Binding proteins recognizing magea4 immunogenic peptides and uses thereof

Abstract

Provided herein are binding proteins recognizing MAGEA4 immunogenic peptides and uses thereof.

Description

[0001] TTC-020

[0002] BINDING PROTEINS RECOGNIZING MAGEA4 IMMUNOGENIC PEPTIDES AND USES THEREOF

[0003] Cross-reference to Related Application

[0004] This application claims the benefit of priority to U. S. Provisional Application Serial No. 63 / 717,717, filed on November 7, 2024, the entire contents of said application are incorporated herein in their entirety by this reference.

[0005] Background of the Invention

[0006] Adoptive cell transfer (ACT) using engineered T cells has demonstrated great efficacy in treating certain types of liquid tumor and holds promise for treating solid tumors. T cell receptor-engineered T cells (TCR-T) are T cells expressing an exogenous TCR that recognizes an antigen that exists in cancer cells. The TCR-antigen interaction is the core component of the targeting mechanism that allows the TCR-T cells to kill cancer cells. One of the challenges for the broad testing and adoption of TCR-T therapy is the lack of TCR-antigen pairs that are applicable to a wide range of patients and indications.

[0007] In addition, the number of pursued antigens is limited due to the difficulty of discovering novel TCR-antigen pairs which commonly require prediction of the MHC presented epitope. However, such epitopes may not be immunogenic, thereby making it difficult to identify a reactive TCR, or the epitope may not be processed and presented physiologically by the cancer cells. Accordingly, there is a great need in the art to identify TCR-antigen pairs in the context of a variety of widely applicable HLA alleles in order to develop useful reagents to diagnose, prognose, treat, and screen agents relevant for disorders characterized by the expression of the antigens.

[0008] of the Invention

[0009] The present invention is based, at least in part, on the discovery of binding proteins, including T cell receptors (TCRs), that recognize MAGEA4 immunogenic peptides, such as those listed in Table 1, in the context of a variety of HLA alleles (e.g., HLA-A*02:01). Such binding proteins have a variety of uses, including treating disorders associated with MAGEA4 expression (e.g., subjects afflicted with melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and / or bladder urothelial carcinoma). MAGEA4 is demonstrated herein to be selectively expressed in cancer and testis tissue, but not in normal somatic tissues,

[0010] FoleyHoagUS 13160800.1 TTC-020

[0011] thereby making it an ideal target for binding proteins described herein. The ability of MAGEA4 binding proteins (e.g., TCRs described herein) to bind MAGEA4 immunogenic peptides and to elicit immune responses that kill cells expressing MAGEA4 e.g., cancer cells) demonstrates the utility of such binding proteins in a diversity of uses, including methods of diagnosis, prognosis, treatment, and screening of agents relevant for disorders characterized by MAGEA4 expression. For example, and without limitation, discovery of TCRs specific to the MAGEA4 epitopes described herein and presented on HLA A02:01 was particularly challenging and the results were unexpected. In particular, analysis of 1.09E9 naive T cells from a variety of 10 different donors only yielded 2,100 target binding clones and it was empirically determined that fewer than 0.33% of such clones (e.g., fewer than 7 clones) displayed substantial cytotoxicity. The two selected clones described further herein were determined to have additional unexpected properties combining superlative cytotoxicity and functionality, no observed alloreactivity, and no observed off-targets of clinical concern.

[0012] In one aspect, provided herein is a binding protein comprising: a) a T cell receptor (TCR) alpha chain CDR sequence with at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence with at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x1 O’4M.

[0013] In another aspect, provided herein is a binding protein comprising: a) a TCR alpha chain variable (Vc<) domain sequence with at least about 80% identity to a TCR Vc< domain sequence selected from the group consisting of TCR Va domain sequences listed in Table 2; and / or b) a TCR beta chain variable (Vp) domain sequence with at least about 80% identity to a TCR Vp domain sequence selected from the group consisting of TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K less than or equal to about 5x1 O’4M.

[0014] In still another aspect, provided herein is a binding protein comprising: a) a TCR alpha chain sequence with at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence with at least about 80% identity to a TCR beta chain sequence selected

[0015] FoleyHoagUS 13160800.1 TTC-020

[0016] from the group consisting of TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5 1 O’4M.

[0017] In yet another aspect, provided herein is a binding protein comprising: a) a TCR alpha chain CDR sequence selected from the group consisting of TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence selected from the group consisting of TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K less than or equal to about 5x1 O’4M.

[0018] In another aspect, provided herein is a binding protein comprising: a) a TCR alpha chain variable (Va) domain sequence selected from the group consisting of TCR Va domain sequences listed in Table 2; and / or b) a TCR beta chain variable (Vp) domain sequence selected from the group consisting of TCR Vp domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5xl0‘4M.

[0019] In still another aspect, provided herein is a binding protein comprising: a) a TCR alpha chain sequence selected from the group consisting of TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5xl0"4M.

[0020] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, 1) the TCR alpha chain CDR, TCR Va domain, and / or TCR alpha chain is encoded by a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCR beta chain CDR, TCR Vp domain, and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof as compared to the cognate reference CDR sequence listed in Table 2. In another embodiment, the MAGEA4 immunogenic peptide comprises an amino acid sequence selected from the group of amino

[0021] - 3 - FoleyHoagUS 13160800.1 TTC-020

[0022] acid sequences listed in Table 1, optionally wherein the MAGEA4 immunogenic peptide sequence comprises the amino acid sequence GVYDGREHTV or KVLEHVVRV. In still another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein is a TCR, an antigen- binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally wherein the binding domain comprises a binding domain having a transmembrane domain and an effector domain that is intracellular. In another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, optionally wherein the TCR alpha chain and the TCR beta chain are covalently linked through a linker peptide. In still another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or a label. In yet another embodiment, the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of an inflammatory agent, cytokine, toxin, cytotoxic molecule, radioactive isotope, or antibody or antigen-binding fragment thereof. In still another embodiment, the binding protein binds to the pMHC complex on a cell surface. In yet another embodiment, the MHC is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In another embodiment, the MHC is an MHC class I molecule. In still another embodiment, the MHC comprises an MHC alpha chain that is an HLA serotype HLA-A*02. In yet another embodiment, the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0205, HLA-A*0206, and HLA-A*0207 allele. In another embodiment, binding of the binding protein to the MAGEA4 peptide-MHC (pMHC) complex elicits an immune response, optionally wherein the immune response. In still another embodiment, the T cell response is selected from the group consisting of a CD8+ T cell response, T cell expansion, cytokine release, and / or cytotoxic killing. In yet another embodiment, the binding protein is capable of specifically and / or selectively binding to the MAGEA4 immunogenic peptide- MHC (pMHC) complex with a Kd less than or equal to about IxlO’4M, less than or equal to about 5x10"''’ M, less than or equal to about IxlO"5M, less than or equal to about 5xl0"6M, less than or equal to about IxlO-6M, less than or equal to about 5x10’7M, less than or equal to about IxlO"7M, less than or equal to about 5x10"8M, less than or equal to about IxlO"8M, less than or equal to about 5xl0"9M, less than or equal to about IxlO’9M, less than or equal - 4 - FoleyHoagUS 13160800.1 TTC-020

[0023] to about 5xlO"10M, less than or equal to about 1x1 O'10M, less than or equal to about 5xl0‘nM, less than or equal to about IxlO"11M, less than or equal to about 5xl0"12M, or less than or equal to about IxlO-12M. In another embodiment, the binding protein has a higher binding affinity to the peptide-MHC (pMHC) than does a known T-cell receptor, optionally wherein the higher binding affinity is at least 1.05-fold higher. In still another embodiment, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing than does a known T-cell receptor when contacted with target cells with a heterozygous expression of MAGEA4, optionally wherein the induction is at least 1.05-fold higher. In yet another embodiment, the cytotoxic killing is a target cancer cell. In another embodiment, the cancer is selected from the group consisting of melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and bladder urothelial carcinoma. In still another embodiment, the binding protein does not bind to a peptide-MHC (pMHC) complex comprising a peptide epitope derived from NALCN, CFI, and MAGE-A8 (e.g., a

[0024] PTAVIRDFGGVMDIFIYLVSLI NALCN epitope, a NDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSL QWGEVKLISNCSKFYGNRFYEKEMECAGTYDGS CFI epitope, and / or a GLLIIVLGMILMEGSRAPEEAIWEALSVMGLYDGREHSVYWKLRK MAGE-A8 epitope.

[0025] These genes are well-known and are recognized to be annotated according to the following NCBI Gene ID numbers, each of which is available on the World Wide Web at ncbi.nlm.nih.gov / gene:

[0026] NALCN: Gene ID 259232 and NM_001350748.2 and NP_001337677.1,

[0027] NM..001350749.2 and NP. 001337678.1, NM..001350750.2 and NP.001337679.1, NM„001350751.2 and NP„001337680.1, and NM_052867.4 and NP__443099.1, as representative clones;

[0028] CFI: Gene ID 3426 and NM_000204.5 and NPJ100195.3, NM__001318057.2 and NP-001304986.2, NMJ101331035.2 and NPJ101317964.1, NMJJ01375278.1 and NP. 001362207.1, NM..001375279.1 and NP.001362208.1, NM...001375280.1 and

[0029] NP-001362209.1, NM_001375281.1 and NP_001362210.1, NM_001375282.1 and NP.001362211.1, NM..001375283.1 and NP...001362212.1, and NM..001375284.1 and NP_001362213.1, as representative clones;

[0030] - 5 - FoleyHoagUS 13160800.1 TTC-020

[0031] MAGE-A8: Gene ID 4107 and NM„001166400.2 and NP„001159872.1, NM„001166401.2 and NP__001159873.1, and NM„005364.5 and NPJ105355.2, as representative clones.

[0032] In another aspect, a TCR alpha chain and / or beta chain selected from the group consisting of TCR alpha chain and beta chain sequences listed in Table 2 is provided.

[0033] In still another aspect, provided herein is an isolated nucleic acid molecule i) that hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, ii) a sequence with at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, and / or iii) a sequence with at least about 80% homology to a nucleic acid encoding listed in Table 2, optionally wherein the isolated nucleic acid molecule comprises 1) a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2 and / or 2) a TRBV, TRBJ, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2.

[0034] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the nucleic acid is codon optimized for expression in a host cell.

[0035] In another aspect, provided herein is a vector comprising the isolated nucleic acid described herein, optionally wherein i) the vector is a cloning vector, expression vector, or viral vector and / or ii) the vector comprises a vector sequence listed in Table 3.

[0036] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the vector further comprises a nucleic acid sequence encoding CD8a, CD8[3, a dominant negative TGF|3 receptor II (DN-TGFpRII), selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In another embodiment, the nucleic acid sequence encoding CD8(X, CD8β, the DN-TGFPRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag, optionally wherein the nucleic acid encoding a tag is at the 5’ upstream of the nucleic acid sequence encoding CD8oc, CD8|3, the DN-TGFpRII, and / or the selectable protein marker such that the tag is fused to the N-terminus of CD8(X, CD8|3, the DN-TGFpRII, and / or the selectable protein marker. In still another embodiment, the tag is a

[0037] - 6 - FoleyHoagUS 13160800.1 TTC-020

[0038] CD34 enrichment tag. In yet another embodiment, the TCRa, TCRβ, and / or the DN- TGFpRII comprises a mutated transmembrane domain and / or a mutated constant domain, optionally wherein the mutated transmembrane domain and / or mutated constant domain enhance cellular surface expression of TCRa, TCRp, and / or the DN-TGFpRII, while decreasing expression of endogenous TCRa, TCRβ, and / or TGFpRII. In another embodiment, the isolated nucleic acid of any one of claims claim 29 or 30, and / or the nucleic acid sequence of any one of claims 32-35 encoding TCRa, TCRp, CD8a, CD8|3, the DN-TGFβRII, and / or the selectable protein marker, is interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide. In still another embodiment, the self-cleaving peptide is P2A, E2A, F2A or T2A.

[0039] In another aspect, provided herein is a host cell which comprises the isolated nucleic acid or vector described herein and / or expresses the binding protein described herein, optionally wherein the cell is genetically engineered.

[0040] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In another embodiment, the host cell comprises a knockout of an HLA gene selected from an al macroglobulin gene, a2 macroglobulin gene, a3 macroglobulin gene, pi microglobulin gene, P2 microglobulin gene, and combinations thereof. In still another embodiment, the host cell comprises a knockout of a TCR gene selected from a TCR a variable region gene, TCR P variable region gene, TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8a, CD8P, a DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, further optionally wherein the CD8a, CD8P, the DN-TGFPRII, and / or the selectable protein marker is fused to a CD34 enrichment tag. In another embodiment, the host cells are enriched using the CD34 enrichment tag. In still another embodiment, the host cell is a hematopoietic progenitor cell, peripheral blood mononuclear cell (PBMC), cord blood cell, or immune cell. In yet another embodiment, the immune cell is a T cell, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, CD4+T cell, CD8+T cell, CD4 / CD8 double negative T cell, gamma delta (y§) T cell, natural killer (NK) cell, NK-T cell, dendritic cell, or a combination thereof. In another embodiment, the T cell is a naive T cell, central memory T cell, effector memory T cell, or a combination thereof. In still another

[0041] - 7 - FoleyHoagUS 13160800.1 TTC-020

[0042] embodiment, the T cell is a primary T cell or a cell of a T cell line. In yet another embodiment, the T cell does not express or has a lower surface expression of an endogenous TCR. In another embodiment, the host cell is capable of producing a cytokine or a cytotoxic molecule when contacted with a target cell that comprises a peptide-MHC (pMHC) complex comprising a MAGEA4 peptide epitope in the context of an MHC molecule, optionally wherein the MHC molecule i) is a MHC class I molecule, ii) comprises an MHC alpha chain that is an HLA serotype HLA-A*02, and / or iii) is encoded by an HLA allele selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 allele. In still another embodiment, the host cell is contacted with the target cell in vitro, ex vivo, or in vivo. In yet another embodiment, the cytokine is TNF-a, IL-2, and / or IFN-y. In another embodiment, the cytotoxic molecule is perforins and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B. In still another embodiment, the host cell is capable of producing a higher level of cytokine or a cytotoxic molecule when contacted with a target cell with a heterozygous expression of MAGEA4. In yet another embodiment, the host cell is capable of producing an at least 1.05-fold higher level of cytokine or a cytotoxic molecule. In another embodiment, the host cell is capable of killing a target cell that comprises a peptide-MHC (pMHC) complex comprising the MAGEA4 peptide epitope in the context of an MHC molecule. In still another embodiment, the killing is determined by a killing assay. In yet another embodiment, the ratio of the host cell and the target cell in the killing assay is from 20:1 to 1:4. In another embodiment, the target cell is a target cell pulsed with 1 pg / mL to 50 pg / mL of MAGEA4 peptide, optionally wherein the target cell is a cell monoallelic for an MHC matched to the MAGEA4 peptide. In still another embodiment, the host cell is capable of killing a higher number of target cells when contacted with target cells with a heterozygous expression of MAGEA4, optionally wherein the cell killing is at least 1.05-fold higher. As used herein, references to fold changes, in some embodiments, may be in comparison to any reference modality of interest, such as comparison to a different binding protein; comparison to the same binding protein under different context like expression of the same binding protein in a different immune cell, at a different level, in combination with other agents described herein; and the like. In still another embodiment, cytotoxic killing is of a target cancer cell. In yet another embodiment, the target cell is cell line or a primary cell, optionally wherein the target cell is selected from the group consisting of a HEK293 derived cell line, a cancer cell line, a primary cancer cell, a transformed cell line, and an immortalized cell line. In another embodiment, the MAGEA4

[0043] - 8 - FoleyHoagUS 13160800.1 TTC-020

[0044] immunogenic peptide is selected from the group of amino acid sequences listed in Table 1, optionally wherein the MAGEA4 immunogenic peptide sequence comprises the amino acid sequence GVYDGREHTV or KVLEHVVRV. In still another embodiment, the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing when contacted with a target cell that comprises a peptide-MHC (pMHC) complex comprising a peptide epitope derived from NALCN, CFL and MAGE-A8. In yet another embodiment, the host cell does not express MAGEA4 antigen, is not recognized by a binding protein of any one of claims 56-78, is not of serotype HLA-A*02 and / or does not express an HLA-A*02 allele.

[0045] In one aspect, provided herein is a population of host cells described herein.

[0046] In another aspect, provided herein is a composition comprising a) a binding protein described herein, b) an isolated nucleic acid or a vector described herein, c) a host cell described herein, and / or d) a population of host cells described herein, and a carrier.

[0047] In still another aspect, provided herein is a device or kit comprising a) a binding protein described herein, b) an isolated nucleic acid or a vector described herein, c) a host cell described herein, and / or d) a population of host cells described herein, said device or kit optionally comprising a reagent to detect binding of a), c) and / or d) to a pMHC complex.

[0048] In yet another aspect, provided herein is a method of producing a binding protein described herein, wherein the method comprises the steps of: (i) culturing a transformed host cell which has been transformed by a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of said binding protein; and (ii) recovering the expressed binding protein.

[0049] In another aspect, provided herein is a method of producing a host cell expressing a binding protein described herein, wherein the method comprises the steps of: (i) introducing a nucleic acid comprising a sequence encoding a binding protein described herein into the host cell; and (ii) culturing the transformed host cell under conditions suitable to allow expression of said binding protein.

[0050] In still another aspect, provided herein is a method of detecting the presence or absence of a MAGEA4 antigen and / or a cell expressing MAGEA4, optionally wherein the cell is a hyperproliferative cell, comprising detecting the presence or absence of said MAGEA4 antigen in a sample by use of at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, wherein detection of the M AGEA4 antigen is indicative of the presence of a MAGEA4 antigen and / or cell expressing MAGEA4.

[0051] - 9 - FoleyHoagUS 13160800.1 TTC-020

[0052] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the at least one binding protein, or the at least one host cell, forms a complex with the MAGEA4 peptide in the context of an MHC molecule, and the complex is detected in the form of fluorescence activated cell sorting (FACS), enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or intracellular flow assay. In another embodiment, the method further comprises obtaining the sample from a subject.

[0053] In another aspect, provided herein is a method of detecting the level of a disorder characterized by MAGEA4 expression in a subject, comprising: a) contacting a sample obtained from the subject with at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) detecting the level of reactivity, wherein the presence or a higher level of reactivity compared to a control level indicates the level of the disorder characterized by MAGEA4 expression in the subject.

[0054] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is a level from a subject without the disorder characterized by MAGEA4 expression.

[0055] In another aspect, provided herein is a method for monitoring the progression of a disorder characterized by MAGEA4 expression in a subject, the method comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; b) repeating step a) at a subsequent point in time; and c) comparing the level of MAGEA4 or the cell of interest expressing MAGEA4 detected in steps a) and b) to monitor the progression of the disorder characterized by MAGEA4 expression in the subject, wherein an absent or reduced MAGEA4 level or the cell of interest expressing MAGEA4 detected in step b) compared to step a) indicates an inhibited progression of the disorder characterized by MAGEA4 expression in the subject and a presence or increased MAGEA4 level or the cell of interest expressing MAGEA4 detected in step b) compared to step a) indicates a progression of the disorder characterized by MAGEA4 expression in the subject.

[0056] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described - 10 - FoleyHoagUS 13160800.1 TTC-020

[0057] herein. For example, in one embodiment, between the first point in time and the subsequent point in time, the subject has undergone treatment to treat the disorder characterized by MAGEA4 expression.

[0058] In another aspect, provided herein is a method for predicting the clinical outcome of a subject afflicted with a disorder characterized by MAGEA4 expression comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity to that from a control, wherein the control is obtained from a subject having a good clinical outcome; wherein the absence or a reduced level of reactivity in the subject sample as compared to the control indicates that the subject has a good clinical outcome.

[0059] In still another aspect, provided herein is a method of assessing the efficacy of a therapy for a disorder characterized by MAGEA4 expression comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, in a first sample obtained from the subject prior to providing at least a portion of the therapy for the disorder characterized by MAGEA4 expression to the subject, and b) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, in a second sample obtained from the subject following provision of the therapy for the disorder characterized by MAGEA4 expression, wherein the absence or a reduced level of reactivity in the second sample, relative to the first sample, is an indication that the therapy is efficacious for treating the disorder characterized by MAGEA4 expression in the subject, and wherein the presence or an increased level of reactivity in the second sample, relative to the first sample, is an indication that the therapy is not efficacious for treating the disorder characterized by MAGEA4 expression in the subject.

[0060] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the level of reactivity is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiments, the T cell binding, activation, and / or effector function is detected using

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[0062] fluorescence activated cell sorting (FACS), enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or intracellular flow assay.

[0063] In another aspect, provided herein is a method of preventing and / or treating a disorder characterized by MAGEA4 expression comprising contacting target cells expressing MAGEA4 with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein, optionally wherein the composition is administered to a subject.

[0064] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the cell is an allogeneic cell, syngeneic cell, or autologous cell. In another embodiment, the cell is a host cell described herein or a population of host cells described herein. In still another embodiment, the target cell is a cancer cell expressing MAGEA4. In yet another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition induces an immune response against the target cell expressing MAGEA4 in the subject. In still another embodiment, the composition induces an antigen- specific T cell immune response against the target cell expressing MAGEA4 in the subject. In yet another embodiment, the antigen- specific T cell immune response comprises at least one of a CD4+helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for the disorder characterized by MAGEA4 expression, optionally wherein the at least one additional treatment for the disorder characterized by MAGEA4 expression is administered concurrently or sequentially with the composition. In still another embodiment, the disorder characterized by MAGEA4 expression is a cancer or relapse thereof, optionally wherein the cancer is selected from the group consisting of melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEA4 expression and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

[0065]

[0066] of the

[0067] Certain working examples and figures refer to certain control TCRs, such as “Comparator” which corresponds to a TCER IMA401 TCR described in U. S. Pat. No.

[0068] 10,889,629, PCT Pat. Appl. No. PCT / EP2017 / 056260, and PCT Publ. No. WO 2017 / 158103

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[0070] (see, e.g. Table 2 of each document), each of which is incorporated herein in its entirety by this reference, and described further herein such as at Table 4.

[0071] Figure 1 shows exemplary MAGE-A4 peptide sequences.

[0072] Figures 2A and 2B show more than 2100 MAGE- A4- specific TCRs were identified using the ReceptorScan platform. Figure 2A shows that CD 14+ monocytes were isolated from PBMCs of HLA-A*02:01 healthy donors on day -4 and differentiated to mature DCs. On day -1, naive CD8 T cells were isolated from autologous PBMCs and rested overnight. Co-culture of naive CD8 T cells and DCs was performed following 3 h pulsing of DCs with 1 pg / mL MAGE-A4 peptide as part of our multiplexed ReceptorScan screens, followed by a 10-day cell expansion phase. Figure 2B shows that dextramer staining was performed with HLA-A*02:01-specific MAGE-A4 dextramers to identify clones. Dextramers were then also used to isolate MAGE- A4-specific cells. Sequencing of isolated T cells and pairing of TCR alpha and beta chains was performing using the 10X Genomics platform.

[0073] Figures 3A-3C show that ActivScan platform identifies 98 TCRs with high expression and affinity. Figure 3 A shows that MAGE-A4- specific TCRs identified by the ReceptorScan platform were synthesized using TScan’s proprietary PISTACHIO cloning method to generate TCR libraries. Figure 3B shows that selection of TCRs with high expression was performed by lentivirally transducing pan T cells with PISTACHIO-cloned TCR libraries, followed by isolation of dextramer-bound cells. Figure 3C shows that identification of TCRs with high affinity was performed by sorting cells activated by titrations of cognate peptide. 98 TCRs were identified that exhibited high expression and affinity.

[0074] Figure 4 shows that cytotoxicity assays identified several potential TCRs with similar functions to comparator TCR. Pan T cells were transduced to express 98 MAGE-A4- specific TCRs individually, and engineered T cells were then co-cultured with NucLight™ Red-labeled A375 target cells at an E: T of 5:1. Target cell survival was quantified by time¬ dependent imaging as a readout of T cell cytotoxicity. Non-transduced cells (NTD) served as a control. 7 out of 98 TCRs were selected for further evaluation for surface expression and cytotoxic potential against additional MAGE-A4-expressing cell lines.

[0075] Figures 5A-5E show potency and function of MAGE-A4 specific TCRs. Pan T cells isolated from two HLA-A*02:01-positive healthy donor PBMCs were transduced to express MAGE-A4-specific TCRs 3, 162, 176, 625, 638, 657, and 916, as well as the comparator TCR, from which CD34+ cells were isolated and expanded, and T cells were assessed for functional responses against target cells that expressed HLA-A*02:01 and vaying levels of - 13 - FoleyHoagUS 13160800.1 TTC-020

[0076] MAGE-A4, as well as a MAGE-A4-negative control line. Figure 5A are dot plots showing expression of MAGE-A4-specific TCRs, as assessed by A*02:01-restricted MAGE-A4 dextramer staining, gated on live cells. Figures 5B-5D show cytotoxic function of the MAGE-A4-specific TCRs to HLA-A*02:01+ MAGE-A4+ target cell lines A375, NCI-H1703, BICR-56, and to the HLA-A*02:01- MAGE-A4-negative control cell line 647V. Engineered T cells were co-cultured with NucLight™ Red-labeled target cell lines at indicated E: T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. Figure 5E shows production of IFN-y was measured in co-culture supernatants at 24 h (E: T 1:1) in response to each indicated cell line.

[0077] Figures 6A-6D show the functional evaluation of MAGE-A4-specific TCRs expressed in nanoplasmid. Pan T cells isolated from PBMCs isolated from 2 healthy donors were electroporated to express the MAGE-A4-specific TCR 3 in nanoplasmid, from which CD34+ cells were selected using methotrexate and expanded, and T cells were assessed for functional responses against target cells that expressed HLA-A*02:01 and varying levels of MAGE-A4, as well as a MAGE-A4-negative control line. Figure 6A are dot plots showing expression of MAGE-A4-specific TCR, as assessed by A*02:01-restricted MAGE-A4 dextramer staining, gated on live cells. Figures 6B and 6C show the cytotoxic function of the MAGE-A4-specific TCRs to HLA-A*02:01+ MAGE-A4+ target cell lines A375, NCI-H1703, BICR-56 and to the HLA-A*02:01- MAGE-A4-negative control cell line 647V. Engineered T cells were co-cultured with NucLight™ Red-labeled target cell lines at indicated E: T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. Figure 6D shows the production of IFN-y in response to the indicated cell lines was measured in co-culture supernatants at 24 h (E: T 1:1).

[0078] Figure 7 shows that TSC-202-A0201 demonstrated in vivo anti-tumor efficacy. NCG female mice were implanted subcutaneously with NCI-H1703 tumor cells (day 1). When tumors reached an average volume of 100 mnF (21 days post tumor inoculation), mice were randomized into 3 treatment groups (10 mice / group) and received two injections, 7 days apart, of TSC-202-A0201 cells (2e7 viable TCR-T cells), un transfected control T cells from matching donor (UTF), or vehicle (PBS) as a control as indicated by arrow. Tumor volume was monitored biweekly up to day 50 of the study.

[0079] Figure 8 is a schematic illustrating the principle of the Target Scan screen.

[0080] Figure 9 A is a graphical representa tion of the results of the Target Scan screens for mechanistically representative TSC-202-A0201 TCR-T cells. Plotted is the enrichment score for each of -500,000 tiles / peptides in the screen calculated from 8 technical replicates,

[0081] - 14 - FoleyHoagUS 13160800.1 TTC-020

[0082] measured relative to the input. Proteins with overlapping tiles that are enriched above background are highlighted in matching colors are indicated on the graph.

[0083] Figure 9B shows that SafetyScan of TScan’s TSC-202-A0201 TCR identified 4 proteins that, when overexpressed as 90-amino acid long fragments, were recognized by the TCR. The physiological relevance of the 3 potential off-targets was then assessed in detail by co-culturing the TCR-T cells with primary cells that naturally express the full-length proteins at normal levels.

[0084] Figure 10 shows alloreactivity profiling of mechanistically representative TSC-202- A0201 TCR-T cells. Mechanistically representative TSC-202-A0201 TCR-T cells were cocultured with MHC-null HEK293T cells re-expressing one of the 110 most frequently encountered Class I HLAs in the US population for the indicated timeframe. A positive control consisting of HEK293T cells expressing both a fragment of MAGE-A4 containing the HLA-A*02:01-restricted epitope and HLA-A*02:01 (red) and a negative control consisting of MHC- / - HEK293T cells (blue) were included in the screen. The inhibition of target cell growth by the TCR-T cells relative to that by the UTD control T cells was measured over the indicated timeframe of coculture as a readout of the reactivity of mechanistically representative version of the therapeutic ICR to allogeneic HLA proteins. Data represented is one of three experiments performed.

[0085] Figures 11A and 11B show that TSC-202-A0201 TCR-T cells display no risk of off- tumor reactivity. Three batches of process-representative TSC-202-A0201 TCR-T cells (PD412, PD413, and PD414) were assessed for risk of off-tumor reactivity. The top of Figure 11A shows that TSC-202-A0201 TCR-T cells showed no reactivity to a panel of HLA-A*02:01-positive cancer cell lines naturally expressing off-targets, or to HEK293T cells transduced to express physiologically relevant levels of CFI, while reactivity to the MAGE-A8-positive (MAGE-A4-negative) cancer cell line H695T was observed for two TCR-T cell batches. The bottom of Figure UA shows that a panel of 70 HLA-A*02:01- positive normal primary or iPSC-derived human cell samples derived from 34 tissues was tested as targets for TSC-202-A0201 to test off-tumor reactivity. Normal primary human cells included epithelial cells, mesenchymal cells, endothelial cells, fibroblasts, and muscle cells derived from multiple vital and non-vital organs, reproductive and nonreproductive organs, and male and female donors. The data is presented as a tabulated summary of the reactivity of the TCR-T cells as assessed by IFN-y measurement; each colored cell in the table illustrates a single lot of cells for the indicated cell type. For each cell type, 1-3 lots of

[0086] - 15 - FoleyHoagUS 13160800.1 TTC-020

[0087] cells (i.e., donors) were tested, depending on the availability of the primary cells. Reactivity is indicated in red, lack of reactivity by gray. Figure 1 IB shows that expression of the putative off-targets of the TSC-202-A0201 therapeutic TCR was determined by RNAseq in the various cell types tested; the average expression of the off-target for each cell type is presented as a heat map to indicate the range of expression across samples. The color scale used in RNAseq heat maps has TPM values of zero set to white, and values above zero follow a continuous color scale up to 100 TPM.

[0088] Figure 12 shows the pMHC dose-dependent function of processes-representative TSC-202-A0201 TCR-T Cells. T2 cells were pulsed with a 10-point titration of MAGE- A4 peptide (0.25 pg / mL - 10 pg / mL) and co-cultured with three batches (PD412-H, PD413-H, PD-414-H) of TSC-202-A0201 process-representative TCR-T cells. Top panels show the relative growth of T2 cells over 72 hours of co-culture with TSC-202-A0201 TCR-T cells at an E: T ratio of 5:1, normalized to t=Oh. For each donor, the co-culture was performed in triplicate (n=3). The error bars at each data point show the standard error of the mean (SEM). In the bottom panel, the area under the curve (AUC) for the T2 cell growth curves over 72 hours was plotted as a function of the peptide concentration.

[0089] Figures 13A and 13B show that TSC-202-A0201 TCR-T cells secreted Granzyme B and pro-inflammatory cytokines IFN-y, IL-2, TNF-a in a target dependent manner. Figure 13A shows that TSC-202-A0201 process-representative materials secreted donor-dependent levels of all 4 cytokines. No cytokine secretion was seen against the negative control cell line, 647-V, and in the T cell alone conditions. Granzyme B levels were maxed out across all three donors when co-cultured with A375 and NCI-H1755. High levels of Granzyme B were also observed when the TSC products were co-cultured with NCI-H1395. The rest of the cytokines showed donor dependent levels when co-cultured with the MAGEA4 positive cell lines. Figure 13B shows that no IFN-y or IL-2 was observed in the donor-matched UTF products. Background levels of TNF-a and Granzyme B were observed in UTF conditions across the three donors.

[0090] Figures 14A and 14B show that TSC-202-A0201 TCR-T cells displayed potent and selective cytotoxicity. Figure 14A shows that three batches of process-representative TSC-202-A0201 TCR-T cells (Red growth curves) and donor- matched untransfected (UTF) control T cells (grey growth curves) were analyzed in the IncuCyte- based cytotoxicity assay for their cytotoxicity potential against an HLA-A*02:01-positive, MAGE-A4-negative control cell line (647-V) or three different HLA-A*02:01 -positive, MAGE-A4-positive target cell lines (A375, NCI-H1755, and NCI-H1395). T cells and target cells were co-cultured - 16 - FoleyHoagUS 13160800.1 TTC-020

[0091] across a range of effector to target ratios (E: T ranging from 5:1 to 0.6:1) and the growth of the target cells was measured over 72 hours. Data presented were obtained with TSC-202-A0201 TCR-T cells and UTF control T cells from batch PD412 and are representative of the data obtained with all 3 batches of process-representative material tested. Target cells cultured alone are displayed as a negative control (blue growth curves). Figure 14B shows the cytotoxic activity of the three batches of process-representative TSC-202-A0201 TCR-T cells over 72h summarized as the area under the curve (AUC) of the growth curves of target cells co-cultured with TSC-202-A0201 TCR-T cells at an E: T of 2.5:1, normalized to the growth curves of target cells co-cultured with the corresponding UTF control cells. Co¬ cultures were performed in triplicate; bars depict mean with standard error.

[0092] Figures 15A and 15B show TSC-202-A0201 TCR-T cells proliferated in a targetdependent manner. TSC-202-A0201 TCR-T cells (Figure 15A) or donor matched transduced control T cells (Figure 15B) from three T cell batches (PD412, PD413 and PD414) were labeled with CTV dye and were cultured in the absence of target cells, or were co-cultured at an E: T of 1:1 with either the HLA-A*A02:01 positive, Mage-A4-negative target cell line 647-v or three different HLA-A*02:01 -positive Mage-A4-positive cell lines (A375, dark burgundy bars; NCIH-1755 (medium burgundy bars); NCIH-1395 (light burgundy bars). After 3.5-day co-culture, cells were stained for flow cytometric quantification of T cell proliferation. Graphs depict the number of dividing cells (identified as CTV dim population) normalized to counting beads. The number of dividing cells is shown for the following T cell subsets: total T cells (left panels); helper T cells (middle panels) and cytotoxic T cells (right panels).

[0093] Figure 16 shows the phospho-flow cytometric analysis of Phospho-SM AD2 or Total SMAD2 in TSC-202-A0201 TCR-T cells. Shown are data obtained with TSC-202-A0201 process-representative TCR-T cells (batches PD412, PD413 and PD414) or Reference Material bench-scale process-similar controls, RM2-TCR-T cells and RM1-UTF cells from the same donors, untreated or treated with 5 ng / mL TGFP-1. Viable single cells are characterized as CD34-positive (TSC-202-A0201 or RM2 TCR-T cells) or CD34-negative (RM1-UTF controls) and these subpopulations were further characterized by histogram analysis for Phospho-SMAD2 or Total SMAD2. Histogram analysis overlays consist of Phospho-SMAD2 or Total SMAD2 in untreated CD34 positive-TCR-T cells (dark red or

[0094] - 17 - FoleyHoagUS 13160800.1 TTC-020

[0095] pink, filled), TGF'P-1 treated CD34 positive-TCR-T cells (dark red or pink, open), untreated CD34 negative-UTF (black, filled) and TGFp-1 treated CD34 negative-UTF (black, open).

[0096] Figure 17 shows the map for a representative Vector pNVVD262_TSC-202-A02.. TCR-3.. MSCV-TCR-3-CD8-EFla-dnTGFbRII-DHFR.

[0097] Figure 18 shows the map for a representative Vector pNVVD265_TSC-202- A02.. TCR-638.. MSCV-TCR-638-CD8-EFla-dnTGFbRII-DHFR.

[0098] Figure 19 shows a flow chart describing the steps and timelines of the cytokine assay to test off-tumor reactivity of TSC-202-A0201 TCR-T cells.

[0099] Figure 20 shows expression of the putative off-targets of the therapeutic TCR used in TSC-202-A0201 TCR-T cells in primary cells. RNA was extracted from the primary cells and sequenced. Heat maps show TPM (transcripts per million) calculated from the counts. The color scale used in RNAseq heatmaps has TPM values of < 1 set to gray and values above 1 follow a continuous color scale up to 100 TPM.

[0100] Figure 21 shows that TSC-202-A0201 TCR-T cells showed no reactivity to HLA-A*02:01+primary cells. TSC-202-A0201 TCR-T cells and donor-matched UTF control T cells were cocultured with a panel of primary cells and supernatants were evaluated for levels of IFN-y as a measure of T cell reactivity. Note that for each target cell tested, three bars are depicted which represent from left to right: IFN-y measured in cocultures of un-pulsed target cells with UTF control T cells (grey) or TSC-202-A0201 TCR-T cells (burgundy); and cocultures of TSC-202-A0201 TCR-T cells with target cells pulsed with the cognate MAGE-A4 peptide GVYDGREHTV (white).

[0101] Figures 22A and 22B show expression of the putative off-targets of the therapeutic TCR used in TSC-202-A0201 TCR-T cells in cancer cell lines. RNA was extracted from the primary cells and sequenced. Heat maps show TPM (transcripts per million) calculated from the counts. Figure 22A shows that the color scale used in RNAseq heatmaps for off-target expression has TPM values of < 1 set to gray and values above 1 follow a continuous color scale up to 100 TPM. Figure 22B shows that the color scale used in RNAseq heatmaps for HLA-A expression above 1 follows a continuous color scale up to 2500 TPM.

[0102] Figure 23 shows that TSC-202-A0201 TCR-T cells showed no reactivity to most HLA-A*02:01+cancer cell lines. TSC-202-A0201 TCR-T cells and donor-matched UTF control T cells were cocultured with a panel of primary cells and supernatants were evaluated for levels of IFN-y as a measure of T cell reactivity. Note that for each target cell tested, three bars are depicted which represent from left to right: IFN-y measured in cocultures of un-pulsed target cells with UTF control T cells (grey) or TSC-202-A0201 TCR-T cells

[0103] - 18 - FoleyHoagUS 13160800.1 TTC-020

[0104] (burgundy); and cocultures of TSC-202-A0201 TCR-T cells with target cells pulsed with the cognate MAGE-A4 peptide GVYDGREHTV (white).

[0105] Figure 24 shows MAGE-A4 expression in 48 normal human organs.

[0106] Figure 25 shows MAGE-A8 expression in 48 normal human organs.

[0107] For any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curve, or other data presented from left to right for each indication correspond directly and in order to the boxes from top to bottom, or from left to right, of the legend unless indicated otherwise.

[0108] Detailed Description of the Invention

[0109] The present invention is based, at least in part, on the discovery of binding proteins (e.g., those having sequences listed in Table 2) that recognize MAGEA4 antigens, and uses thereof. A systematic, comprehensive survey was carried out to map the precise T cell targets recognized by an initial pool of T cells of interest.

[0110] Accordingly, the present invention relates, in part, to the identified binding proteins (e.g., TCRs), host cells expressing binding proteins (e.g., TCRs), compositions comprising binding proteins (e.g., TCRs) and host cells expressing binding proteins (e.g., TCRs), methods of diagnosing, prognosing, and monitoring T cell response to cells expressing MAGEA4, and methods for preventing and / or treating disorders characterized by MAGEA4 expression.

[0111] I. Definitions

[0112] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0113] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, By way of example, “an element” means one element or more than one element. In addition, references to a table provided herein encompass all sub-tables of the table unless otherwise indicated.

[0114] The term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and selfadministering. This involves the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. In some embodiments, routes of administration for binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous.

[0115] - 19 - FoleyHoagUS 13160800.1 spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. Alternatively, a binding protein described herein may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering may also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0116] As used herein, the term “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. An antigen may be a MAGEA4 antigen, or a fragment thereof, against which protective or therapeutic immune responses are desired. An “epitope” is the part of the antigen bound by a natural or synthetic substance.

[0117] The term “adjuvant” as used herein refers to substances, which when administered prior, together or after administration of an antigen accelerates, prolong and / or enhances the quality and / or strength of an immune response to the antigen in comparison to the administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0118] The term “antibody” as used to herein includes whole antibodies and any antigen binding fragments (i.e., “antigen-binding portions”) or single chains thereof. An “antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is compri sed of one domain, CL. The VH and VL. regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL. is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of - 20 - FoleyHoagUS 13160800.1 the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0119] The term “antigen presenting cell” or “APC” includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0120] The term “antigen-binding portion” of a binding protein, such as a TCR, as used herein, refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen (e.g., a MAGEA4 antigen) and cognate MHC / HLA. Such portions are, for example, between about 8 and about 1,500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed within the term “antigen-binding portion” of a TCR, include (i) a Fv fragment consisting of the Va and Vp domains of a TCR, (ii) an isolated complementarity determining region (CDR) or (iii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although Va and Vp, are coded by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Va and Vp regions pair to form monovalent molecules (known as single chain TCR (scTCR)). Such single chain TCRs are also intended to be encompassed within the term “antigen-binding portion” of a TCR. These TCR fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are complete binding proteins.

[0121] Antigen-binding portions may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0122] The terms “complementarity determining region” and “CDR” are synonymous with “hypervariable region” or “HVR” and are known in the art to refer to non-contiguous sequences of amino acids within certain binding proteins, such as TCR variable regions, which confer antigen specificity and / or binding affinity. For TCRs, in general, there are three CDRs in each a-chain variable region (aCDRl, aCDR2, and aCDR3) and three CDRs in

[0123] - 21 - FoleyHoagUS 13160800.1 each P-chain variable region (PCDRl, pCDR2, and (3CDR3). CDR3 is believed to be the main CDR responsible for recognizing processed antigen. CDR1 and CDR2 mainly interact with the MHC.

[0124] The term “body fluid” refers to fluids that are excreted or secreted from the body as well as fluids that are normally not excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the body fluid comprises immune cells, optionally wherein the immune cells are cytotoxic lymphocytes such as cytotoxic T cells and / or NK cells, CD4+ T cells, and the like.

[0125] The term “coding region” refers to regions of a nucleotide sequence comprising codons that are translated into amino acid residues, whereas the term “non-coding region” refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0126] The term “comparator T-cell receptor” refers to at least one benchmark T-cell receptor. In some embodiments, a comparator TCR corresponds to the TCER IM A401 TCR described in U. S. Pat. No. 10,889,629, PCT Pat. Appl. No. PCT / EP2017 / 056260, and PCT Publ. No. WO 2017 / 158103 (see, e.g.. Table 2 of each document), each of which is incorporated herein in its entirety by this reference. Engineered versions of such parental sequences were used in the working examples and sequences of such engineered versions are set forth in Table 4. In some embodiments, the comparator T-cell receptor has sequences set forth in Table 4.

[0127] The term “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is anti-parallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is anti-parallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at

[0128] - 22 - FoleyHoagUS 13160800.1 TTC-020

[0129] least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and, in other embodiments, at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range in between, inclusive, such as at least about 80%-100%, of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0130] As used herein, the term “costimulate” with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non-activating receptor mediated signal (a “costimulatory signal”) that induces proliferation or effector function. For example, a costimulatory signal may result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. Immune cells that have received a cell-receptor mediated signal, e.g., via an activating receptor are referred to herein as “activated immune cells,”

[0131] “CD3” is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, Cellular and Molecular Immunology (9thEdition) (2018): Janeway et al.

[0132] (Immunobiology) (9thEdition) (2016)). In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3E chains, and a homodimer of CD3^ chains. The CD3y, CD38, and CD3e chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD38, and CD3e chains are negatively charged, which is a characteristic that is believed to allow these chains to associate with positively charged regions or residues of T cell receptor chains. The intracellular tails of the CD3y, CD38, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or IT AM, whereas each CD3u, chain has three ITAMs. Without wishing to be bound by theory, it is believed that the IT AMs are important for the signaling capacity of a TCR complex. CD3 used in accordance with the present invention may be from various animal species, including human, mouse, rat, or other mammals.

[0133] A “component of a TCR complex,” as used herein, refers to a TCR chain (i.e., TCRoc, TCRp, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD38, CD3e or CD3Q, or a complex

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[0135] formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRcx and TCRp, a complex of TCRy and TCR8, a complex of CD3e and CD38, a complex of CD3y and CD3e, or a sub-TCR complex of TCRa, TCRp, CD3y, CD38, and two CD3e chains).

[0136] The term “chimeric antigen receptor” or “CAR” refers to a fusion protein that is engineered to contain two or more amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs encompassed by the present invention include an extracellular portion comprising an antigen-binding domain (i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR specific for a MAGEA4 antigen, a single chain TCR-derived binding protein, an scFv derived from an antibody, an antigen binding domain derived or obtained from a killer immunoreceptor from an NK cell, and the like) linked to a transmembrane domain and one or more intracellular signaling domains (such as an effector domain, optionally containing costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; see also Harris and Kranz (2016) Trends Pharmacol. Sci. 37: 220; Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0137] As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CDfe T cells.

[0138] The term “consisting essentially of is not equivalent to “comprising” and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) “consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0139] The term “determining a suitable treatment regimen for the subject” is taken to mean the determination of a treatment regimen (i.e., a single therapy or a combination of different

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[0141] therapies that are used for the prevention and / or treatment of the viral infection in the subject) for a subject that is started, modified and / or ended based or essentially based or at least partially based on the results of the analysis according to the present invention. One example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify the dosage of a particular chemotherapy. The determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most cases, the actual determination of the suitable treatment regimen for the subject will be performed by the attending physician or doctor.

[0142] The term “dominant negative TGFp receptor” or “DN-TGF'PR” refers to a transforming growth factor (TGF) beta receptor variant or mutant that provides resistance to TGI T signaling. There are five type II receptors (activation receptors) and seven type I receptors (signaling propagation receptors). The active TGFp receptor is a hetero tetramer consisting of two TGF p receptors I (TGFpRI) and two TGF p receptors II (TGFpRII). In some embodiments, the DN-TGFpR is a DN-TGFPRII (i.e., a TGF beta receptor II variant or mutant). In some embodiments, resistance is to the suppressive effect of TGF signaling on an immune cell, such as a T cell, which TGFp may be produced by cancer cells or by other immune cells within a cellular environment, such as by stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, and the like. TGFp signaling inhibitors are well- known in the art and include, without limitation, mutant TGFp that sequesters receptors and thereby inhibits signaling, antibodies that bind to TGFp and / or TGFp receptors (e.g., lerdelimumab, metlimumab, fressolimumab, and the like), soluble TGFp-binding proteins such as portions of TGFp receptors that sequester TGFp (e.g., TGFpRII-Fc fusion proteins) or other binders, such as beta-glycans. Any and all known TGFP signaling inhibitors may be used instead of or in addition to DN-TGFPR (e.g., DN-TGFpRII) described herein. In some embodiments, a DN-TGFPR lacks an intracellular portion required for TGFp-mediated signaling, such as the entire intracellular domain, a kinase signaling domain, etc. DN-TGFPR constructs are well-known in the art (see representative, non-limiting embodiments at Brand et al. (1993) J. Biol. Chem. 268:11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bollard et al. (2002) Blood 99::3179-3187; PCT Publ. WO 2009 / 152610; PCT Publ. WO 2017 / 156484; Kloss et al. (2018) Mol. Ther. 26:1855-1866; PCT Publ. WO.

[0143] 2019 / 089884; PCT Publ. WO 2020 / 042647; and PCT Publ. WO 2020 / 042648.

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[0145] “Hematopoietic progenitor cell” is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with a CD24LOLin- CD117+phenotype or those found in the thymus (referred to as progenitor thymocytes).

[0146] “Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and, in other embodiments, at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range in between, inclusive, such as at least about 80%-100%, of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0147] The term "hyperproliferative disorder characterized by expression of a MAGEA4 antigen" can be any hyperproliferative disorder where the MAGEA4 antigen is present in a MHC (e.g., HLA) complex expressed by at least some hyperproliferating cells in the subject. Examples of hyperproliferative disorders characterized by MAGEA4: HLA complexes include solid malignancies, such as those described in detail infra.

[0148] The term “immune response” includes I' cell mediated and / or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.

[0149] An increased ability to stimulate an immune response or the immune system, can result from an enhanced agonist activity of T cell costimulatory receptors and / or an enhanced - 26 - FoleyHoagUS 13160800.1 TTC-020

[0150] antagonist activity of inhibitory receptors. An increased ability to stimulate an immune response or the immune system may be reflected by a fold increase of the ECso or maximal level of activity in an assay that measures an immune response, e.g., an assay that measures changes in cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly via detecting CD107a or granzymes) and proliferation. The ability to stimulate an immune response or the immune system activity may be enhanced by at least 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 110%, 120%, 130%, 140%. 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0151] The term ‘‘immunotherapeutic agent” may include any molecule, peptide, antibody or other agent which can stimulate an immune system to generate an immune response to a cancer cell, such as in a subject. Various immunotherapeutic agents are useful in the compositions and methods described herein.

[0152] The term “immune cell” refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages: a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes); and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+T cell, a CD8+T cell, a CD4 CD8 double negative T cell, a gd T cell, a regulatory I' cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[0153] An “isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of a biomarker polypeptide or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations - 27 - FoleyHoagUS 13160800.1 TTC-020

[0154] of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as a “contaminating protein”), or, in some embodiments, less than about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any range in between inclusive, such as less than about 1% to 5%, of non-biomarker protein. When binding protein, antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it may be substantially free of culture medium, i.e., culture medium represents less than about 20%, 15%, 10%, 5%, 1%, or less, or any range in between inclusive, such as less than about 1% to 5%, of the volume of the protein preparation.

[0155] As used herein, the term “isotype” refers to the antibody class (e.g., IgM, IgGl, IgG2C, and the like) that is encoded by heavy chain constant region genes.

[0156] As used herein, the term “KD” is intended to refer to the dissociation equilibrium constant of a particular binding protein- antigen interaction. The binding affinity of binding proteins encompassed by the present invention may be measured or determined by standard binding protein-target binding assays, for example, competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively lower Kd value indicates a relatively higher binding affinity (e.g., Kd values of less than or equal to about 5xl0~4M (500 uM) include a Kd value of IxlO’4M (100 uM) and a 100 uM Kd indicates a relatively higher binding affinity as compared to a 500 uM Kd).

[0157] A “kit” is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a probe or small molecule, for specifically detecting and / or affecting the expression of a marker encompassed by the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods encompassed by the present invention. The kit may comprise one or more reagents necessary to express a composition useful in the methods encompassed by the present invention. In some embodiments, the kit may further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis. One skilled in the art can envision many such control proteins, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta¬ galactosidase), proteins not classified in any of pathway encompassing cell growth, division, migration, survival or apoptosis by GeneOntology reference, or ubiquitous housekeeping proteins. Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container. In addition, instructional materials which describe the use of the compositions within the kit may be included.

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[0159] As used herein, the term “linked” refers to the association of two or more molecules. The linkage may be covalent or non-covalent. The linkage also may be genetic (i.e., recombinantly fused). Such linkages may be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[0160] A “linker,” in some embodiments, may refer to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex). In some embodiments, a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.

[0161] The terms “MAGEA4” or “MAGE-A4” refer to a particular member of the melanoma antigen gene family clustered on human chromosome Xq28 that is also known as cancer / testis antigen 1.4 (CT1.4), MGC21336, MAGE4, MAGE4A, MAGE4B, MAGE-41, and MAGE-X2 (Sani et al. (2018) J. Cancer Res. Ther. 14:1059-1064; Fujiwara- Kuroda et al. (2018) Int. J. Oncol. 53:713-724; lura et al. (2017) Virchows Arch. 47:383-392; Gunda et al. (2013) Surgery 154:1456-1462: Cabezon et al. (2013) Mol. Cell Proteomics 12:381-394: Bhan et al. (2012) Oncol. Rep. 25:1498-1502; Peikert et al. (2006) Cancer Res. 66:4693-4700; Sarcevic et al. (2003) Oncol. 64:443-449; De Plaen et al. (1997) Genomics 40:305- 313). MAGEA4 is believed to regulate cell proliferation through the inhibition of cell cycle arrest at the G1 phase, as well as negatively regulate p53-mediated apoptosis. MAGEA4 is not expressed in normal tissues, except for testis, and is expressed in tumors of various histological types such as melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and bladder urothelial carcinoma.

[0162] The terms “MAGEA4” or “MAGE-A4” is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human MAGEA4 cDNA and human M AGEA4 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI) (see, for example, ncbi.nlm.nih.gov / gene / 4103). For example, human MAGEA4 (NP_001011548.1) is encodable by any one of several variants encoding the same protein (e.g., NM..001011548.1; NM_001011549.1; NM.001011550.1; NM_001386196.1; NMJ101386197.1;

[0163] NMJ101386198.1; NM„001386199.1; NM_001386200.1; NM_001386202.1;

[0164] NM„001386203.1; and NM_002362.4). Nucleic acid and polypeptide sequences of

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[0166] MAGEA4 orthologs in organisms other than humans are well-known and include, for example, chimpanzee MAGEA4 (XM__003317754.1 and XP__003317802.1). Representative sequences of MAGEA4 sequences are also presented below in Table 3.

[0167] Anti-MAGEA4 antibodies suitable for detecting MAGEA4 protein are well-known in the art and include, for example, antibodies orb534736 and orb 1047003 (Biorbyt, Cambridge, UK), antibodies AP13131PU-N and CF505361 (OriGene, Rockville, MD); antibodies 26-864 and 8191 (ProSci, Poway, CA); antibodies 6C1 and A-l (Santa Cruz Biotechnology, Santa Cruz, CA); antibodies A07175 and M07175 (Boster Bio, Pleasanton, CA), and antibodies ABIN1310045, ABIN1310047, ABIN7123889, and ABIN6964213 (Antibodies-online, Limerick, PA). In addition, reagents are well-known for detecting MAGEA4 expression. Moreover, multiple siRNA, shRNA, CRISPR constructs for modulating M AGEA4 expression can be found in the commercial product lists of a variety of companies, such as open reading frame (ORF) clones LC425301 and LC425302 (OriGene, Rockville, MD), PROTP43358 (Boster Bio, Pleasanton, CA), and orb 1096477 and orb 1099936 (Biorbyt, Cambridge, UK), as well as CRISPR knockouts, such as GA102788 and GA202557 (OriGene, Rockville, MD). It is to be noted that the term can further be used to refer to any combination of features described herein regarding MAGEA4 molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe a MAGEA4 molecule encompassed by the present invention.

[0168] The term “MAGEA4 antigen” or “MAGEA4 peptide antigen” or “MAGEA4-containing peptide antigen” or “MAGEA4 epitope” or “MAGEA4 peptide epitope” or “MAGEA4 peptide” refers to a naturally or synthetically produced immunogenic portion of MAGEA4. In some embodiments, MAGEA4 antigen protein can range in length from about 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids, or any range in between, inclusive, such as 8-15 amino acids. In some embodiments, MAGEA4 antigen protein can form a complex with an MHC e.g., HLA) molecule such that a binding protein of this disclosure that recognizes a MAGEA4 peptide: MHC (e.g., HLA) complex can bind (e.g., specifically and / or selectively) to such a complex. Representative MAGEA4 peptide antigen sequences are shown in Table 1.

[0169] The term “major histocompatibility complex” (MHC) refers to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated b2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins,

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[0171] a and b, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide antigen-MHC (pMHC) complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. Human MHC is referred to as human leukocyte antigen (HLA).

[0172] The terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

[0173] The term “prognosis” includes a prediction of the probable course and outcome of a cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of a cancer in an individual. For example, the prognosis may be surgery, development of a clinical subtype of a cancer, development of one or more clinical factors, or recovery from the disease.

[0174] As used herein, “percent identity” between amino acid sequences is synonymous with “percent homology,” which can be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The noted algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410.

[0175] BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a polynucleotide described herein. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used.

[0176] The phrase “pharmaceutically-acceptable carrier” means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

[0177] The term “ratio” refers to a relationship between two numbers (e.g., scores, summations, and the like). Although, ratios may be expressed in a particular order (e.g., a to b or a:b), one of ordinary skill in the art will recognize that the underlying relationship - 31 - FoleyHoagUS 13160800.1 TTC-020

[0178] between the numbers may be expressed in any order without losing the significance of the underlying relationship, although observation and correlation of trends based on the ratio may be reversed.

[0179] The term “recombinant host cell” (or simply “host cell”) refers to a cell that comprises a nucleic acid that is not naturally present in the cell, such as a cell into which a recombinant expression vector has been introduced. It should be understood that cells according to the present invention is intended to refer not only to the particular subject cell, but also encompasses progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term cell according to the present invention.

[0180] The term “cancer response,” “response to immunotherapy,” or “response to modulators of T-cell mediated cytotoxicity / immunotherapy combination therapy” relates to any response of the hyperproliferative disorder (e.g., cancer) to a cancer agent, such as a modulator of T-cell mediated cytotoxicity, and an immunotherapy, preferably to a change in tumor mass and / or volume after initiation of neoadjuvant or adjuvant therapy. The term “neoadjuvant therapy” refers to a treatment given before the primary treatment. Examples of neoadjuvant therapy may include chemotherapy, radiation therapy, and hormone therapy. Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention may be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria. Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and / or the tumor bed. This is typically three months after initiation of neoadjuvant therapy. In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are - 32 - FoleyHoagUS 13160800.1 TTC-020

[0181] in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the response to cancer therapies are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related): “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen may be administered to a population of subjects and the outcome may be correlated to biomarker measurements that were determined prior to administration of any cancer therapy. The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival may be monitored over a period of time for subjects following cancer therapy for which biomarker measurement values are known. In certain embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored may vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement threshold values that correlate to outcome of a cancer therapy may be determined using well-known methods in the art, such as those described in the Examples section.

[0182] As indicated, the terms may also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause. To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that - 33 - FoleyHoagUS 13160800.1 TTC-020

[0183] a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (i.e., will exhibit a lack of response or be non-responsive).

[0184] The term “resistance” refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy ( i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 5%, 10%, 15%. 20%, 25%, 30%, 35%, 40%, 45%, 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15- fold, 20-fold or more, or any range in between, inclusive. The reduction in response may be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal that is known to have no resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called “multidrug resistance.” The multidrug resistance may be mediated by P-glycoprotein or may be mediated by other mechanisms, or it may occur when a mammal is infected with a multi-drug-resistant microorganism or a combination of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, may be measured by cell proliferative assays and cell death assays as described herein as “sensitizing.” In some embodiments, the term “reverses resistance” means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p<0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g., chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing logarithmically.

[0185] The term “sample” used for detecting or determining the absence, presence, or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of “body fluids”), or a tissue sample (e.g., biopsy) such as a skin, colon sample, or surgical resection tissue. In some embodiments, methods encompassed by the present invention further comprises obtaining the sample from the individual prior to detecting or determining the absence, presence, or level of at least one marker in the sample.

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[0187] The term “sensitize” means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g., anti-immune checkpoint, chemotherapeutic, and / or radiation therapy). In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the therapies. An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res.

[0188] 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A.1 P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). The sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such 2-fold, 3-fold, 4- fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range in between, inclusive, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy may be equally applied to methods for sensitizing hyperproltf era live or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

[0189] The term “small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane el al. (1998) Science 282:63-68), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

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[0191] The term “specific binding” refers to binding protein binding to a predetermined antigen. Typically, the binding protein binds with an affinity (KD) of approximately less than or equal to about 5xlC)"4M, less than or equal to about IxlO"4M, less than or equal to about 5xl0'5M, less than or equal to about 1x105M, less than or equal to about 5x106M, less than or equal to about IxlO"6M, less than or equal to about 5x10"?M, less than or equal to about 1x10 '' M, less than or equal to about 5xl0'8M, less than or equal to about IxlO'8M, less than or equal to about 5xl0"9M, less than or equal to about IxlO'9M, less than or equal to about 5xlO"10M, less than or equal to about IxlO"10M, less than or equal to about 5x10"11M, less than or equal to about IxlO'11M, less than or equal to about 5xl0"12M, less than or equal to about I lO"12M, or even lower, or any range in between, inclusive, such as between about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, and the like, when determined by a binding assay, such as surface plasmon resonance (SPR) technology in a BIAcore™ assay instrument using an antigen of interest as the analyte and the binding protein as the ligand. In some embodiments, the binding protein binds to the predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases “a binding protein recognizing an antigen” and “a binding protein specific for an antigen” are used interchangeably herein with the term “a binding protein which binds specifically to an antigen.” Selective binding is a relative term referring to the ability of a binding protein to discriminate the binding of one antigen over another, such as a particular family member or antigen target over a related family member or antigen target. For example, analytical data provided in the Examples section demonstrate that binding proteins described herein specifically bind MAGEA4 immunogenic epitopes and / or selectively bind a number of related epitopes e.g., MAGEA4 immunogenic epitopes and closely related sequences) discriminating such targets from the vast majority of other possible epitopes available in the human genome.

[0192] The term “subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a disorder characterized by MAGEA4 expression, such as a non-malignant disorder, a hyperproliferative disorder, or a relapse of ahyperpoliferative disorder characterized by MAGEA4 expression. The term “subject” is interchangeable with “patient.”

[0193] The term “survival” includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor - 36 - FoleyHoagUS 13160800.1 TTC-020

[0194] related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0195] The term “synergistic effect” refers to the combined effect of two or more agents e.g., a M AGEA4-related agent described herein and another therapy, such as an additional MAGEA4-targeted TCR, anti-cancer therapy, immunotherapy, etc., for treating a disorder characterized by MAGEA4 expression) that is greater than the sum of the separate effects of the cancer agents / therapies alone.

[0196] As used herein, the term “T cell-mediated response” refers to a response mediated by T cells, including effector T cells (e.g., CD8+cells) and helper T cells (e.g., CD4+cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

[0197] A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g., an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a biomarker nucleic acid and normal post- transcriptional processing (e.g., splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

[0198] A “T cell” is an immune system cell that matures in the thymus and produces T cell receptors (TCRs). T cells may be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). I'M may be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perform as compared to TCM. Other exemplary T cells include regulatory T cells, such as CD4+CD25+(Foxp3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28, and Qa-1 restricted T cells.

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[0200] Conventional T cells, also known as Tconv or Teffs, have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self-recognition, and the like) to increase immune responses by virtue of their expression of one or more T cell receptors. cons or Teffs are generally defined as any T cell population that is not a Treg and include, for example, naive T cells, activated T cells, memory T cells, resting Tcons, or Tcons that have differentiated toward, for example, the Thl or Th2 lineages. In some embodiments, Teffs are a subset of non-Treg T cells. In some embodiments, Teffs are CD4+ Teffs or CD8+ Teffs, such as CD4+ helper T lymphocytes (e.g., ThO, Thl, Tfh, or Th 17) and CD8+ cytotoxic T lymphocytes. As described further herein, cytotoxic T cells are CD8+ T lymphocytes.

[0201] “Naive Tcons” are CD4+T cells that have differentiated in bone marrow, and successfully underwent a positive and negative processes of central selection in a thymus, but have not yet been activated by exposure to an antigen. Naive Tcons are commonly characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44 or CD69, and absence of memory markers such as CD45RO. Naive Tcons are therefore believed to be quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin- 15 (IL- 15) for homeostatic survival (see, at least WO 2010 / 101870). The presence and activity of such cells are undesired in the context of suppressing immune responses. Unlike Tregs, Tcons are not anergic and can proliferate in response to antigenbased T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Land. Biol. Sci.

[0202] 356:625-637).

[0203] “T effector” (“Teff” or “TE”) cells refers to T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activities as well as T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but does not include regulatory T cells (Treg cells).

[0204] “T cell receptor” or “TCR” refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail: see, e.g., Janeway et al. (1997) Cure. Biol. Publ. 4:33) that is capable of binding (e.g., e.g., specifically and / or selectively) to an antigen peptide bound to an MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having alpha and beta chains (also known as TCRoc and TCRβ, respectively), or y and 5 chains (also known as TCRy and TCR8, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portion of TCR chains (e.g., a-chain and p-chain) contain two immunoglobulin domains: a variable domain (e.g., a-chain variable domain or V« and p-chain variable domain or Vp; typically amino acids 1 to 116 based on

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[0206] Kabat numbering (Kabat el al. (1991) “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 5thed.) at the N-terminal end, and one constant domain (e.g., a-chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, P-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) at the C-terminal end and adjacent to the cell membrane. Also like immunoglobulins, the variable domains contain complementary determining regions (“CDRs”, also called hypervariable regions or “HVRs”) separated by framework regions (“FRs”) (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sei. US. A.

[0207] 87:9138; Chothia el al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol.

[0208] 27:55). In some embodiments, a TCR is found on the surface of a T cell (or T lymphocyte) and associates with the CD3 complex. The source of a TCR encompassed by the present invention may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.

[0209] The term “T cell receptor” or “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the «P form or y8 form. In some embodiments, the TCR is an antigen-binding portion that is less than a full- length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen- binding portion contains the variable domains of a TCR, such as variable a chain and variable chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions (CDRs) involved in recognition of the peptide, M HC and / or MHC-peptide complex.

[0210] Nomenclature established by the International Immunogenetics Information System (IMGT) (see also Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; T Cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). The IMGT provides unique sequences used to describe a TCR, and sequences described herein may be identified by reference to such unique sequences provided herein. TCR sequences are publicly available at the IMGT database at imgt.org.

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[0212] As described above, native alpha / beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain comprises variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region comprises three hypervariable CDRs (Complementarity Determining Regions) embedded in a framework sequence. CDR3 is well-known to be the main mediator of antigen recognition. There are several types of alpha chain variable (Va) regions and several types of beta chain variable (VP) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Va types are referred to in IMGT nomenclature by a unique TRAV number. For example, “TRAV4” defines a TCR Va region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR.

[0213] Similarly, “TRBV2” defines a TCR Vp region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence. It is known that there are 54 alpha variable genes, of which 44 are functional, and 67 beta variable genes, of which 42 are functional, within the alpha and beta loci, respectively.

[0214] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature. The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD / TRBJ regions are often considered together as the joining region.

[0215] The gene pools that encode the TCR alpha and beta chains are located on different chromosomes and contain separate V, (D), J and C gene segments, which are brought together by rearrangement during T cell development. This leads to a very high diversity of T cell alpha and beta chains due to the large number of potential recombination events that occur between the 54 TCR alpha variable genes and 61 alpha J genes or between the 67 beta variable genes, two beta D genes and 13 beta J genes. The recombination process is not precise and introduces further diversity within the CDR3 region. Each alpha and beta variable gene may also comprise allelic variants, designated in IMGT nomenclature as TRAVxx*01 and *02, or TRBVx-x*01 and *02 respectively, thus further increasing the amount of variation. In the same way, some of the TRBJ sequences have two known variations. (Note that the absence of a “*” qualifier means that only one allele is known for the relevant sequence). The natural repertoire of human TCRs resulting from recombination - 40 - FoleyHoagUS 13160800.1 TTC-020

[0216] and thymic selection has been estimated to comprise approximately 106unique beta chain sequences, determined from CDR3 diversity (Arstila et al. (1999) Science 286:958-961) and could be even higher (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thus generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0217] The term “TCR alpha variable domain” therefore refers to the concatenation of TRAV and TRAJ regions; a TRAV region only: or TRAV and a partial TRAJ region, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated or full-length TRAC sequence. Likewise the term “TCR beta variable domain” refers to the concatenation of TRBV and TRBD / TRBJ regions: to the TRBV and TRBD regions only; to the TRBV and TRBJ regions only; or to the TRBV and partial TRBD and / or TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBC region, or to a C-terminal truncated or full length TRBC sequence. These TCR alpha variable domain and TCR beta variable domain nomenclature similarly applies to the variable domains of TCR gamma and TCR delta chains, respectively, for gamma / delta TCRs. An ordinarily skilled artisan can obtain TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC gene sequences, such as through the publicly available IMGT database.

[0218] The term “TCR complex” refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex may be composed of a CD3y chain, a CD38 chain, two CD3e chains, a homodimer of CD3^ chains, a TCRa chain, and a TCRp chain.

[0219] Alternatively, a TCR complex may be composed of a CD3y chain, a CD38 chain, two CD3e chains, a homodimer of CD3^ chains, a TCRy chain, and a TCR8 chain.

[0220] The term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.

[0221] The terms “therapeutically effective amount” and “effective amount” means that amount of a substance that produces some desired effect, such as a desired local or systemic therapeutic effect, in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any treatment. In some embodiments, a therapeutically effective amount of a substance will depend on the substance's therapeutic index, solubility, pharmacokinetics, half-life, and the like. Toxicity and therapeutic efficacy of subject compounds may be

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[0223] determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. In some embodiments, compositions that exhibit large therapeutic indices are used. In some embodiments, the LD50 (lethal dosage) may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent Similarly, the ED50 (i.e., the concentration which achieves a half-maximal inhibition of symptoms) may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%. 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, similarly, the IC50 may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. In some embodiments, T cell immune response in an assay may be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a viral load may be achieved.

[0224] The term “treat” refers to the therapeutic management or improvement of a condition (e.g., a disease or disorder) of interest. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments, a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the - 42 - FoleyHoagUS 13160800.1 TTC-020

[0225] disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0226] The term “unresponsiveness” includes refractivity of cancer cells to therapy or refractivity of therapeutic cells, such as immune cells, to stimulation, e.g., stimulation via an activating receptor or a cytokine. Unresponsiveness may occur, e.g., because of exposure to immunosuppressants or exposure to high doses of antigen. As used herein, the term “anergy” or “tolerance” includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate. Anergic T cells may, however, proliferate if cultured with cytokines (e.g., IL-2). For example, T cell anergy may also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct may be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5’ IL-2 gene enhancer or by a multimer of the API sequence that may be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0227] The term “vaccine” refers to a pharmaceutical composition that elicits an immune response to an antigen of interest. The vaccine may also confer protective immunity upon a subject.

[0228] The term “variable region” or “variable domain” refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR Ot-chain or P-chain (or y chain and

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[0230] 3 chain for y8 TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., TCR) to antigen. The variable domains of the a-chain and p-chain (Va and Vp, respectively) of a native TCR generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. The Va domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the Vp domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single Va or Vp domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind a particular antigen may be isolated using a Va or Vp domain from a TCR that binds the antigen to screen a library of complementary V or Vp domains, respectively.

[0231] The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. In some embodiments, vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids” which refer generally to circular double stranded DNA loops, which, in their vector form are not bound to the chromosome. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, as will be appreciated by those skilled in the art, the present invention is intended to include such other forms of expression vectors that serve equivalent functions and which become subsequently known in the art.

[0232] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0233] GENETIC CODE

[0234] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0235] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0236] Asparagine (Asn, N) AAC, AAT

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[0238] Aspartic acid (Asp, D) GAC, GAT

[0239] Cysteine (Cys, C) TGC, TGT

[0240] Glutamic acid (Glu, E) GAA, GAG

[0241] Glutamine (Gin, Q) CAA, CAG

[0242] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0243] Histidine (His, H) CAC, CAT

[0244] Isoleucine (Ile, I) ATA, ATC, ATT

[0245] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0246] Lysine (Lys, K) AAA, AAG

[0247] Methionine (Met, M) ATG

[0248] Phenylalanine (Phe, F ) TTC, TTT

[0249] Proline (Pro, P) CCA, CCC, CCG, CCT

[0250] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0251] Threonine (Thr, T) ACA, ACC, ACG, ACT

[0252] Tryptophan (Trp, W) TGG

[0253] Tyrosine (Tyr, Y) TAC, TAT

[0254] Valine (Val, V) GTA, GTC, GTG, GTT

[0255] Termination signal (end) TAA, TAG, TGA

[0256] An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0257] In view of the foregoing, the nucleotide sequence of a DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) may be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino

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[0259] acid sequence). Thus, description and / or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and / or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0260] I. Binding Proteins

[0261] In an aspect encompassed by the present invention, provided herein are binding proteins that bind e.g., specifically and / or selectively), to a peptide-MHC (pMHC) complex comprising a MAGEA4 immunogenic peptide in the context of an MHC molecule (e.g., a MHC class I molecule), such as a peptide listed in Table 1.

[0262] Table 1: MAGEA4 epitopes

[0263] MAGEA4 epitopes presented by HLA serotype HLA-A*02

[0264] Peptide Epitopes

[0265] GVYDGREHTV

[0266]

[0267] KVLEHVVRV _

[0268] * Included in Table 1, are peptide epitopes, as well as polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any sequence listed in Table 1, or a portion thereof. Such polypeptides may have a function of the full-length peptide or polypeptide as described further herein.

[0269] In some embodiments, the binding protein is capable of binding (e.g., specifically and / or selectively) to a MAGEA4 peptide-MHC complex, such as with a Kd less than or equal to about IO’4M (e.g., about 104, 105, 10’6, 10’7, about 10’8, about 109, about 101°, about 10’11, about 1 O’12, about 10’13, about 10’14, etc.). In some embodiments, the binding protein is capable of binding (e.g., specifically and / or selectively) to a MAGEA4 peptide- MHC (pMHC) complex with a Kd less than or equal to about 5xl0’4M, less than or equal to about IxlO’4M, less than or equal to about 5xl0’3M, less than or equal to about IxlO"5M, less than or equal to about 5xl0"6M, less than or equal to about IxlO’6M, less than or equal to about 5xl0- / M, less than or equal to about IxlO’7M, less than or equal to about 5xl0’8M,

[0270] - 46 - FoleyHoagUS 13160800.1 TTC-020

[0271] less than or equal to about IxlO"8M, less than or equal to about 5xl0"9M, less than or equal to about IxlO"9M, less than or equal to about 5xlO"10M, less than or equal to about IxlO"10M, less than or equal to about 5xl0"nM, less than or equal to about IxlO-11M, less than or equal to about 5xl0"12M, less than or equal to about IxlO12M, or any range in between, inclusive, such as between about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, and the like. In some embodiments, the MHC molecule comprises an MHC alpha chain that is an HLA serotype of HLA-A*02, optionally wherein the HLA allele is HLA-A*02:01. In some embodiments, the binding proteins provided herein are genetically engineered, isolated, and / or purified.

[0272] In some embodiments, the binding proteins have a higher binding affinity to the MAGEA4 peptide-MHC (pMHC) than does a known T-cell receptor (e.g., a comparator TCR or others described herein). For example, the binding proteins may have at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, 5000 fold, 10000 fold, 50000 fold, 100000 fold, 500000 fold, 1000000 fold, or more, or any range in between, inclusive, such as 1.2 fold to 2 fold, higher binding affinity to the MAGEA4 peptide-MHC (pMHC) than does a known T-cell receptor (e.g., a comparator TCR or others described herein).

[0273] In some embodiments, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing than does a known T-cell receptor when contacted with target cells with expression of MAGEA4 at a certain level or below (e.g., see the Examples section for representative cell lines expressing MAGEA4 at varying levels). For example, in some embodiments of any aspect described herein, MAGEA4 level can be expressed in terms of transcripts per million and may be, for example, less than or equal to about 1,000 transcript per million transcripts (TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM. 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM. 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM.

[0274] 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range in between, inclusive, such as less than or equal to - 47 - FoleyHoagUS 13160800.1 TTC-020

[0275] about 1,000 TPM to less than or equal to about 73 TPM). In some embodiments, the low MAGEA4 expression level is termed "heterozygous expression” meaning between about 1 TPM and about 73 TPM, or any range in between, inclusive, such as 73 TPM or 1-73 TPM. A higher expression is 74 TPM and higher. As described further herein, TPM is measured according to well-known techniques, such as RNA-Seq, and gene expression TPM data are well-known in the art for a variety of cell lines, tissue types, and the like (see, for example, the Broad Institute Cancer Cell Line Encyclopedia (CCLE) on the World Wide Web at portals.broadinstitute.org). In some embodiment, the binding protein induces at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, or more, or any range in between, inclusive, such as 1.2 fold to 2 fold, increase in T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing than does a known T-cell receptor (e.g., a comparator TCR described herein) when contacted with target cells expressing MAGEA4 peptide epitope, such as with heterozygous expression of MAGEA4 peptide epitope.

[0276] In some embodiments, the expression of MAGEA4 is detected using RNA-sequencing (RNA-seq). RNA-seq generally comprises the following steps: obtaining a sample containing genetic material, isolating total RNA from the sample obtained, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to assess the expression level of different transcripts. The sample can be a population of cells, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA can be isolated from the biological sample using any method known in the art. In certain embodiments, total RNA is extracted from plasma.

[0277] Plasma RNA extraction is described in Enders et al., “The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia,” Clinr. As described therein, the plasma collected after the centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged and the aqueous layer is transferred to a new tube. Ethanol is added to this aqueous layer. The mixture is then placed in an RNeasy mini column (Qiagen) and processed according to the manufacturer's recommendations.

[0278] In some embodiments, RNA-seq described herein includes the step of preparing amplified cDNA from total RNA. For example, cDNA is prepared and the

[0279] - 48 - FoleyHoagUS 13160800.1 TTC-020

[0280] isolated RNA sample is randomly amplified without dilution, or the mixture of genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, amplification is initiated randomly at the 3 'end and throughout the entire transcriptome in the sample to amplify both mRNA and non-polyadenylated transcripts. In this way, double- stranded cDNA amplification products are optimized for the generation of sequencing libraries for next generation sequencing platforms. A kit suitable for amplification of cDNA by the method encompassed by the present invention includes, for example, Ovation® RNA-Seq System.

[0281] In some embodiments, RNA-seq described herein includes the step of sequencing the amplified cDNA. Any known sequencing method can be used to sequence the

[0282] amplified cDNA mixture including the single molecule sequencing method. In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing can be performed using various next generation sequencing platforms such as Illumina® Genome Analyzer platform, ABT SOLID™ Sequencing platform, or Life Science's 454 Sequencing platform.

[0283] In some embodiments, RNA-seq described herein further comprises performing digital counting and analysis on the cDNA. The number of amplified sequences for each transcript in the amplified sample can be quantified by sequence reading (one reading per amplified strand). In some embodiments, transcript per million (TPM) is used to quantify the expression level of a particular transcript. TPM may be calculated as shown in Wagner et al. (2012 ) Theory in Biosciences 131:281-285, the content of which is incorporated by reference herein in its entirety.

[0284] In certain embodiments, the binding proteins recognize a MAGEA4 immunogenic peptide in a complex with MHC molecules, such as particular HLA molecules having particular HLA alpha chain alleles. For example, binding proteins listed in Table 2 were identified as binders of M AGEA4 immunogenic peptides in association with an MHC whose alpha chain had an HLA-A*02 serotype, such as that encoded by an HLA-A*02:01, as described further in the Examples section. In some embodiments, the binding proteins recognize a complex of MAGEA4 immunogenic peptide and an MHC molecule, wherein the MHC molecule comprises an MHC alpha chain that is an HLA serotype of HLA-A*02, optionally wherein the HLA allele is HLA-A*02:01. In some embodiments, the MAGEA4 immunogenic peptides are derived from a human MAGEA4 protein and / or a MAGEA4 protein shown in Table 3. In some embodiments, one or more MAGEA4 immunogenic peptides are administered alone or in combination with an adjuvant.

[0285] - 49 - FoleyHoagUS 13160800.1 TTC-020

[0286] In some embodiments, the binding proteins do not bind to a peptide-MHC (pMHC) complex, optionally wherein the peptide is derived from an “off-target” described herein.

[0287] In some embodiments, the binding protein does not bind to an “off-target” described herein complexed with an MHC peptide-MHC (pMHC) complex.

[0288] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence with at least about 80%, 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 2; and / or b) a TCR beta chain sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2.

[0289] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2

[0290] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (Va) domain sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR alpha chain variable (Va) domain sequence selected from the group consisting of the TCR V« domain sequences listed in Table 2; and / or b) a TCR beta chain variable (Vp) domain sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR beta chain variable (Vp) domain sequence selected from the group consisting of the TCR Vp domain sequences listed in Table 2.

[0291] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (Va) domain sequence selected from the group consisting of the TCR Va domain sequences listed in Table 2; and / or b) a TCR beta chain variable (Vp) domain sequence selected from the group consisting of the TCR Vp domain sequences listed in Table 2.

[0292] - 50 - FoleyHoagUS 13160800.1 TTC-020

[0293] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two or three, such as CDR3 alone or in combination with a CDR1 and CDR2)) TCR alpha chain complementarity determining region (CDR) sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2. CDR3 is believed to be the main CDR responsible for recognizing processed antigen and CDR1 and CDR2 mainly interact with the MHC, so, in some embodiments, binding protein comprising a CDR3 alone from a TCR alpha chain and / or a CDR3 alone from a TCR beta chain listed in Table 2, each CDR3 having a sequence homology as recited in this paragraph, are provided.

[0294] In some embodiments, the binding proteins provided herein may also include (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two or three, such as CDR3 alone or in combination with a CDR1 and CDR2)) TCR beta chain complementarity determining region (CDR) sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2. As described above, CDR3 is believed to be the main CDR responsible for recognizing processed antigen and CDR1 and CDR2 mainly interact with the MHC, so, in some embodiments, binding protein comprising a CDR3 alone from a TCR beta chain and / or a CDR3 alone from a TCR alpha chain listed in Table 2, each CDR3 having a sequence homology as recited in this paragraph, are provided.

[0295] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two or three)) TCR alpha chain complementarity determining region (CDR) listed in Table 2.

[0296] In some embodiments, the binding proteins provided herein may also include (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two or three)) TCR beta chain complementarity determining region (CDR) listed in Table 2.

[0297] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of) a TCR alpha chain constant region (Ca) sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR Ca sequence listed in Table 2.

[0298] - 51 - FoleyHoagUS 13160800.1 TTC-020

[0299] In some embodiments, the binding proteins provided herein may also include (e.g., comprise, consist essentially of, or consist of) a TCR beta chain constant region (Cp) sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%.

[0300] 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more identity to a TCR Cp sequence listed in Table 2.

[0301] In some embodiments, the binding proteins provided herein include (e.g., comprise, consist essentially of, or consist of) a TCR alpha chain constant region (Ca) sequence selected from the group consisting of the TCR Ca sequences listed in Table 2.

[0302] In some embodiments, the binding proteins provided herein may also include (e.g., comprise, consist essentially of, or consist of) a TCR beta chain constant region (Cp) sequence selected from the group consisting of the TCR Cp sequences listed in Table 2.

[0303] Table 2: TCR sequences recognizing a MAGEA4 antigen presented by HLA serotype HLA-A*02

[0304] M AGEA4-230-3 MGTM codon optimized sequence (also known as “TCR-3”, “TCR-202-A02”, “TCR-202-A0201”, “TCR expressed by TSC-202-A02”, and “TCR expressed by TSC-202-A0201”)

[0305] Alpha chain:

[0306] TRAV14 / DV4 / TRAJ23 / TRAC

[0307] Alpha chain DNA sequence atgtccctgagcagcctgcttaaggtcgtgacagccagcctgtggctcggacctggaatcgcccagaagatcacccagacacagccc ggcatgttcgtgcaagagaaagaagccgtgaccctggactgcacctacgataccagcgatcctagctacggcctgttctggtacaag cagcctagcagcggcgagatgatcttcctgatctaccagggcagctacgaccagcagaatgccaccgagggcagatacagcctg aacttccagaaggcccggaagtccgccaacctggtcatttctgctagccagctgggcgacagcgccatgtacttttgtgccatgggtg ctgggggtaaattgatcttcggacagggaacggagttatctgtgaaacccaacATCCAGAACCCCGACCCCGCC GTGTACCAGCTGAGGGACTCCAAGTCCAGCGACAAGAGCGTGTGTCTGTTTACG GACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGGACAGCGACGTCTACATA ACGGATAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCC GTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGC ATCATCCCCGAGGACACCTTCTTCCCCAGCAGCGACGTGCCCTGCGACGTGAAAC TGGTGGAGAAGTCCTTCGAGACAGACACCAATCTGAACTTTCAGAACCTGCTGGT GATCGTGCTGCGGATTCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACC CTGCGGCTGTGGAGCAGC

[0308] Alpha chain Protein sequence MSLSSLLKVVTASLWLGPGIAOKITOTOPGMFVQEKEAVTLDCTYDTSDPSYGLFW YKOPSSGEMIFLIYOGSYDQONATCGRYSLNFOKARKSANLVLSASOLGDSAMYFC AMGAGGKLIFGOGTELSVKPNIONPDPAVYQLRDSKSSDKSVCLFTDFDSOTNVSO SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVP CDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0309] - 52 - FoleyHoagUS 13160800.1 TTC-020

[0310] Beta chain:

[0311] TRBV11-2 / TRBJ1-4 / TRBC1

[0312] Beta chain DNA sequence atggggacaagactgctttgttgggccgcactgtgcctgttgggagctgaacttactgaagccggcgtggcccagtctccaagataca agatcatcgagaagcggcagtccgtggccttctggtgcaatcctatctccggacacgctaccctctattggtatcagcaaatcctcggc cagggacctaagctgctgattcagttccagaacaacggcgtggtggacgacagccagctgcctaaggacaggttctcagccgaga gactgaaaggcgtggacagcaccctgaagatccagcctgccaagctggaagattccgccgtgtacctgtgcgcctcctctcacggg TCCAGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCACACACAAAA AGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGTCT TGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCCCAGCCT CTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCAGACTG AGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGC AGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCG TGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCACCA GCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTGCT GGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCATG GTCAAGCGGAAGGACTTT

[0313] Beta chain Protein sequence MGTRLLCWAALCLLGAELTEAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOQ ILGOGPKLLIQFONNGVVDDSOLPKDRFSAERLKGVDSTLKIOPAKLEDSAVYLCAS PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYE ILLGKATLYAVLVSALVLMAMVKRKDF

[0314] Complete Beta and Alpha ORF DNA Sequence atggggacaagactgctttgttgggccgcactgtgcctgttgggagctgaacttactgaagccggcgtggcccagtctccaagataca agatcatcgagaagcggcagtccgtggccttctggtgcaatcctatctccggacacgctaccctctattggtatcagcaaatcctcggc cagggacctaagctgctgattcagttccagaacaacggcgtggtggacgacagccagctgcctaaggacaggttctcagccgaga gactgaaaggcgtggacagcaccctgaagatccagcctgccaagctggaagattccgccgtgtacctgtgcgcctcctctcacggg TCCAGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCACACACAAAA AGCCACCCTCGTGTGCCTGGCCACCGGCITErTCCCCGACCACGTGGAACTGTCT TGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCCCAGCCT CTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCAGACTG AGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGC AGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCG TGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCACCA GCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTGCT GGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCATG GTCAAGCGGAAGGACTTTggcagcggcagagccaaaaggtccgggagcggtgcgacaaactttagcctgttgaaa caagccggcgacgttgaagagaaccccggacctatgtccctgagcagcctgcttaaggtcgtgacagccagcctgtggctcggacct ggaatcgcccagaagatcacccagacacagcccggcatgttcgtgcaagagaaagaagccgtgaccctggactgcacctacgat ccagcgatcctagctacggcctgttctggtacaagcagcctagcagcggcgagatgatcttcctgatctaccagggcagctacgac cagcagaatgccaccgagggcagatacagcctgaacttccagaaggcccggaagtccgccaacctggtcatttctgctagccagct gggcgacagcgccatgtacttttgtgccatgggtgctgggggtaaattgatcttcggacagggaacggagttatctgtgaaaccca acATCCAGAACCCCGACCCCGCCGTGTACCAGCTGAGGGACTCCAAGTCCAGCGA

[0315] - 53 - FoleyHoagUS 13160800.1 TTC-020

[0316] CAAGAGCGTG’IGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAG CAAGGACAGCGACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCAT GGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTG CGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCAGC GACGTGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGACACCAAT CTGAACTI CAGAACCTGCTGGTGATCGTGCTGCGGA1 CTGCTGCTGAAAGTGG CCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGC

[0317] Complete Beta and Alpha ORF Protein Sequence MGTRLLCWAALCLLGAELTEAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOQ ILGOGPKLLIOFONNGVVDDSOLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCAS SHGGTREKLFFGSGTOLSVLEDLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFF PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYE ILLGKATLYAVLVSALVLMAMVKRKDFGSGRAKRSGSGATNFSLLKQAGDVEENPG PMSLSSLLKVVTASLWLGPGIAOKITOTOPGMFVOEKEAVTLDCTYDTSDPSYGLFW YKOPSSGEMIFLIYOGSYDOONATEGRYSLNFOKARKSANLVL ASOLGDSAMYFC AMGAGGKLIFGOGTELSVKPNIONPDPAVYQLRDSKSSDKSVCLFTDFDSOTNVSO SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVP CDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS MAGEA4-230-3 wild type sequence

[0318] Alpha chain DNA sequence

[0319] TRAV14 / DV4 / TRAJ23 / TRAC atgtcactttctagcctgctgaaggtggtcacagcttcactgtggctaggacctggcattgcccagaagataactcaaacccaaccagg aatgttegtgcaggaaaaggaggctgtgactctggactgcacatatgacaccagtgatccaagttatggtctattctggtacaagcag cccagcagtggggaaatgatttttcttatttatcaggggtcttatgaccagcaaaatgcaacagaaggtcgctactcattgaatttccag aaggcaagaaaatccgccaaccttgtcatctccgcttcacaactgggggactcagcaatgtacttctgtgcaatgggagcgggagga aagcttatcttcggacagggaacggagttatctgtgaaacccaATATCCAGAACCCTGACCCTGCCGTGTA CCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT GATTCTCAAACAAATGTGTCACAAAGTAAGGATFCTGATGTGTATATCACAGACA AAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCT GGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCC AGAAGACACC'ITCrrCCCCAGCCCAGAAAGTrCCTGTGATGTCAAGCTGGTCGAG AAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGT TCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCT GTGGTCCAGC

[0320] Alpha chain Protein sequence MSLSSLEKVVTASLWLGPGIAOKITOTOPGMFVOEKEAVTLDCTYDTSDPSYGLFW YKOPSSGEMIFLIYOGSYDOONATEGRYSLNFOKARKSANLVISASOLGDSAMYFC AMGAGGKLIFGOGTELSVKPNIONPDPAVYOLRDSKSSDKSVCLFTDFDSOTNVSO SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS CDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0321] Beta chain DNA sequence

[0322] TRB V 11 -2 / TRB J 1 -4 / TRBC 1

[0323] - 54 - FoleyHoagUS 13160800.1 TTC-020

[0324] atgggcaccaggctcctctgctgggcggccctctgtctcctgggagcagaactcacagaagctggagttgcccagtctcccagatata agattatagagaaaaggcagagtgtggctttttggtgcaatcctatatctggccatgctaccctttactggtaccagcagatcctgggac agggcccaaagcttctgatcagtttcagaataacggtgtagtggatgattcacagttgcctaaggatcgattttctgcagagaggctc aaaggagtagactccactctcaagatccagcctgcaaagcttgaggactcggccgtgtatctctgtgccagcagccatggggggac gagagaaaaactgttttttggcagtggaacccagctctctgtcttggAGGACCTGAACAAGGTGTTCCCACC CGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGC CACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGG TGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTC AAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGG GTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCA CCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTrACCTCGGT GTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGG AAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCA AGAGAAAGGATTTC

[0325] Beta chain Protein sequence MGTRLLCWAALCLLGAELTEAGVAOSPRYKIIEKROSVAFWCNPISGHATLYWYOQ ILGOGPKLLIOFQNNGVVDDSOLPKDRFSAERLKGVDSTLKIOPAKLEDSAVYLCAS DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEI LLGKATLYAVLVSALVLMAMVKRKDF MAGEA4_286-638 MGTM codon optimized sequence (also known as “TCR-638”) Alpha chain:

[0326] TRAV8-4 / TRA. I6 / TRAC

[0327] Alpha chain DNA sequence atgctcctgctgctggtgcccgtgctggaagtgatttttaccctcggcggcacacgggcccagtotgttacacaactgggaagccacgt gtccgtgtccgaaggtgctctggtgctgctgagatgcaactacagcagctccgtgcctccatacctcttttggtacgtcaataccccaa ccagggactgcaactcctgctcaagtacacctctgccgccacactggtcaagggaatcaatggcttcgaggctgagtttaagaagtcc gagacatccttccatctgaccaagcctagcgctcacatgtccgacgccgctgagtacttctgcgctgtctcagtcggcgggagctac

[0328] ACAGCCAGACCAACGTGAGTCAAAGCAAGGACAGCGACGTCTACATAACGGATA AGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCT GGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCC CGAGGACACCTTCTTCCCCAGCAGCGACGTGCCCTGCGACGTGAAACTGGTGGA GAAGTCCTrCGAGACAGACACCAATCTGAACTITCAGAACCTGCTGGTGATCGTG CTGCGGATTCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGC TGTGGAGCAGC

[0329] Alpha chain Protein sequence MLLLLVPVLEVIFTLGGTRAOSVTOLGSHVSVSEGALVLLRCNYSSSVPPYLFWYVO YPNOGLOLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSV DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDV KLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0330] - 55 - FoleyHoagUS 13160800.1 TTC-020

[0331] Beta chain:

[0332] TRBV28 / TRBJ1- 1 / TRBC 1

[0333] Beta chain DNA sequence atgggcattcgcctgctgtgcagagtggccttctgttttctggccgtcggcctggtggatgtgaaagtgacccagagcagcagatacct cgtgaagagaaccggcgagaaggtgttcctggaatgcgtgcaggacatggatcacgagaatatgttttggtatagacaagatcccg gcctcggactgaggctgatctatttctcctacgacgtcaagatgaaggaaaagggcgacatccctgagggctactccgtgtctagag agaagaaagagcggtctccctgatcctcgagagc.gccagcacaaaccagacctccatgtatctgtgcgctagttccttcattggcg CCTCCAGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCACACACAA AAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGT CTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCCCAGC CTCTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCAGAC TGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGT GCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCC CGTGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCAC CAGCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTG CTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCA TGGTCAAGCGGAAGGACTTT

[0334] Beta chain Protein sequence MGIRLLCRVAFCFLAVGLVDVKVTOSSRYLVKRTGEKVFLECVODMDHENMFWYR ODPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNOTSMYLCASS FPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDF

[0335] Complete Beta and Alpha ORF DNA Sequence atgggcattcgcctgctgtgcagagtggccttctgttttctggccgtcggcctggtggatgtgaaagtgacccagagcagcagatacct cgtgaagagaaccggcgagaaggtgttcctggaatgcgtgcaggacatggatcacgagaatatgttttggtatagacaagatcccg gcctcggactgaggctgatctattctcctacgacgtcaagatgaaggaaaagggcgacatccctgagggctactccgtgtctagag agaagaaagagcggttctccctgatcctcgagagcgccagcacaaaccagacctccatgtatctgtgcgctagttccttcattggcg ctgggcgcgacaccgaagcattcttcggacaaggcaccagactcacagttgtagAAGATCTGAACAAGGTGTTC CCTCCAGAGGTGGCCGTG'ITCGAGCCTTCTAAGGCCGAGATCGCCCACACACAA AAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGT CTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCCCAGC CTCTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCAGAC TGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGT GCAGTnTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCC CGTGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCAC CAGCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTG CTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCA TGGTCAAGCGGAAGGACTTTggcagcggcagagccaaaaggtccgggagcggtgcgacaaactttagcctgttg aaacaagccggcgacgttgaagagaaccccggacctatgctcctgctgctggtgcccgtgctggaagtgatttttaccctcggcggca cacgggcccagtctgttacacaactgggaagccacgtgtccgtgtccgaaggtgctctggtgctgctgagatgcaactacagcagct ccgtgcctccatacctcttttggtacgttcaataccccaaccagggactgcaactcctgctcaagtacacctctgccgccacactggtca agggaatcaatggcttcgaggctgagtttaagaagtccgagacatccttccatctgaccaagcctagcgctcacatgtccgacgccgct gagtacttctgcgctgtctcagtcggcgggagctacattcctacattcggaagaggaaccagccttattgttcatccgtacATCC

[0336] - 56 - FoleyHoagUS 13160800.1 TTC-020

[0337] AGAACCCCGACCCCGCCGTGTACCAGCTGAGGGACTCCAAGTCCAGCGACAAGA GCGTGTGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGG ACAGCGACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGACT TCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCA ACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCAGCGACGT GCCCTGCGACGTGAAACTGGTGGAGAAGTCCITCGAGACAGACACCAATCTGAA CTTTCAGAACCTGCTGGTGATCGTGCTGCGGATTCTGCTGCTGAAAGTGGCCGGC TTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGC

[0338] Complete Beta and Alpha ORF Protein Sequence MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVODMDHENMFWYR QDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNOTSMYLCASS FPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDFGSGRAKRSGSGATNFSLLKQAGDVEEN PGPMLLLLVPVLEVIFTLGGTRAOSVTOLGSHVSVSEGALVLLRCNYSSSVPPYLFW YVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCA KDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVP CDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS MAGEA4_286-638 wild type sequence

[0339] Alpha chain DNA sequence

[0340] TRAV8-4 / TRAJ6 / TRAC atgctcctgctgctcgtcccagtgctcgaggtgatttttaccctgggaggaaccagagcccagtcggtgacccagcttggcagccacg tctctgtctctgaaggagccctggttctgctgaggtgcaactactcatcgtctgttccaccatatctcttctggtatgtgcaataccccaac caaggactccagcttctcctgaagtacacatcagcggccaccctggttaaaggcatcaacggttttgaggctgaatttaagaagagtg aaacctccttccacctgacgaaaccctcagcccatatgagcgacgcggctgagtacttctgtgctgtgagtgtcggtggaagctaca tacctacatttggaagaggaaccagccttattgttcatccgtATATCCAGAACCCTGACCCTGCCGTGTAC CAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGA1TTTG ATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAA AACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTG GAGCAACAAATCTGACTITGCATGTGCAAACGCCITCAACAACAGCAITATTCCA GAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGA AAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTT CCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG TGGTCCAGC

[0341] Alpha chain Protein sequence MLLLLVPVLEVIFTLGGTRAOSVTOLGSHVSVSEGALVLLRCNYSSSVPPYLFWYVQ YPNOGLOLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSV DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVK LVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0342] Beta chain DNA sequence

[0343] TRB V28 / TRBJ1 - 1 / TRBC 1

[0344] - 57 - FoleyHoagUS 13160800.1 TTC-020

[0345] atgggaatcaggctcctctgtcgtgtggccttttgtttcctggctgtaggcctcgtagatgtgaaagtaacccagagctcgagatatctag tcaaaaggacgggagagaaagttttctggaatgtgtccaggatatggaccatgaaaatatgttctggtatcgacaagacccaggtct ggggctacggctgatctatttctcatatgatgttaaaatgaaagaaaaaggagatattcctgaggggtacagtgtctctagagagaag aaggagcgcttctccctgattctggagtccgccagcaccaaccagacatctatgtacctctgtgccagcagttttataggggcagga CCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAG GCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCT GGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCC TCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGA GGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCA GTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGT CACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTC GGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTA GGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGG TCAAGAGAAAGGATTTC

[0346] Beta chain Protein sequence MGIRLLCRVAFCFLAVGLVDVKVTOSSRYLVKRTGEKVFLECVODMDHENMFWYR ODPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNOTSMYLCASS FIGAGRDTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGF FPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDF

[0347] * Table 2 provides, in part, representative TCR sequences grouped according to MHC serotype presentation and sub-grouped according to different peptides presented by the MHC serotype and bound by the sub-grouped TCRs. Individual TCRs, such as those representatively exemplified in the tables, are described and claimed, as well as the genus of binding proteins that bind a peptide epitope sequence described herein either alone or in a complex with an MHC, such as those grouped in the tables provided herein. In addition, TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and TRBV, TRB J, and TRBC genes for each TCR beta chain described herein, are provided. Sequences for each TCR described herein are provided as pairs of cognate alpha chain and beta chains for each named TCR. TCR sequences described herein are annotated. Variable domain sequences are shown in lower case. Constant domain sequences are capitalized. CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined text. CDR1, CDR2, and CDR3 are shown in standard order of appearance from left (N-terminus) to right (C-terminus). TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein, are annotated according to well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2

[0348] - 58 - FoleyHoagUS 13160800.1 TTC-020

[0349] of TRAV and TRBV are well-known in the art since they are based on well-known and annotated TRAV and TRBV sequences (e.g., as annotated in databases like IMGT available at imt.org and IEDB available at iedb.org).

[0350] Table 3

[0351] Representative Human MAGEA4 cDNA sequence atgtcttctgagcagaagagtcagcactgcaagcctgaggaaggcgttgaggcccaagaagaggccctgggcctggtgggtgcaca ggctcctactactgaggagcaggaggctgctgtctcctcctcctctcctctggtccctggcaccctggaggaagtgcctgctgctgagt cagcaggtcctccccagagtcctcagggagcctctgccttacccactaccatcagcttcacttgctggaggcaacccaatgagggttc cagcagccaagaagaggaggggccaagcacctcgcctgacgcagagtccttgttccgagaagcactcagtaacaaggtggatgag ttggctcattttctgctccgcaagtatcgagccaaggagctggtcacaaaggcagaaatgctggagagagtcatcaaaaattacaagc gctgctttcctgtgatcttcggcaaagcctccgagtccctgaagatgatctttggcattgacgtgaaggaagtggaccccgccagcaac acctacacccttgtcacctgcctgggcctttcctatgatggcctgctgggtaataatcagatctttcccaagacaggccttctgataatcgt cctgggcacaattgcaatggagggcgacagcgcctctgaggaggaaatctgggaggagctgggtgtgatgggggtgtatgatggg agggagcacactgtctatggggagcccaggaaactgctcacccaagattgggtgcaggaaaactacctggagtaccggcaggtacc cggcagtaatcctgcgcgctatgagttcctgtggggtccaagggctctggctgaaaccagctatgtgaaagtcctggagcatgtggtc agggtcaatgcaagagttcgcattgcctacccatccctgcgtgaagcagctttgttagaggaggaagagggagtctga Representative Homan MAGEA4 protein sequence (Representative, non-limiting epitopes underlined) MSSEQKSQHCKPEEGVEAQEEALGLVGAQAPTTEEQEAAVSSSSPLVPGTLEEVPAA ESAGPPQSPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDE LAHFLLRKYRAKELVTKAEMLERVIKNYKRCFPVIFGKASESLKMIFG1DVKEVDPAS NTYTLVTCLGLSYDGLLGNNQIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVY DGREHTVYGEPRKLLTODWVOENYLEYROVPGSNPARYEFLWGPRALAETSYVKV LEHVVRVNARVRIAYPSLREAALEEEEEGV

[0352] Representative Human HLA-A*02:01 DNA sequence atggccgtcatggcgccccgaaccctcgtcctgctactctcgggggctctggccctgacccagacctgggcgggctctcactccatg aggtatttcttcacatccgtgtcccggcccggccgcggggagccccgcttcatcgcagtgggctacgtggacgacacgcagttcgtg cggttcgacagcgacgccgcgagccagaggatggagccgcgggcgccgtggatagagcaggagggtccggagtattgggacgg ggagacacggaaagtgaaggcccactcacagactcaccgagtggacctggggaccctgcgcggctactacaaccagagcgaggc cggttctcacaccgtccagaggatgtatggctgcgacgtggggtcggactggcgcttcctccgcgggtaccaccagtacgcctacga cggcaaggattacatcgccctgaaagaggacctgcgctcttggaccgcggcggacatggcagctcagaccaccaagcacaagtgg gaggcggcccatgtggcggagcagttgagagcctacctggagggcacgtgcgtggagtggctccgcagatacctggagaacggg aaggagacgctgcagcgcacggacgcccccaaaacgcatatgactcaccacgctgtctctgaccatgaagccaccctgaggtgctg ggccctgagcttctaccctgcggagatcacactgacctggcagcgggatggggaggaccagacccaggacacggagctcgtgga gaccaggcctgcaggggatggaaccttccagaagtgggcggctgtggtggtgccttctggacaggagcagagatacacctgccatg tgcagcatgagggtttgcccaagcccctcaccctgagatgggagccgtcttcccagcccaccatccccatcgtgggcatcattgctgg cctggttctctttggagctgtgatcactggagctgtggtcgctgctgtgatgtggaggaggaagagctcagatagaaaaggagggagc tactctcaggctgcaagcagtgacagtgcccagggctctgatgtgtctctcacagcttgtaaagtgtga Representative Human HLA-A*02:01 protein sequence MAVMAP TLVLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSD ASQRMEPRAPWIEQEGPEYWDGETRKVK HSQTHRVDLGTLRGYYNQ SEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQ TTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVS DHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPS

[0353] - 59 - FoleyHoagUS 13160800.1 TTC-020

[0354] GQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMW RRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV*

[0355] Representative Vector (the TCR-encoding protein of which can be interchanged with any TCR sequence of interest): pNVVD262_TSC-202-A02_TCR-3_MSCV-TCR-3-CD8- EF1 a-dnTGFbRII-DHFR GCTAGCTGGCTTGTTGTCCACAACCATTAAACCTTAAAAGCTTTAAAAGCCTTAT ATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGAT TTAIATIAAIG’TTATTGITCAAACATCAGAGCTTAGTACGTGAAACATGAGAGCT TAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAGGGTTTAGTTCATTAA ACATGAGAGCTTAGTACATTAAACATGAGAGCTTAGTACATACTATCAACAGGTT GAACTGCTGATCTGTACAGTAGAA'I GGTAAAGAGAGTrGTGTAAAATATTGAGT TCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGAC AAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGTACCAAT TACAI GCTIGCAA AACCCTmACGGlTATAAGGATCTAGATGAGATAGAAA GATTTGGTTTTCGGATTTGTGTTACATAAGATGCCTAAAATAAAAATTGAGATTC AATTTTTTTTAAACTTTTTTTTAATTGGTGGTAAGAATATTCCCTCTACCTGTTTGA GAGTAATGAAATTGTAGTATGATTTTTCAACAAACTAAAAAAACAACATAAATCT CACATAATAACTTTATTTCAATCACACAATTGAATACCAATAGGTTGACAGTACT TACCAGCCTGCAGGTGAAAGACCCCACCTGTAGGTTTGGCAAGTTAGCTTAA GTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTT CAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATA TCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCC CAGATGCGGTCCCGCCCTCAGCAGTTTCTAGCGAACCATCAGATGTTTCCAG GGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCA GTTTGCTTCTTGCTTCTGTTTGTGTGCTTCTGCTCCCTGAGCTCAATAAAAG AGCCCACAACCCCTCACTTGGTGGGCCAGTCCTCTGATAGACTGTGTCCCCT GGA ACCCGTACGGTACCGClAGCCiCCACCATGGGGACAAGACTGCTTTGTTGGG CCGCACTGTGCCTGTTGGGAGCTGAACTTACTGAAGCCGGCGTGGCCCAGTCTCCA AGATACAAGATCATCGAGAAGCGGCAGTCCGTGGCCTTCTGGTGCAATCCTATCTC CGGACACGCTACCCTCTATTGGTATCAGCAAATCCTCGGCCAGGGACCTAAGCTGC TGATTCAGTTCCAGAACAACGGCGTGGTGGACGACAGCCAGCTGCCTAAGGACAG GTTCTCAGCCGAGAGACTGiAAGGCGTGGACAGCACCCTGAAGATCCAGCCTGCC AAGCTGGAAGATTCCGCCGTGTACCTGTGCGCCTCCTCTCACGGGGGCACTCGCG AGAAACTCTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAAGATCTGAACAAG GTGTTCCCTCCAGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCACAC ACAAAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAAC TGTCTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCCCA GCCTCTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCAGA CTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGT GCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCC GTGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCACCA GCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTGCTG GGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCATGG TCAAGCGGAAGGACTTTGGCAGCGGCAGAGCCAAAAGGTCCGGGAGCGGTGCGA CAAACTTTAGCCTGTTGAAACAAGCCGGCGACGTTGAAGAGAACCCCGGACCTA TGTCCCTGAGCAGCCTGCTTAAGGTCGTGACAGCCAGCCTGTGGCTCGGAC CTGGAATCGCCCAGAAGATCACCCAGACACAGCCCGGCATGTTCGTGCAAG AGAAAGAAGCCGTGACCCTGGACTGCACCTACGATACCAGCGATCCTAGCT ACGGCCTGTTCTGGTACAAGCAGCCTAGCAGCGGCGAGATGATCTTCCTGA

[0356] - 60 - FoleyHoagUS 13160800.1 TTC-020

[0357] TCTACCAGGGCAGCTACGACCAGCAGAATGCCACCGAGGGCAGATACAGCC TGAACTTCCAGAAGGCCCGGAAGTCCGCCAACCTGGTCATTTCTGCTAGCCA GCTGGGCGACAGCGCCATGTACTTTTGTGCCATGGGTGCTGGGGGTAAATT GATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAACATCCAGAACCC CGACCCCGCCGTGTACCAGCTGAGGGACTCCAAGTCCAGCGACAAGAGCGT GTGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGGA CAGCGACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGA CTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTG CGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGC AGCGACGTGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGAC ACCAATCTGAACTTTCAGAACCTGCTGGTGATCGTGCTGCGGATTCTGCTGC TGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGCA GGGCTAAGAGGTCCGGCAGCGGAGCCACCAATTTTTCCCTGCTGAAACAGGCTG GTGACGTGGAAGAAAACCCTGGCCCCATGGCGCTGCCCGTCACCGCGCTGCTGCT GCCCCTGGCGCTGCTGTTACACGCCGCTCGGCCAGAGCTTCCCACCCAGGGCACATr CTCCAACGTGTCCACCAATGTGTCGGGAGGCGGCGGATCGTCCCAGTTCAGAGTGTC CCCTCTGGACCGCACCTGGAACCTGGGCGAGACCGTGGAGCTGAAATGTCAGGTCCT GCTGAGCAACCCGACCTCCGGGTGCAGTTGGCTGTTCCAGCCGCGTGGTGCTGCCGC AAGCCCTACGTTCCTGCTTTACCTGAGCCAGAACAAGCCCAAGGCGGCCGAGGGCCT GGACACCCAGAGArrCTCCGGCAAGCGCCTGGGGGACACAnCGTGCrTACITrGAGC GATTTCCGCAGAGAGAACGAGGGCTACTATTTCTGTTCGGCGCTGAGCAATTCCATCAT GTATTTCAGCCACTTTGTGCCAGTGTTCCTGCCTGCCAAGCCTACCACAACACCAGCTC CCCGTCCCCCGACTCCGGCGCCTACCATCGCGAGTCAACCGTrGAGCCTGAGGCCTG AGGCTTGTCGGCCCGCTGCGGGGGGTGCCGTCCACACCAGGGGCCTCGACTTTGCG TGCGACATCTATArnGGGCGCCTCTGGCGGGTACCTGCGGGGTGCTGCTGCTGTCAT TGGTGAITAC(X: TGT CTGC TCA(X: GCAAC(: GCCGGCGGGTCTGTMGTGC(X: ACG GCCTGTGGTCAAGTCCGGTGACAAACCGTCGCTCTCGGCTCGCTACGTGCGCGCTAA GCGCAGCGG'I CCGGGGCCACCAAC TrCAl GCTGAAGCAGGCCGGTGATGTG GAGGAGAATCCAGGGCCCATGCGCCCCAGGCTTTGGCTCCTTCTTGCTGCTCAGC TCACTGTCTTGCATGGCAACTCCGTTCTGCAGCAGACTCCCGCCTACATCAAGGT GCAGACGAACAAGATGGTGATGCTGTCATGCGAGGCCAAGATCTCTCTTTCAAAT ATGAGAATTTATTGGCTACGACAGCGCCAGGCCCCCTCCAGCGACAGCCACCAC GAGI CCTGGCGC G GGATrCTGCTAAAGGCACCATCCATGGAGAGGAGGTG GAACAGGAGAAGATAGCTGTCTTCCGCGACGCATCCCGCTTCATCCTGAACCTGA CCAGCGTGAAGCCGGAGGACAGCGGCATCTACTTCTGTATGATCGTTGGCTCCCC CGAGCTGACCI CGGCAAAGGCACCCAGCTGTCCGTGGTGGAC1 CCTGCCCACC ACAGCCCAGCCAACCAAGAAATCCACCCTCAAGAAGCGCGTGTGCCGACTGCCC CGCCCTGAAACCCAGAAGGGCCCTCTGTGCTCCCCCATCACCCTTGGACTGCTGG TGGCGGGAGTCCTGGTGCTGCTCGTATCTCTGGGTGTCGCCATCCACCTGTGCTG CCGCCGCCGCCGCGCCCGCCTGAGGTTTATGAAACAGTTTTACAAGTGATAAATC GATGGAAGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAA GTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTT GTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGC AAGGGGCAAGTrGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCA AGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTC AAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGAC CAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCA GGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATT GCTGGGAI ACAGGCGTGAACCACTGCTCCCnVCCTGTCCl CTGAI ACTAGTG GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT

[0358] - 61 - FoleyHoagUS 13160800.1 TTC-020

[0359] TGTGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCC GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGG GTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTT GATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAA GGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCC GCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA GCCAl^rrAAAATrm’GArGACCrGCTGCGACGCrrrn’rrCTGGCAAGArAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGC GGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGA GCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTG GTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCC GGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAG GGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCC ACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGG AGTACCX3GGCGCCG’K 'AGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTC GTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGG GTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTG

[0360] cccTrn,rrrrGAGrrrrrrGGArrcrrrrGGrrTCArrrrcrrcAAGCCrrcAGACAGTGG’rrcAAAGrr TTTTTTCTTCCATTTCAGGTGTCGTGAACTAGTCCAGTGTGGTGGAATTCTGCAGA TATCACGGCTAGCGCCACCATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCT GCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAG TCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCA CAACTGTGTAAAlTI GTGATGTGAGATrrrCCACCTGTGACAACCAGAAATCCT GCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTG TGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATG ACCCCAAGCTCCCCTACCATGACm ITCTGGAAGATGCTGCTTCTCCAAAGTG CATTATGAAGGAGAAGAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGC TCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATC CTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACT GGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTGAACCGGCAG CAGAAGGCTAGTGGTTCAGGCGCAACGAATTTCTCTTTGCTGAAGCAGGCTGGG GATGTCGAAGAAAATCCGGGTCCAATGGTGGGCTCGCTCAACTGCATCGTAGCA GTCTCCCAGAATATGGGCATCGGGAAGAACGGTGATTTCCCGTGGCCCCCACTTC GCAACGAGAGCCGTI n CCAAAGAATGACTACAACCTCCTCCGTGGAGGGTA AGCAGAACCTGGTCATCATGGGGAAGAAGACCTGGTTCTCTATCCCTGAAAAAA ACCGCCCCCTGAAGGGCCGCATCAACCTGGTGCTGAGCAGGGAACTCAAGGAGC CTCCTCAGGGCGCGCATTTTCTGAGCCGCTCATTGGATGACGCTCTCAAACTGAC CGAACAGCCGGAGCTAGCCAACAAGGTGGACATGGTGTGGATCGTCGGAGGCTC CTCCGTGTACAAGGAGGCCATGAATCACCCCGGCCACTrGAAGCTGTTCGTCACC CGGATCATGCAGGACTTCGAGTCGGACACGTTCTTTCCAGAGATTGACCTGGAGA AGTACAAGCTGCTGCCCGAGTACCCGGGAGTTCTTAGTGATGTGCAGGAGGAGA AAGGCATCAAGTACAAAT1TGAGGTGTACGAGAAGAACGACTAACGGTCCGTCC TGACCAATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGG TTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTG CATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACA GGTTACCTCAGTCTCCTAGGTACGTCTTATATCTATGAAAAAACATTCAAAAGCA CAACATCTAGAAGAACTTACCTIGTTTCAC ACTCTATrciCAAAGA’IATGTACCG ATTTCTCTCGAAGTACAAAAAACCGCTAGTTTTCAAATTCACCTCAAGACTTTGA

[0361] - 62 - FoleyHoagUS 13160800.1 TTC-020

[0362] AAAAAAATrGAATCTGTCAATGTCAAATAAAATCAGAAACAAATGTCATAATGT TACGTTAATGTTGTCAGGTCGAAAAATAAAATTGCAAATAGAAATTTTGTTCCTT TTTTATTGGTTTTTATTGGTGGGAAAAATATTCCCTCTAACTGCAAAAGGGTTAAT TATGTTAGAGGTAGAGTCGAC

[0363] * For certain depicted vectors, MSCV promoter is in bold. Beta chain is annotated using bold and italic text. Alpha chain is annotated using bold and underlined text. CD34-enrichment tag (Q tag) is annotated using italic and underlined text. CD8-alpha is in italic. CD8-beta is underlined.

[0364] Representative Vector (the TCR-encoding protein of which can be interchanged with any TCR sequence of interest): pNVVI)265_TSC-202-A02_TCR-638_MSCV-TCR-638-CD8-EFla-dnTGFbRII-DHFR GCTAGCTGGCTTGTTGTCCACAACCATTAAACC’I AAAAGCTTTAAAAGCCTTAT ATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGAT TTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCT TAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAGGGTTTAGTTCATTAA ACATGAGAGCTTAGTACATTAAACATGAGAGCTTAGTACATACTATCAACAGGTT GAACTGCIGATCTGTACAGTAGAATTGGTAAAGAGAGTTGTGTAAAATATTGAGT TCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGAC AAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGTACCAAT TACATTGCTTGCAAIlAACraiTAACGGTTAIAAGGATCTAGATGAGATAGAAA GATTTGGTTTTCGGATTTGTGTTACATAAGATGCCTAAAATAAAAATTGAGATTC AATTTTTTTTAAACTTTTTTTTAATTGGTGGTAAGAATATTCCCTCTACCTGTTTGA GAGTAATGAAATTGTAGTATGATTTTTCAACAAACTAAAAAAACAACATAAATCT CACATAATAACTTTATTTCAATCACACAATTGAATACCAATAGGTTGACAGTACT TACCAGCCTGCAGGTGAAAGACCCCACCTGTAGGTTTGGCAAGTTAGCTTAA GTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTT CAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATA TCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCC CAGATGCGGTCCCGCCCTCAGCAGTTTCTAGCGAACCATCAGATGTTTCCAG GGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCA GTTTGCTTCTTGCTTCTGTTTGTGTGCTTCTGCTCCCTGAGCTCAATAAAAG AGCCCACAACCCCTCACTTGGTGGGCCAGTCCTCTGATAGACTGTGTCCCCT GGATACCCGTACGGTACCGCTAGCGCCACCATGGGCA7TCGCCTGCTGTGCAGAG TGGCCTTCTGTTTTCTGGCCGTCGGCCTGGTGGA TGTGAAA GTGA CCCAGAGCAGC AGATACCTCGTGAAGAGAACCGGCGAGAAGGTGTTCCTGGAATGCGTGCAGGACA TGGATCACGAGAATATGTTTTGGTATAGACAAGAICCCGGCCTCGGACTGAGGCTG ATCTATTTCTCCTACGACGTCAAGATGAAGGAAAAGGGCGACATCCCTGAGGGCTA CTCCGTGTCTAGAGAGAAGAAAGAGCGGTTCTCCCTGATCCTCGAGAGCGCCAGC ACAAACCAGACCTCCATGTATCTGTGCGCTAG1TCC1TCATTGGCGCTGGGCGCGA CACCGAAGCATTCTTCGGACAAGGCACCAGACTCACAGTTGTAGAAGATCTGAACA AGGTGTTCCCTCCAGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCAC ACACAAAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGA ACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTCAACGGATCCC CAGCCTCTGAAAGAACAGCCTGCCCTGAACGACAGCCGGTACTGCCTGAGCTCCA GACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAG

[0365] - 63 - FoleyHoagUS 13160800.1 TTC-020

[0366] GTGCA GTTTTA CGGCCTGAGCGAGAACGACGA GTGGACCCAGGACAGAGCCAAGC CCGTGACACAAATCGTGTCTGCCGAAGCCTGGGGAAGAGCCGATTGCGGCATCAC CAGCGCCTCCTATCACCAGGGCGTGCTGAGCGCCACAATCCTGTACGAAATCCTGC TGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCTCTGGTGCTGATGGCCAT GGTCAAGCGGAAGGACT7 GGCAGCGGCAGAGCCAAAAGGTCCGGGAGCGGTGC GACAAACTTTAGCCTGTTGAAACAAGCCGGCGACGTTGAAGAGAACCCCGGACC TATGCTCCTGCTGCTGGTGCCCGTGCTGGAAGTGATTTTTACCCTCGGCGGC ACACGGGCCCAGTCTGTTACACAACTGGGAAGCCACGTGTCCGTGTCCGAA GGTGCTCTGGTGCTGCTGAGATGCAACTACAGCAGCTCCGTGCCTCCATAC CTCTTTTGGTACGTTCAATACCCCAACCAGGGACTGCAACTCCTGCTCAAGT ACACCTCTGCCGCCACACTGGTCAAGGGAATCAATGGCTTCGAGGCTGAGT T TAAGAAGTCCGAGACATCC TTCCATCTGACCAAGCC TAGCGCTCACATGTC CGACGCCGCTGAGTACTTCTGCGCTGTCTCAGTCGGCGGGAGCTACATTCC TACATTCGGAAGAGGAACC. AGCCTTATTGTTCATCCGTAC. ATCCAGAACCCC GACCCCGCCGTGTACCAGC TGAGGGACTCCAAGTCCAGCGACAAGAGCGTG TGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGGAC AGCGACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGAC T TCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACT TCGCCTGC GCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCA GCGACGTGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGACA CCAATCTGAACTTTCAGAACCTGCTGGTGATCGTGCTGCGGATTCTGCTGCT GAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGCAG GGCTAAGAGGTCCGGCAGCGGAGCCACCAATTTTTCCCTGCTGAAACAGGCTGG TGACGTGGAAGAAAACCCTGGCCCCATGGCGCTGCCCGTCACCGCGCTGCTGCTG CCCCTGGCGCTGCTGrrACACGCCGCTCGGCCAGAGCTrCCCACCCAGGGCACA'nC TCCAACGTGTCCACCAATGTGTCGGGAGGCGGCGGATCGTCCCAGnGAGAGTGTCC CCTCTGGACCGCACCTGGAACCTGGGCGAGACCGTGGAGCTGAAATGTCAGGTCCTG CTGAGC / ACCCGACCTCCGGGTGCAGITGGCTGITCCAGCCGCGTGGTGCTGCCGCA AGCCCTACGTTCCTGCTTTACCTGAGCCAGAACAAGCCCAAGGCGGCCGAGGGCCTG GACACCCAGAGATTCTCCGGCAAGCGCCTGGGGGACACATTCGTGCTTACTTTGAGCG

[0367] AmCCGCAGAGAGAACGAGGGCTACl'ATnCTGnCGGCGCTGAGCAArrCCATCATG TATTTCAGCCACTTTGTGCCAGTGTTCCTGCCTGCCAAGCCTACCACAACACCAGCTCC CCCJirCCCCC CI’CCGGCGCCZACCAT'CGCCJAGT'CAACCGZ X GCCTGAC^GCCIGA GG(:77’G7rGGCCGGC7GCC GC GC G7’GGCG7,CC C CCAGGGGCC7rGAG^7TGCG7,GCGACATCTATATTTGGGCGCCTCTGGCGGGTACCTGCGGGGTGCTGCTGCTGTCATT GGTGA:rrACCCTGlACTGCAATCACCGCAACCGCCGGCGGGTCTGlAAGTGCCCACG GCCTGTGGTCAAGTCCGGTGACAAACCGTCGCTCTCGGCTCGCTACGTGCGCGCTAA GCGCAGCGGTTCCGGGGCCACCAACTTTTCATTGCTGAAGCAGGCCGGTGATGTG GAGGAGAATCCAGGGCCCATGCGCCCCAGGCTTTGGCTCCTTCTTGCTGCTCAGC TCACTGTCTTGCATGGCAACTCCGTTCTGCAGCAGACTCCCGCCTACATCAAGGT GCAGACGAACAAGArGGrGATGClGTCArGCGAGGCCAAGArCFCrCITrCAAAl ATGAGAATTTATTGGCTACGACAGCGCCAGGCCCCCTCCAGCGACAGCCACCAC GAGTTCCTGGCGCTTTGGGATTCTGCTAAAGGCACCATCCATGGAGAGGAGGTG GAACAGGAGAAGATAGCTGTCI CCGCGACGCATCCCGCTrCATCCTGAACCTGA CCAGCGTGAAGCCGGAGGACAGCGGCATCTACTTCTGTATGATCGTTGGCTCCCC CGAGCTGACCTTCGGCAAAGGCACCCAGCTGTCCGTGGTGGACTTCCTGCCCACC ACAGCCCAGCCAACCAAGAAATCCACCCTCAAGAAGCGCGTGTGCCGACTGCCC CGCCCTGAAACCCAGAAGGGCCCTCTGTGCTCCCCCATCACCCTTGGACTGCTGG TGGCGGGAGrCCTCGFGCTGCrCGTATCTCTGGGTCTCGCCATCCACCTGTCCTG CCGCCGCCGCCGCGCCCGCCTGAGGTTTATGAAACAGTTTTACAAGTGATAAATC

[0368] - 64 - FoleyHoagUS 13160800.1 TTC-020

[0369] GATGGAAGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAA GTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTT GTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGC AAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCA AGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTC AAGCGATrCTCCTGCCTCAGCCTCCCGAGTTGI GGGATrCCAGGCATGCATGAC CAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCA GGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATT GCTGGGA3 ACAGGCGTGAACCACTGCTCCC1 CCCTGTCCTTCTGATTACTAGTG GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT TGTGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC'ITn CCCGAGGGTGGGGGAGA ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCC GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGG GTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTT GATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAA GGAGCCCCTrCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCC GCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA GCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT TGTAAATGCGGGCCAAGATCTGCACACTGGTA rTCGG m GGGGCCGCGGGC GGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGA GCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTG GTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCC GGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAG GGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCC ACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGG AGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTC GTCmAGGI GGGGGGAGGGGTITrATGCGATGGAGTTrCCCCACACTGAGTGG GTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTG CCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGT TTTTTTCTTCCATTTCAGGTGTCGTGAACTAGTCCAGTGTGGTGGAATTCTGCAGA TATCACGGCTAGCGCCACCATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCT GCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTrCAGAAG TCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCA CAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCT GCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTG TGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATG ACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTG CATTATGAAGGAGAAGAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGC TCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATC CTGACI GTrGCTAGTCATATITCAAGTGACAGGCATCAGCCTCCTGCCACCACT GGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTGAACCGGCAG CAGAAGGCTAGTGGTTCAGGCGCAACGAATTTCTCTTTGCTGAAGCAGGCTGGG GATGTCGAAGAAAATCCGGGTCCAATGGTGGGCTCGCTCAACTGCATCGTAGCA GTCTCCCAGAATATGGGCATCGGGAAGAACGGTGATTTCCCGTGGCCCCCACTTC GCAACGAGAGCCGTTATTTCCAAAGAATGACTACAACCTCCTCCGTGGAGGGTA AGCAGAACCTGGTCATCATGGGGAAGAAGACCTGGTTCTCTATCCCTGAAAAAA ACCGCCCCCTGAAGGGCCGCATCAACCTGGTGCTGAGCAGGGAACTCAAGGAGC CTCCTCAGGGCGCGCATTTTCTGAGCCGCTCAIIGGATCACGCTCTCAAACTGAC CGAACAGCCGGAGCTAGCCAACAAGGTGGACATGGTGTGGATCGTCGGAGGCTC

[0370] - 65 - FoleyHoagUS 13160800.1 TTC-020

[0371] CTCCGTGTACAAGGAGGCCATGAATCACCCCGGCCACTTGAAGCTGTTCGTCACC CGGATCATGCAGGACTTCGAGTCGGACACGTTCTTTCCAGAGATTGACCTGGAGA AGTACAAGCTGCTGCCCGAGTACCCGGGAGTTCTTAGTGATGTGCAGGAGGAGA AAGGCATCAAGTACAAATTTGAGGTGTACGAGAAGAACGACTAACGGTCCGTCC TGACCAATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGG TTACAAATAAAGCAATAGCATCACAAAIT’TCACAAATAAAGCATTITTTTCACTG CATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACA GGTTACCTCAGTCTCCTAGGTACGTCTTATATCTATGAAAAAACATTCAAAAGCA CAACATCTAGAAGAACTTACCTTTT’ITCACC / XCTCTATTGCAAAGATATGTACCG ATTTCTCTCGAAGTACAAAAAACCGCTAGTTTTCAAATTCACCTCAAGACTTTGA AAAAAAATTGAATCTGTCAATGTCAAATAAAATCAGAAACAAATGTCATAATGT TACGTTAATGTI’GTCAGGTCGAAAAATAAAATrGCAAATAGAAATn GTrCCTT TTTTATTGGTTTTTATTGGTGGGAAAAATATTCCCTCTAACTGCAAAAGGGTTAAT TATGTTAGAGGTAGAGTCGAC

[0372] * For certain depicted vectors, MSCV promoter is in bold. Beta chain is annotated using bold and italic text. Alpha chain is annotated using bold and underlined text. CD34-enrichment tag (Q tag) is annotated using italic and underlined text. CD8-alpha is in italic. CD8-beta is underlined.

[0373] Table 4

[0374] MAGEA4-286 MGTM codon optimized sequence (also known as “Comparator TCR”) Alpha chain:

[0375] TRAV5*01 / TRAJ3*01 / MGTM modified TRAC

[0376] Alpha chain DNA sequence ATGAAAACCTTCGCCGGCTTCAGCTTCCTGTTTCTGTGGCTCCAACTGGACTGCAT GAGCAGAGGCGAGGATGT'CGAGCAGTCACTGTTCCTGAGCGTCAGAGAAGGCGA CAGCAGCGTGATCAACTGCACTTATACCGACAGCTCCAGCACCTACCTTTATTG GTATAAGCAAGAGCCAGGCGCCGGACTCCAGCTGCTGACCTACATCTTCTCCAA CATGGACATGAAGCAGGACCAGCGGCTGACCGTCCTGCTGAACAAGAAGGACA AGCACCTGAGCCTGCGGATCGCCGATACACAGACAGGCGACTCAGCTATCTACTT CTGTGCAGAGTACAGCAGTGCTTCCAAGATAATCTTTGGATCAGGGACCAGA CTCAGCATCCGGCCAAACatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaag agcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtg ctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaa caacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacaga caccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcg gctgtggagcagc

[0377] Alpha chain protein sequence MKTFAGFSFLFLWLQLDCMSRGEDVEOSLFLSVREGDSSVINCTYTDSSSTYLYWYK OEPGAGLOLLTYIFSNMDMKODORLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEY SSASKIIFGSGTRLSIRPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksn savawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlwss (SEQ ID NO: 33)

[0378] Beta chain:

[0379] - 66 - FoleyHoagUS 13160800.1 TTC-020

[0380] TRBV 12-4*01 / TRBJ2- 1 *01 / MGTM modified TRBC

[0381] Beta chain DNA sequence ATGGGCAGCTGGACACTGTGTTGTGTGTCTCTGTGCATTCTGGTCGCTAAGCACA CTGATGCAGGCGTGATACAGAGCCCAAGGCATGAAGTCACCGAAATGGGACAAG AAGTCACCCTGCGTTGTAAACCCATTAGCGGCCACGACTATCTTTTCTGGTATC GCCAAACAATGATGAGGGGACTCGAGCTGTTGATATACTTTAACAACAATGTTC CGATCGATGACTCCGGGAIYK TGAGGACAGGTTTTCCGC AAAATGCCTAATG CCTCCTTCAGCACCCTCAAAATTCAACCCTCCGAGCCTCGGGATTCAGCTGTGTA CTTCTGTGCCAGC. AGAGCCAACACCGGCGAGCTGTTCTTCGGGCCAGGGACA CGGCTCACCGTGCTAGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcg cccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaaga ggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagact gagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagtttacggcctgagcgagaacgacgagtg gacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcc tatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctgg tgctgatggccatggtcaagcggaaggacttt

[0382] Beta chain protein sequence MGSWTLCCVSLCILVAKHTDAGVIOSPRHEVTEMGOEVTLRCKPISGHDYLFWYRQ TMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTEKIQPSEPRDSAVYFCAS RANTGELFFGPGTRLTVLEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvs tdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvl satilyeillgkatlyavlvsalvlmamvkrkdf

[0383] Complete Beta and Alpha ORF DNA Sequence ATGGGCAGCTGGACACTGTCTTTGTGTGTCTCTGTGCATTCTGGTCGCTAAGCACA CTGATGCAGGCGTGATACAGAGCCCAAGGCATGAAGTCACCGAAATGGGACAAG AAGTCACCCTGCGTTGTAAACCCATTAGCGGCCACGACTATCTTTTCTGGTATC GCCAAACAATGATGAGGGGACTCGAGCTGTTGATATACTTTAACAACAATGTTC CGATCGATGACTCCGGGATGCCTGAGGACAGGTTTTCCGCCAAAATGCCTAATG CCTCCTTCAGCACCCTCAAAATTCAACC TCCGAGC TCGGGArirAGCTGTGIA CTTCTGTGCCAGCAGAGCCAACACCGGCGAGCTGTTCTTCGGGCCAGGGACA CGGCTCACCGTGCTAGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcg cccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaaga ggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagact gagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtg gacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcc tatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctgg tgctgatggccatggtcaagcggaaggactttggcagcggc6iga.gccflaiaa.ggfccgg.ga.gc.ggt.gc.g< ca< actnagcct.gff gaaacaagccggcgacgffgaaga.e«gccccggacctATGAAAACCTTCGCCGGCTTCAGCTTCCTG TTTCTGTGGCTCCAACTGGACTGCATGAGCAGAGGCGAGGATGTCGAGCAGTCA CTGTTCCTGAGCGTCAGAGAAGGCGACAGCAGCGTGA’irAACTGCACTTATACC GACAGCTCCAGCACCTACCTTTATTGGTATAAGCAAGAGCCAGGCGCCGGACT CCAGCTGCTGACCTACATCTTCTCCAACATGGACATGAAGCAGGACCAGCGGC TGACCGTCCTGCTGAACAAGAAGGACAAGCACCTGAGCCTGCGGATCGCCGATA CACAGACAGGCGACTCAGCTATCTACTTCTGTGCAGAGTACAGCAGTGCTTCC AAGATAATCTTTGGATCAGGGACCAGACTCAGCATCCGGCCAAACatccagaaccccga ccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggactcgacagccagaccaacgtgag

[0384] - 67 - FoleyHoagUS 13160800.1 TTC-020

[0385] tcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtg gcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgac gtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcgga ttctgctgctgaaagtggccggcttcaatotgctgatgaccctgcggctgtggagcagc

[0386] Complete Beta and Alpha ORF Protein Sequence MGSWTLCCVSLCILVAKHTDAGVIOSPRHEVTEMGOEVTLRCKPISGHDYLFWYRQ TMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCAS RANTGELFFGPGTRLTVLEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvs tdpqplkeqpalndsryclssrlrvsatfwqnpmhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvl satilveillgkatlvavlvsalvlmamvkrkdf^ g / cr g' gn,»? A7Z / cg g<7veenp^pMKTFAGFSFLFLWLO EDCMSRGEDVEOSEFESVREGDSSVINCTYTDSSSTYEYWYKOEPGAGEQELTYIFS RPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsii pedtffps sdvpcd vkl veksfetdtnlnfqnllvivlri lllkvagfnllm tlrlwss

[0387] * Included in Tables 1-4 are peptide epitopes, as well as polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any sequences listed in Tables 1-4, or a portion thereof. Such polypeptides may have a function of the full-length peptide or polypeptide as described further herein.

[0388] * Included in Tables 1-4 are RNA nucleic acid molecules (e.g., thymines replaced with uredines), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any sequence listed in Tables 1-4, or a portion thereof. Such nucleic acid molecules can have a function of the full-length nucleic acid as described further herein.

[0389] In some embodiments, the binding proteins provided herein comprise a constant region that is chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, a human variable region may be chimerized with a murine constant region or a murine variable region may be humanized with a human constant region and / or human framework regions. In some embodiments, the constant regions may be mutated to modify functionality (e.g., introduction of non-naturally occurring cysteine substitutions in opposing residue locations in TCR alpha and beta chains to provide disulfide bonds useful for increasing affinity between the TCR alpha and beta chains). Similarly, mutations may be

[0390] - 68 - FoleyHoagUS 13160800.1 TTC-020

[0391] made in the transmembrane domain of the constant region to modify functionality (e.g., to increase hydrophobicity by introducing a non-naturally occurring substitution of a residue with a hydrophobic amino acid). In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof as compared to a reference CDR sequence. In some embodiments, mutations may be made to the constant region to increase cell surface expression.

[0392] In some embodiments, the binding proteins disclosed herein may be engineered protein scaffolds, an antibody or an anti gen -binding fragment thereof, TCR-mimic antibodies, and the like. Such binding moieties may be designed and / or generated against peptides and / or MHC-peptide complexes described herein using routine immunological methods, such as immunizing a host, obtaining antibody-producing cells and / or antibodies thereof, and generating hybridomas useful for producing monoclonal antibodies (e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. ChemBiol.

[0393] 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272; PCT Publ. Nos. WO 2007 / 143104, PCT / US86 / 02269, and WO 86 / 01533; U. S. Pat. No. 4,816,567; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. U. S. A. 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999- 1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.

[0394] 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; U. S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;

[0395] Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060. If desired, binding moieties may be isolated or purified using conventional procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC) (e.g., Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N. Y.).

[0396] The terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies and multi- specific antibodies, as well as - 69 - FoleyHoagUS 13160800.1 TTC-020

[0397] fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

[0398] In addition, intrabodies are well-known antigen-binding molecules having the characteristic of antibodies, but that are capable of being expressed within cells in order to bind and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods are well-known in the art for adapting antibodies to target (e.g., inhibit) intracellular moieties, such as the use of single-chain antibodies (scFvs), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and / or modulate intracellular localization, and the like. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and / or therapeutic purposes (e.g., as a gene therapy) (see, at least PCT Publ. Nos. WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U. S. Pat. No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer- Verlag pubis.); Kontennann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al.

[0399] (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0400] The term "‘antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically and / or selectively bind to an antigen (e.g., a peptide and / or an MHC-peptide complex described herein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “anti gen -binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by

[0401] - 70 - FoleyHoagUS 13160800.1 TTC-020

[0402] a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g.. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90:6444- 6448; Poljak et al. (1994) Structure 2:1121-1123).

[0403] Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, protein subunit peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab'), fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0404] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the invention bind specifically and / or selectively or substantially specifically and / or selectively to a peptide and / or an MHC-peptide complex described herein. The terms “monoclonal antibodies” and “monoclonal antibody

[0405] - 71 - FoleyHoagUS 13160800.1 TTC-020

[0406] composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

[0407] Similar to other binding moieties described herein, antibodies may also be “humanized,” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro / ex vivo or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, have been grafted onto human framework sequences.

[0408] In some embodiments, the binding proteins disclosed herein may comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding protein disclosed herein may comprise two polypeptide chains, each of which comprises a variable region comprising a CDR3 of a TCR alpha chain and a CDR3 of a TCR beta chain, or a CDR 1, CDR2, and CDR3 of both a TCR alpha chain and a TCR beta chain. In some embodiments, a binding protein comprises a single chain TCR (scTCR), which comprises both the TCR Va and TCR Vp domains, but only a single TCR constant domain (Ca or Cp). The term “chimeric antigen receptor” (CAR) refers to a fusion protein that is engineered to contain two or more naturally- occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs encompassed by the present invention may include an extracellular portion comprising an antigen-binding domain i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as an antibody or TCR, or an antigen binding domain derived or obtained from a killer immunoreceptor from an NK cell)

[0409] - 72 - FoleyHoagUS 13160800.1 TTC-020

[0410] linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co- stimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220, and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0411] In some embodiments, 1) the TCR alpha chain CDR, TCR Va domain, and / or TCR alpha chain is encoded by a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCR beta chain CDR, TCR Vp domain, and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2 and / or Table 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof as compared to the cognate reference CDR sequence listed in Table 2.

[0412] In some embodiments, the binding proteins (e.g., the TCR, antigen-binding fragment of a TCR, or chimeric antigen receptor (CAR)) disclosed herein is chimeric (e.g., comprises amino acid residues or motifs from more than one donor or species), humanized (e.g., comprises residues from a non-human organism that are altered or substituted so as to reduce the risk of immunogenicity in a human), or human.

[0413] Methods for producing engineered binding proteins, such as TCRs, CARs, and antigen-binding fragments thereof, are well-known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 46:3000; U. S. Pat. No. 6,410,319; U. S. Pat. No. 7,446,191; U. S. Pat. Publ. No.

[0414] 2010 / 065818; U. S. Pat. No. 8,822,647; PCT Publ. No. WO 2014 / 031687; U. S. Pat. No.

[0415] 7,514,537; and Brentjens et al. (2007) Clin. Cancer Res. 13:5426).

[0416] In some embodiments, the binding protein described herein is a TCR, or antigen-binding fragment thereof, expressed on a cell surface, wherein the cell surface-expressed TCR is capable of more efficiently associating with a CD3 protein as compared to endogenous TCR. A binding protein encompassed by the present invention, such as a TCR, when expressed on the surface of a cell like a T cell, may also have higher surface expression on the cell as compared to an endogenous binding protein, such as an endogenous TCR. In some embodiments, provided herein is a CAR, wherein the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0417] Also provided are modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) that may be prepared according to well-known methods using a binding

[0418] - 73 - FoleyHoagUS 13160800.1 TTC-020

[0419] protein having one or more of the Va and / or Vp sequences disclosed herein as starting material to engineer a modified binding protein that may have altered properties from the starting binding protein. A binding protein may be engineered by modifying one or more residues within one or both variable regions (i.e., Va and / or Vp), for example within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, a binding protein may be engineered by modifying residues within the constant region(s).

[0420] Another type of variable region modification is to mutate amino acid residues within the Va and / or Vp CDR1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis may be performed to introduce the mutation(s) and the effect on protein binding, or other functional property of interest, may be evaluated in in vitro, ex vivo, or in vivo assays as described herein and provided in the Examples. In some embodiments, conservative modifications (as discussed above) may be introduced. The mutations may be amino acid substitutions, additions or deletions. In some embodiments, the mutations are substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are modified.

[0421] In some embodiments, binding proteins (e.g., TCRs, antigen- binding fragments of TCRs, or CARs) described herein may possess one or more amino acid substitutions, deletions, or additions relative to a naturally occurring TCR. In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof as compared to the cognate reference CDR sequence listed in Table 2. Conservative substitutions of amino acids are well-known and may occur naturally or may be introduced when the binding protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al. supra).

[0422] - 74 - FoleyHoagUS 13160800.1 TTC-020

[0423] A variety of criteria known to the ordinarily skilled artisan indicate whether an amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids may be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GENEWORKS™, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).

[0424] In some embodiments, an encoded binding protein (e.g., TCR, antigen-binding fragment of a TCR, or CAR) may comprise a “signal peptide” (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. A signal peptide may be removed from the polypeptide during or once localization or secretion is completed.

[0425] Polypeptides that have a signal peptide are referred to herein as a “pre-protein” and polypeptides having their signal peptide removed are referred to herein as “mature” proteins or polypeptides. In some embodiments, a binding protein (e.g., TCR, antigen-binding fragment of a TCR, or CAR) described herein comprises a mature Va domain, a mature Vp domain, or both. In some embodiments, a binding protein (e.g., TCR, antigen-binding fragment of a TCR, or CAR) described herein comprises a mature TCR P-chain, a mature TCR (X-chain, or both.

[0426] - 75 - FoleyHoagUS 13160800.1 TTC-020

[0427] In some embodiments, the binding proteins are fusion proteins comprising: (a) an extracellular component comprising a TCR or antigen-binding fragment thereof; (b) an intracellular component comprising an effector domain or a functional portion thereof; and (c) a transmembrane domain connecting the extracellular and intracellular components. In some embodiments, the fusion protein is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising a MAGEA4 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule comprises an MHC alpha chain that is an HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of HLA-A*02:01.

[0428] As used herein, an “effector domain” or “immune effector domain” is an intracellular portion or domain of a fusion protein or receptor that can directly or indirectly promote an immune response in a cell when receiving an appropriate signal. In some embodiments, an effector domain is from an immune cell protein or portion thereof or immune cell protein complex that receives a signal when bound (e.g., CD3C), or when the immune cell protein or portion thereof or immune cell protein complex binds directly to a target molecule and triggers signal transduction from the effector domain in an immune cell.

[0429] An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an intracellular tyrosine-based activation motif (ITAM), such as those found in costimulatory molecules. Without wishing to be bound by theory, it is believed that IT AMs are useful for T cell activation following ligand engagement by a T cell receptor or by a fusion protein comprising a T cell effector domain. In some embodiments, the intracellular component or functional portion thereof comprises an ITAM. Exemplary immune effector domains include but are not limited to those from, CD3e, CD35, CD3i;, CD25, CD79A, CD79B, CARD11, DAP10, FcRcc, FcR£, FcRy, Fyn, HVEM, ICOS, Lek, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, an effector domain comprises a lymphocyte receptor signaling domain (e.g., CD3 or a functional portion or variant thereof7).

[0430] In further embodiments, the intracellular component of the fusion protein comprises a costimulatory domain or a functional portion thereof selected from CD27, CD28, 4- IBB (CD137), 0X40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CDlla / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, NKG2C, SLAMF7, NKp80,

[0431] - 76 - FoleyHoagUS 13160800.1 TTC-020

[0432] CD160, B7-H3, a ligand that binds (e.g., specifically and / or selectively) with CD83, or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 costimulatory domain or a functional portion or variant thereof (which may optionally include a LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB costimulatory domain or a functional portion or variant thereof, or both.

[0433] In some embodiments, an effector domain comprises a CD3e endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises a CD27 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises a CD28 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In still further embodiments, an effector domain comprises a 4- IBB endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises an 0X40 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof In further embodiments, an effector domain comprises a CD2 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises a CD5 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises an ICAM-1 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises a LFA-1 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, an effector domain comprises an ICOS endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof.

[0434] An extracellular component and an intracellular component encompassed by the present invention are connected by a transmembrane domain. A “transmembrane domain,” as used herein, is a portion of a transmembrane protein that can insert into or span a cell membrane. Transmembrane domains have a three-dimensional structure that is thermodynamically stable in a cell membrane and generally range in length from about 15 amino acids to about 30 amino acids. The structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain,

[0435] - 77 - FoleyHoagUS 13160800.1 TTC-020

[0436] a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0437] In some embodiments, the extracellular component of the fusion protein further comprises a linker disposed between the binding domain and the transmembrane domain. As used herein when referring to a component of a fusion protein that connects the binding and transmembrane domains, a “linker” may be an amino acid sequence having from about two amino acids to about 500 amino acids, which can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker. For example, a linker encompassed by the present invention can position the binding domain away from the surface of a host cell expressing the fusion protein to enable proper contact between the host cell and a target cell, antigen binding, and activation (Patel et al. (1999) Gene Therapy 6:412-419). Linker length may be varied to maximize antigen recognition based on the selected target molecule, selected binding epitope, or antigen binding domain size and affinity (see, e.g., Guest et al. (2005) Immunother.

[0438] 28:203-11 and PCT Publ. No. WO 2014 / 031687). Exemplary linkers include those having a glycine- serine amino acid chain having from one to about ten repeats of GlyxSery, wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0 (e.g., (Gly4Ser)2, (Gly4Ser)2, Gly4Ser, or a combination thereof, such as ((GlysSer^GlyzSer)).

[0439] A binding protein may be conjugated to an agent, such as a detection moiety, radiosensitizer, photosensitizer, and the like, and / or may be chemically modified as described above regarding peptides.

[0440] Binding proteins encompassed by the present invention may, in some embodiments, be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or a label. The affinity tag may be selected from the group consisting of Glutathione-S-Transferase (GST), calmodulin binding protein

[0441] (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of an inflammatory agent, an anti-inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioactive isotope, or an antibody such as a single-chain Fv.

[0442] A binding protein may be conjugated to an agent used in imaging, research, therapeutics, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, a binding protein may be conjugated to or fused with detectable agents, such as a fluorophore, a near-infrared dye, a contrast agent, a - 78 - FoleyHoagUS 13160800.1 TTC-020

[0443] nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties may be linked to a binding protein. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dyes are not easily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent emitting electromagnetic radiation at a wavelength between 650 nm and 4000 nm, such emissions being used to detect such agent Non-limiting examples of fluorescent dyes that may be used as a conjugating molecule include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional, non-limiting examples of fluorescent dyes for use as a conjugating molecule in accordance with present invention include acradine orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivative thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO® dye and any derivative thereof, auraminerhodamine stain and any derivative thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivative thereof, 1-chloro-9,10- bis(phenylethynyl)anthracene and any derivative thereof, DAPI, DiOC6, DyLight® Fluors® and any derivative thereof, epicocconone, ethidium bromide, FlAsH-EDT2®, Fluo dye and any derivative thereof, FluoProbe® and any derivative thereof, fluorescein and any derivative thereof, Fura® and any derivative thereof, GelGreen® and any derivative thereof, GelRed® and any derivative thereof, fluorescent proteins and any derivative thereof, m isoform proteins and any derivative thereof such as for example mCherry, heptamethine dye and any derivative thereof, hoeschst stain, iminocoumarin, Indian yellow, indo-1 and any derivative thereof, laurdan, lucifer yellow and any derivative thereof, luciferin and any derivative thereof, luciferase and any derivative thereof, mercocyanine and any derivative thereof, nile dyes and any derivative thereof, perylene, phloxine, phyco dye and any derivative thereof, - 79 - FoleyHoagUS 13160800.1 TTC-020

[0444] propium iodide, pyranine, rhodamine and any derivative thereof, ribogreen, RoGFP, rubrene, stilbene and any derivative thereof, sulforhodamine and any derivative thereof, SYBR and any derivative thereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris, Texas Red, Titan Yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4', 5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green™ dyes (e.g., Oregon Green™ 488, 500, 514., etc.), Texas Red®, Texas Red®-X, SPECTRUM RED®, SPECTRUM GREEN®, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY- 5.5, etc.), Alexa Fluor® dyes (e.g., Alexa Fluor® 350, 488, 532, 546, 568, 594, 633, 660, 680, etc.), BODIPY® dyes (e.g., BODIPY® FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40™, IRD700™, IRD800™, etc. ), and the like. Additional suitable detectable agents are well-known in the art (e.g., PCT Publ. No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0445] Binding proteins may be conjugated to a radiosensitizer or photosensitizer. Examples of radiosensitizers include but are not limited to: ABT-263, ABT- 199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include but are not limited to: fluorescent molecules or beads that generate heat when illuminated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins,

[0446] - 80 - FoleyHoagUS 13160800.1 TTC-020

[0447] chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5- ami no levulinic acid. Advantageously, this approach allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent e.g., drug) and electromagnetic energy (e.g., radiation or light) concurrently. In some embodiments, the binding protein is fused with, or covalently or non-covalently linked to the agent, for example, directly or via a linker.

[0448] In some embodiments, the binding protein may be chemically modified. For example, a binding protein may be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation sites, pH, function, and the like. N-methylation is one example of methylation that can occur in a binding protein encompassed by the present invention. In some embodiments, a binding protein may be modified by methylation on free amines such as by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0449] A chemical modification may comprise a polymer, a polyether, polyethylene glycol, a biopolymer, a zwitterionic polymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain such as palmitate or myristolate, or albumin. The chemical modification of a binding protein with an Fc region may be a fusion Fc-protein. A polyamino acid may include, for example, a poly amino acid sequence with repeated single amino acids (e.g., poly glycine), and a poly amino acid sequence with mixed poly amino acid sequences that may or may not follow a pattern, or any combination of the foregoing.

[0450] In some embodiments, the binding proteins encompassed by the present invention may be modified. In some embodiments, the modifications having substantial or significant sequence identity to a parent binding protein to generate a functional variant that maintains one or more biophysical and / or biological activities of the parent binding protein (e.g., maintain pMHC binding specificity). In some embodiments, the mutation is a conservative amino acid substitution.

[0451] In some embodiments, binding proteins encompassed by the present invention may comprise synthetic amino acids in place of one or more naturally-occurring amino acids.

[0452] - 81 - FoleyHoagUS 13160800.1 TTC-020

[0453] Such synthetic amino acids are well-known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, P-phenylserine P~ hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N'-dibenzyl- lysine, 6-hydroxylysine, ornithine, a- aminocyclopentane carboxylic acid, oc-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornanej-carboxylic acid, aj-diaminobutyric acid,, P-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0454] Binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via a disulfide bridge), or converted into an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0455] In some embodiments, the attachment of a hydrophobic moiety, such as to the N-terminus, the C-terminus, or an internal amino acid, may be used to extend half-life of a peptide encompassed by the present invention. In other embodiments, a binding protein may include post-translational modifications (e.g., methylation and / or amidation), which can affect, for example, serum half-life. In some embodiments, simple carbon chains (e.g., by myristoylation and / or palmitylation) may be conjugated to the binding proteins. In some embodiments, the simple carbon chains may render the binding proteins easily separable from the unconjugated material. For example, methods that may be used to separate the binding proteins from the unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography. The lipophilic moieties can extend half-life through reversible binding to serum albumin. The conjugated moieties may be lipophilic moieties that extend half-life of the peptides through reversible binding to serum albumin. In some embodiments, the lipophilic moiety may be cholesterol or a cholesterol derivative, including cholestenes, cholestanes, cholestadienes and oxysterols. In some embodiments, the binding proteins may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, a binding protein may be coupled (e.g., conjugated) to a half¬ life modifying agent. Examples of half-life modifying agents include but are not limited to: a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a - 82 - FoleyHoagUS 13160800.1 TTC-020

[0456] water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, or a molecule that binds to albumin. In some embodiments, a spacer or linker may be coupled to a binding protein, such as 1, 2, 3, 4, or more amino acid residues that serve as a spacer or linker in order to facilitate conjugation or fusion to another molecule, as well as to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, binding proteins may be conjugated to other moieties that, for example, can modify or effect changes to the properties of the binding proteins.

[0457] A binding protein may be produced recombinantly or synthetically, such as by solidphase peptide synthesis or solution-phase peptide synthesis. Polypeptide synthesis may be performed by known synthetic methods, such as using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. Polypeptide fragments may be joined together enzymatically or synthetically.

[0458] In an aspect encompassed by the present invention, provided herein are methods of producing a binding protein described herein, comprising the steps of: (i) culturing a transformed host cell which has been transformed by a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of said binding protein; and (ii) recovering the expressed binding protein.

[0459] Methods useful for isolating and purifying recombinantly produced binding protein, by way of example, may include obtaining supernatants from suitable host cell / vector systems that secrete the binding protein into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions.

[0460] Purification of the binding protein may be performed according to methods described herein and known in the art.

[0461] In any of the herein disclosed embodiments, the encoded binding protein is capable of bind to a peptide-MHC (pMHC) complex comprising a MAGEA4 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the

[0462] - 83 - FoleyHoagUS 13160800.1 TTC-020

[0463] MHC molecule comprises an MHC alpha chain that is an HLA serotype HLA-A*02. In some embodiments, the HLA allele is HLA-A*02:01.

[0464] A variety of assays are well-known for assessing binding affinity and / or determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand e.g., peptide antigen-MHC complex). It is within the level of a skilled artisan to determine the binding affinity of a binding protein for a target, such as a T cell peptide epitope of a target polypeptide, such as by using any of a number of binding assays that are well-known in the art. For example, in some embodiments, a Biacore™ machine may be used to determine the binding constant of a complex between two proteins. The dissociation constant (KD) for the complex may be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoas says (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, LTV absoiption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore™) analysis (see, e.g., Scatchard et al. (1949) Ann. N. Y. Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U. S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing and other methods for detection of expressed nucleic acids. In one example, apparent affinity for a target is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled multimers, such as MHC-antigen tetramers. In one representative example, apparent KD of a binding protein is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.

[0465] II. Nucleic Acids and Vectors

[0466] In an aspect encompassed by the present invention, provided herein are nucleic acid molecules that encode proteins described herein, such as binding proteins (e.g., TCRs, antigen-binding fragments of the TCRs, CARs, and the like), peptides, fragments, and the like, as described herein.

[0467] In some embodiments, the nucleic acid molecule hybridizes, under stringent conditions, with the complement of a sequence with at least about at least about 80%, 81%,

[0468] - 84 - FoleyHoagUS 13160800.1 TTC-020

[0469] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity, such as over the full length, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0470] In some embodiments, the nucleic acid molecule hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Tables 1-3.

[0471] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Tables 1-3.

[0472] In some embodiments, the nucleic acid sequence encodes a MAGEA4 immunogenic peptides described herein.

[0473] In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR a-chain CDR set forth in Table 2. In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding a TCR Va domain having an amino acid sequence that is at least about at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR Va domain sequence set forth in Table 2. In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding a TCR a-chain having an amino acid sequence that is at least about at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR a-chain sequence set forth in Table 2.

[0474] In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR p- chain CDR set forth in Table 2. In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding a TCR Vp domain having an amino acid sequence that is at least about at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR Vp domain sequence set forth in Table 2. In some embodiments, the nucleic acids comprise (e.g., comprise, consist essentially of, or consist of) a nucleotide sequence encoding a TCR β-chain having an amino acid sequence that is at least

[0475] - 85 - FoleyHoagUS 13160800.1 TTC-020

[0476] about at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a TCR β-chain sequence set forth in Table 2.

[0477] The term “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which may be single¬ stranded or double- stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which may contain natural, non-natural or altered nucleotides, and which may contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In an embodiment, the nucleic acid comprises complementary DNA (cDNA).

[0478] In some embodiments, the nucleic acids encompassed by the present invention are recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that may replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication may be in vitro, ex vivo, or in vivo replication.

[0479] The nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook ei al. supra. For example, a nucleic acid may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).

[0480] Examples of modified nucleotides that may be used to generate the nucleic acids include, but are not limited to, 5-fiuorouracil, 5-bromouracil, 5 -chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N°-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-

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[0482] oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids encompassed by the present invention can be purchased from companies, such as Integrated DNA Technologies (Coralville, IA).

[0483] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound to a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency.

[0484] Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell, such as a T cell).

[0485] The present invention also provides a nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0486] The nucleotide sequence which hybridizes under stringent conditions may hybridize under high stringency conditions. By “high stringency conditions” is meant that the nucleotide sequence specifically and / or selectively hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the inventive TCRs. It is generally appreciated that conditions may be rendered more stringent by the addition of increasing amounts of formamide.

[0487] - 87 - FoleyHoagUS 13160800.1 TTC-020

[0488] The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any of the nucleic acids described herein.

[0489] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0490] The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Thus, a further object encompassed by the present invention relates to a vector comprising a nucleic acid encompassed by the present invention.

[0491] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason J O et al. 1985) and enhancer (Gillies S D et al. 1983) of immunoglobulin H chain and the like.

[0492] Any expression vector for animal cell may be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other representative examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Representative examples of viral vector include adenoviral, retroviral, lentiviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses are well-known in the art and may be found, for instance, in PCT Publ. WO 95 / 14785, PCT. Publ. WO 96 / 22378, U. S. Pat. No. 5,882,877, U. S. Pat. No. 6,013,516, U. S. Pat. No. 4,861,719, U. S. Pat. No. 5,278,056. and PCT Publ. WO 94 / 19478.

[0493] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or a polypeptide described herein or a

[0494] - 88 - FoleyHoagUS 13160800.1 TTC-020

[0495] fragment thereof. In some embodiments, the nucleic acid includes regulatory elements necessary for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers may be included. These elements may be operably linked to a sequence that encodes the binding protein, polypeptide or fragment thereof.

[0496] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8a, CD8P, a dominant negative TGF|3 receptor (e.g., a DN-TGFβRII), selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In certain embodiments, the nucleic acid sequence encoding CD8a, CD8|3, the DN-TGFpR, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag (e.g., a CD34 enrichment tag). In specific embodiments, a nucleic acid sequence described herein, such as a nucleic acid sequence encoding a TCRa, TCRp. CD8a, CD8|3, DN-TGF(3R, and / or selectable protein marker are interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide, such as P2A, E2A, F2A or T2A, etc.

[0497] In some embodiments, the expression vector provided herein comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any of the nucleic acids set forth in Tables 1-3.

[0498] As described above, representative examples of promoters include, but are not limited to, promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein. Examples of suitable polyadenylation signals include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.

[0499] In addition to the regulatory elements required for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include the promoters described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.

[0500] In some embodiments, the nucleic acid may be operably incorporated in a carrier or delivery vector as described further below. Useful delivery vectors include but are not

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[0502] limited to biodegradable microcapsules, immuno- stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers such as viruses or bacteria.

[0503] In some embodiments, the vector is a viral vector, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses. For example, a lentivirus vector may be used to infect T cells.

[0504] In some embodiments, the recombinant expression vector is capable of delivering a polynucleotide to an appropriate host cell, for example, a T cell or an antigen-presenting cell, i.e., a cell that displays a peptide / MHC complex on its cell surface (e.g., a dendritic cell) and lacks CD8. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell may be a CD4+T cell, a CD8+T cell, a CD4 / CD8 double negative T cell, a gd T cell, a natural killer cell, a dendritic cell, or any combination thereof. In some embodiments, wherein a T cell is the host, the T cell may be naive, a central memory T cell, an effector memory T cell, or any combination thereof The recombinant expression vectors may therefore also include, for example, lymphoid tissue- specific transcriptional regulatory elements (TREs), such as a B lymphocyte, T lymphocyte, or dendritic cell specific TREs. Lymphoid tissue specific TREs are known in the art (see, e.g., Thompson et al. (1992) Mol. Cell. Biol. 72:1043, Todd et al. (1993) J. Exp. Med. 777:1663, and Penix et al. (1993), / . Exp. Med. 775:1483).

[0505] In some embodiments, a recombinant expression vector comprises a nucleotide sequence encoding a TCR a chain, a TCR P chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector comprises a nucleotide sequence encoding the full-length TCR alpha and TCR beta chains of the binding protein with a linker positioned between them, wherein the nucleotide sequence encoding the beta chain is positioned 5' of the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequence encodes the full-length TCR alpha and TCR beta chains with a linker positioned between them, wherein the nucleotide sequence encoding the TCR beta chain is positioned 3 ' of the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full-length TCR alpha and / or TCR beta chains are replaced with fragments thereof.

[0506] As described further below, another aspect encompassed by the present invention relates to a cell which has been transfected, infected or transformed by a nucleic acid and / or a vector in accordance with the present invention. A host cell may include any individual cell

[0507] - 90 - FoleyHoagUS 13160800.1 TTC-020

[0508] or cell culture which may receive a vector or the incorporation of nucleic acids and / or proteins, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory)). The term “transformation” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been “transformed.”

[0509] The nucleic acids encompassed by the present invention may be used to produce a recombinant polypeptide encompassed by the present invention in a suitable expression system. The term “expression system” means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[0510] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) is defective (Urlaub G et al (1980), rat YB2 / 3HL. P2. G11.16Ag.2O cell (ATCC CRL 1662, hereinafter referred to as “YB2 / 0 cell”), and the like. In some embodiments, the YB2 / 0 cell is used since ADCC activity of chimeric or humanized binding proteins is enhanced when expressed in this cell.

[0511] The present invention also encompasses methods of producing a recombinant host cell expressing binding proteins, peptides and fragments thereof encompassed by the present invention, said method comprising the steps consisting of (i) introducing in vitro or ex vivo a - 91 - FoleyHoagUS 13160800.1 TTC-020

[0512] recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express said binding proteins, peptides and fragments thereof. Such recombinant host cells may be used for the diagnostic, prognostic, and / or therapeutic method encompassed by the present invention.

[0513] In another aspect, as described above, the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides of this embodiment may be used for isolating, detecting, and / or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides encompassed by the present invention may be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library. In some embodiments, the cDNA library comprises at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range in between, inclusive, such as at least about 80%-100%, full-length sequences. The cDNA libraries may be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions may optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and may be employed to identify orthologous or paralogous sequences. Optionally, polynucleotides encompassed by the present invention will encode at least a portion of a binding protein encoded by the polynucleotides described herein. The polynucleotides encompassed by the present invention embrace nucleic acid sequences that may be employed for selective hybridization to a polynucleotide encoding a binding protein encompassed by the present invention (see, e.g., Ausubel, supra and Colligan, supra).

[0514] III. Engineered cells

[0515] In an aspect encompassed by the present invention, provided herein are host cells that express proteins described herein, such as the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, or fusion proteins comprising a TCR and an effector domain)

[0516] - 92 - FoleyHoagUS 13160800.1 TTC-020

[0517] described herein. In some embodiments, the host cells comprise the nucleic acids or vectors described herein.

[0518] In some embodiments, a polynucleotide encoding a binding protein is used to transform, transfect, or transduce a host cell (e.g., a T cell) for use in adoptive transfer therapy. Advances in nucleic acid sequencing and particular TCR sequencing have been described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med.

[0519] 2:47ra64, Robins et al. (2011) J. 1mm. Meth., and Warren et al. (2011) Genome Res. 21:790) and may be employed in the course of practicing embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with desired nucleic acids are well-known in the art (e.g., U. S. Pat. Publ. No. US 2004 / 0087025) as have adoptive transfer procedures using T cells of desired antigen-specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240, Dossett et al. (2009) Mol. Ther. 17:742, Till et al. (2008) Blood 112:2261, Wang et al. (2007) Hum. Gene Then 18:112, Kuball et al. (2007) Blood 109:2331, U. S. Pat. Publ. 2011 / 0243972, U. S. Pat. Publ. 2011 / 0189141, and Leen et al. (2007) Ann. Rev. Immunol. 25:243).

[0520] Any suitable immune cell may be modified to include a heterologous polynucleotide encompassed by the present invention, including, for example, a T cell, a NK cell, or a NK-T cell. In some embodiments, the cell may be a primary cell or a cell of a cell line. In some embodiments, a modified immune cell comprises a CD4+T cell, a CD8+T cell, or both. For purposes herein, the T cell may be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell may be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells may also be enriched for or purified. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a T cell isolated from a human. The T cell may be any type of T cell and may be of any developmental stage, including but not limited to, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, CD4+ / CD8+double positive T cells, CD4+helper T cells, e.g., Thl and Th2 cells, CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0521] Any appropriate method may be used to transfect or transduce the cells (e.g., T cells), or to administer the nucleotide sequences or compositions encompassed by methods described herein. Methods for delivering polynucleotides to host cells include, for example,

[0522] - 93 - FoleyHoagUS 13160800.1 TTC-020

[0523] use of cationic polymers, lipid-like molecules, and certain commercial products such as, for example, in vivo-jetPEI®. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like. Still further methods of transfecting or transducing host cells employ vectors, described in further detail herein.

[0524] Modified immune cells as described herein may be functionally characterized using methodologies for assaying T cell activity, including determination of T cell binding, activation or induction and also including determination of T cell responses that are antigenspecific. Examples include determination of T cell proliferation, T cell cytokine release, antigen- specific T cell stimulation, MHC restricted T cell stimulation, CTL activity (e.g., by detecting51Cr release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions.

[0525] Procedures for performing these and similar assays may be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998), as well as Current Protocols in Immunology, Weir, (1986) Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) (1979) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA; Green and Reed (1998) Science 281:1309, and references cited therein.

[0526] In some embodiments, apparent affinity for a binding protein, such as a TCR or antigen-binding portion thereof, may be measured by assessing binding to various concentrations of MHC multimers. “MHC-peptide multimer staining” refers to an assay used to detect antigen-specific T cells, which, in some embodiments, features a tetramer of MHC molecules, each comprising an identical peptide having an amino acid sequence that is cognate (e.g., identical or related to) at least one antigen (e.g., a MAGEA4 immunogenic peptide), wherein the complex is capable of binding to a binding protein, such as a TCR or antigen-binding portion thereof, that recognizes the cognate antigen. Each of the MHC molecules may be tagged with a biotin molecule. Biotinylated MHC / peptides may be multimerized (e.g., tetramerized) by the addition of streptavidin, which may be fluorescently labeled.

[0527] The multimer may be detected by flow cytometry via the fluorescent label. In some embodiments, a pMHC multimer assay is used to detect or select enhanced affinity binding protein, such as a TCR or antigen-binding portion thereof, encompassed by the present invention. In some examples, apparent KD of a binding protein, such as a TCR or antigen- - 94 - FoleyHoagUS 13160800.1 TTC-020

[0528] binding portion thereof, is measured using 2-fold dilutions of labeled multimers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.

[0529] Levels of cytokines may be determined using methods described herein, such as ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry).

[0530] Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and / or cell viability, such as by incorporation of tritiated thymidine or non¬ radioactive assays, such as MTT assays and the like. The effect of an immunogen described herein on the balance between a Thl immune response and a Th2 immune response may be examined, for example, by determining levels of Thl cytokines, such as IFN-g, IL- 12, IL-2, and TNF-b, and Type 2 cytokines, such as IL-4, IL-5, IL-9, IL- 10, and IL-13.

[0531] A host cell encompassed by the present invention may comprise a single polynucleotide that encodes a binding protein as described herein, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or may be contained on two or more nucleic acid molecules.

[0532] Moreover, as described further below and in the working examples, a host cell encompassed by the present invention may encode and / or express useful accessory proteins in addition to a binding protein as described herein, either on the same polynucleotide or a different polynucleotide as the binding protein or components thereof. For example, the host cell may encode and / or express CD8a, CD8β, a DN-TGFβR (e.g., a DN-TGFβRII), and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR.

[0533] In some embodiments, a polynucleotide encoding two or more components or portions of a binding protein encompassed by the present invention comprises the two or more coding sequences operatively associated in a single open reading frame. Such an arrangement can advantageously allow coordinated expression of desired gene products, such as, for example, contemporaneous expression of alpha- and beta-chains of a TCR, such that they are produced in about a 1:1 ratio. In some embodiments, two or more substituent gene

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[0535] products of a binding protein encompassed by the present invention, such as a TCR (e.g., alpha- and beta-chains) or CAR, are expressed as separate molecules and associate posttranslation ally. In further embodiments, two or more substituent gene products of a binding protein encompassed by the present invention are expressed as a single peptide with the parts separated by a cleavable or removable segment. For instance, self-cleaving peptides useful for expression of separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, a porcine tescho virus- 1 2 A (P2A) peptide, a thosea asigna virus 2A (T2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide, and a foot-and-mouth disease virus 2A (F2A) peptide.

[0536] In some embodiments, a binding protein encompassed by the present invention comprises one or more junction amino acids. “Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of a construct that encodes a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein encompassed by the present invention (e.g., a P2A peptide disposed between a TCR a-chain and a TCR β-chain, the self-cleavage of which can leave one or more junction amino acids in the a-chain, the TCR β-chain, or both).

[0537] Engineered immune cells encompassed by the present invention may be administered as therapies for, e.g., a disorder characterized by MAGEA4 expression (such as a non-malignant disorder, a hyperproliferative disorder, or a relapse of a hyperproliferative disorder characterized by MAGEA4 expression). In some circumstances, it may be desirable to reduce or stop the activity associated with a cellular immunotherapy. Thus, in some embodiments, an engineered immune cell encompassed by the present invention comprises a heterologous polynucleotide encoding a binding protein and an accessory protein, such as a safety switch protein, which can be targeted using a cognate drug or other compound to selectively modulate the activity (e.g., lessen or ablate) of such cells when desirable. Safety switch proteins used in this regard include, for example, a truncated EGF receptor polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for - 96 - FoleyHoagUS 13160800.1 TTC-020

[0538] pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al. (2011) Blood 118:1255-1263), a caspase polypeptide (e.g., iCasp9; Straathof et al. (2005) Blood 105:4247-4254. Di Stasi et al. (2011) N. Engl. J. Med.

[0539] 365:1673-1683, Zhou and Brenner (2016) Hematol. pii: S0301-472X:30513-30516), RQR8 (Philip et al. (2014) Blood 124:1277-1287), and a human c-myc protein tag (Kieback et al. (2008) Proc. Natl. Acad. Sci. USA 105:623-628).

[0540] Other accessory components useful for therapeutic cells comprise a tag or selection marker (e.g., a CD34 enrichment tag) that allows the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells having desired characteristics (e.g., an antigen-specific TCR and a safety switch protein) may be sorted away from unmarked cells in a sample and more efficiently activated and expanded for inclusion in a therapeutic product of desired purity.

[0541] As used herein, the term “selection marker” comprises a nucleic acid construct that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker. For example, RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16 amino acid CD34 minimal epitope. In some embodiments, such as certain embodiments provided in the examples herein, the CD34 minimal epitope is incorporated at the amino terminal position of the CD8 stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence may be combined with a target epitope for CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al. 2014). This construct allows for the selection of immune cells expressing the construct, with for example, CD34-specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (e.g., Philip et al. (2014) Blood 124:1277-1287, U. S. Pat. Publ. 2015-0093401, and U. S. Pat. Publ. 2018-0051089).

[0542] Further exemplary selection markers include several truncated type I transmembrane proteins normally not expressed on T cells: the truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (e.g., Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Mavilio et al. (1994) Blood 83:1988-1997, and Fehse et al. (2000) Mol. Ther. 7:448- 456). A particularly attractive feature of CD19 and CD34 is the availability of the off-the-shelf Miltenyi CliniMACs™ selection system that can target these markers for clinical-grade sorting. However, CD 19 and CD34 are relatively large surface proteins that may tax the - 97 - FoleyHoagUS 13160800.1 TTC-020

[0543] vector packaging capacity and transcriptional efficiency of an integrating vector. Surface markers containing the extracellular, non-signaling domains or various proteins (e.g., CD19, CD 34, LNGFR, etc.) also may be employed. Any selection marker may be employed and should be acceptable for good manufacturing practices. In some embodiments, selection markers are expressed with a polynucleotide that encodes a gene product of interest (e.g., a binding protein encompassed by the present invention, such as a TCR or CAR, or antigen-binding fragment thereof). Further examples of selection markers include, for example, reporters such as GFP, EGFP, P-gal or chloramphenicol acetyltransferase (CAT). In some embodiments, a selection marker, such as, for example, CD34 is expressed by a cell and the CD34 may be used to select enrich for, or isolate (e.g., by immunomagnetic selection) the transduced cells of interest for use in the methods described herein. As used herein, a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or other antigen recognition moiety that binds to CD34.

[0544] In some embodiments, a selection marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof’.

[0545] By way of background, inclusion of CD4+T cells in an immunotherapy cell product can provide antigen-induced IL-2 secretion and augment persistence and function of transferred cytotoxic CD8+T cells (e.g., Kennedy et al. (2008) Immunol. Rev. 222:129 and Nakanishi et al. Nature (2009) 462:510). In some embodiments, a class I-restricted TCR in CD4+T cells may require the transfer of a CD8 co-receptor to enhance sensitivity of the TCR to class I HLA peptide complexes. CD4 co-receptors differ in structure to CD8 and cannot effectively substitute for CD8 co-receptors (e.g., Stone & Kranz. (2013) Front. Immunol. 4:244 and Cole et al. (2012) Immunology 137:139). Thus, another accessory protein for use in the compositions and methods encompassed by the present invention comprises a CD8 coreceptor or component thereof. Engineered immune cells comprising a heterologous polynucleotide encoding a binding protein encompassed by the present invention may, in some embodiments, further comprise a heterologous polynucleotide encoding a CD8 co¬ receptor protein, or a beta-chain or alpha-chain component thereof.

[0546] A host cell may be efficiently transduced to contain, and may efficiently express, a single polynucleotide that encodes the binding protein, safety switch protein, selection marker, and CDS co-receptor protein.

[0547] In one embodiment, the host cell encompassed by the present invention further includes a nucleic acid encoding a co-stimulatory molecule, such that the modified T cell - 98 - FoleyHoagUS 13160800.1 TTC-020

[0548] expresses the co-stimulatory molecule. In some embodiments, the co- stimulatory domain is selected from CD3, CD27, CD28, CD83, CD86. CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

[0549] In any of the foregoing embodiments, a host cell that express the binding protein described herein may be a universal immune cell. A “universal immune cell” comprises an immune cell that has been modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide product selected from PD-1, LAG-3, CTLA4, TIM3, TIGIT, or other immune checkpoint, an HLA molecule, a TCR molecule, or any combination thereof. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, TIGIT), or may interfere with the binding activity of a heterologously expressed binding protein encompassed by the present invention (e.g., an endogenous TCR that binds a non-MAGEA4 antigen and interferes with the modified immune cell binding to a target cell that expresses a MAGEA4 antigen such as a MAGEA4 immunogenic peptide in the context of an MHC molecule. Further, endogenous proteins (e.g., immune cell proteins, such as an HLA allele) expressed on a donor immune cell may be recognized as foreign by an allogeneic host, which may result in elimination or suppression of the modified donor immune cell by the allogeneic host.

[0550] Accordingly, decreasing or eliminating expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of the modified immune cells in an autologous or allogeneic host setting, and allows universal administration of the cells (e.g., to any recipient regardless of HLA type). In some embodiments, cells in accordance with the present invention are syngeneic, meaning that they are genetically identical or sufficiently identical and immunologically compatible as to allow for transplantation. In some embodiments, a universal immune cell is a donor cell (e.g., allogeneic) or an autologous cell. In some embodiments, a modified immune cell (e.g., a universal immune cell) encompassed by the present invention comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT,, or other immune checkpoint, an HLA component (e.g., a gene that encodes an α1 macroglobulin, an α2 macroglobulin, an α3 macroglobulin, a β1 microglobulin, or a β2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai et al. (2016) Nature Sci. Rep. 6:21757; Torikai et al. (2012) Blood 119:5697; and Torikai et al. (2013) Blood 122:1341, which also provide

[0551] - 99 - FoleyHoagUS 13160800.1 TTC-020

[0552] representative, exemplary gene editing techniques, compositions, and adoptive cell therapies useful according to the present invention).

[0553] As used herein, the term “chromosomal gene knockout” refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout may include, for example, introduced nonsense mutations (including the formation of premature stop codons), mis sense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0554] In some embodiments, a chromosomal gene knock-out or gene knock-in may be made by chromosomal editing of a host cell. Chromosomal editing may be performed using, for example, endonucleases. As used herein “endonuclease” refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain. In some embodiments, an endonuclease is capable of cleaving a targeted gene thereby inactivating or “knocking out” the targeted gene. An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. The nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule may be used for a donor gene “knock-in”, for target gene “knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event. NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide. NHEJ may be used to “knock-out” a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0555] As used herein, a “zinc finger nuclease” (ZFN) refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues may be changed to alter triplet sequence specificity (e.g., Desjarlais et al. (1993) Proc. Natl. Acad. Sci. 90:2256-2260 and Wolfe et al. (1999) J. Mol. Biol. 285:1917-1934). Multiple zinc finger motifs may be linked in tandem to create binding specificity to desired DNA sequences, such as regions having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome,

[0556] - 100 - FoleyHoagUS 13160800.1 TTC-020

[0557] and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair. Alternatively, a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In some embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.

[0558] As used herein, a “transcription activator-like effector nuclease” (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA cleavage domain, such as a Fokl endonuclease. A “TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains / units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids. The TALE repeat domains are involved in binding of the TALE to a target DNA sequence. The divergent amino acid residues, referred to as the repeat variable diresidue (RVD), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histidine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also well-known in the art (e.g., U. S. Pat. Publ. No. US 2011 / 0301073, which atypical RVDs are incorporated by reference herein in their entirety). TALENs may be used to direct site-specific double-strand breaks (DSB) in the genome of T cells. Non-homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene. In some embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.

[0559] As used herein, a “clustered regularly interspaced short palindromic repeats / Cas” (CRISPR / Cas) nuclease system refers to a system that employs a CRISPR RN (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence.

[0560] CRISPR / Cas systems are classified into three types (i.e., type I, type II, and type III) based on - 101 - FoleyHoagUS 13160800.1 TTC-020

[0561] the sequence and structure of the Cas nucleases. The crRNA-guided surveillance complexes in types I and III need multiple Cas subunits. Type II system, the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA comprises a duplex forming region. A crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base¬ pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM. Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions which disrupt expression of the targeted locus. Alternatively, a transgene with homologous flanking sequences may be introduced at the site of DSB via homology directed repair. The crRNA and tracrRNA may be engineered into a single guide RNA (sgRNA or gRNA) (e.g., Jinek et al. (2012) Science 337:816-821). Further, the region of the guide RNA complementary to the target site may be altered or programed to target a desired sequence (Xie et al. (2014) PLOS One 9:el00448, U. S. Pat. Publ. No. US 2014 / 0068797, U. S. Pat. Publ. No. US 2014 / 0186843, U. S. Pat. No. 8,697,359, and PCT Publ. No. WO 2015 / 071474). In some embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR / Cas nuclease system.

[0562] Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al. (2017) Clin. Cancer Res. 23:2255-2266, which provides representative, exemplary gRNAs, CAS9 DNAs, vectors, and gene knockout techniques.

[0563] As used herein, a “meganuclease,” also referred to as a “homing endonuclease,” refers to an endodeoxyribonuclease characterized by a large recognition site (double stranded DNA sequences of about 12 to about 40 base pairs). Meganucleases may be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, Rl-Sce, I-ScelV, I-Csml, I-Panl, I-Scell, I-Ppol, 1-SceIII, I-Crel, I-Tevl, I-TevII and I-TevIII, whose recognition sequences are well-known (e.g., U. S. Pat. Nos. 5,420,032 and 6,833,252, Belfort et al. (1997) Nucl. Acids Res. 25:3379-3388, Dujon et al. (1989) Gene 52:115-118, Perler et al. (1994) Nucl. Acids Res. 22:1125-1127, Jasin (1996) Trends Genet. 72:224-228, Gimble et al. (1996) J. Mol. Biol. 263:163-180, and Argast et al. (1998) J. Mol. Biol. 280:345-353).

[0564] - 102 - FoleyHoagUS 13160800.1 TTC-020

[0565] In some embodiments, naturally-occurring meganucleases may be used to promote site-specific genome modification of a target of interest, such as an immune checkpoint, an HLA-encoding gene, or a TCR component-encoding gene.

[0566] In other embodiments, an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al. (2005) Nat. Biotechnol. 23:961-13, Sussman et al. (2004) J. Mol. Biol. 342:31-41, Epinat et al. (2003) Nucl. Acids Res. 37:2952-2962, Chevalier et al. (2002) Mol. Cell 70:895-905, Ashworth et al. (2006) Nature 441:656-659, Paques et al. (2007) Curr. Gene Ther. 7:49-66, and U. S. Pat. Publ. Nos. US 2007 / 0117128, US 2006 / 0206949, US 2006 / 0153826, US 2006 / 0078552, and US 2004 / 0002092). In further embodiments, a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL.

[0567] MegaTALs may be utilized to not only knock-out one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0568] In some embodiments, a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen- specific receptor that binds (e.g., specifically and / or selectively) to a MAGEA4 antigen, wherein the inhibitory nucleic acid molecule encodes a target- specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (i.e., of PD-1, TIM3, LAG 3, CTLA4, TIGIT,, or other immune checkpoint, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

[0569] A chromosomal gene knockout may be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent.

[0570] Chromosomal gene knockouts may also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.

[0571] In some embodiments, a host cell encompassed by the present invention is capable of specifically and / or selectively killing 50% or more of target cells that comprise a peptide-MHC (pMHC) complex comprising a MAGEA4 immunogenic peptide in the context of an MHC molecule.

[0572] - 103 - FoleyHoagUS 13160800.1 TTC-020

[0573] In some embodiments, the modified immune cell is capable of producing a cytokine when contacted with target cells that comprise a peptide-MHC (pMHC) complex comprising a MAGEA4 immunogenic peptide in the context of an MHC molecule.

[0574] In some embodiments, the cytokine comprises IFN-yor IL2. In some embodiments, the cytokine comprises TNF-a.

[0575] In some embodiments, the host cell is capable of producing a higher level of cytokine or a cytotoxic molecule when contacted with a target cell with expression of MAGEA4 at a level of less than or equal to about 1,000 transcript per million transcripts (TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM. 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM. 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM. 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM. 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range in between, inclusive, such as less than or equal to about 1,000 TPM to less than or equal to about 35 TPM). In some embodiments, the low MAGEA4 expression level is termed “heterozygous expression” meaning between about 1 TPM and about 35 TPM, or any range in between, inclusive, such as 1-32 TPM. For example, the host cell is capable of producing an at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, or more, or any range in between, inclusive, such as 1.2 fold to 2 fold, higher level of cytokine or a cytotoxic molecule.

[0576] In some embodiments, the host cell is capable of specifically and / or selectively killing a target cell expressing MAGEA4 (e.g., a hyperproliferative cell expressing MAGEA4). In certain embodiments, the target cell expresses a MAGEA4 immunogenic peptide in the context of an MHC molecule (e.g., a matched MHC molecule). In certain embodiments, the target cell expresses: (i) a polypeptide comprising or consisting of an amino acid sequence shown in Table 1; and (ii) a matched MHC molecule.

[0577] In some embodiments, host cells do not express MAGEA4 antigen, are not recognized by a binding protein described herein, are not of serotype HLA-A*02, and / or do not express

[0578] - 104 - FoleyHoagUS 13160800.1 TTC-020

[0579] an HLA-A*02 allele, such as HLA-A*02:01. For example, a patient may receive host cells from a healthy donor who is MAGEA4-negative or negative for an MHC that presents a MAGEA4 immunogenic peptide described herein (such as HLA-A*02-negative), or even autologous cells that have selected and / or engineered. Cells, such as stem cells like hematopoietic stem cells, isolated from that donor (or engineered autologous cells) may be used as the source of transplant material. In parallel, T cells isolated from the same donor may be genetically engineered to recognize MAGEA4, such as by expressing a MAGEA4 binding protein described herein. Donor cells, such as stem cells, may be used to engraft cell populations into the patient (e.g., hematopoietic stem cells used to reconstitute an immune system) and host cells may be infused into the patient with the goal of eliciting a highly specific anti-tumor effect. The engineered donor T cells may be designed to recognize and eliminate MAGEC-2 expressing cells, such as all of the patient’s native blood cells, including, for example, cancer cells like residual cancer cells, which are MAGEA4-positive, thereby preventing relapses and promoting complete cures. Because a patient’s new healthy blood cells are derived from the donor and are therefore either MAGEA4-negative, HLA-A*02 serotype-negative, and / or HLA-A*02 allele-negative (e.g., negative for HLA-A*02:01), engineered cells described herein may have minimal toxic side effects. Such patient-matched host cells and treatment methods may be used according to therapeutic methods described further below.

[0580] In some embodiments, the killing is determined by a killing assay. In some embodiment, the killing assay is carried out by co-culturing the host cell and the target cell at a ratio from 20:1 to 0.625:1, for example, from 15:1 to 1.25:1, from 10:1 to 1.5:1, from 8:1 to 3:1, from 6:1 to 5:1, 20:1 to 5:1, 10:1 to 2.5:1 etc.. In some embodiments, the target cell is pulsed with 1 pg / mL to 50 pg / mL of MAGEA4 peptide, for example, from 1 ug / mL to 10 ng / mL, 500 ng / mL to 0.5 ng / mL, from 10 ng / mL to 10 pg / mL from 250 ng / mL to 1 ng / mL, from 50 ng / mL to 5 ng / mL, from 20 ng / mL to 10 ng / mL, etc.

[0581] In some embodiments, the host cell is capable of killing a higher number of target cells when contacted with target cells with a level of MAGEA4 less than or equal to about 1,000 transcript per million transcripts (TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM. 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 - 105 - FoleyHoagUS 13160800.1 TTC-020

[0582] TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range in between, inclusive, such as less than or equal to about 1,000 TPM to less than or equal to about 73 TPM). In some embodiments, the low MAGEA4 expression level is termed "heterozygous expression" meaning between about 1 TPM and about 73 TPM, or any range in between, inclusive, such as 1-73 TPM. For example, the host cell may be capable of killing an at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold. 20 fold. 25 fold. 30 fold. 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, or more, or any range in between, inclusive, such as 1.2 fold to 2 fold, higher number of target cells.

[0583] The present invention further provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also may be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment encompassed by the present invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0584] In an embodiment encompassed by the present invention, the numbers of cells in the population may be rapidly expanded. Expansion of the numbers of T cells may be accomplished by any of a number of methods as are well-known in the art (e.g., U. S. Pat. Nos. 8,034,334 and 8,383,099, U. S. Pat. Publ. No. 2012 / 0244133, Dudley et al. (2003) J. Immunother. 26:332-242, and Riddell et al. (1990) J. Immunol. Methods 128:189-201). For example, expansion of the numbers of T cells may be carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC).

[0585] IV. Pharmaceutical compositions

[0586] - 106 - FoleyHoagUS 13160800.1 TTC-020

[0587] In another aspect encompassed by the present invention, pharmaceutical compositions are provided herein comprising compositions described herein (e.g., binding proteins, nucleic acids, cells, and the like) and a pharmaceutically acceptable carrier, diluent, or excipient The term “pharmaceutically acceptable” refers to those agents, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0588] Agents and other compositions encompassed by the present invention may be specially formulated for administration in solid or liquid form, including those adapted for various routes of administration, such as (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. Any appropriate form factor for an agent or composition described herein, such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas, is contemplated.

[0589] Pharmaceutical compositions encompassed by the present invention may be presented as discrete dosage forms, such as capsules, sachets, or tablets, or liquids or aerosol sprays each containing a pre-determined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non- aqueous liquid, an oil-in-water emulsion, a water-in-oil liquid emulsion, powders for reconstitution, powders for oral consumptions, bottles (including powders or liquids in a bottle), orally dissolving films, lozenges, pastes, tubes, gums, and packs. Such dosage forms may be prepared by any of the methods of pharmacy.

[0590] Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In some embodiments, compositions comprising host cells, binding proteins, or fusion proteins as disclosed herein further comprise a suitable infusion media. Suitable infusion media may be any isotonic medium formulation, typically normal saline, Normosol™-R (Abbott) or Plasma-Lyte™ A (Baxter), 5% dextrose in water, Ringer's lactate may be utilized. An infusion medium may be supplemented...

Claims

TTC-020What is claimed is:

1. A binding protein comprising:a) a T cell receptor (TCR) alpha chain CDR sequence with at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of TCR alpha chain CDR sequences listed in Table 2; and / orb) a TCR beta chain CDR sequence with at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of TCR beta chain CDR sequences li sted in Table 2, wherein the binding protein is capable of binding to a M AGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x1 O’4M.

2. A binding protein comprising:a) a TCR alpha chain variable (V«) domain sequence with at least about 80% identity to a TCR V« domain sequence selected from the group consisting of TCR V« domain sequences listed in Table 2; and / orb) a TCR beta chain variable (Vp) domain sequence with at least about 80% identity to a TCR Vp domain sequence selected from the group consisting of TCR Vp domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x1 O’4M.

3. A binding protein comprising:a) a TCR alpha chain sequence with at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of TCR alpha chain sequences listed in Table 2; and / orb) a TCR beta chain sequence with at least about 80% identity to a TCR beta chain sequence selected from the group consisting of TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x1 O’4M.

4. A binding protein comprising:a) a TCR alpha chain CDR sequence selected from the group consisting of TCR alpha chain CDR sequences listed in Table 2; and / or- 232 - FoleyHoagUS 13160800.1TTC-020b) a TCR beta chain CDR sequence selected from the group consisting of TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x10-4M.

5. A binding protein comprising:a) a TCR alpha chain variable (Va) domain sequence selected from the group consisting of TCR V« domain sequences listed in Table 2; and / orb) a TCR beta chain variable (Vp) domain sequence selected from the group consisting of TCR Vp domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x10-4M.

6. A binding protein comprising:a) a TCR alpha chain sequence selected from the group consisting of TCR alpha chain sequences listed in Table 2; and / orb) a TCR beta chain sequence selected from the group consisting of TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA4 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a Kd less than or equal to about 5x10-4M.

7. The binding protein of any one of claims 1-6, wherein 1) the TCR alpha chain CDR, TCR Va domain, and / or TCR alpha chain is encoded by a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCR beta chain CDR, TCR Vp domain, and / or TCR beta chain is encoded by a TRBV, TRB J, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof as compared to the cognate reference CDR sequence listed in Table 2.

8. The binding protein of any one of claims 1-7, wherein the MAGEA4 immunogenic peptide comprises an amino acid sequence selected from the group of amino acid sequences listed in Table 1, optionally wherein the MAGEA4 immunogenic peptide sequence comprises the amino acid sequence GVYDGREHTV or KVLEHVVRV.- 233 - FoleyHoagUS 13160800.1TTC-0209. The binding protein of any one of claims 1-8, wherein the binding protein is chimeric, humanized, or human.

10. The binding protein of any one of claims 1-9, wherein the binding protein is a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally wherein the binding domain comprises a binding domain having a transmembrane domain and an effector domain that is intracellular.

11. The binding protein of any one of claims 1-10, wherein the TCR alpha chain and the TCR beta chain are covalently linked, optionally wherein the TCR alpha chain and the TCR beta chain are covalently linked through a linker peptide.

12. The binding protein of any one of claims 1-11, wherein the TCR alpha chain and / or the TCR beta chain are covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or a label.

13. The binding protein of claim 12, wherein the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein.

14. The binding protein of any one of claims 1-13, wherein the covalently linked moiety is selected from the group consisting of an inflammatory agent, cytokine, toxin, cytotoxic molecule, radioactive isotope, or antibody or antigen-binding fragment thereof.

15. The binding protein of any one of claims 1-14, wherein the binding protein binds to the pMHC complex on a cell surface.

16. The binding protein of any one of claims 1-15, wherein the MHC is an MHC multimer, optionally wherein the MHC multimer is a tetramer.

17. The binding protein of any one of claims 1-16, wherein the MHC is an MHC class I molecule.- 234 - FoleyHoagUS 13160800.1TTC-02018. The binding protein of any one of claims 1-17, wherein the HC comprises an MHC alpha chain that is an HLA serotype HLA-A*02.

19. The binding protein of any one of claims 1-18, wherein the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0205, HLA-A*0206, and HLA-A*0207 allele.

20. The binding protein of any one of claims 1-19, wherein binding of the binding protein to the MAGEA4 peptide-MHC (pMHC) complex elicits an immune response, optionally wherein the immune response.

21. The binding protein of any one of claims 1-20, wherein the T cell response is selected from the group consisting of a CD8+ T cell response, T cell expansion, cytokine release, and / or cytotoxic killing.

22. The binding protein of any one of claims 1-21, wherein the binding protein is capable of specifically and / or selectively binding to the MAGEA4 immunogenic peptide-MHC (pMHC) complex with a Kd less than or equal to about 1x104M, less than or equal to about 5xl0"3M, less than or equal to about IxlO’5M, less than or equal to about 5xl0’6M, less than or equal to about IxlO"6M, less than or equal to about 5xl0"7M, less than or equal to about IxlO'7M, less than or equal to about 5xl0"8M, less than or equal to about IxlO"8M, less than or equal to about 5xl0"9M, less than or equal to about IxlO"9M, less than or equal to about 5xl0"i0M, less than or equal to about IxlO"10M, less than or equal to about 5xl0"nM, less than or equal to about I lO"11M, less than or equal to about 5x10"12M, or less than or equal to about IxlO"12M.

23. The binding protein of any one of claims 1-22, wherein the binding protein has a higher binding affinity to the peptide-MHC (pMHC) than does a known T-cell receptor, optionally wherein the higher binding affinity is at least 1.05-fold higher.

24. The binding protein of any one of claims 1-23, wherein the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing than does a known T-cell receptor when contacted with target cells with a heterozygous expression of MAGEA4, optionally wherein the induction is at least 1.05-fold higher.

25. The binding protein of claim 24, wherein the cytotoxic killing is a target cancer cell.- 235 - FoleyHoagUS 13160800.1TTC-02026. The binding protein of claim 25, wherein the cancer is selected from the group consisting of melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and bladder urothelial carcinoma.

27. The binding protein of any one of claims 1-26, wherein the binding protein does not bind to a peptide-MHC (pMHC) complex comprising a peptide epitope derived from NALCN, CFI, and MAGE-A8.

28. A TCR alpha chain and / or beta chain selected from the group consisting of TCR alpha chain and beta chain sequences listed in Table 2.

29. An isolated nucleic acid molecule i) that hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, ii) a sequence with at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, and / or iii) a sequence with at least about 80% homology to a nucleic acid encoding listed in Table 2, optionally wherein the isolated nucleic acid molecule comprises 1) a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2 and / or 2) a TRBV, TRBJ, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2.

30. The isolated nucleic acid of claim of claim 29, wherein the nucleic acid is codon optimized for expression in a host cell.

31. A vector comprising the isolated nucleic acid of claim 29 or 30, optionally wherein i) the vector is a cloning vector, expression vector, or viral vector and / or ii) the vector comprises a vector sequence listed in Table 3.

32. The vector of claim 31, wherein the vector further comprises a nucleic acid sequence encoding CD8a, CD8[3, a dominant negative TGF|3 receptor II (DN-TGFpRII), selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR).- 236 - FoleyHoagUS 13160800.1TTC-02033. The vector of claim 32, wherein the nucleic acid sequence encoding CD8a, CD8β, the DN-TGFpRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag, optionally wherein the nucleic acid encoding a tag is at the 5’ upstream of the nucleic acid sequence encoding CD8a, CD8P, the DN-TGFPRII, and / or the selectable protein marker such that the tag is fused to the N-terminus of CD8a, CD8P, the DN- TGF|3RII, and / or the selectable protein marker.

34. The vector of claim 32 or 33, wherein the tag is a CD34 enrichment tag.

35. The vector of any one of claims 31-34, wherein the TCRa, TCRp, and / or the DN-TGFβRII comprises a mutated transmembrane domain and / or a mutated constant domain, optionally wherein the mutated transmembrane domain and / or mutated constant domain enhance cellular surface expression of TCRa, TCRp, and / or the DN-TGFPRII, while decreasing expression of endogenous TCRa, TCRP, and / or TGFPRII.

36. The nucleic acid or vector of any one of claims 29-35, wherein the isolated nucleic acid of any one of claims claim 29 or 30, and / or the nucleic acid sequence of any one of claims 32-35 encoding TCRa, TCRP, CD8a, CD8β, the DN-TGFPRII, and / or the selectable protein marker, is interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide.

37. The nucleic acid or vector of claim 36, wherein the self-cleaving peptide is P2A, E2A, F2A or T2A.

38. A host cell which comprises the isolated nucleic acid or vector of any one of claims 29-37 and / or expresses the binding protein according to any one of claims 1-27, optionally wherein the cell is genetically engineered.

39. The host cell of claim 38, wherein the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both.

40. The host cell of claim 38 or 39, wherein the host cell comprises a knockout of an HLA gene selected from an al macroglobulin gene, a2 macroglobulin gene, a3 macroglobulin gene, pi microglobulin gene, 2 microglobulin gene, and combinations thereof.- 237 - FoleyHoagUS 13160800.1TTC-02041. The host cell of any one of claims 38-40, wherein the host cell comprises a knockout of a TCR gene selected from a TCR a variable region gene, TCR p variable region gene, TCR constant region gene, and combinations thereof.

42. The host cell of any one of claims 38-41, wherein the host cell expresses CD8a, CD8β, a DN-TGFPRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, further optionally wherein the CD8a, CD8|3, the DN-TGFpRII, and / or the selectable protein marker is fused to a CD34 enrichment tag.

43. The host cell of claim 42, wherein host cells are enriched using the CD34 enrichment tag.

44. The host cell of any one of claims 38-43, wherein the host cell is a hematopoietic progenitor cell, peripheral blood mononuclear cell (PBMC), cord blood cell, or immune cell.

45. The host cell of claim 44, wherein the immune cell is a T cell, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, CD4+T cell, CD8+T cell, CD4 / CD8 double negative T cell, gamma delta (y5) T cell, natural killer (NK) cell, NK-T cell, dendritic cell, or a combination thereof.

46. The host cell of any one of claims 38-45, wherein the T cell is a naive T cell, central memory T cell, effector memory T cell, or a combination thereof.

47. The host cell of any one of claims 38-46, wherein the T cell is a primary T cell or a cell of a T cell line.

48. The host cell of any one of claims 38-47, wherein the T cell does not express or has a lower surface expression of an endogenous TCR.

49. The host cell of any one of claims 38-48, wherein the host cell is capable of producing a cytokine or a cytotoxic molecule when contacted with a target cell that comprises a peptide-MHC (pMHC) complex comprising a MAGEA4 peptide epitope in the context of an MHC molecule, optionally wherein the MHC molecule i) is a MHC class I molecule, ii) comprises an MHC alpha chain that is an HLA serotype HLA-A*02, and / or iii) is encoded by an HLA- 238 - FoleyHoagUS 13160800.1TTC-020allele selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 allele.

50. The host cell of claim 49, wherein the host cell is contacted with the target cell in vitro, ex vivo, or in vivo.

51. The host cell of claim 49 or 50, wherein the cytokine is TNF-a, IL-2, and / or IFN-y.

52. The host cell of any one of claims 49-51, wherein the cytotoxic molecule is perforins and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B.

53. The host cell of any one of claims 49-52, wherein the host cell is capable of producing a higher level of cytokine or a cytotoxic molecule when contacted with a target cell with a heterozygous expression of MAGEA4.

54. The host cell of claim 53, wherein the host cell is capable of producing an at least 1.05 -fold higher level of cytokine or a cytotoxic molecule.

55. The host cell of any one of claims 49-54 wherein the host cell is capable of killing a target cell that comprises a peptide-MHC (pMHC) complex comprising the MAGEA4 peptide epitope in the context of an MHC molecule.

56. The host cell of claim 55, wherein the killing is determined by a killing assay.

57. The host cell of claim 55 or 56, wherein the ratio of the host cell and the target cell in the killing assay is from 20:1 to 1:4.

58. The host cell of any one of claims 55-57, wherein the target cell is a target cell pulsed with 1 pg / mL to 50 pg / mL of MAGEA4 peptide, optionally wherein the target cell is a cell monoallelic for an MHC matched to the MAGEA4 peptide.

59. The host cell of any one of claims 55-58, wherein the host cell is capable of killing a higher number of target cells when contacted with target cells with a heterozygous expression of MAGEA4, optionally wherein the cell killing is at least 1.05-fold higher.

60. The host cell of any one of claims 55-59, wherein the target cell is cell line or a primary cell, optionally wherein the target cell is selected from the group consisting of a- 239 - FoleyHoagUS 13160800.1TTC-020HEK293 derived cell line, a cancer cell line, a primary cancer cell, a transformed cell line, and an immortalized cell line.

61. The host cell of any one of claims 55-60, wherein the MAGEA4 immunogenic peptide is selected from the group of amino acid sequences listed in Table 1, optionally wherein the MAGEA4 immunogenic peptide sequence comprises the amino acid sequence GVYDGREHTV or KVEEHVVR V.

62. The host cell of any one of claims 38-61, wherein the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing when contacted with a target cell that comprises a peptide-MHC (pMHC) complex comprising a peptide epitope derived from NALCN, CFI, and MAGE-A8.

63. The host cell of any one of claims 38-62, wherein the host cell does not express MAGEA4 antigen, is not recognized by a binding protein of any one of claims 56-78, is not of serotype HLA-A*02 and / or does not express an HLA-A*02 allele.

64. A population of host cells according to any one of claims 38-63.

65. A composition comprising a) a binding protein according to any one of claims 1-27, b) an isolated nucleic acid or a vector according to any one of claims 29-37, c) a host cell according to any one of claims 38-63, and / or d) a population of host cells according to claim 64, and a carrier.

66. A device or kit comprising a) a binding protein according to any one of cl aims 1-27, b) an isolated nucleic acid or a vector according to any one of claims 29-37, c) a host cell according to any one of claims 38-63, and / or d) a population of host cells according to claim 64, said device or kit optionally comprising a reagent to detect binding of a), c) and / or d) to a pMHC complex.

67. A method of producing a binding protein according to any one of claims 1-27, wherein the method comprises the steps of: (i) culturing a transformed host cell which has been transformed by a nucleic acid comprising a sequence encoding a binding protein according to any one of claims 1-27 under conditions suitable to allow expression of said binding protein; and (ii) recovering the expressed binding protein.- 240 - FoleyHoagUS 13160800.1TTC-02068. A method of producing a host cell expressing a binding protein according to any one of claims 1-27, wherein the method comprises the steps of: (i) introducing a nucleic acid comprising a sequence encoding a binding protein according to any one of claims 1-27 into the host cell; and (ii) culturing the transformed host cell under conditions suitable to allow expression of said binding protein.

69. A method of detecting the presence or absence of a MAGEA4 antigen and / or a cell expressing MAGEA4, optionally wherein the cell is a hyperproliferative cell, comprising detecting the presence or absence of said MAGEA4 antigen in a sample by use of at least one binding protein according to any one of claims 1-27, at least one host cell according to any one of claims 38-63, or a population of host cells according to claim 64, wherein detection of the MAGEA4 antigen is indicative of the presence of a MAGEA4 antigen and / or cell expressing MAGEA4.

70. The method of claim 69, wherein the at least one binding protein, or the at least one host cell, forms a complex with the MAGEA4 peptide in the context of an MHC molecule, and the complex is detected in the form of fluorescence activated cell sorting (FACS), enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or intracellular flow assay.

71. The method of claim 69 or 70, further comprising obtaining the sample from a subject.

72. A method of detecting the level of a disorder characterized by MAGEA4 expression in a subject, comprising:a) contacting a sample obtained from the subject with at least one binding protein according to any one of claims 1-27, at least one host cell according to any one of claims 38- 63, or a population of host cells according to claim 64; andb) detecting the level of reactivity,wherein the presence or a higher level of reactivity compared to a control level indicates the level of the disorder characterized by MAGEA4 expression in the subject.

73. The method of claim 72, wherein the control level is a reference number.

74. The method of claim 72 or 73, wherein the control level is a level from a subject without the disorder characterized by MAGEA4 expression.- 241 - FoleyHoagUS 13160800.1TTC-02075. A method for monitoring the progression of a disorder characterized by MAGEA4 expression in a subject, the method comprising:a) detecting in a subject sample the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 1- 27, at least one host cell according to any one of claims 38-63, or a population of host cells according to claim 64;b) repeating step a) at a subsequent point in time; andc) comparing the level of MAGEA4 or the cell of interest expressing MAGEA4 detected in steps a) and b) to monitor the progression of the disorder characterized by MAGEA4 expression in the subject, wherein an absent or reduced MAGEA4 level or the cell of interest expressing MAGEA4 detected in step b) compared to step a) indicates an inhibited progression of the disorder characterized by MAGEA4 expression in the subject and a presence or increased MAGEA4 level or the cell of interest expressing MAGEA4 detected in step b) compared to step a) indicates a progression of the disorder characterized by MAGEA4 expression in the subject.

76. The method of claim 75, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment to treat the disorder characterized by MAGEA4 expression.

77. A method for predicting the clinical outcome of a subject afflicted with a disorder characterized by MAGEA4 expression comprising:a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 1-27, at least one host cell according to any one of claims 38-63, or a population of host cells according to claim 64; andb) comparing the presence or level of reactivity to that from a control, wherein the control is obtained from a subject having a good clinical outcome;wherein the absence or a reduced level of reactivity in the subject sample as compared to the control indicates that the subject has a good clinical outcome.

78. A method of assessing the efficacy of a therapy for a disorder characterized by MAGEA4 expression comprising:- 242 - FoleyHoagUS 13160800.1TTC-020a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 1-27, at least one host cell according to any one of claims 38-63, or a population of host cells according to claim 64, in a first sample obtained from the subject prior to providing at least a portion of the therapy for the disorder characterized by MAGEA4 expression to the subject, andb) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 1-27, at least one host cell according to any one of claims 38-63, or a population of host cells according to claim 64, in a second sample obtained from the subject following provision of the therapy for the disorder characterized by MAGEA4 expression,wherein the absence or a reduced level of reactivity in the second sample, relative to the first sample, is an indication that the therapy is efficacious for treating the disorder characterized by MAGEA4 expression in the subject, and wherein the presence or an increased level of reactivity in the second sample, relative to the first sample, is an indication that the therapy is not efficacious for treating the disorder characterized by MAGEA4 expression in the subject.

79. The method of any one of claims 69-78, wherein the level of reactivity is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release.

80. The method of any one of claims 69-79, wherein the T cell binding, activation, and / or effector function is detected using fluorescence activated cell sorting (FACS), enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunochemically, Western blot, or intracellular flow assay.

81. A method of preventing and / or treating a disorder characterized by MAGEA4 expression comprising contacting target cells expressing MAGEA4 with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein according to any one of claims 1-27, optionally wherein the composition is administered to a subject.

82. The method of claim 81, wherein the cell is an allogeneic cell, syngeneic cell, or autologous cell.- 243 - FoleyHoagUS 13160800.1TTC-02083. The method of claim 81 or 82, wherein the cell is a host cell according to any one of claims 38-63 or a population of host cells according to claim 64.

84. The method of any one of claims 81-83, wherein the target cell is a cancer cell expressing MAGEA4.

85. The method of any one of claims 81-84, wherein the composition further comprises a pharmaceutically acceptable carrier.

86. The method of any one of claims 81-85, wherein the composition induces an immune response against the target cell expressing MAGEA4 in the subject.

87. The method of any one of claims 81-86, wherein the composition induces an antigen¬ specific T cell immune response against the target cell expressing MAGEA4 in the subject.

88. The method of any one of claims 81-87, wherein the antigen-specific T cell immune response comprises at least one of a CD4+helper T lymphocyte (Th) response and a CD8+ cyto toxic T lymphocyte (CTL) response.

89. The method of any one of claims 81-88, further comprising administering at least one additional treatment for the disorder characterized by MAGEA4 expression, optionally wherein the at least one additional treatment for the disorder characterized by MAGEA4 expression is administered concurrently or sequentially with the composition.

90. The method of any one of claims 81-89, wherein the disorder characterized by MAGEA4 expression is a cancer or relapse thereof, optionally wherein the cancer is selected from the group consisting of melanoma, head & neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, breast invasive carcinoma, and bladder urothelial carcinoma.

91. The method of any one of claims 81-90, wherein the subject is an animal model of a disorder characterized by MAGEA4 expression and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent.- 244 - FoleyHoagUS 13160800.1

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