T-cell receptors targeting PIK3CA mutations and uses thereof

Abstract

The presently disclosed subject matter provides methods and compositions for treating cancer (e.g., breast cancer). The presently disclosed subject matter relates to a mutant PIK3CA-targeting TCR that specifically targets a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide), and an immune response cell comprising such a TCR. The mutant PIK3CA peptide-specific TCR of the present disclosure has enhanced immune activation properties (including anti-tumor activity). For example, the present invention provides a T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises an H1047L mutation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 864,148, filed June 20, 2019, the contents of which are incorporated herein by reference in their entirety and to which priority is claimed. Sequence Listing

[0002] This specification references a Sequence Listing (submitted electronically as a text file named "0727341093SL.txt" on June 19, 2020). This text file was created on June 18, 2020 and is 37,623 bytes in size. The entire contents of this Sequence Listing are incorporated herein by reference.

[0003] Introduction The presently disclosed subject matter provides methods and compositions for treating cancer (e.g., breast cancer, endometrial cancer, cervical cancer, head and neck cancer, colon cancer, glioblastoma multiforme) associated with T cell receptors (TCRs) that specifically target phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) containing mutations. The presently disclosed subject matter further provides immune response cells containing such TCRs and methods of using such TCRs and such cells to treat any human PIK3CA-mutated cancer, including, but not limited to, breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer. [Background technology]

[0004] Cell-based immunotherapy is a potentially curative therapy for cancer treatment.T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding specific TCR for selected antigens.Targeted T cell therapy using specific TCR has shown clinical success in recent years in the treatment of hematological malignancies.

[0005] One-third of breast cancer patients harbor PIK3CA mutations, and the locations of hotspot mutations in PIK3CA are similar across breast cancer subtypes. Furthermore, PIK3CA harbors only a limited number of hotspot mutations that are conserved across breast cancer patients. These characteristics make targeting specific mutations in PIK3CA a promising strategy for targeting and eliminating breast cancer cells. Therefore, there is a need for new therapeutic strategies to identify and generate TCRs that target mutated PIK3CA, as well as strategies that can potently induce cancer eradication with minimal toxicity and immunogenicity. Summary of the Invention [Means for solving the problem]

[0006] The presently disclosed subject matter generally provides a T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises a mutation. In certain embodiments, the mutation is H1047L. In certain embodiments, the PIK3CA peptide is an 8-mer, a 9-mer, or a 10-mer. In certain embodiments, the PIK3CA peptide is a 9-mer. In certain embodiments, the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:51, or SEQ ID NO:52. In certain embodiments, the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:11.

[0007] In certain embodiments, the PIK3CA peptide is associated with an HLA class I complex. In certain embodiments, the HLA class I complex is selected from HLA-A, HLA-B, and HLA-C. In certain embodiments, the HLA class I complex is HLA-A. In certain embodiments, the HLA-A is the HLA-A*03 superfamily. In certain embodiments, the HLA-A*03 superfamily is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74. In certain embodiments, the HLA-A*03 superfamily is HLA-A*03.

[0008] In certain embodiments, the TCR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain binds to the PIK3CA peptide. In certain embodiments, the extracellular domain comprises: a) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof; b) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof; c) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof.

[0009] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof; b) an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof; or c) an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof.

[0010] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof; b) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof; or c) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof.

[0011] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; b) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; and an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or c) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; and an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37.

[0012] In certain embodiments, the extracellular domain comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3.

[0013] In certain embodiments, the extracellular domain comprises: a) a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; c) a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0014] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or c) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0015] In certain embodiments, the extracellular domain comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0016] In certain embodiments, the extracellular domain comprises an alpha chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:31, or SEQ ID NO:41.

[0017] In certain embodiments, the extracellular domain comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 41. In certain embodiments, the extracellular domain comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0018] In certain embodiments, the extracellular domain comprises a β chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:32, or SEQ ID NO:42.

[0019] In certain embodiments, the extracellular domain comprises a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 32, or SEQ ID NO: 42. In certain embodiments, the extracellular domain comprises a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0020] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:7, and a beta chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8; b) an alpha chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:31, and a beta chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:32, or c) an alpha chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:41, and a beta chain variable region comprising an amino acid sequence at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:42.

[0021] In certain embodiments, the extracellular domain comprises: a) an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; b) an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:31; and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:32; or c) an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:41; and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:42. In certain embodiments, the extracellular domain comprises: an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0022] In certain embodiments, the extracellular domain comprises: a) an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:9; and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:10; b) an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:33; and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:34; or c) an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:43; and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:44. In certain embodiments, the extracellular domain comprises: an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:9; and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0023] In certain embodiments, the extracellular domain binds to the same epitope on the human mutant PIK3CA peptide as a reference TCR or functional fragment thereof, wherein the reference TCR or functional fragment thereof comprises: a) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; a) an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or c) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0024] In certain embodiments, the TCR is recombinantly expressed or expressed from a vector. In certain embodiments, the TCR does not target a wild-type PIK3CA peptide.

[0025] In certain embodiments, the TCR comprises a modified alpha chain constant region and / or a modified beta chain constant region. In certain embodiments, the modified alpha chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. In certain embodiments, the modified alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the engineered beta chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 45. In certain embodiments, the engineered beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0026] The presently disclosed subject matter further provides an immune response cell comprising the TCR disclosed herein. In certain embodiments, the immune response cell is transduced with the TCR. In certain embodiments, the TCR is constitutively expressed on the surface of the immune response cell. In certain embodiments, the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells from which lymphoid cells can be differentiated. In certain embodiments, the immune response cell is a T cell. In certain embodiments, the T cell is selected from the group consisting of cytotoxic T cells (CTLs), regulatory T cells, and central memory T cells.

[0027] The presently disclosed subject matter further provides a composition comprising the immune response cells disclosed herein, hi certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0028] The presently disclosed subject matter further provides a vector comprising the isolated nucleic acid molecule disclosed herein. In certain embodiments, the vector is a gamma retroviral vector.

[0029] The presently disclosed subject matter further provides a nucleic acid molecule encoding the T cell receptor (TCR) disclosed herein.The presently disclosed subject matter further provides a method for generating an immune response cell that binds to a human mutant PIK3CA peptide, comprising introducing into an immune response cell a nucleic acid molecule encoding the TCR disclosed herein or a vector containing the nucleic acid molecule.

[0030] The presently disclosed subject matter further provides a host cell comprising the nucleic acid molecule disclosed herein. In certain embodiments, the host cell is a T cell.

[0031] The presently disclosed subject matter further provides a method for treating and / or preventing a neoplasm comprising a PIK3CA mutation in a subject. The presently disclosed subject matter further provides use of the immune response cells or compositions disclosed herein in treating and / or preventing a neoplasm comprising a PIK3CA mutation in a subject. In certain embodiments, the PIK3CA mutation comprises or consists of H1047L. In certain embodiments, the method comprises administering to the subject an effective amount of the immune response cells or compositions disclosed herein. In certain embodiments, the neoplasm is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer. In certain embodiments, the neoplasm is breast cancer. In certain embodiments, the method reduces or eradicates tumor burden in the subject. In certain embodiments, the subject is human.

[0032] The presently disclosed subject matter further provides a kit for treating and / or preventing a neoplasm. In certain embodiments, the kit includes the immune response cell disclosed herein, the nucleic acid molecule disclosed herein, or the vector disclosed herein. In certain embodiments, the kit further includes written instructions for using the immune response cell, nucleic acid molecule, or vector to treat a subject with a neoplasm.

[0033] The following detailed description, given by way of example and not intended to limit the invention to the particular embodiments described, can be understood in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0034] [Figure 1]Figure 1 shows a schematic diagram of obtaining the minimal epitope sequence of mutant PIK3CA by mass spectrometry. COS-7 is a monkey-derived cell line that 1) lacks human HLA molecules and 2) is suitable for electroporation. COS-7 cells are co-electroporated with RNA encoding HLA-A*03:01 and mutant PIK3CA. Wild-type PIK3CA with HLA-A*03:01 and mutant PIK3CA with an irrelevant HLA (HLA-A*69:01) are included as assay controls. Using a pan-anti-MHC class I antibody, all class I peptide-MHC complexes are acid-eluted from the surface of transfected COS-7 cells. The sequences and sizes of the eluted peptides are then obtained by LC / MS / MS.

[0035] [Figure 2] Figure 2 illustrates the analysis of the sequence of the minimal epitope by mass spectrometry. The upper panel shows Skyline analysis showing enriched signals in the transfectant group electroporated with a combination of mutant PIK3CA and HLA-A*03:01. No signals were detected in the two control groups. The lower panel shows the results of peptide fragmentation spectroscopy of the enriched signals detected in the mutant PIK3CA + HLA-A*03:01 group. Based on the fragmentation pattern, the peptide sequence was determined to be "ALHGGWTTK" [SEQ ID NO: 11]. The wild-type peptide consists of the sequence represented by AHHGGWTTK [SEQ ID NO: 46].

[0036] [Figure 3]Figure 3 shows the results of a differential scanning fluorimetry experiment designed to determine the relative stability of the interaction between HLA-A*03:01 and wild-type or mutant PIK3CA minimal peptides. Two technical replicates were performed for each group. This technique used real-time PCR to monitor thermally induced protein denaturation, detecting changes in the fluorescent signal of a dye that preferentially binds to hydrophobic residues exposed as the protein unfolds unstably. The data demonstrated stable binding to the mutated peptide on the HLA-A*03:01 molecule, which was maintained even at temperatures exceeding 54°C. In contrast, the wild-type epitope was less stable to HLA-A*03:01, with a low melting temperature of 36°C. The mutant peptide consisted of the sequence represented by ALHGGWTTK [SEQ ID NO: 11]. The wild-type peptide consisted of the sequence represented by AHHGGWTTK [SEQ ID NO: 46].

[0037] [Figure 4] Figure 4 illustrates the crystal structure of recombinant HLA-A*03:01 complexed with either mutant or wild-type PIK3CA peptides. The X-ray crystal structure on the right shows the topography of the mutant peptide sequence (red) contained within the α-helical domain of HLA-A*03:01 (green and blue).

[0038] [Figure 5]Figure 5 illustrates the identification of HLA-A3 as a restriction element for the PIK3CA mutation-specific TCR (21LT2-2). The 21LT2-2 TCR was retrovirally integrated into the genome of nonspecific healthy donor T cells. Four to six days after transduction, T cells were incubated with monkey-derived antigen-presenting cells co-electroporated with one class I HLA allele and either the wild-type or mutant version of PIK3CA. The mutant version had a histidine-to-leucine substitution at position 1047 (H1047L). Cells were gated for CD8+ TCR+ expression. Changes in the production of inflammatory cytokines (TNF-α) were used to indicate the presence of mutation-specific reactivity with the correct HLA allele. The data demonstrated that 21LT2-2 can recognize the H1047L mutation in the context of HLA-A*03:01.

[0039] [Figure 6] Figure 6 illustrates that the PIK3CA mutant-specific TCR (21LT2-2) recognized endogenously processed antigen and minimal peptide. The 21LT2-2 TCR was retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were incubated with monkey-derived antigen-presenting cells electroporated with RNA encoding HLA-A*03:01 and either wild-type or mutant PIK3CA RNA (left) or pulsed with wild-type or mutant PIK3CA minimal 9-amino acid peptide (1 μg / mL) (right). Cells were gated for CD8+ TCR+ expression. Changes in CD107A and TNFα production were used to indicate the presence of mutant-specific reactivity with HLA-A*03:01. The data demonstrate that 21LT2-2 can recognize both endogenously processed and presented mutant antigen (left) and passively pulsed mutant minimal peptide (right).

[0040] [Figure 7]Figure 7 illustrates that the PIK3CA mutant-specific TCR (21LT2-2) is coreceptor-independent. The 21LT2-2 TCR was retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were incubated with RNA encoding HLA-A*03:01 and monkey-derived antigen-presenting cells electroporated with either wild-type or mutant PIK3CA RNA (left) or pulsed with the minimal 9-amino acid peptide (1 μg / mL) (right). Cells were gated for CD4+ TCR+ expression. Changes in CD107A and TNFα production were used to indicate the presence of mutant-specific reactivity with respect to HLA-A*03:01. The data indicate that 21LT2-2 is also functional within the CD4+ T cell backbone and does not require the presence of a CD8 coreceptor to recognize the H1047L mutation.

[0041] [Figure 8-1] Figure 8 illustrates the alanine substitutions, demonstrating that all positions except P4 are essential for pMHC:TCR interaction. The 21LT2-2 TCR was retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were incubated with monkey-derived antigen-presenting cells electroporated with RNA encoding HLA-A*03:01 and pulsed with the alanine-substituted peptide (10 μg / mL). Cells were gated for CD8+ TCR+ expression. A >50% reduction in recognition when pulsed with the alanine-substituted peptide compared to the unmodified 9-amino acid mutant sequence (Table; mutation at P2) would indicate that the native amino acid at that position is important for TCR recognition. The data presented here demonstrate that, with the exception of the glycine residue at position 4, all amino acids in this 9-amino acid sequence appear to be important for pMHC-TCR interaction. These peptide sequences are SEQ ID NOs: 11-19, respectively, from left to right. [Figure 8-2] Same as above.

[0042] [Figure 9] FIG. 9 illustrates a scan of the human proteome for potential cross-reactivity for 21LT2-2.

[0043] [Figure 10] FIG. 10 illustrates the identification of additional unique T cell receptors that recognize mutant PIK3CA restricted by HLA-A*03:01.

[0044] [Figure 11] Figure 11 illustrates multiple unique T cell receptors capable of recognizing mutant PIK3CA restricted by HLA-A*03:01. Cells were gated on CD8+TCR+ cells.

[0045] [Figure 12] Figure 12 illustrates the coreceptor dependence of mutant PIK3CA-specific T cell receptors. Cells were gated on CD4+TCR+ cells.

[0046] [Figure 13] Figure 13 shows that the PIK3CA neoantigen-specific receptor exhibited cytolytic activity. Cells were gated on CD8+TCR+ cells.

[0047] [Figure 14-1]Figures 14A-14C illustrate alanine-substituted peptides that exhibited different TCR-HLA / peptide interaction profiles. Figure 14A relates to P2-P5; Figure 14B relates to P6-P9; and Figure 14C relates to the native wild-type PIK3CA peptide and the mutant PIK3CA P1 peptide. The P1 peptide consists of the amino acid sequence set forth in SEQ ID NO: 11 (ALHGGWTTK). The P2 peptide consists of the amino acid sequence set forth in SEQ ID NO: 12 (AAHGGWTTK). The P3 peptide consists of the amino acid sequence set forth in SEQ ID NO: 13 (ALAGGWTTK). The P4 peptide consists of the amino acid sequence set forth in SEQ ID NO: 14 (ALHAGWTTK). The P5 peptide consists of the amino acid sequence set forth in SEQ ID NO: 15 (ALHGAWTTK). The P6 peptide consists of the amino acid sequence set forth in SEQ ID NO: 16 (ALHGGATTK). The P7 peptide consists of the amino acid sequence set forth in SEQ ID NO: 17 (ALHGGWATK). The P8 peptide consists of the amino acid sequence set forth in SEQ ID NO: 18 (ALHGGWTAK). The P9 peptide consists of the amino acid sequence set forth in SEQ ID NO: 19 (ALHGGWTTA). The wild-type PIK3CA peptide consists of the amino acid sequence set forth in SEQ ID NO: 46 (AHHGGWTTK). [Figure 14-2] Same as above. [Figure 14-3] Same as above.

[0048] [Figure 15] Figure 15 illustrates the motifs assigned to identify residues important for TCR recognition and cross-reactivity determination. The dotted line indicates 50% of maximum reactivity. Based on this, motifs for determining potential cross-reactivity are shown on the right. An "x" indicates a residue that is not important for TCR recognition. Position 1 has been assigned an "x" as the default because the native amino acid at this position is alanine and therefore not applicable to this test. XLHXGWTTK is SEQ ID NO: 48; XXXXGWTTK is SEQ ID NO: 49, and XLHGGWTTK is SEQ ID NO: 50.

[0049] [Figure 16-1] Figures 16A and 16B illustrate scanning of the human proteome for potential cross-reactivity. Figure 16 shows that no cross-reactive epitopes were detected for TCR 21LT2-2 and 1022T8. One potentially cross-reactive epitope (ivfmGWTTK) was detected for TCR 0606T1-2. Figure 16B shows that HLA*A03:01+ target cells pulsed with a potentially cross-reactive epitope did not trigger 0606T1-2. A mutant PIK3CA peptide was used as a control. ALHGGWTTK is SEQ ID NO: 11. ivfmGWTTK is SEQ ID NO: 47. [Figure 16-2] Same as above.

[0050] [Figure 17] Figure 17 illustrates that PIK3CA mutant-specific TCRs can recognize mutant peptides of different lengths. Mutant PIK3CA-specific TCRs were retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were incubated with HLA-A*03:01+ antigen-presenting cells and pulsed with 8-mer and 10-mer mutant PIK3CA peptide pools (1 μg / mL). Wild-type and 9-mer peptides were used as controls. Cells were gated by CD8+ TCR+ expression. Mutant peptide-specific upregulation of TNFα production indicates that different peptide lengths are recognized. The wild-type 9-mer consists of the amino acid sequence set forth in SEQ ID NO: 46 (AHHGGWTTK). The mutant 9-mer consists of the amino acid sequence represented by ALHGGWTTK (SEQ ID NO: 11). The mutant pool contains an 8-mer peptide consisting of the amino acid sequence represented by LHGGWTTK (SEQ ID NO: 51) and a 10-mer peptide consisting of the amino acid sequence represented by DALHGGWTTK (SEQ ID NO: 52). DETAILED DESCRIPTION OF THE INVENTION

[0051] The presently disclosed subject matter provides TCRs that target PIK3CA (eg, human PIK3CA) that include a mutation, for example, the mutation includes or consists of H1047L.

[0052] The presently disclosed subject matter also provides immune response cells (e.g., T cells (e.g., cytotoxic T cells (CTLs), regulatory T cells, central memory T cells, etc.)), natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells from which lymphoid cells can differentiate) that comprise a PIK3CA-targeting TCR, and methods of using such immune response cells to treat neoplasms (e.g., breast cancer). I. Definition

[0053] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0054] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice of the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0055] As used herein, the term "cell population" refers to a group of at least two cells that express similar or different phenotypes. In a non-limiting example, a cell population may contain at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cells that express similar or different phenotypes.

[0056] As used herein, the term "vector" refers to any genetic element, e.g., a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which, when accompanied by the appropriate control elements, is capable of replication and the transfer of gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral and plasmid vectors.

[0057] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence (e.g., a recombinant DNA molecule) containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site (often along with other sequences). Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0058] As used herein, "CDR" is defined as the complementarity determining region amino acid sequences of a TCR, which are the hypervariable regions of the TCR alpha and beta chains. Generally, a TCR contains at least three CDRs in the alpha chain variable region and at least three CDRs in the beta chain variable region. The CDRs provide the majority of contact residues for binding of the TCR to an antigen or epitope. CDR regions can be delineated using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), the Chothia numbering system (Chothia et al., J Mol Biol. (1987) 196:901-17), the AbM numbering system (Abhinandan et al., Mol. Immunol. 2008, 45, 3832-3839), or the IMGT numbering system (http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html, accessible at http: / / www.imgt.org / IMGTindex / numbering.php). In certain embodiments, the CDR regions are delineated using the IMGT numbering system.

[0059] Nucleic acid molecules useful in the presently disclosed subject matter include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules useful in the presently disclosed subject matter include nucleic acid molecules encoding a TCR or its target binding site. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial homology" or "substantial identity" to an endogenous sequence typically can hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing with a complementary polynucleotide sequence (e.g., a gene described herein) or portion thereof to form a double-stranded molecule under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0060] The terms "substantially homologous" or "substantially identical" refer to a polypeptide or nucleic acid molecule that exhibits at least 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence may be at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or even about 99% homologous or identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0061] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of similarity to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program can be used, e.g., -3 ~ e-100 A probability score of indicates that the sequences are highly related.

[0062] As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0063] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, a ligand binds to a receptor on another cell, allowing for intercellular recognition and / or interaction.

[0064] As used herein, the term "disease" refers to any condition or disorder that inhibits or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasms or pathogenic infection of cells.

[0065] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease (e.g., a neoplasm), or otherwise reduce the pathological consequences of a disease (e.g., a neoplasm). An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of the art. When determining an appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the immune response cells administered.

[0066] As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and subsequent migration or invasion of other tissues or organs. Neoplastic growth is typically uncontrolled and progressive and occurs under conditions that would not induce or arrest the proliferation of normal cells. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, organs or tissues or cell types selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0067] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA molecule, a DNA molecule, or an RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0068] As used herein, the term "immune response cell" refers to a cell that functions in an immune response, or its progenitor, or its progeny.

[0069] As used herein, the term "modulate" refers to changing, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0070] As used herein, the term "increase" refers to a positive alteration by at least about 5%, including, but not limited to, a positive alteration by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0071] As used herein, the term "reduce" refers to a negative alteration by at least about 5%, including, but not limited to, a negative alteration by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0072] As used herein, the term "isolated cell" refers to a cell that has been separated from the molecular and / or cellular components that naturally accompany the cell.

[0073] As used herein, the terms "isolated," "purified," or "biologically pure" refer to a substance that is free, to varying degrees, from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or environment. "Purified" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is free from other substances to the extent that any impurities do not substantially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free from cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free from chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can be modified (eg, phosphorylation or glycosylation), different modifications can result in different isolated proteins that can be separately purified.

[0074] As used herein, the term "secreted" intends polypeptides that are released from the cell by the secretory pathway through the endoplasmic reticulum, the Golgi apparatus, and as vesicles that transiently fuse with the cell plasma membrane, releasing the protein to the exterior of the cell.

[0075] As used herein, the terms "specifically bind" or "specifically binds to" or "specifically targets" refer to a polypeptide or fragment thereof that recognizes and binds a biological molecule of interest (e.g., a polypeptide) but does not substantially recognize and bind other molecules in a sample (e.g., a biological sample containing or expressing a human mutant PIK3CA peptide).

[0076] As used herein, the term "treat" or "treatment" refers to clinical intervention in an attempt to alter the disease course of the treated individual or cell, and can be performed for prophylaxis or during the clinical pathological process. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of the pathology, and amelioration or improvement of prognosis. By preventing the progression of a disease or disorder, treatment can prevent the progression caused by the disorder in an affected or diagnosed subject, or a subject suspected of having the disorder, but treatment can also prevent the occurrence of the disorder or the symptoms of the disorder in a subject at risk of the disorder or suspected of having the disorder.

[0077] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, etc. (e.g., that will be the recipient of a particular treatment or from which cells are collected). II.PIK3CA

[0078] Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; Gene ID: 5290 (also known as MCM, CWS5, MCAP, PI3K, CLOVE, MCMTC, PI3K-alpha, and p110-alpha)) is a gene encoding the catalytic subunit of phosphatidylinositol 3-kinase, which uses ATP to phosphorylate PtdIns, PtdIns4P, and PtdIns(4,5)P2. PIK3CA has been shown to be oncogenic and has been associated with breast and cervical cancer. III. T cell receptor (TCR)

[0079] TCRs are disulfide-linked heterodimeric proteins consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCRs comprise an α chain and a β chain (encoded by TRA and TRB, respectively). In certain embodiments, TCRs comprise a γ chain and a δ chain (encoded by TRG and TRD, respectively).

[0080] Each chain of the TCR contains two extracellular domains: a variable region and a constant region. The constant region is close to the cell membrane and is followed by a transmembrane domain and a short cytoplasmic tail (i.e., the intracellular domain). The variable region binds to the peptide / MHC complex. The variable regions of both chains each contain three complementarity-determining regions (CDRs).

[0081] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex binds to its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0082] In certain embodiments, the subject matter of the present disclosure provides a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the TCR differs from any naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues. In certain embodiments, the TCR has been modified by at least one amino acid residue from a naturally occurring TCR. In certain embodiments, the TCR has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues altered from a naturally occurring TCR.

[0083] In certain embodiments, the TCR specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises or consists of a mutation ("mutant PIK3CA peptide"). In certain embodiments, the mutation is selected from the group consisting of H1047R, E545K, E542K, N345K, H1047L, E726K, C420R, and any combination thereof. In certain embodiments, the mutation is H1047R or H1047L. In certain embodiments, the mutation is H1047L. In certain embodiments, the PIK3CA peptide consists of the mutation H1047L, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the TCR does not target a wild-type PIK3CA peptide. In certain embodiments, the wild-type PIK3CA peptide is a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46. SEQ ID NO: 46 is shown below. [ka]

[0084] In certain embodiments, the TCR specifically targets a PIK3CA peptide listed in Figure 8 and Figures 14A-14C. In certain embodiments, the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 51, or SEQ ID NO: 52. SEQ ID NOs: 11-19, 51, and 52 are shown below. [ka]

[0085] In certain embodiments, the TCR specifically targets a PIK3CA peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the TCR specifically targets a PIK3CA peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0086] In certain embodiments, the TCR specifically targets a PIK3CA peptide that binds to an HLA class I complex (e.g., HLA-A, HLA-B, and HLA-C). In certain embodiments, the TCR specifically targets a PIK3CA peptide that binds to an HLA class II complex (e.g., HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR).

[0087] In certain embodiments, the TCR specifically targets a PIK3CA peptide that binds to the HLA-A*03 superfamily (e.g., in an HLA-A*03 superfamily-dependent manner). In certain embodiments, the HLA*A03 superfamily members include, but are not limited to, alleles and suballeles of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74. TCR clonotype

[0088] In certain embodiments, the TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the TCR comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 9 and SEQ ID NO: 10 are shown in Table 1.

[0089] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the extracellular domain of the TCR comprises a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NOs: 7 and 8 are shown in Table 1.

[0090] In certain embodiments, the TCR is a human TCR that specifically binds to a mutant PIK3CA peptide (eg, a human mutant PIK3CA peptide) and is designated 21LT2-2.

[0091] In certain embodiments, the TCR is a human TCR and comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:9 and / or a beta chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0092] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region and a beta chain variable region, or a CDR selected from Table 1. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 7. For example, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 8. For example, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:7, and a beta chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:8.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0093] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or a conservative modification thereof. SEQ ID NOs: 1 to 3 are shown in Table 1. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof. SEQ ID NOs: 4 to 6 are shown in Table 1. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or a conservative modification thereof, a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. Table 1.(21LT2-2) [Table 1-1] [Table 1-2]

[0094] In certain embodiments, the CDR regions described above (including in Table 1) are delineated using the IMGT numbering system (http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html, accessible at http: / / www.imgt.org / IMGTindex / numbering.php).

[0095] In certain embodiments, the TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the TCR comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 34. SEQ ID NO: 33 and SEQ ID NO: 34 are shown in Table 2.

[0096] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the extracellular domain of the TCR comprises a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. SEQ ID NO: 31 and SEQ ID NO: 32 are shown in Table 2.

[0097] In certain embodiments, the TCR is a human TCR that specifically binds to a mutant PIK3CA peptide (eg, a human mutant PIK3CA peptide) and is designated 0606T1-2.

[0098] In certain embodiments, the TCR is a human TCR and comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 33 and / or a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0099] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region and a beta chain variable region, or CDRs selected from Table 2. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 31. For example, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32. For example, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 31, and a beta chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:31, and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:32.

[0100] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof. SEQ ID NOs: 25-27 as disclosed in Table 2. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. SEQ ID NOs: 28-30 as disclosed in Table 2. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof, a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. Table 2.(0606T1-2) [Table 2-1] [Table 2-2]

[0101] In certain embodiments, the CDR regions described above (including in Table 2) are delineated using the IMGT numbering system (http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html, accessible at http: / / www.imgt.org / IMGTindex / numbering.php).

[0102] In certain embodiments, the TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the TCR comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 44. SEQ ID NOs: 43 and 44 are disclosed in Table 2.

[0103] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the extracellular domain of the TCR comprises a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42. SEQ ID NOs: 41 and 42 are disclosed in Table 3.

[0104] In certain embodiments, the TCR is a human TCR that specifically binds to a mutant PIK3CA peptide (eg, a human mutant PIK3CA peptide) and is designated 1022T8.

[0105] In certain embodiments, the TCR is a human TCR and comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 43 and / or a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 44.

[0106] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region and a beta chain variable region, or CDRs selected from Table 3. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 41. For example, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42. For example, the extracellular domain of the TCR comprises a β chain variable region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 41, and a beta chain variable region comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO:41, and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:42.

[0107] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof. SEQ ID NOs: 35 to 37 are disclosed in Table 3. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof. SEQ ID NOs: 38 to 40 are disclosed in Table 3. In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof, an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof.In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37, a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. Table 3.(1022T8) [Table 3]

[0108] In certain embodiments, the CDR regions described above (including in Table 3) are delineated using the IMGT numbering system (http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html, accessible at http: / / www.imgt.org / IMGTindex / numbering.php).

[0109] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the TCR of the present disclosure, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Amino acids can be classified into groups based on physicochemical properties such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group, and the altered TCR can be tested for retained function (i.e., the functions described in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, two, three, four, or five residues within a particular sequence or CDR region are altered.

[0110] In certain embodiments, the amino acid sequence of the α chain variable region and / or β chain variable region has at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a specific sequence (e.g., SEQ ID NOs: 7, 8, 31, 32, 41, and 42) and contains modifications including, without limitation, substitutions (e.g., conservative substitutions), insertions, or deletions to the specific sequence(s), but retains the ability to bind to a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide). In certain embodiments, such modifications are not within the CDR domains of said variable regions.

[0111] In certain embodiments, the extracellular domain specifically binds to a mutant PIK3CA peptide (eg, a human mutant PIK3CA peptide) but not to the corresponding wild-type peptide sequence.

[0112] In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41, or SEQ ID NO:42. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDRs of the extracellular domain. In certain embodiments, the extracellular domain comprises an α chain variable region and / or a β chain variable region sequence selected from the group consisting of SEQ ID NOs:7, 8, 31, 32, 41, and 42, including post-translational modifications of the sequence (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41, or SEQ ID NO:42).

[0113] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which must be introduced for optimal alignment of the two sequences) (i.e., % homology = number of identical positions / total number of positions x 100). Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm.

[0114] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is incorporated into the ALIGN algorithm (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0115] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can also be used as "query sequences" to search public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to specific sequences disclosed herein. To perform gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0116] In certain embodiments, the extracellular domain of the TCR binds to the same epitope on a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide) or a functional fragment thereof as a reference TCR. For example, the extracellular domain of a TCR of the present disclosure binds to the same epitope on a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide) or a functional fragment thereof as a reference TCR, for example, comprising the α-chain variable region CDR1, CDR2, and CDR3 sequences and the β-chain variable region CDR1, CDR2, and CDR3 sequences of any one of the TCRs of the present disclosure. In certain embodiments, the extracellular domain of a TCR of the present disclosure binds to the same epitope on a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide) or a functional fragment thereof as a reference TCR, for example, comprising the α-chain variable region sequence and the β-chain variable region sequence of any one of the TCRs of the present disclosure.

[0117] It is well known in the art that the CDR3 domain can alone, independently of the CDR1 and / or CDR2 domain(s), determine the binding specificity of a TCR or functional fragment thereof to a cognate antigen, and that multiple TCRs with the same binding specificity can be predictably generated based on a common CDR3 sequence.

[0118] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or a conservative modification thereof; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof; and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof; and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof.

[0119] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof; and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof; and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof.

[0120] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof; and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof; and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof.

[0121] In certain embodiments, the TCR of the presently disclosed subject matter further comprises an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter used for expression of the TCR gene can be a constitutive promoter (e.g., a ubiquitin C (UbiC) promoter).

[0122] The presently disclosed subject matter also provides nucleic acid molecules encoding the mutant PIK3CA-targeted TCRs described herein. In certain embodiments, the nucleic acid molecules encode both the α and β chains of the TCRs of the present disclosure. In certain embodiments, the α and β chains are separated by a self-cleaving peptide (e.g., a 2A-peptide). In certain embodiments, the α and β chains are separated by a furin 2A peptide. In certain embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 24. [ka]

[0123] In certain embodiments, the nucleic acid molecule encodes a functional portion / fragment of the mutant PIK3CA-targeted TCR of the present disclosure. As used herein, the term "functional portion" or "functional fragment" refers to any portion, portion, or fragment of the mutant PIK3CA-targeted TCR of the present disclosure, which portion, portion, or fragment retains the biological activity of the mutant PIK3CA-targeted TCR (parent TCR). For example, a functional portion encompasses a portion, portion, or fragment of the mutant PIK3CA-targeted TCR of the present disclosure that retains the ability to recognize target cells and treat diseases (e.g., breast cancer) to a similar, equal, or even greater extent than the parent TCR. In certain embodiments, the nucleic acid molecule encoding the functional portion of a mutant PIK3CA-targeted TCR of the present disclosure encodes a protein comprising, for example, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more than about 95%, of the parent TCR. TCRs with modifications within the CDRs

[0124] In certain embodiments, a TCR (or functional fragment thereof) of the present disclosure comprises an alpha chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a beta chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise specific amino acid sequences based on, or modifications of, a TCR (or functional fragment thereof) described herein (see Tables 1-3), and wherein the TCR (or functional fragment thereof) retains the desired functional properties of the mutant PIK3CA peptide-specific TCR (or functional fragment thereof) of the presently disclosed subject matter.

[0125] In certain embodiments, a TCR (or functional fragment thereof) of the present disclosure comprises an alpha chain constant region and a beta chain constant region, wherein at least one of the constant regions comprises a specific amino acid sequence based on a TCR (or functional fragment thereof) described herein (see Tables 1-3), or a modification thereof, and wherein the TCR (or functional fragment thereof) retains the desired functional properties of the mutant PIK3CA peptide-specific TCR (or functional fragment thereof) of the presently disclosed subject matter.

[0126] In certain embodiments, such modifications do not significantly affect or change the binding properties of the TCR comprising the amino acid sequence. Non-limiting examples of such modifications include amino acid substitution, addition, and deletion. Modifications can be introduced into the TCR of the present disclosure or its functional fragments by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-based mutagenesis).

[0127] The modifications may be conservative, non-conservative, or a combination of conservative and non-conservative modifications. As discussed above, a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Exemplary conservative amino acid substitutions are shown in Table 4. In certain embodiments, amino acid substitutions may be introduced into a TCR of interest, and the products may be screened for a desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC). Table 4 [Table 4]

[0128] Amino acids can be grouped according to common side chain properties: · Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; ·Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; ·Acidic: Asp, Glu; · Basic: His, Lys, Arg; · Residues that influence chain orientation: Gly, Pro; ·Aromatic: Trp, Tyr, Phe.

[0129] In certain embodiments, one or more amino acid residues in the CDR regions may be replaced with other amino acid residues from the same group, and the altered TCR may be tested using the functional assays described herein.

[0130] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0131] In certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues within a particular sequence or CDR region are altered.

[0132] In certain embodiments, one or more amino acid residues within the constant region of a TCR may be modified to enhance stability and / or cell surface expression of said TCR, hi certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues within a particular sequence or constant region are altered. In certain embodiments, the modifications include, but are not limited to, murineization, cysteine ​​modifications, and transmembrane modifications (Cohen et al., Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR / CD3 stability, Cancer Res. 2006; 66(17):8878-8886; Cohen et al., Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond, Cancer Res. 2007; 67(8):3898-3903; Kuball et al., Facilitating matched pairing and expression of TCR chains introduced into human T cells, Blood 2007; 109(6):2331-2338; Haga-Friedman et al., Incorporation of transmembrane hydrophobic mutations in the TCR enhance its surface expression and T cell functional avidity, Journal of Immunology 2012;188(11):5538-5546, the contents of each of which are incorporated herein by reference in their entireties.

[0133] In certain embodiments, the TCRs disclosed herein comprise a modified TCR alpha chain constant region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. In certain embodiments, the modified TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the modified TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0134] In certain embodiments, the TCRs disclosed herein comprise a modified TCR β chain constant region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 45. In certain embodiments, the modified TCR β chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the modified TCR β chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the modified TCR β chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 45.

[0135] Human alpha chain constant region: [ka] Mouse alpha chain constant region: (cysteine ​​modifications in the transmembrane domain and LVL modifications are underlined) [ka] Human beta chain constant region: [ka] Mouse beta chain constant region: (cysteine ​​modifications are underlined) [ka] Human beta chain constant region: [ka] V. Immune Response Cells

[0136] The presently disclosed subject matter provides cells comprising a TCR (e.g., a TCR disclosed herein) that targets a mutant PIK3CA peptide. Such cells are administered to a human subject in need thereof to treat and / or prevent neoplasia (e.g., breast cancer). In certain embodiments, the cells are immune response cells.

[0137] The presently disclosed subject matter provides immune response cells comprising a TCR that specifically binds to the mutant PIK3CA peptides described above (eg, human mutant PIK3CA peptides).

[0138] The immune response cells can be transduced with a TCR of the present disclosure such that the cells express the TCR. The presently disclosed subject matter also provides methods of using such cells for the treatment of neoplasia (e.g., breast cancer).

[0139] The immune response cells of the presently disclosed subject matter can be cells of the lymphoid lineage. The lymphoid lineage (including B cells, T cells, and natural killer (NK) cells) are responsible for producing TCRs, regulating the cellular immune system, detecting foreign factors in the blood, detecting cells foreign to the host, and the like. Non-limiting examples of immune response cells of the lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., from which lymphoid cells can be differentiated). T cells mature in the thymus and can be lymphocytes that are primarily important in cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells (e.g., T EM Cells and T EMRA Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. In certain embodiments, the TCR-expressing T cells express Foxp3 and achieve and maintain a T regulatory phenotype.

[0140] Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to exert their cytotoxic effect on target cells.

[0141] The immune response cells of the presently disclosed subject matter can express a TCR that specifically binds to a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide) for treating cancer (e.g., breast cancer). Such immune response cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment of cancer (e.g., breast cancer). In certain embodiments, the immune response cells are T cells. The T cells are CD4+ T cells or CD8 + In certain embodiments, the T cells may be CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0142] The immune response cells of the present disclosure may further comprise at least one recombinant or exogenous costimulatory ligand. For example, the immune response cells of the present disclosure may be further transduced with at least one costimulatory ligand such that the immune response cells co-express or are induced to co-express the PIK3CA-targeting TCR and at least one costimulatory ligand. The interaction between the PIK3CA-targeting TCR and at least one costimulatory ligand provides a non-antigen-specific signal important for the full activation of immune response cells (e.g., T cells). Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily ligands and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share many common characteristics. Most members of the TNF superfamily are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Members of the TNF superfamily include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor β (TNFβ) / lymphotoxin α (LTα), lymphotoxin β (LTβ), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of soluble cell surface proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural characteristics with immunoglobulins; they contain immunoglobulin domains (folds).Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86 (both of which are ligands for CD28), and PD-L1 / (B7-H1) (which is a ligand for PD-1). In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the immune response cells comprise one recombinant costimulatory ligand, which is 4-1BBL. In certain embodiments, the immune response cells comprise two recombinant costimulatory ligands, which are 4-1BBL and CD80.

[0143] Furthermore, the immune response cells of the present disclosure may contain at least one exogenous cytokine. For example, the immune response cells of the present disclosure may be further transduced with at least one cytokine so that the immune response cells not only express a mutant PIK3CA-targeting TCR but also secrete the at least one cytokine. In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-18, and IL-21. In certain embodiments, the cytokine is IL-12.

[0144] The mutant PIK3CA peptide-specific or mutant PIK3CA-targeted human lymphocytes may be peripheral donor lymphocytes (e.g., those disclosed in Sadelain, M. et al., 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express TCRs); Morgan, RA et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express the full-length tumor antigen-recognizing T cell receptor complex containing an αβ heterodimer); Panelli, MC et al., 2000 J Immunol 164:495-504; Panelli et al., 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J. et al., 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al., 2003 Blood 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). The immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0145] The raw source of CTLs can be any known in the art, such as bone marrow, fetal, neonatal, or adult, or other hematopoietic cell sources (e.g., fetal liver, peripheral blood, or umbilical cord blood). Various techniques can be employed to separate cells. For example, non-CTLs can be first removed by negative selection methods.

[0146] The majority of terminally differentiated cells can be first removed by a relatively crude separation. For example, magnetic bead separation can be used to first remove many irrelevant cells. Preferably, at least about 80%, usually at least about 70%, of the total hematopoietic cells are removed prior to cell isolation.

[0147] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with TCR-coated magnetic beads; affinity chromatography; cytotoxic agents (including, but not limited to, complement and cytotoxins) conjugated to or in combination with mAbs; and panning with TCRs bound to solid matrices (e.g., plates, chips), elutriation, or any other convenient technique.

[0148] Techniques for separation and analysis include, but are not limited to, flow cytometry, which may have varying degrees of sophistication (e.g., multiple color channels, low and obtuse angle light scatter detection channels, impedance channels).

[0149] The cells may be selected against dead cells by utilizing a dye associated with dead cells, such as propidium iodide (PI). Preferably, the cells are collected in a medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, preferably sterile, isotonic medium. VI. Vectors

[0150] Genetic modification of immune response cells (e.g., T cells, NK cells) can be achieved by transducing a substantially homogeneous cell composition with recombinant DNA or RNA constructs.The vector can be a retroviral vector (e.g., gamma retrovirus), which is used to introduce DNA or RNA constructs into the host cell genome.For example, the polynucleotide encoding a mutant PIK3CA-targeting TCR can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from the long terminal repeat of the retrovirus, or from an alternative internal promoter.

[0151] Non-viral vectors or RNA can also be used. Random chromosomal integration, or targeted integration (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs)), or transgene expression (e.g., using natural or chemically modified RNA) can be used.

[0152] For the initial genetic modification of cells to provide mutant PIK3CA-targeted TCR-expressing cells, retroviral vectors are generally used for transduction, but any other suitable viral vector or non-viral delivery system can be used.For the subsequent genetic modification of cells to provide cells containing an antigen-presenting complex containing at least two costimulatory ligands, retroviral gene transfer (transduction) has been found to be similarly effective.The combination of a retroviral vector and an appropriate packaging line is also suitable, in this case, the capsid protein functions to infect human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles (e.g., particles pseudotyped with VSVG, RD114, or GALV envelopes, and any other particles known in the art) are also suitable.

[0153] Possible methods of transduction also include direct co-culturing of the cells with producer cells (e.g., by the method of Bregni et al. (1992) Blood 80:1418-1422), or by culturing with viral supernatant alone or concentrated vector stocks in the presence or absence of appropriate growth factors and polycations (e.g., by the method of Xu et al. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817).

[0154] Viral vector transduction can be used to express costimulatory ligands and / or secrete cytokines (e.g., 4-1BBL and / or IL-12) in immune response cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors have been particularly well developed and are used in clinical practice (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., US Pat. No. 5,399,346).

[0155] In certain non-limiting embodiments, the vector expressing a mutant PIK3CA-targeting TCR of the present disclosure is a retroviral vector (eg, an oncoretroviral vector).

[0156] Non-viral approaches can also be used to express proteins in cells. For example, nucleic acid molecules can be introduced into cells by lipofection (Feigner et al., Proc. Nat'l. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also be potentially useful for delivering DNA into cells. Transplantation of normal genes into affected tissues of a subject can also be achieved by introducing normal nucleic acids into a culturable cell type ex vivo (e.g., autologous or heterologous primary cells, or their progeny), followed by injection of the cells (or their progeny) into the target tissue or systemically. Recombinant receptors can also be induced or obtained by using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression can be achieved by RNA electroporation.

[0157] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1 alpha enhancer / promoter / intron structure). For example, if desired, enhancers known to direct gene expression preferentially in specific cell types can be used to direct expression of the nucleic acid. Enhancers used can include, but are not limited to, those characterized as tissue-specific or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences from a heterologous source (including any of the promoters or regulatory elements described above).

[0158] The resulting cells can be grown under conditions similar to those for unmodified cells, thereby allowing the modified cells to be expanded and used for a variety of purposes. VII. Genome integration into immune response cells

[0159] In certain embodiments, the TCR of the present disclosure can be integrated into a selected locus in the genome of an immune response cell. Any targeted genome editing method can be used to integrate a TCR into a selected locus in the genome of an immune response cell. In certain embodiments, the expression of the TCR is driven by an endogenous promoter / enhancer in or near the locus. In certain embodiments, the expression of the TCR is driven by an exogenous promoter integrated into the locus. The locus into which the TCR is integrated is selected based on the expression level of the gene in the locus and the timing of gene expression of the gene in the locus. The expression level and timing can vary in various cell differentiation stages and mitogen / cytokine microenvironments (which are among the factors considered when making the selection).

[0160] In certain embodiments, the CRISPR system is used to integrate the TCR into the selected locus of the genome of immune response cells. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide to the correct section of host DNA, along with a region that binds to tracrRNA (usually in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and also forms an active complex with Cas9), and an optional DNA repair template (DNA that guides the cell's repair process, allowing for the insertion of specific DNA sequences). CRISPR / Cas9 often uses a plasmid to transfect target cells. The crRNA is the sequence used by Cas9 to identify and directly bind to target DNA in cells, and therefore must be designed for each application. The repair template containing the TCR expression cassette must also be designed for each application, as it must overlap the sequences on either side of the cleavage and encode the insertion sequence. Multiple crRNAs and the tracrRNA may be combined to form a single guide RNA (sgRNA). This sgRNA can be combined with the Cas9 gene and made into a plasmid for transfection into cells. Methods for using the CRISPR system are described, for example, in WO2014093661A2, WO2015123339A1, and WO2015089354A1 (which are incorporated by reference in their entireties).

[0161] In certain embodiments, zinc finger nucleases are used to integrate the TCR into a selected locus in the genome of an immune response cell. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be designed to target a specific DNA sequence, allowing the zinc finger nuclease to target a desired sequence within the genome. The DNA-binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger domains with known specificities. The most common cleavage domain in ZFNs is the nonspecific cleavage domain derived from the type IIs restriction enzyme FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template bearing the TCR expression cassette, ZFNs can be used to insert the TCR expression cassette into a genome. When the target sequence is cut by ZFN, the HR mechanism searches for the homology between the damaged chromosome and the homologous DNA template, and then replicates the sequence of the template between the two cut ends of the chromosome, thereby the homologous DNA template is integrated into the genome.Methods using ZFN system are described, for example, in WO2009146179A1, WO2008060510A2 and CN102174576A (these are incorporated by reference in their entirety).

[0162] In certain embodiments, the TALEN system is used to integrate the TCR into a selected locus in the genome of an immune response cell. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be designed to cleave specific sequences in DNA. TALEN systems function similarly to ZFNs. They are generated by combining the DNA-binding domain of a transcription activator-like effector with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two variable sites that have strong recognition for specific nucleotides. By constructing an array of these TALEs, the TALE DNA-binding domain can be designed to bind to a desired DNA sequence, thereby guiding the nuclease to cleave a specific location in the genome. Methods for using the TALEN system are described in WO2014134412A1, WO2013163628A2, and WO2014040370A1, which are incorporated by reference in their entireties.

[0163] The method for delivering the genome editing agent may vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered by a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, imperfection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyping of viral vector envelope proteins, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0164] The modification can be made anywhere within the selected locus or anywhere that may affect gene expression of the integrated TCR. In certain embodiments, the modification is introduced upstream of the transcription start site of the integrated TCR. In certain embodiments, the modification is introduced between the transcription start site and the protein coding region of the integrated TCR. In certain embodiments, the modification is introduced downstream of the protein coding region of the integrated TCR. VIII. Polypeptides and Analogs and Polynucleotides

[0165] The presently disclosed subject matter also includes a TCR or functional fragment thereof that specifically binds to a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide), and a polynucleotide encoding the TCR that has been modified in a manner that enhances its anti-tumor activity when expressed in immune response cells. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by making alterations within the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further includes analogs of any naturally occurring polypeptide of the presently disclosed subject matter. Analogs may differ from the naturally occurring polypeptide of the presently disclosed subject matter by differences in amino acid sequence, by post-translational modifications, or both. Analogs of the presently disclosed subject matter generally may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity or homology with all or a portion of a naturally occurring amino acid sequence of the presently disclosed subject matter. The length of sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program can be used, e.g., -3 ~e -100A probability score of 0 indicates high sequence relatedness. Modifications include in vivo and in vitro chemical derivatization of polypeptides (e.g., acetylation, carboxylation, phosphorylation, or glycosylation); such modifications may occur during polypeptide synthesis or processing or after treatment with isolated modifying enzymes. Analogs may also differ from naturally occurring polypeptides of the presently disclosed subject matter by alterations in the primary sequence. These include genetic variants, both natural and induced (e.g., obtained by random mutagenesis, e.g., by exposure to radiation or ethanemethylsulfate, or by site-directed mutagenesis as described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs containing residues other than L-amino acids, e.g., D-amino acids, or non-naturally occurring or synthetic amino acids (e.g., beta (β) or gamma (γ) amino acids).

[0166] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptide or peptide domains of the presently disclosed subject matter. Fragments can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, fragments are at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, fragments are at least about 60 to about 80, about 100, about 200, about 300, or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of skill in the art or can be the result of normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0167] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins of the present invention. Such analogs are administered according to the methods of the presently disclosed subject matter. Such analogs may exceed the physiological activity of the original polypeptide. Methods for designing analogs are well known in the art, and analogs can be synthesized according to such methods by modifying the chemical structure so that the resulting analog increases the antineoplastic activity of the original polypeptide when expressed in immune response cells. These chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation at specific carbon atoms of the reference polypeptide. Protein analogs may be relatively resistant to in vivo degradation and may provide a longer-lasting therapeutic effect when administered. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0168] According to the presently disclosed subject matter, the polynucleotide encoding the extracellular domain that specifically binds to a mutant PIK3CA peptide (e.g., a human mutant PIK3CA peptide) can be modified by codon optimization. Codon optimization can modify both naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system. Factors involved in various stages of protein expression include codon adaptability, mRNA structure, and various cis elements in transcription and translation. Any suitable codon optimization method or technique known to those skilled in the art (including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron) can be used to modify the polynucleotide of the presently disclosed subject matter. X. Administration

[0169] The PIK3CA-targeting TCRs of the present disclosure and immune response cells comprising them can be provided systemically or directly to a subject to treat and / or prevent neoplasms. In certain embodiments, the mutant PIK3CA-targeting TCRs of the present disclosure and immune response cells comprising them are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, or in addition, the mutant PIK3CA-targeting TCRs of the present disclosure and immune response cells comprising them are provided to the organ of interest indirectly (e.g., by administration into the circulatory system (e.g., tumor vasculature)). Proliferation and differentiation agents may be provided prior to, during, or after administration of the cells and compositions to increase T cell generation in vitro or in vivo.

[0170] The mutant PIK3CA-targeting TCRs and immune response cells comprising the same of the presently disclosed subject matter can be administered in any physiologically acceptable vehicle, usually intravascularly, but can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus). In certain embodiments, at least about 1 x 10 5 cells (ultimately about 1 × 10 10 In certain embodiments, at least about 1 x 10 6A single cell population containing immune response cells containing a mutant PIK3CA-targeting TCR may be administered. A cell population containing immune response cells containing a mutant PIK3CA peptide-targeting TCR may comprise a purified population of cells. Those skilled in the art can easily determine the proportion of immune response cells within a cell population using various well-known methods (e.g., fluorescence-activated cell sorting (FACS)). The purity range in a cell population containing genetically modified immune response cells containing a mutant PIK3CA peptide-specific TCR may be about 50% to about 55%, about 55% to about 60%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art (e.g., a decrease in purity may require an increased dosage). The immune response cells may be administered by injection, catheter, or the like. If desired, interleukins such as, but not limited to, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, and other interleukins, colony stimulating factors such as G-, M-, and GM-CSF, interferons (e.g., gamma interferon) may also be included.

[0171] The presently disclosed subject matter further provides a composition comprising the immune response cells of the present disclosure comprising a mutant PIK3CA-targeting TCR. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. Administration may be autologous or non-autologous. For example, immune response cells comprising a mutant PIK3CA-targeting TCR and compositions comprising the same can be obtained from a subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived T cells of the presently disclosed subject matter, or their progeny (e.g., derived in vivo, ex vivo, or in vitro), can be administered by local injection (including catheter administration), systemic injection, local injection, intravenous injection, or parenteral administration. When administered, the pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising immune response cells comprising a mutant PIK3CA-targeting TCR) can be formulated into an injectable unit dosage form (solution, suspension, emulsion). XI. Formulation

[0172] The immune response cells of the present disclosure, comprising a mutant PIK3CA-targeting TCR of the present disclosure, and compositions comprising the same, can be conveniently provided as sterile liquid preparations (e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions), which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat easier to administer (particularly by injection). In contrast, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0173] Sterile injectable solutions can be prepared by combining a composition of the presently disclosed subject matter (e.g., a composition comprising immune response cells comprising a mutant PIK3CA-targeting TCR of the present disclosure) in the required amount of an appropriate solvent, along with various amounts of other ingredients, as desired. Such compositions may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, dyes, and the like, depending on the route of administration and the desired formulation. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985 (incorporated herein by reference), may be consulted to prepare suitable formulations without undue experimentation.

[0174] Various additives (including antimicrobial preservatives, antioxidants, chelating agents, and buffers) may be added to improve the stability and sterility of the composition. Various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) can ensure the inhibition of microbial activity. The prolonged absorption period of injectable pharmaceutical forms can be achieved by using agents that delay absorption (e.g., alum monostearate and gelatin). However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with the immune response cells containing the (generally) mutant PIK3CA-targeting TCR of the subject matter of the present disclosure.

[0175] The composition can be isotonic, i.e., have the same osmotic pressure as blood and tears. The desired isotonicity of the composition of the presently disclosed subject matter can be achieved by using sodium chloride or other pharmaceutically acceptable agents (e.g., dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes). Sodium chloride is particularly preferred for buffers containing sodium ions.

[0176] The viscosity of the composition can be maintained at a selected level, if desired, using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily available, economically available, and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the agent selected. The key is to use an amount that achieves the selected viscosity. Needless to say, the selection of appropriate carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form (e.g., liquid dosage form) (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a sustained-release form or liquid encapsulation form).

[0177] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability and effectiveness of immune response cells as described in the subject matter of the present disclosure. This will not be a problem for those skilled in the art of chemical and pharmaceutical principles, or the problem may be avoided by reference to standard texts or by simple experimentation (without undue experimentation) from this disclosure and the literature cited herein.

[0178] One consideration regarding therapeutic applications of immune response cells of the presently disclosed subject matter is the number of cells needed to achieve optimal efficacy. The number of cells administered will vary depending on the subject being treated. In certain embodiments, about 10 4 ~about 10 10 , about 10 5 ~about 10 9 , or about 10 6 ~about 10 8 of the immune response cells of the presently disclosed subject matter are administered to a subject. More effective cells can be administered in even lower numbers. In some embodiments, at least about 1 x 10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , and approximately 5 × 10 8 The immune response cell of the subject of the present disclosure is administered to human subjects.The precise determination of what is considered to be an effective amount can be based on each subject's specific factors, including the subject's size, age, sex, weight and the condition of the specific subject.Dosage can be easily determined based on the present disclosure and knowledge in the art.

[0179] The amounts of cells, and optional additives, vehicles, and / or carriers, to be administered in the compositions and methods of the presently disclosed subject matter can be readily determined by one of skill in the art. Typically, any additives (in addition to the active cell(s) and / or active agent(s)) are present in an amount of about 0.001% to about 50% by weight of solution in phosphate-buffered saline, with the active ingredient being present in amounts on the order of micrograms to milligrams (e.g., about 0.0001% to about 5% by weight, about 0.0001% to about 1% by weight, about 0.0001% to about 0.05% by weight, about 0.001% to about 20% by weight, about 0.01% to about 10% by weight, or about 0.05% to about 5% by weight). For any composition administered to animals or humans, and for any particular method of administration, toxicity should be determined, for example, by determining the lethal dose (LD) and LD50 in an appropriate animal model (e.g., rodents such as mice); and the dosage of the composition(s), the concentrations of the components therein, and the timing of administration of the composition(s) that will elicit an appropriate response. Such determinations are based on the knowledge of those skilled in the art, this disclosure, and the references cited herein, and do not require undue experimentation. Additionally, the duration of sequential administrations can be ascertained without undue experimentation. XII. Treatment Methods

[0180] Provided herein is a method for treating neoplasms in a subject.The method comprises administering the immune response cells of the present disclosure in an amount sufficient to achieve the desired effect (whether it is the alleviation of existing conditions or the prevention of recurrence).For treatment, the amount administered is an amount effective to produce the desired effect.An effective amount can be provided in one or a series of administrations.An effective amount can be provided by bolus or by continuous perfusion.

[0181] For adoptive immunotherapy using antigen-specific T cells, typically about 10 6 ~about 10 10 (e.g., about 10 9 or about 10 6) is injected. Upon administration of the immune response cells to a subject and subsequent differentiation, immune response cells directed against one specific antigen (e.g., a mutant PIK3CA peptide) are induced. "Induction" of T cells can include inactivation of antigen-specific T cells (e.g., by deletion or anergy). Inactivation is particularly useful for establishing or re-establishing tolerance, such as in autoimmune disorders. The immune response cells of the presently disclosed subject matter can be administered by any method known in the art, including, but not limited to, pleural, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus. In certain embodiments, the immune response cells and compositions comprising the same are administered intravenously to a subject in need thereof.

[0182] The subject matter of the present disclosure provides various methods using immune response cells (e.g., T cells) comprising mutant PIK3CA targeting TCR.For example, the subject matter of the present disclosure provides a method for reducing tumor burden in a subject.In certain embodiments, the method for reducing tumor burden comprises administering an effective amount of immune response cells of the present disclosure to a subject.The immune response cells of the present disclosure can reduce the number of tumors, reduce tumor size, and / or eradicate tumors in a subject.

[0183] The presently disclosed subject matter also provides methods of increasing or extending the survival time of a subject having a neoplasm, hi certain embodiments, the method of increasing or extending the survival time of a subject having a neoplasm comprises administering to the subject an effective amount of an immune response cell of the present disclosure.

[0184] The cancer whose growth is inhibited by using the immune response cell of the subject of the present disclosure includes cancer that typically responds to immunotherapy.Non-limiting examples of cancer for treatment include PIK3CA mutation cancer (including but not limited to breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer).In certain embodiments, the cancer is breast cancer.

[0185] Furthermore, the presently disclosed subject matter provides a method for increasing immune-activating cytokine production in response to cancer cells in a subject. In certain embodiments, the method comprises administering immune response cells of the present disclosure to the subject. The immune-activating cytokines can be granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, IL-18, interferon regulatory factor 7 (IRF7), and combinations thereof. In certain embodiments, immune response cells comprising a mutant PIK3CA peptide-specific TCR of the presently disclosed subject matter increase production of GM-CSF, IFN-γ, and / or TNF-α.

[0186] Human subjects suitable for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "progressive disease" or "anti-tumor burden" are those with clinically measurable tumors (e.g., breast cancer). Clinically measurable tumors are tumors that can be detected based on tumor burden (e.g., by palpation, CT scan, sonogram, mammogram, or X-ray; biochemical or histopathological positive markers of the tumor itself are insufficient to identify this population). Pharmaceutical compositions embodied in the presently disclosed subject matter are administered to these subjects to induce an anti-tumor response with the aim of alleviating their condition. Ideally, this results in a reduction in tumor burden, although any clinical improvement would be beneficial. Clinical improvement includes a reduction in the risk or rate of tumor (e.g., breast cancer) progression or a reduction in pathological consequences.

[0187] A second group of suitable subjects is known in the art as the "adjuvant group." These individuals have a history of neoplasia (e.g., breast cancer) but have responded to other treatments. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected of being at risk for progression of the disease near the original tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk groups. This subdivision is based on characteristics observed before or after initial treatment. These characteristics are known in the clinical field and are appropriately defined for each type of neoplasia. A distinctive feature of the high-risk subgroup is that the tumor (e.g., breast cancer) has invaded adjacent tissues or involved lymph nodes. Another group has a genetic predisposition to neoplasia (e.g., breast cancer) but has not yet shown clinical signs of the neoplasia (e.g., breast cancer). For example, a woman who has tested positive for a genetic mutation involved in breast cancer but is still of childbearing age may wish to receive one or more TCRs described herein in a prophylactic treatment to prevent the development of a neoplasm until prophylactic surgery is appropriate.

[0188] The subject may have an advanced stage of the disease (e.g., breast cancer), in which case the goal of treatment may include slowing or reversing disease progression and / or ameliorating side effects. The subject may also have a history of such a condition and have already been treated for it, in which case the goal of treatment typically includes reducing or delaying the risk of recurrence. XIII. Kit

[0189] The subject matter of the present disclosure provides a kit for treating and / or preventing neoplasms (e.g., breast cancer). In certain embodiments, the kit includes a therapeutic or prophylactic composition in the form of a unit dosage form, the therapeutic or prophylactic composition including an effective amount of immune response cells comprising a mutant PIK3CA-targeting TCR. In certain embodiments, the cells further express at least one costimulatory ligand.

[0190] If desired, the immune response cells may be provided with instructions for administering the cells to a subject having or at risk of developing a neoplasm (e.g., breast cancer). The instructions generally include information about using the composition for the treatment or prevention of a neoplasm (e.g., breast cancer). In other embodiments, the instructions include at least one of the following: dosing schedule and administration for the treatment or prevention of a neoplasm (e.g., breast cancer) or a symptom thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if any) or as a label affixed to the container; or may be a separate sheet, pamphlet, card, or folder provided in or with the container. [Example]

[0191] The practice of the present invention will utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of those skilled in the art. Such techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are available for producing the polynucleotides and polypeptides of the invention, and as such may be considered in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections.

[0192] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions, and assay, screening, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Example 1

[0193] To identify the naturally processed and presented epitopes of PIK3CA, we generated transient transfectants by co-electroporating human HLA-A*03:01 with either wild-type (WT) or mutant PIK3CA into a COS-7 monkey cell line (see Figure 1). Cell surface peptide-MHC complexes were then bound by anti-class I antibodies, shed from the cell surface, and eluted peptides were purified and sequenced by LC / MS / MS.

[0194] As shown in Figure 2 (top panel), Skyline analysis shows enriched signals in the transfectant group electroporated with the combination of mutant PIK3CA and HLA-A*03:01. No signals were detected in either WT+PIK3CA or mutant PIK3CA+HLA-A*69:01. As shown in Figure 2 (bottom panel), the peptide fragmentation spectrum of the enriched signal detected in the mutant PIK3CA+HLA-A*03:01 group indicated that the sequence of the enriched peptide was a 9-amino acid peptide with the sequence "ALHGGWTTK" (SEQ ID NO: 11). The amino acid in the peptide had a histidine-to-leucine substitution at the P2 position.

[0195] Based on the sequence information of the minimal epitope loaded onto HLA-A*03:01, a differential scanning fluorimetry assay was performed to determine the stability of the biophysical interaction of the wild-type and mutant peptides with HLA-A*03:01. The results are shown in Figure 3. As shown in Figure 3, the binding of the mutant peptide was significantly more thermally stable than that of the wild-type peptide. Furthermore, crystal structures of the peptide-MHC complexes were generated for both the wild-type and mutant peptides, as shown in Figure 4. In vitro sensitization of healthy donor PBMCs with mutant PIK3CA resulted in the production of mutant-specific CD8 +A T cell clone (21LT2-2) was identified and isolated, its TCR sequence was obtained by 10x genomics sequencing, and the sequence was cloned into a retroviral vector and transduced into allogeneic donor PBMCs.

[0196] To test the reactivity and restriction elements of the isolated TCRs, COS-7 cells were electroporated with individual class I HLA molecules and either wild-type or mutant PIK3CA and cocultured with donor T cells transduced with the 21LT2-2 TCR (see Figure 5). 21LT2-2 TCR reactivity was shown to be specific for the combination of mutant H1047L PIK3CA in the context of HLA-A*03:01, as evidenced by upregulation of inflammatory cytokines (TNF-α).

[0197] Next, originally CD8 + The 21LT2-2 TCR isolated from T cells binds to CD4 + To determine whether it is also functional in T cells, we performed a test using CD8 + TCR was expressed in the presence of co-receptors. + CD8 and CD4 subsets were isolated and co-cultured with two types of targets: i) processed and presented antigen, or ii) passively pulsed minimal peptide. Isolated TCRs expressed CD8 T cells, as evidenced by mutation-specific upregulation of CD107a and TNF-α. + (See Figure 6) and CD4 + (See Figure 7.) Functionality in both T cell subsets demonstrated CD8 coreceptor independence. Lack of coreceptor dependence is generally associated with high-affinity TCR interactions. Furthermore, the 21LT2-2 TCR was shown to be capable of recognizing both processed and presented antigens as well as passively pulsed minimal epitopes.

[0198] Next, we performed tests to determine the amino acid residues contained within the minimal epitope that were critical for pMHC-TCR interaction and recognition (see Figure 8). Alanine peptide substitutions were made at each position across the nine-amino acid sequence and the loss of recognition compared to the unsubstituted mutant sequence was examined. The data indicate that all positions, except for the glycine at P4, are critical for pMHC-TCR interaction and recognition. As observed here, the predominance of critical residues potentially limits the number of cross-reactive peptides that can bind and trigger the 21LT2-2 TCR. As shown in Figure 9, a search using the ScanProsite tool, which scans the human proteome for matching sequences, did not yield any hits. Example 2

[0199] We aimed to establish a library of HLA-A*03:01 mutant PIK3CA-specific TCRs with unique immunological attributes to identify optimal candidates for clinical development. Based on the findings with 21LT2-2, we created custom dextramer reagents (fluorophore-conjugated peptide-MHC complex conformations) of mutant PIK3CA peptides complexed with HLA-A*03:01. Figure 10 shows the results of retroviral transduction of identified mutant PIK3CA-specific TCRs and CD8 + TCR + Dextramer staining profiles of donor T cells gated on T cells are shown. Three TCRs, 21LT2-2, 0606T1-2, and 1022T8, were derived from three unique healthy donors. All stained positively with dextramer. The staining profile of 1022T8 was different from 21LT2-2 and 0606T1-2, possibly indicating a lower affinity TCR.

[0200] The PIK3CA neoantigen-specific TCRs 21LT2-2, 0606T1-2, and 1022T8 were retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were co-cultured with artificial antigen-presenting cells (aAPCs) electroporated with RNA encoding HLA-A*03:01 carrying either wild-type or mutant PIK3CA RNA. The presence of mutant-specific reactivity was assessed by the production of proinflammatory cytokines (TNF-α). The results are shown in Figures 11 and 12. The cells shown in Figure 11 were CD8 + TCR + The cells shown in Figure 12 were gated on CD4 + TCR + As shown in Figure 11, in addition to 21LT2-2, both 0606T1-2 and 1022T8 can recognize endogenously processed and presented mutant antigens presented by HLA-A*03:01. As shown in Figure 12, like 21LT2-2, 0606T1-2 is coreceptor-independent and can recognize CD4 + It was able to function in T cells; 1022-T8 was a co-receptor dependent TCR (suggesting that it may have had lower affinity compared to 21LT2-2 and 0606T1-2).

[0201] Furthermore, we measured the expression of CD107a (a prototypic marker associated with cytolytic activity). As shown in Figure 13, TCR-transduced cells upregulated the expression of CD107a in response to co-expression of HLA-A*03:01 and mutant PIK3CA.

[0202] Mutant PIK3CA-specific TCRs were retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells were incubated with target cells expressing HLA-A*03:01 and pulsed with alanine-substituted peptides (10 μg / mL). Wild-type and native mutant PIK3CA 9-mer peptides were used as controls to establish the range of reactivity for each individual TCR. Cells were CD8+ TCR + The 9-amino acid sequence was gated at 1000 kJ / s. When pulsed with the alanine-substituted peptide, the recognition was reduced by >50% compared to the unmodified 9-amino acid mutant sequence (Table 1; mutation at P2), indicating that the native amino acid at that position is important for TCR recognition. The results are shown in Figures 14A-14C. As shown in Figures 14A-14C, for 21LT2-2, all amino acids in this 9-amino acid sequence, except for the glycine residue at position 4, appeared to be important for pMHC-TCR interaction. Furthermore, as shown in Figures 14A-14C, for 0606T1-2, the P2, P3, and P4 substitutions appeared to be tolerated for TCR function. Furthermore, as shown in Figures 14A-14C, for 1022T8, alanine substitutions at any position resulted in a >50% reduction in function, indicating that all positions are important for TCR function.

[0203] We derived positional motifs based on reduced TCR reactivity and identified residues important for TCR recognition and cross-reactivity. Data from Figures 14A-14C are shown in graph form in Figure 15. The dotted line indicates 50% of maximum reactivity. Based on this, motifs for determining potential cross-reactivity are shown on the right. "x" indicates residues that are not important for TCR recognition. Position 1 was assigned an "x" as the default because the native amino acid at this position is alanine and therefore not applicable to this test.

[0204] After determining the critical residues in the minimal peptide sequence, we used the ScanProsite tool to search all UniProtKB / Swiss-Prot (release 2019_03 on 10-Apr-19: 559,634 entries) database sequences for proteins containing the motif xLHxGWTTK [SEQ ID NO: 48] for 21T2-2, xxxxGWTTK [SEQ ID NO: 49] for 0606T1-2, and xLHGGWTTK [SEQ ID NO: 50] for 1022T8 in the human proteome. See Figure 16A. For 21LT2-2 and 1022T8, no hits matching potential cross-reactive sequences in the human proteome were detected. For 0606T1-2, one hit was obtained, derived from the transmembrane protein 87B (ivfmGWTTK [SEQ ID NO: 47]). See Figure 16B. We synthesized this peptide and pulsed it onto HLA-A*03:01+ target cells. A native mutant 9-mer peptide (ALHGGWTTK [SEQ ID NO: 11]) was used as a control. The cross-reactive peptide was not recognized by the 0606T1-2 TCR.

[0205] Furthermore, we investigated whether PIK3CA mutant-specific TCRs could recognize mutant peptides of different lengths. Mutant PIK3CA-specific TCRs were retrovirally integrated into the genome of healthy donor T cells. Four to six days after transduction, T cells expressed HLA-A*03:01. + The cells were incubated with antigen-presenting cells and pulsed with 8-mer and 10-mer mutant PIK3CA peptide pools (1 μg / mL). Wild-type and 9-mer peptides consisting of the amino acid sequence set forth in SEQ ID NO: 11 were used as controls. The cells were CD8 + TCR +Expression was gated. The mutant peptide-specific upregulation of TNFα production indicates that alternative peptide lengths are recognized. The 8-mer mutant PIK3CA peptide consists of the amino acid sequence set forth in LHGGWTTK [SEQ ID NO: 51]. The 10-mer mutant PIK3CA peptide consists of the amino acid sequence set forth in DALHGGWTTK [SEQ ID NO: 52]. The results are shown in Figure 17. As shown in Figure 17, all three TCRs can recognize both the 8-mer and 10-mer mutant PIK3CA peptides.

[0206] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various uses and conditions, and such embodiments are also within the scope of the following claims.

[0207] All patents, publications, and sequences cited by accession number or reference number are herein incorporated by reference to the same extent as if each individual patent and publication, and sequence was specifically and individually indicated to be incorporated by reference. In certain embodiments, for example, the following are provided: (Item 1) A T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises the H1047L mutation. (Item 2) The TCR described in item 1, wherein the PIK3CA peptide is an 8-mer, 9-mer, or 10-mer. (Item 3) 3. The TCR of item 1 or 2, wherein the PIK3CA peptide is a 9-mer. (Item 4) A TCR described in any one of items 1 to 3, wherein the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 51, or SEQ ID NO: 52. (Item 5) 5. The TCR according to any one of items 1 to 4, wherein the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. 5. The TCR of any one of items 1 to 4, wherein the PIK3CA peptide is associated with the HLA class I complex. (Item 6) 6. The TCR of item 5, wherein the HLA class I complex is selected from HLA-A, HLA-B, and HLA-C. (Item 7) 7. The TCR of item 5 or 6, wherein the HLA class I complex is HLA-A. (Item 8) 8. The TCR of item 6 or 7, wherein the HLA-A is the HLA-A*03 superfamily. (Item 9) The TCR of item 8, wherein the HLA-A*03 superfamily is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74. (Item 10) 10. The TCR of item 8 or 9, wherein the HLA-A*03 superfamily is HLA-A*03. (Item 11) 11. The TCR according to any one of items 1 to 10, comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain binds to the PIK3CA peptide. (Item 12) the extracellular domain is a) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof; b) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof; or c) an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof 12. The TCR according to any one of items 1 to 11, comprising: (Item 13) the extracellular domain is a) an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof; b) an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof; or c) an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof, and a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof 13. The TCR according to item 12, comprising: (Item 14) the extracellular domain is a) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof; b) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof; or c) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, and a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof 14. The TCR according to item 12 or 13, comprising: (Item 15) the extracellular domain is a) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; b) an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; and an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or c) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; and an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37 15. The TCR according to any one of items 1 to 14, comprising: (Item 16) the extracellular domain is a) a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) a β-chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a β-chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a β-chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or c) a β-chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a β-chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a β-chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 16. The TCR according to any one of items 1 to 15, comprising: (Item 17) the extracellular domain is a) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or c) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 17. The TCR according to any one of items 1 to 16, comprising: (Item 18) 18. The TCR according to any one of items 1 to 17, wherein the extracellular domain comprises an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. (Item 19) 19. The TCR according to any one of items 1 to 18, wherein the extracellular domain comprises an alpha chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 41. (Item 20) 20. The TCR of item 19, wherein the extracellular domain comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 41. (Item 21) 21. The TCR of item 20, wherein the extracellular domain comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. (Item 22) 22. The TCR according to any one of items 1 to 21, wherein the extracellular domain comprises a β chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 32, or SEQ ID NO: 42. (Item 23) 23. The TCR of item 22, wherein the extracellular domain comprises a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 32, or SEQ ID NO: 42. (Item 24) 24. The TCR of item 23, wherein the extracellular domain comprises a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. (Item 25) the extracellular domain is a) an α chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:7, and a β chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8; b) an alpha chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 31, and an alpha chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32; a beta chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to c) an α chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 41, and a β chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42. 25. The TCR according to any one of items 1 to 24, comprising: (Item 26) the extracellular domain is a) an α chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7, and a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; b) an α chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31, and a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32; c) an α chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41, and a β chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42 26. The TCR according to any one of items 1 to 25, comprising: (Item 27) 27. The TCR of item 26, wherein the extracellular domain comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. (Item 28) the extracellular domain is a) an α chain comprising the amino acid sequence set forth in SEQ ID NO: 9, and a β chain comprising the amino acid sequence set forth in SEQ ID NO: 10; b) an α chain comprising the amino acid sequence set forth in SEQ ID NO: 33, and a β chain comprising the amino acid sequence set forth in SEQ ID NO: 34; or c) an α chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a β chain comprising the amino acid sequence set forth in SEQ ID NO: 44 28. The TCR according to any one of items 1 to 27, comprising: (Item 29) 29. The TCR of item 28, wherein the extracellular domain comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 10. (Item 30) the extracellular domain binds to the same epitope on the human mutant PIK3CA polypeptide as a reference TCR or functional fragment thereof, wherein the reference TCR or functional fragment thereof is a) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; b) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or c) an α chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; an α chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36; an α chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; a β chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38; a β chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39; and a β chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 30. The TCR according to any one of items 1 to 29, comprising: (Item 31) 31. The TCR of any one of items 1 to 30, wherein the TCR is recombinantly expressed and / or expressed from a vector. (Item 32) 32. The TCR of any one of items 1 to 31, wherein the TCR does not target a wild-type PIK3CA peptide. (Item 33) 33. The TCR of any one of items 1 to 32, wherein the TCR comprises a modified alpha chain constant region and / or a modified beta chain constant region. (Item 34) 34. The TCR of item 33, wherein the modified alpha chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. (Item 35) 35. The TCR of item 34, wherein the modified alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20. (Item 36) 36. The TCR of item 35, wherein the modified beta chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 45. (Item 37) 37. The TCR of item 36, wherein the modified beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22. (Item 38) 38. An immune response cell comprising the TCR of any one of items 1 to 37. (Item 39) 39. The immune response cell of claim 38, wherein the immune response cell is transduced with the TCR. (Item 40) 40. The immune response cell of item 38 or 39, wherein the TCR is constitutively expressed on the surface of the immune response cell. (Item 41) 41. The immune response cell according to any one of Items 38 to 40, wherein the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells from which lymphoid cells can be differentiated. (Item 42) 42. The immune response cell of claim 41, wherein the immune response cell is a T cell. (Item 43) 43. The immune response cell of item 42, wherein the T cell is selected from the group consisting of a cytotoxic T cell (CTL), a regulatory T cell, and a central memory T cell. (Item 44) A composition comprising the immune response cell according to any one of items 38 to 43. (Item 45) 45. The composition according to item 44, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 46) A nucleic acid molecule encoding the T cell receptor (TCR) according to any one of items 1 to 37. (Item 47) 47. A vector comprising the nucleic acid molecule of item 46. (Item 48) 48. The vector according to item 47, wherein the vector is a gamma retroviral vector. (Item 49) A host cell comprising the nucleic acid molecule of item 46. (Item 50) 50. The host cell of item 49, wherein the host cell is a T cell. (Item 51) A method for producing immune response cells that bind to a human mutant PIK3CA peptide, comprising introducing into immune response cells a nucleic acid molecule encoding a TCR described in any one of Items 1 to 37 or the nucleic acid molecule described in Item 46, or the vector described in Item 47 or 48. (Item 52) A method for treating and / or preventing a neoplasm comprising a PIK3CA mutation in a subject, the method comprising administering to the subject an effective amount of the immune response cell of any one of Items 38 to 43 or the composition of Items 44 or 45. (Item 53) 53. The method of item 52, wherein the PIK3CA mutation comprises or consists of H1047L. (Item 54) 54. The method of item 52 or 53, wherein the neoplasm is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer. (Item 55) 55. The method of item 54, wherein the neoplasm is breast cancer. (Item 56) 56. The method of any one of items 52 to 55, wherein the method reduces or eradicates tumor burden in the subject. (Item 57) 57. The method according to any one of items 52 to 56, wherein the subject is a human. (Item 58) 44. The immune response cell of any one of items 38 to 43 or the composition of item 37 or 38 for use in the treatment and / or prevention of a neoplasm comprising a PIK3CA mutation in a subject. (Item 59) 59. The cell or composition for use according to item 58, wherein the PIK3CA mutation comprises or consists of H1047L. (Item 60) 60. The cell or composition for use according to item 58 or 59, wherein the neoplasm is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer. (Item 61) 61. The cell or composition for use according to item 60, wherein the neoplasm is breast cancer. (Item 62) 62. The cell or composition for use according to any one of items 58 to 61, wherein the subject is a human. (Item 63) A neoplasm comprising a PIK3CA mutation, the neoplasm comprising the immune response cell according to any one of Items 38 to 43, the nucleic acid molecule according to Item 46, or the vector according to Item 47 or 48. Kits for treatment and / or prevention. (Item 64) 64. The kit of item 63, further comprising written instructions for using the immune response cell, nucleic acid molecule, or vector to treat a subject having the neoplasm. (Item 65) 65. The kit of item 64, wherein the neoplasm is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, and salivary gland cancer. (Item 66) Item 66. The kit of item 65, wherein the neoplasm is breast cancer.

Claims

1. A T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises an H1047L mutation; and the TCR comprises an α chain variable region comprising an amino acid sequence that is at least about 93% identical to the amino acid sequence set forth in SEQ ID NO:7 and a β chain variable region comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the α chain variable region comprises an α chain CDR1 having the amino acid sequence of SEQ ID NO:1, an α chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an α chain CDR3 having the amino acid sequence of SEQ ID NO:3, and the β chain variable region comprises a β chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:

6.

2. The TCR of claim 1, further comprising an alpha chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

20.

3. The TCR of claim 1, further comprising an alpha chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

21.

4. The TCR of claim 1, further comprising a β chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

23.

5. The TCR of claim 1, further comprising a beta chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

45.

6. A T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises an H1047L mutation, and the TCR comprises an alpha chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a beta chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

8.

7. The TCR of claim 6, further comprising an alpha chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

20.

8. The TCR of claim 6, further comprising an alpha chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

21.

9. The TCR of claim 6, further comprising a β chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

23.

10. The TCR of claim 6, further comprising a beta chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

45.

11. The TCR of claim 1, wherein the PIK3CA peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 51, or SEQ ID NO:

52.

12. A T cell receptor (TCR) that specifically targets a PIK3CA peptide, wherein the PIK3CA peptide comprises a mutation of H1047L, and the TCR comprises: a) an alpha chain variable region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:7; b) a β-chain variable region comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:8; c) an alpha chain constant region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:21; and d) a β-chain constant region comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 23 wherein the α chain variable region comprises an α chain CDR1 having the amino acid sequence of SEQ ID NO: 1, an α chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and an α chain CDR3 having the amino acid sequence of SEQ ID NO: 3; and the β chain variable region comprises a β chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a β chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:

6.

13. 13. The TCR of any one of claims 1 to 12, wherein the TCR is recombinantly expressed and / or expressed from a vector.

14. A nucleic acid molecule encoding the T cell receptor (TCR) of any one of claims 1 to 12.

15. 1. A composition for use in treating cancer in a subject, said composition comprising engineered immune cells, said engineered immune cells comprising: A TCR according to any one of claims 1 to 12; or A nucleic acid molecule encoding the TCR of any one of claims 1 to 12. A composition comprising:

16. The composition of claim 15, wherein the cancer is a solid tumor cancer.

17. 17. The composition of claim 16, wherein the solid tumor cancer is breast cancer.

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