Anti-Newton Esophageal Ssquamous Cell Cancer 1 (NY-ESO-1) antigen binding proteins and methods of use thereof

Abstract

The present disclosure provides antigen binding proteins that specifically bind to the Newton esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide displayed by the HLA, as well as therapeutic and diagnostic methods of using these binding proteins. 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 from 2, 3, 4, 5 or more amino acids.

Description

[0001] This application is a divisional application of the patent application with application number 202080061474.0, application date July 2, 2020, and title "Anti-New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen-binding proteins and methods of use thereof".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 870,232, filed July 3, 2019, U.S. Provisional Patent Application No. 63 / 020,177, filed May 5, 2020, and U.S. Provisional Patent Application No. 63 / 021,826, filed May 8, 2020. The entire contents of each of the above patent applications are incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on July 2, 2020, named 118003_10520_SL.txt, and is 245,227 bytes in size. Technical Field

[0006] 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 to therapeutic and diagnostic methods using those binding proteins. Background Art

[0007] New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a cancer-testis antigen (CTA), also known as CTAG1B. Expression of NY-ESO-1, encoded by the CTAG1B gene, is restricted to germ cells. However, NY-ESO-1 is frequently aberrantly expressed by different tumor types. The major protein product of the CTAG1B gene is an 18 kDa protein that is 180 amino acids long and has a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. However, the function of NY-ESO-1 remains unclear.

[0008] Spontaneous CD8 + 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, it has been found that the NY - ESO - 1 peptides 157 - 165, 157 - 167, and 155 - 163 are restricted by HLA - A2 in tumor - reactive CD8 + T cell lines, while the peptide 56 - 62 is recognized by HLA - A31 CD8 + T cells (Jager et al. (1998) J Exp Med 187:265 - 270; Wang, R.F. et al. (1998) J Immunol 161:3598 - 3606). Several epitopes restricted by HLA - DR4 in CD4 T cell responses have also been demonstrated, and the response to the peptide 157 - 170 is restricted by HLA - DP4, an allele found in the majority of Caucasians (Jager et al. (2000) J Exp Med 191:625 - 630; Zang et al. (2001) Proc Natl Acad Sci USA 98:3964 - 3969).

[0009] The ability of NY - ESO - 1 to elicit spontaneous humoral and cellular immune responses, together with its restricted expression profile, makes it an attractive candidate target for cancer therapy. Although the NY - ESO - 1 antigen has been evaluated as a cancer vaccine candidate, little complete humoral and cellular immune response has been achieved. In fact, using NY - ESO - 1 as a therapeutic target is highly challenging because it is difficult to design antigen - binding proteins that target HLA - presented antigens and the need to avoid off - target binding to prevent non - specific binding that may lead to 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 the subject).

[0010] Accordingly, the need in the art for new therapeutic strategies that specifically target NY - ESO - 1 and treat NY - ESO - 1 - associated cancers remains unmet. SUMMARY OF THE INVENTION

[0011] The present disclosure provides antigen-binding proteins that specifically bind to conformational epitopes of HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptides. The antigen-binding proteins of the present disclosure bind to HLA-presented NY-ESO-1 with a high degree of specificity, but do not bind to HLA-presented peptides that differ by 2, 3, 4, 5, or more amino acids. The antigen-binding proteins of the present disclosure allow for the specific targeting of NY-ESO-1 peptide-presenting cells (i.e., cells that present NY-ESO-1 peptides bound to MHC molecules (such as 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 the diagnosis and prognosis of NY-ESO-1-positive diseases or disorders that are highly sensitive to changes in the number and distribution of NY-ESO-1 peptide-presenting cells, which is a more relevant measure of disease progression than circulating NY-ESO-1 levels.

[0012] The antigen-binding proteins of the present disclosure can be antibodies, such as full-length (e.g., IgG1 or IgG4 antibodies) antibodies, or can comprise only the antigen-binding portion of an antibody (e.g., Fab, F(ab')2, or scFv fragments), and can be modified to affect functionality, e.g., to eliminate residual effector function (Reddy et al. (2000) J. Immunol. 164:1925-1933). In some embodiments, the antigen-binding proteins of the present disclosure can be antibodies or antigen-binding fragments thereof. In certain embodiments, the antigen-binding protein can be bispecific.

[0013] In a first aspect, the present disclosure provides a recombinant antigen-binding protein that specifically binds to a conformational epitope of an HLA-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide (such as an HLA-presented peptide comprising amino acid residues 157-165 of NY-ESO-1). In certain embodiments, the antigen-binding protein is an antibody. In some embodiments, the antibody is a fully human antibody.

[0014] 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 regions (HCDR1, HCDR2, and HCDR3)), and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3)) of exemplary anti-HLA-A2:NY-ESO-1 antibodies. Table 2 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.

[0015] The present disclosure provides an antigen-binding protein comprising an HCVR, the HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1 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%, at least 99%, at least 99.5%, at least 99.9%, or 100% sequence identity to the amino acid sequence. In some embodiments, the antigen-binding protein with less than 100% sequence identity comprises the CDR sequences of the HCVR selected from 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.

[0016] The present disclosure provides an antigen-binding protein comprising an LCVR, the LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1 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%, at least 99%, at least 99.5%, at least 99.9%, or 100% sequence identity to the amino acid sequence. In some embodiments, the antigen-binding protein with less than 100% sequence identity comprises the CDR sequences of the LCVR selected from 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.

[0017] The present disclosure also provides an antigen-binding protein comprising an HCVR and an LCVR amino acid sequence pair (HCVR / LCVR), wherein the HCVR and LCVR amino acid sequence pair comprises 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 an antigen-binding protein comprising an HCVR / LCVR amino acid sequence pair, wherein the HCVR / LCVR amino acid sequence pair is comprised 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).

[0018] In certain embodiments, the present disclosure provides an anti-HLA-A2:NY-ESO-1 antigen-binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 with no more than five amino acid substitutions, and the LCVR comprises 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 an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence shown in SEQ ID NO:2 with no more than five amino acid substitutions, and the LCVR comprises the amino acid sequence shown 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 an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence shown in SEQ ID NO:2 with at least one amino acid substitution, and the LCVR comprises the amino acid sequence shown in SEQ ID NO:10 with at least one amino acid substitution.

[0019] 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 of the HCDR1s listed in Table 1 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have a sequence identity of 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%.

[0020] 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 of the HCDR2s listed in Table 1 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have a sequence identity of 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%.

[0021] 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 of the HCDR3s listed in Table 1 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have a sequence identity of 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%.

[0022] 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 of the LCDR1s listed in Table 1 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have a sequence identity of 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%.

[0023] 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 of the LCDR2s listed in Table 1 or a sequence substantially similar thereto, and 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 light chain CDR3 (LCDR3), wherein the light chain CDR3 comprises an amino acid sequence selected from any of the LCDR3s listed in Table 1 or a sequence substantially similar thereto, and 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.

[0025] The present disclosure also provides an antigen-binding protein comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), wherein the HCDR3 and LCDR3 amino acid sequence pair comprises 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 an HCDR3 / LCDR3 amino acid sequence pair, wherein the HCDR3 / LCDR3 amino acid sequence pair is comprised in any of the exemplary anti-HLA-A2:NY-ESO-1 antigen-binding proteins listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO:8 / 16 (e.g., mAb24955N), 28 / 36 (e.g., mAb24956N), 48 / 56 (e.g., mAb25958N), and 68 / 76 (e.g., mAb24959N).

[0026] The present disclosure also provides an antigen-binding protein comprising an HCVR and an LCVR, wherein the HCVR comprising 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 an antigen-binding protein comprising an HCVR and an LCVR, wherein the LCVR comprising 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 an HCVR and an LCVR, wherein the HCVR comprises: HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:4; HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:6; and HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:8. In another exemplary embodiment, the present disclosure provides an antigen-binding protein comprising an HCVR and an LCVR, wherein the HCVR comprises: LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:12; LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:14; and LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence that differs by 1 amino acid from SEQ ID NO:16.

[0027] The present disclosure also provides antigen-binding proteins that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) from any of the exemplary antigen-binding proteins listed in Table 1. In certain embodiments, the group of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences 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).

[0028] In related embodiments, the present disclosure provides antigen-binding proteins that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) from an HCVR / LCVR amino acid sequence pair defined by any of the exemplary antigen-binding proteins listed in Table 1. For example, the present disclosure includes antigen-binding proteins that comprise the group of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences from an 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).

[0029] 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 the CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to define the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, 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 methods. See, e.g., 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.

[0030] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins having a modified glycosylation profile. In some embodiments, modifications that remove unwanted glycosylation sites, or antibodies that have a fucose moiety deletion on the oligosaccharide chain, may be beneficial, for example, in increasing antibody-dependent cell cytotoxicity (ADCC) function (see, e.g., Shield et al. (2002) "J. Biol. Chem." 277:26733). In other applications, modifications of galactosylation can be made to modify complement-dependent cytotoxicity (CDC).

[0031] In certain embodiments, the antigen-binding proteins of the present disclosure are monoclonal antibodies comprising a pair of HCVR and LCVR amino acid sequences (HCVR / LCVR), wherein the pair of HCVR and LCVR amino acid sequences comprises any 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.

[0032] The present disclosure provides an antigen-binding protein comprising a heavy chain or an antigen-binding fragment thereof, wherein the heavy chain comprises an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have 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 with less than 100% sequence identity comprises the CDR sequences of the HC in Table 3. For example, such an antigen-binding protein may comprise these CDR sequences but has differences in the framework regions compared to the HC in Table 3.

[0033] The present disclosure provides an antigen-binding protein comprising a light chain or an antigen-binding fragment thereof, wherein the light chain comprises an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3 or a sequence substantially similar thereto, and the substantially similar sequence and the amino acid sequence have a sequence identity of 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%. In some embodiments, the antigen-binding protein with less than 100% sequence identity comprises the CDR sequences of the LC in Table 3. For example, such an antigen-binding protein may comprise these CDR sequences but has differences in the framework regions compared to the LC in Table 3.

[0034] The present disclosure further provides an antigen-binding protein comprising an HC and LC amino acid sequence pair (HC / LC) or an antigen-binding fragment thereof, wherein the HC and LC amino acid sequence pair comprises any pairing of an 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 an antigen-binding fragment thereof comprising an HC / LC amino acid sequence pair, wherein the HC / LC amino acid sequence pair is comprised 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.

[0035] On the one hand, the present disclosure provides an antigen-binding protein or an antigen-binding fragment thereof that binds to an HLA-peptide complex, wherein the antigen-binding protein or the 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 the antigen-binding fragment thereof "covers" or contacts all of the amino acid residues of the peptide contained in the HLA-peptide complex. In certain embodiments, the antigen-binding protein or the antigen-binding fragment thereof binds to the HLA-peptide complex with high affinity and specificity, wherein the antigen-binding protein or the 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 the antigen-binding fragment thereof binds to an 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 concentrated substantially on the peptide in the peptide-binding groove of HLA-A2, thereby "covering" (physically blocking) the HLA-A2-peptide complex. In another embodiment, the antigen-binding protein or the antigen-binding fragment thereof binds to an 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 concentrated substantially on the peptide in the peptide-binding groove of HLA-A2, thereby "covering" (physically blocking) the HLA-A2-peptide complex. In certain embodiments, the antigen-binding protein or the antigen-binding fragment thereof comprises 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. 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.

[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 shown in SEQ ID NO: 269. In one embodiment, the antigen-binding protein binds to at least 3 amino acids shown in SEQ ID NO: 269.

[0037] 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 shown in SEQ ID NO: 270 or 291. In one embodiment, the antigen-binding protein binds to at least 3 amino acids shown 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 shown in SEQ ID NO: 270.

[0038] In certain embodiments, the present disclosure provides an 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, wherein the conformational epitope comprises one or more amino acids shown in SEQ ID NO: 269.

[0039] In certain embodiments, the present disclosure provides an 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, wherein the conformational epitope comprises one or more amino acids shown 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 shown in SEQ ID NO: 270 or 291.

[0040] The present disclosure also provides an antigen-binding protein that competes with an antigen-binding protein comprising the CDRs of HCVR and the CDRs of LCVR for specific binding to HLA-A2:NY-ESO-1, wherein 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 an antigen-binding protein that cross-competes with a reference antigen-binding protein comprising the CDRs of HCVR and the CDRs of LCVR for binding to HLA-A2:NY-ESO-1, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0042] The present disclosure also provides an antigen-binding protein that binds to the same epitope as a reference antigen-binding protein comprising the CDRs of HCVR and the CDRs of LCVR, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present disclosure provides an antigen-binding protein that binds to the same epitope as a reference antigen-binding protein comprising the CDRs of HCVR and the CDRs of LCVR, wherein HCVR is selected from the group consisting of: SEQ ID NO:2, 22, 42, and 62, and LCVR is selected from the group consisting of: SEQ ID NO:10, 30, 50, and 70.

[0043] In one embodiment, the present disclosure provides a recombinant antigen-binding protein that specifically binds to the conformational epitope of the HLA-A2-presented New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide, wherein the antigen-binding protein has the following properties selected from the group consisting of: (a) binding dissociation equilibrium constant (K D ) less than about 1 nM as measured in a surface plasmon resonance assay at 25 °C with monomeric HLA-A2:NY-ESO-1 157-165 peptide complex (C165 or V165); (b) EC 50 less than about 10 nM as measured in a flow cytometry assay with cells expressing the HLA-A2:NY-ESO-1 157-165 peptide complex, but not binding to cells expressing a predicted off-target peptide; and (c) the epitope comprises one or more amino acids shown in SEQ ID NO:269, 270, or 291. As disclosed elsewhere herein, an "off-target peptide" refers to a peptide that differs from the target peptide (e.g., 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.

[0044] In a second aspect, the present disclosure provides a nucleic acid molecule encoding an anti-HLA-A2:NY-ESO-1 antigen-binding protein. For example, the present disclosure provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2 or a substantially similar sequence, and 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.

[0045] 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 thereto, and 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 HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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 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 thereto, and 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 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 thereto, and 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 of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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 of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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 nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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.

[0052] The present disclosure also provides nucleic acid molecules encoding HCVR, wherein HCVR comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), and 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.

[0053] The present disclosure also provides nucleic acid molecules encoding LCVR, wherein LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of LCDR1-LCDR2-LCDR3 amino acid sequences is defined by any exemplary anti-HLA-A2:NY-ESO-1 antigen-binding protein listed in Table 1.

[0054] The present disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, wherein HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 1, and wherein LCVR comprises the 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 of the HCVR nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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; and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2 or a substantially similar sequence thereto, and 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 of this aspect according to the present disclosure, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-HLA-A2:NY-ESO-1 antigen-binding protein listed in Table 1.

[0055] The present disclosure provides nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in Table 3. The present disclosure also provides nucleic acid molecules encoding any of the light chain amino acid sequences listed in Table 3.

[0056] The present disclosure also provides nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), wherein HC comprises the amino acid sequence of any one of the HC amino acid sequences listed in Table 3, and wherein LC comprises the amino acid sequence of any one of the LC amino acid sequences listed in Table 3.

[0057] In relevant aspects, the present disclosure provides recombinant expression vectors capable of expressing polypeptides comprising heavy and / or light chain variable regions of an anti-HLA-A2:NY-ESO-1 antigen-binding protein. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences listed in Table 1. The present disclosure also provides recombinant expression vectors capable of expressing polypeptides comprising heavy and / or light chain polypeptides of an anti-HLA-A2:NY-ESO-1 antigen-binding protein. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the heavy or light chain sequences listed in Table 2. Also within the scope of the present disclosure are host cells into which such vectors have been introduced, and methods of producing an antigen-binding protein by culturing the host cells under conditions permitting the production of the antigen-binding protein and recovering the antigen-binding protein so produced.

[0058] In a third aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a recombinant antigen-binding protein, and a pharmaceutically acceptable carrier, wherein the recombinant antigen-binding protein specifically binds to a conformational epitope of an NY-ESO-1 peptide presented by HLA-A2 (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1). In relevant aspects, the present disclosure features a composition that is a combination of an anti-HLA-A2:NY-ESO-1 antigen-binding protein and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-HLA-A2:NY-ESO-1 antigen-binding protein. Exemplary agents that can be advantageously combined with the anti-HLA-A2:NY-ESO-1 antigen-binding protein include, but are not limited to, other agents that modulate the activation of immune cells. Additional therapies that can be used in combination with the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure are disclosed elsewhere herein.

[0059] In a fourth aspect, the present disclosure provides a method of treating a subject having a NY-ESO-1 related disease or disorder, 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 disorder being treated is any disease or disorder that is ameliorated, improved, 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 in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent can be selected from the group consisting of: an antibody to a T cell co-inhibitor, an antibody against a tumor cell antigen, an antibody against a T cell receptor, a cytotoxic agent, an anticancer drug, an anti-inflammatory drug (e.g., corticosteroid), a chemotherapeutic agent, surgery, radiation therapy, an immunosuppressant, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent can 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.

[0060] In certain embodiments, the present disclosure provides a method for inhibiting the growth of a NY-ESO-1 related cancer. For example, the present disclosure provides a method for inhibiting tumor growth resulting from a primary or metastatic tumor in a subject. In certain embodiments, the present disclosure provides a method for increasing the survival rate (e.g., progression-free survival rate or overall survival rate) of a subject having a NY-ESO-1 related cancer. Examples of cancers include, but are not limited to: liposarcoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, breast cancer, astrocytic tumor, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, malignant tumor, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid, round cell liposarcoma, metastatic melanoma, or recurrent non-small cell lung cancer.

[0061] In certain embodiments, the present disclosure provides a method for arresting 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.

[0062] An antigen-binding protein (such as an antibody) or an antigen-binding fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The antigen-binding protein (such as an antibody) or an antigen-binding fragment thereof can be administered at a dose of from about 0.1 mg / kg of the subject's body weight to about 100 mg / kg of the subject's body weight.

[0063] 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 the CAR on its surface). The CAR can include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to a conformational epitope of a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide presented 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 of the present disclosure are any of the antigen-binding proteins described herein.

[0064] For example, in certain embodiments, the antigen-binding protein suitable for the CAR of the present disclosure comprises: three heavy-chain complementarity-determining regions CDR (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.

[0065] In other embodiments, the antigen-binding protein suitable for the CAR of the present disclosure comprises: an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and / or an HCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

[0066] In some embodiments, the antigen-binding protein suitable for the CAR of the present disclosure comprises: (a) an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and (b) an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

[0067] In one embodiment, the antigen-binding protein of a CAR suitable for the present disclosure comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213, and 232; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 182, 199, 215, and 234; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, and 236; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, and 240; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, and 242; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224, and 244.

[0068] In a further embodiment, the antigen-binding protein of a CAR suitable for the present disclosure comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 42 / 50, 62 / 70, 82 / 90, 102 / 110, 122 / 130, 142 / 150, 162 / 170, 180 / 186, 196 / 203, 211 / 219, and 230 / 238.

[0069] In some embodiments, the antigen-binding protein for use in a CAR of the present disclosure is an scFv.

[0070] In other aspects, the present disclosure provides a vector comprising an isolated CAR nucleic acid molecule; and an immune effector cell comprising such a vector.

[0071] In other aspects of the present disclosure, methods are provided for treating a subject having an NY-ESO-1 related disease or disorder, such as an NY-ESO-1 positive cancer, the NY-ESO-1 related disease or disorder being, 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 tumors, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant tumors, myelodysplastic syndromes, 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. The method includes administering to the subject a population of immune effector cells comprising a CAR of the present disclosure.

[0072] In some aspects, the present disclosure provides methods for detecting NY-ESO-1 positive cells in, for example, a subject or a sample obtained from a subject. The method includes: 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 the subject, and detecting the presence of the detectable moiety.

[0073] Other embodiments will become apparent by reading the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1A Diagrams depicting three different CAR constructs, including promoters and vector elements.

[0075] Figure 1B Depicts the tumor volumes of mice treated with T cells 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 28 / z CAR from 0 to 21 days, where the left panel: mean tumor volume; the right panel: tumor volume of individual mice.

[0076] Figure 1C Depicts the tumor volumes of mice treated with T cells 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 with T cell treatment of any of the 28 / z CARs from 0 to 38 days, where the left panel shows the average tumor volume and the right panel shows the tumor volume of individual mice. Detailed implementation

[0077] Before describing the method, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are now described, but any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference in their entirety.

[0079] 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 re-expressed in multiple cancer types and encoded by the CTAG1B gene.

[0080] The amino acid sequence of full-length NY-ESO-1 is provided in GenBank under accession number NP_001318.1 (SEQ ID NO:271). The term "NY-ESO-1" includes recombinant NY-ESO-1 or fragments thereof. The term also includes NY-ESO-1 or fragments thereof conjugated to, for example, a histidine tag, murine or human Fc, or a signal sequence such as ROR1. In certain embodiments, the term includes NY-ESO-1 or fragments thereof in the context of HLA-A2, linked to HLA-A2, or presented as HLA-A2.

[0081] In certain embodiments, the NY-ESO-1 peptide comprises amino acids 157-165 (SLLMWITQC (SEQ ID NO:269)) shown in SEQ ID NO:271 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 to have a C or V at position 165). In other embodiments, the NY-ESO-1 peptide comprises amino acid residues 157-165 (SLMMWITQV (SEQ ID NO:270)) shown in SEQ ID NO:271 where the cysteine at residue 165 has been replaced by valine, and is referred to herein as “NY-ESO-1_157-165V peptide” or “NY-ESO-1(157-165V) peptide”, or otherwise designated as “C165V” or “V165”. For simplicity, it is understood that unless otherwise stated, the terms “NY-ESO-1_157-165 peptide”, “NY-ESO-1(157-165)”, and “peptide of NY-ESO-1 comprising amino acid residues 157-165” include NY-ESO-1_157-165C peptide and NY-ESO-1_157-165V peptide.

[0082] The term “HLA” refers to the human leukocyte antigen (HLA) system or complex, which is the gene complex encoding the major histocompatibility complex (MHC) proteins of humans. These cell surface proteins are responsible for regulating the human immune system. HLA corresponding to class I MHC (A, B, and C) presents peptides from within the cell.

[0083] 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 major 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 the invariant β2-microglobulin molecule.

[0084] The term “HLA-A2” is a specific class I major histocompatibility complex (MHC) allele group located at the HLA-A locus; the α-chain is encoded by the HLA-A*02 gene, and the β-chain is encoded by the β2-microglobulin or B2M locus.

[0085] As used herein, the terms "antigen-binding protein", "binding protein", or "binding molecule" include a molecule that contains at least one antigen-binding site that specifically binds to a target molecule, such as a conformational epitope of the New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) presented by HLA-A2 (e.g., the peptide presented by HLA-A2 that comprises 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.

[0086] The terms "HLA-A2:NY-ESO-1 antigen-binding protein" or "HLA-A2:NY-ESO-1 antigen-binding protein", etc. refer to an antigen-binding protein (such as an antibody) or an antigen-binding portion thereof that specifically binds to a conformational epitope by a peptide fragment of NY-ESO-1 presented by HLA-A2 (e.g., amino acid residues 157-165). In certain embodiments, the conformational epitope is generated on the cell surface by the NY-ESO-1 peptide presented by HLA-A2. As used herein, the terms "HLA-A2:NY-ESO-1 157-165 peptide complex", etc. refer to a complex between HLA-A2 and an NY-ESO-1 polypeptide, wherein HLA-A2 presents amino acid residues 157-165 of NY-ESO-1. In such cases, NY-ESO-1 can be a full-length NY-ESO-1 polypeptide or a truncated form, as long as amino acids 157-165 (referenced 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 as short as 157-165 of NY-ESO-1 (relative to SEQ ID NO:271) or longer peptides up to and including the full-length NY-ESO-1 protein (e.g., the sequence shown in SEQ ID NO:271).

[0087] 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 can have more than one epitope. Thus, different antigen-binding proteins may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen that elicits a response from B cells and / or T cells. It also refers to the region of an antigen that binds to an antigen-binding protein. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have residues that directly contribute to the interaction affinity. Epitopes can also be "conformational", i.e., composed of non-linear amino acids. In certain embodiments, an epitope can include a determinant that is a chemically reactive surface group of a molecule such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group and can have specific three-dimensional structural characteristics and / or specific charge characteristics in certain embodiments. In some embodiments, the antigen-binding proteins of the present disclosure interact 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 shown in SEQ ID NO:271. To determine the amino acids corresponding to one or more of M160, W161, or Q164, 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 shown in SEQ ID NO:271, as determined by or higher X-ray crystallography methods. For the avoidance of doubt, or higher resolution includes 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 a resolution of or higher.

[0088] 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.

[0089] As used herein, the term “antibody” is intended to mean an immunoglobulin molecule (i.e., a “full antibody molecule”) composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and its multimer (e.g., IgM) or an antigen-binding fragment thereof. Each heavy chain is composed of a heavy chain variable region (“HCVR” or “V H ”) and a heavy chain constant region (composed of domains C H 1, C H 2 and C H 3). Each light chain is composed of a light chain variable region (“LCVR” or “V L ”) and a light chain constant region (C L ). V H and V L regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or an antigen-binding fragment thereof) can be identical to the human germline sequences, or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on the alignment analysis of two or more CDRs.

[0090] 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. (FASEB J. 9:133-139, 1995) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which the amino acids of one or two CDRs did not contact the antigen (see also Vajdos et al., J Mol Biol 320:415-428, 2002).

[0091] CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically based on previous studies of Kabat CDR regions outside the Chothia CDRs (e.g., residues H60-H65 in CDRH2 that are not usually required). If a CDR or its residues are omitted, it is typically replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus sequence of such sequences. The positions of substitutions within the CDR and the amino acids for substitution can also be chosen empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0092] 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, can contain 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. This disclosure includes antigen-binding proteins (e.g., antibodies), or antigen-binding fragments or CARs derived from any of the amino acid sequences disclosed herein, in which one or more amino acids within one or more of the 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 variations are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily produce a variety of antigen-binding proteins (e.g., antibodies), or antigen-binding fragments or CARs containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are mutated back to the residues found in the original germline sequence from which the antigen-binding protein (e.g., an antibody) is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as mutated residues found only within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Additionally, an antigen-binding protein (e.g., an antibody), or an antigen-binding fragment thereof, or a CAR of the present disclosure can contain any combination of two or more germline mutations within the framework and / or CDR domains, e.g., where certain individual residues are mutated to the corresponding residues of a specific germline sequence while certain other residues different from the original germline sequence are retained or mutated to the corresponding residues of a different germline sequence. Once obtained, one or more desired properties of an antigen-binding protein (e.g., an antibody) and antigen-binding fragments containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity, etc. Antigen-binding proteins (e.g., antibodies), or antigen-binding fragments thereof, or CARs obtained in this general manner are included within the present disclosure.

[0093] 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, that contain 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 that have 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.

[0094] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies (mAbs) of the present disclosure may include, for example, amino acid residues in the CDRs, particularly in CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). 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., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in a human subject.

[0095] As used herein, the term "recombinant" refers to an antigen-binding protein (e.g., an antibody) of the present disclosure or an antigen-binding fragment thereof that is produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology (including, for example, DNA splicing and transgenic expression). The term refers to, for example, an antigen-binding protein (e.g., an antibody) expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse) or a cell (e.g., a CHO cell) expression system or isolated from a recombinant combinatorial human antibody library.

[0096] As used herein, the terms "chimeric antigen receptor" or "CAR" are used interchangeably herein and both 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 presented by HLA-A2, e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1), a transmembrane domain, and at least one intracellular signaling domain.

[0097] 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, induction of ADCC and / or CDC). In one embodiment, the immune effector cells used with the CARs described herein are T lymphocytes, particularly cytotoxic T cells (CTLs; CD8+ T cells) and helper T cells (HTLs; CD4+ T cells). Other T cell populations can also be used herein, such as naive T cells and memory T cells. As will be understood by those skilled in the art, other cells can also be used as immune effector cells having a CAR as described herein. Specifically, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitors of effector cells, where the progenitors can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in this regard, immune effector cells include progenitors of immune effector cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells after administration to a subject or can be induced to differentiate into mature immune effector cells in vitro.

[0098] As disclosed herein, the term "off-target peptide" refers to a peptide that differs from a target peptide (e.g., the NY-ESO-1_157-165 peptide) by 2, 3, 4, 5, or more amino acids. In certain embodiments, the term includes peptides that differ from the 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 not identical to the target peptide, it is considered an "off-target" peptide. In certain embodiments, amino acid identity is expressed as "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 DoS ≤ 6 are considered "off-target" peptides. The term "off-target" peptide also refers to a peptide that is predicted to bind to HLA-A2 and is contained in a protein expressed in essential normal tissues, based on sequence homology to the target peptide.

[0099] The term "specifically binds" or "binds specifically to", etc. 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 less (e.g., a smaller K D indicating 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)It is identified that it specifically binds to the conformational epitope of the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide presented by HLA-A2 (for example, a peptide containing amino acid residues 157-165 of NY-ESO-1).

[0100] The term "high-affinity" antigen-binding protein, such as an antibody, refers to those antigen-binding proteins (such as mAbs) that have a binding affinity for the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2 (for example, a peptide containing amino acid residues 157-165 of NY-ESO-1), with a K D expressed, as measured by surface plasmon resonance (such as BIACORE TM or solution affinity ELISA), of 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.

[0101] The terms "slow dissociation rate", "Koff", or "kd" refer to the rate constant of dissociation of the antigen-binding protein from HLA-A2:NY-ESO-1 of 1×10 TM s -3 or less, preferably 1×10 -1 s -4 or less, as measured by surface plasmon resonance (such as BIACORE -1 .

[0102] As used herein, the "antigen-binding portion" of an antigen-binding protein (such as an antibody), the "antigen-binding fragment" of an antigen-binding protein (such as 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 "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2 (for example, a peptide containing amino acid residues 157-165 of NY-ESO-1 conjugated to HLA-A2).

[0103] In a specific embodiment, an antigen-binding protein of the present disclosure, such as an antibody or an antibody fragment, or a CAR, can be conjugated to a moiety, such as a ligand, a detectable moiety, or a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a second anti-HLA-A2:NY-ESO-1 antigen-binding protein, an antibody against a tumor-specific antigen, an anti-cancer drug, or any other therapeutic moiety that can be used to treat diseases or conditions including NY-ESO-1-related diseases or conditions (such as NY-ESO-1-positive cancers).

[0104] As used herein, an “isolated antigen-binding protein”, such as an isolated antibody, is intended to mean an antigen-binding protein (such as an antibody) that is substantially free of other antigen-binding proteins having different antigen specificities, such as antibodies (Abs) (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, such as antibodies, that specifically bind to an antigen other than the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2.

[0105] As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows the analysis of real-time biomolecular interactions by detecting changes in the protein concentration within a biosensor matrix, for example, using a BIACORE TM system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) to analyze real-time biomolecular interactions.

[0106] As used herein, the term “K D ” is intended to mean the equilibrium dissociation constant of a specific antigen-binding protein-antigen interaction.

[0107] As used herein, the term "cross-competition" refers to an antigen-binding protein (e.g., an antibody) or an antigen-binding fragment thereof that 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 both directions, i.e., the first antigen-binding protein (e.g., antibody) binds to the second antigen-binding protein (e.g., antibody) and blocks the binding between them, 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) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) can bind to different but overlapping epitopes such that the binding of one antigen-binding protein, e.g., via steric hindrance, inhibits or blocks the binding of the second antigen-binding protein. Cross-competition between antigen-binding proteins (e.g., antibodies) can 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 the binding of the second antigen-binding protein (e.g., antibody) being less than the background signal due to self-binding (where the first and second antigen-binding proteins (e.g., antibodies) are the same antigen-binding protein (e.g., antibody)). Cross-competition between two antigen-binding proteins (e.g., antibodies) can be expressed as the % binding of the second antigen-binding protein (e.g., antibody) being less than the baseline self-background binding (where the first and second antigen-binding proteins (e.g., antibodies) are the same antigen-binding protein (e.g., antibody)).

[0108] The terms "substantially identical" or "substantially the same", when referring to a nucleic acid or a fragment thereof, mean that when optimally aligned with the appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand), as measured by any well-known sequence identity algorithm, 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, which will be discussed below. In some cases, a nucleic acid molecule that is substantially identical to a reference nucleic acid molecule can encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0109] Algorithms can be used to calculate sequence identity, such as the Needleman Wunsch algorithm for global alignment (Needleman and Wunsch 1970 Journal of Molecular Biology 48:443-453) or the Smith Waterman algorithm for local alignment (Smith and Waterman 1981 Journal of Molecular Biology 147:195-197). Another preferred algorithm was described by Dufresn et al. in 2002 in Nature Biotechnology (Volume 20, pages 1269-71) and is used in the software GenePAST (GQ Life Sciences, Inc., Boston, Massachusetts).

[0110] When applied to polypeptides, the term "substantially similar" or "substantially similarity" means that two peptide sequences, when optimally aligned, e.g., using the programs GAP or BESTFIT with default gap weights, share at least 90% sequence identity, and even more preferably share at least 95%, 96%, 97%, 98% or 99% sequence identity. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent similarity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, 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 having 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 having a non-negative value in the PAM250 log-likelihood matrix.

[0111] Sequence analysis software is typically used to measure the sequence similarity of polypeptides. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different organism species, or between a wild-type protein and its mutant. See, for example, GCG version 6.1. Polypeptide 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 an alignment of the best overlapping regions between a query and a search sequence and a percentage of sequence identity (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm, in which an affine gap search is used, where the gap opening penalty is 12, the gap extension penalty is 2, and the 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 with default parameters, especially BLASTP or TBLASTN. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, which are incorporated herein by reference.

[0112] The phrase “therapeutically effective amount” means an amount administered to produce a desired effect. The exact amount will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0113] As used herein, the term “subject” refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as an NY-ESO-1 related disease or disorder, such as an NY-ESO-1 related cancer (e.g., NY-ESO-1 positive cancer). The term includes human subjects suffering from or at risk of suffering from an NY-ESO-1 related disease or disorder, such as an NY-ESO-1 related cancer or metastatic NY-ESO-1 related cancer.

[0114] As used herein, "anticancer drug" refers to any agent that can be used to treat, ameliorate, or inhibit cancer, including but not limited to cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, 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 a chemotherapeutic agent and means any agent that is harmful to cells. Examples include: (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dantron, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs.

[0115] As used herein, the term "antiviral drug" refers to any drug or therapy used to treat, prevent, or ameliorate a 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.

[0116] An immunogen comprising any of the following can be used to generate an antigen-binding protein, such as an antibody, that binds to a conformational epitope of an NY-ESO-1 peptide presented by HLA-A2 (e.g., a peptide comprising amino acid residues 157-165 of NY-ESO-1 linked to HLA-A2). In certain embodiments, the antigen-binding proteins of the present disclosure, such as antibodies, can be obtained from mice immunized with: full-length native NY-ESO-1 protein (see, GenBank accession number NP_001318.1) (SEQ ID NO: 271); or a recombinant NY-ESO-1 peptide having GenBank accession number 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 (SLMMWITQV; SEQ ID NO: 270) having a C165V substitution with a GenBank accession and binding to an HLA protein such as HLA-A2.

[0117] Alternatively, NY-ESO-1 or a fragment thereof can be produced using standard biochemical techniques and modified in the context of HLA-A2 and used as an immunogen.

[0118] In some embodiments, the immunogen can be a recombinant NY-ESO-1 peptide (possibly a recombinant NY-ESO-1 peptide presented by HLA) expressed in Escherichia coli or any other eukaryotic or mammalian cell such as Chinese hamster ovary (CHO) cells.

[0119] In certain embodiments, an antigen-binding protein that specifically binds to the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2 can be prepared using the above polypeptide or a fragment thereof. In some embodiments, the NY-ESO-1 peptide presented by HLA-A2 can extend about 5 to about 20 amino acid residues beyond one or both of the N- or C-termini of the regions described herein. In certain embodiments, any combination of the above regions or fragments thereof can be used to prepare an HLA-A2:NY-ESO-1 specific antigen-binding protein, such as an antibody.

[0120] The peptide can be modified to include the addition or substitution of certain residues for purposes of labeling or for conjugation to a carrier molecule such as KLH. For example, 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.

[0121] Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in the examples herein. In Example 3, the binding affinity and kinetic constants of a human anti-HLA-A2:NY-ESO-1 specific antigen-binding protein (such as an antibody) were determined by surface plasmon resonance and measured on a Biacore 4000 or T200 instrument. Example 4 describes the binding of an antibody to cells overexpressing an NY-ESO-1 fragment.

[0122] For example, antigen-binding proteins (such as antibodies) against 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 binds. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface. In one embodiment, the label can be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antigen-binding proteins can be used in diagnostic assays, including imaging assays.

[0123] Antigen-binding protein

[0124] The present disclosure provides antigen-binding proteins, which include antibodies or antigen-binding fragments thereof and CARs (e.g., nucleic acid molecules encoding the CARs of the present disclosure) (as described below). Unless otherwise specifically indicated, as used herein, the term "antibody" should be understood to encompass antibody molecules (i.e., "full antibody molecules") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as antigen-binding fragments thereof. As used herein, terms such as "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 retain the ability to specifically bind to the conformational epitope of an NY-ESO-1 peptide presented by HLA-A2. Antigen-binding proteins (such as antibody fragments) can include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding proteins (such as antigen-binding fragments of antibodies) can be obtained from full antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable domains and (optionally) constant domains of the antibody. Such DNA is known and / or readily obtainable from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The 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 in a suitable configuration or to introduce codons, generate cysteine residues, modify, add, or delete amino acids, etc.

[0125] Non-limiting examples of antigen-binding fragments of antibodies include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included within the expression "antigen-binding fragment" as used herein.

[0126] Antigen-binding fragments of antigen-binding proteins (such as antibodies) typically contain at least one variable domain. The variable domain can have any size or amino acid composition and typically contains at least one CDR adjacent to or in-frame with one or more framework sequences. In an antigen-binding protein having a V L domain associated with a V H domain, the V H and V L domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0127] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antigen-binding proteins of the present disclosure include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains can be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region can consist 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. Additionally, the antigen-binding fragments of the antibodies of the present disclosure can comprise homodimers or heterodimers (or other multimers) of any of the above-listed variable and constant domain configurations that non-covalently associate with each other and / or non-covalently associate with one or more monomeric V H or V L domains (e.g., via disulfide bonds).

[0128] Like whole antibody molecules, antigen-binding proteins (e.g., antigen-binding fragments of antibodies) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will generally comprise at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be applicable for use in the context of the antigen-binding fragments of the antibodies of the present disclosure using conventional techniques available in the art.

[0129] Preparation of Antigen-Binding Proteins

[0130] Methods for generating 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 the conformational epitope of the New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) presented by HLA-A2.

[0131] By using techniques (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, ), or any other known methods for generating antigen-binding proteins, such as monoclonal antibodies, antigen-binding proteins with high affinity for the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2, such as chimeric antibodies, are initially isolated and have human variable regions and murine constant regions. The technology relates to generating transgenic mice having a genome that includes human heavy and light chain variable regions operably linked to an endogenous murine constant region locus such that the mice produce antigen-binding proteins, such as antibodies, that contain human variable regions and murine constant regions in response to antigen stimulation. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0132] Generally, mice are challenged with a target antigen, and lymphocytes, such as B cells, are recovered from the mice that express the antigen-binding protein (e.g., antibody). The lymphocytes can be fused with a myeloma cell line to produce an immortalized hybridoma cell line, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the relevant antigen. DNA encoding the heavy and light chain variable regions can be isolated and linked to heavy and light chain constant regions of a desired isotype. Such antigen-binding proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding an antigen-specific antigen-binding protein (e.g., a chimeric antibody), or the variable domains of the light and heavy chains, can be isolated directly from antigen-specific lymphocytes.

[0133] Initially, high-affinity antigen-binding proteins having human variable regions and murine constant regions, such as chimeric antibodies, are isolated. As in the experimental section below, the antigen-binding proteins are characterized and selected for desired properties, including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to produce antigen-binding proteins of the present disclosure, such as fully human antibodies, e.g., wild-type or modified IgG1 or IgG4. Although the constant regions selected can vary depending on the particular use, the high-affinity antigen-binding and target-specificity characteristics reside in the variable regions.

[0134] Bioequivalence

[0135] The anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure include proteins having an amino acid sequence different from those of the antigen-binding proteins (e.g., antibodies) but retaining the ability to bind to the conformational epitope of the NY-ESO-1 peptide presented by HLA-A2. Such variant antigen-binding proteins contain an addition, deletion, or substitution of one or more amino acids as compared to the parental sequence, but exhibit a biological activity substantially equivalent to that of the antigen-binding proteins. Similarly, the DNA sequences encoding the antigen-binding proteins of the present disclosure include sequences that contain an addition, deletion, or substitution of one or more nucleotides as compared to the disclosed sequences but encode antigen-binding proteins that are substantially bioequivalent to the antigen-binding proteins of the present disclosure.

[0136] An antigen-binding protein or antibody is considered a biologic equivalent if, for example, two antigen-binding proteins or antibodies are pharmaceutical equivalents or pharmaceutical alternatives that do not show a significant difference in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions (single dose or multiple doses). If certain antigen-binding proteins or antibodies have the same extent of absorption but different absorption rates, they will be considered equivalents or pharmaceutical alternatives, but may still be considered biologic equivalents because such differences in absorption rate are intentional and reflected in the label, are not necessary for achieving an effective in vivo drug concentration (e.g., for long-term use), and are considered medically insignificant for the particular drug product under study.

[0137] In one embodiment, two antigen-binding proteins (or antibodies) are bioequivalent if they do not have clinically significant differences in their safety, purity, or potency.

[0138] In one embodiment, two antigen-binding proteins (or antibodies) are bioequivalent if a patient can switch between a reference product and a biological product one or more times without an increased risk of expected adverse reactions, including clinically significant changes in immunogenicity or decreased efficacy, compared to continued treatment without such a switch.

[0139] In one embodiment, two antigen-binding proteins (or antibodies) are bioequivalent if they both act on one or more conditions of use through one or more common mechanisms of action, provided that such mechanism is known.

[0140] 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 metabolite is measured over time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that are related to and reasonably predictive of in vivo bioavailability data in humans; (c) in vivo tests in humans or other mammals, where the appropriate acute pharmacological effect of the antibody (or its target) is measured over time; and (d) in well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0141] Bioequivalent variants of the antigen-binding proteins (or antibodies) of the present disclosure can be constructed, for example, by making various substitutions or deleting terminal or internal residues or sequences necessary for biological activity in the residues or sequences. 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 upon refolding. In other cases, bioequivalent antigen-binding proteins can include antigen-binding protein variants that contain amino acid changes that modify the glycosylation profile of the antigen-binding protein, such as mutations that eliminate or remove glycosylation.

[0142] Anti-HLA-A2:NY-ESO-1 antigen-binding protein comprising an Fc variant

[0143] According to certain embodiments of the present disclosure, there is provided an anti-HLA-A2:NY-ESO-1 antigen-binding protein, such as an antibody, comprising an Fc domain having one or more mutations, wherein the mutations enhance or reduce the binding of the antigen-binding protein to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that comprise mutations in the C H 2 or C H 3 region, wherein the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes at a pH range from about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increase in the serum half-life of the antigen-binding protein. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); modifications at positions 250 and 428 (e.g., L or F); modifications at positions 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 modifications 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 modifications at position 250 and / or position 428; or modifications 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) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L (e.g., T250Q and M428L) modifications; and 307 and / or 308 (e.g., 308F or 308P) modifications. In yet another embodiment, the modification includes 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0144] For example, the present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that comprise an Fc domain having one or more pairs or sets 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 N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that comprise an Fc domain having an S108P mutation in the hinge region of IgG4 to promote dimer stability. All possible combinations of the foregoing Fc domain mutations and other mutations within the variable domains of the antigen-binding proteins disclosed herein are contemplated within the scope of the present disclosure.

[0145] The present disclosure also includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that comprise a chimeric heavy-chain constant (C H ) region, wherein the chimeric C H region comprises segments of C H regions derived from more than one immunoglobulin isotype. For example, the antigen-binding proteins of the present disclosure may comprise a chimeric C H region that comprises portions or all of the C H 2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule, which is combined with portions or all of the C H 3 domain derived from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, the antigen-binding proteins of the present disclosure comprise a chimeric C H region having a chimeric hinge region. By way of example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region, which is combined with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from 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 C H region as described herein is included HAntigen-binding proteins of the 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).

[0146] Biological properties of the antigen-binding protein

[0147] Generally, the antigen-binding proteins of the present disclosure function by binding to the conformational epitope of the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide presented by HLA-A2.

[0148] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that bind highly specifically to the NY-ESO-1 peptide in the context of HLA-A2. In the absence of HLA-A2, the anti-HLA-A2:NY-ESO-1 antigen-binding protein does not bind to the NY-ESO-1 peptide. In addition, the anti-HLA-A2:NY-ESO-1 antigen-binding protein does not bind to off-target peptides in the context of HLA-A2.

[0149] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that bind with high affinity to monomeric HLA-A2:NY-ESO-1 (157-165) peptide, where the NY-ESO-1 157-165 peptide can contain C165 or V165; i.e., the antigen-binding proteins of the present disclosure can be specific for either form or non-specific for which form. For example, the present disclosure includes a K measured by surface plasmon resonance in the assay format defined in Example 3 herein that is less than about 1 nM. D Antigen-binding proteins that bind to monomeric HLA-A2:157-165 peptide (optionally having a C165V substitution) (e.g., at 25 °C or at 37 °C). In certain embodiments, the antigen-binding protein has a K measured by surface plasmon resonance in the assay format defined in Example 3 herein or a substantially similar assay that is 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. D Bind to monomeric HLA-A2:NY-ESO-1_157-165 peptide.

[0150] The present disclosure also includes an EC determined by flow cytometry assays defined in Examples 4 and 5 herein or substantially similar assays that is less than about 10 nM. 50An antigen-binding protein that binds to cells expressing the HLA-A2:NY-ESO-1:157-165 peptide complex (wherein the NY-ESO-1 157-165 peptide can comprise C165 or V165; that is, the antigen-binding proteins of the present disclosure can be specific for either form, or non-specific for which form) and does not bind to cells expressing a predicted off-target peptide. In certain embodiments, the antigen-binding protein has an EC determined by, for example, a flow cytometry assay as defined in Examples 4 and 5 herein or a substantially similar assay in the form of the assays in Examples 4 and 5 herein or a substantially similar assay 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 50 Binds to cells expressing the HLA-A2:NY-ESO-1_157-165 peptide, but does not bind to cells expressing a predicted off-target peptide.

[0151] In certain embodiments, the antigen-binding proteins of the present disclosure can be used to inhibit tumor growth or delay cancer progression and can increase the survival rate of a subject when prophylactically administered to a subject in need thereof. For example, administration of the antigen-binding proteins of the present disclosure can result in shrinkage of a primary tumor and can prevent the metastasis or development of secondary tumors. In certain embodiments, the antigen-binding proteins of the present disclosure can be used to inhibit tumor growth and can increase the survival rate of a subject when therapeutically administered to a subject in need thereof. For example, administration of a therapeutically effective amount of the antigen-binding proteins of the present disclosure to a subject can result in shrinkage and disappearance of an established tumor in the subject.

[0152] In one embodiment, the present disclosure provides a separated recombinant antigen-binding protein that binds to the conformational epitope of an NY-ESO-1 peptide presented by HLA-A2, wherein the antigen-binding protein exhibits one or more of the following characteristics: (i) comprising an HCVR having an amino acid sequence selected from SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 142, 162, 180, 196, 211, and 230 or a sequence substantially similar thereto, and 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; (ii) comprising an LCVR having an amino acid sequence selected from SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150, 170, 186, 203, 219, and 238 or a sequence substantially similar thereto, and 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 an HCDR3 domain having an amino acid sequence selected from SEQ ID NO: 8, 28, 48, 68, 88, 108, 128, 148, 168, 184, 201, 217, and 236 or a sequence substantially similar thereto, and 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;and an LCDR3 domain, having an amino acid sequence selected from SEQ ID NO: 16, 36, 56, 76, 96, 116, 136, 156, 190, 205, 224, and 244 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; (iv) comprising an HCDR1 domain, having an amino acid sequence selected from SEQ ID NO: 4, 24, 44, 64, 84, 104, 124, 144, 164, 213, and 232 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; an HCDR2 domain, having an amino acid sequence selected from SEQ ID NO: 6, 26, 46, 66, 86, 106, 126, 146, 166, 215, and 234 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; an LCDR1 domain, having an amino acid sequence selected from SEQ ID NO: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, and 240 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;and the LCDR2 domain, having an amino acid sequence selected from SEQ ID NO: 14, 34, 54, 74, 94, 114, 134, 154, 174, and 242 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; (v) binds to the monomeric HLA-A2:NY-ESO-1 157-165 (C165 or V165) peptide complex with a binding dissociation equilibrium constant (KD) of less than about 1 nM as measured in surface plasmon resonance assays at 25°C; (vi) binds to the 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 surface plasmon resonance assays at 25°C; (vii) has 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 off-target peptides presented by HLA-A2, wherein the peptide differs from SEQ ID NO: 271 by 2, 3, 4, 5, or more amino acids.

[0153] The antigen-binding proteins of the present disclosure can have one or more of the above biological characteristics, or any combination thereof. By reading the present disclosure, including the working examples herein, other biological properties of the antigen-binding proteins of the invention will be apparent to those of ordinary skill in the art.

[0154] Epitope mapping and related techniques

[0155] 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 the NY-ESO-1 peptide presented by HLA-A2. The epitope can 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).

[0156] A variety of 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 within a polypeptide or protein". Exemplary techniques include, for example, conventional cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis, peptide blotting (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 employed (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify the amino acids within a polypeptide that interact with an antigen-binding protein is to detect hydrogen / deuterium exchange by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium-labeling the target protein, and then binding the antigen-binding protein to the deuterium-labeled protein. Next, the protein / antigen-binding protein complex is transferred to water, and the exchangeable protons within the amino acids protected by the antigen-binding protein complex undergo deuterium-hydrogen back-exchange at a slower rate than the exchangeable protons within the amino acids that are not part of this interface. As a result, the amino acids that form part of the protein / antigen-binding protein interface can retain deuterium and thus have a relatively higher mass compared to the amino acids not included in this interface. After dissociation of the antigen-binding protein, the target protein is subjected to protease cleavage and mass spectrometry, 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 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0157] The term "epitope" refers to the site on an antigen to which B cells and / or T cells react. B cell epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by contiguous amino acids generally remain upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are generally lost upon treatment with a denaturing solvent. Epitopes presenting a unique spatial conformation generally include at least 3 and more typically at least 5 or 8 to 10 amino acids.

[0158] Modified avidity profiling (MAP), also known as antigen-structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antigen-binding proteins (such as antibodies (mAbs)) against the same antigen based on the similarity of the binding characteristics of each antibody to the surface of a chemically or enzymatically modified antigen (see US2004 / 0101920, which is incorporated herein by reference in its entirety). Each class may reflect a distinct epitope that is clearly different or partially overlapping from the epitope represented by another class. This technique allows for the rapid filtration of antigen-binding proteins that are identical in gene, such that characterization can be focused on antigen-binding proteins that are different in gene. When applied to hybridoma screening, MAP may assist in the identification of rare hybridoma clones that produce antigen-binding proteins with desired characteristics. MAP can be used to classify the antigen-binding proteins of the present disclosure into groups of antigen-binding proteins that bind different epitopes.

[0159] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that bind to the same epitope or a portion of an 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. Similarly, the present disclosure also includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that compete with any of the specific exemplary antigen-binding proteins 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, for binding to HLA-A2:NY-ESO-1 or a fragment thereof.

[0160] By using conventional methods known in the art, it can be readily determined whether an antigen-binding protein binds to the same epitope or competes for binding with a reference anti-HLA-A2:NY-ESO-1 antigen-binding protein. 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 the HLA-A2:NY-ESO-1 protein or peptide under saturating conditions. Next, the ability of the test antigen-binding protein to bind to the 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 a different epitope than the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein. On the other hand, if the test antigen-binding protein is unable to bind to the HLA-A2:NY-ESO-1 protein after saturation binding with the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein, the test antigen-binding protein may bind to the same epitope as the reference anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure.

[0161] To determine whether an antigen-binding protein competes with a reference anti-HLA-A2:NY-ESO-1 antigen-binding protein for binding, the above binding method is carried out in two directions: In the first direction, the reference antigen-binding protein is allowed to bind to the HLA-A2:NY-ESO-1 protein under saturated conditions, and then the binding of the test antigen-binding protein to the HLA-A2:NY-ESO-1 molecule is evaluated. In the second direction, the test antigen-binding protein is allowed to bind to the HLA-A2:NY-ESO-1 molecule under saturated conditions, and then the binding of the reference antigen-binding protein to the HLA-A2:NY-ESO-1 molecule is evaluated. If in both directions 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 understood by those of ordinary skill in the art, an antigen-binding protein that competes with a reference antigen-binding protein for binding may not necessarily bind to the same epitope as the reference antigen-binding protein, but can sterically block the binding of the reference antigen-binding protein by binding to overlapping or adjacent epitopes.

[0162] If two antigen-binding proteins competitively inhibit (block) the binding of the other to an antigen, 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 one antigen-binding protein inhibits the binding of the other antigen-binding protein by at least 50%, but preferably 75%, 90%, or even 99% (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if substantially all of the amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other antigen-binding protein, the two antigen-binding proteins have the same epitope. Alternatively, if some of the amino acid mutations that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other antigen-binding protein, the two antigen-binding proteins have overlapping epitopes.

[0163] Additional conventional experiments (e.g., peptide mutagenesis and binding assays) can then be carried out 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 carried out using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antigen-binding protein binding assay available in the art.

[0164] Immunoconjugate

[0165] The present disclosure includes anti-HLA-A2:NY-ESO-1 antigen-binding proteins that are conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin or chemotherapeutic agent, for treating cancer. As used herein, the term “immunoconjugate” refers to an antigen-binding protein that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety (such as a detectable moiety), enzyme, toxin, peptide or protein, or therapeutic agent. The antigen-binding protein can be linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or therapeutic agent at any position along the molecule, so long as the antigen-binding protein is capable of binding to its target. Examples of immunoconjugates include antigen-binding protein-drug conjugates and antigen-binding protein-toxin fusion proteins. In one embodiment, the reagent can be a second, different antibody that is specific for NY-ESO-1 or HLA-A2:NY-ESO-1. In certain embodiments, the antigen-binding protein can be conjugated to a reagent 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; see, for example, PCT Publication No. WO 05 / 103081.

[0166] Chimeric antigen receptor (CAR)

[0167] Chimeric antigen receptor (CAR) redirects T cell specificity to antibody-recognized antigens expressed on the surface of cancer cells, while the T cell receptor (TCR) expands the target range to include intracellular tumor antigens. CARs specific for the B cell differentiation antigen CD19 have shown significant efficacy in treating B cell malignancies, while TCR-redirected T cells have shown benefit in patients with solid cancers. Stauss et al. described strategies for modifying therapeutic CARs and TCRs for use in cancer treatment to, for example, enhance antigen-specific effector functions and limit the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24:113-118).

[0168] One aspect of the present disclosure includes a chimeric antigen receptor (CAR) that is specific for an NY-ESO-1 peptide presented on the surface of tumor cells via HLA-A2, such as a peptide comprising amino acid residues 157-165 of NY-ESO-1. In one embodiment of the present disclosure, the CAR 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 co-stimulatory 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 region 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 shown in SEQ ID NO:305. In some embodiments, the transmembrane domain comprises the CD28 sequence shown in SEQ ID NO:304. In some embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:297. In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:299. In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:298.

[0169] 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 the antigen-binding protein of the present disclosure.

[0170] As used herein, the binding domain or extracellular domain of a CAR provides the CAR with the ability to bind to a target antigen. The binding domain can be any protein, polypeptide, oligopeptide, or peptide having the ability to specifically recognize and bind a biomolecule (e.g., a cell surface receptor or tumor protein or a component thereof). The binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding conjugate of the target biomolecule. For example, as further described herein, the binding domain can be the variable regions of the antibody light and heavy chains, or the variable regions of the light and heavy chains can be joined together as a single chain and in either orientation (e.g., VL-VH or VH-VL). A variety of assays for identifying the binding domains of the present disclosure that specifically bind to a particular target are known, including Western blotting, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis), and are described herein. The target can be any antigen of clinical interest for which it is desirable to trigger an effector immune response leading to tumor killing. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is a conformational epitope of the NY-ESO-1 peptide (such as the peptide NY-ESO-1 comprising amino acid residues 157-165) presented by HLA-A2 on the surface of tumor cells.

[0171] Exemplary binding domains include antigen-binding proteins (such as antigen-binding fragments of antibodies), such as scFv, scTCR, the extracellular domain of a receptor, a ligand of a cell surface molecule / receptor or its receptor-binding domain, and tumor-binding proteins. In certain embodiments, the antigen-binding domain included in the CARs of the present disclosure can be a variable region (Fv), CDR, Fab, scFv, VH, VL, domain antibody variant (dAb), camelid antibody (VHH), fibronectin type III domain variant, ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds.

[0172] In one embodiment, the binding domain of the CAR is an anti-HLA-A2:NY-ESO-1 single-chain antibody (scFv), and can be murine, human, or humanized scFv. The single-chain antibody can be cloned from the V-region genes of a hybridoma specific for the desired target. Techniques for cloning the variable region heavy chain (VH) and variable region light chain (VL) have been described (e.g., Orlandi et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1989; 86:3833-3837). Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but can also be a non-antibody-derived binding domain. The antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody that are involved in binding to an antigen.

[0173] In certain embodiments, the CARs of the present disclosure may include linkers between various domains, which add appropriate spacing and conformation for the molecule. For example, in some embodiments, there may be a linker between the VH and VL regions of the binding domain, and the length of the linker 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 further embodiments, the length of the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Ranges including the numbers described herein are also included herein, for example, linkers having a length of 10 to 30 amino acids.

[0174] In certain embodiments, the linkers suitable for the CARs described herein are flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable variety of lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0175] 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, where n is an integer of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). Additionally, the linker can comprise multiple units of the above sequences, such as ((G)nS)m), where 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 can thus act as neutral tethers between domains of a fusion protein, such as the CARs described herein. Compared to alanine, glycine can access more phi-psi space and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173 / -142 (1992)). One of ordinary skill in the art will recognize that the design of the CAR can include all or part of a flexible linker such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure to provide the desired CAR structure.

[0176] There may be a "spacer" or "hinge" following the binding domain of the CAR, which refers to the region that moves the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen-binding, and activation (Patel et al., Gene Therapy, 1999; 6:412-419). The hinge region in a CAR is typically located between the transmembrane (TM) and binding domains. In certain embodiments, the hinge region is an immunoglobulin hinge region and can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, and CD7, which can be the wild-type hinge regions from these molecules or can vary. In one embodiment, the hinge region comprises the CD8α hinge.

[0177] The "transmembrane" region or domain is the part of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell and facilitates the binding of the domain to the target antigen. The transmembrane domain can be the 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 primarily contain hydrophobic residues such as leucine and valine.

[0178] The "intracellular signaling domain" refers to the part of the chimeric antigen receptor protein that is involved in transducing the information of the effective CAR binding to the target antigen into the interior of the immune effector cell to initiate effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity. The effector cell functions include releasing cytotoxic factors to the target cells binding the CAR, or other cellular responses triggered by the binding of the antigen to the extracellular CAR domain. The term "effector function" refers to the specific functions of the cell. The effector functions of T cells can be, for example, cytolytic activity or helper or activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to the protein part that transduces the signals of effector functions and directs the cell to perform specific functions. Although usually the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire domain. In terms of using truncated parts of the intracellular signaling domain, such truncated parts can be used instead of the entire domain as long as they can transduce the signals of effector functions. The term intracellular signaling domain is intended to include any truncated part of the intracellular signaling domain that is sufficient to transduce the signals of effector functions. The intracellular signaling domain is also referred to as the "signaling domain" and is usually derived from parts of human CD3 or FcRy chains.

[0179] It is known that the signals generated only through the T cell receptor are not sufficient to fully activate T cells, and co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those cytoplasmic signaling sequences that initiate antigen-dependent primary activation through the T cell receptor (primary cytoplasmic signal sequences); and those cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signal sequences). The primary cytoplasmic signal sequences regulate the primary activation of the T cell receptor complex in an inhibitory manner. The primary cytoplasmic signal sequences that act in a co-stimulatory manner may contain signal motifs called immunoreceptor tyrosine-based activation motifs or ITAMs.

[0180] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly used in the present disclosure include those sequences derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, 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 CD3ζ or FcRγ.

[0181] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the portion of a CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for the effective activation and function of T lymphocytes upon binding 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 the ligand that specifically binds CD83. Thus, while the present disclosure provides exemplary costimulatory domains derived from CD3ζ, CD28, and 4-1BB, other costimulatory domains are also contemplated for use in the CARs described herein. Inclusion of one or more costimulatory signaling domains can enhance the efficacy and expansion of T cells expressing the CAR receptor. The intracellular signaling domain and the costimulatory signaling domain can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0182] Although CARs based on scFv engineered to contain signaling domains from CD3 or FcRγ have been shown to provide effective signals for T cell activation and effector functions, they are not sufficient to initiate signals that promote T cell survival and expansion in the absence of an accompanying costimulatory signal. Other CARs that contain a binding domain, hinge, transmembrane, and a signaling domain derived from CD3ζ or FcRγ, as well as one or more costimulatory signaling domains (e.g., intracellular costimulatory 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, within 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., Proceedings of the National Academy of Sciences of the United States of America, 2009; 106:3360-3365).

[0183] In one embodiment, the HLA-A2:NY-ESO-1 CAR of the present disclosure comprises: (a) an anti-HLA-A2:NY-ESO-1 scFv as a binding domain (e.g., an scFv having a binding region (e.g., a CDR or variable domain) of any one or more of the 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) an intracellular signaling domain of the human T cell receptor CD3ζ chain (CD3), and optionally one or more co-stimulatory signaling domains derived from CD28, CD137, CD134, and CD278. In one embodiment, the different protein domains are arranged in the following order from the amino to the carboxyl terminus: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and the optional co-stimulatory signaling domain are tandemly linked to the transmembrane carboxyl terminus in any order to form a single-chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule that contains different coding sequences, e.g., coding sequences of a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8α hinge, a human CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ intracellular signaling domain (5' to 3'). In another embodiment, the nucleic acid construct encoding the HLA-A2:NY-ESO-1 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule that contains different coding sequences, e.g., coding sequences of a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8α hinge, a human CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ co-stimulatory domain (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 that contains different coding sequences, e.g., coding sequences of a human anti-HLA-A2:NY-ESO-1 scFv, a human CD8α hinge, a human CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ co-stimulatory domain (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 pair: 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 a nucleic acid molecule encoding an HLA-A2:NY-ESO-1 CAR selected from the sequences of Table 2.

[0184] In certain embodiments, a polynucleotide encoding the CAR described herein is inserted into a vector. As used herein, the term "vector" refers to an agent into which a polynucleotide encoding a protein can be covalently inserted such that expression of the protein and / or cloning of the polynucleotide is effected. Such vectors may also be referred to as "expression vectors". Any suitable method known in the art can be used to insert the isolated polynucleotide into the vector, for example, but not limited to, the vector can be digested with an appropriate restriction enzyme and then ligated to the isolated polynucleotide having matching restriction ends. An 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 capable of being transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. An expression vector may contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences having other functions and are discussed below. An expression vector may contain additional elements, for example, an expression vector may have two replication systems such that it is maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host.

[0185] Expression vectors can have the necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences like CMV, PGK, and EF1α promoters, ribosome recognition and binding TATA boxes, and 3' UTR AAUAAA transcription termination sequences 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 can 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 part of the vector for expressing chimeric antigen receptors. This provides a molecular switch capable of turning on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0186] Expression vectors can have additional sequences, such as 6x - histidine (SEQ ID NO:292), c - Myc, and FLAG tags, which are incorporated into the expressed CAR. Thus, expression vectors can be engineered to contain 5' and 3' untranslated regulatory sequences that can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences, which can facilitate or enhance the efficient transcription of the target nucleic acid carried on the expression vector. Expression vectors can also be engineered for replication and / or expression functions (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors can include selectable markers for maintaining the vector in host or recipient cells.

[0187] 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 (YACs), bacterial artificial chromosomes (BACs), or P1 - derived artificial chromosomes (PACs), phages such as λ phage or M13 phage, and animal viruses. Examples of categories of animal viruses that can be used as vectors include but are not limited to: retroviruses (including lentiviruses), adenoviruses, adeno - associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (e.g., SV40). An example of an expression vector is Lenti - X for expression in mammalian cells TMDicistronic expression system (Neo) vector (Clontrch), pClneo vector (Promega); pLenti4 / V5-DEST.TM., pLenti6 / V5-DEST.TM. and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the CARs disclosed herein can be ligated into such expression vectors for expressing chimeric proteins in mammalian cells.

[0188] In certain embodiments, the nucleic acid encoding the CARs of the present disclosure is provided in a viral vector. The viral vector can be those viral vectors derived from retroviruses, lentiviruses or foamy viruses. As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the ability to be packaged into viral vector particles. The viral vector can contain the coding sequences of the various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particle can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Multiple forms of viral vectors are known in the art.

[0189] In certain embodiments, the viral vector containing the CAR coding sequences 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.

[0190] The 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, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV)). The "retroviruses" of the present disclosure also include human T cell leukemia virus HTLV-1 and HTLV-2, as well as the lentivirus 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.

[0191] The lentiviral vectors used herein refer to vectors derived from lentiviruses, a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included in this group include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Maedi-Visna disease of sheep; 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 the methods of 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).

[0192] Retroviral vectors (i.e., lentiviral and non-lentiviral) for use in the present disclosure can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F.M. et al.) (The English Dialect Society) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0193] Suitable sources for obtaining retroviral (i.e., lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNA obtainable from commercially available sources, including the American Type Culture Collection (ATCC), Rockville, Maryland. The sequences can also be chemically synthesized.

[0194] For the expression of HLA-A2:NY-ESO-1 CAR, a vector can be introduced into a host cell to allow for the expression of the polypeptide within the host cell. The expression vector can 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 can be appropriately selected by those of ordinary skill in the art. For example, a promoter sequence can be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include but are not limited to the T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence can be selected to enhance transcription of the polynucleotide. A selectable marker can be selected to allow for the selection of host cells that have inserted the vector from host cells that have not, e.g., the selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside of the host cell.

[0195] To clone a polynucleotide, a vector can be introduced into a host cell (an isolated host cell) to allow for the replication of the vector itself and thus the amplification of copies of the polynucleotide contained therein. The cloning vector can contain sequence components that generally include but are not limited to an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those of ordinary skill in the art. For example, an origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0196] In certain embodiments, the present disclosure provides isolated host cells containing the vectors provided herein. Host cells containing the vector can be used for the expression or cloning of the polynucleotide contained in the vector. Suitable host cells can 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: eubacteria such as Gram-negative or Gram-positive organisms; Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia such as Serratia marcescens and Shigella; and Bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa, and Streptomyces.

[0197] The CARs of the present disclosure are 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 an exogenous nucleic acid sequence into a host cell. The nucleic acid can be integrated into the host cell DNA or can be maintained episomally. The nucleic acid can be maintained transiently or can be stably introduced. Transfection can be accomplished by a variety of methods known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and particle bombardment. Transduction refers to the delivery of one or more genes using a viral or retroviral vector by viral infection rather than by transfection. In certain embodiments, the retroviral vector is transduced by packaging the vector into virions prior to contact with the cells. For example, a nucleic acid encoding an anti-HLA-A2:NY-ESO-1 CAR carried by a retroviral vector can be transduced into cells by infection and proviral integration.

[0198] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms "genetically modified cell", "modified cell", and "redirected cell" are used interchangeably.

[0199] 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, such as a conformational epitope of the NY-ESO-1 peptide presented by HLA-A2, e.g., amino acid residues 157-165.

[0200] The present disclosure provides methods for preparing immune effector cells that express a CAR as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject suffering from an NY-ESO-1-related disease or disorder, such that the immune effector cells express one or more of the CARs described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. These cells can then be directly re-administered to the same individual (so-called autologous therapy) or to a different individual (so-called allogeneic therapy). In other embodiments, the immune effector cells are first activated and stimulated in vitro to proliferate and are subsequently genetically modified to express the CAR. In this regard, the immune effector cells can be cultured before or after genetic modification (i.e., transduced or transfected as described herein to express the CAR).

[0201] Prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, a cell source can be obtained from a subject. In particular, the immune effector cells used in conjunction with the CARs described herein include T cells. Such recombinant T cells are referred to herein as "T bodies."

[0202] 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 co-stimulatory signaling domain derived from a co-stimulatory 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 region between the extracellular binding domain and the transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3ζ or FcRγ), and / or one or more co-stimulatory signaling domains derived from a co-stimulatory molecule. In yet another embodiment of the present disclosure, the T body includes a T body construct CAR, which comprises an extracellular target-specific binding domain and a T cell receptor constant domain. The extracellular target-specific binding domain suitable for use in a T body comprising any CAR described herein may comprise a Fab, Fab', (Fab')2, Fv, or single-chain Fv (scFv) of an antigen-binding protein of the present disclosure.

[0203] There are many sources for obtaining T cells, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those of skill in the art (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 typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma portion and the cells can be placed in an appropriate buffer or medium for further processing. In one embodiment of the present disclosure, the cells are washed with PBS. In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. As will be understood by one of ordinary skill in the art, the washing step can be accomplished by methods known to those of skill in the art (e.g., by using a semi-automated flow-through centrifuge). After washing, the cells can be resuspended in a variety of biocompatible buffers or other salt solutions with or without a buffer. In certain embodiments, the undesired components of the apheresis sample can be removed in the medium in which the cells are directly resuspended.

[0204] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes (e.g., by TM gradient centrifugation with PERCOLL). Specific T cell subsets, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished by a combination of antibodies directed against surface markers specific for the cells to be negatively selected. One method for use herein is cell sorting and / or cell selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the target cell population for the present disclosure.

[0205] PBMCs can be used directly for genetic modification with CARs using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before and / or after genetic modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In embodiments, memory T cells are present in the CD62L+ and CD62L- subgroups of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are separated into CD62L-CD8+ and CD62L+CD8+ fractions. In some 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 are 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.

[0206] In certain embodiments, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are positive for CD62L and CD45RO. In some embodiments, effector CD4+ cells are negative for CD62L and CD45RO.

[0207] Immune effector cells such as T cells can be genetically modified using known methods after isolation, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In another embodiment, immune effector cells such as T cells are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR), and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and described, e.g., in 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. Generally, such methods include contacting PBMCs or isolated T cells with stimulators and co-stimulators (such as anti-CD3 and anti-CD28 antibodies) that are typically attached to beads or other surfaces in a medium having appropriate cytokines such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead act as "surrogate" antigen presenting cells (APCs). In other embodiments, methods such as those described in U.S. Patent No. 6,040,177; U.S. Patent 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.

[0208] The present disclosure provides populations of modified immune effector cells for treating NY-ESO-1 related diseases or disorders such as cancer, which comprise HLA-A2:NY-ESO-1 CAR as disclosed herein.

[0209] The CAR-expressing immune effector cells prepared as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that are obvious to those skilled in the art based on the present disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; see also U.S. Patent No. 4,690,915 to Rosenberg.

[0210] In some embodiments, the cells are formulated by first harvesting the cells from their medium and then washing and concentrating the cells in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier) in a therapeutically effective amount. Suitable infusion media can be any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% aqueous dextrose or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin.

[0211] A 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 cells. The number of cells will depend on the desired end use of the composition, as will the types of cells included.

[0212] The cells can be autologous or allogeneic to the patient undergoing the therapy. If desired, the treatment can 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 the immune response.

[0213] The population of CAR-expressing immune effector cells of the present disclosure can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components (such as IL-2) or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present disclosure can comprise a population of CAR-expressing immune effector cells, such as the T cells described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0214] The anti-tumor immune responses induced in a subject by administering the CAR-expressing T cells described herein using the methods described herein or other methods known in the art can include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses capable of killing infected cells. Humoral immune responses primarily mediated by helper T cells capable of activating B cells to result in antibody production can also be induced. A variety of techniques can be used to analyze the types of immune responses induced by the compositions of the present disclosure, and these techniques 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, N.Y.).

[0215] Accordingly, the present disclosure provides methods of treating an individual diagnosed with, suspected of having, or at risk of developing an NY-ESO-1 related disease or disorder, such as an 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.

[0216] In one embodiment, the present disclosure provides a method of treating a subject diagnosed with an NY-ESO-1 positive cancer, comprising removing immune effector cells from a subject diagnosed with an 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 to produce a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In one embodiment, the immune effector cells comprise T cells.

[0217] Methods for administering the cell compositions described herein include any method having the following effects: reintroducing into a subject ex vivo genetically modified immune effector cells that directly express the CAR of the present disclosure; or reintroducing into a subject gene-modified progenitor cells of immune effector cells that differentiate into mature immune effector cells expressing the CAR upon introduction into the subject. One method comprises ex vivo transducing peripheral blood T cells with a nucleic acid construct according to the present disclosure and returning the transduced cells to the subject's body.

[0218] Therapeutic Administration and Formulations

[0219] The present disclosure provides therapeutic compositions comprising an anti-HLA-A2:NY-ESO-1 antigen-binding protein (such as an antibody), or an antigen-binding fragment or CAR thereof, of the present disclosure. The therapeutic compositions according to the present disclosure will be administered together with suitable carriers, excipients, and other reagents, all of which are incorporated into the formulation to improve transfer, delivery, tolerability, etc. Many suitable formulations can be found in all the formularies 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 absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See, for example, Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) Journal of Pharmaceutical Sciences and Technology 52:238-311.

[0220] The dosage of an antigen-binding protein (such as an antibody) or an antigen-binding fragment thereof can vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antigen-binding proteins of the present disclosure are used to treat a disease or disorder in an adult patient, or to prevent such a disease, it is generally advantageous to administer the antigen-binding protein (such as an antibody) or an antigen-binding fragment thereof 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 (such as an antibody) or an antigen-binding fragment thereof 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 subsequent dose of the antigen-binding protein (such as an antibody) or an antigen-binding fragment thereof can be administered in an amount that is substantially the same as or less than the initial dose after the initial dose, wherein subsequent doses are separated by at least 1 day 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.

[0221] A variety of 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, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Introduction methods 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, such as by infusion or bolus injection, absorption through the epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in combination with other bioactive agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in vesicles (especially liposomes) (see, e.g., Langer (1990) Science 249:1527-1533).

[0222] This document also contemplates the use of nanoparticles to deliver the antigen-binding proteins (such as antibodies) of the present disclosure or antigen-binding fragments thereof. Antigen-binding protein-conjugated nanoparticles can be used for therapeutic and diagnostic applications. Arruebo, M. et al., 2009 describes in detail antigen-binding protein-conjugated nanoparticles and methods of preparation and use ("Antibody-conjugated nanoparticles for biomedical applications", Journal of Nanomaterials (J. Nanomat), 2009 volume, 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 with the antigen-binding proteins contained in pharmaceutical compositions to target tumor cells or autoimmune tissue cells or virus-infected cells. Nanoparticles for drug delivery have also been described, for example, in U.S. Patent No. 8,257,740 or U.S. Patent No. 8,246,995, each of which is incorporated herein in its entirety.

[0223] In certain cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed near the target of the composition, so that only a portion of the systemic dose is required.

[0224] Injectables can include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intracranial injection, intraperitoneal injection, intramuscular injection, infusion, etc. 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 an oily medium conventionally used for injection. As the 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 appropriate solubilizers, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (adduct of polyethylene oxide (50 mol) and hydrogenated castor oil)), etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, etc. The injectables thus prepared are preferably filled into appropriate ampoules.

[0225] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen-type delivery devices are readily usable for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily 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 within the reservoir is emptied, the entire device is discarded.

[0226] Many reusable pens and auto-injector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. By way of example only, examples include but are not limited to: AUTOPEN 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 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt am Main, Germany). By way of example only, examples of disposable pen delivery devices applicable to subcutaneous delivery of pharmaceutical compositions useful in accordance with the methods described herein include, but are not limited to: SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPENTM (Eli Lilly and Company), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, California), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA TM Pen (Abbott Labs, Abbott Park, Illinois).

[0227] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dosage forms suitable for combining with a certain dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. In the unit dosage, the content of the above antigen-binding protein is usually about 5 to about 500 mg / dosage form; especially in the injection dosage form, preferably the content of the above antigen-binding protein is about 5 to about 100 mg, and in other dosage forms it is about 10 to about 250 mg.

[0228] Therapeutic uses of antigen-binding proteins

[0229] The antibodies of the present disclosure can be used, in particular, to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by NY-ESO-1. The present disclosure provides methods for treating NY-ESO-1-related diseases or disorders, such as NY-ESO-1-related cancers (e.g., NY-ESO-1-positive cancers) (tumor growth inhibition) by administering to a patient in need thereof an anti-HLA-A2:NY-ESO-1 antigen-binding protein (or a pharmaceutical composition comprising an anti-HLA-A2:NY-ESO-1 antigen-binding protein) described herein, and for treating NY-ESO-1-related 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 can be used to treat, prevent, and / or ameliorate a disease or disorder or condition, such as NY-ESO-1-related cancer and / or to ameliorate at least one symptom associated with such disease, disorder, or condition. In the context of the treatment methods described herein, the anti-HLA-A2:NY-ESO-1 antigen-binding protein can be administered as a single therapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0230] In some embodiments, the antibodies described herein can be used to treat subjects suffering from primary or recurrent cancer, including but not limited to: NY-ESO-1 related cancers 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 tumors, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant teratoma, 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.

[0231] The antigen-binding protein can be used to treat the early or late symptoms of NY-ESO-1 related cancers. In one embodiment, the antibodies or fragments thereof of the present disclosure can be used to treat advanced or metastatic cancers. The antigen-binding protein can be used to reduce or inhibit or shrink tumor growth. In certain embodiments, treatment with the antigen-binding protein of the present disclosure results in tumor regression in more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% of the subjects. In certain embodiments, the antigen-binding protein can be used to prevent tumor recurrence. In certain embodiments, the antigen-binding protein can be used to prolong the progression-free survival or overall survival of subjects suffering from NY-ESO-1 related cancers. In some embodiments, the antibody can be used to reduce the toxicity caused by chemotherapy or radiotherapy while maintaining the long-term survival of patients suffering from NY-ESO-1 related cancers.

[0232] One or more antibodies of the present disclosure can be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

[0233] The present disclosure also contemplates prophylactic use of one or more antibodies of the present disclosure in patients at risk of developing a disease or disorder such as an NY-ESO-1 related disease or disorder such as an NY-ESO-1 related cancer.

[0234] 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 an NY-ESO-1 related disease or disorder such as an NY-ESO-1 related cancer. In another embodiment of the present disclosure, the antibodies of the present invention are used as an adjuvant therapy in combination with any other agent or any other therapy known to those skilled in the art for treating NY-ESO-1 related cancers.

[0235] Combination Therapies and Formulations

[0236] Combination therapies can include the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure, such as the CARs of the present disclosure (e.g., immune effector cells comprising the CARs of the present disclosure) or the pharmaceutical compositions of the present disclosure, and any additional therapeutic agents that can be advantageously combined with the antigen-binding proteins of the present disclosure. The antigen-binding proteins of the present disclosure can be synergistically combined with one or more anti-cancer drugs or therapies for treating or inhibiting NY-ESO-1-related diseases or disorders, 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 tumors, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumors, soft tissue sarcoma, melanoma, malignant teratoma, 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.

[0237] The use of the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure in combination with immune-stimulatory and / or immune-supportive therapies is contemplated herein to inhibit tumor growth and / or improve the survival rate of cancer patients. Immune-stimulatory therapies include direct immune-stimulatory therapies that enhance immune cell activity by "releasing the brake" or "stepping on the gas" of inhibited immune cells to activate the immune response. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Immune-supportive 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.

[0238] In various embodiments, one or more antigen-binding proteins of the present disclosure can be used in combination with the following: PD-1 inhibitors (e.g., anti-PD-1 antibodies such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810); PD-L1 inhibitors (e.g., anti-PD-L1 antibodies such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504); CTLA-4 inhibitors (e.g., ipilimumab); TIM3 inhibitors; BTLA inhibitors; TIGIT inhibitors; CD47 inhibitors; GITR inhibitors; antagonists of another T cell co-inhibitor or ligand (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA); indoleamine-2,3-dioxygenase (IDO) inhibitors; vascular endothelial growth factor (VEGF) antagonists (e.g., "VEGF-Trap" such as aflibercept or other VEGF-inhibiting fusion proteins described in US 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); 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; radiotherapy; 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 non-steroidal anti-inflammatory drugs); dietary supplements, such as antioxidants; or any other cancer therapy. In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present invention can be used in combination with cancer vaccines including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc. to enhance the anti-tumor response. Examples of cancer vaccines that can be used in combination with the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure include: MAGE3 vaccine for melanoma and bladder cancer, MUC1 vaccine for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers).;

[0239] In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure can be co-administered with radiotherapy in methods for generating long-lasting anti-tumor responses and / or increasing 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 radiotherapy is administered to a cancer patient. For example, radiotherapy 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 radiotherapy can be performed on a tumor lesion to enhance the local immunogenicity (adjuvant radiotherapy) and / or kill tumor cells (ablative radiotherapy) of a patient's tumor, 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 co-administered with systemic administration of the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure to a patient with brain cancer (e.g., glioblastoma multiforme). In certain embodiments, the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure can be co-administered in combination with radiotherapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0240] Additional therapeutic active agents / components can be administered before, simultaneously with, or after the anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the present disclosure are administered. For the purposes of the present disclosure, such administration regimens are considered to be "combination" administration of the anti-HLA-A2:NY-ESO-1 antigen-binding proteins with a second therapeutic active ingredient.

[0241] Additional therapeutic active components can be administered to a subject prior to administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure. For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes or less than 1 minute prior to administration of the second component, the first component can be considered to be administered "prior to" the second component. In other embodiments, additional therapeutic active components can be administered to a subject after administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein 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 administration of the second component, the first component can be considered to be administered "after" the second component. In still other embodiments, the additional therapeutic active components can be administered to the subject concurrently with the administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure. For the purposes of the present disclosure, "concurrent" administration includes, for example, administering the anti-HLA-A2:NY-ESO-1 antigen-binding protein and the additional therapeutic 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 can be administered by the same route (e.g., both the anti-HLA-A2:NY-ESO-1 antigen-binding protein and the additional therapeutic active ingredient can be administered intravenously, subcutaneously, etc.); or, each dosage form can also be administered by a different route (e.g., the anti-HLA-A2:NY-ESO-1 antigen-binding protein can be administered intravenously and the additional therapeutic active ingredient can be administered subcutaneously). In any case, for the purposes of the present disclosure, administration of the components in a single dosage form, by the same route in separate dosage forms or by different routes in separate dosage forms is considered to be "concurrent administration". For the purposes of the present disclosure, administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein "prior to", "concurrently" or "after" (as defined by those terms above) the additional therapeutic active ingredient is considered to be administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein "in combination" with the additional therapeutic active ingredient.

[0242] The present disclosure includes pharmaceutical compositions, wherein 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 in multiple dose combinations.

[0243] Dosing regimen

[0244] 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) can be administered to a subject over a defined period of time. The method according to this aspect of the present disclosure comprises sequentially administering multiple doses of the anti-HLA-A2:NY-ESO-1 antigen-binding protein of the present disclosure to the subject. As used herein, "sequentially administering" means administering each dose of the 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 include sequentially administering a single initial dose of the anti-HLA-A2:NY-ESO-1 antigen-binding protein to the patient, followed by one or more second doses of the anti-HLA-A2:NY-ESO-1 antigen-binding protein, and optionally subsequently 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.

[0245] The terms "initial dose", "second dose", and "third dose" refer to the chronological order of administration of the anti-HLA-A2:NY-ESO-1 antigen-binding protein that can be used herein. Thus, the "initial dose" is the dose administered at the start of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second, and third doses can all contain the same amount of the anti-HLA-A2:NY-ESO-1 antigen-binding protein, but can generally differ from each other in terms of the frequency of administration. However, in certain embodiments, the amounts of the anti-HLA-A2:NY-ESO-1 antigen-binding protein contained in the initial, second, and / or third doses differ from each other during the course of the treatment (e.g., appropriately adjusted up or down). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are given as a "loading dose" at the start of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") at a lower frequency.

[0246] In certain embodiments, the amount of anti-HLA-A2:NY-ESO-1 antigen-binding protein included 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 administered at a very low level to produce a therapeutic effect or an antibody dose below the level required to treat a disease such as cancer.

[0247] 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 / 2 or more weeks). 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 a patient before the next dose in the sequence is administered, without an intervening dose.

[0248] The method according to this aspect of the disclosure may comprise administering to a 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.

[0249] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments, the frequency of administering the second and / or third doses to the patient may vary during the course of the treatment regimen. The frequency of administration may also be adjusted based on the needs of the individual patient after a clinical examination during the course of treatment by a physician.

[0250] Diagnostic uses of the antigen-binding protein

[0251] The anti-HLA-A2:NY-ESO-1 antigen-binding proteins of the disclosure can be used to detect and / or measure NY-ESO-1 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate using one or more of the antigen-binding proteins of the disclosure in an assay to detect a disease or disorder, such as a NY-ESO-1-related disease or disorder, such as NY-ESO-1-positive cancer. Exemplary diagnostic assays 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 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 isolate 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 radioisotope, 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).

[0252] Samples that can be used in the NY-ESO-1 diagnostic assays according to the present disclosure include any tissue or fluid sample that can be obtained from a subject and that contains a detectable amount of NY-ESO-1 protein or a fragment thereof in a normal or pathological state. Typically, the level of NY-ESO-1 in a specific sample obtained from a healthy patient (e.g., a patient not suffering from an NY-ESO-1-related disease or disorder, such as an 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.

[0253] Antigen-binding proteins that are specific for 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 binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

[0254] 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 evaluate the prognosis of a patient's cancer and predict survival.

[0255] Examples

[0256] The following examples are presented to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the methods and compositions of the invention, and the following examples are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressures are at or near atmospheric pressure.

[0257] Example 1: Generation of Human Antibodies Against HLA-A2:NY-ESO-1_157-165

[0258] Human antibodies against HLA-A2:NY-ESO-1 were generated using an HLA-A2-coupled NY-ESO-1 peptide fragment of GenBank accession number NP_001318.1 (SEQ ID NO:271), which includes amino acids 157-165 (SLLMWITQV; "NY-ESO-1_V") in which cysteine (C) at position 165 is replaced by valine (V); SEQ ID NO:291). For example, as described in U.S. Patent No. 8,502,018, the immunogen was administered directly to mice (i.e., engineered mice containing DNA encoding the variable regions of the human immunoglobulin heavy and κ light chains) together with an adjuvant that stimulates an immune response. Antibody immune responses were monitored by HLA-A2:NY-ESO-1-specific immunoassays. When the desired immune response was achieved, spleen cells were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce HLA-A2:NY-ESO-1-specific antibodies. Using this technique and the above immunogen, several anti-NY-ESO-1 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained. As described in U.S. Patent 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 any of the immunized mice) without fusion to myeloma cells. Using this method, 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 named as follows: mAb24955N; mAb24956N; mAb24958N; mAb24959N; mAb28042P; mAb28035P; mAb28037P2; mAb28075P; mAb28105P; mAb28113P; mAb28128P; mAb29814P; mAb24955N; and mAb29822P2.

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

[0261] Example 2: Amino Acid and Nucleotide Sequences of Heavy and Light Chain Variable Regions

[0262]

[0263] ​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. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0264] Table 1: Amino Acid Sequence Identifiers

[0265]

[0266]

[0267] Table 2: Nucleic Acid Sequence Identifiers

[0268]

[0269] 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, an antibody can be referred to herein as, for example, "mAb17670P", "mAb17930N", "mAb17368N2", etc. As will be understood by those of ordinary skill in the art, an antibody having a particular Fc isotype can be converted into an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted into an antibody having a human IgG4, etc.), but in any case, the variable domains (including CDRs) represented by the numerical identifiers shown in Table 1 will remain the same, and regardless of the nature of the Fc domain, the binding characteristics to the antigen are expected to be the same or substantially similar.

[0270] In certain embodiments, selected antibodies having a murine IgG1 Fc are converted into antibodies having a human IgG4 Fc. In certain embodiments, the antibody comprises a human IgG4 Fc having two or more amino acid changes, as disclosed in U.S. Patent Publication No. 20100331527, the entire contents of which are incorporated herein. In one embodiment, the IgG4 Fc domain contains a serine-to-proline mutation (S108P) in the hinge region to promote dimer stability.

[0271] Table 3 lists the amino acid sequence identifiers of the heavy and light chain sequences of selected antibodies of the present disclosure.

[0272] Table 3: Heavy and Light Chain Sequence Identifiers

[0273]

[0274] *As described in U.S. Patent 9,359,437.

[0275] Example 3: Surface Plasmon Resonance-Derived Binding Affinity and Kinetic Constants of Human Monoclonal Anti-HLA-A2:NY-ESO-1 Monospecific Antibodies

[0276] The binding affinity 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 were 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. Patent 9,359,437)), hIgG2a constant region, mIgG2a constant region, or mIgG1 constant region). Reference (Ref) antibodies 1, 2, and 3 were generated from the antibodies described by Stewart-Jones et al., Proceedings of the National Academy of Sciences 106(14):5784-5788 (2009) and International Patent Publication No. WO2010106431. Ref Ab 1 contains the VH and VL domains of the 3M4E5 antibody with human IgG1 Fc and human λ light chain described in those publications; Ref Ab 2 contains the 3M4E5 VH domain and the T1 Fab VL domain with human IgG1 Fc and human λ light chain from the said publications; Ref Ab 3 contains the 3M4E5 VH domain and the T1Fab VL domain with human IgG4 with reduced effector function Fc (as described in U.S. Patent 9,359,437) and human λ VL domain from the said publications. Antibodies were captured onto a CM5 Biacore sensor surface (GE Healthcare Life Sciences) derivatized by amine coupling with a monoclonal anti-human Fc antibody (Jackson Immunoresearch) or onto 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 complex and 2906 (SEQ ID NO:272) were injected at a flow rate of 50 μL / min (MASS-1) or 30 μL / min (Biacore 4000) onto the anti-HLA-A2:NY-ESO-1 antibody capture surface. Antibody-reagent binding 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).

[0277] 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 association-dissociation equilibrium constant (KD) and dissociation half-life (t 1 1 / 2) were calculated from the kinetic rate constants as follows:

[0278] K D (M) = (kd / ka) and t 1 1 / 2 (min) = ((ln (2)) 2) / (60*kd))

[0279] The binding kinetic parameters of the monospecific anti-HLA-A2:NY-ESO-1 antibody to the monomeric HLA-A2:NY-ESO-1 peptide complex are shown in Table 4 below.

[0280] Table 4: Biacore binding affinity of anti-HLA-A2:NY-ESO-1 antibody at 25 °C

[0281]

[0282] Table 5: Biacore binding affinity of anti-HLA-A2:NY-ESO-1 antibody at 37 °C

[0283]

[0284]

[0285] The data indicate that most of the tested anti-HLA-A2:NY-ESO-1 antibodies bind selectively to the soluble HLA-A2:NY-ESO-1 peptide complex, some of which show nanomolar or sub-nanomolar affinities, and many antibodies have higher affinities for the complex than the reference antibody.

[0286] Example 4: FACS binding of anti-HLA-A2:NY-ESO-1 antibody to T2 cells pulsed with NY-ESO-1_157-165 peptide

[0287] The relative binding of the NY-ESO-1:157-165 antibody was evaluated by flow cytometry on T2 (174CEM.T2) cells pulsed with the NY-ESO-1_157-165 (C165) peptide (SEQ ID NO: 269). To achieve pulsing, T2 cells (174CEM.T2) were seeded at 1 × 10 6Cells / ml (Fetal Bovine Serum Cat#31035 - 025) were resuspended at the density in AIM V medium. The cells were pulsed with 10 μg / ml hB2M (EMD Millipore Cat#475828) and 100 μg / ml of the indicated peptides. Then the T2 cells were incubated overnight at 37 °C, washed in staining buffer and subsequently stained.

[0288] To stain the cells, cells were harvested from the flask using cell dissociation buffer (Millipore, Cat#S - 004 - C) and counted. In a 96 - well V - bottom plate, the 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 serial 3 - fold dilutions (1.7 pM - 100 nM) of the primary antibody for 30 minutes at 4 °C. After incubation with the primary antibody, the cells were washed once in staining buffer and then stained with an APC - conjugated secondary antibody (Jackson ImmunoResearch, Cat#109 - 136 - 170) at 5 μg / ml for 30 minutes at 4 °C. Then the cells were washed and fixed using 50% of BD Cytofix solution (BD, Cat#554655). Samples were analyzed on an intellicyt iQue flow cytometer to calculate the 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 the EC 50 value. A separate secondary antibody (i.e., no primary antibody) for each dose - response curve was also included in the analysis as a continuation of the serial 3 - fold dilutions and represented as the lowest dose. As described above, Ref Ab 2 and Ref Ab 3 were used as controls. The EC 50 values (M) are shown in Table 6.

[0289] Table 6: FACS Binding of Anti - HLA - A2:NY - ESO - 1 Antibodies

[0290]

[0291]

[0292] Example 5: Binding Specificity of Anti-HLA-A2:NY-ESO-1 Antibody to T2 Cells Pulsed with Predicted Off-Target Peptides The binding specificity of the NY-ESO-1:157-165 antibody was evaluated by flow cytometry 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 pulsing, T2 cells (174CEM.T2) were resuspended in AIMV medium at a density of 1×10 6 cells / 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 peptide. The T2 cells were then incubated overnight at 37 °C, washed in staining buffer, and stained with the indicated antibody at a concentration of 10 μg / ml according to the protocol described in Example 4 above. The MFI values were calculated and expressed as the ratio of T2 pulsed cells to unpulsed cells. The results of these assays are presented in Table 8. Effective peptide loading was determined by comparing the increase in HLA-A2 surface staining of pulsed cell lines with unpulsed cell lines using anti-HLA-A2 antibody. Any increase of 1.4-fold or more was considered to be peptide-loaded. Loading of one peptide (ITCH(807-815)) could not be confirmed. However, as described above, the two comparator NY-ESO-1 mAb Ref Ab 1 and Ref Ab 2 bound to cells pulsed with this peptide, indicating that some peptide had been loaded onto the cells.

[0293] Table 7: NY-ESO-1_157-165 Peptide and Predicted Off-Target Peptides

[0294]

[0295]

[0296] Table 8: Ratio of Anti-HLA-A2:NY-ESO-1 Antibody Bound to T2 Cells Pulsed with Off-Target Peptides to Unpulsed Cells

[0297]

[0298] 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 two comparator antibodies which had significant binding to multiple off-target peptides. Nonspecific binding can 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 a subject). Thus, as described herein, the identification of antigen-binding proteins (such as antibodies) with minimal off-target binding can be beneficial for the development of therapeutics targeting MAGE-A4.

[0299] 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 alanine-scanned 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 were payloaded by HLA.A2 surface staining (as described above), except for NY-ESO-1:157-165, C165V, S157A and NY-ESO-1:157-165, C165V, I162A. The following residues were important for the 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 the binding of the comparator monoclonal antibody Ref Ab 3. Methionine 160 was particularly important for the binding of mAb24956N, mAb24958N, and mAb28105P.

[0300] Table 9: Ratio of anti-HLA-A2:NY-ESO-1 antibody binding to T2 cells pulsed with alanine-scanned peptides to unpulsed cells

[0301]

[0302] Example 6: Reformatting anti-HLA-A2:NY-ESO-1 antibodies into ScFv for chimeric antigen receptors

[0303] 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. The NY-ESO-1:157-165 CARs were cloned into a lentiviral expression vector (Lenti-X TMIn the dual-cistronic lentiviral expression system, Clontech Cat#632181), and according to the manufacturer's protocol, lentiviral particles were generated using the Lenti-X Packaging Single-Shot (VSV-G) system (Clontech Cat#631276). Then, according to the manufacturer's protocol, 8 different CARs were used to transduce Jurkat / NFATLuc cl.3C7 cells (cells expressing the NFAT-luciferase reporter gene) using RetroNectin pre-coated culture dishes (Clontech, Cat#T110a). 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-T 24955N VL-VH; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VH-VL; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24956N VL-VH; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24958N VH-VL; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24958N VL-VH; Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24959N VH-VL; and Jurkat / NFATLuc cl 3C7 / NY-ESO CAR-T 24959N VL-VH. Then the activity of the CAR-T cell lines was evaluated in a CAR-T / APC (antigen-presenting cell) bioassay. For the bioassay, 50,000 CAR-T cells were added to 50 μl of assay medium (RPMI medium containing 10% FBS and 1% P / S / G) in a Thermo-Nunc 96-well white plate (Thermo Scientific, Cat#136101), and then 3-fold serial dilutions of APC (from 200,000 cells to 274 cells) were added in 50 μl of 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 number P01892), human β2M (accession number NP_004039.1), and the ubiquitin peptide cassette as described above, which contains 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 Med 189(6):895-906); containing the 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 a 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 values. The zero APC condition for each dose-response curve was also included in the analysis as a continuation of the three-fold serial dilution and was represented as the lowest dose. The maximum activation fold was 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-1:157-165WT cells, all eight NY-ESO-1 CAR cell lines were activated. As shown in Table 10, three NY-ESO-1 CAR cell lines were activated in the presence of the following IM9 cells (Jurkat / NFATLuc cl 3C7 / NY-ESOCAR-T 24955N VH-VL; Jurkat / NFATLuc cl 3C7 / NY-ESOCAR-T 24955N VL-VH; and Jurkat / NFATLuc cl3C7 / NY-ESOCAR-T 24956N VL-VH).

[0304] Table 10: Activation of NY-ESO-1:157-165 CAR-T in the CAR-T / APC bioassay

[0305]

[0306]

[0307] Example 7: Structural Analysis of a Fab That Binds to the HLA2:NY-ESO-1_157-165 Peptide

[0308] To better understand the specific interactions between antibodies and HLA-peptide complexes, the X-ray crystal structure of the Fab fragment of antibody mAb28105P that binds to HLA-A2 / hB2M was determined. This fragment displays a peptide containing residues 157-165 from the cancer-testis antigen 1 (CTAG1B; NY-ESO-1). The peptide was modified by replacing its native cysteine residue 165 with valine. All nine residues of the NY-ESO-1(C165V) peptide presented by HLA were clearly visible in the electron density map of this structure, and the residues of HLA and Fab surrounding the peptide were also well resolved. This structure was refined at a resolution of , but updated crystal refinement techniques (deformable elastic network or "jelly body" refinement) helped prevent overfitting and ensured the accuracy of the resulting model.

[0309] The mAb28105P Fab binds to the top of the HLA-peptide complex in a manner similar to TCR binding. The Fab is centered on the bound peptide, with HCDR3 contacting the C-terminal half of the bound peptide and the light chain CDRs contacting the N-terminal half of the peptide. The peptide residues M160 and W161 are located at the center of the Fab binding interface and contact heavy and light chain CDR residues, as described below. Other published structures of groove peptide-antibody complexes (e.g., PDB codes 1W72 and 4WUU) indicate that antibodies do not have to cover the entire HLA-presented peptide; however, antibodies with only partial peptide coverage have lower specificity and can tolerate larger variations in the uncontacted peptide portion with little loss of binding affinity.

[0310] 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. The peptide residues 157, 158, 159, 162, and 165 all point towards the HLA molecule. The Fab light chain completely screens residue 163 from the solvent but does not directly contact the antibody residue. The bound peptide is numbered according to the residue positions in SEQ ID NO:271, as shown in SEQ ID NO:291:

[0311] Amino acid S L L M W I T Q V Position 157 158 159 160 161 162 163 164 165

[0312] (SEQ ID NO:291)

[0313] All Fab contacts are made with the side chains of the HLA-bound peptide, not its backbone.

[0314] The peptide contacts made by mAb28105P Fab are concentrated in HCDR3, with little contribution 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 "interact" can include direct or water-mediated hydrogen bonds, charge-charge interactions, or hydrophobic / Van der Waals interactions.

[0315] Four of the NY-ESO-1_157-165 antibodies (mAb24955N, mAb24956N, mAb24958N, and mAb24959N) are highly similar in their light chain sequences but differ in their heavy chain sequences in HCDR2 and HCDR3. Seven peptide-binding residues are generally conserved: three residues are the same in all four antibodies, and more than three of the four sequences are the same, indicating a common binding mode among the antibodies.

[0316] Example 8: Analysis of Recombinant Antigen Receptor Signaling Domains

[0317] Chimeric antigen receptors use anti-HLA-A2 / NY-ESO-1 157-165 antibody mAb28105P (V L :SEQ ID NO:294; V H :SEQ ID NO:293) V L and V H sequences to construct, and the chimeric antigen receptor contains anti-HLA-A2 / NY-ESO-1 in the V H -V L orientation 157-165scFv; and 1) a human CD8 (huCD8) hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ signaling domain (BB / z CAR) (full-length CAR sequence: SEQ ID NO: 301); or 2) a huCD28 hinge / transmembrane / co-stimulatory domain and CD3ζ signaling domain (28 / z CAR) (full-length CAR sequence: SEQ ID NO: 302). As a non-binding control, the BB / z CAR was designed using an irrelevant scFv plus the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. These CARs were cloned into the pLVX lentiviral vector with an EF1a promoter and P2A:eGFP sequence (SEQ ID NO: 300) for tracking CAR-transduced cells and generating VSV pseudotyped lentiviruses. Figure 1A For construct design, Table 11 is a summary of the constructs.

[0318] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) from normal donors, stimulated with CD3 / CD28 beads + 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI = 5. The transduced cells were expanded with CD3 / CD28 beads + 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use during in vivo experiments.

[0319] To determine the in vivo efficacy of anti-HLA-A2 / NY-ESO-1 157-165-targeted chimeric antigen receptor (CAR) T cells, xenograft tumor studies were conducted. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg 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, 20 × 10 6 T cells, which expressed the non-binding control BB / zCAR (control CART), anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR, or anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR from two different donors (as determined by the frequency of GFP-expressing cells, a marker of those cells transduced with CAR) were injected intravenously into the mice (n = 5 per group). Tumor growth was evaluated on day 21 by measuring tumor volume.

[0320] To determine the tumor volume by external calipers, the maximum longitudinal diameter (in mm) and the maximum transverse diameter (in mm) were determined. The tumor volume based on caliper measurements was calculated using the following formula: Volume (mm 3 ) = (length × width 2 ) / 2.

[0321] Table 11: CAR constructs

[0322]

[0323] Overall, the results showed that compared with anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR T cells, anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR T cells exhibited superior in vivo anti-tumor activity and anti-tumor kinetic characteristics, but both types of CARs showed anti-tumor activity compared with the non-binding control. A375 tumors gradually grew in mice receiving control CART cells. Compared with control mice receiving CAR T treatment, mice receiving anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR T cells had slower tumor growth on day 21 (p < 0.04), day 28 (p < 0.0001), and day 38 (p < 0.0001) (statistical data analyzed by 2-way ANOVA). Anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR T treatment also led to inhibition of the growth of established A375 tumors on day 16 (p = 0.03), day 19 (p = 0.0002), day 21 (p < 0.0001), day 28 (p < 0.0001), and day 38 (p < 0.0001) (statistical data analyzed by 2-way ANOVA). Thus, the enhanced efficacy of anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR compared with anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR was confirmed because, as determined by 2-way ANOVA, the tumor sizes on day 21 (p = 0.002), day 28 (p < 0.0001), and day 38 (p < 0.0001) were statistically significant (with p < 0.0001 for two days). See Figure 1B and Figure 1C , and Tables 12 to 22.

[0324] Table 12: CAR efficacy, day 3

[0325]

[0326] Table 13: CAR efficacy, day 5

[0327]

[0328] Table 14: CAR efficacy, day 7

[0329]

[0330] Table 15: CAR efficacy, day 10

[0331]

[0332] Table 16: CAR efficacy, day 12

[0333]

[0334] Table 17: CAR efficacy, day 14

[0335]

[0336] Table 18: CAR efficacy, day 16

[0337]

[0338] Table 19: CAR efficacy, day 19

[0339]

[0340] Table 20: CAR efficacy, day 21

[0341]

[0342] Table 21: CAR efficacy, day 28

[0343]

[0344] Table 22: CAR efficacy, day 38

[0345]

[0346] The present invention should not be limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention will also become apparent to those skilled in the art from the foregoing description and the accompanying drawings, in addition to those modifications described herein. Such modifications are intended to be within the scope of the appended claims.

Claims

1. An antigen-binding protein that specifically binds to an HLA-A2: New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) complex, wherein at least one amino acid of the complex corresponds to an amino acid selected from the group consisting of M160, W161, and Q164 of SEQ ID NO:

271.

2. The antigen-binding protein according to claim 1, wherein the isolated antigen-binding protein specifically binds to a conformational epitope of an NY-ESO-1 polypeptide presented by HLA-A2.

3. The antigen-binding protein according to claim 1 or 2, wherein the at least one amino acid is at least two amino acids.

4. The antigen-binding protein according to claim 3, wherein the at least one amino acid is three amino acids.

5. An antigen-binding protein that specifically binds to the HLA-A2:NY-ESO-1 157-165 peptide complex, wherein the isolated antigen-binding protein interacts with the amino acids M160, W161, and Q164 of the NY-ESO-1 157-165 peptide, as determined by X-ray crystallography at a resolution of or better.

6. The antigen-binding protein according to claim 5, wherein the resolution is or higher.

7. The antigen-binding protein according to claim 6, wherein the resolution is or higher.

8. An antigen-binding protein that specifically binds to an HLA-A2:NY-ESO-1 157-165 peptide complex but does not specifically bind to one or more off-target peptides selected from the group consisting of BCL9L 1351-1359 (SEQ ID NO: 273), GRID1 7-15 (SEQ ID NO: 274), ZDHHC1 376-384 (SEQ ID NO: 276), ITCH 807-815 (SEQ ID NO: 277), and URB1 1853-1861 (SEQ ID NO: 280), wherein the ratio of i) the antigen-binding protein that binds to cells pulsed with the one or more off-target peptides to ii) unpulsed cells is less than about 9.

9. The antigen-binding protein according to claim 8, wherein the ratio is less than about 8.

10. The antigen-binding protein according to claim 9, wherein the ratio is less than about 7.

11. The antigen-binding protein according to claim 10, wherein the ratio is less than about 6.

12. The antigen-binding protein according to claim 11, wherein the ratio is less than about 5.

13. The antigen-binding protein according to claim 12, wherein the ratio is less than about 4.

14. The antigen-binding protein according to claim 13, wherein the ratio is less than about 3.

15. The antigen-binding protein according to claim 14, wherein the ratio is less than about 2.

16. The antigen-binding protein according to any one of claims 1 to 15, wherein the antigen-binding protein binds to a monomeric HLA-A2:NY-ESO-1 157-165 peptide complex, and the binding dissociation equilibrium constant (K D ) is less than about 1 nM as measured in a surface plasmon resonance assay at 25 °C.

17. The antigen-binding protein according to any one of claims 1 to 16, wherein the NY-ESO-1 peptide comprises the amino acid sequence of SLLMWITQC (SEQ ID NO: 269) or SLLMWITQV (SEQ ID NO: 291).

18. The antigen-binding protein according to any one of claims 1 to 17, wherein the antigen-binding protein is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), T-body construct, or chimeric antigen receptor (CAR).

19. The antigen-binding protein according to claim 19, wherein the antigen-binding protein is a CAR.

20. The CAR according to claim 19, wherein the CAR comprises a heavy chain variable region (LCVR), a light chain variable region (LCVR), a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain.

21. The CAR according to claim 20, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

22. The CAR according to claim 20, wherein the co-stimulatory domain is a CD28 co-stimulatory domain.

23. The antigen-binding protein according to any one of claims 1 to 22, wherein the antigen-binding protein is a human monoclonal antibody or an antigen-binding fragment thereof.

24. The antigen-binding protein according to any one of claims 1 to 23, which comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in any one of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any one of the light chain variable region (LCVR) sequences listed in Table 1.

25. The antigen-binding protein according to claim 24, wherein the HCVR sequence is selected from the group consisting of: SEQ ID NO:2, 22, 42, 142, and 250.

26. The antigen-binding protein according to claim 24, wherein the LCVR sequence is selected from the group consisting of: SEQ ID NO:10, 30, 50, 150, and 260.

27. The antigen-binding protein according to claim 24, wherein the HCVR / LCVR sequence is selected from the group consisting of: SEQ IDNO: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.

28. The antigen-binding protein according to claim 24, wherein the HCVR / HCVR sequence is selected from the group consisting of: SEQ IDNO:2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258.

29. The antigen-binding protein according to any one of claims 1 to 25, which comprises an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1.

30. The antigen-binding protein according to claim 29, wherein the HCVR sequence is selected from the group consisting of: SEQ ID NO:2, 22, 42, 142, and 250.

31. The antigen-binding protein according to any one of claims 1 to 30, which comprises an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

32. The antigen-binding protein according to claim 31, wherein the LCVR sequence is selected from the group consisting of: SEQ ID NO:10, 30, 50, 150, and 260.

33. The antigen-binding protein according to any one of claims 1 to 32, comprising: (a) an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and (b) an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

34. The antigen-binding protein according to claim 33, wherein the HCVR / LCVR sequences are 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, 230 / 238, and 250 / 258.

35. The antigen-binding protein according to claim 34, wherein the HCVR / HCVR sequences are selected from the group consisting of: SEQ ID NO: 2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258.

36. The antigen-binding protein according to any one of claims 1 to 35, comprising: (a) an 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, 232, and 252; (b) an 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, 234, and 254; (c) an 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, 236, and 256; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of: SEQ ID NO: 12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, 240, and 260; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 34, 54, 74, 94, 114, 134, 154, 174, 242, and 262; and (f) an 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, 244, and 264.

37. The antigen-binding protein according to any one of claims 1 to 28 and 36, wherein the antigen-binding protein comprises an HCVR having at least 80% sequence identity with the HCVR sequences listed in Table 1.

38. The antigen-binding protein according to claim 37, wherein the antigen-binding protein comprises an HCVR having at least 90% sequence identity with the HCVR sequences listed in Table 1.

39. The antigen-binding protein according to claim 38, wherein the isolated antigen-binding protein comprises an HCVR having at least 95% sequence identity with the HCVR sequences listed in Table 1.

40. The antigen-binding protein according to any one of claims 1 to 28 and 36 to 39, wherein the antigen-binding protein comprises an LCVR having at least 80% sequence identity with the LCVR sequences listed in Table 1.

41. The antigen-binding protein according to claim 40, wherein the isolated antigen-binding protein comprises an LCVR having at least 90% sequence identity with the LCVR sequences listed in Table 1.

42. The antigen-binding protein according to claim 38, wherein the antigen-binding protein comprises an LCVR having at least 95% sequence identity with the LCVR sequences listed in Table 1.

43. An antigen-binding protein that competes for binding with the antigen-binding protein according to any one of claims 1 to 42.

44. An antigen-binding protein that binds to the same epitope as the antigen-binding protein according to any one of claims 1 to 42.

45. The antigen-binding protein according to any one of claims 1 to 44, which comprises a detectable moiety.

46. A pharmaceutical composition comprising the antigen-binding protein that binds to HLA-A2:NY-ESO-1 according to any one of claims 1 to 45 and a pharmaceutically acceptable carrier or diluent.

47. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding an HCVR of the antigen-binding protein according to any one of claims 1 to 45.

48. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding an LCVR of the antigen-binding protein according to any one of claims 1 to 45.

49. A vector comprising the polynucleotide molecule of claim 47 or 48.

50. A cell that expresses the polynucleotide molecule of claim 47 or 48 or the vector of claim 49.

51. A method of treating a subject having an NY-ESO-1-related disease or disorder, comprising administering to the subject a therapeutically effective amount of the antigen-binding protein according to any one of claims 1 to 45, or the pharmaceutical composition according to claim 46, or the cell according to claim 50, thereby treating the subject.

52. The method according to claim 51, wherein the NY-ESO-1-related disease or disorder is NY-ESO-1-related cancer.

53. The method according to claim 52, wherein the NY-ESO-related cancer is selected from the group consisting of: 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, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, brain tumor, fallopian tube cancer, ovarian epithelial cancer, primary peritoneal cancer, advanced solid tumor, soft tissue sarcoma, melanoma, sarcoma, myelodysplastic syndrome, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Hodgkin disease, multiple myeloma, synovial sarcoma, metastatic solid tumor, esophageal cancer, rhabdomyosarcoma, advanced myxoid disease, round cell liposarcoma, metastatic melanoma or recurrent non-small cell lung cancer.

54. The method according to any one of claims 51 to 53, wherein the antigen-binding protein is administered to the subject in combination with a second therapeutic agent.

55. The method according to claim 54, wherein the second therapeutic agent is selected from the group consisting of: PD-1 inhibitor, CTLA-4 inhibitor, antibody against a tumor-specific antigen, antibody against a virus-infected cell antigen, PD-L1 inhibitor, CD20 inhibitor, bispecific antibody against CD20 and CD3, dietary supplement such as antioxidant, VEGF antagonist, chemotherapeutic agent, cytotoxic agent, surgery, radiotherapy, NSAID, corticosteroid and any other therapy that can be used to improve at least one symptom associated with the disease or disorder.

56. The method according to any one of claims 51 to 55, wherein the antigen-binding protein is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.

57. The method according to any one of claims 51 to 56, wherein the antigen-binding protein is administered at a dose of from about 0.1 mg / kg of the subject's body weight to about 100 mg / kg of the subject's body weight.

58. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular binding domain, a transmembrane domain and an intracellular signaling domain that specifically binds to a conformational epitope of a New York esophageal squamous cell carcinoma 1 peptide (NY-ESO-1 peptide) presented by HLA-A2.

59. The isolated nucleic acid molecule according to claim 58, wherein the extracellular binding domain is an anti-HLA-A2:NY-ESO-1 antigen-binding protein.

60. The isolated nucleic acid molecule according to claim 59, wherein the antigen-binding protein comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in any one of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any one of the light chain variable region (LCVR) sequences listed in Table 1.

61. An isolated nucleic acid molecule according to any one of claims 58 to 60, wherein the antigen-binding protein comprises an HCVR and has an amino acid sequence selected from the HCVR sequences listed in Table 1.

62. An isolated nucleic acid molecule according to any one of claims 58 to 61, wherein the antigen-binding protein comprises an LCVR and has an amino acid sequence selected from the LCVR sequences listed in Table 1.

63. An isolated nucleic acid molecule according to any one of claims 58 to 62, wherein the antigen-binding protein comprises (a) an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1; and (b) an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

64. An isolated nucleic acid molecule according to any one of claims 58 to 63, wherein the antigen-binding protein comprises: (a) an 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, 232, and 252; (b) an 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, 234, and 254; (c) an 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, 236, and 256; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of: SEQ ID NO:12, 32, 52, 72, 92, 112, 132, 152, 172, 188, 221, 240, and 260; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of: SEQ ID NO:14, 34, 54, 74, 94, 114, 134, 154, 174, 242, and 262; and (f) an 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, 244, and 264.

65. An isolated nucleic acid molecule according to any one of claims 58 to 64, wherein the antigen-binding protein comprises an 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, 230 / 238, and 250 / 258.

66. An isolated nucleic acid molecule according to any one of claims 58 to 65, wherein the antigen-binding protein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO:2 / 10, 22 / 30, 42 / 50, 142 / 150, and 250 / 258.

67. An isolated nucleic acid molecule according to any one of claims 58 to 66, which comprises any one of the sequences listed in Table 2.

68. An isolated nucleic acid molecule according to any one of claims 58 to 67, wherein the antigen-binding protein is a scFv.

69. A vector which comprises an isolated nucleic acid molecule according to any one of claims 58 to 68.

70. An isolated immune effector cell which comprises an isolated nucleic acid molecule according to any one of claims 58 to 68 or a vector according to claim 69.

71. The isolated immune effector cell according to claim 70, which is a T body.

72. The isolated immune effector cell according to claim 70 or 71, wherein the cell expresses the CAR.

73. A method of treating a subject having a NY-ESO-1 related disease or disorder, which comprises administering to the subject an immune effector cell according to any one of claims 70 to 72.

74. The method according to claim 73, wherein the NY-ESO-1 related disease or disorder is NY-ESO-1 related cancer.

75. The method according to claim 73 or 74, wherein the antigen-binding protein is administered to the subject in combination with a second therapeutic agent.

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