CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/665,234, filed May 1 , 2018, which is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND

DEVELOPMENT

[0002] This invention was made with Government support under project number

Z01BC011651-03 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 85,179 Byte ASC1I (Text) file named“742l94_ST25.txt,” dated April 22, 2019.

BACKGROUND OF THE INVENTION

[0004] Some cancers may have very limited treatment options, particularly when the cancer becomes metastatic and unresectable. Despite advances in treatments such as, for example, surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as, for example, non-small-cell lung cancer (NSCLC), may be poor. Accordingly, there exists an unmet need for additional treatments for cancer.

BRIEF SUMMARY OF THE INVENTION

[0005] An embodiment of the invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for the mutated epidermal growth factor receptor (EGFR) amino acid sequence of AIKTSPKANKEIL (SEQ ID NO: 36).

[0006] Further embodiments of the invention provide polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the inventive TCRs. [0007] Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are further provided by embodiments of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0008] Figure 1 is a graph showing the amount of IFN-g (pg/ml) secreted upon co-culture of cells transduced with Construct 1 (EGFR TCR alpha P2A beta), Construct 2 (EGFR TCR beta P2A alpha), an anti-KRAS G12V TCR, or a mixture of vectors encoding the a and b chains (EGFR TCR alpha+beta) with dendritic cells which had been pulsed with WT EGFR peptide or mutated EGFR (E746_A750 del) peptide. Transduced cells cultured alone (medium) served as a control.

[0009] Figure 2 is a graph showing the amount of IFN-g (pg/ml) secreted upon co-culture of cells transduced with Construct 1 (EGFR TCR alpha P2A beta), Construct 2 (EGFR TCR beta P2A alpha), an anti-KRAS G12V TCR, or a mixture of vectors encoding the a and b chains (EGFR TCR alpha+beta) with PC-9 cells which had been retrovirally transduced with a combination of (i) HLA-DPA1 *01 :03 and DPBl *0l :0l or (ii) HLA-DP A 1 *02:01 and DPB1 *01 :0l . Transduced PBL were co-cultured with untransduced PC-9 cells as a control.

[0010] Figure 3 is a graph showing the amount of IFN-g (pg/ml) secreted upon co-culture of cells transduced with Construct 1 (EGFR TCR alpha P2A beta), Construct 2 (EGFR TCR beta P2A alpha), an anti-KRAS G12V TCR, or a mixture of vectors encoding the a and b chains (EGFR TCR alpha+beta) with HCC827 cells which had been retrovirally transduced with a combination of (i) HLA-DPA1 *01 :03 and DPB1 *01 :01 or (ii) HLA-DPA1 *02:01 and DPB1 *01 :0l . Transduced PBL were co-cultured with untransduced HCC827 cells as a control.

[0011] Figure 4A is a graph showing the amount of IFN-g (pg/ml) secreted upon co culture of cells transduced with Construct 1 (5’-TCR alpha chain-linker-TCR beta chain-3’) (SEQ ID NO: 30) with dendritic cells which had been pulsed with one of the mutated EGFR (E746_A750 del) peptides of SEQ ID NOs: 35 and 40-50.

[0012] Figure 4B is a schematic showing the location of the deleted wild-type EGFR amino acid residues 746-750 (ELREA (SEQ ID NO: 51)) in relation to the mutated EGFR E746_A750del peptide AIKTSPKANKEIL (SEQ ID NO: 36). DETAILED DESCRIPTION OF THE INVENTION

[0013] EGFR (also referred to as ERBB1 or HER1) is a transmembrane glycoprotein that belongs to the receptor tyrosine kinase (RTK) super-family of cell surface receptors, which mediate cell signaling by extra-cellular growth factors. EGFR is a cell surface protein that binds to epidermal growth factor (EGF). Binding of EGFR to EGF induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. Examples of wild-type (WT), unmutated human EGFR amino acid sequences include those disclosed in Genbank Accession Nos. NP_00l 333826.1 (isoform e precursor), NP_00l 333827.1 (isoform f precursor), NP_001333828.l (isoform g precursor), NP_00l333829.l (isoform h precursor), NP_00l333870.l (isoform i precursor), NP_005219.2 (isoform a precursor), NP_958439.l (isoform b precursor), NP_958440.l (isoform c precursor), and NP_95844l .l (iso form d precursor).

[0014] Mutations in EGFR may be associated with cancer. For example, EGFR mutations may be found in about 10% ofNSCLC patients in the United States and in about 50% ofNSCLC patients in Asia. The deletion of amino acid residues E746-A750 may account for about 30 to about 40% of EGFR mutations.

[0015] Amino acid residue position numbers of EGFR are defined herein by reference to the amino acid sequence of the full-length, WT, unmutated human EGFR amino acid sequence of SEQ ID NO: 1. The actual positions of the amino acid residues of a particular embodiment of an EGFR amino acid sequence are defined relative to the corresponding positions of SEQ ID NO: 1 and may represent different residue position numbers than the residue position numbers of SEQ ID NO: 1. An EGFR amino acid sequence (e.g., a EGFR peptide) may comprise fewer than all of the amino acid residues of the full-length, WT EGFR protein. For example, positions 746-750 are defined herein by reference to the WT full- length EGFR protein (namely, SEQ ID NO: 1) with the understanding that the actual position of the corresponding residue in a particular example of a EGFR amino acid sequence may be different. For example, when a particular example of a WT EGFR amino acid sequence is PEGEKVKIPVAIKELREATSPKANK (SEQ ID NO: 34) (an exemplary WT EGFR peptide corresponding to contiguous amino acid residues 733 to 757 of SEQ ID NO: 1), the deletion of EGFR amino acid residues 746-750 refer to a deletion of the underlined residues in SEQ ID NO: 34, even though the actual positions of the underlined residues in SEQ ID NO: 34 are 14-18, respectively. [0016] The terms“EGFR E746_A750del” and“EGFR E746-A750 deletion” refer to a mutated EGFR amino acid sequence (i) in which all of the contiguous amino acid residues normally present at positions 746-750 of EGFR SEQ ID NO: 1 are absent and (ii) which comprises a suitable number of contiguous amino acid residues which flank each of the amino side and the carboxyl side of positions 746-750 of WT EGFR SEQ ID NO: 1. The number of contiguous amino acids from the WT EGFR protein flanking each side of the deleted amino acid residues 746-750 is not limited and may be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or a range defined by any two of the foregoing values. In a preferred embodiment, the mutated EGFR amino acid sequence with the EGFR E746-A750 deletion is AIKTSPKANKEIL (SEQ ID NO: 36).

[0017] An embodiment of the invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for the mutated epidermal growth factor receptor (EGFR) amino acid sequence of AIKTSPKANKEIL (SEQ ID NO: 36).

[0018] In an embodiment of the invention, the mutated EGFR E746 A750dcl peptide has any length suitable for binding to any of the HLA Class II molecules described herein. For example, the TCR may have antigenic specificity for a mutated EGFR peptide with the E746- A750 deletion, the mutated EGFR peptide having a length of about 11 to about 30 amino acid residues, about 15 to about 25 amino acid residues, about 18 to about 22 amino acid residues, or about 8 to about 15 amino acid residues. In an embodiment of the invention, the TCR may have antigenic specificity for a EGFR peptide with the E746-A750 deletion, the mutated EGFR peptide having a length of about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, 11 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, about 15 amino acid residues, about 16 amino acid residues, about 17 amino acid residues, about 18 amino acid residues, about 19 amino acid residues, about 20 amino acid residues, about 21 amino acid residues, about 22 amino acid residues, about 23 amino acid residues, about 24 amino acid residues, about 25 amino acid residues, about 26 amino acid residues, about 27 amino acid residues, about 28 amino acid residues, about 29 amino acid residues, or about 30 amino acid residues.

Examples of specific mutated EGFR peptides with the E746-A750 deletion, which may be recognized by the inventive TCR, include those set forth in Table A. TABLE A

[0019] In an embodiment of the invention, the inventive TCRs are able to recognize EGFR E746_A750del presented by an HLA Class II molecule. In this regard, the TCR may elicit an immune response upon binding to EGFR E746_A750del within the context of an HLA Class II molecule. The inventive TCRs may bind to the HLA Class II molecule in addition to EGFR E746_A750del.

[0020] In an embodiment of the invention, the HLA Class II molecule is an HLA-DP molecule. The HLA-DP molecule is a heterodimer of an a chain (DPA) and b chain (DPB). The HLA-DPA chain may be any HLA-DPA chain. The HLA-DPB chain may be any HLA- DPB chain. In an embodiment of the invention, the HLA Class II molecule is a heterodimer of an HLA-DPA1 chain and an HLA-DPB 1 chain. Examples of HLA-DPA 1 molecules may include, but are not limited to, those encoded by the HLA-DPA1 *01 :03, HLA-DPA1 *01 :04, HLA-DPA 1 *01 :05, HLA-DPA 1 *01 :06, HLA-DPA 1 *01 :07, HLA-DPA1 *01 :08, HLA- DPA1 *01 :09, HLA-DPAl *01 : l 0, HLA-DPA1 *02:01 , HLA-DPA1 *02:02, HLA- DPA1 *02:03, HLA-DPA1 *02:04, HLA-DPA1 *03 :01 , HLA-DPA1 *03 :02, HLA- DPA1 *03 :03, and HLA-DPA1 *04:01 alleles. Examples of HLA-DPB 1 molecules may include, but are not limited to, those encoded by the HLA-DPB 1 *01 :01 , HLA-DPB 1 *02:01 , HLA-DPB1 *02:02, HLA-DPB1 *03:01 , HLA-DPB1 *04:01 , HLA-DPB 1 *04:02, HLA- DPBl *05:01 , HLA-DPBl *06:0l , HLA-DPB1 *07:01 , HLA-DPB1 *08:01 , HLA- DPB1 *09:01 , and HLA-DPB1 *10:01 alleles. Preferably, the HLA Class II molecule is a heterodimer of an HLA-DPA1 *02:01 chain and an HLA-DPB1 *01 :01 chain.

[0021] The TCRs of the invention may provide any one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. EGFR with the E746-A750 deletion is expressed by cancer cells and is not expressed by normal, noncancerous cells. Without being bound to a particular theory or mechanism, it is believed that the inventive TCRs advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancerous cells, thereby reducing, for example, by minimizing or eliminating, toxicity. Moreover, the EGFR E746-A750 deletion is a“driver mutation,” which drives the development of the cancer. Because the driver mutation is needed for the cancer cells to stay cancerous, substantially all of the cancer cells will have the driver mutation. A challenge in cancer treatment is the heterogeneity of cancer cells. Non-driver mutations, so called“passenger mutations,” may exist in some cancer cells but not in all of the cancer cells. Even if a cancer treatment were to target and eliminate passenger mutation-positive cancer cells, the passenger mutation-negative cancer cells could still survive, which could limit the benefit for the patient. Without being bound to a particular theory or mechanism, it is believed that because the EGFR E746-A750 deletion is a driver mutation, the targeting of cells with this mutation will kill substantially all of the cancer cells.

[0022] Moreover, the inventive TCRs may, advantageously, successfully treat or prevent cancers which express EGFR with the E746-A750 deletion that do not respond to other types of treatment such as, for example, chemotherapy, surgery, or radiation. The inventive TCRs may provide highly avid recognition of EGFR with the E746-A750 deletion, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-y; transfected with a vector encoding one or more of EGFR E746_A750del peptide, HLA-DPA1 *02:01 chain, and HLA-DPB1 *01 :01 chain; pulsed with a EGFR E746_A750del peptide; or a combination thereof). Moreover, the F1LA- DPA1 *02:01 chain and HLA-DPB1 *01 :01 chain alleles are expressed by about 10% of Caucasians and about 50% of African Americans in the U.S. Accordingly, the inventive TCRs may increase the number of immunotherapy-eligible cancer patients to include those patients that express the HLA-DPA1 *02:01 and HLA-DPB1 *01 :01 alleles who may not be eligible for immunotherapy using TCRs that recognize EGFR E746_A750del presented by other MFIC molecules.

[0023] The phrase“antigenic specificity,” as used herein, means that the TCR can specifically bind to and immunologically recognize mutated EGFR with the E746-A750 deletion with high avidity. For example, a TCR may be considered to have“antigenic specificity” for EGFR E746_A750del if about 1 x 10 ⁴ to about 1 x 10 ⁵ T cells expressing the TCR secrete at least about 200 pg/mL or more (e.g., 200 pg/mL or more, 300 pg/mL or more, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more, 1000 pg/mL or more, 5,000 pg/mL or more, 7,000 pg/mL or more, 10,000 pg/mL or more, 20,000 pg/mL or more, or a range defined by any two of the foregoing values) of IFN-g upon co culture with (a) antigen-negative, HLA Class II molecule positive target cells pulsed with a low concentration of EGFR E746_A750del peptide (e.g., about 0.05 ng/mL to about 10 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, 8 ng/mL, 10 ng/mL, or a range defined by any two of the foregoing values) or (b) antigen-negative, HLA Class II molecule positive target cells into which a nucleotide sequence encoding EGFR E746_A750del has been introduced such that the target cell expresses EGFR E746__A750del. Cells expressing the inventive TCRs may also secrete IFN-g upon co-culture with antigen-negative, HLA Class II molecule positive target cells pulsed with higher concentrations of EGFR E746__A750del. The HLA Class II molecule may be a heterodimer of an HLA-DPA1 *02:01 chain and an HLA-DPB1 *01 :0l chain.

[0024] Alternatively or additionally, a TCR may be considered to have“antigenic specificity” for EGFR E746 A750del if T cells expressing the TCR secrete at least twice as much IFN-g upon co-culture with (a) antigen-negative, HLA Class II molecule positive target cells pulsed with a low concentration of EGFR E746_A750del peptide or (b) antigen negative, HLA Class II molecule positive target cells into which a nucleotide sequence encoding EGFR E746_A750del has been introduced such that the target cell expresses EGFR E746 A750dcl as compared to the amount of IFN-g expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR, co-cultured with (a) antigen-negative, HLA Class II molecule positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the EGFR E746_A750del peptide) or (b) antigen-negative, HLA Class II molecule positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cell expresses the irrelevant peptide, or (ii) untransduced T cells (e.g., derived from PBMC, which do not express the TCR) co-cultured with (a) antigen negative, HLA Class II molecule positive target cells pulsed with the same concentration of EGFR E746_A750del peptide or (b) antigen-negative, HLA Class II molecule positive target cells into which a nucleotide sequence encoding EGFR E746_A750del has been introduced such that the target cell expresses EGFR E746_A750del. The HLA Class II molecule expressed by the target cells of the negative control would be the same HLA Class II molecule expressed by the target cells that are co-cultured with the T cells being tested. The HLA Class II molecule may be a heterodimer of an HLA-DPA1 *02:01 chain and an HLA- DPB1 *01 :0l chain. IFN-g secretion may be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (EL1SA).

[0025] Alternatively or additionally, a TCR may be considered to have“antigenic specificity” for mutated EGFR with the E746-A750 deletion if at least twice as many of the numbers of T cells expressing the TCR secrete IFN-g upon co-culture with (a) antigen negative, FILA Class II molecule positive target cells pulsed with a low concentration of EGFR E746_A750del peptide or (b) antigen-negative, HLA Class II molecule positive target cells into which a nucleotide sequence encoding EGFR E746_A750del has been introduced such that the target cell expresses EGFR E746_A750del as compared to the numbers of negative control T cells that secrete IFN-g. The HLA Class II molecule, concentration of peptide, and the negative control may be as described herein with respect to other aspects of the invention. The numbers of cells secreting IFN-g may be measured by methods known in the art such as, for example, ELISPOT.

[0026] Alternatively or additionally, a TCR may be considered to have“antigenic specificity” for EGFR E746_A750del if T cells expressing the TCR upregulate expression of one or more T-cell activation markers as measured by, for example, flow cytometry after stimulation with target cells expressing mutated EGFR. Examples of T-cell activation markers include 4-1BB, 0X40, CDl07a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

[0027] An embodiment of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (a) chain of a TCR, a beta (b) chain of a TCR, a gamma (g) chain of a TCR, a delta (d) chain of a TCR, or a combination thereof. The polypeptides of the inventive TCR can comprise any amino acid sequence, provided that the TCR has antigenic specificity for EGFR E746_A750del.

[0028] In an embodiment of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR)l, a CDR2, and a CDR3 of a TCR. In an embodiment of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 3 (CDR1 of a chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 4 (CDR2 of a chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5 (CDR3 of a chain), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 (CDR1 of b chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 7 (CDR2 of b chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (CDR3 of b chain). In this regard, the inventive TCR can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 3-8. In an embodiment of the invention, the TCR comprises the amino acid sequences of: (a) all of SEQ ID NOs: 3-5, (b) all of SEQ ID NOs: 6-8, or (c) all of SEQ ID NOs: 3-8. In an especially preferred embodiment, the TCR comprises the amino acid sequences of all of SEQ ID NOs: 3-8.

[0029] In an embodiment of the invention, the TCR comprises an amino acid sequence of a variable region of a TCR comprising the CDRs set forth above. The TCR may comprise a human variable region, e.g., a human a chain variable region and a human b chain variable region. In this regard, the TCR can comprise the amino acid sequence of: SEQ ID NO: 9 (variable region of a chain); SEQ ID NO: 10 (variable region of b chain); SEQ ID NO: 11 (variable region of a chain); SEQ ID NO: 12 (variable region of b chain); both of SEQ ID NOs: 9 and 10, or both of SEQ ID NO: 11 and 12. Preferably, the TCR comprises the amino acid sequences of both of SEQ ID NOs: 9 and 10 or both of SEQ ID NO: 11 and 12.

[0030] The inventive TCRs may further comprise an a chain constant region and a b chain constant region. The constant region may be derived from any suitable species such as, e.g., human or mouse. In an embodiment of the invention, the TCRs further comprise murine a and b chain constant regions or human a and b chain constant regions. As used herein, the term“murine” or“human,” when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, a chain, and/or b chain), means a TCR (or component thereof) which is derived from a mouse or a human, respectively, i.e., a TCR (or component thereof) that originated from or was, at one time, expressed by a mouse T cell or a human T cell, respectively.

[0031] In an embodiment of the invention, the TCR comprises a human constant region.

The TCR may comprise the amino acid sequence of SEQ ID NO: 13 (wild-type (WT) human a chain constant region), SEQ ID NO: 14 (WT human b chain constant region), or both SEQ ID NOs: 13 and 14. Preferably, the inventive TCR comprises the amino acid sequences of both of SEQ ID NOs: 13 and 14. The TCR may comprise any of the human constant regions described herein in combination with any of the CDR regions as described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequences of: (a) all of SEQ ID NOs: 3-5 and 13; (b) all of SEQ ID NOs: 6-8 and 14; or (c) all of SEQ ID NOs: 3-8 and 13-14. In another embodiment of the invention, the TCR may comprise any of the human constant regions described herein in combination with any of the variable regions described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequences of: (i) both of SEQ ID NOs: 9 and 13; (ii) both of SEQ ID NOs: 10 and 14; (iii) all of SEQ ID NOs: 9-10 and 13-14; (iv) both of SEQ ID NOs: 11 and 13; (v) both of SEQ ID NOs: 12 and 14; or (vi) all of SEQ ID NOs: 11- 12 and 13-14.

[0032] In another embodiment of the invention, the TCR comprises the amino acid sequence(s) of: SEQ ID NO: 19 (a chain with human constant region), SEQ ID NO: 20 (b chain with human constant region), or both of SEQ ID NO: 19-20.

[0033] An embodiment of the invention provides a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR has antigenic specificity for the mutated epidermal growth factor receptor (EGFR) amino acid sequence of

AIKTSPKANKEIL (SEQ ID NO: 36). The murine constant region may provide any one or more advantages. For example, the murine constant region may diminish mispairing of the inventive TCR with the endogenous TCRs of the host cell into which the inventive TCR is introduced. Alternatively or additionally, the murine constant region may increase expression of the inventive TCR as compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 17 (wild-type (WT) murine a chain constant region), SEQ ID NO: 18 (WT murine b chain constant region), or both SEQ ID NOs: 17 and 18. Preferably, the inventive TCR comprises the amino acid sequences of both of SEQ ID NOs: 17 and 18. The chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the CDR regions as described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequences of: (a) all of SEQ ID NOs: 3-5 and 17; (b) all of SEQ ID NOs: 6-8 and 18; or (c) all of SEQ ID NOs: 3-8 and 17-18. In another embodiment of the invention, the chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequences of: (i) both of SEQ ID NOs: 9 and 17; (ii) both of SEQ ID NOs: 10 and 18; (iii) all of SEQ ID NOs: 9-10 and 17-18; (iv) both of SEQ ID NOs: 11 and 17; (v) both of SEQ ID NOs: 12 and 18; or (vi) all of SEQ ID NOs: 11-12 and 17-18. [0034] In an embodiment of the invention, the TCR comprises a substituted constant region. In this regard, the TCR may comprise the amino acid sequence of any of the TCRs described herein with one, two, three, or four amino acid substitution(s) in the constant region of one or both of the a and b chain. Preferably, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of one or both of the a and b chains. In an especially preferred embodiment, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of the a chain and one amino acid substitution in the murine constant region of the b chain. In some embodiments, the TCRs comprising the substituted constant region advantageously provide one or more of increased recognition of targets which express mutated EGFR with the E746-A750 deletion, increased expression by a host cell, diminished mispairing with endogenous TCRs, and increased anti-tumor activity as compared to the parent TCR comprising an unsubstituted (wild-type) constant region. In general, the substituted amino acid sequences of the murine constant regions of the TCR a and b chains, SEQ ID NOs: 15 and 16, respectively, correspond with all or portions of the unsubstituted murine constant region amino acid sequences SEQ ID NOs: 17 and 18, respectively, with SEQ ID NO: 15 having one, two, three, or four amino acid substitution(s) when compared to SEQ ID NO: 17 and SEQ ID NO: 16 having one amino acid substitution when compared to SEQ ID NO: 18. In this regard, an embodiment of the invention provides a TCR comprising the amino acid sequences of (a) SEQ ID NO: 15 (constant region of a chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 1 12 is Ser, Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 16 (constant region of b chain), wherein X at position 57 is Ser or Cys; or (c) both of SEQ ID NOs: 15 and 16. In an embodiment of the invention, the TCR comprising SEQ ID NO: 15 does not comprise SEQ ID NO: 17 (unsubstituted murine constant region of a chain). In an embodiment of the invention, the TCR comprising SEQ ID NO: 16 does not comprise SEQ ID NO: 18

(unsubstituted murine constant region of b chain).

[0035] In an embodiment of the invention, the TCR comprises an a chain comprising a variable region and a constant region and a b chain comprising a variable region and a constant region. In this regard, the TCR may comprise (a) an a chain comprising the amino acid sequence of SEQ ID NO: 21 , wherein: (i) X at position 181 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 245 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 247 of SEQ ID NO: 21 is Met, Ala, Val, Leu, lie, Pro, Phe, or Trp; and (iv) X at position 248 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; (b) a b chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 188 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, the TCR comprising SEQ ID NO: 21 does not comprise SEQ ID NO: 17 (unsubstituted murine constant region of a chain). In an embodiment of the invention, the TCR comprising SEQ ID NO: 22 does not comprise SEQ ID NO: 18 (unsubstituted murine constant region of b chain).

[0036] In an embodiment of the invention, the substituted constant region includes cysteine substitutions in the constant region of one or both of the a and b chains to provide a cysteine-substituted TCR. Opposing cysteines in the a and the b chains provide a disulfide bond that links the constant regions of the a and the b chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted murine constant regions. In this regard, the TCR may be a cysteine-substituted TCR in which one or both of the native Thr at position 48 (Thr48) of SEQ ID NO: 17 and the native Ser at position 57 (Ser57) of SEQ ID NO: 18 may be substituted with Cys. Preferably, both of the native Thr48 of SEQ ID NO: 17 and the native Ser57 of SEQ ID NO: 18 are substituted with Cys.

Examples of cysteine-substituted TCR constant regions sequences are set forth in Table 1. In an embodiment of the invention, the cysteine-substituted TCR comprises (i) SEQ ID NO: 15, (ii) SEQ ID NO: 16, or (iii) both of SEQ ID NOs: 15 and 16, wherein both of SEQ ID NOs: 15 and 16 are as defined in Table 1. The cysteine-substituted TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.

[0037] In an embodiment of the invention, the cysteine-substituted, chimeric TCR comprises a full length alpha chain and a full-length beta chain. Examples of cysteine- substituted, chimeric TCR alpha chain and beta chain sequences are set forth in Table 1. In an embodiment of the invention, the TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22, or (iii) both of SEQ ID NO: 21 and 22, wherein SEQ ID NOs: 21-22 are as defined in Table 1.

TABLE 1

[0038] In an embodiment of the invention, the substituted amino acid sequence includes substitutions of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the a and b chains with a hydrophobic amino acid to provide a hydrophobic amino acid-substituted TCR (also referred to herein as an“LVL- modified TCR”). The hydrophobic amino acid substitution(s) in the TM domain of the TCR may increase the hydrophobicity of the TM domain of the TCR as compared to a TCR that lacks the hydrophobic amino acid substitution(s) in the TM domain. In this regard, the TCR is an LVL-modified TCR in which one, two, or three of the native Seri 12, Metl 14, and Glyl 15 of SEQ ID NO: 17 may, independently, be substituted with Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. Preferably, all three of the native Serl 12, Metl 14, and Glyl 15 of SEQ ID NO: 17 may, independently, be substituted with Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. In an embodiment of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 15, (ii) SEQ ID NO: 16, or (iii) both of SEQ ID NOs: 15 and 16, wherein both of SEQ ID NOs: 15 and 16 are as defined in Table 2. The LVL-modified TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.

[0039] In an embodiment of the invention, the LVL-modified TCR comprises a full length alpha chain and a full-length beta chain. Examples of LVL-modified TCR alpha chain and beta chain sequences are set forth in Table 2. In an embodiment of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 21 , (ii) SEQ ID NO: 22, or (iii) both of SEQ 1D NO: 21 and 22, wherein SEQ ID NOs: 21-22 are as defined in Table 2. TABLE 2

[0040] In an embodiment of the invention, the substituted amino acid sequence includes the cysteine substitutions in the constant region of one or both of the a and b chains in combination with the substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the a and b chains with a hydrophobic amino acid (also referred to herein as“cysteine-substituted, LVL-modified TCR”). In this regard, the TCR is a cysteine-substituted, LVL-modified, chimeric TCR in which the native Thr48 of SEQ ID NO: 17 is substituted with Cys; one, two, or three of the native Serl 12, Metl 14, and Glyl 15 of SEQ ID NO: 17 are, independently, substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, He, or Val; and the native Ser57 of SEQ ID NO: 18 is substituted with Cys. Preferably, all three of the native Serl 12, Metl 14, and Glyl 15 of SEQ ID NO: 17 may, independently, be substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. In an embodiment of the invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 15, (ii) SEQ ID NO: 16, or (iii) both of SEQ ID NOs: 15 and 16, wherein both of SEQ ID NOs: 15 and 16 are as defined in Table 3. The cysteine-substituted, LVL-modified TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.

[0041] In an embodiment, the cysteine-substituted, LVL-modified TCR comprises a full- length alpha chain and a full-length beta chain. In an embodiment of the invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 21 , (ii) SEQ ID NO: 22, or (iii) both of SEQ ID NO: 21 and 22, wherein SEQ ID NOs: 21-22 are as defined in Table 3. In a preferred embodiment, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 23 (a full-length alpha chain), (ii) SEQ ID NO: 24 (full-length beta chain), or (iii) both of SEQ ID NOs 23-24. Preferably, the cysteine-substituted, LVL-modified TCR comprises both of SEQ ID NOs: 23-24.

TABLE 3

[0042] Also provided by an embodiment of the invention is a polypeptide comprising a functional portion of any of the TCRs described herein. The tenn "polypeptide," as used herein, includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds.

[0043] With respect to the inventive polypeptides, the functional portion can be any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the functional portion specifically binds to EGFR E746_A750del. The term“functional portion,” when used in reference to a TCR, refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Functional portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to EGFR E746_A750del (e.g., within the context of a FILA Class II molecule), or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a higher extent, as the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or more, of the parent TCR.

[0044] The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to EGFR E746_A750del; and/or having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent TCR.

[0045] The polypeptide can comprise a functional portion of either or both of the a and b chains of the TCRs of the invention, such as a functional portion comprising one or more of the CDR1, CDR2, and CDR3 of the variable region(s) of the a chain and/or b chain of a TCR of the invention. In an embodiment of the invention, the polypeptide can comprise the amino acid sequence of SEQ ID NO: 3 (CDR1 of a chain), SEQ ID NO: 4 (CDR2 of a chain), SEQ ID NO: 5 (CDR3 of a chain), SEQ ID NO: 6 (CDR1 of b chain), SEQ ID NO: 7 (CDR2 of b chain), SEQ ID NO: 8 (CDR3 of b chain), or a combination thereof.

[0046] In this regard, the inventive polypeptide can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 3-8. In an embodiment of the invention, the TCR comprises the amino acid sequences of: (a) all of SEQ ID NOs: 3-5, (b) all of SEQ ID NOs: 6-8, or (c) all of SEQ ID NOs: 3-8. In a preferred embodiment, the polypeptide comprises the amino acid sequences of all of SEQ ID NOs: 3-8.

[0047] In an embodiment of the invention, the inventive polypeptide can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the polypeptide can comprise the amino acid sequence of (i) SEQ ID NO: 9 (variable region of a chain), (ii) SEQ ID NO: 10 (variable region of b chain), (iii) both of SEQ ID NOs: 9 and 10, (iv) SEQ ID NO: 1 1 (variable region of a chain), (v) SEQ ID NO: 12 (variable region of b chain), or (vi) both of SEQ ID NOs: 11 and 12. Preferably, the polypeptide comprises the amino acid sequences of both of SEQ ID NOs: 9 and 10 or both of SEQ ID NOs: 1 1 and 12.

[0048] In an embodiment of the invention, the inventive polypeptide can further comprise the constant region of the inventive TCR set forth above. In this regard, the polypeptide can further comprise the amino acid sequence of SEQ 1D NO: 17 (WT murine constant region of a chain), SEQ ID NO: 18 (WT murine constant region of b chain), SEQ ID NO: 15,

(substituted murine constant region of a chain), SEQ ID NO: 16 (substituted murine constant region of b chain), both SEQ ID NOs: 15 and 16, or both SEQ ID NOs: 17 and 18.

Preferably, the polypeptide further comprises the amino acid sequences of both of SEQ ID NOs: 15 and 16 or both of SEQ ID NO: 17 and 18 in combination with any of the CDR regions or variable regions described herein with respect to other aspects of the invention.

[0049] In an embodiment of the invention, the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 15, wherein: (i) X at position 48 of SEQ ID NO: 15 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 15 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 15 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 16, wherein X at position 57 of SEQ ID NO: 16 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, one or both of SEQ ID NOs: 15 and 16 of the polypeptide are as defined in any one of Tables 1-3.

[0050] In an embodiment of the invention, the inventive polypeptide can comprise the entire length of an a or b chain of the TCR described herein. In this regard, the inventive polypeptide can comprise the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, both of SEQ ID NOs: 21 and 22, or both of SEQ ID NOs: 23 and 24. Preferably, the polypeptide comprises the amino acid sequences of both of SEQ ID NOs: 21 and 22 or both of SEQ ID NOs: 23 and 24.

[0051] In an embodiment of the invention, the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 21 , wherein: (i) X at position 181 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 245 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 247 of SEQ ID NO: 21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 248 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 22, wherein X at position 188 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, any one or more of SEQ ID NOs: 21-22 of the polypeptide are as defined in any one of Tables 1-3.

[0052] An embodiment of the invention further provides a protein comprising at least one of the polypeptides described herein. By "protein" is meant a molecule comprising one or more polypeptide chains.

[0053] In an embodiment, the protein of the invention can comprise a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 3-5 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 6-8.

[0054] In another embodiment of the invention, the protein may comprise (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NO: 9 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NO: 10; or (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NO: 11 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NO: 12.

[0055] The inventive protein may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in an embodiment of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 18. In an embodiment of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 15 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 16.

[0056] In an embodiment of the invention, the protein comprises: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 15, wherein: (i) X at position 48 of SEQ ID NO: 15 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 15 is Ser, Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 15 is Met, Ala, Val, Leu, lie, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 15 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16, wherein X at position 57 of SEQ ID NO: 16 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, one or both of SEQ ID NOs: 15 and 16 of the protein are as defined in any one of Tables 1-3.

[0057] Alternatively or additionally, the protein of an embodiment of the invention can comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:

21 , wherein: (i) X at position 181 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 245 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 247 of SEQ 1D NO: 21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 248 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 188 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, one or both of SEQ ID NOs: 21-22 are as defined in any one of Tables 1 -3. In an embodiment of the invention, the protein may comprise a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 24.

|0058] The protein of the invention can be a TCR. Alternatively, if, for example, the protein comprises a single polypeptide chain comprising the amino acid sequences of both SEQ ID NOs: 21 and 22, both SEQ ID NOs: 23 and 24, or if the first and/or second polypeptide chain(s) of the protein further comprise(s) other amino acid sequences, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof, then the inventive protein can be a fusion protein. In this regard, an embodiment of the invention also provides a fusion protein comprising at least one of the inventive polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CDla, CDlb, CDlc, CDld, etc.

[0059] The fusion protein can comprise one or more copies of the inventive polypeptide and/or one or more copies of the other polypeptide. For instance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copies of the inventive polypeptide and/or of the other polypeptide. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods.

[0060] In some embodiments of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the a chain and the b chain. In this regard, the TCRs, polypeptides, and proteins of the invention may further comprise a linker peptide. The linker peptide may advantageously facilitate the expression of a recombinant TCR, polypeptide, and/or protein in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may be a P2A linker comprising the amino acid sequence of SEQ ID NO:25. Upon expression of the construct including the linker peptide by a host cell, the linker peptide may be cleaved, resulting in separated a and b chains. In an embodiment of the invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length a chain, a full-length b chain, and a linker peptide positioned between the a and b chains.

[0061] The protein of the invention can be a recombinant antibody, or an antigen binding portion thereof, comprising at least one of the inventive polypeptides described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or an antigen binding portion thereof. The polypeptide of an antibody, or antigen binding portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment of an antibody, etc. The polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention. The polypeptide of an antibody, or an antigen binding portion thereof, can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0062] Included in the scope of the invention are functional variants of the inventive TCRs, polypeptides, or proteins described herein. The term“functional variant,” as used herein, refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, which functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants encompass, for example, those variants of the TCR, polypeptide, or protein described herein (the parent TCR, polypeptide, or protein) that retain the ability to specifically bind to EGFR E746_A750del for which the parent TCR has antigenic specificity or to which the parent polypeptide or protein specifically binds, to a similar extent, the same extent, or to a higher extent, as the parent TCR, polypeptide, or protein. In reference to the parent TCR, polypeptide, or protein, the functional variant can, for instance, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein, respectively.

[0063] The functional variant can, for example, comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc. [0064] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid

substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent TCR, polypeptide, or protein.

[0065] The TCR, polypeptide, or protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not materially change the biological activity of the TCR, polypeptide, or protein. In this regard, the inventive TCR, polypeptide, or protein can, for example, consist essentially of the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, both of SEQ ID NOs: 21-22 or both of SEQ ID NO: 23-24. Also, for instance, the inventive TCRs, polypeptides, or proteins can consist essentially of the amino acid sequence(s) of (i) SEQ ID NO: 9, (ii) SEQ ID NO: 10, (iii) both of SEQ ID NOs: 9 and 10, (iv) SEQ ID NO: 11, (v) SEQ ID NO: 12, or (vi) both of SEQ ID NO: 1 1 and 12. Furthermore, the inventive TCRs, polypeptides, or proteins can consist essentially of the amino acid sequences of (a) any one or more of SEQ ID NOs: 3-8; (b) all of SEQ ID NO: 3-5; (c) all of SEQ ID NO: 6-8; or (d) all of SEQ ID NOs: 3-8.

[0066] The TCRs, polypeptides, and proteins of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the TCRs, polypeptides, or proteins retain their biological activity, e.g., the ability to specifically bind to EGFR E746_A750del; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc. For example, the polypeptide can be in the range of from about 50 to about 5000 amino acids long, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides.

[0067] The TCRs, polypeptides, and proteins of the invention can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are 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, b-phenylserine b-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, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbomane)-carboxylic acid, a,g-diaminobutyric acid, a,b-diaminopropionic acid, homophenylalanine, and a-tert- butyl glycine.

[0068] The TCRs, polypeptides, and proteins of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.

[0069] The TCR, polypeptide, and/or protein of the invention can be obtained by methods known in the art such as, for example, de novo synthesis. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 ^th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and/or proteins described herein can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp.

(Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this respect, the inventive TCRs, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.

[0070] Included in the scope of the invention are conjugates, e.g., bioconjugates, comprising any of the inventive TCRs, polypeptides, or proteins (including any of the functional portions or variants thereof), nucleic acids, recombinant expression vectors, host cells, populations of host cells, or antibodies, or antigen binding portions thereof.

Conjugates, as well as methods of synthesizing conjugates in general, are known in the art.

[0071] An embodiment of the invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide 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). It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.

[0072] Preferably, the nucleic acids of the 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 can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.

For purposes herein, the replication can be in vitro replication or in vivo replication.

[0073] 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 et ah, supra. For example, a nucleic acid can 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). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N ⁶ -isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2, 2-dimethyl guanine, 2- methyladenine, 2-methylguanine, 3 -methyl cytosine, 5-methylcytosine, N ⁶ -substituted adenine, 7-methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N ⁶ -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0074] The nucleic acid can comprise any nucleotide sequence which encodes any of the TCRs, polypeptides, or proteins described herein. In an embodiment of the invention, the nucleic acid may comprise the nucleotide sequences of any one of SEQ ID NOs: 26-29 (Table 4). In an embodiment of the invention, the nucleic acid comprises the nucleotide sequences of both of SEQ ID NOs: 26-27 or both of SEQ ID NO: 28-29.

TABLE 4

[0075] In an embodiment of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound to any 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.

Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. For example, SEQ ID NOs: 28-29 are codon optimized for expression in human cells.

[0076] The 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.

[0077] The nucleotide sequence which hybridizes under stringent conditions preferably hybridizes under high stringency conditions. By“high stringency conditions” is meant that the nucleotide sequence specifically 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 can be rendered more stringent by the addition of increasing amounts of formamide.

[0078] The invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid may consist essentially of any of the nucleotide sequences described herein.

[0079] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. In an embodiment of the invention, the recombinant expression vector comprises a nucleotide sequence encoding the a chain, the b chain, and linker peptide.

[0080] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non- naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

[0081] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as lOTIO, lOT 1 1 , ZapIl (Stratagene), lEMBE4, and lNMI 149, also can be used. Examples of plant expression vectors include pBIOl, pBHOl .2, pBHOl .3, pBH2l and rB1N19

(Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector. In an especially preferred embodiment, the recombinant expression vector is an MSGV1 vector.

[0082] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook et ah, supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 m plasmid, l, SV40, bovine papillomavirus, and the like.

[0083] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0084] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G4l 8 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0085] The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the TCR, polypeptide, or protein, or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the TCR, polypeptide, or protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.

[0086] The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0087] Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0088] In an embodiment of the invention, the recombinant expression vector comprises a nucleotide sequence encoding the a chain, the b chain, and linker peptide. For example, in an embodiment, the recombinant expression vector comprises a nucleotide sequence encoding the full-length alpha and beta chains of the inventive TCR, polypeptide, or protein with a linker positioned between them. In an embodiment, the recombinant expression vector comprises a nucleotide sequence encoding an alpha chain and a beta chain of any of the TCRs, polypeptides, or proteins described herein, wherein the nucleotide sequence encoding the beta chain is positioned 5’ of the nucleotide sequence encoding the alpha chain. In this regard, the nucleotide sequence encoding the alpha chain may be positioned 3’ of the nucleotide sequence encoding the beta chain. Examples of nucleotide sequences, wherein the nucleotide sequence encoding the beta chain is positioned 5’ of the nucleotide sequence encoding the alpha chain include SEQ ID NO: 31 (expression cassette) and SEQ ID NO: 33 (MSGVI vector including the expression cassette).

[0089] In another embodiment, the nucleotide sequence encoding the beta chain is positioned 3’ of the nucleotide sequence encoding the alpha chain. In this regard, the nucleotide sequence encoding the alpha chain may be positioned 5’ of the nucleotide sequence encoding the beta chain. Examples of nucleotide sequences, wherein the nucleotide sequence encoding the alpha chain is positioned 5’ of the nucleotide sequence encoding the beta chain include SEQ ID NO: 30 (expression cassette) and SEQ ID NO: 32 (MSGVI vector including the expression cassette).

[0090] Another embodiment of the invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0091] For purposes herein, the T cell can 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 can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4 ⁺ /CD8 ⁺ double positive T cells, CD4 ⁺ helper T cells, e.g.,

Thi and Th ₂ 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.

[0092] Also provided by the invention is a population of cells comprising at least one host cell described herein. The population of cells can 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 can 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 can 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 of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0093] In an embodiment of the invention, the numbers of cells in the population may be rapidly expanded. Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent

8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012/0244133; Dudley et ah, J. Immunother., 26:332-42 (2003); and Riddell et ah, J. Immunol. Methods, 128:189-201 (1990). In an embodiment, expansion of the numbers of T cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC).

[0094] The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof), can be isolated and/or purified. The term "isolated," as used herein, means having been removed from its natural environment. The term "purified," as used herein, means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or can be about 100%.

[0095] The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof), all of which are

collectively referred to as "inventive TCR materials" hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof), described herein, and a pharmaceutically acceptable earner. The inventive pharmaceutical compositions containing any of the inventive TCR materials can comprise more than one inventive TCR material, e.g., a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition can comprise an inventive TCR material in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0096] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the particular inventive TCR material under consideration. Methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22 ^nd Ed.,

Pharmaceutical Press (2012). It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0097] The choice of carrier will be determined in part by the particular inventive TCR material, as well as by the particular method used to administer the inventive TCR material. Accordingly, there are a variety of suitable formulations of the phannaceutical composition of the invention. Suitable formulations may include any of those for parenteral,

subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or interperitoneal administration. More than one route can be used to administer the inventive TCR materials, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0098] Preferably, the inventive TCR material is administered by injection, e.g., intravenously. When the inventive TCR material is a host cell (or population thereof) expressing the inventive TCR, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0099] For purposes of the invention, the amount or dose (e.g., numbers of cells when the inventive TCR material is one or more cells) of the inventive TCR material administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the inventive TCR material should be sufficient to bind to a cancer antigen (e.g., EGFR E746_A750del), or detect, treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive TCR material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[00100] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or IFN-g is secreted by T cells expressing the inventive TCR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-g is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0100] The dose of the inventive TCR material also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR material. Typically, the attending physician will decide the dosage of the inventive TCR material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR material to be administered, route of administration, and the severity of the cancer being treated. In an embodiment in which the inventive TCR material is a population of cells, the number of cells administered per infusion may vary, e.g., from about 1 x 10 ⁶ to about 1 x 10 ¹² cells or more. In certain embodiments, fewer than 1 x 10 ⁶ cells may be administered.

[0101] One of ordinary skill in the art will readily appreciate that the inventive TCR materials of the invention can be modified in any number of ways, such that the therapeutic or prophylactic efficacy of the inventive TCR materials is increased through the modification. For instance, the inventive TCR materials can be conjugated either directly or indirectly through a bridge to a chemotherapeutic agent. The practice of conjugating compounds to a chemotherapeutic agent is known in the art. One of ordinary skill in the art recognizes that sites on the inventive TCR materials, which are not necessary for the function of the inventive TCR materials, are suitable sites for attaching a bridge and/or a chemotherapeutic agent, provided that the bridge and/or chemotherapeutic agent, once attached to the inventive TCR materials, do(es) not interfere with the function of the inventive TCR materials, i.e., the ability to bind to EGFR E746__A750del or to detect, treat, or prevent cancer. [0102] It is contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells can be used in methods of treating or preventing cancer. Without being bound to a particular theory, the inventive TCRs are believed to bind specifically to EGFR E746_A750del, such that the TCR (or related inventive polypeptide or protein), when expressed by a cell, is able to mediate an immune response against a target cell expressing EGFR E746_A750del. In this regard, the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0103] An embodiment of the invention provides any of the pharmaceutical

compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0104] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect !n this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.

Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset of the cancer, or a symptom or condition thereof. Alternatively or additionally,“prevention” may encompass preventing or delaying the recurrence of cancer, or a symptom or condition thereof.

[0105] Also provided is a method of detecting the presence of cancer in a mammal. The method comprises (i) contacting a sample comprising one or more cells from the mammal with any of the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions described herein, thereby forming a complex, and (ii) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal.

[0106] With respect to the inventive method of detecting cancer in a mammal, the sample of cells can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction.

[0107] For purposes of the inventive method of detecting cancer, the contacting can take place in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro.

[0108] Also, detection of the complex can occur through any number of ways known in the art. For instance, the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells, described herein, can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).

[0109] For purposes of the inventive methods, wherein host cells or populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0110] With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer. A preferred cancer is lung cancer. Preferably, the lung cancer is NSCLC. In an embodiment of the invention, the cancer is a cancer which expresses a mutated EGFR amino acid sequence with a deletion of amino acid residues 746-750, wherein amino acid residues 746-750 are defined by reference to SEQ ID NO: 1.

[0111] The mammal referred to in the inventive methods can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order

Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0112] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0113] This example demonstrates the isolation of a TCR having antigenic specificity for the mutated human EGFR amino acid sequence of AIKTSPKANKEIL (SEQ ID NO: 36).

[0114] TIL were isolated from a NSCLC patient. T cells which recognize EGFR E746_A750del were isolated from the TIL. Nucleotide sequences which encode the full length alpha and beta chains (SEQ ID NOs: 26-27, respectively) were isolated from the anti- EGFR E746 A750del T cells by single cell reverse transcription polymerase chain reaction (RT-PCR). The amino acid sequences of the TCR are set forth in Table 5.

TABLE 5

EXAMPLE 2

[0115] This example demonstrates the construction of a retroviral vector encoding the TCR of Example 1 with the modifications described in this Example.

[0116] To facilitate cloning of the TCR expression cassette into the MSGV1 vector site, an alanine was inserted into the second position of each the alpha and the beta chains, resulting in the alpha and beta chain variable region amino acid sequences of SEQ ID NOs:

11 and 12, respectively.

[0117] The human b chain constant region was replaced with the murine b chain constant region. The human a chain constant region was replaced with the murine a chain constant region. Without being bound to a particular theory or mechanism, it is believed that replacing the constant regions of the human TCRa and TCRfl chains with the corresponding murine constant regions improves TCR expression and functionality (Cohen et ah, Cancer Res., 66(17): 8878-86 (2006)).

[0118] In addition, the murine TCRa and TCRb constant chains were cysteine-modified. Transmembrane hydrophobic mutations were also introduced into the murine TCRa constant chain. Without being bound to a particular theory or mechanism, it is believed that these modifications result in preferential pairing of the introduced TCR chains and enhanced TCR surface expression and functionality (Cohen et ah, Cancer Res., 67(8):3898-903 (2007); Haga-Friedman et ah, J. Immu., 188: 5538-5546 (2012)).

[0119] After the modifications described in this Example were made, the full-length TCR a chain comprised the amino acid sequence of SEQ ID NO: 23, and the full-length TCR b chain comprised the amino acid sequence of SEQ ID NO: 24.

[0120] The nucleotide sequences encoding the modified TCR a and b chains were codon optimized for expression in human cells, resulting in the nucleotide sequences of SEQ ID NO: 28 (codon optimized full-length modified a chain) and SEQ ID NO: 29 (codon- optimized full-length modified b chain).

[0121] Nucleotide sequences encoding the modified TCR a and b chains (SEQ ID NOs: 28 and 29) were cloned into an MSGV1 retroviral vector with one of the following two expression cassette configurations: (1) 5’-TCR alpha chain-linker-TCR beta chain-3’ (Construct 1 ; SEQ ID NO: 30) or (2) 5’-TCR beta chain-linker-TCR alpha chain-3’

(Construct 2; SEQ ID NO: 31). The P2A linker comprised the amino acid sequence of SEQ ID NO: 25. The vector comprising construct 1 comprised the nucleotide sequence of SEQ ID NO: 32. The vector comprising construct 2 comprised the nucleotide sequence of SEQ ID NO: 33.

EXAMPLE 3

[0122] This example demonstrates that peripheral blood T cells transduced with the modified TCR a and b chain of Example 2 specifically recognize autologous dendritic cells pulsed with mutated EGFR peptide.

[0123] Peripheral blood lymphocytes (PBL) were retrovirally transduced as described in one of (l)-(4) below:

1. The cells were transduced with a vector encoding an HLA- A3 -restricted KRAS Gl2V-reactive TCR (irrelevant TCR, negative control);

2. Retrovirus containing a vector encoding the modified TCR alpha chain of

Example 2 (SEQ ID NO: 23) and retrovirus containing a vector encoding the modified TCR beta chain of Example 2 (SEQ ID NO: 24) were separately produced and mixed. The cells were transduced with the mixture of retroviruses.

3. The cells were transduced with a vector comprising Construct 1 of Example 2 (alpha chain and beta chain were cloned in the MSGV1 vector in the order of alpha chain-linker-beta chain) (SEQ ID NO: 32).

4. The cells were transduced with a vector comprising Construct 2 of Example 2 (alpha chain and beta chain were cloned in the MSGV1 vector in the order of beta chain-linker-alpha chain) (SEQ ID NO: 33).

[0124] The transduced cells were co-cultured with autologous dendritic cells which had been pulsed with 1 mM of the wild-type (WT) EGFR peptide of

PEGEKVKIPVAIKELREATSPKANK (SEQ ID NO: 34) or the mutated EGFR (E746_A750 del) peptide of PEGEKVKIPVAIKTSPKANKEILDE (SEQ 1D NO: 35). Transduced cells cultured in medium alone served as a control.

[0125] IFN-g secretion was measured. The results are shown in Figure 1. Each of transductions (2)-(4) above conferred recognition of the mutated EGFR (E746 A750 del) peptide-pulsed autologous dendritic cells. Negative control transduction (1) failed to confer recognition of the mutated EGFR (E746_A750 del) peptide-pulsed autologous dendritic cells. In this experiment, the order of TCR alpha-link er-TCR beta (Construct 1) performed better than the others.

EXAMPLE 4

[0126] This example demonstrates that peripheral blood T cells transduced with the modified TCR a and b chain of Example 2 specifically recognize NSCLC cell lines, which express the E746_A750 deletion, in an HLA-DPA1 *02:01, DPBl *0l :0l-restricted manner.

[0127] PC-9 and HCC827 are NSCLC cell lines which express EGFR with the

E746_A750 deletion. The cell lines were retro virally transduced with a combination of (i) HLA-DPA1 *01 :03 and DPB1 *01 :01 or (ii) HLA-DPA1 *02:01 and DPB1 *01 :01.

[0128] PBL were retro virally transduced as described in one of (l)-(4) of Example 3.

[0129] The transduced cells were co-cultured with each one of the transduced cell lines. Transduced PBL were co-cultured with untransduced cell line as a control.

[0130] IFN-g was measured. The results are shown in Figure 2 (PC-9) and Figure 3 (HCC827). As shown in Figures 2-3, the TCR is HLA-DPA1 *02:01, DPB 1 *01 :01 -restricted. The T cells transduced with the modified TCR a and b chain of Example 2 specifically recognized NSCLC cell lines, which express the E746_A750 deletion, in an FILA- DPA1 *02:01, DPBl *01 :01-restricted manner.

EXAMPLE 5

[0131] This example demonstrates that peripheral blood T cells transduced with the modified TCR a and b chain of Example 2 recognize the EGFR E746_A750del peptide of AIKTSPKANKEIL (SEQ ID NO: 36).

[0132] A series of truncated EGFR E746_A750del peptides SEQ ID NOs: 35 and 40-50 (shown in Figure 4A and Table B) were synthesized. Figure 4B shows the location of the deleted wild-type EGFR amino acid residues 746-750 (ELREA (SEQ ID NO: 51)) in relation to the mutated EGFR E746_A750del peptide AIKTSPKANKEIL (SEQ ID NO: 36). TABLE B

[0133] PBL were retrovirally transduced with a vector comprising Construct 1 of Example 2 (alpha chain and beta chain were cloned in the MSGY1 vector in the order of alpha chain-linker-beta chain) (SEQ ID NO: 32). The transduced cells were co-cultured with autologous dendritic cells which had been pulsed with 1 mM of one of the EGFR

E746_A750del peptides of SEQ ID NOs: 35 and 40-50. IFN-g was measured. The results are shown in Figure 4A.

[0134] As shown in Figure 4A, the shortest peptide recognized by the transduced cells was AIKTSPKANKEILDE (SEQ ID NO: 44). When the N-terminal AI is removed (see KTSPKANKE1LDE (SEQ ID NO: 45), recognition was lost. The peptide

PEGEKVKIPVAIKTSPKANKEIL (SEQ ID NO: 46) was also recognized by the transduced cells. But when the C-terminal“IL” were removed (see PEGEKVKIPVAIKTSPKANKE (SEQ ID NO: 47)), recognition was lost.

[0135] Based on these data, the TCR epitope was narrowed down to the l3-mer peptide of AIKTSPKANKEIL (SEQ ID NO: 36). It is believed that there are four possibilities for the minimal epitope: AIKTSPKANKEIL (SEQ ID NO: 36), AIKTSPKANKEI (SEQ ID NO: 37), IKTSPKANKEIL (SEQ ID NO: 38), and IKTSPKANKEI (SEQ ID NO: 39). So, the minimal epitope may be as short as 1 1 amino acids.

[0136] Although the minimal epitope may provide the minimum sequence for recognition, the minimal peptide might not provide the strongest recognition. As shown in Figure 4A, the N-terminal PV of PVAIKTSPKANKEILDE (SEQ ID NO: 43) is not necessary for recognition, but removing PV lowers the recognition (see

AIKTSPKANKEILDE (SEQ ID NO: 44). Without being bound to a particular theory or mechanism, it is believed that the N-terminal PV of PVAIKTSPKANKEILDE (SEQ ID NO: 43) may contribute to the recognition by the TCR.

[0137] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0138] The use of the terms“a” and“an” and“the” and“at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term“at least one” followed by a list of one or more items (for example,“at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms“comprising,”“having,”“including,” and“containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0139] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.