains a detectable amount of NY-ESO-1 protein or a fragment thereof. Generally, the NY-ESO-1 level in a particular sample obtained from a healthy patient (e.g., a patient not suffering from an NY-ESO-1-associated disease or disorder, such as an NY-ESO-1-positive cancer) is measured to first establish a baseline or standard level of NY-ESO-1. This baseline level of NY-ESO-1 can then be compared with the level of NY-ESO-1 measured in a sample obtained from an individual suspected of having a cancer-related condition or symptoms associated with such a condition.

[0235] Antigen binding proteins specific for NY-ESO-1 may contain no additional labels or moieties, or they may contain N- or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin is positioned so that the C-terminal portion of the peptide is distal to the surface.

[0236] Aspects of the present disclosure relate to the use of the disclosed antigen binding proteins as markers for predicting the prognosis of NY-ESO-1 positive cancer in patients. The antigen binding proteins of the present disclosure can be used in diagnostic assays to assess the prognosis and predict survival of cancer in patients. [Example]

[0237] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressures are at or near atmospheric pressure.

[0238] Example 1. Generation of human antibodies against HLA-A2:NY-ESO-1_157-165 Human antibodies against HLA-A2:NY-ESO-1 were generated using the HLA-A2-binding NY-ESO-1 peptide fragment of GenBank accession NP_001318.1 (SEQ ID NO: 271), containing amino acids 157-165 (SLLMWITQV, "NY-ESO-1_V") with cysteine ​​(C) at position 165 substituted with valine (V), SEQ ID NO: 291. The immunogen was administered directly to VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions) along with an adjuvant to stimulate the immune response, as described, for example, in U.S. Patent No. 8,502,018. Antibody immune responses were monitored by HLA-A2:NY-ESO-1-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing HLA-A2:NY-ESO-1 specific antibodies. Using this technique and the immunogen described above, several anti-NY-ESO-1 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained.

[0239] Anti-HLA-A2:NY-ESO-1 antibodies were also isolated directly from antigen-positive B cells (from one of the immunized mice) without fusion to myeloma cells, as described in U.S. Patent No. 7,582,298, which is incorporated herein by reference in its entirety. Using this method, several anti-HLA-A2:NY-ESO-1 antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained.

[0240] Exemplary antibodies generated according to the foregoing methods were designated mAb24955N, mAb24956N, mAb24958N, mAb24959N, mAb28042P, mAb28035P, mAb28037P2, mAb28075P, mAb28105P, mAb28113P, mAb28128P, mAb29814P, mAb24955N, and mAb29822P2.

[0241] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples below.

[0242] Example 2. Amino acid and nucleic acid sequences of heavy and light chain variable regions Table 1 shows the heavy and light chain variable regions and CDRs, heavy and light chain amino acid sequence identifiers of selected anti-HLA-A2:NY-ESO-1 antibodies of the present disclosure. The corresponding nucleic acid sequence identifiers are shown in Table 2. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0243] Antibodies herein are typically designated according to the following nomenclature: an Fc prefix (e.g., "mAb"), followed by a numerical identifier (e.g., "17670," "17930," etc., as shown in Table 1), followed by a "P," "N," or "N2" suffix. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "mAb17670P," "mAb17930N," "mAb17368N2," etc. As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc), but in any event, the variable domains (including the CDRs) designated by the numerical identifiers shown in Table 1 remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0244] In certain embodiments, a selected antibody having a mouse IgG1 Fc was converted to an antibody having a human IgG4 Fc. In certain embodiments, the antibody comprises a human IgG4 Fc with two or more amino acid changes as disclosed in U.S. Patent Publication No. 2010 / 0331527 (incorporated herein in its entirety). In one embodiment, the IgG4 Fc domain contains a serine to proline mutation (S108P) in the hinge region to promote dimer stabilization.

[0245] Table 3 shows the amino acid sequence identifiers for the heavy and light chain sequences of selected antibodies of the present disclosure. [Table 3-1] [Table 3-2]

[0246] Example 3. Binding Affinity and Rate Constants of Human Monoclonal Anti-HLA-A2:NY-ESO-1 Monospecific Antibodies by Surface Plasmon Resonance The binding affinity and rate constants of human anti-HLA-A2:NY-ESO-1 antibodies were determined at 25°C and 37°C by real-time surface plasmon resonance (SPR; Biacore 4000 by GE Healthcare Life Science or MASS-1 by Sierra Sensors). The anti-HLA-A2:NY-ESO-1 antibodies tested in this example were bivalent, monospecific binders for HLA-A2:NY-ESO-1 (expressed with a constant region (e.g., hIgG4 constant region), hIgG2a constant region, mIgG2a constant region, or mIgG1 constant region with reduced effector function (e.g., as described in U.S. Pat. No. 9,359,437)). Reference antibodies 1, 2, and 3 were derived from the antibody described in Stewart-Jones et al., Proc Natl Acad Sci USA 106(14):5784-5788 (2009) and International Patent Publication No. 2010 / 106431. Ref Ab1 contains the VH and VL domains from the 3M4E5 antibody described in these publications with a human IgG1 Fc and a human lambda light chain, Ref Ab2 contains the 3M4E5 VH domain and T1 Fab VL domain from a publication with a human IgG1 Fc and a human lambda light chain, and Ref Ab3 contains the 3M4E5 VH domain and T1 Fab VL domain from a publication with a human IgG4 and a human lambda VL domain with reduced effector function Fc (as described in U.S. Pat. No. 9,359,437). Antibodies were captured on a CM5 Biacore sensor surface (GE Healthcare Life Sciences) derivatized by amine coupling with a monoclonal anti-human Fc antibody (Jackson Immunoresearch) or a high-capacity amine sensor surface (Sierra Sensors) derivatized by amine coupling with a polyclonal anti-mouse Fc antibody (GE Life Sciences). Various concentrations of the monomeric HLA-A2:NY-ESO-1(156-165) (SEQ ID NO: 270 or 291, V at position 165) peptide complex, 2906 (SEQ ID NO: 272), were injected over the anti-HLA-A2:NY-ESO-1 antibody-captured surface at a flow rate of 50 μL / min (MASS-1) or 30 μL / min (Biacore 4000). Antibody-reagent association was monitored for 4–5 min, and dissociation was monitored for 10 min. All binding studies were performed in HBS-ET buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20).

[0247] The kinetic association (ka) and dissociation (kd) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. The binding-dissociation equilibrium constant (KD) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants: K D(M) = (kd / ka), and t1 / 2 (min) = ((ln (2)) ) / (60 × kd)).

[0248] The binding kinetic parameters of monospecific anti-HLA-A2:NY-ESO-1 antibodies to the monomeric HLA-A2:NY-ESO-1 peptide complex are shown below in Table 4. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0249] The data show that the majority of anti-HLA-A2:NY-ESO-1 antibodies tested selectively bound to the soluble HLA-A2:NY-ESO-1 peptide complex, some exhibiting nanomolar or subnanomolar affinity, and many with higher affinity for the complex than the reference antibody.

[0250] Example 4. FACS binding of anti-HLA-A2:NY-ESO-1 antibody to T2 cells pulsed with NY-ESO-1 157-165 peptide The relative binding of the NY-ESO-1:157-165 antibody was assessed by flow cytometry in T2 (174CEM.T2) cells pulsed with the NY-ESO-1_157-165 (C165) peptide (SEQ ID NO: 269). To pulse, T2 cells (174CEM.T2) were incubated at 1 x 10 cells. 6The cells were resuspended in AIM V medium (Gibco, catalog number 31035-025) at a density of 100 μg / ml. The cells were pulsed with 10 μg / ml of hB2M (EMD Millipore catalog number 475828) and 100 μg / ml of the indicated peptide. T2 cells were then incubated overnight at 37°C, washed with staining buffer, and then stained.

[0251] For cell staining, cells were harvested from the flasks using cell dissociation buffer (Millipore, Cat. No. S-004-C) and counted. Cells were seeded at a density of 200,000 cells per well in 96-well V-bottom plates in staining buffer (calcium- and magnesium-free PBS (Corning, ref. 21-031-CV) + 2% FBS (Seradigm, lot no. 238B15)) and stained with a 3-fold dilution series of primary antibodies (1.7 pM to 100 nM) for 30 min at 4°C. After primary antibody incubation, cells were washed once in staining buffer and stained with APC-conjugated secondary antibodies (Jackson ImmunoResearch, catalog no. 109-136-170) for 30 min at 4°C. Cells were then washed and fixed using a 50% solution of BD Cytofix (BD, catalog no. 554655). Samples were analyzed on an intellicyt iQue flow cytometer to calculate mean fluorescence intensity (MFI). MFI values ​​were calculated using a 4-parameter logistic equation over a 12-point response curve using Graphpad. Plot in Prism and EC 50 Values ​​were calculated. Secondary antibody alone (i.e., no primary antibody) in each dose-response curve was also included in the analysis as a series of 3-fold dilutions and is represented as the lowest dose. Ref Ab2 and Ref Ab3 were used as controls, as above. EC 50 The values ​​(M) are shown in Table 6. [Table 6-1] [Table 6-2]

[0252] Example 5. FACS binding of anti-HLA-A2:NY-ESO-1 antibodies to T2 cells pulsed with predicted off-target peptides The binding specificity of the NY-ESO-1:157-165 antibody was assessed by flow cytometry in T2 (174CEM.T2) cells pulsed with the NY-ESO-1 (157-165) peptide (C165; SEQ ID NO: 269) or a predicted off-target peptide (Table 7). To pulse, T2 cells (174CEM.T2) were incubated at 1 x 10 cells. 6 T2 cells were resuspended in AIM V medium (Gibco, catalog no. 31035-025) at a density of 100 μg / ml. Cells were pulsed by adding 10 μg / ml of hB2M (EMD Millipore catalog no. 475828) and 100 μg / ml of the indicated peptide. T2 cells were then incubated overnight at 37°C, washed with staining buffer, and stained with the indicated antibodies at a concentration of 10 μg / ml according to the protocol described above in Example 4. MFI values ​​were calculated and expressed as the ratio of pulsed T2 cells to non-pulsed cells. The results of these assays are shown in Table 8. Effective peptide loading was determined by comparing the increase in HLA-A2 surface staining from pulsed cell lines to non-pulsed cell lines using anti-HLA-A2 antibody. Any increase of 1.4-fold or greater was considered to be peptide-loaded. Although loading was not confirmed for one peptide (ITCH(807-815)), the two controls mentioned above, NY-ESO-1mAb Ref Ab1 and Ref Ab2, bound to cells pulsed with this peptide, indicating that some amount of peptide was loaded into the cells. [Table 7-1] [Table 7-2] [Table 8-1] [Table 8-2] [Table 8-3]

[0253] As shown in Table 8, many of the antibodies tested were identified as having no significant binding to T2 cells pulsed with any of the predicted off-targets, particularly compared to two control antibodies that had significant binding to multiple off-target peptides. Non-specific binding can result in reduced therapeutic efficacy and / or increased side effects (e.g., non-specific cytotoxicity that reduces tumor cell killing activity and / or causes side effects in the subject). Therefore, identifying antigen-binding proteins (e.g., antibodies) with minimal off-target binding can be beneficial in the development of therapeutic agents targeting MAGE-A4 as described herein.

[0254] Alanine scanning was performed to determine which residues in the NY-ESO-1(157-165) peptide were important for cell binding. T2 cells were pulsed with the alanine scanning peptide (Table 7) and stained with the NY-ESO-1(157-165) antibody as described above. The fold change relative to unpulsed cells is shown in Table 9. As determined by HLA.A2 surface staining (described above), all peptides except NY-ESO-1:157-165,C165V,S157A and NY-ESO-1:157-165,C165V,I162A loaded effectively. The following residues were important for binding (defined as a 90% or greater reduction in binding) of the anti-NY-ESO-1 mAbs tested: leucine 158, tryptophan 161, threonine 163, and glutamine 164. As noted above, threonine 163 and glutamine 164 were dispensable for binding of the control monoclonal antibody Ref Ab3, whereas methionine 160 was particularly important for binding of mAb24956N, mAb24958N, and mAb28105P. [Table 9-1] [Table 9-2] [Table 9-3]

[0255] Example 6. Reconstitution of anti-HLA-A2:NY-ESO-1 antibody into scFv for use in chimeric antigen receptors Four NY-ESO-1 (157-165) antibodies (mAb24955N, mAb24956N, mAb24958N, and mAb24959N) were reconstituted into VL-VH or VH-VL single-chain variable fragment chimeric antigen receptors (CARs) using the CD8α hinge and transmembrane domains, the 4-1BB costimulatory domain, and the CD3ζ stimulatory domain. The NY-ESO-1:157-165 CARs were cloned into a lentiviral expression vector (Lenti-X™ Bicistronic Expression System (Neo), Clontech, catalog number 632181), and lentiviral particles were generated using the Lenti-X Packaging Single-Shot (VSV-G) system (Clontech, catalog number 631276) according to the manufacturer's protocol. Jurkat / NFATLuc cl.3C7 cells (cells expressing an NFAT-luciferase reporter) were then transduced with eight different CARs using RetroNectin Precoated Dishes (Clontech, Cat. No. T110a) according to the manufacturer's protocol. After at least 2 weeks of selection in 500 μg / ml G418 (Gibco, Cat. No. 11811-098), the following CAR-T cell lines were generated: Jurkat / NFATLuc cl.3C7 / NY-ESO CAR-T 24955N VH-VL, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24955N VL-VH, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VH-VL, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VL-VH, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24958N VH-VL, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T The CAR-T cell lines were: 24958N VL-VH, Jurkat / NFAT Luc cl 3C7 / NY-ESO CAR-T 24959N VH-VL, and Jurkat / NFAT Luc cl 3C7 / NY-ESO CAR-T 24959N VL-VH. The activity of these CAR-T cell lines was then assessed in a CAR-T / APC (antigen-presenting cell) bioassay. To perform the bioassay, 50,000 CAR-T cells were added to a Thermo-Nunc 96-well white plate (Thermo Scientific, catalog number 136101) in 50 ml of assay medium (RPMI medium supplemented with 10% FBS and 1% P / S / G), followed by the addition of a 3-fold dilution series of APCs (200,000–274 cells) in 50 ml of assay medium. APCs were NIH3T3 cells engineered to express 3T3 / HLA.A2 / hB2M / NY-ESO-1:157~165WT (a ubiquitin peptide cassette containing human HLA.A2 (accession number P01892), human B2M (accession number NP_004039.1), and the NY-ESO-1:157~165 peptide) and 3T3 / HLA.A2 / hB2M / HPV16E7 11~19 (a ubiquitin peptide cassette containing the HPV16E7:11~19 peptide) (as described above; Levy F, et al. (1996) Proc. Nat. Acad. Sci. USA 93(10):4907-4912; Valmori D, et al. (1999) J Exp. Med. 189(6):895-906), IM9 (HLA.A2 positive, NY-ESO-1:157-165 positive), and HEK293 (HLA.A2 positive and NY-ESO-1:157-165 negative) cells were used. The cell mixture was incubated at 37°C, 5% CO2, in a humidified incubator for 5 hours. NFAT-luciferase activity was measured using Promega One-Glo (catalog no. E6130) and a Perkin Elmer Envision plate reader. Relative luciferase units (RLU) were plotted in GraphPad Prism using a four-parameter logistic equation over an 8-point response curve to determine EC. 50Values ​​were calculated. The zero APC condition in each dose-response curve was also included in the analysis as a series of 3-fold dilutions and is represented as the lowest dose. Maximum fold activation was determined by obtaining the ratio of the maximum RLU to the minimum on the curve. All eight NY-ESO-1 CAR cell lines were activated in the presence of 3T3 / HLA.A2 / hB2M / NY-ESO-1:157-165WT cells. As shown in Table 10, three NY-ESO-1 CAR cell lines were activated in the presence of IM9 cells (Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24955N VH-VL, Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24955N VL-VH, and Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VL-VH). [Table 10]

[0256] Example 7. Structural analysis of Fab binding to HLA2:NY-ESO-1_157-165 peptide To better understand the specific interactions between antibodies and HLA-peptide complexes, an X-ray crystal structure was determined for the Fab fragment of antibody mAb28105P bound to HLA-A2 / hB2M, which presents a peptide containing residues 157–165 from cancer-testis antigen 1 (CTAG1B; NY-ESO-1). This peptide was modified by substituting residue 165 from its native cysteine ​​with valine. All nine residues of the HLA-presented NY-ESO-1 (C165V) peptide were clearly visible in the electron density map of this structure, and the HLA and Fab residues surrounding the peptide were also well resolved. This structure was refined to 3.3 Å resolution; however, newer crystallographic refinement techniques (deformable elastic network, or "jelly body" refinement) helped prevent overfitting and ensure the accuracy of the resulting model.

[0257] The mAb28105P Fab bound to the top of the HLA-peptide complex in a manner similar to that of TCR binding. This Fab was centered on the bound peptide, with HCDR3 contacting the C-terminal half of the bound peptide and the light chain CDR contacting the N-terminal half of the peptide. Peptide residues M160 and W161 were central to the Fab binding interface and contacted both the heavy and light chain CDR residues, as described below. Other published intra-groove peptide-antibody complex structures (e.g., PDB codes 1W72 and 4WUU) indicate that antibodies do not coat the entire HLA-presented peptide, but that antibodies that only partially coat the peptide have poor specificity and tolerate extensive changes in the portion of the peptide that is not contacted with little loss of binding affinity.

[0258] The structure showed that the mAb28105P Fab heavy chain contacted residues 160, 161, and 164 in the HLA-bound NY-ESO-1 peptide, while the Fab light chain contacted residues 160 and 161. Peptide residues 157, 158, 159, 162, and 165 all pointed toward the HLA molecule. Residue 163 was completely shielded from solvent by the Fab light chain but did not directly contact antibody residues. The bound peptide was numbered according to the residue position in SEQ ID NO:271, as shown in SEQ ID NO:291. [Table 23]

[0259] All Fab contacts were made with the side chains of the HLA-binding peptide and not its backbone.

[0260] Peptide contacts made by MAb28105P Fab were concentrated at HCDR3, with minor contributions from LCDR1 and LCDR2. In particular, Fab heavy chain residues 100, 101, 104, 105, and 111 (SEQ ID NO: 162) and light chain residues 32 and 49 (SEQ ID NO: 170) interacted with the bound peptide, while Fab heavy chain residues 100, 101, 107, and 109 (SEQ ID NO: 162) and light chain residue 92 (SEQ ID NO: 170) interacted with HLA. As used above, the term "interacted with" can include direct or water-mediated hydrogen bonding, charge-charge interactions, or hydrophobic / van der Waals interactions.

[0261] Four of the NY-ESO-1_157-165 antibodies (mAb24955N, mAb24956N, mAb24958N, and mAb24959N) were highly similar in their light chain sequences, but their heavy chain sequences diverged in HCDR2 and HCDR3. Seven peptide-binding residues were generally conserved, with three residues identical in all four antibodies and three more identical in three of the four sequences, suggesting a common binding mode between the antibodies.

[0262] Example 8. Analysis of chimeric antigen receptor signaling domains V H -V L Anti-HLA-A2 / NY-ESO-1 in indications 157~165 A chimeric antigen receptor comprising an scFv and either 1) a human CD8 (huCD8) hinge / transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain (BB / z CAR) (full-length CAR sequence: SEQ ID NO: 301), or 2) a huCD28 hinge / transmembrane / costimulatory domain and a CD3z signaling domain (28 / z CAR) (full-length CAR sequence: SEQ ID NO: 302) was used to express anti-HLA-A2 / NY-ESO-1 157~165 V of the antibody mAb28105P L and V H Array(V L : SEQ ID NO: 294, V H:SEQ ID NO: 293). As a non-binding control, a BB / z CAR was designed using an irrelevant scFv with the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain. These CARs were cloned into the pLVX lentiviral vector with the EF1a promoter and the P2A:eGFP sequence (SEQ ID NO: 300) for tracking CAR-transduced cells, and VSV-pseudotyped lentivirus was produced. See Figure 1A for construct design and Table 11 for a summary of the constructs.

[0263] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of normal donors, stimulated with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were expanded with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use in in vivo experiments.

[0264] Anti-HLA-A2 / NY-ESO-1 157~165 To determine the in vivo efficacy of targeted chimeric antigen receptor (CAR) T cells, a xenogeneic tumor study was performed. On day 0, immunodeficient NOD.Cg-Prkdc SCID Il2rg(trademark) 1WJl / SzJ(NSG) mice, 5 × 10 6 HLA-A2 + NY-ESO-1 + A375 human melanoma tumor cells were injected subcutaneously. Three days after tumor establishment, non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / NY-ESO-1 (anti-HLA-A2 / NY-ESO-1) from two different donors were injected. 157~165 BB / z CAR, or anti-HLA-A2 / NY-ESO-1 157~165 20 × 10 expressing either 28 / z CAR (as determined by the frequency of cells expressing GFP, a marker for those cells transduced with CAR) 6 T cells were injected intravenously into mice (n=5 / group). Tumor growth was assessed over 21 days by measuring tumor volume.

[0265] To determine tumor volume by external caliper, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were determined. Tumor volume based on caliper measurements was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2. [Table 11]

[0266] In summary, the results showed that anti-HLA-A2 / NY-ESO-1 157~165 28 / z CAR T cells are anti-HLA-A2 / NY-ESO-1 157~165 We demonstrate that both types of CARs exhibited antitumor activity compared to the non-binding control, although the BB / z CAR T cells showed superior in vivo antitumor activity and antitumor kinetics. A375 tumors grew progressively in mice receiving the control CAR T cells. 157~165 Mice receiving BB / z CAR T cells showed some tumor control, with reduced tumor growth at days 21 (p<0.04), 28 (p<0.0001), and 38 (p<0.0001) compared to control CAR T-treated mice (statistics analyzed by two-way ANOVA). 157~165 28 / z CAR T treatment also resulted in suppression of established A375 tumor growth on days 16 (p=0.03), 19 (p=0.0002), 21 (p<0.0001), 28 (p<0.0001), and 38 (p<0.0001) (statistics analyzed by two-way ANOVA). 157~165 28 / z CAR and anti-HLA-A2 / NY-ESO-1 157~165Enhanced efficacy with BB / z CAR was confirmed as tumor size at days 21 (p=0.002), 28 (p<0.0001), and 38 (p<0.0001), and was statistically significant by two-way ANOVA (both days p<0.0001). See Figures 1B and 1C and Tables 12-22. [Table 12] [Table 13-1] [Table 13-2] [Table 14] [Table 15] [Table 16-1] [Table 16-2] [Table 17] [Table 18] [Table 19-1] [Table 19-2] [Table 20] [Table 21] [Table 22]

[0267] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such variations are intended to be included within the scope of the appended claims. The present invention provides, for example, the following items. (Item 1) An antigen binding protein that specifically binds to the HLA-A2:New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) complex at at least one amino acid corresponding to an amino acid selected from the group consisting of M160, W161, and Q164 of SEQ ID NO: 271. (Item 2) 2. The antigen-binding protein of item 1, wherein the isolated antigen-binding protein specifically binds to a conformational epitope of an HLA-A2-presented NY-ESO-1 polypeptide. (Item 3) 3. The antigen-binding protein of item 1 or 2, wherein the at least one amino acid is at least two amino acids. (Item 4) 4. The antigen-binding protein of item 3, wherein the at least one amino acid is three amino acids. (Item 5) 1. An antigen binding protein that specifically binds to the HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the isolated antigen binding protein interacts with amino acids M160, W161, and Q164 of the NY-ESO-1 157-165 peptide as determined by X-ray crystallography at a resolution of 4.0 Å or better. (Item 6) 6. The antigen-binding protein according to item 5, wherein the resolution is 3.5 Å or greater. (Item 7) 7. The antigen-binding protein according to item 6, wherein the resolution is 3.3 Å or greater. (Item 8) 1. An antigen binding protein that specifically binds to the HLA-A2:NY-ESO-1 157-165 peptide complex but does not specifically bind to one or more off-target peptides selected from the group consisting of BCL9L 1351-1359 (SEQ ID NO: 273), GRID1 7-15 (SEQ ID NO: 274), ZDHHC1 376-384 (SEQ ID NO: 276), ITCH 807-815 (SEQ ID NO: 277), and URB1 1853-1861 (SEQ ID NO: 280), wherein the ratio of i) the antigen binding protein binding to cells pulsed with said one or more off-target peptides to ii) non-pulsed cells is less than about 9. (Item 9) 9. The antigen-binding protein of item 8, wherein the ratio is less than about 8. (Item 10) 10. The antigen-binding protein of item 9, wherein the ratio is less than about 7. (Item 11) 11. The antigen-binding protein of item 10, wherein the ratio is less than about 6. (Item 12) 12. The antigen-binding protein of item 11, wherein the ratio is less than about 5. (Item 13) 13. The antigen-binding protein of item 12, wherein the ratio is less than about 4. (Item 14) 14. The antigen-binding protein of item 13, wherein the ratio is less than about 3. (Item 15) 15. The antigen-binding protein of item 14, wherein the ratio is less than about 2. (Item 16) The antigen-binding protein has a binding dissociation equilibrium constant (K) of less than about 1 nM for a monomeric HLA-A2:NY-ESO-1 157-165 peptide complex as measured at 25° C. in a surface plasmon resonance assay. D 16. The antigen-binding protein according to any one of items 1 to 15, wherein the antigen-binding protein binds to the nucleotide sequence at the nucleotide level. (Item 17) 17. The antigen-binding protein of any one of items 1 to 16, wherein the NY-ESO-1 peptide comprises the amino acid sequence of SLLMWITQC (SEQ ID NO: 269) or SLLMWITQV (SEQ ID NO: 291). (Item 18) 18. The antigen-binding protein of any one of items 1 to 17, wherein the antigen-binding protein is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), T-body construct, or chimeric antigen receptor (CAR). (Item 19) 20. The antigen-binding protein of item 19, wherein the antigen-binding protein is a CAR. (Item 20) 20. The CAR of item 19, wherein the CAR comprises a heavy chain variable region (LCVR), a light chain variable region (LCVR), a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain. (Item 21) 21. The CAR of paragraph 20, wherein the costimulatory domain is a 4-1BB costimulatory domain. (Item 22) 21. The CAR of paragraph 20, wherein the costimulatory domain is a CD28 costimulatory domain. (Item 23) 23. The antigen-binding protein according to any one of items 1 to 22, wherein the antigen-binding protein is a human monoclonal antibody or an antigen-binding fragment thereof. (Item 24) 24. The antigen-binding protein of any one of items 1 to 23, comprising three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised in any one of the heavy chain variable region (HCVR) sequences listed in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in any one of the light chain variable region (LCVR) sequences listed in Table 1. (Item 25) 25. The antigen binding protein of item 24, wherein the HCVR sequence is selected from the group consisting of SEQ ID NOs: 2, 22, 42, 142, and 250. (Item 26) 25. The antigen binding protein of item 24, wherein the LCVR sequence is selected from the group consisting of SEQ ID NOs: 10, 30, 50, 150, and 260. (Item 27) 25. The antigen binding protein of item 24, wherein the HCVR / LCVR sequences are selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, 230 / 238, and 250 / 258. (Item 28) 25. The antigen-binding protein of item 24, wherein the HCVR / LCVR sequences are selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258. (Item 29) 26. The antigen-binding protein of any one of items 1 to 25, comprising an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1. (Item 30) 30. The antigen binding protein of item 29, wherein the HCVR sequence is selected from the group consisting of SEQ ID NOs: 2, 22, 42, 142, and 250. (Item 31) 31. The antigen-binding protein of any one of items 1 to 30, comprising an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 32) 32. The antigen binding protein of item 31, wherein the LCVR sequence is selected from the group consisting of SEQ ID NOs: 10, 30, 50, 150, and 260. (Item 33) 33. The antigen-binding protein of any one of items 1 to 32, comprising: (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and (b) an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 34) 34. The antigen binding protein of item 33, wherein the HCVR / LCVR sequences are selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, 230 / 238, and 250 / 258. (Item 35) 35. The antigen-binding protein of item 34, wherein the HCVR / LCVR sequences are selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258. (Item 36) (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213, 232, and 252; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 182, 199, 215, 234, and 254; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, 236, and 256; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, 240, and 260; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, 242, and 262; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224, 244, and 264. (Item 37) 37. The antigen binding protein of any one of items 1 to 28 and 36, wherein the antigen binding protein comprises an HCVR comprising at least 80% sequence identity to an HCVR sequence listed in Table 1. (Item 38) 38. The antigen binding protein of item 37, wherein the antigen binding protein comprises an HCVR comprising at least 90% sequence identity to an HCVR sequence listed in Table 1. (Item 39) 39. The antigen binding protein of item 38, wherein the isolated antigen binding protein comprises an HCVR comprising at least 95% sequence identity to an HCVR sequence listed in Table 1. (Item 40) 40. The antigen-binding protein of any one of items 1 to 28 and 36 to 39, wherein the antigen-binding protein comprises an LCVR comprising at least 80% sequence identity to an LCVR sequence listed in Table 1. (Item 41) 41. The antigen binding protein of item 40, wherein the isolated antigen binding protein comprises an LCVR comprising at least 90% sequence identity to an LCVR sequence listed in Table 1. (Item 42) 39. The antigen binding protein of item 38, wherein the antigen binding protein comprises an LCVR comprising at least 95% sequence identity to an LCVR sequence listed in Table 1. (Item 43) 43. An antigen-binding protein that competes for binding with the antigen-binding protein of any one of items 1 to 42. (Item 44) An antigen-binding protein that binds to the same epitope as the antigen-binding protein according to any one of Items 1 to 42. (Item 45) 45. The antigen-binding protein of any one of items 1 to 44, comprising a detectable moiety. (Item 46) 46. ​​A pharmaceutical composition comprising an antigen-binding protein that binds to HLA-A2:NY-ESO-1 according to any one of items 1 to 45, and a pharmaceutically acceptable carrier or diluent. (Item 47) 46. ​​An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antigen-binding protein of any one of items 1 to 45. (Item 48) 46. ​​An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antigen-binding protein of any one of items 1 to 45. (Item 49) 49. A vector comprising the polynucleotide molecule of item 47 or 48. (Item 50) A cell expressing the polynucleotide molecule of item 47 or 48, or the vector of item 49. (Item 51) 50. A method of treating a subject having a NY-ESO-1 associated disease or disorder, comprising administering to the subject a therapeutically effective amount of the antigen-binding protein of any one of items 1 to 45, or the pharmaceutical composition of item 46, or the cell of item 50, thereby treating the subject. (Item 52) 52. The method of claim 51, wherein the NY-ESO-1-associated disease or disorder is NY-ESO-1-associated cancer. (Item 53) The NY-ESO-associated cancers are liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, and myelodysplasia. 53. The method of claim 52, wherein the cancer is selected from the group consisting of: acute myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, synovial sarcoma, metastatic solid tumors, esophageal cancer, rhabdomyosarcoma, advanced myxoid round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer. (Item 54) 54. The method of any one of items 51 to 53, wherein the antigen-binding protein is administered to the subject in combination with a second therapeutic agent. (Item 55) 55. The method of item 54, wherein the second therapeutic agent is selected from the group consisting of a PD-1 inhibitor, a CTLA-4 inhibitor, an antibody against a tumor-specific antigen, an antibody against a viral-infected cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, a bispecific antibody against CD20 and CD3, a nutritional supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, surgery, radiation, an NSAID, a corticosteroid, and any other therapy useful for ameliorating at least one symptom associated with the disease or disorder. (Item 56) 56. The method of any one of items 51 to 55, wherein the antigen-binding protein is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. (Item 57) 57. The method of any one of items 51 to 56, wherein the antigen-binding protein is administered at a dose of about 0.1 mg / kg to about 100 mg / kg of body weight of the subject. (Item 58) 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular binding domain that specifically binds to a conformational epitope of HLA-A2-presented New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide), a transmembrane domain, and an intracellular signaling domain. (Item 59) 59. The isolated nucleic acid molecule of claim 58, wherein the extracellular binding domain is an anti-HLA-A2:NY-ESO-1 antigen binding protein. (Item 60) 60. The isolated nucleic acid molecule of Item 59, wherein the antigen binding protein comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised in any one of the heavy chain variable region (HCVR) sequences listed in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in any one of the light chain variable region (LCVR) sequences listed in Table 1. (Item 61) 61. The isolated nucleic acid molecule of any one of items 58 to 60, wherein the antigen binding protein comprises an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1. (Item 62) 62. The isolated nucleic acid molecule of any one of items 58 to 61, wherein the antigen-binding protein comprises an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 63) 63. The isolated nucleic acid molecule of any one of Items 58 to 62, wherein the antigen-binding protein comprises (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and (b) an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 64) the antigen-binding protein (a) SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 21 an HCDR1 domain having an amino acid sequence selected from the group consisting of: 3, 232, and 252; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 182, 199, 215, 234, and 254; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, 236, and 256; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, 240, and 260; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, 242, and 262; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224, 244, and 264. (Item 65) 65. The isolated nucleic acid molecule of any one of paragraphs 58 to 64, wherein the antigen-binding protein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, 230 / 238, and 250 / 258. (Item 66) 66. The isolated nucleic acid molecule of any one of items 58 to 65, wherein the antigen-binding protein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258. (Item 67) 67. The isolated nucleic acid molecule of any one of items 58 to 66, comprising any one of the sequences listed in Table 2. (Item 68) 68. The isolated nucleic acid molecule of any one of items 58 to 67, wherein the antigen-binding protein is an scFv. (Item 69) 69. A vector comprising the isolated nucleic acid molecule of any one of Items 58 to 68. (Item 70) 69. An isolated immune effector cell comprising the isolated nucleic acid molecule of any one of items 58 to 68 or the vector of item 69. (Item 71) 71. The isolated immune effector cell of item 70, which is a T body. (Item 72) 72. The isolated immune effector cell of paragraph 70 or 71, wherein the cell expresses the CAR. (Item 73) A method for treating a subject having an NY-ESO-1-associated disease or disorder, comprising administering to the subject an immune effector cell described in any one of items 70 to 72. (Item 74) The NY-ESO-1-associated disease or disorder is a NY-ESO-1-associated cancer. 73. The method according to claim 73. (Item 75) 75. The method of claim 73 or 74, wherein the antigen binding protein is administered to the subject in combination with a second therapeutic agent.

Claims

[Claim 1] The invention described in the specification.

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