Anti-New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) antigen binding proteins and methods of use thereof

Abstract

The present disclosure provides antigen binding proteins that specifically bind to HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptides, and therapeutic and diagnostic methods using these binding proteins. The antigen binding proteins of the present disclosure bind to HLA-displayed NY-ESO-1 with high specificity, but do not bind to HLA-displayed peptides that differ by 2, 3, 4, 5 or more amino acids.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 870,232 filed on July 3, 2019, U.S. Provisional Patent Application No. 63 / 020,177 filed on May 5, 2020, and U.S. Provisional Patent Application No. 63 / 021,826 filed on May 8, 2020. The entire contents of each of the above patent applications are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on July 2, 2020, is named 118003_10520_SL.txt, and is 245,227 bytes in size. Technical Field

[0005] The present disclosure relates to antigen binding proteins that specifically bind to HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptides and therapeutic and diagnostic methods using those binding proteins. Background Art

[0006] New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a cancer testis antigen (CTA), also known as CTAG1B. The expression of NY-ESO-1, encoded by the CTAG1B gene, is restricted to germ cells. However, NY-ESO-1 is frequently de novo expressed by different tumor types. The major protein product of the CTAG1B gene is an 180 amino acid long, 18 kDa protein with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. However, the function of NY-ESO-1 remains unclear.

[0007] Spontaneous CD8 expression of NY-ESO-1 in cancer patients has been observed and analyzed + and CD4 + T cell responses, 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 were found to be active in tumor-reactive CD8 + T cell lines are restricted by HLA-A2, while peptides 56-62 are restricted by HLA-A31 CD8 +T cell recognition (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 are restricted by HLA-DP4, an allele found in most Caucasians (Jager et al. (2000) J. Exp. Med. 191:625-630; Zang et al. (2001) Proc Natl Acd Sci USA 98:3964-3969).

[0008] The ability of NY-ESO-1 to elicit spontaneous humoral and cellular immune responses, together with its restricted expression profile, makes it a good candidate target for cancer therapy. Although the NY-ESO-1 antigen has been evaluated as a cancer vaccine candidate, few complete humoral and cellular immune responses have been obtained. In fact, the use of NY-ESO-1 as a therapeutic target is very 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 that may lead to reduced therapeutic efficacy and / or increased side effects (e.g., reduced tumor cell killing activity and / or nonspecific cytotoxicity that causes side effects in subjects).

[0009] Therefore, there is an unmet need in the art for new therapeutic strategies to target NY-ESO-1 with high specificity and treat NY-ESO-1-associated cancers. Summary of the invention

[0010] The present disclosure provides antigen binding proteins that specifically bind to conformational epitopes of HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptides. The antigen binding proteins of the present disclosure bind to HLA-displayed NY-ESO-1 with a high degree of specificity, but do not bind to HLA-displayed peptides that differ by 2, 3, 4, 5 or more amino acids. The antigen binding proteins of the present disclosure allow for specific targeting of NY-ESO-1 peptide-presenting cells (i.e., cells that present NY-ESO-1 peptides bound to MHC molecules (e.g., HLA-A2) on their surface), such as cancer cells that express NY-ESO-1 and, in some embodiments, stimulate T cell activation, to, for example, stimulate T cell-mediated killing of such cells. In addition, when fused to a detectable moiety, the antigen binding proteins of the present disclosure enable diagnosis and prognosis of NY-ESO-1-positive diseases or conditions with a high sensitivity to changes in the number and distribution of NY-ESO-1 peptide-presenting cells, a more relevant measure of disease progression than circulating NY-ESO-1 levels.

[0011] 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., Fab, F(ab')2, or scFv fragments), and may be modified to affect functionality, e.g., to eliminate residual effector functions (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.

[0012] In a first aspect, the present disclosure provides a recombinant antigen binding protein that specifically binds to a conformational epitope of an HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptide, such as an HLA-displayed 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 a fully human antibody.

[0013] Exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure are listed in Tables 1 and 2 herein. Table 1 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR1, HCDR2 and HCDR3) and light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) of exemplary anti-HLA-A2:NY-ESO-1 antibodies. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of exemplary anti-HLA-A2:NY-ESO-1 antibodies.

[0014] The present disclosure provides an antigen binding protein comprising a HCVR comprising an amino acid sequence selected from any one of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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, an antigen binding protein having a sequence identity of less than 100% comprises a CDR sequence selected from a HCVR in Table 1. For example, such an antigen binding protein may comprise these CDR sequences, but have differences in the framework regions compared to the HCVR of Table 1.

[0015] The present disclosure provides antigen binding proteins comprising a LCVR comprising an amino acid sequence selected from any one of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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, an antigen binding protein having less than 100% sequence identity comprises a CDR sequence selected from a LCVR in Table 1. For example, such an antigen binding protein may comprise these CDR sequences, but have differences in the framework regions compared to the LCVR of Table 1.

[0016] The present disclosure also provides antigen binding proteins comprising a HCVR and LCVR amino acid sequence pair (HCVR / LCVR), the HCVR and LCVR amino acid sequence pair comprising a pairing of any HCVR amino acid sequence listed in Table 1 with any LCVR amino acid sequence listed in Table 1. According to certain embodiments, the present disclosure provides antigen binding proteins comprising a HCVR / LCVR amino acid sequence pair, the HCVR / LCVR amino acid sequence pair being included in 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 NO: 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 the following groups: SEQ ID NO: 2 / 10 (e.g., mAb24955N), 22 / 30 (e.g., mAb24956N), 42 / 50 (e.g., mAb24958N), and 62 / 70 (e.g., mAb24959N).

[0017] In certain embodiments, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising a HCVR and a LCVR, the HCVR comprising an amino acid sequence listed in Table 1 with no more than five amino acid substitutions, and the LCVR comprising an amino acid sequence listed in Table 1 with no more than five amino acid substitutions. For example, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising a HCVR and a LCVR, the HCVR comprising an amino acid sequence as set forth in SEQ ID NO:2 with no more than five amino acid substitutions, and the LCVR comprising an amino acid sequence as set forth in SEQ ID NO:10 with no more than five amino acid substitutions. In another exemplary embodiment, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen binding protein comprising a HCVR and a LCVR, the HCVR comprising an amino acid sequence as set forth in SEQ ID NO:2 with at least one amino acid substitution, and the LCVR comprising an amino acid sequence as set forth in SEQ ID NO:10 with at least one amino acid substitution.

[0018] The present disclosure also provides an antigen binding protein comprising a heavy chain CDR1 (HCDR1), wherein the heavy chain CDR1 comprises an amino acid sequence selected from any HCDR1 listed in Table 1 or a sequence substantially similar thereto, wherein the substantially similar sequence and the amino acid sequence have 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 an antigen binding protein comprising a heavy chain CDR2 (HCDR2), wherein the heavy chain CDR2 comprises an amino acid sequence selected from any HCDR2 listed in Table 1 or a substantially similar sequence thereof, wherein the substantially similar sequence and the amino acid sequence have 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 an antigen binding protein comprising a heavy chain CDR3 (HCDR3), wherein the heavy chain CDR3 comprises an amino acid sequence selected from any HCDR3 listed in Table 1, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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 with the amino acid sequence.

[0021] The present disclosure also provides an antigen binding protein comprising a light chain CDR1 (LCDR1), wherein the light chain CDR1 comprises an amino acid sequence selected from any LCDR1 listed in Table 1 or a sequence substantially similar thereto, wherein the substantially similar sequence and the amino acid sequence have 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 an antigen binding protein comprising a light chain CDR2 (LCDR2), wherein the light chain CDR2 comprises an amino acid sequence selected from any LCDR2 listed in Table 1 or a sequence substantially similar thereto, wherein the substantially similar sequence and the amino acid sequence have 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.

[0023] The present disclosure also provides an antigen binding protein comprising a light chain CDR3 (LCDR3), wherein the light chain CDR3 comprises an amino acid sequence selected from any LCDR3 listed in Table 1 or a sequence substantially similar thereto, wherein the substantially similar sequence and the amino acid sequence have 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.

[0024] The present disclosure also provides an antigen binding protein comprising a HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), the HCDR3 and LCDR3 amino acid sequence pair comprising a pairing of any HCDR3 amino acid sequence listed in Table 1 with any LCDR3 amino acid sequence listed in Table 1. According to certain embodiments, the present disclosure provides an antigen binding protein comprising a HCDR3 / LCDR3 amino acid sequence pair, the HCDR3 / LCDR3 amino acid sequence pair being included in any exemplary anti-HLA-A2:NY-ESO-1 antigen binding protein listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8 / 16 (e.g., mAb24955N), 28 / 36 (e.g., mAb24956N), 48 / 56 (e.g., mAb25958N), and 68 / 76 (e.g., mAb24959N).

[0025] The present disclosure also provides antigen binding proteins comprising a HCVR and a LCVR, wherein the HCVR comprising a HCDR1 comprises an amino acid sequence that differs by 1 amino acid from the amino acid sequence listed in Table 1, HCDR2 comprises an amino acid sequence that differs by 1 amino acid from the amino acid sequence listed in Table 1, and HCDR3 comprises an amino acid sequence that differs by 1 amino acid from the amino acid sequence listed in Table 1. In certain embodiments, the present disclosure provides antigen binding proteins comprising a HCVR and a LCVR, wherein the LCVR comprising a LCDR1 comprises an amino acid sequence that differs by 1 amino acid from the amino acid sequence listed in Table 1, LCDR2 comprises an amino acid sequence that differs by 1 amino acid from the amino acid sequence listed in Table 1, and LCDR3 comprises an amino acid sequence that differs by 1 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 a HCVR and a LCVR, wherein the HCVR comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence that differs from SEQ ID NO:4 by 1 amino acid; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 1 amino acid; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that differs from SEQ ID NO:8 by 1 amino acid. In another exemplary embodiment, the present disclosure provides an antigen binding protein comprising a HCVR and a LCVR, wherein the HCVR comprises: a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by 1 amino acid; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that differs from SEQ ID NO: 14 by 1 amino acid; and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 by 1 amino acid.

[0026] The present disclosure also provides an antigen binding protein comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of any exemplary antigen binding protein 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 NO: 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).

[0027] In a related embodiment, the present disclosure provides an antigen binding protein comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) in a 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 an antigen binding protein comprising a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set in a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 2 / 10 (e.g., mAb24955N), 22 / 30 (e.g., mAb24956N), 42 / 50 (e.g., mAb24958N), and 62 / 70 (e.g., mAb24959N).

[0028] 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 a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify the boundaries of a CDR include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence variability, 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, Maryland, (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 can also be used to identify CDR sequences within antigen binding proteins.

[0029] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins with modified glycosylation profiles. In some embodiments, modifications to remove unwanted glycosylation sites, or the presence of antibodies with missing fucose moieties on oligosaccharide chains, may be beneficial, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function, (see Shield et al. (2002) Journal of Biological Chemistry (JBC) 277:26733). In other applications, modifications of galactosylation may be performed to modify complement-dependent cytotoxicity (CDC).

[0030] In certain embodiments, the antigen binding protein of the present disclosure is a monoclonal antibody comprising a HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising a pairing of any HCVR amino acid sequence listed in Table 1 with any LCVR amino acid sequence 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.

[0031] The present disclosure provides an antigen binding protein or antigen binding fragment thereof comprising a heavy chain, the heavy chain comprising an amino acid sequence selected from any one of the HC amino acid sequences listed in Table 3 or a substantially similar sequence thereof, the substantially similar sequence 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. In some embodiments, the antigen binding protein having a sequence identity of less than 100% comprises the CDR sequence of the HC in Table 3. For example, such an antigen binding protein may comprise these CDR sequences, but have differences in the framework region compared to the HC of Table 3.

[0032] The present disclosure provides an antigen binding protein or antigen binding fragment thereof comprising a light chain, the light chain comprising an amino acid sequence selected from any one of the LC amino acid sequences listed in Table 3 or a sequence substantially similar thereto, the substantially similar sequence 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 with the amino acid sequence. In some embodiments, the antigen binding protein having a sequence identity of less than 100% comprises the CDR sequence of the LC in Table 3. For example, such an antigen binding protein may comprise these CDR sequences, but have differences in the framework region compared to the LC of Table 3.

[0033] The present disclosure also provides an antigen binding protein or antigen binding fragment thereof comprising a HC and LC amino acid sequence pair (HC / LC), the HC and LC amino acid sequence pair comprising a pairing of any HC amino acid sequence listed in Table 3 with any LC amino acid sequence listed in Table 3. According to certain embodiments, the present disclosure provides an antibody or antigen binding fragment thereof comprising a HC / LC amino acid sequence pair, the HC / LC amino acid sequence pair being included in 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 NO: 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 NO: 18 / 20, 38 / 40, 58 / 60 and 78 / 80.

[0034] 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 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 full length of the displayed peptide. As used herein, "contact" includes direct or water-mediated hydrogen bonds, charge-charge interactions, or hydrophobic / van der Waals interactions. In one embodiment, the 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 3 of the 9 amino acid residues of peptide 157-165 (SEQ ID NO: 269) and HLA-A2, such that the antigen binding protein is approximately concentrated on the peptide in the HLA-A2 peptide binding groove, thereby "covering" (physically blocking) the HLA-A2-peptide complex. In another embodiment, the 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 3 of the 9 amino acid residues of peptide 157-165 (SEQ ID NO: 270 or 291) and HLA-A2, such that the antigen binding protein is approximately concentrated on the peptide in the HLA-A2 peptide binding groove, thereby "covering" (physically blocking) the HLA-A2-peptide complex. In certain embodiments, the antigen binding protein or antigen binding fragment thereof comprises a CDR of a HCVR and a CDR of a 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 a fully human protein. In certain embodiments, the fully human antigen binding protein is 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.

[0035] In certain embodiments, the present disclosure provides an antigen binding protein or an antigen binding fragment thereof that binds to an HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to one or more amino acids set forth in SEQ ID NO: 269. In one embodiment, the antigen binding protein binds to at least 3 amino acids set forth in SEQ ID NO: 269.

[0036] In certain embodiments, the present disclosure provides an antigen binding protein or an antigen binding fragment thereof that binds to an HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the antigen binding protein binds to one or more amino acids set forth in SEQ ID NO: 270 or 291. In one embodiment, the antigen binding protein binds to at least 3 amino acids set forth in 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 set forth in SEQ ID NO: 270.

[0037] In certain embodiments, the present disclosure provides an antigen binding protein that specifically binds to a conformational epitope of an HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptide, wherein the conformational epitope comprises one or more amino acids set forth in SEQ ID NO:269.

[0038] In certain embodiments, the present disclosure provides an antigen binding protein that specifically binds to a conformational epitope of an HLA-displayed New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptide, wherein the conformational epitope comprises one or more amino acids set forth in 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 set forth in SEQ ID NO: 270 or 291.

[0039] The present disclosure also provides antigen binding proteins that compete for specific binding to HLA-A2:NY-ESO-1 with an antigen binding protein comprising the CDRs of the HCVR and the CDRs of the LCVR, wherein the HCVR and the LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0040] 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 CDRs of a HCVR and CDRs of a LCVR, wherein the HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0041] The present disclosure also provides antigen binding proteins that bind to the same epitope as a reference antigen binding protein comprising a CDR of a HCVR and a CDR of a LCVR, wherein the HCVR and the 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 a CDR of a HCVR and a CDR of a 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.

[0042] In one embodiment, the present disclosure provides a recombinant antigen binding protein that specifically binds to a conformational epitope of a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide presented by HLA-A2, wherein the antigen binding protein has the following properties selected from the group consisting of: (a) a binding dissociation equilibrium constant (K) of less than about 1 nM as measured in a surface plasmon resonance assay at 25°C; D ) binds to a monomeric HLA-A2:NY-ESO-1 157-165 peptide complex (C165 or V165); (b) binds to a monomeric HLA-A2:NY-ESO-1 157-165 peptide complex (C165 or V165) with an EC of less than about 10 nM as measured by flow cytometry 50 binds to cells expressing the HLA-A2:NY-ESO-1 157-165 peptide complex but does not bind to cells expressing a predicted off-target peptide; and (c) the epitope comprises one or more amino acids set forth in 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., a NY-ESO-1 157-165 peptide (SEQ ID NO: 269 and / or SEQ ID NO: 270 or 291)) by 2, 3, 4, 5, or more amino acids.

[0043] 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 HCVR amino acid sequence listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0044] The present disclosure also provides nucleic acid molecules encoding 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 LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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 to the polynucleotide sequence.

[0045] The present disclosure also provides nucleic acid molecules encoding any HCDR1 amino acid sequence listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR1 nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0046] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; 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, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0047] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; 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, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0048] The present disclosure also provides nucleic acid molecules encoding any LCDR1 amino acid sequence listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR1 nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0049] The present disclosure also provides nucleic acid molecules encoding any LCDR2 amino acid sequence listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR2 nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0050] The present disclosure also provides nucleic acid molecules encoding any LCDR3 amino acid sequence listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR3 nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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.

[0051] The present disclosure also provides a nucleic acid molecule encoding a HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined as any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1.

[0052] The present disclosure also provides nucleic acid molecules encoding LCVRs, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined as any of the exemplary anti-HLA-A2:NY-ESO-1 antigen binding proteins listed in Table 1.

[0053] The present disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises an amino acid sequence of any one of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any one of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence listed in Table 2, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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% similarity to the polynucleotide sequence. and a polynucleotide sequence selected from any LCVR nucleic acid sequence listed in Table 2 or a substantially similar sequence thereof, wherein the substantially similar sequence and the polynucleotide sequence have 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. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes the HCVR and the LCVR, wherein the HCVR and the LCVR are both derived from the same anti-HLA-A2:NY-ESO-1 antigen binding protein listed in Table 1.

[0054] The present disclosure provides nucleic acid molecules encoding any heavy chain amino acid sequence listed in Table 3. The present disclosure also provides nucleic acid molecules encoding any light chain amino acid sequence listed in Table 3.

[0055] The present disclosure also provides nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence of any one of the HC amino acid sequences listed in Table 3, and wherein the LC comprises an amino acid sequence of any one of the LC amino acid sequences listed in Table 3.

[0056] In a related aspect, the present disclosure provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain and / or light chain variable region of an anti-HLA-A2:NY-ESO-1 antigen binding protein. For example, the present disclosure includes a recombinant expression vector comprising any of the nucleic acid molecules mentioned above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR and / or CDR sequences listed in Table 1. The present disclosure also provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain and / or light chain of an anti-HLA-A2:NY-ESO-1 antigen binding protein. For example, the present disclosure includes a recombinant expression vector comprising any of the nucleic acid molecules mentioned above, i.e., a nucleic acid molecule encoding any of the heavy chain or light chain sequences listed in Table 2. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods of producing antigen binding proteins by culturing host cells under conditions that allow the production of antigen binding proteins and recovering the produced antigen binding proteins.

[0057] In a third aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a recombinant antigen binding protein that specifically binds to a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2 (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1), and a pharmaceutically acceptable carrier. In a related aspect, the 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 an anti-HLA-A2:NY-ESO-1 antigen binding protein. Exemplary agents that may be advantageously combined with an 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 may be used in combination with the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the disclosure are disclosed elsewhere herein.

[0058] In a fourth aspect, the present disclosure provides a method for treating a subject with a NY-ESO-1-related disease or condition, such as a NY-ESO-1-positive cancer. The method comprises administering to a subject in need thereof 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. The condition 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 an antibody to a T cell co-inhibitor, an antibody to a tumor cell antigen, an antibody to a T cell receptor, a cytotoxic agent, an anticancer drug, an anti-inflammatory drug (e.g., a corticosteroid), a chemotherapeutic agent, surgery, radiotherapy, an immunosuppressant, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effects associated with the antigen binding protein of the present disclosure, if such side effects occur.

[0059] In certain embodiments, the present disclosure provides methods for inhibiting the growth of NY-ESO-1-related cancers. For example, the present disclosure provides methods for inhibiting the growth of tumors due to primary tumors or metastatic tumors in a subject. In certain embodiments, the present disclosure provides methods for improving the survival rate (e.g., progression-free survival rate or overall survival rate) of a subject suffering from a NY-ESO-1-related 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 tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumors, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cavity cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant sarcoma, myelodysplastic syndrome, 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.

[0060] In certain embodiments, the present disclosure provides a method for preventing or inhibiting 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.

[0061] Antigen binding proteins (e.g., antibodies) or antigen binding fragments thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially. Antigen binding proteins (e.g., antibodies) or antigen binding fragments thereof can be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of subject body weight.

[0062] 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 CAR on its surface). CAR may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain that specifically bind to a conformational epitope of a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide displayed by HLA (e.g., amino acid residues 157-165 of NY-ESO-1). 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 disclosed herein are any antigen binding proteins described herein.

[0063] For example, in certain embodiments, an antigen binding protein suitable for use in a CAR of the present disclosure comprises: three heavy chain complementarity determining regions CDRs (HCDR1, HCDR2, and HCDR3) in any one of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) in any one of the light chain variable region (LCVR) sequences listed in Table 1.

[0064] In other embodiments, the antigen binding protein suitable for use in the CAR of the present disclosure comprises: a HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and / or a HCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

[0065] In some embodiments, an antigen binding protein suitable for use in a CAR of the present disclosure comprises: (a) a HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and (b) a LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

[0066] In one embodiment, the antigen binding protein suitable for CAR of the present disclosure comprises: (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213 and 232; (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 26, 46, 66, 86, 106, 126, 146, 166, 182, 199, 215 and 234; (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217 and 236; (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221 and 240; (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:14, 34, 54, 74, 94, 114, 134, 154, 174 and 242; and (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224 and 244.

[0067] In a further embodiment, the antigen binding protein suitable for use in the CAR of the present disclosure comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 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.

[0068] In some embodiments, the antigen binding protein used in the CAR of the present disclosure is a scFv.

[0069] In other aspects, the present disclosure provides vectors comprising isolated CAR nucleic acid molecules; and immune effector cells comprising such vectors.

[0070] In other aspects 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 tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cavity cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin 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. The method comprises administering to the subject a population of immune effector cells comprising a CAR of the present disclosure.

[0071] In some aspects, the present disclosure provides a method for detecting NY-ESO-1 positive cells in, for example, a subject or in a sample obtained from a subject. The method comprises 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 of the present disclosure comprising a detectable moiety to a subject, and detecting the presence or absence of the detectable moiety.

[0072] Other embodiments will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1A Schematic representation of three different CAR constructs, including promoter and vector elements, is depicted.

[0074] Figure 1B Depicted are the results of a CAR T cell line expressing a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / NY-ESO-1 157- 165 BB / z CAR or anti-HLA-A2 / NY-ESO-1 157-165 Tumor volumes of mice treated with T cells of either 28 / z CAR from day 0 to day 21, where left panel: average tumor volume; right panel: tumor volume of individual mice.

[0075] Figure 1C Depicted are the results of a CAR T cell line expressing a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / NY-ESO-1 157- 165 BB / z CAR or anti-HLA-A2 / NY-ESO-1 157-165Tumor volumes of mice treated with T cells of either 28 / z CAR from day 0 to day 38, where left panel: average tumor volume; right panel: tumor volume of individual mice. DETAILED DESCRIPTION

[0076] Before describing the present method, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, because these methods and conditions can be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present disclosure will be limited only by the appended claims.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the disclosure belongs. Preferred methods and materials are now described, but any methods and materials similar or equivalent to the methods and materials described herein may also be used in the practice or testing of the disclosure. All disclosures mentioned herein are incorporated herein by reference in their entirety.

[0078] The terms "NY-ESO-1", "NY-ESO-1", "New York Esophageal Squamous Cell Carcinoma 1" and "CTAG1B" refer to the well-known cancer testis antigen (CTA), which is de novo expressed in multiple cancer types and is encoded by the CTAG1B gene.

[0079] The amino acid sequence of full-length NY-ESO-1 is provided in GenBank as accession number NP_001318.1 (SEQ ID NO: 271). The term "NY-ESO-1" encompasses recombinant NY-ESO-1 or fragments thereof. The term also includes NY-ESO-1 or fragments thereof coupled to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1. In certain embodiments, the term encompasses NY-ESO-1 or fragments thereof in the context of, linked to, or displayed as HLA-A2.

[0080] In certain embodiments, the NY-ESO-1 peptide includes amino acids 157-165 as set forth in SEQ ID NO:271 (SLLMWITQC (SEQ ID NO:269)) and is referred to herein as "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 includes amino acid residues 157-165 as set forth in SEQ ID NO:271 wherein the cysteine ​​at residue 165 has been substituted with valine (SLMMWITQV (SEQ ID NO:270)), and is referred to herein as "NY-ESO-1_157-165V peptide" or "NY-ESO-1 (157-165V) peptide", or otherwise indicated as "C165V" or "V165". For simplicity, it should be understood that the terms "NY-ESO-1_157-165 peptide", "NY-ESO-1 (157-165)" and "NY-ESO-1 peptide comprising amino acid residues 157-165" include NY-ESO-1_157-165C peptide and NY-ESO-1_157-165V peptide unless otherwise specified.

[0081] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, which is a complex of genes encoding a person's major histocompatibility complex (MHC) proteins. These cell surface proteins are responsible for regulating a person's immune system. HLAs corresponding to class I MHC (A, B, and C) present peptides from within cells.

[0082] The term "HLA-A" refers to the group of human leukocyte antigens (HLA) encoded by the HLA-A locus. HLA-A is one of the three main types of human MHC class I cell surface receptors. The receptor is a heterodimer and consists of a heavy α chain and a smaller β chain. The α chain is encoded by a variant HLA-A gene, and the β chain (β2-microglobulin) is an invariant β2 microglobulin molecule.

[0083] The term "HLA-A2" refers to a specific class I major histocompatibility complex (MHC) allele group located at the HLA-A locus; the alpha chain is encoded by the HLA-A*02 gene and the beta chain is encoded by the beta2-microglobulin or B2M locus.

[0084] As used herein, the term "antigen binding protein", "binding protein" or "binding molecule" includes a molecule containing at least one antigen binding site that specifically binds to a target molecule such as a conformational epitope of a New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) presented by HLA-A2 (e.g., a peptide displayed by HLA-A2 comprising amino acid residues 157-165). The binding protein can be an antibody, such as 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.

[0085] The terms "HLA-A2:NY-ESO-1 antigen binding protein" or "HLA-A2:NY-ESO-1 antigen binding protein" and the like refer to an antigen binding protein (such as an antibody) or an antigen binding portion thereof that specifically binds to a conformational epitope through presentation of a peptide fragment (e.g., amino acid residues 157-165) of NY-ESO-1 by HLA-A2. In certain embodiments, the conformational epitope is produced on the cell surface by NY-ESO-1 peptide presented by HLA-A2. As used herein, the term "HLA-A2:NY-ESO-1 157-165 peptide complex" and the like refer to a complex between HLA-A2 and a NY-ESO-1 polypeptide, wherein HLA-A2 presents amino acid residues 157-165 of NY-ESO-1. In this case, NY-ESO-1 may be a full-length NY-ESO-1 polypeptide or a truncated form, as long as amino acids 157-165 (with reference to SEQ ID NO:271) are present in the NY-ESO-1 polypeptide and presented by HLA-A2. For the avoidance of doubt, the term "NY-ESO-1 157-165 peptide" encompasses peptides ranging as short as 157-165 of NY-ESO-1 (relative to SEQ ID NO:271) or longer, up to and including the full-length NY-ESO-1 protein (e.g., the sequence shown in SEQ ID NO:271).

[0086] 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 called a paratope. A single antigen may have more than one epitope. Therefore, different antigen binding proteins may bind to different regions on the antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen that reacts to B cells and / or T cells. It also refers to the region of the antigen that binds to the antigen binding protein. Epitopes can be defined as structural or functional. Functional epitopes are usually subsets of structural epitopes and have 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, an epitope may include a determinant, which is a chemically active surface group of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group and may have specific three-dimensional structural properties and / or specific charge properties in certain embodiments. In some embodiments, the antigen binding protein of the present disclosure interacts with a conformational epitope of an HLA-A2:NY-ESO-1 peptide complex. In some embodiments, the conformational epitope comprises one or more amino acids (e.g., one, two, or three amino acids) corresponding to M160, W161, and Q164 as set forth in SEQ ID NO: 271. To determine the amino acids corresponding to one or more of M160, W161, or Q164, a sequence alignment 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 comprising amino acids corresponding to amino acids 157-165 of SEQ ID NO: 271, as determined by resolution of or higher as determined by X-ray crystallographic methods. For the avoidance of doubt, or higher resolution including or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or higher, or or higher resolution.

[0087] In some embodiments, the binding protein is an antibody or an antigen-binding fragment thereof, such as a full-length antibody or an antigen-binding fragment thereof.

[0088] As used herein, the term "antibody" is intended to refer to immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds (i.e., "whole antibody molecules"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "V H ”) and the heavy chain constant region (composed of 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 ) H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). H and V L It is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or its antigen-binding fragment) 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 side-by-side analysis of two or more CDRs.

[0089] It is also possible to replace one or more CDR residues or omit one or more CDRs. Antigen binding proteins, such as antibodies, have been described in the scientific literature, in which one or two CDRs can be assigned for binding purposes. Padlan et al. (1995 Journal of the Federation of American Societies for Experimental Biology (FASEB J.) 9: 133-139) analyzed the contact area between an antibody and its antigen based on a disclosed crystal structure 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 the amino acids of one or two CDRs did not contact the antigen (see also Vajdos et al., 2002 Journal of Molecular Biology (J Mol Biol) 320: 415-428).

[0090] CDR residues that do not contact the antigen can be identified by molecular modeling and / or experience based on previous studies of Kabat CDR regions outside the Chothia CDR (e.g., residues H60-H65, which are not usually required in CDRH2). If a CDR or its residues are omitted, it is usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or a consensus sequence of such sequences. The position of the substitution within the CDR and the amino acid for substitution can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0091] The anti-HLA-A2:NY-ESO-1 antigen binding proteins disclosed herein, such as fully human anti-HLA-A2:NY-ESO-1 monoclonal antibodies, or antigen-binding fragments thereof, or CARs, may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR domains of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antigen-binding proteins (e.g., antibodies), or antigen-binding fragments thereof or CARs derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework and / or CDR domains are mutated to the corresponding residues of the germline sequence from which the antigen-binding protein is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily produce a variety of antigen binding proteins (e.g., antibodies) or antigen binding fragments thereof or CARs comprising one or more individual germline mutations or combinations thereof. H and / or V LAll frameworks and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence, and the antigen-binding proteins (eg, antibodies) are derived from the germline sequence. In other embodiments, only some residues are mutated back to the original germline sequence, such as only the mutated residues found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more frameworks and / or CDR residues are mutated to the corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences of the antibodies derived initially). In addition, antigen-binding proteins (eg, antibodies) of the present disclosure, or their antigen-binding fragments, or CARs, can include any combination of two or more germline mutations in the framework and / or CDR domains, for example, wherein some individual residues are mutated to the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence are retained or mutated to the corresponding residues of different germline sequences. Once obtained, antigen binding proteins (e.g., antibodies) and antigen binding fragments comprising 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 in this general manner are encompassed within the present disclosure.

[0092] The present disclosure also includes antigen binding proteins, such as fully human anti-HLA-A2:NY-ESO-1 monoclonal antibodies, or antigen binding fragments thereof, or CARs, comprising variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins having HCVR, LCVR, and / or CDR amino acid sequences having 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any one of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0093] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies (mAbs) disclosed herein may include, for example, in CDR, particularly in CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo). 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., mice) have been transplanted to human FR sequences. The term is included in antibodies recombinantly produced in cells of non-human mammals or non-human mammals. The term is not intended to include antibodies separated from human subjects or generated in human subjects.

[0094] As used herein, the term "recombinant" refers to an antigen-binding protein (e.g., antibody) or antigen-binding fragment thereof of the present disclosure that is produced, expressed, separated, or obtained by a technique or method known in the art as recombinant DNA technology (including, for example, DNA splicing and transgenic expression). The term refers to an antigen-binding protein (e.g., antibody) expressed, for example, in a non-human mammal (including a transgenic non-human mammal, such as a transgenic mouse) or in a cell (e.g., CHO cell) expression system or separated from a recombinant combinatorial human antibody library.

[0095] 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 a NY-ESO-1 peptide displayed by HLA-A2, such as a peptide comprising amino acid residues 157-165 of NY-ESO-1), a transmembrane domain, and at least one intracellular signaling domain.

[0096] As used herein, "immune effector cell" refers to any cell of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, ADCC and / or CDC induction). In one embodiment, the immune effector cell used with CAR as described herein is a T lymphocyte, particularly a cytotoxic T cell (CTL; ​​CD8+T cell) and a helper T cell (HTL; CD4+T cell). Other T cell groups may also be used herein, such as primary T cells and memory T cells. As will be appreciated by technicians, other cells may also be used as immune effector cells with CAR as described herein. Specifically, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitor cells of effector cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune effector cells. Thus, in this regard, immune effector cells include progenitor cells of immune effector cells, such as hematopoietic stem cells (HSCs) contained in a CD34+ cell population derived from umbilical cord blood, bone marrow or mobilized peripheral blood, which differentiate into mature immune effector cells after administration in a subject, or can be induced to differentiate into mature immune effector cells in vitro.

[0097] As disclosed herein, the term "off-target peptide" refers to a peptide that differs from a target peptide (e.g., a NY-ESO-1_157-165 peptide) by 2, 3, 4, 5 or more amino acids. In certain embodiments, the term includes peptides that differ from a target peptide by less than or equal to 3 amino acids. For example, for a 9-mer peptide, if 2, 3 or 4 amino acids are inconsistent with the target peptide, it is considered an "off-target" peptide. In certain embodiments, amino acid identity is expressed as a "degree of similarity" (DoS). If 6 amino acids in a 9-mer peptide are the same, the DoS is 6. In certain embodiments, peptides with a DoS≤6 are considered "off-target" peptides. The term "off-target" peptide also refers to a peptide that is similar to the target peptide based on sequence homology, is predicted to bind to HLA-A2 and is contained in a protein expressed in the necessary normal tissues.

[0098] The term "specific binding" or "specifically binds to" and the like means that an antigen binding protein (e.g., an antibody), or an antigen binding fragment thereof, or a CAR, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is characterized by an equilibrium dissociation constant of at least about 1×10 -8 M or smaller (e.g., smaller K D =Indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antigen binding proteins, such as antibodies, have been characterized by surface plasmon resonance (e.g., BIACORE TM) was identified that it specifically binds to a conformational epitope of a 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) presented by HLA-A2.

[0099] The term "high affinity" antigen binding protein, such as an antibody, refers to those antigen binding proteins (e.g., mAbs) that have binding affinity, measured as K, for a conformational epitope of a NY-ESO-1 peptide (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1) presented by HLA-A2. D Indicated by surface plasmon resonance (e.g. BIACORE TM or solution affinity ELISA) measured by at least 10 -8 M; preferably 10 -9 M; more preferably 10 -10 M, even more preferably 10 -11 M, even more preferably 10 -12 M.

[0100] The term "slow off rate", "Koff" or "kd" refers to a rate of dissociation measured by surface plasmon resonance (e.g., BIACORE TM ) measured 1×10 -3 s -1 or smaller, preferably 1×10 -4 s -1 The antigen binding protein dissociates from HLA-A2:NY-ESO-1 with a rate constant of 1:1 or less.

[0101] As used herein, 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 include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen binding fragment" or "antibody fragment" of an antibody refer to one or more fragments of an antibody that retain the ability to bind to a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2 (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1 coupled to HLA-A2).

[0102] In specific embodiments, an antigen binding protein of the disclosure, e.g., an antibody or antibody fragment, or a CAR, can 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 to a tumor-specific antigen, an anti-cancer drug, or any other therapeutic moiety that can be used to treat a disease or condition, including a NY-ESO-1-associated disease or condition (e.g., a NY-ESO-1-positive cancer).

[0103] As used herein, "isolated antigen-binding protein," e.g., isolated antibody, is intended to mean an antigen-binding protein (e.g., 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); and is substantially free of antigen-binding proteins, e.g., antibodies, that specifically bind to antigens other than conformational epitopes of NY-ESO-1 peptides presented by HLA-A2.

[0104] As used herein, the term "surface plasmon resonance" refers to a technique that allows the detection of changes in protein concentration within a biosensor matrix, such as using BIACORE TM systems (Pharmacia Biosensors AB, Uppsala, Sweden and Piscataway, NJ) to analyze optical phenomena of biomolecular interactions in real time.

[0105] As used herein, the term "K D ” is intended to mean the equilibrium dissociation constant for a particular antigen binding protein-antigen interaction.

[0106] As used herein, the term "cross-competition" refers to an antigen-binding protein (e.g., an antibody) or an antigen-binding fragment thereof, which binds to an antigen and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody) or an antigen-binding fragment thereof. The term also includes competition between two antigen-binding proteins (e.g., antibodies) in two directions, i.e., a first antigen-binding protein (e.g., an antibody) binds to a second antigen-binding protein (e.g., an antibody) and blocks the binding therebetween, and vice versa. In certain embodiments, the first antigen-binding protein (e.g., an antibody) and the 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 an antigen-binding protein, e.g., via steric hindrance, inhibits or blocks the binding of a second antigen-binding protein. The cross-competition between antigen-binding proteins (e.g., antibodies) may be measured by methods known in the art, e.g., by real-time, label-free biolayer interferometry. Cross-competition between two antigen-binding proteins (e.g., antibodies) can be expressed as binding of the second antigen-binding protein (e.g., antibody) due to self-binding less than the background signal (wherein the first and second antigen-binding proteins (e.g., antibodies) are the same antigen-binding protein (e.g., antibodies)). Cross-competition between two antigen-binding proteins (e.g., antibodies) can be expressed as % binding of the second antigen-binding protein (e.g., antibody) less than the baseline self-background binding (wherein the first and second antigen-binding proteins (e.g., antibodies) are the same antigen-binding protein (e.g., antibodies)).

[0107] The term "substantial identity" or "substantially identical" in reference to a nucleic acid or fragment thereof means that when optimally aligned with appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known sequence identity algorithm, which will be discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to a polypeptide encoded by the reference nucleic acid molecule.

[0108] Algorithms can be used to calculate sequence identity, such as the Needleman Wunsch algorithm for global alignment (Needleman and Wunsch 1970 "J. Mol. Biol. " 48:443-453) or the SmithWaterman algorithm for local alignment (Smith and Waterman 1981 "J. Mol. Biol. " 147:195-197). Another preferred algorithm is described by Dufresn et al. in Nature Biotechnology (Vol. 20, pp. 1269-71) in 2002 and used in the software GenePAST (GQ Life Sciences, Inc., Boston, MA).

[0109] When applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that, when optimally aligned, share at least 90% sequence identity, even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity, for example, by using the program GAP or BESTFIT with default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which one amino acid residue is substituted by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially change the functional properties of proteins. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the similar percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. The means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains of 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, tryptophan; 5) basic side chains: lysine, arginine, 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 with 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.

[0110] Sequence analysis software is usually used to measure the sequence similarity of polypeptides. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications including conservative amino acid substitutions to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT, which can be used under default parameters to measure sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different organism species) or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA with default or recommended parameters, see the program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlapping region between query and search sequences (Pearson (2000) the same). Sequences can also be compared using the Smith-Waterman homology search algorithm, in which an affine gap search is used, wherein a gap open penalty is 12, a gap extension penalty is 2, and a BLOSUM matrix is ​​62. When comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, both of which are incorporated herein by reference.

[0111] The phrase "therapeutically effective amount" means an amount administered to produce the desired effect. The exact amount will depend on the purpose of the treatment, and will be determined by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0112] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of improvement, prevention and / or treatment of a disease or condition, e.g., a NY-ESO-1-related disease or condition, such as a NY-ESO-1-related cancer (e.g., a NY-ESO-1-positive cancer). The term includes human subjects who have or are at risk of having a NY-ESO-1-related disease or condition, such as a NY-ESO-1-related cancer or a metastatic NY-ESO-1-related cancer.

[0113] As used herein, "anticancer drug" refers to any agent that can be used to treat or improve or inhibit cancer, including but not limited to cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mitotane (O, P'-(DDD)), biological agents (such as antibodies and interferons) and radiopharmaceuticals. As used herein, "cytotoxin or cytotoxic agent" also refers to chemotherapeutic agents, and means any agent that is harmful to cells. Examples include: (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitronquinone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs.

[0114] As used herein, the term "antiviral drug" refers to any drug or therapy used to treat, prevent or improve viral infection in a host subject. The term "antiviral drug" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics and corticosteroids.

[0115] An immunogen comprising any of the following can be used to generate antigen binding proteins, such as antibodies, against a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2 (eg, a peptide comprising amino acid residues 157-165 of NY-ESO-1 linked to HLA-A2). In certain embodiments, antigen binding proteins, such as antibodies, of the present disclosure can be obtained from mice immunized with: 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 having Genbank Accession No. NP_001318.1 (SEQ ID NO: 271) (e.g., a peptide comprising amino acid residues 157-165 (SLLMWITQC; SEQ ID NO: 269)); or a recombinant NY-ESO-1 peptide having a GenBank-accessioned C165V substitution sequence and binding to an HLA protein (such as HLA-A2) (SLMMWITQV; SEQ ID NO: 270).

[0116] Alternatively, NY-ESO-1 or fragments thereof can be produced using standard biochemical techniques and modified in the context of HLA-A2 and used as immunogens.

[0117] In some embodiments, the immunogen may be a recombinant NY-ESO-1 peptide expressed in E. coli or any other eukaryotic or mammalian cells, such as Chinese Hamster Ovary (CHO) cells (potentially a recombinant NY-ESO-1 peptide presented by HLA).

[0118] In certain embodiments, the above polypeptides or fragments thereof can be used to prepare antigen binding proteins that specifically bind to conformational epitopes of NY-ESO-1 peptides presented by HLA-A2. In certain embodiments, the NY-ESO-1 peptide presented by HLA-A2 can extend from one or both of the N- or C-termini of the regions described herein beyond the specified region by about 5 to about 20 amino acid residues. In certain embodiments, any combination of the above regions or fragments thereof can be used to prepare HLA-A2:NY-ESO-1 specific antigen binding proteins, such as antibodies.

[0119] The peptides can be modified to include addition or substitution of certain residues for labeling or conjugation to a carrier molecule such as KLH. For example, a cysteine ​​can be added to the N-terminus or C-terminus of the peptide, or a linker sequence can be added to prepare a peptide conjugated to, for example, KLH for immunization.

[0120] 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) are determined by surface plasmon resonance and measured on a Biacore 4000 or T200 instrument. Example 4 describes the binding of antibodies to cells overexpressing NY-ESO-1 fragments.

[0121] For example, antigen binding proteins (e.g., antibodies) for HLA-A2:NY-ESO-1 may not contain additional labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if any) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented so that the C-terminal portion of the peptide will be on the far side of 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 for diagnostic assays, including imaging assays.

[0122] Antigen Binding Protein

[0123] The present disclosure provides antigen binding proteins, which include antibodies or their antigen binding fragments and CAR (e.g., nucleic acid molecules encoding CAR of the present disclosure) (as described below). Unless otherwise specifically indicated, as used herein, the term "antibody" should be understood to cover antibody molecules (i.e., "complete antibody molecules") comprising two heavy chains of immunoglobulins and two light chains of immunoglobulins, and antigen binding fragments thereof. As used herein, the term "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2. Antigen binding proteins (such as antibody fragments) may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or separated CDRs. Antigen binding proteins (such as, antigen binding fragments of antibodies) can be obtained from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and (optionally) constant domains. Such DNA is known and / or easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0124] 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) the smallest recognition unit consisting of amino acid residues or constrained FR3-CDR3-FR4 peptides that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide). Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included in the expression "antigen-binding fragment" as used herein.

[0125] An antigen binding fragment of an antigen binding protein (e.g., an antibody) generally comprises at least one variable domain. A variable domain can have any size or amino acid composition and generally comprises at least one CDR adjacent to or in the same frame as one or more framework sequences. L Domain-associated V H In antigen binding proteins with domains, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and comprise V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L Structural domain.

[0126] In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of an antigen-binding protein of the present disclosure include: (i) 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 H3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present disclosure may comprise non-covalent associations with each other and / or with one or more monomeric V H or V L The polypeptides may be homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in which the domains are non-covalently associated (e.g., via disulfide bonds).

[0127] As with full antibody molecules, antigen binding proteins (e.g., antigen binding fragments of antibodies) can be monospecific or multispecific (e.g., bispecific). A multispecific antigen binding fragment of an antibody will generally comprise at least two different variable domains, each of which is 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 the context of antigen binding fragments of antibodies disclosed herein using conventional techniques available in the art.

[0128] Preparation of antigen binding proteins

[0129] Methods for producing antigen binding proteins such as human antibodies in transgenic mice are known in the art. In the context of the present disclosure, any such known methods can be used to prepare human antibodies that specifically bind to a conformational epitope of the New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) presented by HLA-A2.

[0130] By using Technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, ) or any other known method for producing an antigen binding protein, such as a monoclonal antibody, having a high affinity for a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2, such as a chimeric antibody, is initially isolated having a human variable region and a mouse constant region. Technology involves producing a transgenic mouse with a genome comprising a human heavy chain and light chain variable region operably connected to an endogenous mouse constant region locus, so that the mouse produces an antigen-binding protein, such as an antibody, comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the heavy chain and light chain variable regions of the antibody is separated and operably connected to the DNA encoding the human heavy chain and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0131] generally, The mouse is attacked with the antigen of the target, and lymphocytes (such as B cells) are recovered from the mouse expressing antigen-binding proteins (such as antibodies). Lymphocytes can be fused with myeloma cell lines to prepare infinite hybridoma cell lines, and this type of hybridoma cell line is screened and selected to identify the hybridoma cell line that produces antibodies specific to the relevant antigen. The DNA encoding the heavy chain and light chain variable regions can be separated and connected to the heavy chain and light chain constant regions of the desired isotype. Such antigen-binding proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific antigen-binding proteins (e.g., chimeric antibodies), or the variable domains of light and heavy chains can be directly separated from antigen-specific lymphocytes.

[0132] Initially, a high-affinity antigen-binding protein, such as a chimeric antibody, having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antigen-binding protein is characterized and selected for desired properties, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to produce an antigen-binding protein, such as a fully human antibody, of the present disclosure, such as a wild-type or modified IgG1 or IgG4. Although the constant region selected may vary depending on the specific use, the high-affinity antigen binding and target-specific characteristics are present in the variable region.

[0133] Bioequivalence

[0134] The anti-HLA-A2:NY-ESO-1 antigen binding proteins disclosed herein include proteins having amino acid sequences that differ from those of the antigen binding proteins (e.g., antibodies) but retain the ability to bind to conformational epitopes of NY-ESO-1 peptides presented by HLA-A2. Such variant antigen binding proteins comprise one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity substantially equivalent to that of the antigen binding protein. Similarly, the DNA sequences encoding the antigen binding proteins disclosed herein comprise sequences that comprise one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences but encode antigen binding proteins that are substantially bioequivalent to the antigen binding proteins disclosed herein.

[0135] If, for example, two antigen-binding proteins or antibodies are pharmaceutical equivalents or drug substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose under similar experimental conditions (single dose or multiple doses), then the antigen-binding proteins or antibodies are considered bioequivalents. If certain antigen-binding proteins or antibodies are absorbed to the same extent, but at different rates, they will be considered equivalents or drug substitutes, but may still be considered bioequivalents because this difference in absorption rate is intentional and reflected in the label, is not necessary to achieve effective in vivo drug concentrations (e.g., long-term use), and is considered medically insignificant for the specific drug product under study.

[0136] In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if they have no clinically meaningful differences in their safety, purity, or potency.

[0137] In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if patients can switch one or more times between the reference product and the biological product without an increased risk of expected adverse reactions, including clinically significant changes in immunogenicity or reduced effectiveness, compared to continued treatment without such switching.

[0138] In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if they both act under one or more conditions of use by one or more common mechanisms of action, provided such mechanisms are known.

[0139] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example: (a) in vivo tests in humans or other mammals, where the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum or other biological fluids; (b) in vitro tests that are correlated with and reasonably predictive of bioavailability data in humans; (c) in vivo tests in humans or other mammals, where appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) in well-controlled clinical trials to establish the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0140] Bioequivalent variants of the antigen-binding proteins (or antibodies) disclosed herein can be constructed by, for example, making various substitutions to residues or sequences or deleting terminal or internal residues or sequences necessary for biological activity. For example, cysteine ​​residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other cases, bioequivalent antigen-binding proteins can include antigen-binding protein variants that include amino acid changes that modify the glycosylation characteristics of the antigen-binding protein, such as mutations that eliminate or remove glycosylation.

[0141] Anti-HLA-A2:NY-ESO-1 antigen binding protein containing Fc variants

[0142] According to certain embodiments of the present disclosure, anti-HLA-A2:NY-ESO-1 antigen binding proteins, such as antibodies, are provided that include an Fc domain having one or more mutations that enhance or reduce binding of the antigen binding protein to an FcRn receptor, such as at an acidic pH, compared to a neutral pH. For example, the present disclosure includes an anti-HLA-A2:NY-ESO-1 antigen binding protein that includes a C in the Fc domain. H 2 or C H Mutations in region 3, wherein the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antigen binding protein when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modification at position 250 (e.g., E or Q); modification of positions 250 and 428 (e.g., L or F); modification at position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modification at position 428 and / or position 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification at position 250 and / or position 428; or modification at position 307 or position 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modification comprises 428L (e.g., M428L) and 434S (e.g., N434S) modification; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modification; 433K (e.g., H433K) and 434 (e.g., 434Y) modification; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; 250Q and 428L (e.g., T250Q and M428L) modification; and 307 and / or 308 (e.g., 308F or 308P) modification. In another embodiment, the modification comprises 265A (e.g., D265A) and / or 297A (e.g., N297A) modification.

[0143] For example, the present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins comprising an Fc domain having one or more pairs or groups of mutations selected from the group consisting of 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 N 434S); 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 having an S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the aforementioned Fc domain mutations and other mutations within the variable domains of the antigen binding proteins disclosed herein are contemplated to be within the scope of the present disclosure.

[0144] The present disclosure also includes chimeric heavy chain constant (C H ) region of the anti-HLA-A2: NY-ESO-1 antigen binding protein, wherein the chimeric C H The region comprises C sequences derived from more than one immunoglobulin isotype H For example, the antigen-binding protein of the present disclosure may comprise a chimeric C H region, the chimeric C H The region comprises part or all of the C region derived from human IgG1, human IgG2 or human IgG4 molecules. H 2 domains, which are derived from part or all of the C H According to certain embodiments, the antigen-binding protein of the present disclosure comprises a chimeric C having a chimeric hinge region. H For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 216 to 227 according to EU numbering), combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 228 to 236 according to EU numbering). According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2 as described herein. HAntigen binding proteins containing the Fc region can exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antigen binding protein (see, e.g., U.S. Patent Publication No. 20140243504, the entire disclosure of which is incorporated herein by reference).

[0145] Biological properties of antigen binding proteins

[0146] In general, the antigen binding proteins of the present disclosure function by binding to a conformational epitope of the New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) peptide presented by HLA-A2.

[0147] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind to NY-ESO-1 peptides with high specificity in the context of HLA-A2. In the absence of HLA-A2, the anti-HLA-A2:NY-ESO-1 antigen binding proteins do not bind to NY-ESO-1 peptides. In addition, the anti-HLA-A2:NY-ESO-1 antigen binding proteins do not bind to off-target peptides in the context of HLA-A2.

[0148] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind with high affinity to a monomeric HLA-A2:NY-ESO-1 (157-165) peptide (wherein the NY-ESO-1 157-165 peptide can comprise C165 or V165; i.e., the antigen binding proteins of the present disclosure can be specific for either form, or non-specific for either form). For example, the present disclosure includes an anti-HLA-A2:NY-ESO-1 antigen binding protein that binds with high affinity to a monomeric HLA-A2:NY-ESO-1 (157-165) peptide with a K of less than about 1 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 herein. D Antigen binding proteins that bind to a monomeric HLA-A2: 157-165 peptide (optionally with a C165V substitution) (e.g., at 25° C. or at 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 as measured by surface plasmon resonance using an assay format as defined in Example 3 herein or a substantially similar assay. D Binds to monomeric HLA-A2:NY-ESO-1_157-165 peptide.

[0149] The present disclosure also includes assays with an EC of less than about 10 nM as determined, for example, by a flow cytometry assay as defined in Examples 4 and 5 herein, or a substantially similar assay. 50An antigen binding protein that binds to cells expressing an HLA-A2:NY-ESO-1:157-165 peptide complex (wherein the NY-ESO-1 157-165 peptide may comprise C165 or V165; i.e., the antigen binding proteins of the present disclosure may be specific for either form, or non-specific for either form) but not to cells expressing a predicted off-target peptide. In certain embodiments, the antigen binding protein has an EC 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 as determined, for example, using an assay format as in Examples 4 and 5 herein or a substantially similar assay, by a flow cytometry assay as defined in Examples 4 and 5 herein or a substantially similar assay. 50 Binds to cells expressing the HLA-A2:NY-ESO-1_157-165 peptide but not to cells expressing the predicted off-target peptide.

[0150] In certain embodiments, the antigen-binding proteins of the present invention can be used to inhibit the growth of tumors or delay the progression of cancer when prophylactically administered to a subject in need and can increase the survival rate of the subject. For example, the administration of the antigen-binding proteins of the present invention can cause the primary tumor to shrink and can prevent the metastasis or development of secondary tumors. In certain embodiments, the antigen-binding proteins of the present invention can be used to inhibit the growth of tumors and can increase the survival rate of the subject when therapeutically administered to a subject in need. For example, administering a therapeutically effective amount of the antigen-binding proteins of the present invention to a subject can cause the already formed tumor in the subject to shrink and disappear.

[0151] In one embodiment, the present disclosure provides an isolated recombinant antigen binding protein that binds to a conformational epitope of a NY-ESO-1 peptide presented by HLA-A2, wherein the antigen binding protein exhibits one or more of the following features: (i) comprising a HCVR having 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 a substantially similar sequence thereof, wherein the substantially similar sequence has 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; (ii) comprising a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: NO:10, 30, 50, 70, 90, 110, 130, 150, 170, 186, 203, 219 and 238, or a substantially similar sequence thereof, wherein the substantially similar sequence and the amino acid sequence have 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; (iii) comprising a HCDR3 domain having a nucleotide sequence selected from SEQ ID NO:8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217 and 236, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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 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, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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; (iv) comprising a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: NO:4, 24, 44, 64, 84, 104, 124, 144, 164, 213 and 232, or a substantially similar sequence thereof, wherein the substantially similar sequence and the amino acid sequence have 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; HCDR2 domain, having a sequence selected from SEQ ID NO:6, 26, 46, 66, 86, 106, 126, 146, 166, 215 and 234, or a substantially similar sequence thereof, wherein the substantially similar sequence and the amino acid sequence have 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; LCDR1 domain, having a sequence selected from SEQ ID NO:12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221 and 240, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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 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, or a substantially similar sequence thereof, wherein the substantially similar sequence has 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 (v) has an association dissociation equilibrium constant (KD) of less than about 1 nM as measured in a surface plasmon resonance assay at 25°C to monomeric HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex; (vi) binds to monomeric HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex with a KD of less than about 1 nM as measured in a surface plasmon resonance assay at 25°C; (vii) binds to monomeric HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex with an EC of less than about 10 nM; 50 binds to cells expressing the HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex; and (viii) does not bind to an off-target peptide displayed by HLA-A2, wherein the peptide differs from SEQ ID NO: 271 by 2, 3, 4, 5 or more amino acids.

[0152] The antigen binding proteins of the present disclosure may have one or more of the above biological characteristics, or any combination thereof. By reading this disclosure, including the working examples herein, other biological properties of the antigen binding proteins of the present invention will be apparent to those of ordinary skill in the art.

[0153] Epitope mapping and related technologies

[0154] 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 NY-ESO-1 peptides displayed by HLA-A2. An epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within one or both of the above domains of the NY-ESO-1 molecule (e.g., a conformational epitope).

[0155] Various techniques known to those of ordinary skill in the art can be used to determine whether an antigen binding protein "interacts with one or more amino acids in a polypeptide or protein". Exemplary techniques include, for example, conventional cross-blocking analysis, such as that described in Antibodies, Harlow and Lane (Cold 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 cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and antigen chemical modification can be used (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids in a polypeptide that interact with an antigen binding protein is to detect hydrogen / deuterium exchange by mass spectrometry. In general, the hydrogen / deuterium exchange method includes deuterium-labeled target proteins, and then antigen-binding proteins are bound to deuterium-labeled proteins. Next, the protein / antigen-binding protein complex is transferred to water, and the exchangeable protons in the amino acids protected by the antigen-binding protein complex are subjected to deuterium-hydrogen reverse exchange at a slower rate than the exchangeable protons in the amino acids that do not belong to the interface. As a result, the amino acids that form a part of the protein / antigen-binding protein interface can retain deuterium, and therefore have a relatively high quality compared to the amino acids not included in the interface. After the antigen-binding protein dissociates, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids that interact with the antigen-binding protein. See, for example, Ehring (1999) "Analytical Biochemistry (Analytical Biochemistry)" 267:252-259; Engen and Smith (2001) "Analytical Chemistry (Anal.Chem.)" 73:256A-265A.

[0156] The term "epitope" refers to a site on an antigen to which B cells and / or T cells react. B cell epitopes can be formed by continuous amino acids or non-continuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes in a unique spatial conformation usually include at least 3 and more usually at least 5 or 8 to 10 amino acids.

[0157] Modification-assisted analysis (MAP), also known as antigen structure-based antibody analysis (ASAP), is a method for classifying a large number of monoclonal antigen-binding proteins (e.g., antibodies (mAbs)) against the same antigen based on the similarity of the binding characteristics of each antibody to a chemically or enzymatically modified antigen surface (see US 2004 / 0101920, which is incorporated herein by reference in its entirety). Each class can reflect a unique epitope that is significantly different from or partially overlaps with the epitope represented by another class. The technology allows rapid filtering of genetically identical antigen-binding proteins, thereby focusing characterization on genetically different antigen-binding proteins. When applied to hybridoma screening, MAP may help identify 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.

[0158] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that bind to the same epitope or a portion of the epitope, such as any of the specific exemplary antigen binding proteins described in Table 1 herein, or antigen binding proteins having the CDR sequences of any of the exemplary antigen binding proteins described in Table 1. Likewise, the present disclosure also includes anti-HLA-A2:NY-ESO-1 antigen binding proteins that compete for binding to HLA-A2:NY-ESO-1 or a fragment thereof with any of the specific exemplary antigen binding proteins described in Table 1 herein, or antigen binding proteins having the CDR sequences of any of the exemplary antigen binding proteins described in Table 1.

[0159] Whether an antigen binding protein binds to or competes for binding to the same epitope as a reference anti-HLA-A2:NY-ESO-1 antigen binding protein can be readily determined using conventional 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 saturation conditions. Next, the ability of the test antigen binding protein to bind to an HLA-A2:NY-ESO-1 molecule is evaluated. If the test antigen binding protein is able to bind to HLA-A2:NY-ESO-1 after saturation binding with the reference anti-HLA-A2:NY-ESO-1 antigen binding protein, it can be concluded that the test antigen binding protein binds to different epitopes 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 the HLA-A2:NY-ESO-1 protein 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.

[0160] 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 method is performed in two orientations: In the first orientation, the reference antigen binding protein is allowed to bind to the HLA-A2:NY-ESO-1 protein under saturating conditions, and then the binding of the test antigen binding protein to the HLA-A2:NY-ESO-1 molecule is evaluated. In the second orientation, the test antigen binding protein is allowed to bind to the HLA-A2:NY-ESO-1 molecule under saturating conditions, and then the binding of the reference antigen binding protein to the HLA-A2:NY-ESO-1 molecule is evaluated. If, in both orientations, only the first (saturated) antigen binding protein is able to bind to the HLA-A2:NY-ESO-1 molecule, then it can be concluded that the test antigen binding protein competes with the reference antigen binding protein for binding to HLA-A2:NY-ESO-1. As will be appreciated by one of ordinary 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.

[0161] If two antigen-binding proteins competitively inhibit (block) the binding of another to the antigen, then the two antigen-binding proteins bind to the same epitope or overlapping epitopes. That is, as measured in a competitive binding assay, a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of antigen-binding proteins inhibits at least 50% of another antigen-binding protein, but preferably 75%, 90% or even 99% of the binding (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, if substantially all amino acid mutations that reduce or eliminate the binding of one antigen-binding protein in the antigen will reduce or eliminate the binding of another antigen-binding protein, then the two antigen-binding proteins have the same epitope. Alternatively, if some amino acid mutations that reduce or eliminate the binding of one antigen-binding protein will reduce or eliminate the binding of another antigen-binding protein, then the two antigen-binding proteins have overlapping epitopes.

[0162] Additional 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 actually due to binding to the same epitope as the reference antigen binding protein, or whether steric hindrance (or other phenomena) is the cause of the observed lack of binding. Such 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.

[0163] Immunoconjugates

[0164] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen binding proteins conjugated to a therapeutic moiety ("immunoconjugate") such as a cytotoxin or a chemotherapeutic agent to treat cancer. As used herein, the term "immunoconjugate" refers to an antigen binding protein chemically or biologically linked to a cytotoxin, a radioactive agent, a cytokine, an interferon, a target or reporter moiety (such as a detectable moiety), an enzyme, a toxin, a peptide or protein, or a therapeutic agent. The antigen binding protein can be linked to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or therapeutic agent at any position along the molecule, as long as the antigen binding protein is able to bind to its target. Examples of immunoconjugates include antigen binding protein-drug conjugates and antigen binding protein-toxin fusion proteins. In one embodiment, the agent can be a second different antibody to NY-ESO-1 or HLA-A2:NY-ESO-1. In certain embodiments, the antigen binding protein can be conjugated to an agent that is 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 suitable reagents for forming immunoconjugates are known in the art; for example, see PCT Publication No. WO 05 / 103081.

[0165] Chimeric Antigen Receptor (CAR)

[0166] Chimeric antigen receptors (CARs) redirect T cells specifically to antibody-recognized antigens expressed on the surface of cancer cells, while T cell receptors (TCRs) expand the target range to include intracellular tumor antigens. CAR-redirected T cells specific for the B cell differentiation antigen CD19 have shown significant efficacy in the treatment of B cell malignancies, while TCR-redirected T cells have shown benefits in patients with solid cancers. Stauss et al. describe strategies for modifying therapeutic CARs and TCRs for use in cancer therapy, such as to enhance antigen-specific effector functions and limit the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24: 113-118).

[0167] One aspect of the present disclosure includes a chimeric antigen receptor (CAR) that is specific for NY-ESO-1 peptides displayed on the surface of tumor cells through HLA-A2, such as peptides containing amino acid residues 157-165 of NY-ESO-1. In one embodiment of the present disclosure, the CAR as described herein comprises: an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain derived from CD3ζ or FcRγ) and / or a costimulatory signaling domain derived from a co-stimulatory molecule such as, but not limited to, CD28, CD137, CD134, or CD278. In one embodiment, the CAR includes a hinge or spacer between the extracellular binding domain and the transmembrane domain, such as a CD8α hinge or a CD28 hinge. In another embodiment of the present disclosure, the CAR as described herein comprises an extracellular target-specific binding domain and a T cell receptor constant domain ("T body construct"). In some embodiments, the hinge / transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 296. In some embodiments, the hinge region comprises the CD28 sequence set forth in SEQ ID NO: 305. In some embodiments, the transmembrane domain comprises the CD28 sequence set forth in SEQ ID NO: 304. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 297. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 299. In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 298.

[0168] It should be understood that for any CAR described herein, the extracellular target-specific binding domain may comprise a Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv) of an antigen binding protein of the present disclosure.

[0169] As used herein, the binding domain or extracellular domain of CAR provides CAR with the ability to bind to the target target antigen. The binding domain can be any protein, polypeptide, oligopeptide or peptide with the ability to specifically recognize and bind biomolecules (e.g., cell surface receptors or tumor proteins or components thereof). The binding domain includes any naturally occurring, synthetic, semi-synthetic or recombinantly produced binding partners of the target biomolecule. For example, as further described herein, the binding domain can be an antibody light chain and heavy chain variable region, or the light chain and heavy chain variable region can be connected together in a single chain and in either direction (e.g., VL-VH or VH-VL). A variety of assays for identifying the binding domains of the present disclosure specifically bound to a specific target are known, including Western blot, ELISA, flow cytometry or surface plasmon resonance analysis (e.g., using BIACORE analysis), and are described herein. The target can be any clinically interesting antigen, and it is desirable to trigger an effector immune response that causes tumor killing for the antigen. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is a conformational epitope of a NY-ESO-1 peptide (such as a peptide NY-ESO-1 comprising amino acid residues 157-165) presented by HLA-A2 on the surface of a tumor cell.

[0170] Exemplary binding domains include antigen binding proteins (such as antigen binding fragments of antibodies), such as scFv, scTCR, the extracellular domain of receptors, cell surface molecules / ligands of receptors or their receptor binding domains, and tumor binding proteins. In certain embodiments, the antigen binding domains included in the CAR of the present disclosure can be variable regions (Fv), CDR, Fab, scFv, VH, VL, domain antibody variants (dAb), camelid antibodies (VHH), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen-specific binding domains derived from other protein scaffolds.

[0171] In one embodiment, the binding domain of CAR is an anti-HLA-A2: NY-ESO-1 single-chain antibody (scFv), and may be a mouse, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas that are specific to the desired target. Techniques that can be used to clone variable region heavy chains (VH) and variable region light chains (VL) have been described (e.g., Orlandi et al., Proceedings of the National Academy of Sciences (PNAS), 1989; 86: 3833-3837). Therefore, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but may also be a non-antibody-derived binding domain. The antibody-derived binding domain may be a genetically engineered product of an antibody fragment or one or more fragments of an antibody, which fragment is involved in binding to an antigen.

[0172] In certain embodiments, the CAR of the present disclosure may include a linker between various domains, which adds appropriate spacing and conformation for the molecule. For example, in some embodiments, there may be a linker between the binding domain VH and VL regions, and its length may be 1-10 amino acids. In other embodiments, the length of the linker between any domains of the chimeric antigen receptor may be between 1-20 or 20 amino acids. In this regard, the length of 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 a further embodiment, the length of the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. The range including the numbers described herein is also included herein, for example, linkers of 10 to 30 amino acid lengths.

[0173] In certain embodiments, the linker applicable to CAR described herein is a flexible linker. Suitable linkers can be easily selected and can be any one of suitable different lengths, such as 1 amino acid (eg, Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0174] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (G)nS, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, wherein n is an integer of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In addition, the linker may comprise multiple units of the above sequence, such as ((G)nS)m), wherein n is an integer of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and m is an integer of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, n is 4, and the linker comprises the sequence GGGGS (SEQ ID NO: 295). In some embodiments, n is 4 and m is 3, and the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 303). Glycine and glycine-serine polymers are relatively unstructured and therefore may act as fusion proteins, such as neutral tethers between domains of CAR described herein. Compared with alanine, glycine can enter more phi-psi spaces and is much less restricted than residues with longer side chains (see Scheraga, "Review of Computational Chemistry (Rev. Computational Chem.)" 11173 / -142 (1992)). Ordinary technicians will recognize that the design of CAR can include all or part of the flexible linker, so that the linker can include a flexible linker and one or more parts that give a lower flexible structure to provide the desired CAR structure.

[0175] There may be a "spacer" or "hinge" behind the binding domain of CAR, which refers to the region where the antigen binding domain is removed from the effector cell surface to achieve appropriate cell / cell contact, antigen-binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in CAR is generally located between the transmembrane (TM) 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 changed wild-type immunoglobulin hinge region. Other exemplary hinge regions used in CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be variable. In one embodiment, the hinge region comprises a CD8α hinge.

[0176] "Transmembrane" region or domain is the CAR part that anchors the extracellular binding part to the plasma membrane of immune effector cells and promotes the binding of the domain to the target antigen. The transmembrane domain can be a CD3ζ transmembrane domain, however, other transmembrane domains that can also be used include those obtained from CD8α, 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 synthetic, in which case it will mainly contain hydrophobic residues, such as leucine and valine.

[0177] "Intracellular signaling domain" refers to a portion of a chimeric antigen receptor protein that is involved in transducing information that binds an effective CAR to a target antigen into an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to target cells that bind to CAR, or other cellular responses that are elicited by binding of antigens to the extracellular CAR domain. The term "effector function" refers to a specific function of a cell. The effector function of a T cell may be, for example, cytolytic activity or help or activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be used, in many cases the entire domain does not need to be used. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of the entire domain, as long as it transduces an effector function signal. The term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal. The intracellular signaling domain is also referred to as a "signaling domain" and is typically derived from a portion of the human CD3 or FcRy chain.

[0178] It is known that signals generated through the T cell receptor alone are not sufficient to fully activate T cells, and auxiliary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the T cell receptor (primary cytoplasmic signaling sequences); and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate primary activation of the T cell receptor complex in an inhibitory manner. Primary cytoplasmic signaling sequences that act in a co-stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0179] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used in the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain specific embodiments, the intracellular signaling domain of the anti-HLA-A2:NY-ESO-1 CAR described herein is derived from CD3 ζ or FcR γ.

[0180] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the portion of the intracellular domain of the CAR containing a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal required for the effective activation and function of T lymphocytes when bound to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands that specifically bind to CD83. Therefore, although the present disclosure provides exemplary costimulatory domains derived from CD3ζ, CD28, and 4-1BB, other costimulatory domains are also contemplated for use in CAR described herein. Including one or more costimulatory signaling domains can enhance the efficacy and amplification of T cells expressing CAR receptors. The intracellular signaling domain and the co-stimulatory signaling domain can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0181] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcRγ have been shown to provide potent signals for T cell activation and effector function, they are insufficient to elicit signals that promote T cell survival and expansion in the absence of accompanying costimulatory signals. Other CARs that contain a binding domain, a hinge, a transmembrane and a signaling domain derived from CD3ζ or FcRγ and one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134, and CD278) may be more effective in directing the anti-tumor activity of CAR-expressing T cells in vitro, in animal models, and in cancer patients, as well as increasing cytokine secretion, lytic activity, survival, and proliferation (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., Proc. Natl. Acad. Sci. USA, 2009; 106: 3360-3365).

[0182] In one embodiment, the HLA-A2:NY-ESO-1 CAR of the present disclosure comprises: (a) anti-HLA-A2:NY-ESO-1 scFv as a binding domain (e.g., a scFv having a binding region (e.g., CDR or variable domain) from any one or more HLA-A2:NY-ESO-1 antibodies described in Table 1); (b) a hinge region derived from human CD8α; (c) a human CD8α transmembrane domain; and (d) a human T cell receptor CD3 ζ 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 in the following order from amino to carboxyl terminus: a binding domain, a hinge region, and a transmembrane domain. The intracellular signaling domain and the optional costimulatory signaling domain are connected in series to the transmembrane carboxyl terminus in any order to form a single-chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding HLA-A2:NY-ESO-1CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule, and the nucleic acid molecule comprises different coding sequences, for example, human anti-HLA-A2:NY-ESO-1scFv, human CD8α hinge, human CD8α transmembrane domain, CD137 co-stimulatory domain and CD3ζ intracellular signaling domain coding sequence (5' to 3'). In another embodiment, the nucleic acid construct encoding HLA-A2:NY-ESO-1CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule, and the nucleic acid molecule comprises different coding sequences, for example, human anti-HLA-A2:NY-ESO-1scFv, human CD8α hinge, human CD8α transmembrane domain, CD137 co-stimulatory domain and CD3ζ co-stimulatory domain coding sequence (5' to 3'). In certain embodiments, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences, for example, human anti-HLA-A2:NY-ESO-1 scFv, human CD8α hinge, human CD8α transmembrane domain, CD137 co-stimulatory domain, and CD3ζ co-stimulatory domain coding sequence (5' to 3'), wherein the anti-HLA-A2:NY-ESO-1 scFv comprises a V selected from the group consisting of H : SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 142, 162, 180, 196, 211, 230 and 250; or wherein the anti-HLA-A2:NY-ESO-1 scFv comprises a V selected from the group consisting of L: SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150, 170, 186, 203, 219, 238 and 258; or wherein the anti-HLA-A2:NY-ESO-1 scFv comprises a V selected from the group consisting of H / V L Pairs: SEQ ID NO: 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. In some embodiments, the present disclosure includes nucleic acid molecules encoding HLA-A2:NY-ESO-1 CARs selected from the sequences of Table 2.

[0183] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be covalently inserted so as to cause expression of the protein and / or cloning of the polynucleotide. Such vectors may also be referred to as "expression vectors". The isolated polynucleotides may be inserted into a vector using any suitable method known in the art, such as, but not limited to, the vector may be digested with an appropriate restriction enzyme, and then it may be connected to an isolated polynucleotide with matching restriction ends. The expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence encoding 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 protein. The expression vector may contain a variety of control sequences, which refer to the transcription and possible translation of the operably connected coding sequence in a specific host organism. In addition to the control sequences for controlling transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions, and are discussed below. The expression vector may include additional elements, for example, the expression vector may have two replication systems so that it is maintained in two organisms, such as expressed in human cells and cloned and amplified in a prokaryotic host.

[0184] The expression vector may have necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences such as CMV, PGK and EF1α promoters, ribosome recognition and binding TATA box, and 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), 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 considered to be part of the vector expressing the chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters or tetracycline promoters.

[0185] The expression vector may have additional sequences, such as 6x-histidine (SEQ ID NO: 292), c-Myc and FLAG tags, which are incorporated into the expressed CAR. Therefore, the expression vector may be engineered to include 5' and 3' non-translated regulatory sequences that may sometimes be used as enhancer sequences, promoter regions and / or terminator sequences, which may promote or enhance the effective transcription of the target nucleic acid carried on the expression vector. The expression vector may also be engineered for replication and / or expression function (e.g., transcription and translation) in a specific cell type, cell position or tissue type. The expression vector may include a selectable marker for maintaining the vector in a host or recipient cell.

[0186] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes (PAC) derived from P1, phages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus classes that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are Lenti-X for expression in mammalian cells. TMBicistronic expression system (Neo) vector (Clontrch), pClneo vector (Promega); pLenti4 / V5-DEST.TM., pLenti6 / V5-DEST.TM. and pLenti6.2N5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. The coding sequence of CAR disclosed herein can be connected to such expression vectors for expression of chimeric proteins in mammalian cells.

[0187] In certain embodiments, the nucleic acid encoding the CAR of the present disclosure is provided in a viral vector. The viral vector may be those derived from a retrovirus, a lentivirus or a foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may contain the coding sequence of various chimeric proteins described herein to replace non-essential viral genes. The vector and / or particle may be used for the purpose of transferring DNA, RNA or other nucleic acids to cells in vitro or in vivo. Various forms of viral vectors are known in the art.

[0188] In certain embodiments, the viral vector comprising the CAR coding sequence described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector containing structural and functional genetic elements mainly derived from retroviruses. The term "lentiviral vector" refers to a vector containing structural and functional genetic elements mainly derived from lentiviruses outside the LTR.

[0189] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend virus (Friend), murine stem cell virus (MSCV) and Rous sarcoma virus (RSV)). "Retroviruses" of the present disclosure also include human T-cell leukemia viruses HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV) and other types of retroviruses.

[0190] Lentiviral vectors for use herein refer to vectors derived from a group (or genus) of retroviruses that cause slowly progressing diseases. The viruses included in this group include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); ovine madie-visna disease; caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). Recombinant lentiviruses can be prepared using methods according to Dull et al. and Zufferey et al. (Dull et al., Journal of Virology (J. Virol.), 1998; 72: 8463-8471 and Zufferey et al., Journal of Virology (J. Virol.), 1998; 72: 9873-9880).

[0191] Retroviral vectors (i.e., lentiviral and non-lentiviral) for use in the present disclosure can be generated using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, FM et al.) (English Dialect Society) Greene Publishing Association Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals; Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) PNAS 85:6460-6464; Wilson et al. (1988) PNAS 85:3014-3018; Armentano et al. (1990) PNAS 87:6141-6145; Huber et al. (1991) PNAS 88:8039-8043; Ferry et al. (1991) PNAS 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0192] Suitable sources for obtaining retroviral (i.e., lentiviral and non-lentiviral) sequences for use in forming vectors include, for example, genomic RNA and cDNA available from commercially available sources including the American Type Culture Collection (ATCC), Rockville, Md. The sequences may also be chemically synthesized.

[0193] For the expression of HLA-A2:NY-ESO-1CAR, the vector can be introduced into the host cell to allow the polypeptide to be expressed in the host cell. The expression vector may contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers and signal sequences. As described above, these elements may be appropriately selected by those of ordinary skill in the art. For example, a promoter sequence may be selected to promote transcription of polynucleotides in the vector. Suitable promoter sequences include but are not limited to T7 promoters, T3 promoters, SP6 promoters, β-actin promoters, EF1a promoters, CMV promoters and SV40 promoters. Enhancer sequences may be selected to enhance transcription of polynucleotides. Selectable markers may be selected to allow host cells to be selected from host cells without inserting the vector to insert the vector, for example, selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside the host cell.

[0194] In order to clone a polynucleotide, the vector can be introduced into a host cell (isolated host cell) to allow replication of the vector itself, and thereby increase the copy of the polynucleotide contained therein. The cloning vector can contain sequence components, which generally include but are 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 a person of ordinary skill in the art. For example, a replication origin can be selected to promote the autonomous replication of the vector in the host cell.

[0195] In certain embodiments, the disclosure provides a host cell containing an isolation of a vector provided herein. The host cell containing the vector can be used to express or clone the 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, true bacteria such as gram-negative or gram-positive organisms; Enterobacteriaceae such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus; Salmonella, such as Salmonella typhimurium; Serratia such as Serratia marcescens and Shigella; and Bacillus such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa and Streptomyces.

[0196] 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 of introducing exogenous nucleic acid sequences into host cells. Nucleic acids can be integrated into host cell DNA or can be maintained outside the chromosome. Nucleic acids can be transiently maintained or can be stably introduced. Transfection can be accomplished by various methods 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 use of a virus or retroviral vector to deliver one or more genes by viral infection rather than by transfection. In certain embodiments, a retroviral vector is transduced by packaging the vector into a virion before contacting the cell. For example, the nucleic acid encoding anti-HLA-A2:NY-ESO-1CAR carried by a retroviral vector can be transduced into the cell by infection and proviral integration.

[0197] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0198] In particular, the CARs of the present disclosure are introduced and expressed in immune effector cells in order to redirect their specificity to a target antigen of interest, such as a conformational epitope of the NY-ESO-1 peptide displayed by HLA-A2, e.g., amino acid residues 157-165.

[0199] The present disclosure provides a method for preparing immune effector cells expressing CAR as described herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from a subject, such as a subject with NY-ESO-1 related diseases or disorders, so that immune effector cells express one or more CARs described herein. In certain embodiments, immune effector cells are separated from an individual and genetically modified without further manipulation in vitro. These cells can then be directly re-applied to the same individual (described autologous therapy) or different individuals (described allogeneic therapy). In other embodiments, immune effector cells are first activated and stimulated in vitro to proliferate, and then genetically modified to express CAR. In this regard, immune effector cells can be cultured before or after genetic modification (i.e., transduced or transfected to express CAR as described herein).

[0200] Prior to in vitro manipulation or genetic modification of immune effector cells described herein, cell sources can be obtained from subjects. In particular, immune effector cells used with CAR described herein include T cells. Such recombinant T cells are referred to herein as "T bodies".

[0201] In one embodiment of the present disclosure, the T body includes a CAR of the present disclosure, the CAR comprising: an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3ζ or FcRγ), and / or a costimulatory signaling domain derived from a costimulatory molecule such as, but not limited to, CD28, CD137, CD134, or CD278. In another embodiment of the present disclosure, the T body includes a CAR of the present disclosure, the CAR comprising: an extracellular target-specific binding domain, a transmembrane domain, a hinge or spacer between an extracellular binding domain and a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3ζ or FcRγ), and / or one or more costimulatory signaling domains derived from costimulatory molecules. In another embodiment of the present disclosure, the T body includes a T body construct CAR, which includes 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 may comprise a Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv) of an antigen binding protein of the present disclosure.

[0202] There are many sources for obtaining T cells, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments, any number of techniques known to technicians (such as FICOLL separation) can be used to obtain T cells from a unit of blood collected from a subject. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in a suitable buffer or culture medium for subsequent processing. In one embodiment of the present disclosure, cells are washed with PBS. In an alternative embodiment, the washing solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. As will be understood by those skilled in the art, the washing step can be achieved by methods known to those skilled in the art (e.g., by using a semi-automated flow-through centrifuge). After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffers.In certain embodiments, undesirable components of the apheresis sample may be removed in the medium in which the cells are directly resuspended.

[0203] In certain embodiments, the monocytes are depleted by lysing the red blood cells (e.g., by using PERCOLL TM Gradient centrifugation) is used to separate T cells from peripheral blood mononuclear cells (PBMC). Specific T cell subsets, such as CD28+, CD4+, CD8+, CD45RA+ and CD45RO+T cells, can be further separated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be accomplished by a combination of antibodies for surface markers specific to cells selected negatively. A method for use herein is to perform cell sorting and / or cell selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies, which are directed to cell surface markers present on cells selected negatively. For example, in order to enrich CD4+ cells by negative selection, a monoclonal antibody mixture typically includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to separate target cell populations for use in the present disclosure.

[0204] PBMC can be used directly for genetic modification with CAR using the methods described herein. In certain embodiments, after the separation of PBMC, T lymphocytes are further separated, and in certain embodiments, cytotoxic and helper T lymphocytes can be classified into primary, memory and effector T cell subsets before or after genetic modification and / or amplification. CD8+ cells can be obtained by using standard methods. In certain embodiments, by identifying cell surface antigens associated with each of these types of CD8+ cells, CD8+ cells are further classified into primary cells, central memory cells and effector cells. In an embodiment, memory T cells are present in CD62L+ and CD62L- subgroups of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are divided into CD62L-CD8+ and CD62L+CD8+ fractions. In certain embodiments, the expression of phenotypic markers of central memory TCM 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 the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0205] In certain embodiments, CD4+T cells are further classified into subgroups. For example, by identifying a cell population with a cell surface antigen, CD4+T helper cells can be divided into naive cells, central memory cells, and effector cells. 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 positive for CD62L and CD45RO. In some embodiments, effector CD4+ cells are negative for CD62L and CD45RO.

[0206] Immune effector cells such as T cells can be genetically modified using known methods after separation, or immune effector cells can be activated and amplified in vitro (or differentiated in the case of progenitor cells) before genetic modification. In another embodiment, immune effector cells such as T cells are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector comprising a nucleic acid encoding CAR), and then activated and amplified in vitro. Methods for activating and amplifying T cells are known in the art and described, such as U.S. Patent No. 6,905,874; U.S. Patent No. 6,867,041; U.S. Patent No. 6,797,514; WO2012079000, US 2016 / 0175358. Typically, such methods are included in a culture medium with appropriate cytokines such as IL-2, and PBMC or separated T cells are contacted with stimulators and co-stimulators (such as, anti-CD3 and anti-CD28 antibodies) that are generally attached to beads or other surfaces. Anti-CD3 and anti-CD28 antibodies attached to the same beads act as "surrogate" antigen presenting cells (APCs). In other embodiments, methods such as those described in U.S. Pat. No. 6,040,177; U.S. Pat. No. 5,827,642; and WO2012129514 can be used to activate and stimulate T cells to proliferate with feeder cells and appropriate antibodies and cytokines.

[0207] The present disclosure provides a population of modified immune effector cells for treating a NY-ESO-1 related disease or disorder, such as cancer, comprising an HLA-A2:NY-ESO-1 CAR as disclosed herein.

[0208] Immune effector cells expressing CAR prepared as described herein can be used in methods and compositions of adoptive immunotherapy according to known techniques or variations thereof that are obvious to those skilled in the art based on the present disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; see also U.S. Patent No. 4,690,915 to Rosenberg.

[0209] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a therapeutically effective amount in a culture medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0210] The therapeutically effective amount of cells in the composition is at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset) or more typically greater than 10 2 cells, and up to 10 6 cells, including 10 8 or 10 9 cells, and can exceed 10 10 The number of cells will depend on the desired end use of the composition, as will the type of cells included therein.

[0211] Cells can be autologous or allogeneic for patients undergoing therapy. If desired, treatment may also include administration of mitogens (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.) as described herein to enhance the induction of immune responses.

[0212] The immune effector cell group expressing CAR disclosed herein can be administered alone, or as a pharmaceutical composition combined with a diluent and / or with other components (such as IL-2) or other cytokines or cell groups. In short, the pharmaceutical composition disclosed herein may include immune effector cell groups expressing CAR, such as T cells as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) and preservatives. The disclosed compositions are preferably formulated for intravenous administration.

[0213] The anti-tumor immune response induced in a subject by administering the T cells expressing CAR described herein using the methods described herein or other methods known in the art may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. It is also possible to induce a humoral immune response mediated mainly by helper T cells that can activate B cells to cause antibody production. A variety of techniques can be used to analyze the type of immune response induced by the compositions disclosed herein, which are well described in the art; for example, Current Protocols in Immunology, edited by John E.Coligan, Ada M.Kruisbeek, David H.Margulies, Ethan M.Shevach, Warren Strober (2001) John Wiley and Sons (New York) NY.

[0214] Thus, the present disclosure provides methods of treating an individual diagnosed with, suspected of having, or at risk of developing a NY-ESO-1-related disease or disorder, such as a NY-ESO-1-positive cancer, comprising administering to the individual a therapeutically effective amount of an immune effector cell expressing a CAR as described herein.

[0215] In one embodiment, the present disclosure provides a method of treating a subject diagnosed with NY-ESO-1 positive cancer, comprising removing immune effector cells from a 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 generating a population of modified immune effector cells, and administering the modified population of immune effector cells to the same subject. In one embodiment, the immune effector cells comprise T cells.

[0216] Methods for administering cell compositions described herein include any method having the following effects: reintroducing into a subject an immune effector cell genetically modified ex vivo that directly expresses the CAR disclosed herein; or reintroducing into a subject a genetically modified progenitor cell of an immune effector cell that differentiates into a mature immune effector cell expressing the CAR when introduced into the subject. A method comprises transducing peripheral blood T cells ex vivo using a nucleic acid construct according to the present disclosure and returning the transduced cells to the subject.

[0217] Therapeutic administration and formulations

[0218] The present disclosure provides therapeutic compositions comprising anti-HLA-A2:NY-ESO-1 antigen binding proteins (e.g., antibodies), or antigen binding fragments thereof or CARs of the present disclosure. The therapeutic compositions according to the present disclosure will be administered together with suitable carriers, excipients, and other agents, all of which are incorporated into the formulation to improve transfer, delivery, tolerance, and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic lipids or anionic lipids) (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See, e.g., Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) "Journal of Pharmaceutical Science and Technology ( ...

[0219] The dosage of antigen binding protein (e.g., antibody) or its antigen binding fragment can vary according to the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antigen binding protein of the present disclosure is used to treat a disease or disorder in an adult patient, or for preventing such a disease, it is generally advantageous to administer the antigen binding protein (e.g., antibody) or its antigen binding fragment of the present disclosure in a single dose of about 0.1 to about 60 mg / kg body weight, more preferably about 5 to about 60 mg / kg body weight, about 20 to about 50 mg / kg body weight, about 10 to about 50 mg / kg body weight, about 1 to about 10 mg / kg body weight, or about 0.8 to about 11 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antigen binding protein (e.g., antibody) or its antigen binding fragment of the present disclosure can be administered in 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 10 to about 100 mg, or about 10 to about 50 mg. In certain embodiments, a second or more subsequent doses of an antigen binding protein (e.g., antibody) or antigen binding fragment thereof may be administered after an initial dose in approximately the same or less amount than the initial dose, wherein 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.

[0220] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, 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 composition can be administered by any convenient route, for example, by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0221] It is also contemplated herein to use nanoparticles to deliver antigen-binding proteins (e.g., antibodies) or antigen-binding fragments thereof of the present disclosure. Antigen-binding protein-conjugated nanoparticles can be used for therapeutic and diagnostic applications. Arruebo, M. et al., 2009 described in detail antigen-binding protein-conjugated nanoparticles and preparation and use methods ("Antibody-conjugated nanoparticles for biomedical applications", Journal of Nanomaterials (J. Nanomat) 2009, Article ID: 439389, 24 pages, Digital Object Identifier: 10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can also be developed and conjugated to antigen-binding proteins contained in pharmaceutical compositions to target tumor cells or autoimmune tissue cells or virally infected cells. Nanoparticles for drug delivery have also been described in, for example, U.S. Patent No. 8,257,740 or U.S. Patent No. 8,246,995, each of which is incorporated herein in its entirety.

[0222] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, a controlled release system can be placed near the target of the composition, so only a portion of the systemic dose is required.

[0223] Injection can include intravenous injection, subcutaneous injection, intradermal injection, intracranial injection, intraperitoneal injection, intramuscular injection, drip and other dosage forms. These injectable preparations can be prepared by well-known methods. For example, injectable preparations can be prepared by, for example, dissolving, suspending or emulsifying the above-mentioned antigen-binding protein or its salt in a sterile aqueous medium or a conventional oily medium for injection. As aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers, such as alcohol (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (adduct of polyethylene oxide (50mol) and hydrogenated castor oil)), etc. As oily medium, sesame oil, soybean oil, etc., are used, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

[0224] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily used to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered, and the cartridge is empty, 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 a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0225] Many reusable pen and auto-injector delivery devices have applications in subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are by no means limited to, AUTOPEN, to name a few. TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Co. Indianapolis), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM PEN (Becton Dickinson, Franklin Lakes, New Jersey), OPTIPEN TM 、OPTIPENPRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany). To name a few, examples of disposable pen delivery devices that may be used for subcutaneous delivery of pharmaceutical compositions useful according to the methods described herein include, but are in no way limited to: SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPENTM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM PEN (Abbott Labs, Abbott Technology Park, IL).

[0226] Advantageously, the pharmaceutical composition for oral or parenteral use is prepared into a unit dosage form suitable for matching a certain dose of active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In a unit dose, the content of the above antigen-binding protein is usually about 5 to about 500 mg / dosage form; particularly in an injection dosage form, the content of the above antigen-binding protein is preferably about 5 to about 100 mg, and in other dosage forms, it is about 10 to about 250 mg.

[0227] Therapeutic uses of antigen binding proteins

[0228] The antibodies of the present disclosure are particularly useful for treating, preventing and / or ameliorating any disease or condition associated with or mediated by NY-ESO-1. The present disclosure provides methods for treating NY-ESO-1-associated diseases or conditions, such as NY-ESO-1-associated cancers (e.g., NY-ESO-1-positive cancers) (tumor growth inhibition) by administering 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 to a patient in need thereof and for treating NY-ESO-1-associated cancers (tumor growth inhibition) by administering 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). The antigen binding proteins of the present disclosure are useful for treating, preventing and / or ameliorating a disease or condition or pathology, such as NY-ESO-1-associated cancer and / or for ameliorating at least one symptom associated with such a disease, disorder or pathology. In the context of the treatment methods described herein, the anti-HLA-A2:NY-ESO-1 antigen binding proteins can be administered as monotherapy (ie, as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0229] In some embodiments, the antibodies described herein can be used to treat a subject with 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 tumors, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cavity cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin 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.

[0230] The antigen binding proteins can be used to treat early or late symptoms of NY-ESO-1-related cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure can be used to treat advanced or metastatic cancer. The antigen binding proteins can be used to reduce or inhibit or shrink 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% tumor regression in a subject. In certain embodiments, the antigen binding proteins can be used to prevent tumor recurrence. In certain embodiments, the antigen binding proteins can be used to extend progression-free survival or overall survival of a subject with NY-ESO-1-related cancer. In some embodiments, the antibodies can be used to reduce toxicity caused by chemotherapy or radiation therapy while maintaining long-term survival of patients with NY-ESO-1-related cancer.

[0231] One or more antibodies of the disclosure may be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

[0232] Also contemplated herein is the prophylactic use of one or more antibodies of the disclosure in a patient 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.

[0233] In another embodiment of the present disclosure, the antibodies of the present invention are used to prepare a pharmaceutical composition for treating a patient suffering from a NY-ESO-1 related disease or disorder, such as a NY-ESO-1 related cancer. In another embodiment of the present disclosure, the antibodies of the present invention are used as an adjuvant therapy together with any other agent or any other therapy known to those skilled in the art that can be used to treat a NY-ESO-1 related cancer.

[0234] Combination therapies and formulations

[0235] Combination therapy may include an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure, such as a CAR of the present disclosure (e.g., an immune effector cell comprising a CAR of the present disclosure) or a pharmaceutical composition of the present disclosure, and any additional therapeutic agent that may be advantageously combined with the antigen binding protein of the present disclosure. The antigen binding protein of the present disclosure may be synergistically combined with one or more anticancer drugs or therapies for treating or inhibiting NY-ESO-1-related diseases or conditions, such as NY-ESO-1-positive cancers, including, for example: 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 tumors, Glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, epithelial ovarian cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin 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.

[0236] It is contemplated herein to use the anti-HLA-A2:NY-ESO-1 antigen binding proteins disclosed herein in combination with immunostimulatory and / or immune supportive therapies to inhibit tumor growth and / or improve the survival of cancer patients. Immunostimulatory therapies include direct immunostimulatory therapies, which enhance immune cell activity by "releasing the brakes" or "stepping on the gas" to activate immune responses against suppressed immune cells. Examples include targeting other checkpoint receptors, vaccinations, and adjuvants. Immune support modalities can increase the antigenicity of tumors by promoting immunogenic cell death, inflammation, or having other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0237] In various embodiments, one or more antigen binding proteins of the present disclosure can be used 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 REGN2810); 3504); a CTLA-4 inhibitor (e.g., ipilimumab); a TIM3 inhibitor; a BTLA inhibitor; a TIGIT inhibitor; a CD47 inhibitor; a GITR inhibitor; an antagonist of another T-cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA); an indoleamine-2,3-dioxygenase (IDO) inhibitor; a vascular endothelial growth factor (VEGF) antagonist (e.g., a "VEGF-Trap" such as Aflibercept or US Other fusion proteins that inhibit VEGF as described in 7,087,411, 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., nesvacumab); transforming growth factor β (TGFβ) inhibitors; epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab, ximab); 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., Bacillus Calmette-Guérin, cancer vaccines); adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor); bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies); 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 drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs); dietary 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 invention can 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 cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers). ;

[0238] In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be administered in combination with radiation therapy in methods of producing a long-lasting anti-tumor response and / or improving the survival rate of cancer patients. In some embodiments, the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure can be administered before, simultaneously with, or after the administration of radiation therapy to a cancer patient. For example, radiation therapy can be administered to a tumor lesion in one or more doses, followed by administration of one or more doses of the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure. In some embodiments, local radiation therapy can be performed on the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablative radiation), followed by systemic administration of the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure. For example, intracranial radiation can be administered to a patient with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure. In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure may be administered in combination with radiation therapy and chemotherapeutic agents (eg, temozolomide) or VEGF antagonists (eg, aflibercept).

[0239] The additional therapeutically active agent / component may be administered prior to, concurrently with, or after administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. For the purposes of the present disclosure, such administration regimens are considered to be administration of an anti-HLA-A2:NY-ESO-1 antigen binding protein "in combination" with a second therapeutically active ingredient.

[0240] The additional therapeutically active component may be administered to the subject prior to administration of the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure. For example, a first component may be considered to be administered "before" a second component if the first component is administered 1 week prior to administration of the second component, 72 hours prior to administration, 60 hours prior to administration, 48 hours prior to administration, 36 hours prior to administration, 24 hours prior to administration, 12 hours prior to administration, 6 hours prior to administration, 5 hours prior to administration, 4 hours prior to administration, 3 hours prior to administration, 2 hours prior to administration, 1 hour prior to administration, 30 minutes prior to administration, 15 minutes prior to administration, 10 minutes prior to administration, 5 minutes prior to administration, or less than 1 minute prior to administration of the second component. In other embodiments, the additional therapeutically active component may be administered to the subject after administration of the anti-HLA-A2:NY-ESO-1 antigen binding proteins of the present disclosure. For example, if the first component 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 the administration of the second component, then the first component can be considered to be administered "after" the second component. In yet other embodiments, the additional therapeutically active component can be administered to the subject simultaneously with the administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure. For purposes of the present disclosure, "concurrent" administration includes, for example, administering the anti-HLA-A2:NY-ESO-1 antigen binding protein and the additional therapeutically active ingredient to the subject in a single dosage form (e.g., co-formulated) or in separate dosage forms within about 30 minutes or less. If administered in separate dosage forms, each dosage form may be administered by 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 by different routes (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 case, for the purposes of this disclosure, administration of the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes is considered "simultaneous administration." For the purposes of this disclosure, administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein "before," "concurrently," or "after" the additional therapeutically active ingredient (as those terms are defined above) is considered administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein "in combination with" the additional therapeutically active ingredient.

[0241] 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 additional therapeutically active components as described elsewhere herein using various dosage combinations.

[0242] Dosage regimen

[0243] According to certain embodiments of the present disclosure, multiple doses of an 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 other therapeutically active agent mentioned herein) may be administered to a subject within a defined period of time. The method according to this aspect of the present disclosure comprises sequentially administering multiple doses of an anti-HLA-A2:NY-ESO-1 antigen binding protein of the present disclosure to a subject. "Sequential administration" as used herein means administering each dose of an anti-HLA-A2:NY-ESO-1 antigen binding protein to the subject at different time points (e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months)). The methods described herein comprise sequentially administering to the patient a single initial dose of an anti-HLA-A2:NY-ESO-1 antigen binding protein, followed by one or more second doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein, and optionally subsequently administering one or more third doses of the anti-HLA-A2:NY-ESO-1 antigen binding protein. The anti-HLA-A2:NY-ESO-1 antigen binding protein can be administered at a dose between 0.1 mg / kg and 100 mg / kg of the subject's body weight.

[0244] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the temporal order of administration of the anti-HLA-A2:NY-ESO-1 antigen binding protein useful herein. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary dose" is the dose administered after the initial dose; and the "tertiary dose" is the dose administered after the second dose. The initial, second and third doses may all contain the same amount of anti-HLA-A2:NY-ESO-1 antigen binding protein, but may generally differ from one another in terms of the frequency of administration. However, in certain embodiments, the amount of anti-HLA-A2:NY-ESO-1 antigen binding protein contained in the initial, second and / or third doses differs from one another during the course of treatment (e.g., adjusted upward or downward as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered at the beginning of the treatment regimen as "loading doses", followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0245] In certain embodiments, the amount of anti-HLA-A2:NY-ESO-1 antigen binding protein contained in the initial, second and / or third dose may be suboptimal or subtherapeutic. As used herein, the term "subtherapeutic" or "suboptimal" refers to an antibody dose that is administered at a very low level to produce a therapeutic effect or an antibody dose that is below the level required to treat a disease, such as cancer.

[0246] In certain exemplary embodiments of the present disclosure, each second and / or third dose is administered 1 to 26 weeks after the immediately preceding dose (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 As used herein, the phrase "immediately preceding dose" means that in a sequence of multiple administrations, a dose of anti-HLA-A2:NY-ESO-1 antigen binding protein is given to the patient prior to administration of the next dose in the sequence, without an intervening dose.

[0247] The method according to this aspect of the present disclosure may comprise administering to the patient any number of second and / or third doses of an anti-HLA-A2:NY-ESO-1 antigen binding protein. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0248] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks or 1 to 2 months immediately after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 12 weeks immediately after the previous dose. In certain embodiments, the frequency of administering the second and / or third dose to the patient can vary during the course of the treatment regimen. The frequency of administration can also be adjusted according to the needs of individual patients after clinical examination during the course of treatment by a physician.

[0249] Diagnostic Uses of Antigen Binding Proteins

[0250] 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 an assay to detect a disease or condition, such as a NY-ESO-1-associated disease or condition, such as a NY-ESO-1-positive cancer. An exemplary diagnostic assay for NY-ESO-1 can comprise, 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, wherein 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 separate NY-ESO-1 from the subject sample. Alternatively, an unlabeled anti-HLA-A2:NY-ESO-1 antigen binding protein can be combined with a second antigen binding protein (e.g., an antibody) that is itself detectably labeled for diagnostic applications. The detectable label or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125I; 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).

[0251] Samples useful for NY-ESO-1 diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a subject that contains a detectable amount of NY-ESO-1 protein or fragments thereof under normal or pathological conditions. Typically, the level of NY-ESO-1 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a NY-ESO-1-related disease or condition, such as a NY-ESO-1-positive cancer) will be measured to initially establish a baseline or standard level of NY-ESO-1. The baseline level of NY-ESO-1 can then be compared to the level of NY-ESO-1 measured in a sample obtained from an individual suspected of having a cancer-related disease or symptoms associated with such a disease.

[0252] Antigen binding proteins specific for NY-ESO-1 may not contain additional tags or moieties, or they may contain N-terminal or C-terminal tags or moieties. In one embodiment, the tag or moiety is biotin. In a binding assay, the position of the tag, if any, can determine the orientation of the peptide relative to the surface to which the peptide binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide will be distal to the surface.

[0253] 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 cancers in patients. The antigen binding proteins of the present disclosure can be used in diagnostic assays to assess the prognosis of cancer in patients and predict survival.

[0254] Examples

[0255] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0256] Example 1: Generation of human antibodies against HLA-A2:NY-ESO-1_157-165

[0257] Human antibodies to HLA-A2:NY-ESO-1 were generated using an HLA-A2-conjugated NY-ESO-1 peptide fragment of GenBank Accession No. NP_001318.1 (SEQ ID NO:271), which includes amino acids 157-165 (SLLMWITQV; "NY-ESO-1_V"); SEQ ID NO:291) in which the cysteine ​​(C) at position 165 is substituted with valine (V). For example, as described in U.S. Pat. No. 8,502,018, the immunogen is administered directly to the subject together with an adjuvant that stimulates an immune response. The invention relates to the administration of human immunoglobulin heavy chain and κ light chain variable regions to mice (i.e., engineered mice containing DNA encoding human immunoglobulin heavy chain and κ light chain variable regions). The antibody immune response is monitored by an HLA-A2:NY-ESO-1 specific immunoassay. When the desired immune response is achieved, the spleen cells are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines are screened and selected to identify cell lines that produce HLA-A2:NY-ESO-1 specific antibodies. Using this technology and the above-mentioned immunogen, several anti-NY-ESO-1 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) are obtained.

[0258] As described in U.S. Pat. No. 7,582,298, which is incorporated herein by reference in its entirety, anti-HLA-A2:NY-ESO-1 antibodies were also isolated directly from antigen-positive B cells (from either immunized mouse) without fusion with myeloma cells. Using this approach, several fully human anti-HLA-A2:NY-ESO-1 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained.

[0259] Exemplary antibodies generated according to the foregoing methods are designated as follows: mAb24955N; mAb24956N; mAb24958N; mAb24959N; mAb28042P; mAb28035P; mAb28037P2; mAb28075P; mAb28105P; mAb28113P; mAb28128P; mAb29814P; mAb24955N; and mAb29822P2.

[0260] The biological properties of exemplary antibodies produced according to the methods of this example are described in detail in the examples set forth below.

[0261] Example 2: Heavy and light chain variable region amino acid and nucleotide sequences

[0262] Table 1 lists the heavy and light chain variable regions, CDRs, and amino acid sequence identifiers of the heavy and light chains of selected anti-HLA-A2:NY-ESO-1 antibodies of the present disclosure. Table 2 lists the corresponding nucleic acid sequence identifiers.

[0263] Table 1: Amino Acid Sequence Identifiers

[0264]

[0265] Table 2: Nucleotide sequence identifiers

[0266]

[0267] Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (e.g., "mAb", etc.), followed by a numerical identifier (e.g., "17670", "17930", etc., as shown in Table 1), and then 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 appreciated by one of ordinary skill in the art, an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted into an antibody with a human IgG4, etc.), but in any case, the variable domains (including CDRs) represented by the numerical identifiers shown in Table 1 will remain unchanged, and the binding properties to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0268] In certain embodiments, a selected antibody with a mouse IgG1 Fc is converted to an antibody with a human IgG4 Fc. In certain embodiments, the antibody comprises a human IgG4 Fc with 2 or more amino acid changes, as disclosed in U.S. Patent Publication No. 20100331527 (incorporated herein in its entirety). In one embodiment, the IgG4 Fc domain comprises a serine to proline mutation in the hinge region (S108P) to promote dimer stability.

[0269] Table 3 lists the amino acid sequence identifiers of the heavy and light chain sequences of selected antibodies of the present disclosure.

[0270] Table 3: Heavy and light chain sequence identifiers

[0271]

[0272] *As described in U.S. Patent 9,359,437.

[0273] Example 3: Surface Plasmon Resonance-Derived Binding Affinity and Kinetic Constants of Human Monoclonal Anti-HLA-A2:NY-ESO-1 Monospecific Antibodies

[0274] Binding affinities and kinetic constants of human anti-HLA-A2:NY-ESO-1 antibodies were determined by real-time surface plasmon resonance (SPR; Biacore 4000 from GE Healthcare Life Sciences, or MASS-1 from Sierra Sensors) at 25°C and 37°C. The anti-HLA-A2:NY-ESO-1 antibodies tested in this example are bivalent monospecific binders of HLA-A2:NY-ESO-1 (expressed with a constant region with reduced effector function (e.g., hIgG4 constant region) (e.g., as described in U.S. Pat. No. 9,359,437), hIgG2a constant region, mIgG2a constant region, or mIgG1 constant region). Reference (Ref) antibodies 1, 2, and 3 were generated by antibodies described in Stewart-Jones et al., Proc. Natl. Acad. Sci. USA 106(14):5784-5788 (2009) and International Patent Publication No. WO2010106431. Ref Ab 1 contained the VH and VL domains of the 3M4E5 antibody with human IgG1 Fc and human lambda light chain described in those publications; Ref Ab 2 contained the 3M4E5 VH domain and T1 Fab VL domain with human IgG1 Fc and human lambda light chain from said publication; Ref Ab 3 contained the 3M4E5 VH domain and T1 Fab VL domain with human IgG4 (as described in U.S. Pat. No. 9,359,437) with reduced effector function Fc and human lambda VL domain from said publication. 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 on a high capacity amine sensor surface (Sierra Sensors) derivatized by amine coupling with a polyclonal anti-mouse Fc antibody (GE Healthcare Life Sciences). Various concentrations of monomeric HLA-A2:NY-ESO-1 (156-165) (SEQ ID NO: 270 or 291; V at position 165) peptide complexes and 2906 (SEQ ID NO: 272) were injected onto the anti-HLA-A2:NY-ESO-1 antibody capture surface at a flow rate of 50 μL / min (MASS-1) or 30 μL / min (Biacore 4000). Antibody-reagent association was monitored for 4 to 5 minutes and dissociation was monitored for 10 minutes. 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).

[0275] 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 (t 1 / 2) is calculated based on the kinetic rate constant:

[0276] K D (M) = (kd / ka) and t 1 / 2(min)=((ln (2)) / (60*kd))

[0277] The binding kinetic parameters of the monospecific anti-HLA-A2:NY-ESO-1 antibody and the monomeric HLA-A2:NY-ESO-1 peptide complex are shown in Table 4 below.

[0278] Table 4: Biacore binding affinity of anti-HLA-A2:NY-ESO-1 antibodies at 25°C

[0279]

[0280] Table 5: Biacore binding affinity of anti-HLA-A2:NY-ESO-1 antibodies at 37°C

[0281]

[0282] The data demonstrate that most of the tested anti-HLA-A2:NY-ESO-1 antibodies selectively bind to soluble HLA-A2:NY-ESO-1 peptide complexes, with some displaying nanomolar or subnanomolar affinities and many having higher affinity for the complexes than the reference antibody.

[0283] Example 4: FACS binding of anti-HLA-A2:NY-ESO-1 antibody to NY-ESO-1_157-165 peptide-pulsed T2 cells

[0284] The relative binding of NY-ESO-1:157-165 antibody was evaluated by flow cytometry on T2 (174CEM.T2) cells pulsed with NY-ESO-1_157-165 (C165) peptide (SEQ ID NO:269). To achieve the pulse, T2 cells (174CEM.T2) were cultured at 1×10 6Cells were resuspended in AIM V medium at a density of 10 μg / ml (fetal bovine serum Cat#31035-025). Cells were pulsed by adding 10 μg / ml hB2M (EMD Millipore Cat#475828) and 100 μg / ml of the indicated peptides. T2 cells were then incubated overnight at 37°C, washed in staining buffer and subsequently stained.

[0285] To stain cells, cells were harvested from flasks using cell dissociation buffer (Millipore, Cat# S-004-C) and counted. In 96-well V-bottom plates, cells were plated at a density of 200,000 cells per well in staining buffer (PBS without calcium and magnesium (Corning, Ref#21-031-CV) + 2% FBS (Seradigm, Lot#238B15) and stained with 3-fold serial dilutions (1.7 pM-100 nM) of primary antibody at 4°C for 30 minutes. After primary antibody incubation, cells were washed once in staining buffer and then stained with APC-conjugated secondary antibody (Jackson ImmunoResearch, Cat#109-136-170) at 5 μg / ml for 30 minutes at 4°C. Cells were then washed and fixed using 50% BD Cytofix solution (BD, Cat#554655). Samples were analyzed on an intelliciyt iQue flow cytometer to calculate mean fluorescence intensity (MFI). MFI values ​​were plotted on a 12-point response curve using a four-parameter logistic equation in Graphpad Prism to calculate EC 50 Values. Secondary antibody alone (i.e., no primary antibody) for each dose-response curve was also included in the analysis as a continuation of the three-fold serial dilutions and is expressed as the lowest dose. Ref Ab 2 and Ref Ab 3 were used as controls as described above. EC 50 The values ​​(M) are shown in Table 6.

[0286] Table 6: FACS binding of anti-HLA-A2:NY-ESO-1 antibodies

[0287] Antibody <![CDATA[EC of the T2 pulse NY-ESO-1_157-165)]]> 50 (M)]]> mAb24955N 6.00E-10 mAb24956N 4.70E-10 mAb24958N 1.90E-08 mAb24959N 1.10E-09 mAb28035P 2.70E-09 mAb28037P2 7.80E-10 mAb28042P 1.60E-09 mAb28075P 3.80E-10 mAb28105P 1.80E-09 mAb28113P 6.00E-10 mAb28128P 3.20E-10 mAb29814P 1.10E-09 mAb29822P2 5.40E-09 Reference Ab 2 2.20E-10 Reference Ab 3 4.90E-10

[0288] Example 5: FACS binding of anti-HLA-A2:NY-ESO-1 antibodies to T2 cells pulsed with predicted off-target peptides

[0289] The binding specificity of the NY-ESO-1:157-165 antibody was evaluated by flow cytometry analysis of T2 (174CEM.T2) cells pulsed with NY-ESO-1 (157-165) peptide (C165; SEQ ID NO: 269) or predicted off-target peptides (Table 7). To achieve the pulse, T2 cells (174CEM.T2) were cultured at 1×106 The cells were resuspended in AIM V medium at a density of 10 μg / ml (fetal bovine serum Cat#31035-025). The cells were pulsed by adding 10 μg / ml hB2M (EMD Millipore Cat#475828) and 100 μg / ml of the indicated peptides. T2 cells were then incubated overnight at 37°C, washed in staining buffer, and stained with the indicated antibodies at a concentration of 10 μg / ml according to the protocol described in Example 4 above. MFI values ​​were calculated and expressed as the ratio of T2 pulsed cells to unpulsed cells. The results of these assays are listed in Table 8. Effective peptide loading was determined by comparing the increase in HLA-A2 surface staining of cell lines pulsed with anti-HLA-A2 antibodies to unpulsed cell lines. Any increase of 1.4-fold or more was considered to be loaded with peptide. Loading of one peptide (ITCH (807-815)) could not be confirmed. However, as described above, two comparator NY-ESO-1 mAbs, Ref Ab 1 and Ref Ab 2, bound to cells pulsed with the peptide, indicating that some peptide had been loaded onto the cells.

[0290] Table 7: NY-ESO-1_157-165 peptide and predicted off-target peptides

[0291]

[0292]

[0293] Table 8: Ratio of anti-HLA-A2:NY-ESO-1 antibody binding to T2 cells pulsed with off-target peptides versus unpulsed cells

[0294]

[0295]

[0296] As seen in Table 8, many of the tested antibodies were determined to have no significant binding to T2 cells pulsed with any of the predicted off-target peptides, particularly compared to the two comparative antibodies, which had significant binding to multiple off-target peptides. Non-specific binding may result in reduced therapeutic efficacy and / or increased side effects (e.g., reduced tumor cell killing activity and / or non-specific cytotoxicity that causes side effects in a subject). Therefore, as described herein, identification of antigen binding proteins (e.g., antibodies) with minimal off-target binding may be beneficial in the development of therapeutic agents targeting MAGE-A4.

[0297] 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 peptides (Table 7) and stained with the NY-ESO-1 (157-165) antibody as described above. Table 9 shows the fold change of unpulsed cells. All peptides except NY-ESO-1: 157-165, C165V, S157A and NY-ESO-1: 157-165, C165V, I162A were effectively loaded by HLA.A2 surface staining (as described above). The following residues were important for binding of the tested anti-NY-ESO-1 mAbs (defined as a 90% or greater reduction in binding): Leucine 158, Tryptophan 161, Threonine 163, and Glutamine 164. As described above, Threonine 163 and Glutamine 164 were dispensable for binding of the comparative monoclonal antibody Ref Ab 3. Methionine 160 is particularly important for the binding of mAb24956N, mAb24958N, and mAb28105P.

[0298] Table 9: Ratio of anti-HLA-A2:NY-ESO-1 antibody binding to T2 cells pulsed with alanine scanning peptides versus unpulsed cells

[0299]

[0300]

[0301] Example 6: Reformatting anti-HLA-A2:NY-ESO-1 antibodies into ScFv for chimeric antigen receptors

[0302] Four NY-ESO-1 (157-165) antibodies (mAb24955N, mAb24956N, mAb24958N, and mAb24959N) were reformatted into VL-VH or VH-VL single-chain variable fragment chimeric antigen receptors (CARs) using the CD8α hinge, transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ stimulatory domain. NY-ESO-1:157-165CAR was cloned into a lentiviral expression vector (Lenti-X TMBicistronic Lentivirus Expression System, Clontech Cat#632181) and lentiviral particles were generated by Lenti-X Packaging Single-Shot (VSV-G) System (Clontech Cat#631276) according to the manufacturer's protocol. Jurkat / NFATLuc cl.3C7 cells (cells expressing the NFAT-luciferase reporter gene) were then transduced with 8 different CARs using RetroNectin pre-coated culture dishes (Clontech, Cat#T110a) according to the manufacturer's protocol. After selection with 500 μg / ml G418 (Gibco, Cat#11811-098) for at least 2 weeks, 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-T24955N 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; VL-VH;Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24959N VH-VL;With Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24959N VL-VH.The activity of the CAR-T cell line was then evaluated in a CAR-T / APC (antigen presenting cell) bioassay.For bioassay, 50,000 CAR-T cells were added to 50 ml of assay medium (RPMI medium containing 10% FBS and 1% P / S / G) in a Thermo-Nunc 96-well white plate (ThermoScientific, Cat#136101), and then 3-fold serial dilutions of APC (200,000 cells to 274 cells) were added to the 50 ml assay medium.The following APCs were used: 3T3 / HLA.A2 / hB2M / NY-ESO-1:157-165WT (NIH3T3 cells engineered to express human HLA.A2 (Accession No. P01892), human B2M (Accession No. NP_004039.1), and a ubiquitin peptide cassette (such as the ubiquitin peptide cassette described above), wherein the ubiquitin peptide cassette comprises the NY-ESO-1:157-165 peptide, 3T3 / HLA.A2 / hB2M / HPV16E7 11-19 (Lévy F et al. (1996) Proc. Natl. Acad. Sci. USA 93(10):4907-4912; Valmori D et al. (1999) J Exp. Sci. Med.)》189(6):895-906); containing HPV16E7:11-19 peptide, IM9 (HLA.A2 positive, NY-ESO-1:157-165 positive) and HEK293 (HLA.A2 positive and NY-ESO-1:157-165 negative). The cell mixture was cultured in a humidified incubator at 37°C, 5% CO2 for 5 hours. NFAT-luciferase activity was measured using Promega One-Glo (Cat#E6130) and Perkin Elmer Envision plate reader. Relative luciferase units (RLU) were generated and plotted on an 8-point response curve using a four-parameter logistic equation to calculate EC. 50 Value. The zero APC condition of each dose-response curve is also included in the analysis as a continuation of three-fold serial dilutions and is expressed as the lowest dose. The maximum activation multiple is determined by the ratio of the highest RLU to the lowest RLU on the curve. In the presence of 3T3 / HLA.A2 / hB2M / NY-ESO-1:157-165 WT cells, all eight NY-ESO-1CAR cell lines are activated. As shown in Table 10, three NY-ESO-1CAR cell lines are activated in the presence of the following IM9 cells (Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24955N VH-VL; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T24955N VL-VH; and Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VL-VH).

[0303] Table 10: Activation of NY-ESO-1:157-165 CAR-T in CAR-T / APC Bioassay

[0304]

[0305]

[0306] Example 7: Structural Analysis of Fabs Binding to HLA2:NY-ESO-1_157-165 Peptide

[0307] To better understand the specific interactions between the antibody and the HLA-peptide complex, the X-ray crystal structure of the Fab fragment of the antibody mAb28105P, bound to HLA-A2 / hB2M, displaying a peptide comprising residues 157-165 from cancer testis antigen 1 (CTAG1B; NY-ESO-1) was determined. The peptide was modified by replacing residue 165 of its original cysteine ​​with valine. All nine residues of the HLA-displayed NY-ESO-1 (C165V) peptide are clearly visible in the electron density map of the structure, and the residues of the HLA and Fab surrounding the peptide are also well resolved. This structure is shown in Figure 2. The model was refined at a resolution of 1.3 GHz, but newer crystallographic refinement techniques (deformable elastic network or “jelly-body” refinement) help prevent overfitting and ensure the accuracy of the resulting model.

[0308] The mAb28105P Fab binds to the top of the HLA-peptide complex in a manner similar to how TCRs bind. The Fab is 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 are located at the center of the Fab binding interface, contacting the heavy and light chain CDR residues as described below. Other published structures of grooved peptide-antibody complexes (e.g., PDB codes 1W72 and 4WUU) indicate that antibodies do not have to cover the entire HLA-displayed peptide; however, antibodies with only partial peptide coverage are less specific and can tolerate large changes in the uncontacted portion of the peptide with little loss in binding affinity.

[0309] The structure shows that the mAb28105P Fab heavy chain contacts residues 160, 161 and 164 in the NY-ESO-1 peptide bound to HLA, while the Fab light chain contacts residues 160 and 161. Peptide residues 157, 158, 159, 162 and 165 all point toward the HLA molecule. The Fab light chain completely screens residue 163 from the solvent but does not make direct contacts with the antibody residues. The binding peptide is numbered according to the residue positions in SEQ ID NO: 271, as shown in SEQ ID NO: 291:

[0310] Amino Acids S L L M W I T Q V Location 157 158 159 160 161 162 163 164 165

[0311] (SEQ ID NO:291)

[0312] All Fab contacts are made with the side chains of the HLA-binding peptide and not its backbone.

[0313] The peptide contacts made by mAb28105P Fab are concentrated in HCDR3, with small 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) interact with the binding peptide, while Fab heavy chain residues 100, 101, 107 and 109 (SEQ ID NO: 162) and light chain residue 92 (SEQ ID NO: 170) interact with HLA. As used herein, the term "interaction" herein may include direct or water-mediated hydrogen bonds, charge-charge interactions, or hydrophobic / van der Waals interactions.

[0314] Four of the NY-ESO-1_157-165 antibodies (mAb24955N, mAb24956N, mAb24958N, and mAb24959N) are highly similar in light chain sequence, but differ in heavy chain sequence in HCDR2 and HCDR3. Seven peptide binding residues are generally conserved: three residues are identical in all four antibodies, and more than three of the four sequences are identical, indicating a common binding mode between the antibodies.

[0315] Example 8: Analysis of recombinant antigen receptor signaling domains

[0316] Chimeric Antigen Receptor Using Anti-HLA-A2 / NY-ESO-1 157-165 Antibody mAb28105P (V L :SEQ ID NO:294; V H : SEQ ID NO: 293) L and V H The chimeric antigen receptor contains V H -V L Anti-HLA-A2 / NY-ESO-1 157-165scFv; and 1) human CD8 (huCD8) hinge / transmembrane domain, 4-1BB costimulatory domain and CD3ζ signaling domain (BB / z CAR) (full-length CAR sequence: SEQ ID NO: 301); or 2) huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR) (full-length CAR sequence: SEQ ID NO: 302). As a non-binding control, BB / z CAR was designed using an unrelated scFv plus huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain and CD3z signaling domain. These CARs were cloned into pLVX lentiviral vectors with EF1a promoter and P2A: eGFP sequence (SEQ ID NO: 300) for tracking CAR-transduced cells and producing VSV pseudotyped lentivirus. Figure 1A For construct design, Table 11 is a summary of the constructs.

[0317] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) from normal donors, stimulated with CD3 / CD28 microbeads + 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI = 5. Transduced cells were expanded with CD3 / CD28 microbeads + 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use during in vivo experiments.

[0318] To determine the in vivo efficacy of anti-HLA-A2 / NY-ESO-1157-165 targeted chimeric antigen receptor (CAR) T cells, a xenograft study was performed. scid Iq tm1Wjl / SzJ (NSG) mice were injected subcutaneously with 5×10 6 HLA-A2 + NY-ESO-1 + A375 human melanoma tumor cells. On day 3 after tumor formation, mice (n=5 per group) were intravenously injected with 20×10 6 T cells expressing non-binding control BB / zCAR (control CART), anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR, or anti-HLA-A2 / NY-ESO-1 from two different donors 157-165 28 / z CAR (as determined by the frequency of cells expressing GFP, a marker for those cells that were transduced with CAR). Tumor growth was assessed on day 21 by measuring tumor volume.

[0319] To determine tumor volume by external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were determined. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0320] Table 11: CAR constructs

[0321]

[0322] Overall, the results showed that anti-HLA-A2 / NY-ESO-1 157-165 Compared with BB / z CAR T cells, anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR T cells showed superior in vivo antitumor activity and antitumor kinetics, but both types of CARs showed antitumor activity compared to non-binding controls. A375 tumors gradually grew in mice receiving control CAR T cells. Compared with control mice receiving CAR T treatment, mice receiving anti-HLA-A2 / NY-ESO-1 157-165 Mice receiving BB / zCAR T cells had slower tumor growth at day 21 (p<0.04), day 28 (p<0.0001), and day 38 (p<0.0001) (statistical data analyzed by 2-way ANOVA). 157-165 28 / z CAR T treatment also resulted in inhibition 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) (statistical data analyzed by 2-way ANOVA). 157-165 28 / z CAR and anti-HLA-A2 / NY-ESO-1 157-165 Enhanced efficacy of BB / z CAR, as tumor size was statistically significant on days 21 (p=0.002), 28 (p<0.0001), and 38 (p<0.0001) by 2-way ANOVA (p<0.0001 on two days). Figure 1B and Figure 1C , and Tables 12 to 22.

[0323] Table 12: CAR Efficacy, Day 3

[0324]

[0325] Table 13: CAR Efficacy, Day 5

[0326]

[0327] Table 14: CAR Efficacy, Day 7

[0328]

[0329] Table 15: CAR Efficacy, Day 10

[0330]

[0331] Table 16: CAR Efficacy, Day 12

[0332]

[0333] Table 17: CAR Efficacy, Day 14

[0334]

[0335] Table 18: CAR Efficacy, Day 16

[0336]

[0337] Table 19: CAR Efficacy, Day 19

[0338]

[0339] Table 20: CAR Efficacy, Day 21

[0340]

[0341] Table 21: CAR Efficacy, Day 28

[0342]

[0343] Table 22: CAR Efficacy, Day 38

[0344]

[0345] The present invention should not be limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will also become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. A human monoclonal antibody or antigen-binding fragment thereof that specifically binds to a conformational epitope of a NY-ESO-1 peptide complex presented by HLA-A2, The human monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the heavy chain complementary determining region 1 of SEQ ID NO:4, the heavy chain complementary determining region 2 of SEQ ID NO:6, and the heavy chain complementary determining region 3 of SEQ ID NO:8, and the light chain variable region comprises the light chain complementary determining region 1 of SEQ ID NO:12, the light chain complementary determining region 2 of SEQ ID NO:14, and the light chain complementary determining region 3 of SEQ ID NO:

16. 2 . The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 , wherein the heavy chain variable region / the light chain variable region comprises SEQ ID NO: 2 / 10.

3. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the human monoclonal antibody or antigen-binding fragment thereof interacts with amino acids M160, W161 and Q164 of the NY-ESO-1 157-165 peptide, and the interaction is through a resolution of or higher as determined by X-ray crystallography.

4. The human monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the resolution is or higher.

5. The human monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the resolution is or higher.

6. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which specifically binds to the NY-ESO-1 157-165 peptide complex presented by HLA-A2, but does not specifically bind to one or more off-target peptides selected from the group consisting of SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 277 and SEQ ID NO: 280, wherein the ratio of i) the human monoclonal antibody or antigen-binding fragment thereof bound to cells pulsed with the one or more off-target peptides to ii) unpulsed cells is less than 9.

7. The human monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein the ratio is less than 8.

8. The human monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein the ratio is less than 7.

9. The human monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the ratio is less than 6.

10. The human monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the ratio is less than 5. The human monoclonal antibody or antigen-binding fragment thereof according to claim 10 , wherein the ratio is less than 4.

12. The human monoclonal antibody or antigen-binding fragment thereof according to claim 11, wherein the ratio is less than 3.

13. The human monoclonal antibody or antigen-binding fragment thereof according to claim 12, wherein the ratio is less than 2.

14. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the human monoclonal antibody or antigen-binding fragment thereof binds to the monomeric HLA-A2-presented NY-ESO-1 157-165 peptide complex with an association dissociation equilibrium constant (K) of less than 1 nM. D ) binding, the binding dissociation equilibrium constant (K D ) was measured in a surface plasmon resonance assay at 25°C.

15. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the NY-ESO-1 peptide comprises an amino acid sequence of SLLMWITQC (SEQ ID NO: 269) or SLLMWITQV (SEQ ID NO: 291).

16. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv or T-body construct. The human monoclonal antibody or antigen-binding fragment thereof according to claim 16 , wherein the human monoclonal antibody or antigen-binding fragment thereof is a single-chain Fv.

18. A chimeric antigen receptor comprising the human monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

19. The chimeric antigen receptor of claim 18, wherein the chimeric antigen receptor comprises a heavy chain variable region, a light chain variable region, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain.

20. The chimeric antigen receptor of claim 19, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

21. The chimeric antigen receptor of claim 19, wherein the costimulatory domain is a CD28 costimulatory domain.

22. The chimeric antigen receptor of claim 18, wherein the human monoclonal antibody or antigen-binding fragment thereof is a single chain Fv.

23. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a detectable moiety.

24. A pharmaceutical composition comprising the human monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 17 or the chimeric antigen receptor according to any one of claims 18 to 22 and a pharmaceutically acceptable carrier or diluent.

25. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy chain variable region and / or light chain variable region according to any one of claims 1 and 3 to 17. A vector comprising the polynucleotide molecule of claim 25 .

27. A cell expressing the polynucleotide molecule according to claim 25 or the vector according to claim 26.

28. Use of the human monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 and 3 to 17, or the chimeric antigen receptor according to any one of claims 18-22, or the pharmaceutical composition according to claim 24, or the cell according to claim 27 in the preparation of a medicament for treating melanoma in a subject.

29. The use according to claim 28, wherein the human monoclonal antibody or antigen-binding fragment thereof, or the chimeric antigen receptor, or the pharmaceutical composition, or the cell is used in combination with a second therapeutic agent.

30. The use according to claim 29, 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 virus-infected cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, a bispecific antibody against CD20 and CD3, a dietary supplement, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, an NSAID, and a corticosteroid.

31. The use according to claim 28, wherein the human monoclonal antibody or antigen-binding fragment thereof, or the chimeric antigen receptor, or the pharmaceutical composition, or the cell is used in combination with surgery, radiotherapy, and any other therapy that can be used to improve at least one symptom associated with the melanoma.

32. The use according to claim 30, wherein the dietary supplement is an antioxidant.

33. The use according to any one of claims 28 to 31, wherein the human monoclonal antibody or antigen-binding fragment thereof, or the chimeric antigen receptor, or the pharmaceutical composition, or the cell is for subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular or intracranial administration.

34. The use according to any one of claims 28 to 31, wherein the human monoclonal antibody or antigen-binding fragment thereof, or the chimeric antigen receptor, or the pharmaceutical composition, or the cell is for administration at a dose of 0.1 mg / kg to 100 mg / kg of the subject's body weight.

35. The isolated polynucleotide molecule of claim 25, wherein the human monoclonal antibody or antigen-binding fragment thereof is a single chain Fv.

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