T cell receptors targeting e545k or n345k mutation in PIK3ca

Abstract

Disclosed are isolated or purified T cell receptors (TCRs) having antigenic specificity for human PIK3CAE545K or human PIK3CAN345K. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Also disclosed are methods of treating or preventing cancer in a mammal.

Description

[0001]Leydig 772435 HHS E-076-2024-0-PC-01 1 T CELL RECEPTORS TARGETING E545K OR N345K MUTATION IN PIK3CA CROSS-REFERENCE TO RELATED APPLICATION This patent application claims the benefit of U.S. Provisional Patent Application No.63 / 565,764, filed March 15, 2024, which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under project number BC010985 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 61,735 Byte XML file named “772435.XML,” dated February 10, 2025. BACKGROUND OF THE INVENTION 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, pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Accordingly, there exists an unmet need for additional treatments for cancer. BRIEF SUMMARY OF THE INVENTION An aspect of the invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence, wherein the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 2-7; (2) all of SEQ ID NOs: 16-21; or (3) all of SEQ ID NOs: 30-35. Leydig 772435 HHS E-076-2024-0-PC-01 2 Further aspects of the invention polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the TCRs of the invention. Still further aspects of the invention provide methods of inducing an immune response against a cancer in a mammal, and methods of treating or preventing cancer in a mammal. Additional aspects of the invention provide methods of producing a host cell expressing the TCR and methods of producing the TCR, polypeptide, or protein. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Figures 1A-1D are a set of graphs showing the percentage of effector cells (positive for expression of murine TCR (mTCR) and cluster of differentiation 8 (CD8)) that upregulated 4-1BB expression following co-culture with target cells. The effector cells were healthy donor peripheral blood lymphocytes (PBL) independently transduced with a retroviral vector encoding the 4367_N345K_TCR-1 (1A, 1C) or TCR-2 (1B, 1D) and tested for specificity against the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation N345K. For Figures 1A-1B, the effector cells were cultured overnight with autologous antigen presenting cells pulsed with titrated concentrations of mutated PIK3CA (circles) or wild type PIK3CA long peptides (squares). For Figures 1C-1D, the effector cells were cultured overnight with COS-7 cells transduced with each of the 4367 (autologous) human leukocyte antigen (HLA) molecules and pulsed with N345K peptides or vehicle dimethyl sulfoxide (DMSO). The parent gates for Figures 1A-1B were TCR-1 mTCR+ / CD3+ / CD4+ and TCR-2 CD3+mTCR+. The parent gates for Figures 1C-1D were TCR-1 CD3+mTCR+ and TCR-2 CD3+. Figure 1E is a graph showing the number of INF-γ spots (ELISPOT assay) measured following co-culture with target cells. The effector cells were healthy donor PBL independently transduced with a retroviral vector encoding the 4367_N345K_TCR-1, -TCR- 2, or mock TCR-transduced T cells (T cells subjected to same procedure of retoviral transduction but without TCR plasmid, used as negative control), and tested for specificity against the PIK3CA mutation N345K. The effector cells were cultured overnight with modified cells from cell line HTB-114TM. Cell line HTB-114TMwas modified as indicated in figure: irrelevant HLA introduced (irr HLA), HLA DPB1*04:01 (HLA-DP+), HLA DP and wild type full length PIK3CA gene (HLA-DP+wt gene), HLA DP and mutated full length Leydig 772435 HHS E-076-2024-0-PC-01 3 PIK3CA gene (HLA-DP+mut gene), HLA and DMSO (HLA-DP+DMSO), HLA DP and an irrelevant peptide control (HLA-DP+irr pept), HLA DP and wild type PIK3CA peptide (HLA-DP+wt pept), HLA DP and PIK3CA N345K mutant peptide (HLA-DP+mut pept); control (media). TNTC = too numerous to count. Figure 1F-1I are a set of graphs showing the results of a cytotoxicity assay of mock or TCR-transduced lymphocytes with cell lines HTB-114TM(1F, 1G) and CCL-225TM(1H, 1I) which were modified to express human allele DPB1*04:01 and N345K full length PIK3CA gene. Mock modified cell lines (1G, 1I) were used as controls. Figure 2A is a graph showing the percentage of effector cells (positive for expression of mTCR and CD8) that upregulated 4-1BB expression following co-culture with target cells. The effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4211_E545K_TCR-1 and tested for specificity against the PIK3CA mutation E545K. The effector cells were cultured overnight with autologous antigen presenting cells pulsed with titrated concentrations of mutated (EITKQEKDFLW, SEQ ID NO: 55) or wild type (EITEQEKDFLW, SEQ ID NO: 52) PIK3CA epitope 3. Figure 2B is a graph showing the number of IFN-gamma spots produced following co-culture of effector cells with target cells. The effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4211_E545K_TCR-1 and tested for specificity against the PIK3CA mutation E545K. The effector cells were cultured overnight with COS-7 cells transduced with each of the 4211 (autologous) HLA molecules and pulsed with E545K peptide (EITKQEKDFLW, SEQ ID NO: 55) or vehicle (DMSO). Figure 2C is a graph showing the number of IFN-gamma spots produced following co-culture of effector cells with target cells. The effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4211_E545K_TCR-1 and tested for specificity against the PIK3CA mutation E545K. The effector cells were cultured overnight with modified, as indicated, cell line MCF7 which natively expresses the E545K PIK3CA protein. Figures 2D-2G are a set of graphs showing the results of a cytotoxicity assay of mock or 4211_E545K_TCR-1-transduced lymphocytes modified to express DRB1*04:01 (2D, 2F) allele or A03+ (control) cell line MCF7 (2E, 2G). Figure 3 is a schematic diagram of a process that can be used to generate retroviral products to transduce PBL for patient treatment using transiently transfected cells. Leydig 772435 HHS E-076-2024-0-PC-01 4 Figure 4 is a graph showing the of TCR transduced cells in post- treatment PBL from patient 4367 who had not been previously treated with ACT. The black bar at day 0 indicates the percentage of TCR transduced cells in the patient’s final infusion product. Figure 5 is a graph showing the functional characterization of infused cells: multiple cytokine analyses for Patient 4367. Supernatants from co-cultures of infused T cell products and autologous DCs pulsed with wild type and mutant peptides were evaluated for levels of the indicated cytokines and chemokines using MACSPLEX™ kits from Miltenyi Biotech. The concentration of peptide was selected based on IFNγ assays: 4367 PIK3CA (LP 1 μg / ml). Figure 6 is a plot showing the level of recovery of T regulatory cells at 1 week and 1 month post ACT for Patient 4367. PBL samples from time points as close to 1 week and 1 month as possible were thawed and rested overnight without IL-2 prior to FACS. Cells were stained with antibodies against human CD3, CD4, and CD8. After permeabilization with the BD PHARMINGEN™ Transcription Factor Buffer Set, cells were stained with anti-human FoxP3. Analyses were performed on lymphocyte-size gated, CD3+ T cells. Figures 7A-7B are a set of graphs showing the level of cytokines in post-treatment serum samples (IFNγ (7A) and IL-2 (7B)) for Patient 4367. IFNγ and IL-2 protein levels in consecutive serum samples from patient 4367 were determined using commercially available ELISA kits (Invitrogen). DETAILED DESCRIPTION OF THE INVENTION Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the catalytic subunit of the enzyme phosphatidylinositol 3-kinase. Wild-type WT (normal) human full-length PIK3CA comprises the amino acid sequence of SEQ ID NO: 1. PIK3CA is encoded by the PIK3CA gene. PIK3CA is the third most common mutated gene in epithelial cancers with a high prevalence in endometrial (about 42%) and breast (about 36%) cancers. The E545K mutation is among the most frequent PIK3CA mutations (about 23% of all PIK3CA gene alterations). About 8% of all patients with advanced breast cancer exhibit the E545K mutation. The N345K mutation is among the most common PIK3CA mutations (about 3% among all PIK3CA gene alterations). About 3% of all patients with advanced breast cancer express the N345K mutation. Leydig 772435 HHS E-076-2024-0-PC-01 5 An aspect of the invention an isolated or purified T cell receptor (TCR) having antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence (hereinafter, “mutated PIK3CA”). Hereinafter, references to a “TCR” also refer to functional portions and functional variants of the TCR, unless specified otherwise. Human full-length PIK3CA with the N345K mutation comprises the amino acid sequence of SEQ ID NO: 60. Human full-length PIK3CA with the E545K mutation comprises the amino acid sequence of SEQ ID NO: 61. Mutations of PIK3CA are defined herein by reference to the amino acid sequence of full-length, WT PIK3CA (SEQ ID NO: 1). Mutations of PIK3CA are described herein by reference to the amino acid residue present at a particular position, followed by the position number, followed by the amino acid with which that residue has been replaced in the particular mutation under discussion. A PIK3CA amino acid sequence (e.g., a PIK3CA peptide) may comprise fewer than all of the amino acid residues of the full-length, WT PIK3CA protein. Accordingly, the position numbers are defined herein by reference to the WT full-length PIK3CA protein (namely, SEQ ID NO: 1) with the understanding that the actual position of the corresponding residue in a particular example of a PIK3CA amino acid sequence may be different. Because the positions are as defined by SEQ ID NO: 1, the term “E545K” indicates that the glutamic acid present at position 545 of SEQ ID NO: 1 is replaced by lysine, and “N345K” indicates that the asparagine present at position 345 of SEQ ID NO: 1 has been replaced with lysine. For example, when a particular example of a PIK3CA amino acid sequence is, e.g., ALRIKILCATYVNVNIRDIDKIYVR (SEQ ID NO: 49) (an exemplary WT PIK3CA peptide corresponding to contiguous amino acid residues 333 to 357 of SEQ ID NO: 1), “N345K” refers to a substitution of the underlined asparagine in SEQ ID NO: 49 with lysine, even though the actual position of the underlined tyrosine in SEQ ID NO: 49 is 13. Human PIK3CA amino acid sequences with the E545K mutation are hereinafter referred to as “E545K” or “PIK3CAE545K.” Human PIK3CA amino acid sequences with the N345K mutation are hereinafter referred to as “N345K” or “PIK3CAN345K.” As used herein, “mutated PIK3CA” refers to human PIK3CAE545Kor human PIK3CAN345K, unless specified otherwise. In an aspect of the invention, the TCR has antigenic specificity for human PIK3CA with a mutation at position 545, as defined by SEQ ID NO: 1. The PIK3CA mutation at position 545 may be a missense mutation. Accordingly, the mutation at position 545 may be a substitution of the native (WT) glutamic acid residue present at position 545 Leydig 772435 HHS E-076-2024-0-PC-01 6 with any amino acid residue other than acid. In an aspect of the invention, the TCR has antigenic specificity for a human PIK3CAE545Kamino acid sequence. For example, the TCR may have antigenic specificity for the human PIK3CAE545Kamino acid sequence of EITKQEKDFLW (SEQ ID NO: 55). In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human PIK3CA amino acid sequence of EITEQEKDFLW (SEQ ID NO: 52). In an aspect of the invention, the TCR has antigenic specificity for human PIK3CA with a mutation at position 345, as defined by SEQ ID NO: 1. The PIK3CA mutation at position 345 may be a missense mutation. Accordingly, the mutation at position 345 may be a substitution of the native (WT) asparagine residue present at position 345 with any amino acid residue other than asparagine. In an aspect of the invention, the TCR has antigenic specificity for a human PIK3CAN345Kamino acid sequence. For example, the TCR may have antigenic specificity for the human PIK3CAN345Kamino acid sequence of ALRIKILCATYVKVNIRDIDKIYVR (SEQ ID NO: 48). In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human PIK3CA amino acid sequence of ALRIKILCATYVNVNIRDIDKIYVR (SEQ ID NO: 49). In an aspect of the invention, the inventive TCRs may be able to recognize mutated PIK3CA in an HLA (human leukocyte antigen)-molecule-dependent manner. “HLA-molecule-dependent manner,” as used herein, means that the TCR elicits an immune response upon binding to mutated PIK3CA presented by a HLA molecule, which HLA molecule is expressed by the patient from which the TCR was isolated. The inventive TCRs may be able to recognize mutated PIK3CA that is presented by the applicable HLA molecule and may bind to the HLA molecule in addition to mutated PIK3CA. In an aspect of the invention, the inventive TCRs are able to recognize E545K presented by an HLA Class II molecule. In this regard, the TCR may elicit an immune response upon binding to E545K presented by a HLA Class II molecule. The inventive TCRs are able to recognize E545K that is presented by an HLA Class II molecule and may bind to the HLA Class II molecule in addition to E545K. In an aspect of the invention, the HLA Class II molecule is an HLA-DR heterodimer. The HLA-DR heterodimer is a cell surface receptor including an α chain and a β chain. The HLA-DR α chain is encoded by the HLA-DRA gene. The HLA-DR β chain is encoded by the HLA-DRB1 gene, the HLA-DRB3 gene, HLA-DRB4 gene, or the HLA- DRB5 gene. Examples of molecules encoded by the HLA-DRB1 gene may include, but are Leydig 772435 HHS E-076-2024-0-PC-01 7 not limited to, HLA-DR1, HLA-DR2, HLA- HLA-DR4, HLA-DR5, HLA-DR6, HLA- DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA- DR14, HLA-DR15, HLA-DR16, and HLA-DR17. The HLA-DRB3 gene encodes HLA- DR52. The HLA-DRB4 gene encodes HLA-DR53. The HLA-DRB5 gene encodes HLA- DR51. In an aspect, the alpha chain of the HLA Class II molecule is expressed by the HLA-DRA1*01:01 allele. In an aspect, the beta chain of the HLA Class II molecule is expressed by the HLA-DRB1*04:01 allele. In an aspect of the invention, the HLA Class II molecule is an HLA-DRB1:HLA-DRA heterodimer. In a preferred aspect, the HLA Class II molecule is a heterodimer of an HLA-DRA1*01:01 chain and an HLA-DRB1*04:01 chain. In an especially preferred aspect, the mutated PIK3CA is E545K and the HLA Class II molecule is a heterodimer of an HLA-DRA1*01:01 chain and an HLA-DRB1*04:01 chain. The HLA-DRB1*04:01 allele is expressed by about 17 to about 20% of the U.S. Caucasian population. Accordingly, the inventive TCRs may broaden the population of patients that may benefit from treatment with adoptive cell therapy to include those patients with cancer that expresses E545K PIK3CA and the HLA-DRB1*04:01 allele. In an aspect of the invention, the inventive TCRs are able to recognize N345K presented by an HLA Class II molecule. In this regard, the TCR may elicit an immune response upon binding to N345K presented by a HLA Class II molecule. The inventive TCRs are able to recognize N345K that is presented by an HLA Class II molecule and may bind to the HLA Class II molecule in addition to N345K. In an aspect of the invention, the HLA Class II molecule is an HLA-DP heterodimer. The HLA-DP heterodimer is a cell surface receptor including an α chain and a β chain. The HLA-DP α chain is encoded by the HLA-DPA1 gene. The HLA-DP β chain is encoded by the HLA-DPB1 gene. In an aspect, the alpha chain of the HLA Class II molecule is expressed by the HLA-DPA1*01:03:01 allele. In an aspect, the beta chain of the HLA Class II molecule is expressed by the HLA-DPB1*04:01:01 allele. In an aspect of the invention, the HLA Class II molecule is an HLA-DPB1:HLA-DPA1 heterodimer. In a preferred aspect, the HLA Class II molecule is a heterodimer of an HLA-DPA1*01:03:01 chain and an HLA-DPB1*04:01:01 chain. In an especially preferred aspect, the mutated PIK3CA is N345K and the HLA Class II molecule is a heterodimer of an HLA-DPA1*01:03:01 chain and an HLA-DPB1*04:01:01 chain. The HLA-DPA1*01:03:01 / HLA-DPB1*04:01:01 heterodimer is expressed by about Leydig 772435 HHS E-076-2024-0-PC-01 8 the DPA1*01:03:01 / HLA-DPB1*04:01:01 heterodimer. The TCRs of the invention may provide any one or more of many advantages, including when expressed by cells used for adoptive cell therapy. Mutated PIK3CA 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 inventive TCRs may, advantageously, successfully treat or prevent mutated PIK3CA-positive cancers that do not respond to other types of treatment such as, for example, chemotherapy, surgery, or radiation. Additionally, the inventive TCRs may provide highly avid recognition of mutated PIK3CA, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with a vector encoding one or both of mutated PIK3CA and the applicable HLA molecule, pulsed with a PIK3CA peptide with the PIK3CA mutation, or a combination thereof). As explained above, PIK3CA is a common mutated gene in epithelial cancers. Accordingly, the inventive TCRs may increase the number of patients who may be eligible for treatment with immunotherapy. The inventive TCRs may provide improved in vivo efficacy in treating cancer expressing mutated PIK3CA as compared to other strategies that inhibit the activity of mutated PIK3CA, e.g., small molecule inhibitors or monoclonal antibodies. The phrase “antigenic specificity,” as used herein, means that the TCR can specifically bind to and immunologically recognize mutated PIK3CA with high avidity. For example, a TCR may be considered to have “antigenic specificity” for mutated PIK3CA if about 1 x 104to about 1 x 105T 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-γ upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated PIK3CA peptide (e.g., about 0.1 ng / mL to about 10,000 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 Leydig 772435 HHS E-076-2024-0-PC-01 9 ng / mL, 1,000 ng / mL, 5,000 ng / mL, 10,000 or a range defined by any two of the foregoing values) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated PIK3CA has been introduced such that the target cell expresses mutated PIK3CA. Cells expressing the inventive TCRs may also secrete IFN-γ upon co-culture with antigen-negative, applicable HLA molecule positive target cells pulsed with higher concentrations of mutated PIK3CA peptide. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated PIK3CA if T cells expressing the TCR secrete at least twice as much IFN-γ upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated PIK3CA peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated PIK3CA has been introduced such that the target cell expresses mutated PIK3CA as compared to the amount of IFN-γ expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR, co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the mutated PIK3CA peptide) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding an irrelevant protein has been introduced such that the target cell expresses the irrelevant protein, or (ii) untransduced T cells (e.g., derived from PBMC, which do not express the TCR) co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of mutated PIK3CA peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated PIK3CA has been introduced such that the target cell expresses mutated PIK3CA. IFN-γ secretion may be measured by methods known in the art such as, for example, enzyme- linked immunosorbent assay (ELISA). The concentration of pulsed peptide may be as described herein with respect to other aspects of the invention. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated PIK3CA if at least twice as many of the numbers of T cells expressing the TCR secrete IFN-γ upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated PIK3CA peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated PIK3CA has been introduced such that the target cell expresses mutated PIK3CA as compared to the numbers of negative control T cells that secrete IFN-γ. The concentration of Leydig 772435 HHS E-076-2024-0-PC-01 10 peptide and the negative control may be as herein with respect to other aspects of the invention. The numbers of cells secreting IFN-γ may be measured by methods known in the art such as, for example, enzyme-linked immunospot (ELISPOT) assay. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated PIK3CA if at least twice as many spots are detected by ELISPOT for the T cells expressing the TCR upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated PIK3CA peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated PIK3CA has been introduced such that the target cell expresses mutated PIK3CA as compared to the number of spots detected by ELISPOT for negative control T cells co- cultured with the same target cells. The concentration of peptide and the negative control may be as described herein with respect to other aspects of the invention. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated PIK3CA if T cells expressing the TCR upregulate expression of one or both of 4-1BB and OX40 as measured by, for example, flow cytometry after stimulation with target cells expressing mutated PIK3CA. An aspect of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) 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 mutated PIK3CA. In an aspect of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR)1, a CDR2, and a CDR3 of a TCR. In an aspect of the invention, the TCR comprises a first polypeptide chain comprising an α chain CDR1 (CDR1α), an α chain CDR2 (CDR2α), and an α chain CDR3 (CDR3α), and a second polypeptide chain comprising a β chain CDR1 (CDR1β), a β chain CDR2 (CDR2β), and a β chain CDR3 (CDR3β). In an aspect of the invention, the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 2-7 (4211_E545K_TCR-1); (2) all of SEQ ID NOs: 16-21 (4367_N345K_TCR-1); or (3) all of SEQ ID NOs: 30-35 (4367_N345K_TCR-2). Each one of the foregoing three collections of amino acid sequences in this paragraph sets forth the six CDR regions of each of three different TCRs having antigenic specificity for mutated human PIK3CA. The six amino acid Leydig 772435 HHS E-076-2024-0-PC-01 11 sequences in each collection correspond to CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of a TCR, respectively. The TCR may comprise the amino acid sequences of any one or more of: SEQ ID NOs: 2-7, 16-21, or 30-35. In an aspect of the invention, the TCR comprises an isolated or purified T cell receptor (TCR) having antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence, wherein the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 2-7; (2) all of SEQ ID NOs: 16-21; or (3) all of SEQ ID NOs: 30-35. In an aspect of the invention, the TCR comprises an α chain variable region amino acid sequence and a β chain variable region amino acid sequence, which together comprise one of the collections of CDRs set forth above. In this regard, the TCR can comprise the amino acid sequences of: (1) both of SEQ ID NOs: 8 and 9 (4211_E545K_TCR-1); (2) both of SEQ ID NOs: 10 and 11 (4211_E545K_TCR-1); (3) both of SEQ ID NOs: 22 and 23 (4367_N345K_TCR-1); (4) both of SEQ ID NOs: 24 and 25 (4367_N345K_TCR-1); (5) both of SEQ ID NOs: 36 and 37 (4367_N345K_TCR-2); or (6) both of SEQ ID NOs: 38 and 39 (4367_N345K_TCR-2). Each one of the foregoing collections of amino acid sequences in this paragraph sets forth the two variable regions of each of three different TCRs having antigenic specificity for mutated human PIK3CA. The two amino acid sequences in each collection correspond to the variable region of the α chain and the variable region of the β chain of a TCR, respectively. The TCR may, e.g., comprise the amino acid sequence of any one or more of SEQ ID NOs: 8, 9, 10, 11, 22, 23, 24, 25, 36, 37, 38, and 39. In an aspect of the invention, the TCR comprises the amino acid sequence(s) of: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) both of SEQ ID NOs: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) both of SEQ ID NOs: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) both of SEQ ID NOs: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) both of SEQ ID NOs: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NOs: 37; (15) both of SEQ ID NOs: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; or (18) both of SEQ ID NOs: 38 and 39. The inventive TCRs may further comprise a constant region. The constant region may be derived from any suitable species such as, e.g., human or mouse. In an aspect of the invention, the TCRs further comprise a murine constant region. 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, alpha Leydig 772435 HHS E-076-2024-0-PC-01 12 chain, and / or beta chain), means a TCR (or 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. In an aspect of the invention, the TCR may comprise a murine α chain constant region and a murine β chain constant region. The murine α chain constant region may be modified or unmodified. A modified murine α chain constant region may be, e.g., cysteine-substituted, LVL-modified, or both cysteine-substituted and LVL-modified, as described, for example, in U.S. Patent No. 10,174,098. The murine β chain constant region may be modified or unmodified. A modified murine β chain constant region may be, e.g., cysteine-substituted, as described, for example, in U.S. Patent No.10,174,098. In an aspect of the invention, the TCR comprises a cysteine-substituted, LVL-modified murine α chain constant region comprising the amino acid sequence of SEQ ID NO: 44 or 45. In an aspect of the invention, the TCR comprises a cysteine-substituted murine β chain constant region comprising the amino acid sequence of SEQ ID NO: 46. In an aspect of the invention, the inventive TCR can comprise an α chain of a TCR and a β chain of a TCR. The α chain of the TCR may comprise a variable region of an α chain and a constant region of an α chain. An α chain of this type can be paired with any β chain of a TCR. The β chain may comprise a variable region of a β chain and a constant region of a β chain. In an aspect of the invention, the TCR can comprise the amino acid sequences of: (1) both of SEQ ID NOs: 12 and 13 (4211_E545K_TCR-1); (2) both of SEQ ID NOs: 14 and 15 (4211_E545K_TCR-1); (3) both of SEQ ID NOs: 26 and 27 (4367_N345K_TCR-1); (4) both of SEQ ID NOs: 28 and 29 (4367_N345K_TCR-1); (5) both of SEQ ID NOs: 40 and 41 (4367_N345K_TCR-2); or (6) both of SEQ ID NOs: 42 and 43 (4367_N345K_TCR-2). Each one of the foregoing collections of amino acid sequences in this paragraph sets forth the α chain and β chain of each of three different TCRs having antigenic specificity for mutated human PIK3CA. The two amino acid sequences in each collection correspond to the α chain and the β chain of a TCR, respectively. The TCR may comprise the amino acid sequence of any one or more of SEQ ID NOs: 12, 13, 14, 15, 26, 27, 28, 29, 40, 41, 42, and 43. In an aspect of the invention, the TCR comprises the amino acid sequences of: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) both of SEQ ID NOs: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) both of SEQ ID NOs: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) both of SEQ ID NOs: 26 Leydig 772435 HHS E-076-2024-0-PC-01 13 and 27; (10) SEQ ID NO: 28; (11) SEQ ID 29; (12) both of SEQ ID NOs: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) both of SEQ ID NOs: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; or (18) both of SEQ ID NOs: 42 and 43. Included in the scope of the invention are functional variants of the inventive TCRs 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 mutated PIK3CA 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%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein, respectively. 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, Ile, 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, Gln, Ser, Thr, Tyr, etc.), etc. 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 Leydig 772435 HHS E-076-2024-0-PC-01 14 the functional variant, such that the of the functional variant is increased as compared to the parent TCR, polypeptide, or protein. 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. Also provided by the invention is a polypeptide comprising a functional portion of any of the TCRs described herein. The term “polypeptide,” as used herein, includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds. 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 mutated PIK3CA. 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 mutated PIK3CA (e.g., in an applicable HLA molecule- dependent manner), or 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. 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 mutated PIK3CA; and / or having the ability to 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. The polypeptide can comprise a functional portion of either or both of the α and β chains of the TCRs of the invention, such as a functional portion comprising one of more of CDR1, CDR2, and CDR3 of the variable region(s) of the α chain and / or β chain of a TCR of the invention. In an aspect of the invention, the polypeptide comprises the amino acid sequences of: (1) all of SEQ ID NOs: 2-7; (2) all of SEQ ID NOs: 16-21; or (3) all of SEQ Leydig 772435 HHS E-076-2024-0-PC-01 15 ID NOs: 30-35.( The polypeptide may the amino acid sequences of any one or more of: SEQ ID NOs: 2-7, 16-21, and 30-35 In an aspect 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, e.g., the amino acid sequences of: (1) both of SEQ ID NOs: 8 and 9; (2) both of SEQ ID NOs: 10 and 11; (3) both of SEQ ID NOs: 22 and 23; (4) both of SEQ ID NOs: 24 and 25; (5) both of SEQ ID NOs: 36 and 37; or (6) both of SEQ ID NOs: 38 and 39. The polypeptide may, e.g., comprise the amino acid sequence of any one or more of SEQ ID NOs: 8, 9, 10, 11, 22, 23, 24, 25, 36, 37, 38, and 39. In an aspect of the invention, the polypeptide comprises the amino acid sequence(s) of: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) both of SEQ ID NOs: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) both of SEQ ID NOs: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) both of SEQ ID NOs: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) both of SEQ ID NOs: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NOs: 37; (15) both of SEQ ID NOs: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; or (18) both of SEQ ID NOs: 38 and 39 In an aspect 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 comprise, e.g., the amino acid sequence of (i) one of SEQ ID NOs 44-46 or (ii) SEQ ID NO: 46 and one of SEQ ID NOs: 44 and 45. In an aspect of the invention, the inventive polypeptide may comprise an α chain and a β chain of the inventive TCR. In this regard, the polypeptide can comprise, e.g., the amino acid sequences of: (1) both of SEQ ID NOs: 12 and 13; (2) both of SEQ ID NOs: 14 and 15; (3) both of SEQ ID NOs: 26 and 27; (4) both of SEQ ID NOs: 28 and 29; (5) both of SEQ ID NOs: 40 and 41; or (6) both of SEQ ID NOs: 42 and 43. The polypeptide may comprise the amino acid sequence of any one or more of SEQ ID NOs: 12, 13, 14, 15, 26, 27, 28, 29, 40, 41, 42, and 43. In an aspect of the invention, the polypeptide comprises the amino acid sequences of: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) both of SEQ ID NOs: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) both of SEQ ID NOs: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) both of SEQ ID NOs: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) both of SEQ ID NOs: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) both of SEQ ID NOs: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; or (18) both of SEQ ID NOs: 42 and 43. Leydig 772435 HHS E-076-2024-0-PC-01 16 An aspect of the invention a protein comprising a functional portion of any of the inventive TCRs described herein. By “protein” is meant a molecule comprising one or more polypeptide chains. In an aspect, the protein of the invention can comprise: first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 2-4; (2) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 5-7; (3) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 2-4 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 5-7; (4) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 16-18; (5) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 19-21; (6) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 16-18 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 19-21; (7) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 30-32; (8) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 33-35; or (9) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 30-32 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 33-35. In an aspect of the invention, the protein comprises first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 25; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24 and the second Leydig 772435 HHS E-076-2024-0-PC-01 17 polypeptide chain comprises the amino acid of SEQ ID NO: 25; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39; or (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39. In an aspect of the invention, the protein comprises first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43; or (18) the first Leydig 772435 HHS E-076-2024-0-PC-01 18 polypeptide chain comprises the amino acid of SEQ ID NO: 42 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43. The protein of the invention may be a TCR. Alternatively, 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 aspect 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., CD1a, CD1b, CD1c, CD1d, etc. 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. In some aspects of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single polypeptide comprising a linker peptide linking the α chain and the β 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 comprise the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 47). Upon expression of the construct including the linker peptide by a host cell, the linker peptide may be cleaved, resulting in separated α and β chains. In an aspect of the invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide positioned between the α and β chains. In some aspects, the TCR, polypeptide or protein disclosed herein comprises an α chain and / or a β chain, as disclosed herein, comprising a signal peptide. In some aspects, the sequence of the signal peptide of any of the α chains and / or β chains disclosed herein Leydig 772435 HHS E-076-2024-0-PC-01 19 comprises an leucine, lysine, alanine or residue substituted for the wild-type residue at position 2. In some aspects, the TCR, polypeptide or protein disclosed herein comprises a mature version of an α chain and / or a β chain, as disclosed herein, that lacks a signal peptide. 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. 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 mutated PIK3CA; 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 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides. The TCRs, polypeptides, and proteins of the invention 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, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl- cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, Leydig 772435 HHS E-076-2024-0-PC-01 20 aminomalonic acid monoamide, N’-benzyl- methyl-lysine, N’,N’-dibenzyl-lysine, 6- hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert- butylglycine. The TCRs, polypeptides, and proteins of the invention can be, e.g., 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. The inventive TCRs, polypeptides, and proteins described herein (including any of the functional portions or variants thereof) are also contemplated to be useful as the soluble TCR component of bispecific engager TCR fusion proteins (e.g., IMMTAC (immune- mobilizing monoclonal TCRs against cancer) molecules). Bispecific engager TCR fusion proteins have two components. One component comprises a soluble TCR. The other component comprises an anti-CD3 effector. The anti-CD3 effector may be any molecule that engages with a CD3 molecule on T cells and activates a T cell immune response. For example, the anti-CD3 effector may be an anti-CD3 antibody or anti-CD3 antibody fragment. The soluble TCR component of the bispecific engager TCR fusion protein binds to the target antigen presented on the surface of cancer cells presented by an HLA molecule. The anti- CD3 effector component engages a CD3 molecule on T cells. The engagement of these components of the bispecific engager TCR fusion protein triggers the activation and recruitment of T cells and redirects T-cell killing to tumor cells. An aspect of the invention provides a bispecific engager TCR fusion protein comprising (i) any of the inventive TCRs, polypeptides, or proteins (including any of the functional portions or variants thereof) described herein and (ii) an anti-CD3 effector. Hereinafter, references to “protein(s)” also encompass the bispecific engager TCR fusion proteins described herein, unless specified otherwise. 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, 4thed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein can be synthesized by Leydig 772435 HHS E-076-2024-0-PC-01 21 any of a variety of commercial entities. In respect, the inventive TCRs, polypeptides, and proteins can be synthetic, recombinant, isolated, and / or purified. An aspect 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 aspect, 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. An aspect of the invention provides an isolated or purified nucleic acid comprising, from 5’ to 3’, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequence, respectively, encode the amino sequences of SEQ ID NOs: 8 and 9; 9 and 8; 10 and 11; 11 and 10; 12 and 13; 13 and 12; 14 and 15; 15 and 14; 22 and 23; 23 and 22; 24 and 25; 25 and 24; 26 and 27; 27 and 26; 28 and 29; 29 and 28; 36 and 37; 37 and 36; 38 and 39; 39 and 38; 40 and 41; 41 and 40; 42 and 43; or 43 and 42. In an aspect of the invention, the nucleic acid further comprises a third nucleotide acid sequence interposed between the first and second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide. For example, the cleavable linker peptide may comprise the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 47). 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. 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 Leydig 772435 HHS E-076-2024-0-PC-01 22 Sambrook et al., supra. For example, a 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, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 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 synthesized by any of a variety of commercial entities. In an aspect 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. An aspect of 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. The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, an aspect of the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. In an aspect of the Leydig 772435 HHS E-076-2024-0-PC-01 23 invention, the recombinant expression a nucleotide sequence encoding the α chain, the β chain, and linker peptide. 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. 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 transposon / transposase series, 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 λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a transposon or a viral vector, e.g., a lentiviral vector or a retroviral vector. The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook et al., 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 μ plasmid, λ, SV40, bovine papillomavirus, and the like. Leydig 772435 HHS E-076-2024-0-PC-01 24 Desirably, the recombinant 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. 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 / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. 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 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, e.g., a human elongation factor-1α 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. 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. 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 daminase, purine nucleoside phosphorylase, and nitroreductase. Leydig 772435 HHS E-076-2024-0-PC-01 25 Another aspect of the invention an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Still another aspect of the invention provides an isolated or purified TCR, polypeptide, or protein that results from expression of any of the nucleic acids or vectors, described herein with respect to other aspects of the invention, in a cell. Another aspect of the invention further provides a host cell comprising any of the nucleic acids or 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, DH5α 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 DH5α 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. For example, the host cell may be a human lymphocyte. In an aspect of the invention, the host cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a natural killer (NK) cell, a macrophage, a pluripotent cell, and a multipotent 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. 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, SupT1, 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., Th1 and Th2 cells, CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating Leydig 772435 HHS E-076-2024-0-PC-01 26 lymphocytes (TILs), memory T cells (e.g., memory T cells and effector memory T cells), naïve T cells, and the like. In an aspect of the invention, the T cell has a CD39-CD69- phenotype. In an aspect of the invention, the host cell is a pluripotent cell or a multipotent cell. Pluripotent cells have the capacity to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent cells may comprise, for example, stem cells, e.g., embryonic stem cells, nuclear transfer derived embryonic stem cells, induced pluripotent stem cells (iPSC), etc. Multipotent cells may comprise, for example, hematopoietic stem cells. Modifying, e.g., reprogramming, cells to a pluripotent state refers to the reversion of a cell to a pluripotent cell and is described for example, in Crompton et al., Trends Immunol., 35(4): 178-185 (2014). Exemplary techniques may include somatic cell nuclear transfer (SCNT), cell–cell fusion, and direct reprogramming. Examples of methods for carrying out cell-cell fusion are described, for example, in Ogle et al., Nat. Rev. Mol. Cell Biol.6: 567-75 (2005) and Zhou et al., Cell Stem Cell, 3: 382-388 (2008). Examples of methods for carrying out SCNT are described, for example, in Hanna et al., Cell, 143: 508- 525 (2010); Stadtfeld et al., Genes Dev., 24: 2239-2263 (2010); Wilmut et al., Nature, 385: 810-813 (1997); Vizcardo et al., Cell Stem Cell, 12: 31-36 (2013); and Crompton et al., Cell Stem Cell, 12: 6-8 (2013). In an aspect of the invention, the host cell is an iPSC that was prepared by reprogramming, any of the host cells described herein (e.g., T cells, NK cells, or invariant natural killer T cells) to a pluripotent state. Also provided by an aspect of 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 cell, 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 aspect of the invention, Leydig 772435 HHS E-076-2024-0-PC-01 27 the population of cells is a clonal host cells comprising a recombinant expression vector as described herein. In an aspect 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 Nos. 8,034,334; 8,383,099; and 11,401,503; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In an aspect, 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). An aspect of the invention provides a method of producing any of the TCRs, polypeptides, or proteins described herein, the method comprising culturing any of the host cells or populations of host cells described herein, so that the TCR, polypeptide, or protein is produced. Another aspect of the invention is a method of producing a host cell expressing a TCR that has antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence, the method comprising contacting a cell with any of the inventive recombinant expression vectors described herein under conditions that allow introduction of the vector into the cell. Another aspect of the invention provides a method of producing an engineered human cell (or an engineered population of human cells), the method comprising introducing any of the inventive nucleic acids or recombinant expression vectors described herein to an isolated human cell (or an isolated population of human cells), wherein the nucleic acid or recombinant expression vector comprises a nucleotide sequence encoding any of the inventive TCRs, polypeptides, or proteins described herein. The isolated human cell, or isolated population of human cells, to which the nucleic acid or recombinant expression vector is introduced, may be as described herein with respect to other aspects of the invention. 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. Leydig 772435 HHS E-076-2024-0-PC-01 28 For example, the purity can be at least at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or can be about 100%. 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, an aspect of 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 carrier. 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 agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. 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, 23rdEd., Academic Press (2020). It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use. 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 pharmaceutical 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. Preferably, the inventive TCR material is administered by injection, e.g., intravenously. When the inventive TCR material is a host cell expressing the inventive TCR, Leydig 772435 HHS E-076-2024-0-PC-01 29 the pharmaceutically acceptable carrier for 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 aspect, the pharmaceutically acceptable carrier is supplemented with human serum albumin. 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., mutated PIK3CA), or 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 aspects, 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. Many assays for determining an administered dose are known in the art. For example, an assay, which comprises comparing the extent to which target cells are lysed or IFN-γ 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-γ is secreted upon administration of a certain dose can be assayed by methods known in the art. In an aspect in which the inventive TCR material is a population of cells, the number of cells administered per infusion may vary, e.g., from about 3 x 1010to about 1.5 x 1011cells or more. In certain aspects, fewer than 1 x 106cells may be administered. 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 ideal sites for attaching a bridge and / or a chemotherapeutic Leydig 772435 HHS E-076-2024-0-PC-01 30 agent, provided that the bridge and / or 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 mutated PIK3CA or to treat, or prevent cancer. It is contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or 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 mutated PIK3CA, 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 mutated PIK3CA. In this regard, an aspect of 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. For example, a mammal’s T cells may be genetically engineered to express the inventive TCR using, for example, retroviral, lentiviral, or non-viral methods, such as the sleeping beauty transposon or CRISPR-based genome editing. The number of these TCR-transduced cells can be rapidly expanded to large numbers using established rapid expansion protocols and can be administered back to pre-conditioned mammals. Once administered, these PIK3CA- specific-TCR-transduced T cells can recognize and readily destroy the cancer cells expressing the targeted mutated PIK3CA protein. Therefore, these TCRs may be used to treat a broad spectrum of mammals with epithelial cancers expressing the respective PIK3CA mutation and applicable HLA molecule. An aspect 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. 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 Leydig 772435 HHS E-076-2024-0-PC-01 31 varying degrees of treatment or prevention one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In 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. It is also contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells can be used in methods of inducing an immune response against a cancer in a mammal. In this regard, an aspect of the invention provides a method of inducing an immune response against a 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 induce an immune response against the cancer in the mammal. An aspect 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 inducement of an immune response against a cancer in a mammal. 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. With respect to the inventive methods, the cancer can be any cancer, including, e.g., 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 Leydig 772435 HHS E-076-2024-0-PC-01 32 of the eye, cancer of the intrahepatic bile 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, colocrectal 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. In a preferred aspect, the cancer is a cancer which expresses mutated PIK3CA. The cancer may express PIK3CA with a mutation at one or both of positions 345 and 545, as defined by SEQ ID NO: 1. The cancer may express PIK3CA with one or both of the following human PIK3CA mutations: E545K and N345K. In an aspect of the invention, the cancer is an epithelial cancer. In an aspect of the invention, the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, uterine cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer. The cancer may be known to comprise a E545K or N345K mutation in human PIK3CA. 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 Lagomorpha, 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. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. Leydig 772435 HHS E-076-2024-0-PC-01 33 1 This example demonstrates This example demonstrates the isolation of a TCR from tumor-infiltrating lymphocytes (TIL) from Patient 4367. Patient 4367 was a 40 year old male patient with colon cancer with metastases to the liver, lungs, and soft tissue. He had four prior cancer therapies. The therapies were as follows: (1) capecitabine and oxaliplatin, (2) capecitabine, (3) folinic acid, fluorouracil and irinotecan (FOLFIRI) and pembrolizumab, and (4) capecitabine. Treatments 1 and 2 were prior to resection of the TCR source material. The 4367_N345K_TCR-1 was isolated from TIL following screening against long peptides (=<25AA) that expressed autologous somatic mutations, including the PIK3CA N345K. Specifically, TIL upregulated expression of CD137 (4-1BB) and secreted IFN-γ when co-cultured with autologous dendritic cells pulsed with the N345K long peptide (ALRIKILCATYVKVNIRDIDKIYVR) (SEQ ID NO: 48). RNA from these cells was isolated, and cDNA was reverse transcribed. cDNA was then sequenced using a nested PCR with primers specific for the TCR alpha and beta chains. The alpha and beta chain variable region amino acid sequences (with and without the N-terminal signal peptide sequences for the TCR) are shown in Table 1. The CDRs are underlined and the N-terminal signal peptide sequences are in bold font. TABLE 1 TCR Name TCR chain Amino acid sequence W S R F Leydig 772435 HHS E-076-2024-0-PC-01 34 TCR Name TCR chain Amino acid sequence (complementarity determining regions are E T Y S This example demonstrates the isolation of a TCR from peripheral blood from Patient 4367. A peripheral blood sample was obtained from Patient 4367. To evaluate the presence of PIK3CA-specific T cells in the peripheral blood, memory T lymphocytes were sorted and were subjected to in vitro sensitization (IVS) with long peptides or TMGs, an approach that has been previously described (Levin et al., Clin. Cancer Res., 2021;27(18): 5084-95; Cafri et al., Nat. Commun., 2019;10(1): 449). Briefly, memory T cells were isolated from PBL by sorting the CD62L+CD45RO+ (Tcm), CD62L-CD45RO+ (Tem) and CD62L-CD45RO- (Temra) T cells and then were subjected to IVS using either long peptides, predicted minimal epitopes or TMGs. Reactive cells were sorted by FACS and either were subjected to a second round of stimulation or a rapid expansion protocol (REP) (Dudley et al., J. Immunother., 2003;26(4):332-42). Finally, in vitro functional assays determined the presence of PIKCA-reactive T cells in the stimulated cells. For patient 4367, the memory T cells from the peripheral blood were subjected to one round of IVS and the resulting population was screened for reactivity against PIK3CAN345Kmutation. CD4+ T cells were found to be reactive against the PIK3CAN345K25mer (ALRIKILCATYVKVNIRDIDKIYVR) (SEQ ID NO: 48), and following sorting, they were subjected to a rapid expansion protocol to increase the cell number. The resulting Leydig 772435 HHS E-076-2024-0-PC-01 35 population was verified to recognize the 25AA-long peptide by flow cytometry and INF-γ secretion. Following sort by FACS and TCR sequencing of the reactive cells, the isolated TCR (4367_TCR-2) was found to be unique, with distinct alpha and beta-TCR chain from the TIL-derived TCR (4367_TCR-1) (Table 2). The alpha and beta chain variable region amino acid sequences (with and without the N-terminal signal peptide sequences for the TCR) are shown in Table 2. The CDRs are underlined and the N-terminal signal peptide sequences are in bold font. TABLE 2 TCR Name TCR chain Amino acid sequence (complementarity determining regions are W S G S L E G S Leydig 772435 HHS E-076-2024-0-PC-01 36 3 This example demonstrates the isolation of a TCR from peripheral blood from Patient 4211. Identification of T cells targeting the common PIK3CA mutation E545K was attempted in TIL and the peripheral blood from a patient with rectal cancer (Patient 4211). No reactive T cells were detected in the pipeline TIL screen against all autologous somatic mutations, including TMG and 25mer peptide encoding the PIK3CAE545K mutation. Next, memory T cells from the same patient were sorted by FACS based on the CD62L and CD45RO surface expression as described for Patient 4367 in Example 2. Sorted T cells were subjected to two subsequent rounds of IVS with long peptides, predicted minimals and TMGs expressing the PIK3CAE545K mutation. At the end of the IVS, the memory CD4+ T cells, which were stimulated twice with a pool of short peptides, showed upregulation of the activation surface markers OX-40 and 4-1BB following overnight co-culture with autologous APCs pulsed with the respective peptide pool. The pool of peptides was derived from the mutated E545K long peptide AISTRDPLSEITKQEKDFLWSHRHY (SEQ ID NO: 50); the corresponding WT long peptide is AISTRDPLSEITEQEKDFLWSHRHY (SEQ ID NO: 51). The pool of peptides is shown in Table 3. The co-culture was repeated with the short peptides within the peptide pool pulsed individually on APCs, revealing that the CD4+ T cells recognized only the E545K_epit3 (EITKQEKDFLW) (SEQ ID NO: 55). This recognition was specific to the mutated and not the wild type peptide (EITEQEKDFLW) (SEQ ID NO: 52). TABLE 3 Name Amino acid sequence SEQ ID NO: Leydig 772435 HHS E-076-2024-0-PC-01 37 RNA from reactive cells was and cDNA was reverse transcribed. cDNA was then sequenced using a nested PCR with primers specific for the TCR alpha and beta chains. The alpha and beta chain variable region amino acid sequences (with and without the N-terminal signal peptide sequences for the TCR) are shown in Table 4. The CDRs are underlined and the N-terminal signal peptide sequences are in bold font. TABLE 4 TCR Name TCR chain Amino acid sequence (complementarity determining regions are D Q E I D I K F This example demonstrates the construction of retroviral vectors encoding the respective TCRs of Tables 1, 2, and 4. Leydig 772435 HHS E-076-2024-0-PC-01 38 Nucleotide sequences encoding variable regions of the α and β chains of the TCRs of Tables 1, 2, and 4 were obtained and codon-optimized. The TCRβ VDJ regions were fused to the mouse TCRβ constant chain. The TCRα VJ regions were fused to the mouse TCRα constant chain. Without being bound to a particular theory or mechanism, it is believed that replacing the constant regions of the human TCRα and TCRβ chains with the corresponding murine constant regions improves TCR expression and functionality (Cohen et al., Cancer Res., 66(17): 8878-86 (2006)). In addition, the murine TCRα and TCRβ constant chains were cysteine-modified. Transmembrane hydrophobic mutations were introduced into the murine TCRα 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 al., Cancer Res., 67(8): 3898-903 (2007); Haga-Friedman et al., J. Immu., 188: 5538–5546 (2012)). The full length α and β chains of each of the TCRs, including these modifications to the constant region, are shown in Table 5. In Table 5, the CDRs are underlined and the N-terminal signal peptides are in bold font. TABLE 5 SEQ ID NO: Sequence SEQ ID NO: 12 MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSV R Q T I L G R S A G K Leydig 772435 HHS E-076-2024-0-PC-01 39 SEQ ID NO: Sequence sequence without N- LTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWS t i l i l S P G F S G T V L V D N M G D S R A G Leydig 772435 HHS E-076-2024-0-PC-01 40 SEQ ID NO: Sequence FEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSEGDYKLSFG C b tit t d AGTTVTVRANI NPEPAVY LKDPRS DSTLCLFTDFDS IN V Q P L C C P L N M P G F S Nucleotide sequences encoding the variable regions of the α and β chains of the TCRs of Table 5 were independently cloned into MSGV1-based retroviral vectors. The TCRβ and TCRα chains were separated by a Furin Ser / Gly P2A linker peptide (SEQ ID NO: 47). Without being bound to a particular theory or mechanism, it is believed that the linker peptide provides comparable expression efficiency of the two chains (Szymczak et al., Nat. Biotechnol., 22(5): 589-94 (2004)). Leydig 772435 HHS E-076-2024-0-PC-01 41 5 This example demonstrates the specificity of 4367_N345K_TCR-1 and 4367_N345K_TCR-2 for the PIK3CA mutation N345K. Healthy donor PBL were independently transduced with the retroviral vector encoding the 4367_N345K_TCR-1 or 4367_N345K_TCR-2 of Example 4 to produce effector cells. The effector cells were co-cultured overnight with autologous antigen presenting cells pulsed with titrated concentrations of mutated (ALRIKILCATYVKVNIRDIDKIYVR) (SEQ ID NO: 48) or wild type (ALRIKILCATYVNVNIRDIDKIYVR) (SEQ ID NO: 49) PIK3CA long peptides. The results are shown in Figures 1A-1B. As shown in Figures 1A- 1B, the 4367_N345K_TCR-1 and 4367_N345K_TCR-2 specifically recognized the PIK3CA mutation N345K. EXAMPLE 6 This example demonstrates that 4367_N345K_TCR-1 and 4367_N345K_TCR-2 recognize the N345K mutation presented by a HLA-DPA1*01:03:01 HLA-DPB1*04:01:01 heterodimer. Healthy donor PBL were independently transduced with the retroviral vector encoding the 4367_N345K_TCR-1 or 4367_N345K_TCR-2 of Example 4 to produce effector cells. The effector cells were co-cultured overnight with COS7 cells transduced with each of the (autologous) HLA molecules expressed by Patient 4367 and pulsed with N345K peptides or vehicle (DMSO). The results are shown in Figures 1C-1D. In a separate experiment, the effector cells were co-cultured overnight with cell line HTB114. The cell line HTB114 had been modified to express an HLA molecule (an irrelevant HLA molecule or an HLA-DP molecule) and PIK3CA (wild type or N345K) (or an irrelevant target peptide), as indicated in Figure 1E. The results are shown in Figure 1E. As shown in Figures 1C-1E, the 4367_N345K_TCR-1 and 4367_N345K_TCR-2 recognize the N345K mutation presented by a HLA-DPA1*01:03:01 HLA-DPB1*04:01:01 heterodimer. Leydig 772435 HHS E-076-2024-0-PC-01 42 7 This example demonstrates that 4367_N345K_TCR-1 and 4367_N345K_TCR-2 recognize the N345K mutation presented by a HLA-DPA1*01:03:01 HLA-DPB1*04:01:01 heterodimer. Healthy donor PBL were independently transduced with the retroviral vector encoding the 4367_N345K_TCR-1 or 4367_N345K_TCR-2 of Example 4 to produce effector cells. Mock-transduced PBL and an irrelevant TCR served as effector cell controls. Target cell lines HTB-114TMand CCL-225TMwere modified to express DPB1*04:01 and N345K PIK3CA. Mock modified cell lines were used as target cell controls. Effector cells were co-cultured with target cell lines overnight. Cytotoxicity of target cell lines was measured. The results are shown in Figures 1F-1I. Figures 1F-1I shows the results of the cytotoxicity assay of mock or TCR-transduced lymphocytes with cell lines HTB-114TMand CCL-225TMwhich were modified to express human allele DPB1*04:01 and N345K PIK3CA. As shown in Figures 1F-1I, 4367_N345K_TCR-1 and 4367_N345K_TCR-2 recognize the N345K mutation presented by a HLA-DPA1*01:03:01 HLA-DPB1*04:01:01 heterodimer. EXAMPLE 8 This example demonstrates the specificity of the 4211_E545K_TCR-1 for the PIK3CA mutation E545K. Healthy donor PBL were independently transduced with the retroviral vector encoding the 4211_E545K_TCR-1 of Example 4 to produce effector cells. The effector cells were co-cultured overnight with autologous antigen presenting cells pulsed with titrated concentrations of mutated (EITKQEKDFLW) (SEQ ID NO: 55) or wild type (EITEQEKDFLW) (SEQ ID NO: 52) PIK3CA epitope 3. The results are shown in Figure 2A. As shown in Figure 2A, the 4211_E545K_TCR-1 specifically recognized the PIK3CA mutation E545K. EXAMPLE 9 This example demonstrates that the 4211_E545K_TCR-1 recognizes the PIK3CA mutation E545K presented by a HLA-DRB1*04:01 HLA-DRA1*01:01 heterodimer. Leydig 772435 HHS E-076-2024-0-PC-01 43 Healthy donor PBL were transduced with the retroviral vector encoding the 4211_E545K_TCR-1 of Example 4 to produce effector cells. The effector cells were co-cultured overnight with COS7 cells transduced with each of the (autologous) HLA molecules expressed by Patient 4211 and pulsed with E545K peptide (EITKQEKDFLW) (SEQ ID NO: 55) or vehicle (DMSO). The results are shown in Figure 2B. In a separate experiment, target cell line MCF7 was modified to express an HLA molecule (or mock HLA), as indicated in Figure 2C. MCF7 natively expresses the E545K PIK3CA protein. The effector cells were co-cultured with the modified target cell line MCF7 overnight. Secretion of interferon gamma was measured. The results are shown in Figure 2C. As shown in Figures 2B-2C, the 4211_E545K_TCR-1 recognized the PIK3CA mutation E545K presented by a HLA-DRB1*04:01 HLA-DRA1*01:01 heterodimer. EXAMPLE 10 This example demonstrates that the 4211_E545K_TCR-1 recognizes the PIK3CA mutation E545K presented by a HLA-DRB1*04:01 HLA-DRA1*01:01 heterodimer. Healthy donor PBL were independently transduced with the retroviral vector encoding the 4211_E545K_TCR-1 of Example 4 to produce effector cells. Mock-transduced PBL and an irrelevant TCR served as effector cell controls. Target cell line MCF7 was modified to express HLA-DRB1*04:01 or HLA-A03+ (control). Effector cells were co-cultured with target cell lines overnight. Cytotoxicity of target cell lines was measured. The results are shown in Figures 2D-2G. Figures 2D-2G shows the results of the cytotoxicity assay of mock-transduced or effector cells which were co-cultured with target cell line MCF7 that was modified to express HLA-DRB1*04:01. As shown in Figures 2D-2G, the 4211_E545K_TCR-1 recognized the PIK3CA mutation E545K presented by a HLA-DRB1*04:01 HLA-DRA1*01:01 heterodimer. EXAMPLE 11 This example demonstrates a method of treating a cancer patient with cells transduced with a recombinant expression vector encoding the 4367_N345K_TCR-1. Leydig 772435 HHS E-076-2024-0-PC-01 44 Patients and Methods Patient eligibility The patient (4367) had treatment-refractory solid epithelial cancer, was age 18 years or older, and had an Eastern Cooperative Oncology Group performance status ≤ 1 and no evidence of active major medical diseases. Study design and treatment The patient was enrolled into an ongoing early phase II pilot study designed to evaluate the safety and efficacy of adoptive cell therapy (ACT) of autologous PBL genetically modified via retroviral transduction to express personalized neoantigen reactive TCRs after nonmyeloablative lymphodepleting chemotherapy in patients with metastatic epithelial cancer (ClinicalTrials identifier NCT03412877). With cell infusion designated as day 0, lymphodepleting chemotherapy consisted of cyclophosphamide (60 mg / kg once daily) administered on days –7 and –6. Fludarabine (25 mg / m2once daily) was administered from day –7 to day –3. After cell infusion, aldesleukin (720,000 IU / kg) was given every 8 hours to tolerance. The arm of the study reported here includes the administration of pembrolizumab 2 days before cell infusion with up to three subsequent doses delivered at 3-week intervals, when clinically appropriate. This study was approved by the Institutional Review Board of the National Cancer Institute (NCI). Informed consent was obtained from the patient, and the study was conducted in accordance with the International Conference on Harmonization Good Clinical Practice and the applicable portions of the US Code of Federal Regulations. Patient 4367 had 4 doses of IL-2 and 2 doses of pembrolizumab during TCR ACT. 1.5e11 cells were infused during TCR ACT.4367_N345K_TCR-1 was used as the TCR during ACT. End points The primary end point of this clinical study was the objective response rate as measured using RECIST 1.1. Cross-sectional imaging was performed pretreatment, at 6 weeks post-treatment, and at regular intervals thereafter until progression. Evaluation of potential neoantigen reactive T cell receptors 4367_N345K_TCR-1 was introduced into autologous or allogeneic PBL as previously described (Parkhurst et al., Cancer Discov., 9: 1022-1035 (2019); Tran et al., Science, 344: 641-645 (2014)). Briefly, cryopreserved apheresis samples were thawed and stimulated with 50 ng / ml soluble agonistic anti-CD3 (OKT3; Miltenyi Biotec) and 300-1200 Leydig 772435 HHS E-076-2024-0-PC-01 45 IU rhu IL-2 (Chiron) for 2 days prior to transduction. To generate transient retroviral supernatants, the retroviral vector MSGV1 encoding the 4367_N345K_TCR-1 of Example 4 and the envelope vector encoding plasmid RD114 were co-transfected into the retroviral packaging cell line 293GP using Lipofectamine 2000 (Life Technologies). Retroviral supernatants were collected ~48 hours after transfection, diluted 1:1 with DMEM media containing 10% FCS, and then centrifuged onto RETRONECTIN reagent (Takara Bio)-coated, non–tissue culture–treated plates at 2,000 x g for 2 h at 32 °C. Activated T cells were then spun onto the retrovirus coated plates for 10 min at 300 x g. GFP and mock transduction controls were included in transduction experiments. Cells were typically assayed 10-14 days post-retroviral transduction for recognition of tandem minigene (TMG)- electroporated and / or peptide-pulsed autologous DCs by measuring IFNa secretion by ELISA and expression of 4-1BB by FACS. Manufacture and characterization of clinical grade retroviral supernatants Retroviral vectors were manufactured using plasmids procured from Genscript, Inc. (Piscataway NJ, USA). The transfer plasmid containing the individual TCR was an MSGV1 derivative. The TCR and RD114 envelope plasmids were transiently transfected into 293GP cells. The 293GP cell line is a derivative of the 293T line and stably expresses the MoMLV (Moloney murine leukemia virus) gag and pol proteins. The cell line was expanded into a Master Cell Bank at Indiana University Vector Production Facility in compliance with GMP regulations and tested to meet specifications for use in GMP manufacturing: sterility, mycoplasma, adventitious viruses, human viral contaminants, identity and replication competent retrovirus. A new vial of 293GP cells was thawed for each vector production run and scaled up to at least one 10-layer stack over 10 days in DMEM (Thermo Fisher Scientific) + 10% FBS (Hyclone, Inc). Confluence, cell number and viability were assessed at each split / manipulation as in-process controls. Mixtures of plasmids (~1:1.5 ratio of envelope to transfer vector and LIPOFECTAMINE 2000 CD reagent (Thermo Fisher Scientific) were incubated in OPTI-MEM Reduced Serum Medium (Thermo Fisher Scientific) according to the manufacturer’s recommendations and added to 10-layer stacks containing 293GP cells overnight. Media was exchanged after 24h. Benzonase endonuclease (Millipore, Burlington MA, USA) was added on the day of harvest (Day 14) at 50 units / ml of vector supernatant and incubated for at least one hour. Following benzonase treatment, the vector supernatant was filtered through a Leukocyte Reduction Filter (LRF) (Pall Medical) and the product was transferred in 100g aliquots to freezing bags. Vector supernatant Leydig 772435 HHS E-076-2024-0-PC-01 46 (product and testing samples) were frozen - °C. End-of-production cells were collected, counted, aliquoted and viably frozen per testing plans. Vector supernatant was tested to meet Certificate of Analysis (COA) specifications as follows: Titer / TCR Expression: Wells in a 24-well plate were coated with RETRONECTIN reagent (Takara Bio)) (100 µg / ml) overnight at 4 °C. Plates were blocked with 10% Human Serum Albumin (Grifols). Vector supernatant was titrated in DMEM + 10% FBS, added to each well, and spun for 2 hours (h) at 2000 x g. Supernatant was aspirated and 2.5e5 activated T cells (48 h with 50 ng / mL OKT3 (Thermo Fisher Scientific); 300 IU / ml IL-2) were added to each well. FACS analysis was performed 4 days later. Cells were stained with PI (BD Bioscience), PE anti-mouse TCRβ (BD Bioscience), and APC anti- CD3 (Biolegend). Percent positive was reported on live lymphocytes expressing CD3 and murine TCRβ. Titer was calculated by multiplying the percent positive by the number of cells in the well at transduction and the dilution factor to give transducing units per milliliter (TU / ml). The reported TU / ml is an average titer of the dilutions which yielded a percent positive between 2% and 30% to capture the majority of cells with one integrated copy. COA specification required ≥30% TCR expression and ≥1e5 TU / ml. PBL Specificity / Potency: Autologous APCs (DCs or B cells) or APCs presenting the appropriate HLA-complex were pulsed with mutant 25 amino acid peptides (>95% HPLC-purified) or 9-10 amino acid minimal epitopes (>95% HPLC-purified) or wild-type (>95% HPLC-purified) counterparts for 2h – 4h. Pulsed APCs were co-cultured with transduced T cells at a ratio of 1e5 effector to 1e5 target cells in U-bottom 96 well plate wells overnight. Plates were gently spun and supernatants were transferred to new plates. IFNa released into the supernatant was measured by ELISA (R&D Systems). COA specification required ≥200 pg / mL IFNa release in mutant peptide co-culture and 2x background (APC + T cells, no peptide). Sterility: Vector supernatant was tested in the NIH Department of Laboratory Medicine Sterility Service using BACT / ALERT system (bioMerieux) for aerobic and anerobic microbial contaminants. In addition, a Sabouraud dextrose agar (SDA) (Hardy Diagnostics) plate was streaked for augmented detection of fungal contaminants. Mycoplasma was detected by using the MYCOSEQ Mycoplasma Real-Time PCR kit (Thermo Fisher Scientific). COA specification was no growth detected or negative. Leydig 772435 HHS E-076-2024-0-PC-01 47 Endotoxin: Endotoxin levels determined using the Limulus Amebocyte Lysate (LAL) PYROGENT™-5000 detection system (Lonza) with the ELX808 microplate reader (Lonza). The assay was run according to manufacturer’s instructions. COA specification was ≤0.5EU / mL. Replication competent retrovirus (RCR): DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germantown MD, USA). (Integrated DNA Technologies, Coralville Iowa, USA) were mixed with extracted DNA and TAQMAN Fast Universal PCR Master Mix (2x), No AMPERASE UNG reagent solution (Thermo Fisher Scientific), and amplified. Samples measured included vector supernatant and transduced cells to meet the specification of ≤10 copies / μl (assay LOD) or decreasing signal between samples / time points. Manufacture and testing of TCR transduced autologous PBL for treatment All processes were conducted in a GMP cleanroom in ISO 7 suites and ISO 5 biosafety cabinets (BSCs) and executed in accordance with approved batch records with review and oversight by NCI SB Management, Quality Assurance, and Quality Control. Cryopreserved PBL were thawed (Stim 1 day 0; S1D0) and resuspended at 1x106cells / ml in RPMI media containing 10% human AB serum (BioIVT), 300 IU / ml rhIL-2 (Proleukin, Clinigen) and 50 ng / ml OKT3 (Miltenyi Biotech). Cells were distributed to T- 175 flasks (100 ml per flask) and incubated at 37 °C for 2 days until the start of the transduction process (S1D2). For some samples, PBL were enriched for CD4 or CD8 cells depending on the identified TCR coreceptor prior to transduction. Enrichment was performed via negative selection using the CIINIMACS system (Miltenyi Biotech) on the day the PBL were thawed (S1D0). CD4 cells were enriched by depleting CD8 cells and vice versa. After thawing PBL, up to 4x109of the recovered cells were resuspended in CIINIMACS PBS / EDTA buffer containing 0.5% human serum albumin (HSA) (Grifols). Gamma-Gard (1.3 ml per 200 ml of cells; Baxalta US Inc.) and either the CD4 or CD8 CIINIMACS reagent were added to the cells. The mixture was incubated with rocking for a minimum of 30 minutes at room temperature. The CLINIMACS system was set up with the appropriate tubing set and buffers, according to the manufacturer’s recommendations. At the end of the labeling process, the CIINIMACS selection was run using a Miltenyi optimized program with cells from the positive and negative fraction collected into separate bags with CLINIMACS Buffer / 0.5% HSA. After the selection, cells from the positive and negative fractions were collected and analyzed by FACS to determine the purity and efficiency of the selection. The Leydig 772435 HHS E-076-2024-0-PC-01 48 enriched fraction was suspended in media IL-2 (300 IU / ml) and OKT3 (50 ng / ml) and seeded into T-175 flasks at 1x106cells per ml at 100 ml per flask. Cells were incubated at 37 °C until the start of the transduction process. Prior to transduction, 8 non-tissue culture treated 6 well plates were coated with 20 μg / well of RETRONECTIN reagent (Takara) and incubated for 2 hrs at room temperature (RT). Plates were then blocked with 2.5% HSA in PBS for 30 min at RT. The wells were aspirated and washed with HBSS containing 2.5% HEPES. On S1D2, the RETRONECTIN reagent coated plates were coated with TCR vector supernatant. The frozen retroviral vector supernatant was thawed and diluted 1:1 with RPMI containing 10% human AB serum without IL-2. The wash buffer was aspirated from the wells, and 4 ml of 1:1 diluted vector supernatant was placed in each well. The vector coated plates were centrifuged at 2000 x g for 2 hours at 32 °C. Four of these plates were used immediately for transduction #1 (Td#1), and the remaining four plates were stored overnight at 4 °C for transduction #2 (Td#2) the following day. To transduce the stimulated PBL, cells were resuspended in RPMI containing 10% human AB serum with 300 IU IL-2 at 0.5x106 / ml. The vector supernatant and media were aspirated from the respective plates, leaving a thin layer of vector supernatant in each well. Four ml of the resuspended PBL were added to each well for a total of 2 x 106cells per well. The four plates for Td#1 were centrifuged at 1000xg for 10 minutes at 32 °C and then incubated overnight at 37 °C. Td#2 was performed the following day (S1D3) using the 4 vector coated plates stored at 4 °C. After allowing the Td#2 plates to come to RT, transduced PBL from Td#1 were harvested, distributed evenly between all wells of Td#2 plates, centrifuged at 1000 x g for 10 minutes at 32 °C, and then incubated overnight at 37 °C. The day following Td#2 (S1D4), cells were harvested, centrifuged, and resuspended in fresh RPMI containing 10% human AB serum with 300 IU / ml IL-2 for plating in T-175 flasks at 0.5x106cells per ml in 50-100 ml per flask. After transduction, cells were maintained in T-175 flasks at 0.7x106- 2x106cells per ml in RPMI containing 10% human AB serum and 300 IU / ml IL-2 until S1D10 where cells entered a rapid expansion protocol (REP: R2D0). To initiate the REP, 50:50 media was prepared by mixing equal volumes of RPMI containing 10% human AB serum and AIM-V media and was supplemented with PenStrep (ThermoFisher), IL-2 (3000 IU / ml), and OKT3 (50 ng / ml). Transduced PBL were seeded into 6 GREX 100 flasks (WilsonWolf) at 5x106cells per flask with irradiated (6000 rads) donor feeder PBL at 5x108for a 1:100 ratio. Leydig 772435 HHS E-076-2024-0-PC-01 49 Untransduced cells were maintained at the patient PBL to feeder cell ratio in T-175 flasks. Cells were maintained at 0.5-2.0e6cells / ml for 7 days. Approximately 1 week after the start of the REP on R2D7, each GREX 100 flask was split at a 1:5 ratio, if possible, at a cell density of 0.5-2.0e6cells / ml with AIM V media containing GLUTAMAX supplement (2mM) (Thermo), Penstrep (Thermo), and IL-2 (3000 IU / ml). Starting on R2D7, cells were maintained at 0.5-2.0e6cells / ml. The AIM V media listed above was used for the final media exchange on R2D11. All cultures remained undisturbed from R2D11 to R2D14 when cells were pooled and harvested for final formulation and release for infusion. Cells were sampled for release testing criteria in accordance with the COA at various stages of the manufacturing process. The following assays were carried out as described above for vector manufacturing with the same release criteria applied on the days noted: samples for TCR expression, PBL specificity / potency and sterility including mycoplasma, were taken on or before R2D11 with all but sterility reporting final results; samples for RCR were taken on or about S1D4, S1D10 / R2D0, and R2D11. Endotoxin testing (≤5 E.U. / kg) and Gram stain (negative) were performed on the morning of cell harvest, prior to final formulation. Cell counts for the final product (i.e., infusion bag) were required to be within 1 x 109– 1.5 x 1011total cells with >70% viability. Additional samples were taken from the infusion bag for post manufacturing testing, including final product sterility, RCR (S+L-), residual benzonase, residual plasmid DNA, and vector copy number, but were not required for product release and infusion. Vector copy number in cell products given to patients was estimated from extracted DNA using the QIAamp DNA Blood Mini Kit (Qiagen). Two separate qPCRs were executed to quantify LTR copies from the MSGV1 vector and albumin gene copies as an endogenous housekeeping gene. Since 2 LTRs are expected per integrant, and 2 albumin gene copies are expected per cell, LTR copies divided by albumin gene copies yielded an average vector copy number per cell across a cell population. Extracted DNA was mixed with TAQMAN Fast Universal PCR Master Mix (2x), No AMPERASE UNG reagent solution (Thermo Fisher Scientific) and LTR primers / probe. Phenotypic evaluation of PBL and TCR transduced cells Pre-treatment PBL, TCR transduced cells, and post-treatment PBL were analyzed by FACS using fluorescently labeled antibodies against human CD3 (clone SK7), CD4 (clone SK3), CD8 (clone RPA-T8), CD45RO (clone UCHL1), CD62L (clone DREG-56), CD27 Leydig 772435 HHS E-076-2024-0-PC-01 50 (clone M-T271), CD39 (clone A1), and (clone FN50), and against murine TCRβ chain (clone H57-597) (BD Biosciences). For analyses of PBL, cryopreserved samples were thawed and rested overnight in the absence of IL-2 prior to FACS. For analyses of TCR transduced cells, cryopreserved samples were thawed and rested overnight in the presence of 600 IU / ml IL-2 prior to FACS. The percentages of T regulatory cells in post-treatment PBL using anti-human FoxP3 (clone 259D / C7) and anti-human CD4 (clone RPA-T4) (BD Biosciences) were evaluated. Since FoxP3 is an intracellular transcription factor, cells were permeabilized prior to staining using the BD PHARMINGEN™ Transcription Factor Buffer Set according to the manufacturer’s instructions. Figure 6 shows the level of recovery of T regulatory cells at 1 week and 1 month post ACT for patient 4367. The phenotypic characterization of the infused cell products is provided in Table 6 below. TABLE 6 Resection # 4367 Leydig 772435 HHS E-076-2024-0-PC-01 51 Functional evaluation of TCR transduced Cells that were adoptively transferred into patients were functionally evaluated by measuring cytokines and chemokines in supernatants from cocultures of the T cells with peptide-pulsed autologous DCs. For analyses of PBL, cryopreserved samples were thawed and rested overnight in the presence or absence of IL-2 prior to functional testing. For analyses of TCR transduced cells, cryopreserved samples were thawed and rested overnight in the presence of 600 IU / ml IL-2 prior to testing, but cells were cocultured in the absence of any exogenous IL-2. IFNa secretion was measured by ELISA using paired antibodies from Invitrogen: primary anti-human IFNa monoclonal antibody (clone 2G1: M700A) and biotin labeled anti-human IFNa (clone B133.5: M701B). Secretion of other cytokines and chemokines was measured using FACS based kits from Miltenyi Biotech (MACSPLEX Cytotoxic T / NK Cell Kit, human and MACSPLEX Cytokine 12 Kit, human) according to manufacturer’s instructions. Figures 5 and 7A-7B show the results of the cytokine and chemokine analysis. Expression of neoantigens and HLA in resected tumors To determine if the identified TCRs had the potential to recognize tumor cells in vivo, the expression level of the specific mutated genes in cancer cells derived from the original tumor resections was evaluated. This was done by estimating the percent of tumor cells bearing the mutation in each specimen via WES (cancer cell fraction; CCF) and by estimating the level of mutant gene expression via RNA-seq (Table 7). Of the 10 neoantigens that were targeted in seven T cell treatment naïve patients, 5 appeared to be clonal with CCF values greater than 0.97 in every specimen analyzed (4367 PIK3CA). T cell treatment naïve patient 4367 appeared to target at least one clonal mutation. Patient 4367 did not respond to T cell therapy. TABLE 7 Resection # 4367 Leydig 772435 HHS E-076-2024-0-PC-01 52 Mean expression** -1 Expression range -10.7 *** LOH (loss of heterozygosity in HLA class I locus) Analyses of post-transfer PBL A clinical study evaluating TIL for melanoma demonstrated that persistence of transferred cells was associated with response to therapy (Rosenberg et al., Clin. Cancer Res., 17: 4550-4557 (2011)), but in other studies of ACT with NY-ESO-1 reactive TCR transduced cells, no such correlation was observed (Robbins et al., Clin. Cancer Res., 21: 1019-1027 (2015); Robbins et al., J. Clin. Oncol., 29: 917-924 (2011)). For every patient in the current study, PBMCs were collected at various time points after ACT and estimated the degree of persistence of the adoptively transferred TCR transduced cells. For these analyses, cryopreserved PBMC were thawed and rested overnight in the absence of IL-2 prior to FACS. For patients who received one TCR, persistence was measured by FACS using the anti-murine TCRβ constant region antibody. For patient 4367, TCR transduced cells in day 41 post-ACT PBL were not reliably detected (see Figure 4). No clear correlation was observed between persistence of TCR transduced cells and response to therapy. EXAMPLE 12 This example demonstrates the successful manufacture and characterization of clinical grade retroviral supernatants. One major difference between this clinical trial and every other gene therapy trial in which gamma retroviruses have been used for gene delivery was the use of retroviral products generated from transiently transfected 293GP cells (Figure 3). In every other trial, large volumes of supernatants were required to treat multiple patients with a single product, and the FDA mandated the generation of stable packaging cell lines (e.g., PG13). The production and validation of such stable packaging cell lines and subsequent retroviral products are impractical for generating small-scale personalized products intended for the treatment of a single patient. Therefore, FDA approved processes were developed for Leydig 772435 HHS E-076-2024-0-PC-01 53 producing small batches of retroviral in-house via transient transfection of 293GP cells with TCR and RD114 env containing plasmids under good manufacturing practices (GMP). Individual vector supernatants were generated for each TCR, and all required quality control testing for immediate clinical use was completed in-house. All vectors passed all quality control tests (Figure 3). Patient PBL was used for transductions to inform expected performance in clinical cell productions. The TCR expression on day seven following transduction ranged from 31 – 88% for all vectors generated. TCR expression was the top (undiluted) point of an 11-point titration curve used to calculate the transducing units per ml (titer) which ranged from 1.7e5 to 6.7e6. TCR specificity was also evaluated by measuring IFNa secretion by TCR transduced cells in response to antigen presenting cells (APCs: DCs or B cells) pulsed with mutant and wild type peptides. All products passed specifications requiring ≥200 pg / ml IFNa and ≥2 times background (Table 8). In addition, required specifications of no growth or no detection for sterility, mycoplasma, and replication competent retrovirus were met for each vector. TABLE 8 Characterization of clinical grade supernatants IFNg (pg / ml)e 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. 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 Leydig 772435 HHS E-076-2024-0-PC-01 54 contradicted by context. The terms “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. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects 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.

Claims

Leydig 772435 HHS E-076-2024-0-PC-01 55 CLAIM(S):

1. An isolated or purified T cell receptor (TCR) having antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence, wherein the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 2-7; (2) all of SEQ ID NOs: 16-21; or (3) all of SEQ ID NOs: 30-35.

2. The TCR of claim 1, wherein the TCR comprises the amino acid sequence(s) of: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) both of SEQ ID NOs: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) both of SEQ ID NOs: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) both of SEQ ID NOs: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) both of SEQ ID NOs: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NOs: 37; (15) both of SEQ ID NOs: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; or (18) both of SEQ ID NOs: 38 and 39.

3. The TCR of claim 1 or 2, wherein the TCR comprises the amino acid sequences of: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13;Leydig 772435 HHS E-076-2024-0-PC-01 56 (3) both of SEQ ID NOs: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) both of SEQ ID NOs: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) both of SEQ ID NOs: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) both of SEQ ID NOs: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) both of SEQ ID NOs: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; or (18) both of SEQ ID NOs: 42 and 43.

4. The TCR of any one of claims 1-3, wherein the human PIK3CAE545Kamino acid sequence is EITKQEKDFLW (SEQ ID NO: 55).

5. The TCR of any one of claims 1-4, wherein the TCR does not have antigenic specificity for the wild-type human PIK3CA amino acid sequence of EITEQEKDFLW (SEQ ID NO: 52).

6. The TCR of any one of claims 1-3, wherein the human PIK3CAN345Kamino acid sequence is ALRIKILCATYVKVNIRDIDKIYVR (SEQ ID NO: 48).

7. The TCR of any one of claims 1-3 and 6, wherein the TCR does not have antigenic specificity for the wild-type human PIK3CA amino acid sequence of ALRIKILCATYVNVNIRDIDKIYVR (SEQ ID NO: 49).

8. An isolated or purified polypeptide comprising a functional portion of the TCR of any one of claims 1-7, wherein the polypeptide comprises the amino acid sequences of:Leydig 772435 HHS E-076-2024-0-PC-01 57 (1) all of SEQ ID NOs: 2-7; (2) all of SEQ ID NOs: 16-21; or (3) all of SEQ ID NOs: 30-35.

9. The polypeptide of claim 8, wherein the polypeptide comprises the amino acid sequences of: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) both of SEQ ID NOs: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) both of SEQ ID NOs: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) both of SEQ ID NOs: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) both of SEQ ID NOs: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NOs: 37; (15) both of SEQ ID NOs: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; or (18) both of SEQ ID NOs: 38 and 39.

10. The polypeptide of claim 8 or 9, wherein the polypeptide comprises the amino acid sequences of: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) both of SEQ ID NOs: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) both of SEQ ID NOs: 14 and 15;Leydig 772435 HHS E-076-2024-0-PC-01 58 (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) both of SEQ ID NOs: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) both of SEQ ID NOs: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) both of SEQ ID NOs: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; or (18) both of SEQ ID NOs: 42 and 43.

11. An isolated or purified protein comprising first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 2-4; (2) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 5-7; (3) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 2-4 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 5-7; (4) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 16-18; (5) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 19-21; (6) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 16-18 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 19-21; (7) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 30-32; (8) the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 33-35; orLeydig 772435 HHS E-076-2024-0-PC-01 59 (9) the first polypeptide chain the amino acid sequences of all of SEQ ID NOs: 30-32 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 33-35.

12. The protein of claim 11, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 25; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 25; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37;Leydig 772435 HHS E-076-2024-0-PC-01 60 (16) the first polypeptide chain the amino acid sequence of SEQ ID NO: 38; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39; or (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

39.

13. The protein of claim 11 or 12, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41;Leydig 772435 HHS E-076-2024-0-PC-01 61 (15) the first polypeptide chain the amino acid sequence of SEQ ID NO: 40 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43; or (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

43.

14. A bispecific engager TCR fusion protein comprising (i) the TCR according to any one of claims 1-7, the polypeptide according to any one of claims 8-10, or the protein according to any one of claims 11-13 and (ii) an anti-CD3 effector.

15. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR of any one of claims 1-7, the polypeptide of any one of claims 8-10, the protein of any one of claims 11-14.

16. An isolated or purified nucleic acid comprising, from 5’ to 3’, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequence, respectively, encode the amino sequences of SEQ ID NOs: 8 and 9; 9 and 8; 10 and 11; 11 and 10; 12 and 13; 13 and 12; 14 and 15; 15 and 14; 22 and 23; 23 and 22; 24 and 25; 25 and 24; 26 and 27; 27 and 26; 28 and 29; 29 and 28; 36 and 37; 37 and 36; 38 and 39; 39 and 38; 40 and 41; 41 and 40; 42 and 43; or 43 and 42.

17. The isolated or purified nucleic acid of claim 16, further comprising a third nucleotide acid sequence interposed between the first and second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

18. The isolated or purified nucleic acid of claim 17, wherein the cleavable linker peptide comprises the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 47).Leydig 772435 HHS E-076-2024-0-PC-01 62 19. A recombinant expression the nucleic acid of any one of claims 15-18.

20. The recombinant expression vector of claim 19, which is a transposon or a lentiviral vector.

21. An isolated or purified TCR, polypeptide, or protein encoded by the nucleic acid of any one of claims 15-18 or the vector of claim 19 or 20.

22. An isolated or purified TCR, polypeptide, or protein that results from expression of the nucleic acid of any one of claims 15-18 or the vector of claim 19 or 20 in a cell.

23. An in vitro method of producing a host cell expressing a TCR that has antigenic specificity for a human PIK3CAE545Kor human PIK3CAN345Kamino acid sequence, the method comprising contacting a cell with the vector of claim 19 or 20 under conditions that allow introduction of the vector into the cell.

24. An isolated or purified host cell comprising the nucleic acid of any one of claims 15-18 or the recombinant expression vector of claim 19 or 20.

25. The host cell of claim 24, wherein the cell is a human lymphocyte.

26. The host cell of claim 24, wherein the cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a natural killer (NK) cell, a macrophage, a pluripotent cell, and a multipotent cell.

27. An isolated or purified population of cells comprising the host cell of any one of claims 24-26.

28. A method of producing the TCR of any one of claims 1-7, 21, or 22, the polypeptide of any one of claims 8-10, 21, or 22, or the protein of any one of claims 11-14, 21, or 22, the method comprising culturing the host cell of any one of claims 24-26, or the population of host cells of claim 27, so that the TCR, polypeptide, or protein is produced.Leydig 772435 HHS E-076-2024-0-PC-01 63 29. A pharmaceutical composition comprising (a) the TCR of any one of claims 1-7, 21, or 22, the polypeptide of any one of claims 8-10, 21, or 22, the protein of any one of claims 11-14, 21, or 22, the nucleic acid of any one of claims 15-18, the recombinant expression vector of claim 19 or 20, the host cell of any one of claims 24-26, or the population of host cells of claim 27 and (b) a pharmaceutically acceptable carrier.

30. The TCR of any one of claims 1-7, 21, or 22, the polypeptide of any one of claims 8-10, 21, or 22, the protein of any one of claims 11-14, 21, or 22, the nucleic acid of any one of claims 15-18, the recombinant expression vector of claim 19 or 20, the host cell of any one of claims 24-26, the population of host cells of claim 27, or the pharmaceutical composition of claim 29, for use in the inducment of an immune response against the cancer in the mammal.

31. The TCR of any one of claims 1-7, 21, or 22, the polypeptide of any one of claims 8-10, 21, or 22, the protein of any one of claims 11-14, 21, or 22, the nucleic acid of any one of claims 15-18, the recombinant expression vector of claim 19 or 20, the host cell of any one of claims 24-26, the population of host cells of claim 27, or the pharmaceutical composition of claim 29, for use in the treatment or prevention of cancer in the mammal.

32. The population of host cells for the use of claim 30 or 31, wherein the population of cells is autologous to the mammal.

33. The population of host cells for the use of claim 30 or 31, wherein the population of cells is allogeneic to the mammal.

34. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of host cells, or pharmaceutical composition for the use of any one of claims 31-33, wherein the cancer is an epithelial cancer.

35. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of host cells, or pharmaceutical composition for the use of any one of claims 31-33, wherein the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer,Leydig 772435 HHS E-076-2024-0-PC-01 64 ovarian cancer, endometrial cancer, non- cell lung cancer (NSCLC), glioblastoma, uterine cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

36. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of host cells, or pharmaceutical composition for the use of any one of claims 31-35, wherein the cancer is known to comprise an E545K or N345K mutation in human PIK3CA.

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