Dual-action antigen-presenting gamma delta car t-cells

Abstract

Compositions and methods disclosed herein can comprise a cell (e.g., gamma delta T cell) engineered to comprise a nucleic acid molecule that encodes an immunizing polypeptide, and the engineered cell can present one or more T cell epitopes from the immunizing polypeptide via HLA, thereby inducing activation of endogenous T cells in a subject (e.g., host or recipient of the engineered cell). The cells can further express a CAR, facilitating induction of (1) a direct immune response mediated by the CAR, and (2) induction of immune responses (e.g., anti-cancer) mediated by the subject's endogenous T cells, for example, against different target antigens or T cell epitopes than those targeted by the CAR. Such a cell can be described as a Dual-action Antigen-presenting CAR γδ T-cell (DACART). DACART can combine the strengths of CAR T-cell therapy with those of cancer vaccination.

Description

WSGR Docket No. 57428-711.601DUAL-ACTION ANTIGEN-PRESENTING GAMMA DELTA CAR T-CELLSCROSS REFERENCE

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 735,144, filed December 17, 2024, which is incorporated herein by reference in its entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Cooperative Agreement number AY 1 AX000075 awarded by the Advanced Research Projects Agency for Health (ARPA-H). The government has certain rights in the invention.BACKGROUND

[0003] The treatment of solid tumors including colorectal cancer (CRC), non-small cell lung cancer (NSCLC), prostate cancer, and breast cancer, remains a formidable challenge. Although chimeric antigen receptor (CAR) T-cell therapies have achieved remarkable success in treating some hematologic cancers, these therapies can fail due to antigen escape, and often show limited effectiveness against solid tumors.SUMMARY

[0004] Disclosed herein, in some aspects, is an engineered cell comprising a heterologous nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope, wherein the engineered cell is a gamma delta T cell.

[0005] Disclosed herein, in some aspects, is an engineered cell comprising: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope.

[0006] In some embodiments, upon administration of the engineered cell to a subject, the engineered cell induces activation of an endogenous T cell in the subject. In some embodiments, upon administration of the engineered cell to a subject, the engineered cell induces an anticancer immune response in the subject mediated by endogenous T cells of the subject. In some embodiments, the engineered cell is capable of inducing activation of an endogenous T cell of a subject upon administration of the engineered cell to the subject. In some embodiments, theWSGR Docket No. 57428-711.601 engineered cell is capable of inducing an anti-cancer immune response mediated by endogenous T cells in the subject upon administration of the engineered cell to the subject. In some embodiments, the immunizing polypeptide comprises a CD4+ T cell epitope. In some embodiments, the immunizing polypeptide comprises a CD8+ T cell epitope. In some embodiments, the immunizing polypeptide comprises a CD4+ T cell epitope and a CD8+ T cell epitope. In some embodiments, the immunizing polypeptide comprises a tumor-associated antigen (TAA). In some embodiments, the immunizing polypeptide comprises a solid tumor antigen. In some embodiments, the immunizing polypeptide comprises a neoantigen. In some embodiments, the immunizing polypeptide comprises a neoepitope. In some embodiments, the immunizing polypeptide comprises at least four T cell epitopes. In some embodiments, the immunizing polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 1-36, 115, and 120. In some embodiments, the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-36, 115, and 120. In some embodiments, the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 37-42 and 52-54. In some embodiments, the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 137-42 and 52-54. In some embodiments, the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class I. In some embodiments, the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class II. In some embodiments, the immunizing polypeptide comprises a targeting sequence to promote HLA presentation of the T cell epitope. In some embodiments, the targeting sequence comprises a human tissue plasminogen activator signal peptide. In some embodiments, the targeting sequence comprises a li-CLIP- replacement peptide. In some embodiments, the targeting sequence comprises a lysosome targeting sequence. In some embodiments, the targeting sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 43-51. In some embodiments, the targeting sequence comprises the amino acid sequence of any one of SEQ ID NOs: 43-51. In some embodiments, the nucleic acid molecule that encodes the immunizing polypeptide is DNA. In some embodiments, the nucleic acid molecule that encodes the immunizing polypeptide is a tandem minigene. In some embodiments, the nucleic acid molecule that encodes the immunizing polypeptide is genomically integrated. In some embodiments, the nucleic acid molecule that encodes the immunizing polypeptide is not genomically integrated. In some embodiments, the nucleic acid molecule that encodes the immunizing polypeptide is RNA. In some embodiments, the CAR comprises an extracellular binding domain that binds to a target associated with a solidWSGR Docket No. 57428-711.601 tumor. In some embodiments, the CAR comprises an extracellular binding domain that binds to a target associated with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer. In some embodiments, the CAR comprises an extracellular binding domain that binds to mesothelin. In some embodiments, the CAR comprises an extracellular binding domain that binds to carcinoembryonic antigen (CEA), CD70, CSPG4, B7-H3, CD133, EGFR, or MUC1. In some embodiments, the extracellular binding domain of the CAR does not bind to the immunizing polypeptide or the T cell epitope. In some embodiments, the CAR is a split CAR or dual CAR. In some embodiments, the CAR comprises an FcsRIy cytoplasmic signaling domain. In some embodiments, the CAR is a second, third, fourth, or fifth generation CAR. In some embodiments, the CAR comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 59-60. In some embodiments, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 59-60. In some embodiments, upon administration of the engineered cell to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide. In some embodiments, upon administration of the engineered cell to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide. In some embodiments, the engineered cell further comprises a safety switch. In some embodiments, the safety switch comprises a truncated EGFR

[0007] Disclosed herein, in some aspects, is a population of cells comprising a plurality of the engineered cell of any one of the preceding embodiments, wherein the population of cells comprises: (i) at least 10% Vdl+ cells and at least 10% Vd2+ cells, (ii) at least 1% Vdl- Vd2- gamma delta T cells, or (iii) at least 11% Vd3+ cells.

[0008] Disclosed herein, in some aspects, is a method of treating a subject in need thereof, the method comprising administering to the subject the engineered cell of any one of the preceding embodiments. In some embodiments, the method treats cancer in the subject. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the engineered cell is allogeneic to the subject. In some embodiments, the engineered cell is matched to the subject for at least one HLA allele. In some embodiments, the engineered cell is autologous to the subject. In some embodiments, the engineered cell induces activation of an endogenous TWSGR Docket No. 57428-711.601 cell in the subject. In some embodiments, the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject. In some embodiments, if the engineered cell is administered to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide. In some embodiments, if the engineered cell is administered to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

[0009] Disclosed herein, in some aspects, is a method of making an engineered cell, comprising introducing into the cell: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope. In some embodiments, the cell is a gamma delta T cell.INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 provides schematics of vectors that comprise ITRs for transposon-mediated genomic integration, a MND promoter, anti-mesothelin CARs, and an immunizing polypeptide encoded by a tandem minigene and with a TP A targeting sequence.

[0012] FIG. 2 is a schematic of DACART cells with MHC-I presentation of TMG neoantigen peptides to activate host CD8+ T cells and direct tumor targeting via a MSLN CAR.

[0013] FIG. 3 provides schematics of vectors that comprise ITRs for transposon-mediated genomic integration, a MND promoter, anti-mesothelin CARs, and an immunizing polypeptide comprising an li-CLIP replacement.

[0014] FIG. 4 is a schematic of DACART cells with MHC-II presentation neoantigen peptides to activate host CD4+ T cells and direct tumor targeting via a MSLN CAR.

[0015] FIG. 5 is a schematic of expression constructs comprising sequences for expression of a CAR and / or immunizing polypeptide, and other elements disclosed herein.WSGR Docket No. 57428-711.601

[0016] FIG. 6 is a schematic of expression constructs comprising sequences for expression of a CAR and / or immunizing polypeptide, and other elements disclosed herein.

[0017] FIG. 7 plots liquid chromatography retention time versus m / z for an immunopeptidomics assay.

[0018] FIG. 8 shows detection of an MHC-I presented T cell epitope from an immunizing polypeptide via tandem mass spectrometry.

[0019] FIG. 9 is a schematic of a dual action antigen-presenting CAR-T cell disclosed herein. Expression constructs encoding a chimeric antigen receptor and an immunizing polypeptide comprising neoantigens and / or TAAs are genomically integrated, e.g., into an allogeneic gamma delta T cell. The CAR can induce direct attack of tumor cells by the CAR-T cell. In addition, the immunizing polypeptide can be processed and presented via MHC class I to activate CD8+ cytotoxic T lymphocytes, and / or via MHC class II to activate CD4+ helper T cells. The non-engineered T cells can in turn provide anti-tumor activity, for example, cytotoxic activity of CD8+ T cells with release of perforin and granzyme to lyse tumor cells, and / or production of cytokines by CD4+ T cells to coordinate further anti-tumor activity.

[0020] FIG. 10 is a schematic of a reporter assay used to measure T cell activation in response to a neoantigen presented by an engineered gamma delta T cell.

[0021] FIG. 11 shows relative activation of reporter T cells with or without a TP53 R175H- specific TCR, in response to co-culture with engineered gamma delta T cells with or without expression of an immunizing polypeptide comprising the TP53 R175H peptide.

[0022] FIG. 12 provides flow cytometry dotplots for CD69 and CD25 expression by reporter T cells with TP53 R175H-specific TCRs. The “yb POC9” condition involved co-culture with engineered gamma delta T cells with expression of an immunizing polypeptide comprising the TP53 R175H peptide. The “yb pulse” condition involved pulsing of non-engineered gamma delta T cells without any mRNA or DNA prior to co-culture.DETAILED DESCRIPTION

[0023] Compositions and methods disclosed herein can comprise or utilize an immunizing polypeptide to induce activation of an endogenous T cell response in a subject. For example, a cell (e.g., gamma delta T cell) can be engineered to comprise a nucleic acid molecule that encodes the immunizing polypeptide, and the engineered cell can present one or more T cell epitopes from the immunizing polypeptide via HLA, thereby inducing activation of endogenous T cells in a subject (e.g., host or recipient of the engineered cell). Accordingly, cells disclosed herein, such as engineered gamma delta T cells, can act as a cellular vaccine to induce or beWSGR Docket No. 57428-711.601 capable of inducing an immune response by a subject’s T cells (e.g., an anti -cancer immune response). Engineered cells disclosed herein can be administered to a subject and can induce or be capable of inducing an anti-cancer immune response mediated by the subject’s endogenous T cells.

[0024] Optionally, the cells can further express a heterologous immune receptor such as a CAR, facilitating induction of (1) a direct immune response mediated by the heterologous immune receptor and engineered cell (e.g., anti-cancer), and (2) induction of immune responses (e.g., anti-cancer) mediated by the subject’s endogenous T cells, for example, against different target antigen(s) or T cell epitope(s) than those targeted by the heterologous immune receptor. Such a cell can be described as a Dual-action Antigen-presenting CAR y6 T-cell (DACART). D AC ART can combine the strengths of CAR T-cell therapy with those of cancer vaccination, simultaneously activating endogenous / host T cells in addition to direct attack of tumor cells recognized by the CAR (FIG. 9).

[0025] Chimeric antigen receptor (CAR) T-cell therapies have shown promise and achieved good results in treating some cancers, however loss of the targeted antigen expression or mutation of the target antigen can result in loss of efficacy, and additionally, CAR-T in most cases have been ineffective for treating solid tumors. Compositions and methods disclosed herein can combine expression of a CAR with expression of an immunizing polypeptide to activate endogenous alpha beta T cells. Cells can be engineered, for example, to express the immunizing polypeptide and present peptides comprising T cell epitopes to the host’s endogenous CD4+ and CD8+ T cells. Supplementing the direct cytotoxic effect of CAR-T cells with activation of endogenous T cells against different target antigens has the potential to achieve complete responses with higher reproducibility than current therapies.

[0026] Most established CAR T-cell therapies use autologous aP T cells. aP T cells are the predominant subtype of circulating T cells and are easier to isolate. However, aP T cells recognize a diversity of MHC-restricted antigens and thus can cause graft-versus-host disease (GvHD) if the donor cells and host cells have a different HLA haplotype.

[0027] y6 T cells are a unique T-cell subset able to function both as direct cytotoxic immune cells and professional antigen- presenting cells (e.g., unlike conventional aP T cells often used in CAR-T therapies). Accordingly, gamma delta T cells cam be engineered to directly kill cancer cells via a cancer-specific CAR while also capitalizing on their ability to activate a patient’s own immune system by displaying tumor-associated antigens (TAA) on major histocompatibility complex (MHC).

[0028] Presenting antigens to activate endogenous CD4+ T cells, endogenous CD8+ T cells, or both sets of endogenous T cells can enhance tumor fighting ability of this cell therapy andWSGR Docket No. 57428-711.601 complement the direct tumor killing via the CAR. Activation of CD8+ T cells can add an additional group of directly cytotoxic T cells to enhance direct tumor killing effects. Activating CD4+ T cells can provide complementary activity by modifying the immunosuppressive TME typically found in solid tumors to become less immunosuppressive and allow better overall control of the tumor.

[0029] While yb T cells can present antigens via MHC display, they do not recognize antigens in an MHC-restricted fashion. Thus, in some embodiments yb T cells are less prone to causing graft-versus-host disease and allow for safe dosing using cells from an allogenic donor, allowing production of less expensive, more readily available “off-the- shelf’ cell therapy products.I. ENGINEERED CELLS

[0030] Compositions and methods disclosed herein can comprise or utilize cells and / or populations thereof, for example, immune cells, such as T cells or gamma delta (yb, gd, or yd) T cells. A population of cells disclosed herein can comprise gamma delta T cells. Gamma delta T cells can be T cells (e.g., CD3+) that express a T cell receptor (TCR) comprising gamma and delta chains. The cells or populations thereof can be engineered as disclosed herein.

[0031] As disclosed herein, gamma delta T cells can act as antigen-presenting cells to induce HLA class-I restricted CD8+ T cell responses and / or class-II restricted CD4+ T cell responses. Interactions between gamma delta T cells (e.g., subsets thereof) and other immune cells such as aP T cells, NK cells, dendritic cell and macrophages can also promote a synergistic immune response.

[0032] Compositions and methods disclosed herein can comprise or utilize one or more polyclonal populations of gamma delta T cells. A polyclonal population of gamma delta T cells can describe a diverse mixture of gamma delta T cells with various TCR gamma & delta variable subunits, specificities, and functional properties. Given the dynamic and heterogenous complexity of the tumor microenvironment, a diverse polyclonal population of gamma delta T cells may exhibit greater adaptability allowing subsets with distinct functional properties to overcome immune suppression.

[0033] The diversity and functional plasticity of a polyclonal population of gamma delta T cells can provide several advantages in terms of antigen recognition, immune responsiveness, and adaptability to the tumor microenvironment, making them promising cell populations for immunotherapy against cancer and other (e.g., autoimmune) diseases.

[0034] Polyclonal gamma delta T cells populations can collectively recognize a wider range of antigens which allows them to target various pathogens, cancer cells, and infected cells,WSGR Docket No. 57428-711.601 thereby enhancing the versatility and efficacy of gamma delta T cell based immunotherapy. Additionally, different subsets of gamma delta T cells within a polyclonal population can exhibit distinct effector functions which allows for complementary immune responses against different types of tumor cells, improving the overall effectiveness of gamma delta mediated immune response. A heterogeneous population of gamma delta T cells with diverse TCR specificities also reduces the likelihood of immune escape by tumor cells through antigen loss or downregulation. Even if target antigens are no longer recognized by a subset of gamma delta T cells (or, e.g., expression of a CAR target antigen is lost), other clones within the polyclonal population can still exert immune surveillance and a potent cytotoxic response against target cells.

[0035] A polyclonal population of gamma delta T cells can exhibit advantageous properties over, for example, a population of gamma delta T cells with less diversity of TCR gamma / delta variable subunits.

[0036] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise any suitable combination of gamma and delta chains, “y” “gamma” “g” and “y” can be used interchangeably herein referring to gamma, for example, “Vy2” “Vg2” V gamma 2” and “Vy2) can all refer to the same gamma chain or gamma chain variable domain. “6” “delta” and “d” can all be used interchangeably herein to refer to delta, for example, “V51” “Vdl” and “V delta 1” can all refer to the same delta chain or delta chain variable domain.

[0037] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR gamma chain, for example, with a Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chain variable domain. In some embodiments, the TCR gamma chain is a Vyl, Vy6, Vy7, VylO, Vyl 1, Vyl2, Vyl3, or Vyl4 gamma chain variable domain.

[0038] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR delta chain, for example, with a V6 I , V62, or V63 delta chain variable domain. In some embodiments, the delta chain is a V64, V85, V86, V67, or V68 delta chain variable domain.

[0039] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising (a) a gamma chain comprising a Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chain variable domain, and (b) a delta chain comprising a V81, V62, or V63 delta chain variable domain.

[0040] A cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising (a) a gamma chain comprising a Vyl, Vy6, Vy7, VylO, Vyl 1, Vyl2, Vyl3, Vyl4, Vy2, Vy3, Vy4, Vy5, Vy8, or Vy9 gamma chainWSGR Docket No. 57428-711.601 variable domain, and (b) a delta chain comprising a V64, V85, V86, V67, V88, V81, V62, or V63 delta chain variable domain.

[0041] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V6 I variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V81 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V6 I variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 5- chain comprises a V6 I variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V6 I variable domain.

[0042] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V62 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V62 variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V62 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V62 variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 5- chain comprises a V62 variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V62 variable domain.

[0043] In some embodiments, the y-chain comprises a Vy2 variable domain and the 8-chain comprises a V63 variable domain. In some embodiments, the y-chain comprises a Vy3 variable domain and the 8-chain comprises a V63 variable domain. In some embodiments, the y-chain comprises a Vy4 variable domain and the 8-chain comprises a V63 variable domain. In some embodiments, the y-chain comprises a Vy5 variable domain and the 8-chain comprises a V63 variable domain. In some embodiments, the y-chain comprises a Vy8 variable domain and the 5- chain comprises a V63 variable domain. In some embodiments, the y-chain comprises a Vy9 variable domain and the 8-chain comprises a V63 variable domain.

[0044] Methods disclosed herein can comprise enriching, selecting, and / or expanding gamma delta T cells from a heterogeneous population of cells in a starting material. Methods disclosed herein can comprise reducing the number or proportion of non-gamma delta T cells, for example, reducing the number or proportion of alpha beta T cells in a population.

[0045] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%,WSGR Docket No. 57428-711.601 at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR gamma chain, for example, as determined by flow cytometry.

[0046] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR delta chain, for example, as determined by flow cytometry.

[0047] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells express a TCR gamma chain and a TCR delta chain, for example, as determined by flow cytometry.

[0048] In some embodiments, of a population of cells disclosed herein or a subpopulation thereof, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are not gamma delta T cells.

[0049] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are alpha beta T cells.

[0050] In some embodiments, of a population of T cells disclosed herein, at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at mostWSGR Docket No. 57428-711.601 about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80% of the cells are alpha beta T cells.

[0051] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express a TCR comprising a TCR gamma or delta chain variable domain disclosed herein, for example, Vyl, Vy2, Vy3, Vy4, Vy5, Vy6, Vy7, Vy8, Vy9, VylO, Vyl l, Vyl2, Vyl3, Vyl4, V51, V62, V83, V64, V85, V86, V87, or V68 variable domain.

[0052] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express V81 and at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of cells express V82.

[0053] In some embodiments, of a population of cells (e.g., a polyclonal population of gamma delta T cells generated by a method disclosed herein) or a subpopulation thereof, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% do not express V6 I or V82.

[0054] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a TCR comprising a constant domain from a y-chain and / or a constant domain from a 8-chain. Constant domains can be indicated by a C preceding the y-chain and 8-chain designations, e.g., Cy2, Cy3, Cy4, Cy5, Cy8, Cy9, Cyl l, C51, C82, C83, and C85.

[0055] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a Vy9V62 TCR or functional fragment thereof.WSGR Docket No. 57428-711.601

[0056] In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing an EPCR protein on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing annexin A2 on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing aberrant HLA protein expression on a cell surface of a target cell. In some embodiments, a cell, population of cells, or subpopulation (e.g., in a polyclonal population of gamma delta T cells) can comprise a gdTCR capable of recognizing cancers in an MHC / HLA-unrestricted manner.

[0057] In some embodiments, a cell, population of cells, or subpopulation comprises, consists essentially of, or consists of a clonal or monoclonal population of gamma delta T cells.

[0058] Cells can be selected or enriched for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence of one or more factors). Selection techniques include positive selection and negative selection techniques, e.g., fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS). In some cases, cells can be selected before engineering, for example, to enrich for a population of cells disclosed herein (e.g., immune cells, such as T cells or a T cell subset disclosed herein, such as gamma delta T cells or alpha beta T cells). The cells can also be selected independent of engineering, e.g., the cells are not subject to engineering modifications before or after sorting and other methods disclosed herein. Cells can be selected after engineering, for example, to enrich for a population of cells disclosed herein (e.g., engineered cells that express a polypeptide (e.g., CAR) or additional polypeptide). Engineered cells can be selected or enriched based on a tag or marker, such as an epitope tag. The tag or marker can be appended to the polypeptide (e.g., CAR). In some embodiments, the tag or marker is not appended to the polypeptide (e.g., CAR). The tag or marker can be co-expressed with the polypeptide (e.g., CAR) as disclosed herein. The tag or marker can comprise a reporter gene, such as a fluorescent protein.

[0059] Heterogeneous populations of cells (e.g., peripheral blood mononuclear cells (PBMCs) or leukapheresis products) can be sorted to enrich or select for gamma delta T cells, e.g., using magnetic and / or fluorescent activated cell sorting. Illustrative products and protocols that can be used include Miltenyi manual and CliniMACs purification of gamma delta T cells. In some embodiments, an anti-TCRaP agent (e.g., antibody) is used to negatively select or reduce the proportion of alpha beta T cells in a population of cells. In some embodiments, an anti-CD14 agent (e.g., antibody) is used to negatively select or reduce the proportion of myeloid cells or monocytes in a population of cells. In some embodiments, an anti-CD19 agent (e.g., antibody) isWSGR Docket No. 57428-711.601 used to negatively select or reduce the proportion of B cells in a population of cells. In some embodiments, an anti-gdTCR agent (e.g., antibody) is used to positively select or increase the proportion of gamma delta T cells in a population of cells. The anti-TCRaP agent, anti-CD14, anti-CD19, or anti-gdTCR agent can be coupled to an agent suitable for negative or positive selection as appropriate, e.g., via direct or indirect magnetic binding, or fluorescent sorting.

[0060] In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a nucleic acid molecule, after gene editing or delivery of a nucleic acid molecule, before selection, after selection, before expansion, after expansion, or a combination thereof. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a nucleic acid molecule. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after gene editing or delivery of a nucleic acid molecule. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before expansion. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after expansion.

[0061] Cells can be selected, enriched, or expanded on the basis of being positive or negative for a given factor. In some embodiments, cells are selected, enriched, or expanded on the basis of being positive for two or more factors. In some embodiments, cells can be selected, enriched, or expanded on the basis of being positive for one or more factors, and negative for one or more factors.

[0062] A cell in a composition or method disclosed herein can be a mammalian cell. A cell in a composition or method disclosed herein can be a human cell. A cell in a composition or method disclosed herein can be a non-rodent cell. A cell in a composition or method disclosed herein can be a primate cell, e.g., human or non-human primate. In some cases, a cell is a primary cell. In some cases, a cell is not a primary cell. A cell can be a therapeutic cell, for example, suitable for use in a therapeutic application in a suitable subject, such as a human.

[0063] A population of cells disclosed herein can comprise, consist essentially of, or consist of mammalian cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of human cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of non-rodent cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of primate cells, e.g., human or non-human primate cells. A population of cells disclosed herein can comprise, consist essentially of, or consist of primary cells, or in some embodiments, non-primary cells (e.g., a cell line). A population of cells can be a population of therapeutic cells, for example, suitable for use in a therapeuticWSGR Docket No. 57428-711.601 application in s suitable subject, such as a human. A population of therapeutic cells can comprise any cell type(s) or combinations thereof disclosed herein, and in some embodiments comprises a population of polyclonal gamma delta T cells (e.g., engineered to express a CAR / transgene, or non-engineered).

[0064] A cell in a composition, population, or method disclosed herein can be an immune cell. A cell in a composition or method disclosed herein can be a lymphocyte, T cell, alpha-beta T cell, gamma-delta T cell, CD4+ T cell, CD8+ T cell, a T effector cell, naive T cell, memory T cell (e.g., central memory, effector memory, or resident memory), lymphoid cell, innate lymphoid cell (ILC), a regulatory T-cell, a thymocyte, or any mixture or combination of cells thereof.

[0065] In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises a dendritic cell, an eosinophil, a granulocyte, a Langerhans cell, a macrophage, a neutrophil, a mast cell, a megakaryocyte, a monocyte, a myeloid cell, a plasma cell, B cell, an NK cell, an NKT cell or any mixture or combination of cells thereof.

[0066] In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises a precursor of an immune cell. In some embodiments, a cell in a composition or method disclosed herein, or in a population of cells disclosed herein, comprises or is derived from a stem cell, e.g., an iPSC or hematopoietic stem cell.

[0067] A cell in a composition, population, or method disclosed herein can be engineered, for example, modified to comprise a transgene, expression construct, nucleotide, and / or genomic alteration (e.g., insertion, deletion, knockout, translocation) as compared to a native cell. In some embodiments, a cell in a composition, population, or method disclosed herein can be nonengineered, for example, lack an artificially introduced transgene, expression construct, nucleotide, and / or genomic alteration, or can be non-genetically or genomically modified.

[0068] In some embodiments, an engineered cell comprises a disruption or deletion of one or more TCR-encoding genes, such as TRAC, TRB, TRG, and / or TRD. In some embodiments, an engineered cell comprises a disruption or deletion of a variable region of one or more TCR- encoding genes, such as a disruption or deletion in TRAC, TRB, TRG, and / or TRD.

[0069] In some embodiments, an engineered cell comprises a disruption or deletion of TRAC and / or TRB, and comprises an exogenously introduced gamma delta TCR and / or CAR

[0070] Cells can be harvested, for example, formulated in a pharmaceutical composition suitable for administration to a subject, and / or cryopreserved. In some embodiments, a harvested population of cells is suspended in a suitable cryopreservation buffer (e.g., CryoStor CS5 orWSGR Docket No. 57428-711.601CS10). The harvested population of cells can be formulated at a suitable concentration for storage, e.g., about 10-50 xlOA6 cells / mL.II. IMMUNIZING POLYPEPTIDE

[0071] Compositions and methods disclosed herein can comprise or utilize an immunizing polypeptide to induce activation of an endogenous T cell response in a subject. For example, a cell (e.g., gamma delta T cell) can be engineered to comprise a nucleic acid molecule that encodes the immunizing polypeptide, and the engineered cell can present one or more T cell epitopes from the immunizing polypeptide via HLA, thereby inducing activation of endogenous T cells in a subject (e.g., host or recipient of the engineered cell). Accordingly, cells disclosed herein, such as engineered gamma delta T cells, can act as a cellular vaccine to induce or be capable of inducing an immune response by a subject’s T cells (e.g., an anti-cancer immune response). Engineered cells disclosed herein can be administered to a subject and can induce or be capable of inducing an anti-cancer immune response mediated by the subject’s endogenous T cells.

[0072] Optionally, the cells can further express a heterologous immune receptor such as a CAR, facilitating induction of (1) a direct immune response mediated by the heterologous immune receptor and engineered cell (e.g., anti-cancer), and (2) induction of immune responses (e.g., anti-cancer) mediated by the subject’s endogenous T cells, for example, against different target antigens or T cell epitopes than those targeted by the heterologous immune receptor. Such a cell can be described as a Dual-action Antigen-presenting CAR y6 T-cell (DACART).

[0073] An immunizing polypeptide can comprise one or more T cell epitopes, for example, CD4+ and / or CD8+ T cell epitopes.

[0074] A CD8+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class I or MHC class I. Accordingly, an engineered immune cell can express the immunizing polypeptide and present the T cell epitope on HLA class I. A CD8+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class I of a common haplotype. A CD8+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class I of haplotype present in an engineered cell disclosed herein. A CD8+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class I of haplotype present in a subject disclosed herein.

[0075] A CD4+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class II or MHC class II. Accordingly, an engineered immune cell can express the immunizing polypeptide and present the T cell epitope on HLA class II. A CD4+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class IIWSGR Docket No. 57428-711.601 of a common haplotype. A CD4+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class II of haplotype present in an engineered cell disclosed herein. A CD4+ T cell epitope can be an epitope that is presented by or predicted to be presented by HLA class II of haplotype present in a subject disclosed herein.

[0076] Human leukocyte antigens (HLA), also broadly referred to as Major histocompatibility complex (MHC) antigens, can be protein molecules expressed on the surface of a cell that can confer an antigenic identity to that cell, and that can present antigens (e.g., including T cell epitopes) to T cells. HLA class I antigens (A, B, and C in humans) can be expressed by the vast majority of cells, while HLA class II antigens (DR, DP, and DQ in humans) can be expressed primarily on professional antigen presenting cells, including, e.g., gamma delta T cells.

[0077] HLA antigens are encoded by highly polymorphic genes; a range of alleles exist for each HLA class I and II gene. Allelic gene products can differ in one or more amino acids in the a and / or p domain(s). Panels of specific antibodies or nucleic acid reagents can be used to determine HLA haplotypes of individuals, for example, using leukocytes that express class I and class II molecules. HLA alleles can be described at various levels of detail. Most designations begin with HLA- and the locus name, then * and some (even) number of digits specifying the allele. The first two digits can specify a group of alleles. The third through fourth digits, when present, can specify a synonymous allele. Digits five through six, when present, can denote any synonymous mutations within the coding frame of the gene. The seventh and eighth digits, when present, can distinguish mutations outside the coding region. Letters such as L, N, Q, or S may follow an allele's designation to specify an expression level or other non-genomic data known about it. Thus, a completely described allele may be up to 9 digits long, not including the HLA- prefix and locus notation.

[0078] The set of HLA alleles present in a cell (for example, an engineered cell disclosed herein) can determine the range of T cell epitopes that the cell is capable of presenting to T cells to induce immune responses. For example, different HLA alleles can have different affinity for a peptide, such as a peptide processed from an immunizing polypeptide. A given HLA molecule (e.g., encoded by a given HLA allele), can present peptides that comprise a motif, e.g., a pattern of residues in an amino acid sequence of defined length, such as a motif comprising anchor residues that facilitate binding to the HLA.

[0079] An HLA class I motif can comprise, consist essentially of, or consist of, e.g., a peptide of about 8-14 amino acids in length, e.g., 8, 9, 10, 11, 12, 13, or 14 amino acids in length, which can comprise one or more CD8+ T cell epitopes. In some embodiments, an HLA class I motif identifies a peptide of 9, 10, or 11 amino acid residues in length.WSGR Docket No. 57428-711.601

[0080] An HLA class II motif can comprise, consist essentially of, or consist of, e.g., a peptide of about 6-25 amino acids in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, which can comprise one or more CD4+ T cell epitopes.

[0081] Motifs can be different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchor residues.

[0082] Tools can be used to predict affinity of epitopes for HLA or MHC, for example, Net MHC 4.0, NetMHCII, NetMHCIIpan, NetMHCIIpan 4.1, MHCflurry, epitopepredict, DockTope, HLAthena, MHCnuggets, and ConvNeXt-MHC.

[0083] An immunizing polypeptide can comprise one or more peptides that comprise T cell epitopes (e.g., CD4+ and / or CD8+ T cell epitopes), wherein the one or more peptides bind or are predicted to bind an HLA protein encoded by a given allele, for example, an allele present in an engineered cell disclosed herein, such as an engineered gamma delta T cell comprising a nucleic acid molecule encoding the immunizing polypeptide.

[0084] In some embodiments, an immunizing polypeptide comprises a peptide that comprises a T cell epitope, and the peptide that comprises the T cell epitope has a suitable affinity for an HLA protein or type (e.g., encoded by allele disclosed herein). In some embodiments, the peptide that comprises the T cell epitope binds or is predicted to bind an HLA protein with an IC50 of less than 5000 nM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, or less than 10 nM. The peptide that comprises the T cell epitope can be of a suitable length, for example, comprising, consisting essentially of, or consisting of 8-14, 6-25, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. The peptide that comprises the T cell epitope can comprise an HLA class I or HLA class II motif to facilitate HLA binding. In some embodiments the peptide that comprises the T cell epitope is 8 amino acid residues in length. In some embodiments the peptide that comprises the T cell epitope is 9 amino acid residues in length.

[0085] The peptide comprising the T cell epitope can bind or be predicted to bind to an HLA protein encoded by any suitable allele.

[0086] In some embodiments, the peptide comprising the T cell epitope binds or is predicted to bind to an HLA protein encoded by HLA of the allele / type A*01:01, A*02:01, A*02:03, A*02:04, A*02:07, A*03:01, A*24:02, A*29:02, A*31:01, A*68:02, B*35:01, B*44:02, B*44:03, B*51:01, B*54:01 or B57:01.

[0087] In some embodiments, the peptide comprising the T cell epitope binds or is predicted to bind to an HLA protein encoded by HLA of the allele / type A*02:01, A*01:01, A*03:01, A*24:02, A*l l:01, A*68:01, A*26:01, A*32:01, A*31:01, A*29:02, A*23:01, A*25:01,WSGR Docket No. 57428-711.601A*30:01, A*33:03, A*30:02, B*07:02, B*08:01, B*44:02, B*35:01, B*51:01, B*15:01, B*18:01, B*44:03, B*40:01, B*27:05, B*57:01, B*14:02, B*13:02, B*38:01, B*35:03, C*07:01, C*07:02, C*04:01, C*06:02, C*05:01, C*03:04, C*12:03, C*03:03, C*01:02, C*02:02, C*16:01, C*08:02, C*15:02, C*07:04, or C*17:01.

[0088] In some embodiments, the peptide comprising the T cell epitope binds or is predicted to bind to an HLA protein encoded by HLA of the allele / type HLA-A*02, HLA-A30*, HLA- B*15, HLA-B*07, HLA-O07, HLA-DRBl*03, HLA-DRB1*15, or HLA-DRB115*.

[0089] In some embodiments, the peptide comprising the T cell epitope binds or is predicted to bind to an HLA protein encoded by HLA of the allele / type HLA-A01 :01, HLA-A02:01. HLA-A03:01, HLA-A11 :01, HLA-A24:02, HLA-B07:02, and HLA-B08:0L

[0090] In some embodiments, the peptide comprising the T cell epitope binds or is predicted to bind to an HLA protein encoded by HLA of the allele / type HLA-C*08-02, HLA-A*03:01, HLA-A* 11 :01, HLA-C*01:02, HLA-A*02:01, HLA-A*02, HLA-A*03, HLA-DRpi*04, HLA- DRpi*07, or HLA-DRpi*08.

[0091] An immunizing polypeptide can comprise one or multiple T cell epitopes. For example, the immunizing polypeptide can be processed into smaller peptides, including one or more peptides that comprise T cell epitope(s), some or all of which can then be presented by HLA.

[0092] HLA presentation of a peptide comprising a T cell epitope by HLA can be determined by, for example, immunoprecipitation and tandem mass spectrometry.

[0093] In some embodiments, presentation of T cell epitopes to endogenous T cells may reduce persistence of the engineered cells, for example, due to immune activation targeting the engineered cells. However, in some embodiments reduced persistence of the engineered cells also reduces incidence or severity of graft-versus-host disease for allogeneic engineered cells, and / or can reduce the need for immunosuppressive drugs to prevent or treat GVHD.

[0094] An immunizing polypeptide can comprise multiple T cell epitopes and multiple peptides that comprise the T cell epitopes. In some embodiments an engineered cell expresses one immunizing polypeptide, which can for example contain or comprise one or multiple T cell epitopes, and the immunizing polypeptide can be processed and presented as multiple peptides each comprising one or more T cell epitopes. For example, a nucleic acid molecule can comprise a tandem minigene (TMG) that is expressed as a contiguous polypeptide chain, with each antigen peptide separated by a linker, such as a glycine-serine linker. The TMG can comprise, e.g., up to 5, up to 10, up to 15, up to 20, up to 50, at least 5, at least 10, at least 15, or at least 20 minigenes. The TMG can encode, e.g., up to 5, up to 10, up to 15, up to 20, up to 50, at least 5, at least 10, at least 15, or at least 20 peptides that each comprise a T cell epitope. TMGs can beWSGR Docket No. 57428-711.601 effective to introduce multiple antigens or T cell epitopes simultaneously into cells for HLA- presentation.

[0095] In some embodiments, an immunizing polypeptide comprises peptide sequences that each comprise T cell epitope(s), and the peptide sequences are separated by linkers or spacers, for example, flexible linkers (e.g., flexible linkers described herein). In some embodiments, an immunizing polypeptide comprises peptide sequences that each comprise T cell epitope(s), and the peptide sequences are separated by cleavable linkers, for example, furin-sensitive or 2A linkers. In some embodiments, an immunizing polypeptide comprises linkers or flanking sequences conducive to processing of the immunizing polypeptide for presentation via HLA.

[0096] In some embodiments an engineered cell expresses two or more immunizing polypeptides, each of which can contain or comprise one or multiple T cell epitopes and can be processed and presented as peptides each comprising one or more T cell epitopes. For example, peptides comprising T cell epitopes can be transcribed and / or translated separately.

[0097] Non-limiting examples of immunizing polypeptides are provided in TABLE 5 and TABLE 7.A. T cell epitopes

[0098] A T cell epitope in an immunizing polypeptide can be an epitope present in a tumor- associated antigen (TAA). In some embodiments, a TAA is an antigen present in a wild type protein that is abnormally expressed (e.g., expressed more highly) in a cancerous cell. In some embodiments, a TAA comprises a mutation, for example, a neoantigen or neoepitope. In some embodiments, a TAA comprises a viral-associated antigen or epitope, for example, from a cancer-associated or oncogenic virus, such as a herpesvirus, Human Papillomavirus (HPV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Herpesvirus 8 (HHV-8), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Human T-cell Lymphotropic Virus-1 (HTLV-1), or Merkel Cell Polyomavirus (MCPyV).

[0099] A T cell epitope in an immunizing polypeptide can be an epitope present in a neoantigen. A T cell epitope in an immunizing polypeptide can be or comprise a neoepitope.

[0100] Cancer-specific mutations can arise, for example, as a result of missense, splice-site, frameshift or read-through point mutations, or from the fusion of two genes (or within the same gene. Splice-site, frameshift, and read-through mutations and gene fusions can generate novel stretches of amino acids that are normally not translated, but now are expressed and translated as a result of mutation. Missense mutations can lead to neoepitopes with single amino acid changes.WSGR Docket No. 57428-711.601

[0101] A T cell epitope in an immunizing polypeptide can comprise a cancer-specific mutation flanked by a suitable number of amino acid codons from the context of the mutated gene on either side of the mutated codon. For example, in some embodiments, the cancerspecific mutation (e.g., point mutation) is flanked by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 amino acids from the wild type protein upstream of the mutation. In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from the wild type protein downstream of the mutation.

[0102] In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids from the wild type protein upstream of the mutation. In some embodiments, the cancerspecific mutation (e.g., point mutation) is flanked by 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids from the wild type protein downstream of the mutation.

[0103] In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 amino acids from the wild type protein upstream of the mutation. In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most17, at most 18, at most 19, or at most 20 amino acids from the wild type protein downstream of the mutation.

[0104] In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from the wild type protein upstream and downstream of the mutation.

[0105] In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids from the wild type protein upstream and downstream of the mutation.

[0106] In some embodiments, the cancer-specific mutation (e.g., point mutation) is flanked by at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 amino acids from the wild type protein upstream and downstream of the mutation.WSGR Docket No. 57428-711.601

[0107] Immunizing polypeptides disclosed herein can comprise T cell epitopes useful to treat or prevent a disorder of interest, for example, a cancer or infectious disease.

[0108] A T cell epitope in an immunizing polypeptide can comprise a T cell epitope that is associated with cancer. For example, a T cell epitope in an immunizing polypeptide can comprise mutation, such as a point mutation, that is frequently observed cancer generally, solid tumors, hematological cancers, or in a particular type of cancer, such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer. The T cell epitope can be present in context that facilitates HLA presentation as disclosed herein (for example, comprising suitable flanking sequences with anchor residues). Suitable sequences can be identified using databases and in silico resources, for example, the cancer genome atlas (TCGA).

[0109] Non-limiting examples of T cell epitopes (e.g., mutations, point mutations, neoepitopes) that can be used in immunizing polypeptides disclosed herein include those described in TABLE 1 (colorectal cancer) and TABLE 2 (NSCLC / lung adenocarcinoma). Further non-limiting examples of peptides comprising T cell epitopes are provided in TABLE 4.

[0110] TABLE 1 common somatic mutations in colorectal cancerWSGR Docket No. 57428-711.601

[0111] TABLE 2 common somatic mutations in NSCLS / lung adenocarcinoma

[0112] T cell epitope in an immunizing polypeptide can be an epitope from a microbe, for example, an infectious bacterium, virus, or parasite.

[0113] An immunizing polypeptide can comprise a copy of the invariant chain (li), in which the CLIP peptide has been replaced by a single cancer testis antigen (CTA). An immunizing polypeptide can comprise a CTA. CTAs can be expressed only in germline cells in most circumstances, but can be overexpressed in cancers. Additionally although presentation on MHC-II is generally for exogenous antigens, the li-CLIP- replacement approach can facilitate loading an endogenous peptide onto MHC-II complexes. li-CLIP replacement genes containingWSGR Docket No. 57428-711.601CTA peptides known to be overexpressed in cancers can be utilized, for example, the CTAs shown in TABLE 3.

[0114] An immunizing polypeptide can comprise one or more CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises one CD4+ T cell epitope. In some embodiments, an immunizing polypeptide comprises two CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises three CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises four CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises five CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises seven CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises ten CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 15 CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 20 CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 25 CD4+ T cell epitopes.

[0115] An immunizing polypeptide can comprise one or more CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises one CD8+ T cell epitope. In some embodiments, an immunizing polypeptide comprises two CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises three CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises four CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises five CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises seven CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises ten CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 15 CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 20 CD8+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 25 CD8+ T cell epitopes.WSGR Docket No. 57428-711.601

[0116] An immunizing polypeptide can comprise one or more CD8+ T cell epitopes and one or more CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises one CD8+ T cell epitope and one CD4+ T cell epitope. In some embodiments, an immunizing polypeptide comprises two CD8+ T cell epitopes and two CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises three CD8+ T cell epitopes and three CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises four CD8+ T cell epitopes and four CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises five CD8+ T cell epitopes and five CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises seven CD8+ T cell epitopes and seven CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises ten CD8+ T cell epitopes and ten CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 15 CD8+ T cell epitopes and 15 CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 20 CD8+ T cell epitopes and 20 CD4+ T cell epitopes. In some embodiments, an immunizing polypeptide comprises 25 CD8+ T cell epitopes and 25 CD4+ T cell epitopes.B. Targeting sequence

[0117] In some embodiments, an immunizing polypeptide comprises a targeting sequence to promote antigen processing and presentation, for example, HLA presentation of the T cell epitope.

[0118] An immunizing polypeptide can comprise a human tissue plasminogen activator signal peptide (TP A) to enhance HLA / MHC loading. A TPA activator signal peptide can, for example, help to guide the immunizing polypeptide into the endoplasmic reticulum.

[0119] An immunizing polypeptide can comprise a copy of the invariant chain (li), in which the CLIP peptide has been replaced by a single cancer testis antigen (CTA). The li-CLIP- replacement approach can be useful, for example, to promote loading of a peptide expressed by a cell onto HLA / MHC class II.

[0120] A targeting sequence to promote antigen processing and presentation can comprise an element that targets the immunizing polypeptide for proteasomal degradation, for example, ubiquitin or a mutant or fragment thereof. A targeting sequence to promote antigen processing and presentation can comprise an element that targets the immunizing polypeptide for proteasome-independent processing, for example, proprotein processing operated by furin in the trans-Golgi compartment of the secretory pathway, such as a TPA signal peptide.

[0121] In some embodiments, a targeting sequence to promote antigen processing and presentation comprises an endoplasmic reticulum targeting sequence, for example, a targetingWSGR Docket No. 57428-711.601 sequence from MHC class I signal peptide fragment (sec), E3 leader sequence of adenovirus (E3), or Human tissue plasminogen activator signal peptide (TP A).

[0122] In some embodiments, a targeting sequence to promote antigen processing and presentation comprises a lysosome targeting sequence, for example, a targeting sequence from Lysosome-associated membrane protein 1 (LAMP-1), Dendritic cell lysosome-associated membrane protein (DC-LAMP), MHC class I trafficking signal (MITD), or MHC class II- associated invariant chain (li).

[0123] A targeting sequence to promote antigen processing and presentation can comprise membrane translocating sequence (MTS) from the HIV-1 derived Tat gene at the C-terminus, e.g., for its ability to deliver exogenous antigens into the intracellular compartments where processing into MHC -binding peptides occurs.

[0124] An immunizing polypeptide can comprise one targeting sequence. In some embodiments, an immunizing polypeptide comprises two or more targeting sequences, for example, a combination of two or more targeting sequences disclosed herein, such as those provided in TABLE 6.

[0125] Illustrative, non-limiting examples of targeting sequences are provided in TABLE 6.III. CHIMERIC ANTIGEN RECEPTOR AND HETEROLOGOUS IMMUNE RECEPTORS

[0126] Populations of cells disclosed herein (e.g., gamma delta T cells) can be engineered to express a heterologous immune receptor, such as a chimeric antigen receptor (CAR) or T cell receptor. An expression construct, cell, or nucleic acid molecule disclosed herein can comprise a transgene that encodes a heterologous immune receptor.

[0127] A heterologous immune receptor can comprise an extracellular domain (including an extracellular binding domain), a transmembrane domain, and a cytoplasmic signaling domain.

[0128] A heterologous immune receptor can be expressed by an immune cell and configured to induce activation of and / or signaling in the immune cell upon contacting a target cell that expresses a cell surface molecule. A target cell can be a cell that is associated with a disease or condition. A target cell can be a cancer cell. A target cell can be a solid tumor cell. A target cell can be an immune cell. A target cell can be a hematologic cancer cell. A target cell can be a leukemia cell. A target cell can be a lymphoma cell. A target cell can be a myeloma cell. A target cell can be a B cell. A target cell can be a CD19+ cell. A target cell can be a cell that is associated with an autoimmune or inflammatory disease.

[0129] In some embodiments, a heterologous immune receptor is a chimeric antigen receptor (CAR). In some embodiments, a heterologous immune receptor is a first, second, third,WSGR Docket No. 57428-711.601 fourth, or fifth generation CAR. A first generation CAR can contain a single CD3 zeta cytoplasmic signaling domain (e.g., and lack a co-stimulatory cytoplasmic signaling domain). A second generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a costimulatory cytoplasmic signaling domain, such as a CD28 or 4 IBB costimulatory domain. A third generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and two costimulatory cytoplasmic signaling domains, for example, two of CD28, 4 IBB, and 0X40. A fourth generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a costimulatory cytoplasmic signaling domain (such as a CD28 or 4 IBB costimulatory domain), and a protein, such as interleukin 12 (IL-12), that is constitutively or inducibly expressed upon CAR activation. A fourth generation CAR can be, for example, a T cell redirected for universal cytokine-mediated killing (TRUCK). A fifth generation CAR can be based on a second generation CAR and contain a truncated cytoplasmic IL-2 receptor P-chain domain with a binding site for the transcription factor STAT3.

[0130] A heterologous immune receptor can comprise a component of a TCR signaling complex, for example, an extracellular domain, transmembrane domain, and / or cytoplasmic domain of a TCR signaling complex, such as a human TCR signaling complex.

[0131] In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex comprises two TCR chains (e.g., an alpha chain and a beta chain, or a gamma chain and a delta chain). In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex comprises a single chain TCR (scTCR), e.g., comprising a TCR alpha chain variable domain and a TCR beta chain variable domain joined by a suitable linker.

[0132] In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex is a TCR, e.g., comprises an extracellular binding domain that comprises TCR variable regions and TCR CDRs. In some embodiments, a heterologous immune receptor that comprises a component of a TCR signaling complex is not TCR, for example, comprises a non-TCR extracellular binding domain, and comprises a component of a TCR signaling complex.

[0133] A heterologous immune receptor can comprise a full length or substantially full length CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta. A heterologous immune receptor can comprise a full length or substantially full length TCR alpha chain (e.g., constant regions, or variable plus constant regions), TCR beta chain (e.g., constant regions, or variable plus constant regions), TCR gamma chain (e.g., constant regions, or variable plus constant regions), or TCR delta chain (e.g., constant regions, or variable plus constant regions).WSGR Docket No. 57428-711.601

[0134] In some embodiments, a system disclosed herein allows use of two or more heterologous immune receptors (e.g., CARs) delivered alone or in combination, concurrently or sequentially.

[0135] A heterologous immune receptor disclosed herein can comprise an extracellular domain. The extracellular domain can comprise an extracellular binding domain that can specifically bind to a cell surface molecule on a target cell, thereby modulating signaling by the heterologous immune receptor.

[0136] An extracellular binding domain can utilize one or more antigen-binding domains, for example, an antigen-binding domain of or derived from an antibody. In some embodiments, an extracellular binding domain disclosed herein comprises an antigen-binding domain or fragment from an antibody, such as an scFv or a nanobody.

[0137] An extracellular binding domain of the disclosure can comprise complementarity determining regions (CDRs). For example, an antibody, antigen-binding fragment thereof, or antigen-binding domain can comprise CDRs. In some embodiments, the CDRs determine or substantially determine binding specificity and / or affinity for a surface molecule on a target cell. For example, the CDRs can be grafted onto a different suitable framework, or the framework region can be altered (e.g., via amino acid substitutions, deletions, and / or insertions), and the antigen-binding fragment or domain can retain binding for the target, and the extracellular binding domain remains functional despite the alterations outside of the CDRs.

[0138] An extracellular binding domain can comprise an antibody fragment, antigen-binding domain, or antigen-binding fragment of an antibody. Non-limiting examples of antibody fragments, antigen-binding fragments, and antigen-binding domains include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, nanobodies, minibodies, dia-, tria-, and tetrabodies, and linear antibodies. Fab and Fab' are antigen-binding fragments that can comprise the VH and CHI domains of the heavy chain linked to the VL and CL domains of the light chain via a disulfide bond. A F(ab')2 can comprise two Fab or Fab' that are joined by disulfide bonds. A Fv can comprise the VH and VL domains held together by non-covalent interactions. A scFv (single-chain variable fragment) is a fusion protein that can comprise the VH and VL domains connected by a peptide linker. Manipulation of the orientation of the VH and VL domains and the linker length can be used to create different forms of molecules that can be monomeric, dimeric (diabody), trimeric (triabody), or tetrameric (tetrabody). Minibodies can be scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0139] The extracellular binding domain can be or can comprise a single domain antibody. The single domain antibody can be or can comprise a variable region of a heavy chain only antibody. Such a single domain antibody can also be known as a nanobody or VHH. The singleWSGR Docket No. 57428-711.601 domain antibody can be, for example, a variable region from or derived from a heavy chain only antibody from a camelid (e.g., camels: one-humped Camelus dromedaries and two-humped Camelus bactrianus; llamas: Lama glama, Lama guanicoe, and Lama vicugna; and alpacas: Vicugna pacos), a shark (e.g., a nurse shark), a wobbegong, or a spotted ratfish. Such animals have a special type of antibody called heavy chain Abs (HCAbs), that lack the entire light chain and the first heavy chain C region (CHI) compared to regular antibodies.

[0140] An extracellular binding domain can comprise an antigen-binding domain or fragment of a chimeric, humanized, or fully human antibody. An extracellular binding domain can comprise CDRs grafted onto a humanized or fully human framework sequence.

[0141] In addition to antibodies and antigen-binding fragments or domains thereof, other compounds can also comprise antigen-binding domains that can be used in compositions, systems, and methods of the disclosure, such as in an extracellular binding domain of a heterologous immune receptor. Non-limiting examples of non-antibody antigen-binding compounds include ankyrin proteins, ankyrin repeat proteins, designed ankyrin repeat proteins (DARPins), affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, P- hairpin mimetics, and receptors and derivatives thereof.

[0142] In some embodiments, an extracellular binding domain binds to a target (e.g., antigen) expressed on or associated with a solid tumor cell or cell type. In some embodiments, an extracellular binding domain binds to a target (e.g., antigen) expressed on or associated with a hematologic cancer cell or cell type. In some embodiments, an extracellular binding domain or CAR disclosed in Tomar al. "Development of highly effective anti-mesothelin hYP218 chimeric antigen receptor T cells with increased tumor infiltration and persistence for treating solid tumors." Molecular cancer therapeutics 21.7 (2022): 1195-1206, which is incorporated herein by reference, is used.

[0143] In some embodiments, an extracellular binding domain binds to a target associated with a solid tumor. In some embodiments, an extracellular binding domain binds to a target associated with colorectal cancer (CRC). In some embodiments, an extracellular binding domain binds to a target associated with non-small cell lung cancer (NSCLC). In some embodiments, an extracellular binding domain binds to a target associated with lung adenocarcinoma. In some embodiments, an extracellular binding domain binds to a target associated with prostate cancer. In some embodiments, an extracellular binding domain binds to a target associated with breast cancer.

[0144] In some embodiments, an extracellular binding domain binds to mesothelin. A known mesothelin-binding domain, such as the SSI scFv, can be used as an extracellular binding domain to bind mesothelin.WSGR Docket No. 57428-711.601

[0145] In some embodiments, an extracellular binding domain binds to carcinoembryonic antigen (CEA), CD70, CSPG4, B7-H3, CD133, EGFR, or MUCl. In some embodiments, an extracellular binding domain binds to CD 19, BAFF, CSPG4, GD2, CD70, mesothelin, carcinoembryonic antigen (CEA), B7-H3, CD 133, EGFR, MUC1, CD20, CD22, CD23, CD79a, or CD79b.

[0146] In some embodiments, an extracellular binding domain binds to CD 19, ACE2, an Fc domain, APRIL, BAFFR, B7H6, B7H3, BCMA, CA9, CAIX, carcinoembryonic antigen, CD133, CD16, CD174, CD22, CD23, CD27, CD274, CD276, CD33, CD38, CD44, CD5, CD70, CEACAM5, CSPG4, CTLX, DNAM-1, Dsg3, E l 3 Y IL13, E3 adnectin, EGFR, EGFRvIII, Envs, EPCAM, EPHA2, EPHB4, EPHRIN B2, ErbB, ERBB2, FAP, fibroblast activation protein, FLT3, FLT3L, FOLH1, FOLR1, FSH, FSHR, GD2, glycoprotein B, glycoprotein E2, GMCSF, GMR, gpl20, gp41, GPC3, GPNMB, HBsAg, HER2, ICAM-I, IL10, IL10R, IL11, ILl lRa, IL13Ra2, IL1RAP, IL3RA, Insulin-B chain, Islet-specific glucose-6- phosphatase catalytic subunit-related protein, KDR, LI CAM, LFA-1, M2e, mesothelin, MET, MICA, MICB, MPL, MS4A1, MSLN, MUC1, myelin oligodendrocyte glycoprotein, NCAM1, Nectin-2, NKG2D, NKp30, PDCD1, PSCA, PSMA, PVR, ROR1, SARS-CoV2 S protein, SDC1, SLAMF7, SSTR, TIE, TACI, TEM1, TNFRSF17, TNFRSF8, TPO, transmembrane form of IgE, TriPRIL, ULBP1, ULBP1-6, ULBP2, or VEGFR2. In some embodiments, an extracellular binding domain binds to CD 19. In some embodiments, the extracellular binding domain comprises an FMC63 scFv.

[0147] In some embodiments, an extracellular binding domain is or comprises a component of a receptor or a receptor ligand, for example, utilizes the naturally occurring specificity of a receptor or ligand. For example, an extracellular binding domain can comprise a receptorbinding domain or ligand-binding domain of B7H6, an Fc domain, APRIL, BAFF, BCMA, CD16, CD27, CD70, CTLX, DNAM-1, E l 3 Y IL13, E3 adnectin, EGFR, EPHB4, EPHRIN B2, ErbBl, ErbB2, ErbB3, ErbB4, FLT3, FLT3L, FSH, FSHR, GMCSF, GMR, ICAM-I, IL10, IL10R, IL11, ILl lRa, IL13Ra2, LFA-1, MICA, MICB, MPL, Nectin-2, orNKG2D.

[0148] In some embodiments, an extracellular binding domain does not bind to an immunizing polypeptide, for example, does not bind to an antigen or epitope (e.g., T cell epitope) present in an immunizing polypeptide or that is processed from the immunizing polypeptide and presented via HLA. In some embodiments, an extracellular binding domain does not bind to an immunizing polypeptide, for example, does not bind to any antigen or epitope (e.g., T cell epitope) present in an immunizing polypeptide or that is processed from the immunizing polypeptide and presented via HLA. For example, the heterologous immune receptor (e.g., CAR) can induce a direct anti-cancer immune response against cancer cells viaWSGR Docket No. 57428-711.601 recognition of the target bound by the extracellular binding domain, and in parallel the engineered cell can activate the host’ s endogenous immune response against different cancer- associated targets via HLA-I and / or HLA-II presentation of T cell epitopes distinct from the CAR target.

[0149] In some embodiments, an extracellular binding domain binds to an immunizing polypeptide or an antigen or epitope present in the immunizing polypeptide.

[0150] The heterologous immune receptor can comprise one or more additional extracellular domains as well as the extracellular binding domain.

[0151] In some embodiments, a heterologous immune receptor comprises an additional extracellular domain or amino acid sequence that is a linker or spacer. In some embodiments, a heterologous immune receptor comprises a hinge, such as an IgG hinge or a CD8 hinge.

[0152] A heterologous immune receptor can comprise a transmembrane domain. Any suitable transmembrane domain can be used. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of CD8. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of CD28. In some embodiments, the heterologous immune receptor comprises a transmembrane domain of 0X40, 4 IBB, or CD86.

[0153] The transmembrane domain can be a transmembrane domain of an immune receptor or TCR signaling complex component disclosed herein, for example, of a mammalian or a human TCR signaling complex. The transmembrane domain can comprise, for example, a transmembrane domain of TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta. In some embodiments, a transmembrane domain is not from an immune receptor or is not from a TCR signaling complex component.

[0154] A heterologous immune receptor can comprise a cytoplasmic domain or a mutant, variant, or derivative thereof. A cytoplasmic domain can comprise a cytoplasmic signaling domain or a mutant, variant, or derivative thereof. The cytoplasmic signaling domain can contribute to the ability of the heterologous immune receptor to elicit a response. For example, the cytoplasmic signaling domain can contribute to induction of signaling and / or immune cell activation upon of binding of the heterologous immune receptor (e.g., an extracellular binding domain thereof) to a surface molecule of a target cell. In some cases, the cytoplasmic signaling domain can contribute to the induction of a pro-inflammatory response, an anti-cancer immune response, an immune tolerance-promoting response, a transcriptional response, TCR signaling, T cell activation, T cell proliferation, cytokine production, a cytotoxic response against the target cell, or a combination thereof. In some cases, the cytoplasmic signaling domain can contribute to the activation of bystander immune cells that do not comprise a heterologousWSGR Docket No. 57428-711.601 immune receptor of the disclosure. A cytoplasmic signaling domain can enhance the proliferation, survival, and / or function of immune cells, and / or development of effector and / or memory immune responses (e.g., memory T cells).

[0155] A cytoplasmic signaling domain can partake in an immune cell activation pathway that involves, for example, phosphorylation, dephosphorylation, calcium release, ubiquitination, de-ubiquitination, proteolytic cleavage, protein-protein interactions, a transcriptional response, or a combination thereof. An immune cell activation pathway can comprise, for example, an innate, adaptive, STING, NFkB, inflammasome, TCR, BCR, JAK / STAT, TLR, NLR, RLR, costimulatory, co-inhibitory, cytokine, or chemokine signaling pathway. A cytoplasmic signaling domain can comprise one or more immunoreceptor tyrosine-based activation motifs (IT AMs). A cytoplasmic signaling domain can comprise one or more immunoreceptor tyrosine-based inhibition motifs (ITIMs).

[0156] In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof. In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta with 1, 2, or 3 functional or active IT AMs. In some embodiments, the heterologous immune receptor contains a cytoplasmic signaling domain of CD3 zeta with one inactivated IT AM or two inactivated ITAMs. In some embodiments, the heterologous immune receptor does not contain a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof.

[0157] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain, or a functional fragment thereof. In some embodiments, a heterologous immune receptor does not contain a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain.

[0158] A cytoplasmic domain or cytoplasmic signaling domain can be derived from and / or interact with a kinase, (e.g., a protein kinase, a tyrosine kinase or a serine / threonine kinase, a receptor tyrosine kinase, a lipid kinase, a phosphoinositide kinase, a carbohydrate kinase, or a combination thereof), a phosphatase, a ubiquitin ligase, a caspase, an adapter protein, a transcription factor, an ion channel, or a combination thereof. A cytoplasmic domain or cytoplasmic signaling domain can contribute to interaction of the heterologous immune receptor with additional proteins or factors (e.g., members of a complex and / or signal transduction pathway).

[0159] A heterologous immune receptor can comprise a cytoplasmic signaling domain that enhances antigen processing and / or presentation, for example, by yb T cells that express the immunizing polypeptide. For example, in some embodiments, inclusion of an FcsRIy cytoplasmic signaling domain contributes to enhancing antigen presentation by yb T cells, forWSGR Docket No. 57428-711.601 example, via increased MHC-II / HLA-II expression. A heterologous immune receptor can comprise a cytoplasmic signaling domain of FcsRIy (Fc Epsilon Receptor Ig).

[0160] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a costimulatory immune receptor, or a functional fragment thereof. Non-limiting examples of costimulatory immune receptors include CD28, 2B4 (CD244, SLAMF4), 4-1BB (CD137), CD2 (LFA2, 0X34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM (CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LTpR (TNFRSF3), Lyl08 (NTBA, CD352, SLAMF6), Ly9 (CD229,SLAMF3), 0X40 (CD134), SLAM (CD150, SLAMF1), TIM1 (HAVCR1, KIMI), TIM2, CD56, DAP10, DAP12, IGSF8, J AML, LFA1- alpha, LF Al -beta, and NKRPla. In some embodiments, a heterologous immune receptor does not contain a cytoplasmic signaling domain of a costimulatory immune receptor.

[0161] A heterologous immune receptor can comprise a cytoplasmic signaling domain of a co-inhibitory immune receptor, or a functional fragment thereof. A heterologous immune receptor can comprise a cytoplasmic signaling domain of an activating NK receptor or inhibitory NK receptor, or a functional fragment thereof.

[0162] A heterologous immune receptor can comprise a cytoplasmic signaling domain that is a component of a TCR signaling complex, for example, of a mammalian or a human TCR signaling complex. The cytoplasmic signaling domain can comprise, for example, a cytoplasmic signaling domain of CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, a functional fragment thereof, or a combination thereof.

[0163] In some cases, a heterologous immune receptor of the disclosure does not contain a cytoplasmic signaling domain, but can nonetheless elicit an immune cell activation signal, for example, via a cytoplasmic signaling domain in another protein that can associate with the heterologous immune receptor. In some embodiments, a heterologous immune receptor of the disclosure that comprises constant regions from one or more TCR chains can transmit an immune cell activation signal via associated CD3 proteins that comprise cytoplasmic signaling domains (e.g., CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 eta, or a combination thereof).

[0164] A heterologous immune receptor can comprise one or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. A heterologous immune receptor can comprise, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. A heterologous immune receptor can comprise at least one, at least two, at least three, at least four, or at least five cytoplasmic signaling domains. AWSGR Docket No. 57428-711.601 heterologous immune receptor can comprise at most one, at most two, at most three, at most four, at most five, or at most ten cytoplasmic signaling domains.

[0165] A cytoplasmic signaling domain can be from a mammalian protein. In some cases, a cytoplasmic signaling domain is from a murine (mouse) protein. In some cases, a cytoplasmic signaling domain is from a human protein. In some cases, a cytoplasmic signaling domain can comprise modifications compared to a wild type sequence.

[0166] A heterologous immune receptor of the disclosure can comprise one or more linkers for example, between different domains of the protein. A linker can be a chemical bond, for example, a covalent bond or a non-covalent bond. A linker as described herein can include a flexible or rigid linker. A linker can be a peptide. A linker can be selected to achieve a desired functionality of the heterologous immune receptor. A linker can comprise a linker sequence, for example, a linker peptide sequence. The length a linker can be adjusted to allow for proper folding or to increase or decrease biological activity of the heterologous immune receptor.

[0167] A flexible linker can have a sequence containing glycine residues. The small size of the glycine residues can provide flexibility, and allow for mobility of the connected protein domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous conditions by forming hydrogen bonds with the water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. In some cases, flexible linkers can also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and / or polar amino acids such as lysine and glutamine, to improve solubility.

[0168] A rigid linker can have, for example, an alpha helix-structure. An alpha-helical rigid linker can act as a spacer between protein domains. A rigid linker can have a proline-rich sequence, (XP)n, with X designating alanine, lysine, glutamine, or any amino acid, and n designating a number of repeats. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

[0169] A linker can comprise a hinge region, for example an amino acid sequence derived from a hinge region of an antibody or immune receptor. In some embodiments, a linker comprises a hinge region from CD8a, IgGl, or IgG4.

[0170] In some embodiments, a heterologous immune receptor is a chimeric antigen receptor known as or within an engineered cell or construct known as Tisagenlecleucel (Kymriah®), Axicabtagene Ciloleucel (Yescarta®), Brexucabtagene Autoleucel (Tecartus™), Lisocabtagene maraleucel, Idecabtagene Vicleucel, or KTE-X19.

[0171] In some embodiments, a heterologous immune receptor is a dual CAR, a split CAR, or an inducible split CAR. A dual CAR can comprise two CARs with different extracellular binding domains, and thus signal induction based on two target antigens. A split CAR canWSGR Docket No. 57428-711.601 comprise two CARs with different extracellular binding domains and separation of costimulatory domains (e.g., CD28 and 4 IBB) from CD3zeta on the distinct CAR polypeptides, thereby requiring engagement of both CARs for T cell activation.

[0172] In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets (for example, comprises an extracellular binding domain that binds) CD19, BAFF, CSPG4, GD2, CD70, mesothelin, carcinoembryonic antigen (CEA), B7-H3, CD 133, EGFR, MUC1, CD20, CD22, CD23, CD79a, or CD79b and a second CAR that targets (for example, comprises an extracellular binding domain that binds) CD 19, BAFF, CSPG4, GD2, CD70, mesothelin, CEA, B7-H3, CD 133, EGFR, MUC1, CD20, CD22, CD23, CD79a, or CD79b.

[0173] In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets (for example, comprises an extracellular binding domain that binds) CD 19 and a second CAR that targets (for example, comprises an extracellular binding domain that binds) BAFF. In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets CSPG4 and a second CAR that targets GD2. In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets CSPG4 and a second CAR that targets CD70. In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets CSPG4 and a second CAR that targets mesothelin. In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets CSPG4 and a second CAR that targets B7H3. In some embodiments, the heterologous immune receptor is a dual CAR comprising a first CAR that targets GD2 and a second CAR that targets B7H3.

[0174] In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets (for example, comprises an extracellular binding domain that binds) CD19, BAFF, CSPG4, GD2, CD70, mesothelin, carcinoembryonic antigen (CEA), B7-H3, CD 133, EGFR, MUC1, CD20, CD22, CD23, CD79a, or CD79b and a second CAR that targets (for example, comprises an extracellular binding domain that binds) CD 19, BAFF, CSPG4, GD2, CD70, mesothelin, CEA, B7-H3, CD 133, EGFR, MUC1, CD20, CD22, CD23, CD79a, or CD79b.

[0175] In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets CD 19 and a second CAR that targets BAFF. In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets CSPG4 and a second CAR that targets GD2. In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets CSPG4 and a second CAR that targets CD70. In some embodiments, the heterologous immune receptor is a split CAR comprising a first CARWSGR Docket No. 57428-711.601 that targets CSPG4 and a second CAR that targets mesothelin. In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets CSPG4 and a second CAR that targets B7H3. In some embodiments, the heterologous immune receptor is a split CAR comprising a first CAR that targets GD2 and a second CAR that targets B7H3.

[0176] In some embodiments, the heterologous immune receptor is a split CAR disclosed in WO2022094314A1 or W02021038036A1, each of which is incorporated herein by reference in its entirety.

[0177] In some embodiments, a heterologous immune receptor is a universal CAR, for example, an extracellular binding domain can be combined with amino acid sequence(s) from one or more components of a TCR signaling complex and / or a chimeric antigen receptor (CAR) to generate a “universal” heterologous immune receptor that can be armed and disarmed based on the presence of adapter molecule(s). An adapter molecule can direct an immune cell expressing the heterologous immune receptor to a target cell (e.g., a cancer cell), and upregulate activation of the immune cell upon encountering the target cell (e.g., leading to a cytotoxic response against the target cell). A universal CAR can be capable of binding to various adapter molecules that can confer target specificity.IV. SAFETY SWITCH

[0178] An engineered cell disclosed herein can comprise a safety switch. For example, a nucleic acid molecule can encode a safety switch to allow deletion, killing of, or induction of apoptosis of engineered immune cells. In some embodiments, the safety switch comprises an epitope that a therapeutic antibody can bind to induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC). The safety switch can comprise, for example, an epitope that is recognized by cetuximab. For example, the safety switch can comprise a truncated epidermal growth factor receptor (tEGFR) domain, antigen, or epitope to easily detect CAR expression and provide an inducible safety switch. The safety switch can comprise, for example, an epitope that is recognized by rituximab. The safety switch can be combined with another domain or tag. For example, the safety switch can be part of RQR8 which further comprises a CD34 epitope that facilitates identification, isolation, sorting, or enrichment of engineered immune cells.

[0179] In some embodiments, the safety switch comprises a factor that induces apoptosis of the engineered immune cell, for example, an inducible caspase such as inducible caspase 9 that triggers apoptosis upon contacting an inducing agent, such as rapamycin a rapalog, an another chemical induce of dimerization.WSGR Docket No. 57428-711.601

[0180] In some embodiments, a safety switch comprises a cytotoxic protein, such as an inducible cytotoxic protein. A safety switch can comprise, for example, a caspase or a catalytic domain thereof. A safety switch can be or comprise a caspase, for example, caspase 1, caspase 3, caspase 8, caspase 9, or a catalytic domain thereof. A safety switch can be, comprise, consist essentially of, or consist of a non-inducible caspase, such as a non-inducible caspase 1, caspase 3, caspase 8, or caspase 9, or a non-inducible protein that comprises a catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9. A safety switch can be or comprise a selfactivating caspase, such as a self-activating caspase 1, self-activating caspase 3, self-activating caspase 8, or self-activating caspase 9, or a self-activating protein that comprises a catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9. A safety switch can be, comprise, consist essentially of, or consist of an inducible caspase, such as an inducible caspase 1, inducible caspase 3, inducible caspase 8, or inducible caspase 9 (iCasp9), or an inducible protein that comprises a catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9. An inducible cytotoxic protein, such as an inducible caspase disclosed herein, can be in an inactive state until contacting with a chemical or biological compound that activates the cytotoxic protein. An inducible cytotoxic protein, such as an inducible caspase disclosed herein, can be activated by contacting with a macrolide. An inducible cytotoxic protein, such as an inducible caspase disclosed herein, can be activated by contacting with rapamycin or a structural analogue thereof. An inducible cytotoxic protein, such as an inducible caspase disclosed herein, can be activated by contacting with another inducing agent, such as AP20187. An inducible cytotoxic protein can comprise caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using the small molecule AP20187 (which can be a synthetic analog of FK506). An inducible cytotoxic protein can be a rapamycin-inducible cytotoxic protein. For example, a cytotoxic protein can comprise, consist essentially of, or consist of a rapamycin-inducible caspase, such as a rapamycin-inducible caspase 1, rapamycin-inducible caspase 3, rapamycin- inducible caspase 8, or rapamycin-inducible caspase 9, or a rapamycin-inducible protein that comprises a catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9.V. NUCLEIC ACIDS, VECTORS, AND CELL ENGINEERING

[0181] In some embodiments, compositions and methods disclosed herein comprise an engineered cell or population thereof. Methods can comprise engineering the cells, or previously engineered cells can be used. Methods disclosed herein and other suitable known methods can be used to generate engineered cells, for example, gamma delta T cells comprising a nucleic acid molecule that encodes a CAR, one or more immunizing polypeptides, or a combination thereof. For example, cell engineering techniques disclosed herein and / or known to a skilled person canWSGR Docket No. 57428-711.601 be used to modify cells to comprise a nucleic acid molecule that encodes a heterologous immune receptor (e.g., CAR) and / or an immunizing polypeptide of the disclosure, thereby generating engineered cells (such as engineered gamma delta T cells).

[0182] A nucleic acid molecule can be a heterologous nucleic acid molecule, for example, that is heterologous with respect to the cell, e.g., introduced into the cell, not natively present in the cell, and / or encoding a product that is not naturally occurring or not natively produced by the cell.

[0183] In some embodiments, a nucleic acid molecule comprising a nucleic acid sequence that encodes a CAR is introduced into a cell or population thereof. In some embodiments, a nucleic acid molecule comprising a nucleic acid sequence that encodes an immunizing polypeptide is introduced into a cell or population thereof. In some embodiments, a nucleic acid molecule comprising a nucleic acid sequence that encodes an exogenous TCR is introduced into a cell or population thereof. In some embodiments, a nucleic acid molecule comprising a nucleic acid sequence that encodes an exogenous gdTCR can be introduced into a cell, such as a lymphocyte, lymphoid cell, T cell (e.g., alpha-beta T cell), or myeloid cell.

[0184] Compositions and methods disclosed herein can comprise a nucleic acid molecule, for example, comprising a nucleic acid sequence (e.g., transgene) that encodes a chimeric antigen receptor or other heterologous immune receptor (e.g., CAR). A nucleic acid molecule can comprise, for example, one or more expression regulatory regions (e.g., a promoter, enhancer, intron, and / or exon), one or more transgenes (e.g., a nucleic acid sequence encoding a heterologous immune receptor (e.g., CAR), an immunizing polypeptide, or a combination thereof), a polyadenylation signal, or a combination thereof. In some embodiments one nucleic acid molecule comprises a first nucleotide sequence that encodes a heterologous immune receptor, and a second nucleotide sequence that encodes an immunizing polypeptide. The heterologous immune receptor and immunizing polypeptide can be expressed as one polypeptide that is then cleaved to separate the heterologous immune receptor and immunizing polypeptide. In some embodiments, the heterologous immune receptor and immunizing polypeptide are expressed as separate polypeptides. The heterologous immune receptor and immunizing polypeptide can be separated by, for example, a 2A linker, a self-cleaving linker, a ribosomal skip element, or an IRES.

[0185] In some embodiments a first nucleic acid molecule comprises a first nucleotide sequence that encodes a heterologous immune receptor, and a second nucleic acid molecule comprises a second nucleotide sequence that encodes an immunizing polypeptide. For example, the first nucleic acid molecule and the second nucleic acid molecule can each comprise their own promoter.WSGR Docket No. 57428-711.601

[0186] A nucleic acid molecule can be a substance comprising, consisting essentially of, or consisting of nucleotides linked in a chain. Non-limiting examples of the nucleic acid molecule include a circular nucleic acid, a DNA, a single stranded DNA, a double stranded DNA, a genomic DNA, a plasmid, a nanoplasmid, a plasmid DNA, a viral DNA, a minicircle (e.g., lacking a bacterial origin of replication), and an RNA.

[0187] In some embodiments, a nucleic acid molecule encodes two or more polypeptides linked by one or more 2A linkers or self-cleaving peptides, which can be processed into separate polypeptides co-translationally or after translation (e.g., P2A, T2A, F2A, E2A). Inclusion of a 2A linker can increase the likelihood that an appropriate ratio of components are produced (e.g., a 1 : 1, 1 :2, 1:3, 1 :4, or 1 :5 ratio of two components).

[0188] For example, in some embodiments a heterologous immune receptor (e.g., CAR) can comprise a TCR gamma chain constant region and TCR delta chain constant region, and inclusion of a 2A linker can increase the likelihood that equal or close to equal levels of TCR gamma chain and TCR delta chain, are produced. In some cases, use of a 2A linker can allow for fewer components in a system for transgene expression and / or genome modification, e.g., inclusion of multiple components in one vector rather than separate vectors, such as inclusion of a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding one or more immunizing polypeptides.

[0189] An expression construct or nucleic acid molecule disclosed herein can be or can comprise DNA. An expression construct or nucleic acid molecule disclosed herein can be or can comprise double stranded DNA. For example, an expression construct or nucleic acid molecule disclosed herein can be or comprise a plasmid, such as a nanoplasmid. In some embodiments, an expression construct or nucleic acid molecule disclosed herein is or comprises a minicircle, a midge, a MIP, or a doggy bone.

[0190] In some embodiments an expression construct or nucleic acid molecule lacks an origin of replication. An expression construct or nucleic acid molecule disclosed herein can be or can comprise a circular nucleic acid molecule. An expression construct or nucleic acid molecule disclosed herein can be or can comprise a linear nucleic acid molecule. An expression construct or nucleic acid molecule disclosed herein can comprise one or more transgenes or open reading frames.

[0191] A nucleic acid molecule can be or be present in an expression construct. A nucleic acid molecule or expression construct can comprise a promoter, enhancer, or combination thereof that drive or upregulate expression of a nucleic acid sequence or transgene, for example, that encodes a heterologous immune receptor (e.g., CAR), immunizing polypeptide, or aWSGR Docket No. 57428-711.601 combination thereof. The nucleic acid sequence or transgene can be operatively linked to and / or under regulatory control of the promoter and / or enhancer.

[0192] A promoter disclosed herein can be a mammalian promoter or derived from a mammalian promoter. A promoter disclosed herein can be a human promoter or derived from a human promoter. The promoter can be a promoter as found in a naturally-occurring genome. In some embodiments, a promoter is not found in a naturally-occurring genome. In some embodiments, the promoter is a synthetic or engineered promoter. The promoter can be a minimal promoter. A promoter can be a constitutive, viral, inducible, or tissue-specific promoter. In some embodiments the promoter is an MND promoter.

[0193] A promoter can be an immune-cell selective promoter, for example, a promoter that results in preferential expression in immune cells as compared to non-immune cells. An immune cell-selective promoter can result in preferential expression in, for example, lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, NK cells, or NKT cells. The expression can be preferential compared to control cells, such as fibroblasts, neurons, epithelial cells, keratinocytes, or hepatocytes, etc.

[0194] A promoter can be a T-cell selective promoter, for example, a promoter that results in preferential expression in T cells as compared to non-T cells. In some embodiments, a T cell- selective promoter can limit off-target effects, e.g., limit off target effects resulting from expression of a heterologous immune receptor (e.g., CAR) in non-T cells. Non-limiting examples of T-cell selective promoters include promoters that natively drive expression of CD3 (e g., CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta), CD4, CD8, CD28, TCRB, TRAC, TRG, or TRD.

[0195] In some embodiments, a nucleic acid molecule disclosed herein comprises RNA, for example, mRNA.

[0196] In some embodiments, an expression construct or nucleic acid molecule disclosed herein is or comprises single stranded DNA. In some embodiments, an expression construct or nucleic acid molecule disclosed herein comprises a component of a viral genome or a viral packaging element, for example, a 5' and / or 3' inverted terminal repeat (ITR). In some embodiments, an expression construct or nucleic acid molecule disclosed herein is not single stranded DNA. In some embodiments, an expression construct or nucleic acid molecule disclosed herein lacks a component of a viral genome or lacks a viral packaging element, for example, lacks a 5' and / or 3' inverted terminal repeat (ITR).

[0197] In some embodiments, an expression construct or nucleic acid molecule disclosed herein is integrating, e.g., integrates or is integrated into the genome of an engineered cell. InWSGR Docket No. 57428-711.601 some embodiments, an expression construct or nucleic acid molecule disclosed herein is nonintegrating, e.g., does not integrate or is not integrated into the genome of an engineered cell.

[0198] A nucleic acid molecule can include one or more homology arms, for example, comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination or homology directed repair). A nucleic acid molecule can comprise one or more promoter regions, barcodes, restriction sites, cleavage sites, endonuclease recognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites may be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance properties (e.g., antibiotic resistance for selection), or any combination thereof. A nucleic acid molecule may also include transcriptional or translational regulatory sequences, for example, one or more promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A linkers and / or polyadenylation signals.

[0199] In some embodiments, a nucleic acid molecule or expression construct disclosed herein comprises natural, synthetic, and / or artificial nucleotide analogues or bases. In some embodiments, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of a deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0200] In some embodiments, a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Illustrative alkyl moieties include, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[0201] A nucleic acid molecule, gene editing component, or other cargo can be delivered to a cell by any suitable method, for example, using any suitable vector. In some embodiments a composition or method utilizes a vector comprising any of the nucleic acid molecules described herein.WSGR Docket No. 57428-711.601

[0202] Methods to introduce nucleic acid molecules and / or gene editing components into a cell include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including lentiviral, adenoviral, AAV, and retroviral vectors.

[0203] Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the Xenon Electroporation System (ThermoFisher Scientific), or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids into a cell. Electroporation parameters may be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type can have a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and / or cell viability, e.g., for gamma delta T cells.

[0204] A vector disclosed herein can be a non-viral, lipid-based vector. A non-viral, lipid- based vector can be, for example, a liposome, a lipoplex, a lipid nanoparticle, a vesicle, or a micelle. In some embodiments, a vector is or comprises a poloxamer, nanoparticle, polyplex, or dendrimer. A vector can be a nanoparticle, for example, an inorganic nanoparticle, such as a gold, silica, iron oxide, titanium, calcium phosphate, PLGA, poly(B-amino ester) (PBAE, e.g., PBAE-447), or hydrogel nanoparticle. In some embodiments a vector is not a nanoparticle, e.g., is not an inorganic nanoparticle.

[0205] A vector can be or can comprise a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, a transposon, and the like. Any vector systems can be used including, but not limited to, DNA vectors, RNA vectors, ribonucleoprotein vectors, hybrid DNA-RNA vectors, plasmid vectors, minicircle vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors and adeno-associated virus vectors, etc. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. In some cases, one vector is used. In some cases, two vectors are used. In some cases, three or more vectors are used.

[0206] In some cases, the vector is a viral vector, such as a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the vector is a non-WSGR Docket No. 57428-711.601 viral vector, for example, a plasmid, nanoplasmid, minicircle, a midge, a MIP, or a doggybone, a lipid-based nanoparticle, a liposome, a circular nucleic acid molecule (e.g., DNA or RNA), a linear nucleic acid molecule (e.g., a DNA or RNA), or a combination thereof. In some embodiments, viral vectors are not used in a method disclosed herein.

[0207] In some cases, a nucleic acid molecule, gene editing component, or other can be delivered to cells without the use of vectors. In some cases, one or more nucleic acid molecules, gene editing components, or other cargos of the disclosure can be delivered to cells via vectors, and one or more nucleic acid molecules, gene editing components, or other cargos can be delivered without the use of vectors.

[0208] Engineering methods can comprise contacting a cell with a nucleic acid molecule, or with a vector that comprises the nucleic acid molecule, under conditions that permit uptake of the nucleic acid molecule by the cell. A nucleic acid molecule can comprise a nucleotide sequence that encodes a heterologous immune receptor (e.g., CAR) disclosed herein or a component thereof, an immunizing polypeptide, or a combination thereof. In some cases, a nucleic acid molecule is utilized to alter a genome of a cell. An engineered cell can be generated by a method that comprises contacting a cell with a nucleic acid molecule or vector disclosed herein.

[0209] For targeted integration, a nucleic acid molecule sequence to be inserted can be flanked by homology arms comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination and / or homology- directed repair, HDR). A double stranded break can be introduced at a target site in the genome, and the homology arms can promote insertion of the nucleic acid molecule. In some cases, a nucleic acid molecule can be excised from a vector, such as a nanoplasmid (e.g., via a nuclease), and inserted into the genome of the cell.

[0210] A nucleic acid molecule can be inserted in a safe harbor locus. A safe harbor can comprise a genomic location where a nucleic acid molecule can integrate and function without substantially perturbing endogenous activity, for example, with a relatively low impact on local or global gene expression. For example, one or more nucleic acid molecules can be inserted into any one of HPRT, an AAVS site (E.G., AAVS1, AAVS2, etc.), CCR5, hROSA26, and / or any combination thereof. A nucleic acid molecule can be inserted in an intergenic region. A nucleic acid molecule can be inserted in a non-coding region. A nucleic acid molecule can be inserted within a gene. In some cases, a nucleic acid molecule can disrupt a gene it is inserted into (e.g., reduce or eliminate expression of the disrupted gene). A disrupted gene can be for example, an endogenous TCR gene (e.g., TRAC, TCRB, TCRBC1, TRBC2, TRG, TRD), or an immuneWSGR Docket No. 57428-711.601 checkpoint gene (e.g., PD-1, CTLA-4). A nucleic acid molecule can be inserted adjacent to or near to a promoter.

[0211] In some cases, one or more nucleic acid molecules of the disclosure can be inserted randomly into the genome of a cell. For instance, a nucleic acid molecule can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively or additionally, a nucleic acid molecule can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a non-coding region.

[0212] A variety of enzymes can catalyze generation of a double-stranded break in the genome and / or insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR systems, CRISPR-associated polypeptide (Cas), TALEN, zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptide, meganuclease, Mega-TAL, transposon-based systems, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0213] A transposon-based system can be utilized for insertion of a nucleic acid molecule encoding a polypeptide (e.g., CAR) of the disclosure or a component thereof into a genome. A transposon can comprise a nucleic acid molecule that can be inserted into a DNA sequence. A class I transposon can be transcribed into an RNA intermediate, then reverse transcribed and inserted into a DNA sequence. A class II transposon can comprise a DNA sequence that is excised from one DNA sequence and / or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector that contains a nucleic acid sequence (e.g., comprising a transgene) flanked by inverted terminal repeats, and (ii) a source for the transposase enzyme. A transposon system (e.g., class II transposon system) can direct the integration of a nucleic acid molecule sequence encoding a polypeptide (e.g., CAR and / or immunizing polypeptide) or a component thereof, while leaving behind the rest of the vector. A transposon and a transposase can be introduced into a cell. In some cases, a vector that encodes a transposase and comprises a nucleic acid molecule is introduced into a cell, and the transposase is expressed and mediates insertion of the transposon into the genome.

[0214] Examples of transposon-based systems that can be used include, but are not limited to, TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.WSGR Docket No. 57428-711.601

[0215] In some embodiments, a nucleic acid molecule disclosed herein comprises or is flanked by inverted terminal repeats (ITR) to facilitate recognition by a transposase enzyme and genomic integration.

[0216] TcBuster (TcB) and hyperactive TcBuster (TcB-M) can be obtained from Bio- Techne (Minneapolis, MN). In some embodiments, a TcB transposase mRNA and transposon plasmid comprising a nucleic acid sequence or transgene to be genomically integrated are used, and, e.g., delivered via electroporation. Non-limiting examples of TcBuster systems that can be used are described in US Patent Application Nos. US20210277366A1, US20200323902A1, and US20180216087A1, each of which is incorporated herein by reference in its entirety.

[0217] A CRISPR system can be utilized to facilitate insertion of a nucleic acid molecule encoding a heterologous immune receptor (e.g., CAR) or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome or a random site of a genome.

[0218] In some cases, a CRISPR system comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR- associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, or type VI CRISPR-associated (Cas) polypeptides a derivative, variant, or functional fragment thereof.

[0219] In some embodiments, a CRISPR system comprises a Class I system or endonuclease (e.g., Type I, Type III or Type IV Cas proteins). A class I system can be of the LA, I-B, I-C, I- U, I-D, I-E, I-F, IV-A, IV-B, III-A, III-D, III-C, or III-B subtype.

[0220] In some embodiments, a CRISPR system comprises a Class II system or endonuclease (e.g., Type II, Type V, or Type VI). A class II, Type II system can be of the ILA, ILB, ILC1, or II-C2 subtype. A class II, Type V systems can of the V-A, V-Bl, V-B2, V-C, V- D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-Ul, V-U2, or V-U4 subtype. A Class II, Type IV systems can be of the: VI-A, VI-B1, VI-B2, VI-C, or VI-D subtype.

[0221] In some embodiments, a Cas protein used in a method disclosed herein is a class II endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V Cas endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V-A Cas endonuclease.

[0222] Non-limiting examples of Cas proteins that can be used in the CRISPR systems include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO,WSGR Docket No. 57428-711.601Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof, and modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A Cas protein can be a high-fidelity Cas protein. Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpfl family that display cleavage activity in mammalian cells.

[0223] In some embodiments, a gene editing system comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the gene editing moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein.

[0224] In some cases, a dual nickase approach may be used to introduce a double stranded break. Cas proteins can be mutated at certain amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break. A nickase along with two distinct guide RNAs targeting opposite strands may be utilized to generate a DSB within a target site (often referred to as a “double nick” or “dual nickase” CRISPR system).

[0225] In some embodiments a polypeptide (e.g., CAR and / or immunizing polypeptide) can be expressed in an engineered cell without genomic integration of a nucleic acid molecule comprising a nucleic acid sequence or transgene. For example, a nucleic acid sequence or transgene can be expressed from an episomal vector, such as a DNA, RNA, circular DNA, circular RNA, minicircle, or the like. A polypeptide (e.g., CAR and / or immunizing polypeptide) can be transiently expressed. For example, expression of a polypeptide (e.g., CAR and / or immunizing polypeptide) can be reduced as a nucleic acid that encodes it is degraded. One method of generating engineered cells is through the use of a ribonucleic acid (RNA) system, e.g., a system that involves delivering one or more nucleic acid molecules as an RNA. In some cases, the use of RNA can minimize DNA-induced toxicity and immunogenicity sometimes observed with the use of DNA.

[0226] Cells can be genetically engineered to comprise a nucleic acid molecule that encodes a polypeptide (e.g., CAR and / or immunizing polypeptide) ex vivo. For example, cells can be taken from a subject in one or more blood draws and / or apheresis procedures, modified ex vivo, optionally selected and / or expanded before and / or after genetic modification, and optionally reintroduced into the subject or a different subject by infusion or injection.WSGR Docket No. 57428-711.601

[0227] In some cases, a selectable marker is introduced to a cell, e.g., together with or as part of a nucleic acid molecule encoding a polypeptide (e.g., CAR and / or immunizing polypeptide), so that cells that comprise the polypeptide (e.g., CAR and / or immunizing polypeptide) or modification express the selectable marker and can be selected, enriched, or expanded. In some cases, a selectable marker is an antibiotic resistance gene, and cells that do not express the antibiotic resistance gene can be killed by treatment with the antibiotic (e.g., to select or enrich for cells that comprise a polypeptide (e.g., CAR and / or immunizing polypeptide)). In some embodiments, the selectable marker is an epitope tag.

[0228] Expression of a polypeptide (e.g., CAR and / or immunizing polypeptide) can be quantified, for example, by qPCR, RNA sequencing, western blot, or flow cytometry.

[0229] In some embodiments, cells are engineered to express a cytokine or chemokine to exhibit autocrine or paracrine signaling to modulate (e.g., enhance) function of the engineered cells, for example, IL-15, IL-2, IL-7, IL-12, IFN-alpha, IFN-gamma, IL-lbeta, or a functional variant thereof.

[0230] Compositions and methods disclosed herein can comprise an expansion culture medium that can be suitable for expanding a population of therapeutic cells, for example, gamma delta T cells or polyclonal gamma delta T cells.

[0231] An expansion culture medium can comprise a suitable basal component for culturing T cells, for example, OpTimizer T-cell expansion Basal Medium, TheraPEAK T-Vivo, AIM-V, X-VIV015, TexMACS, or RPMI.

[0232] An expansion culture medium can comprise one or more antibiotics, for example, penicillin and streptomycin. In some embodiments, an expansion culture medium used in one or more steps of a method disclosed herein does not contain antibiotics, for example, lacks penicillin and streptomycin.

[0233] An expansion culture medium can comprise one or more cell culture supplements, for example, L-glutamine, Glutamax, non-essential amino acids, HEPES, 2-mercaptoethanol, Sodium Bicarbonate (NaHCO3), trace elements, vitamins, inorganic salts, and the like.

[0234] An expansion culture medium can comprise serum, for example, human AB serum or fetal bovine serum. In some embodiments an expansion culture medium is serum free, for example, comprises a serum substitute such as Physiologix™ Cell-Vive™ CTS™ Immune Cell SR, Proliferum LSR, or other available serum substitutes. In some embodiments an expansion culture medium is a xeno-free formulation.

[0235] An expansion culture medium can comprise one or more cytokines or growth factors, for example, interleukin 2 (IL-2), interleukin 7 (IL-7), and / or interleukin 15 (IL- 15). In some embodiments, an expansion culture medium comprises IL-2. In some embodiments, anWSGR Docket No. 57428-711.601 expansion culture medium comprises IL-7. In some embodiments, an expansion culture medium comprises IL-15. In some embodiments, an expansion culture medium comprises IL-2 and IL-7. In some embodiments, an expansion culture medium comprises IL-2 and IL-15. In some embodiments, an expansion culture medium comprises IL-7 and IL-15. In some embodiments, an expansion culture medium comprises IL-2, IL-7, and IL-15. In some embodiments, an expansion culture medium lacks IL-2, IL-7, and / or IL-15.

[0236] An expansion culture medium can comprise one or more T cell and / or gamma delta T cell stimulating agents, for example, a gamma delta T cell receptor (gdTCR) stimulating agent, a CD3 stimulating agent, or a CD28 stimulating agent.

[0237] A gamma delta T cell receptor (gdTCR) stimulating agent can be or comprise a gdTCR stimulating antibody or a pan-gdTCR stimulating antibody. A gdTCR stimulating agent can specifically or preferentially induce signaling by a gamma delta TCR (e.g., complex), for example, compared to an alpha beta TCR. In some embodiments, the gdTCR stimulating agent is a pan-gdTCR that stimulates gdTCRs that comprise multiple gamma chain variable domains and / or delta chain variable domains. In some embodiments, the gdTCR stimulating agent stimulates gdTCRs that comprise one or more gamma chain variable domains and / or delta chain variable domains disclosed herein. Illustrative gdTCR stimulating agents include antibodies (e.g., clone REA591 from Miltenyi), phosphoantigens (e.g., isopentenyl pyrophosphate, IPP), and aminobisphosphonates (e.g., zoledronic acid).VI. PHARMACEUTICAL COMPOSITIONS

[0238] Pharmaceutical compositions of the present disclosure can comprise a composition disclosed herein and a pharmaceutically acceptable excipient. A pharmaceutical composition can comprise, for example, a pharmaceutically acceptable excipient, vehicle, carrier, or diluent, and an expression construct or nucleic acid molecule, vector, and / or a cell (e.g., engineered cell) disclosed herein. A pharmaceutical composition can be formulated, for example, for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration. These compositions can take the form of solutions or suspensions. IN some embodiments, a pharmaceutical composition is in the form of an emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.

[0239] A pharmaceutical composition can comprise a population of cells in a unit dosage form. A pharmaceutical composition can comprise a nucleic acid molecule or vector comprising the nucleic acid molecule in a unit dosage form.WSGR Docket No. 57428-711.601

[0240] In some cases, unit dosage forms, include, but are not limited to, sterile or substantially sterile parenteral solutions or suspensions, tablets, capsules, pills, powders, granules, oral solutions or suspensions, and oil water emulsions.

[0241] A formulation or composition described herein can be an aqueous solution.Compositions in some examples herein are provided as sterile or substantially sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Compositions described herein can also comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions containing the cells can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0242] Formulations of cells can include those for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration, for example, injection or infusion.VII. METHODS

[0243] Disclosed herein are methods and uses that comprise administering to a subject a population of cells, for example, gamma delta T cells, such as gamma delta T cells comprising a heterologous nucleic acid molecule that encodes an immunizing polypeptide. The immunizing polypeptide can comprise a T cell epitope and / or a nucleic acid molecule that encodes a heterologous immune receptor (e.g., CAR). The cells can be administered as part of a pharmaceutical composition.

[0244] An engineered cell (e.g., gamma delta T cell) comprising a nucleic acid molecule that encodes an immunizing polypeptide can be administered to the subject, and the engineered cell can act as an antigen presenting cell and present one or more T cell epitopes from the immunizing polypeptide via HLA / MHC to T cells, thereby inducing activation of endogenous T cells in the subject (e.g., host or recipient of the engineered cell). Accordingly, cells disclosed herein, such as engineered gamma delta T cells, can act as a cellular vaccine to induce anticancer immune responses by a subject’s endogenous T cells.

[0245] The methods can comprise treating a subject in need thereof. The subject can have a disease or condition, such as a cancer. The cancer can be a solid tumor. The cancer can be a liquid tumor. The cancer can be a hematologic tumor. The cancer can be an immune cell cancer. The cancer can be a B cell cancer.WSGR Docket No. 57428-711.601

[0246] A population of engineered cells disclosed herein can be employed in treating a subject with cancer, an infectious disease, or an immune disorder. In some embodiments, the subject has a solid tumor that the method treats, for example, colorectal cancer (CRC), nonsmall cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer.

[0247] In some embodiments, the subject has a hematological cancer that the method treats. In some embodiments, the cancer is lymphoma, (e.g., mantle cell, diffuse large B cell, follicular, lymphoplasmacytic, marginal zone B-cell, small-cell lymphocytic, Burkitt, primary central nervous system, primary intraocular lymphoma, etc.), leukemia (e.g., chronic lymphocytic, acute lymphoblastic, hairy cell, chronic myeloid, etc), myelodysplastic syndromes, myeloproliferative disorder, or multiple myeloma.

[0248] In some embodiments, a population of engineered cells is used to treat a subject with a B cell related leukemia such as acute lymphoblastic leukemia, myeloma, the non-Hodgkin lymphomas (NHL) mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL).

[0249] A population of engineered cells disclosed herein can reduce tumor growth in a subject, for example, reduce tumor volume / burden or reduce expansion of tumor volume / burden.

[0250] In some embodiments, administering a population of engineered cells disclosed herein to a subject results in an at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold reduction in tumor volume.

[0251] The vaccine aspect of the present disclosure can result in improved control of cancers. For example, in some cases engineered cells disclosed herein that comprise (i) a heterologous immune receptor (e.g., CAR) and (ii) a nucleic acid molecule encoding the immunizing polypeptide can elicit improved anti-cancer immunity compared to corresponding engineered cells that comprise the heterologous immune receptor but lack the nucleic acid molecule encoding the immunizing polypeptide, for example, due to activation of endogenous T cell responses against the cancer.

[0252] In some embodiments, administering an engineered cell or population thereof disclosed herein to a cohort of subjects results in an at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold more reduction in tumor volume than a control engineered cell or population thereof administered to a control cohort of subjects. The control engineeredWSGR Docket No. 57428-711.601 cell or population thereof can lack the nucleic acid molecule that encodes the immunizing polypeptide.

[0253] In some embodiments, administering an engineered cell or population thereof disclosed herein to a cohort of subjects results in an at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold increased average survival than a control engineered cell or population thereof administered to a control cohort of subjects. The control engineered or population thereof cell can lack the nucleic acid molecule that encodes the immunizing polypeptide.

[0254] In some embodiments, if an engineered cell or population thereof disclosed herein is administered to a cohort of subjects, tumor volume is reduced on average by an at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold than in a control cohort of subjects administered a control engineered cell or population thereof. The control engineered cell or population thereof can lack the nucleic acid molecule that encodes the immunizing polypeptide.

[0255] In some embodiments, if an engineered cell or population thereof disclosed herein is administered to a cohort of subjects, average survival is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold longer than average survival of a control cohort of subjects administered a control engineered cell or population thereof. The control engineered or population thereof cell can lack the nucleic acid molecule that encodes the immunizing polypeptide.

[0256] In some embodiments, if an engineered cell or population thereof disclosed herein is administered to a cohort of subjects, average survival is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold longer than a control group not administered the engineered cell or population thereof.

[0257] In some embodiments, the subject has an infectious disease. In some embodiments, a method disclosed herein can be used for treating and / or or preventing an infectious disease, for example, a viral, bacterial, or parasitic infection.WSGR Docket No. 57428-711.601

[0258] The population of cells can be administered in an amount effective to treat or prevent a disease or condition. “Treatment” (and grammatical variations thereof such as “treat” or “treating”) can refer to clinical intervention in an attempt to alter the natural course of the individual (subject) being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment can include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0259] A population of cells, nucleic acid molecule, vector, or pharmaceutical composition can be administered to a subject, for example, by parenteral administration. A population of cells, nucleic acid molecule, vector, or pharmaceutical composition can be administered to a subject, for example, by intravenous, intraperitoneal, intramuscular, subdermal, intracerebral, intracerebroventricular, intra-articular, intraarterial, intrathecal, intracapsular, subcapsular, intraorbital, intracardiac, intradermal, subcutaneous, subarachnoid, or intracranial injection or infusion. The administration can be via localized injection or infusion. The administration can be via systemic injection or infusion. The administration can be via intravenous injection or infusion. The administration can be via intratumoral injection or infusion. The administering can be local. The administering can be systemic.

[0260] Various dosing schedules can be used. In some embodiments, a population of cells, nucleic acid molecule, vector, or pharmaceutical composition is administered to a subject once. In some embodiments, the population of cells, nucleic acid molecule, vector, or pharmaceutical composition is administered to a subject two or more times.

[0261] The population of cells can be autologous to the subject. The population of cells can be allogeneic to the subject, for example, from a related or unrelated donor. The population of cells can be haploidentical to the subject. The population of cells can be HLA-matched to the subject. In some embodiments, the population of cells is HLA-matched to the subject, for example, HLA matched at all typed HLA alleles. In some embodiments, a population of cells and a subject can be HLA-typed HLA- A, HLA-B, HLA-C, and / or HLA-DR alleles. In some embodiments, the population of cells and subject are matched for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 HLA alleles. In some embodiments, the population of cells is haploidentical to the subject.

[0262] The set of HLA alleles inherited from one parent forms a haplotype. HLA haploidentical can refer to a donor-recipient pair where one chromosome is matched at least at HLA-A; HLA-B, and HLA-DR between the donor and recipient. The haploidentical pair may or may not be matched at other alleles, e.g., other HLA genes on the other chromosome, orWSGR Docket No. 57428-711.601 additional histocompatibility loci on either chromosome. Such donors can frequently occur in families, e.g. a parent can be haploidentical to a child; and siblings may be haploidentical.

[0263] A cell population can HLA-typed at any number of HLA alleles for a recipient that will receive the engineered cell population. An engineered cell population and a subject can be HLA matched, e.g., matched at all typed HLA alleles. An engineered cell population and a subject can be HLA mismatched, e.g., at least one HLA antigen can be mismatched between the donor and recipient.

[0264] In some embodiments, an engineered cell population and a subject can be HLA-typed at six alleles consisting of HLA- A, HLA-B, and HLA-DR alleles. The engineered cell population and subject can be matched at, for example 3 / 6 4 / 6, 5 / 6, or 6 / 6 of the alleles. In some embodiments, the engineered cell population and subject are matched at least at 5 / 6 alleles. In some embodiments, the engineered cell population and subject are matched at 6 / 6 alleles.

[0265] In some embodiments, an engineered cell population and a subject can be HLA-typed at eight alleles consisting of HLA- A, HLA-B, HLA-C, and HLA-DR alleles (e g., HLA-DRB1 alleles). The engineered cell population and subject can be matched at, for example 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 of the alleles. In some embodiments, the engineered cell population and subject are matched at least at 6 / 8 alleles. In some embodiments, the engineered cell population and subject are matched at least at 7 / 8 alleles. In some embodiments, the engineered cell population and subject are matched at 8 / 8 alleles.VIII. EMBODIMENTS

[0266] Embodiment 1. An engineered cell comprising a heterologous nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope, wherein the engineered cell is a gamma delta T cell.

[0267] Embodiment 2. An engineered cell comprising: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope.

[0268] Embodiment 3. The engineered cell of any one of the preceding embodiments, wherein upon administration of the engineered cell to a subject, the engineered cell induces activation of an endogenous T cell in the subject.

[0269] Embodiment 4. The engineered cell of any one of the preceding embodiments, wherein upon administration of the engineered cell to a subject, the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject.WSGR Docket No. 57428-711.601

[0270] Embodiment 5. The engineered cell of any one of the preceding embodiments, wherein the engineered cell is capable of inducing activation of an endogenous T cell of a subject upon administration of the engineered cell to the subject.

[0271] Embodiment 6. The engineered cell of any one of the preceding embodiments, wherein the engineered cell is capable of inducing an anti-cancer immune response mediated by endogenous T cells in the subject upon administration of the engineered cell to the subject.

[0272] Embodiment 7. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a CD4+ T cell epitope.

[0273] Embodiment 8. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a CD8+ T cell epitope.

[0274] Embodiment 9. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a CD4+ T cell epitope and a CD8+ T cell epitope.

[0275] Embodiment 10. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a tumor-associated antigen (TAA).

[0276] Embodiment 11. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a solid tumor antigen.

[0277] Embodiment 12. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a neoantigen.

[0278] Embodiment 13. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a neoepitope.

[0279] Embodiment 14. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises at least four T cell epitopes.

[0280] Embodiment 15. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

[0281] Embodiment 16. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 1-36, 115, and 120.

[0282] Embodiment 17. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-36, 115, and 120.

[0283] Embodiment 18. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 37-42 and 52-54.WSGR Docket No. 57428-711.601

[0284] Embodiment 19. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 137-42 and 52-54.

[0285] Embodiment 20. The engineered cell of any one of the preceding embodiments, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class I.

[0286] Embodiment 21. The engineered cell of any one of the preceding embodiments, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class II.

[0287] Embodiment 22. The engineered cell of any one of the preceding embodiments, wherein the immunizing polypeptide comprises a targeting sequence to promote HLA presentation of the T cell epitope.

[0288] Embodiment 23. The engineered cell of embodiment 22, wherein the targeting sequence comprises a human tissue plasminogen activator signal peptide.

[0289] Embodiment 24. The engineered cell of embodiment 22, wherein the targeting sequence comprises a li-CLIP- replacement peptide.

[0290] Embodiment 25. The engineered cell of embodiment 22, wherein the targeting sequence comprises a lysosome targeting sequence.

[0291] Embodiment 26. The engineered cell of embodiment 22, wherein the targeting sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 43-51.

[0292] Embodiment 27. The engineered cell of embodiment 22, wherein the targeting sequence comprises the amino acid sequence of any one of SEQ ID NOs: 43-51.

[0293] Embodiment 28. The engineered cell of any one of the preceding embodiments, wherein the nucleic acid molecule that encodes the immunizing polypeptide is DNA.

[0294] Embodiment 29. The engineered cell of any one of the preceding embodiments, wherein the nucleic acid molecule that encodes the immunizing polypeptide is a tandem minigene.

[0295] Embodiment 30. The engineered cell of any one of the preceding embodiments, wherein the nucleic acid molecule that encodes the immunizing polypeptide is genomically integrated.

[0296] Embodiment 31. The engineered cell of any one of the preceding embodiments, wherein the nucleic acid molecule that encodes the immunizing polypeptide is not genomically integrated.WSGR Docket No. 57428-711.601

[0297] Embodiment 32. The engineered cell of any one of embodiments 1-27, wherein the nucleic acid molecule that encodes the immunizing polypeptide is RNA.

[0298] Embodiment 33. The engineered cell of any one of the preceding embodiments, wherein the CAR comprises an extracellular binding domain that binds to a target associated with a solid tumor.

[0299] Embodiment 34. The engineered cell of any one of the preceding embodiments, wherein the CAR comprises an extracellular binding domain that binds to a target associated with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer.

[0300] Embodiment 35. The engineered cell of any one of the preceding embodiments, wherein the CAR comprises an extracellular binding domain that binds to mesothelin.

[0301] Embodiment 36. The engineered cell of any one of embodiments 1-34, wherein the CAR comprises an extracellular binding domain that binds to carcinoembryonic antigen (CEA), CD70, CSPG4, B7-H3, CD 133, EGFR, or MUCl.

[0302] Embodiment 37. The engineered cell of any one of the preceding embodiments, wherein the extracellular binding domain of the CAR does not bind to the immunizing polypeptide or the T cell epitope.

[0303] Embodiment 38. The engineered cell of any one of the preceding embodiments, wherein the CAR is a split CAR or dual CAR.

[0304] Embodiment 39. The engineered cell of any one of the preceding embodiments, wherein the CAR comprises an FcsRIy cytoplasmic signaling domain.

[0305] Embodiment 40. The engineered cell of any one of the preceding embodiments, wherein the CAR is a second, third, fourth, or fifth generation CAR.

[0306] Embodiment 41. The engineered cell of any one of the preceding embodiments, wherein the CAR comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 59-60.

[0307] Embodiment 42. The engineered cell of any one of the preceding embodiments, wherein CAR comprises the amino acid sequence of any one of SEQ ID NOs: 59-60.

[0308] Embodiment 43. The engineered cell of any one of the preceding embodiments, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

[0309] Embodiment 44. The engineered cell of any one of the preceding embodiments, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cellWSGR Docket No. 57428-711.601 improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

[0310] Embodiment 45. The engineered cell of any one of the preceding embodiments, wherein the engineered cell further comprises a safety switch.

[0311] Embodiment 46. The engineered cell of any one of the preceding embodiments, wherein the safety switch comprises a truncated EGFR.

[0312] Embodiment 47. A population of cells comprising a plurality of the engineered cell of any one of the preceding embodiments, wherein the population of cells comprises: (i) at least 10% Vdl+ cells and at least 10% Vd2+ cells, (ii) at least 1% Vdl- Vd2- gamma delta T cells, or (iii) at least 11% Vd3+ cells.

[0313] Embodiment 48. A method of treating a subject in need thereof, the method comprising administering to the subject the engineered cell of any one of embodiments 1-46.

[0314] Embodiment 49. The method of embodiment 48, wherein the method treats cancer in the subject.

[0315] Embodiment 50. The method of embodiment 49, wherein the cancer is a solid tumor.

[0316] Embodiment 51. The method of embodiment 49, wherein the cancer is colorectal cancer (CRC).

[0317] Embodiment 52. The method of embodiment 49, wherein the cancer is non-small cell lung cancer (NSCLC).

[0318] Embodiment 53. The method of embodiment 49, wherein the cancer is lung adenocarcinoma.

[0319] Embodiment 54. The method of embodiment 49, wherein the cancer is prostate cancer.

[0320] Embodiment 55. The method of embodiment 49, wherein the cancer is breast cancer.

[0321] Embodiment 56. The method of any one of embodiments 48-55, wherein the engineered cell is allogeneic to the subject.

[0322] Embodiment 57. The method of any one of embodiments 48-56, wherein the engineered cell is matched to the subject for at least one HLA allele.

[0323] Embodiment 58. The method of any one of embodiments 48-55, wherein the engineered cell is autologous to the subject.

[0324] Embodiment 59. The method of any one of embodiments 48-58, wherein the engineered cell induces activation of an endogenous T cell in the subject.WSGR Docket No. 57428-711.601

[0325] Embodiment 60. The method of any one of embodiments 48-59, wherein the engineered cell induces an anti -cancer immune response in the subject mediated by endogenous T cells of the subject.

[0326] Embodiment 61. The method of any one of embodiments 48-60, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

[0327] Embodiment 62. The method of any one of embodiments 48-61, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

[0328] Embodiment 63. A method of making an engineered cell, comprising introducing into the cell: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope.

[0329] Embodiment 64. The method of embodiment 63, wherein the cell is a gamma delta T cell.IX. EXAMPLESEXAMPLE 1: TMG design

[0330] Peptides were designed containing antigens from common tumor mutations (TABLE 4). Peptides containing point mutations like Glycine 506 to valine in the gene BRAF were flanked by twelve wildtype amino acids on both ends (SEQ ID NO: 4). Peptides containing frameshift mutations like the mutation in Lysine 437 of the ACVR2A gene included twelve wild type amino acids on the N-terminal side of the mutation and a sequence of mutant amino acids after the frameshift up through either the first stop codon (SEQ ID NO: 1) or thirteen total mutated amino acids. Peptides containing in-frame deletion mutations like the deletion of glutamic acid 746 through alanine 750 in the gene EGFR were constructed with twelve amino acids on either side of the deletion (SEQ ID NO: 9).

[0331] Tandem minigenes were constructed by combining sets of mutation-containing peptides (TABLE 5) either directly or with Glycine-Serine (e.g., GGS) linkers separating each mutant peptide.WSGR Docket No. 57428-711.601

[0332] An immunizing polypeptide containing the mutant peptides KRAS G12D (SEQ ID NO: 14), KRAS G12V (SEQ ID NO: 16), TP53 R175H (SEQ ID NO: 32), and PIK3CA H1047R (SEQ ID NO: 23) was designed with peptides separated by Glycine-Serine (e.g., GGS) linkers (SEQ ID NO: 37).

[0333] An immunizing polypeptide containing the mutant peptides KRAS G12D (SEQ ID NO: 14), KRAS G12V (SEQ ID NO: 16), TP53 R175H (SEQ ID NO: 32), and PIK3CA H1047R (SEQ ID NO: 23) was designed without linkers (SEQ ID NO: 38).

[0334] An immunizing polypeptide containing the mutant peptides KRAS G12D (SEQ ID NO: 14), BRAF V640E (SEQ ID NO: 6), RNF43 G659Vfs*41 (SEQ ID NO: 25), KRAS G12V (SEQ ID NO: 16), PIK3CA E545K (SEQ ID NO: 22), KRAS G13D (SEQ ID NO: 18), ACVR2A K437Rfs*5 (SEQ ID NO: 1), TP53 R175H (SEQ ID NO: 32), RPL22 K15Rfs*5 (SEQ ID NO: 26), PIK3CA H1047R (SEQ ID NO: 23), LARP4B T163Hfs*47 (SEQ ID NO: 19), PIK3CA E542K (SEQ ID NO: 21), BCORL1 P1681Qfs*20 (SEQ ID NO: 3), TP53 R273H (SEQ ID NO: 34), KRAS A146T (SEQ ID NO: 11), PIK3CA R88Q (SEQ ID NO: 24), TP53 R282W (SEQ ID NO: 36), TP53 R248Q (SEQ ID NO: 33), APC T1556Nfs*3 (SEQ ID NO: 2), and CSMD3 F3640Lfs*61 (SEQ ID NO: 7) was designed without linkers (SEQ ID NO: 39).

[0335] An immunizing polypeptide containing the mutant peptides KRAS G12C (SEQ ID NO: 13), KRAS G12V (SEQ ID NO: 16), EGFR L858R (SEQ ID NO: 10), KRAS G12D (SEQ ID NO: 14), KRAS G12A (SEQ ID NO: 12), EGFR E746_A750del (SEQ ID NO: 9), BRAF V640E (SEQ ID NO: 6), PIK3CA E545K (SEQ ID NO: 22), EGFR E746_A750del (SEQ ID NO: 9), KRAS G13C (SEQ ID NO: 17), TP53 R273L (SEQ ID NO: 35), TP53 R158L (SEQ ID NO: 31), KRAS G12S (SEQ ID NO: 15), BRAF G506V (SEQ ID NO: 4), TP53 G245V (SEQ ID NO: 29), BRAF G509V (SEQ ID NO: 5), CTNNB1 S37F (SEQ ID NO: 8), MB21D2 Q311E (SEQ ID NO: 20), TP53 R110L (SEQ ID NO: 30), TP53 A159P (SEQ ID NO: 28), and STK11 D53Tfs*l 1 (SEQ ID NO: 27) was designed without linkers (SEQ ID NO: 40).

[0336] An immunizing polypeptide containing the mutant peptides KRAS G12D (SEQ ID NO: 14), BRAF V640E (SEQ ID NO: 6), RNF43 G659Vfs*41 (SEQ ID NO: 25), KRAS G12V (SEQ ID NO: 16), PIK3CA E545K (SEQ ID NO: 22), KRAS G13D (SEQ ID NO: 18), ACVR2A K437Rfs*5 (SEQ ID NO: 1), TP53 R175H (SEQ ID NO: 32), RPL22 K15Rfs*5 (SEQ ID NO: 26), PIK3CA H1047R (SEQ ID NO: 23), LARP4B T163Hfs*47 (SEQ ID NO: 19), PIK3CA E542K (SEQ ID NO: 21), BCORL1 P1681Qfs*20 (SEQ ID NO: 3), TP53 R273H (SEQ ID NO: 34), KRAS A146T (SEQ ID NO: 11), PIK3CA R88Q (SEQ ID NO: 24), TP53 R282W (SEQ ID NO: 36), TP53 R248Q (SEQ ID NO: 33), APC T1556Nfs*3 (SEQ ID NO: 2), and CSMD3 F3640Lfs*61 (SEQ ID NO: 7) was designed with peptides separated by Glycine- Serine (e.g., GGS) linkers (SEQ ID NO: 41).WSGR Docket No. 57428-711.601

[0337] An immunizing polypeptide containing the mutant peptides KRAS G12C (SEQ ID NO: 13), KRAS G12V (SEQ ID NO: 16), EGFR L858R (SEQ ID NO: 10), KRAS G12D (SEQ ID NO: 14), KRAS G12A (SEQ ID NO: 12), EGFR E746_A750del (SEQ ID NO: 9), BRAF V640E (SEQ ID NO: 6), PIK3CA E545K (SEQ ID NO: 22), EGFR E746_A750del (SEQ ID NO: 9), KRAS G13C (SEQ ID NO: 17), TP53 R273L (SEQ ID NO: 35), TP53 R158L (SEQ ID NO: 31), KRAS G12S (SEQ ID NO: 15), BRAF G506V (SEQ ID NO: 4), TP53 G245V (SEQ ID NO: 29), BRAF G509V (SEQ ID NO: 5), CTNNB1 S37F (SEQ ID NO: 8), MB21D2 Q311E (SEQ ID NO: 20), TP53 R110L (SEQ ID NO: 30), TP53 A159P (SEQ ID NO: 28), and STK11 D53Tfs*l 1 (SEQ ID NO: 27) was designed with peptides separated by Glycine-Serine (e.g., GGS) (SEQ ID NO: 42).

[0338] TABLE 4 : Illustrative peptides containing antigens / T cell epitopes from common tumor mutations. Mutations relative to wild type are indicated by underline (e.g., substitutions, insertions, frameshift mutations) or bold (deletions).WSGR Docket No. 57428-711.601

[0339] TABLE 5 : Illustrative immunizing polypeptides.WSGR Docket No. 57428-711.601

[0340] Tandem minigenes with targeting sequence

[0341] Some versions of tandem minigene immunizing polypeptides were constructed to include a targeting sequence appended to either the N-terminal or C-terminal end. Illustrative targeting sequences used are listed in TABLE 6.

[0342] One design included the TPA expression peptide (SEQ ID NO: 51) followed by Prototype 4 (SEQ ID NO: 38) producing the polypeptide SEQ ID NO: 52. Another designWSGR Docket No. 57428-711.601 includes Prototype 4 (SEQ ID NO: 38) followed by the LAMP 1 peptide (SEQ ID NO: 43) producing the sequence SEQ ID NO: 53. Another design includes the TPA peptide (SEQ ID NO: 51), Prototype 4 (SEQ ID NO: 38) and LAMP 1 (SEQ ID NO: 43) giving the polypeptide sequence SEQ ID NO: 54.

[0343] TABLE 6 : Illustrative targeting sequences.

[0344] TABLE 7 : Illustrative immunizing polypeptides with targeting sequences.WSGR Docket No. 57428-711.601EXAMPLE 2: CAR designs

[0345] One CAR construct contains an anti-mesothelin scFv domain with a signal peptide, hinge sequence, and transmembrane domains from CD8 (SEQ ID NO: 55), with an intracellular CD28 costimulatory domain (SEQ ID NO: 56) and a CD3(^ stimulatory domain (SEQ ID NO:57). Combining these elements creates the complete CAR sequence SEQ ID NO: 59.

[0346] One CAR construct contains an anti-mesothelin scFv domain with a signal peptide, hinge sequence, and transmembrane domains from CD8 (SEQ ID NO: 55) with an intracellular CD28 costimulatory domain (SEQ ID NO: 56) and a FcsRIy stimulatory domain (SEQ ID NO:58). Combining these elements creates the complete CAR sequence SEQ ID NO: 60.

[0347] TABLE 8: Illustrative CAR components.

[0348] TABLE 9 : Illustrative CAR sequences.WSGR Docket No. 57428-711.601EXAMPLE 3: Expression constructs

[0349] Complete expression constructs to be integrated into immune cells include a promoter like MND (SEQ ID NO: 64), a gene coding for a multicistronic polypeptide constructed by joining a CAR polypeptide and a TMG immunizing polypeptide using a Furin cleavage site (SEQ ID NO: 63) and / or a 2A element (SEQ ID NO: 61 or SEQ ID NO: 62) to facilitate the cleavage of the two polypeptides, and a poly adenylation site like bGH (SEQ ID NO: 67). The whole construct is flanked by TcBuster inverted tandem repeat (ITR) sequences to facilitate integration into the target immune cell genome by TcBuster transposase. Some iterations also include additional genes such as tEGFR (SEQ ID NO: 66) as a safety switch.

[0350] One example of a complete expression construct (SEQ ID NO: 70, FIG. 1, lower diagram) contains 3’ TcB ITR1 (SEQ ID NO: 69); MND promoter (SEQ ID NO: 64); a protein coding sequence coding for the mesothelin CAR (SEQ ID NO: 55), CD28 costimulatory domain (SEQ ID NO: 56), FcsRIy stimulatory domain (SEQ ID NO: 58), Furin cleavage site (SEQ ID NO: 63), T2A cleavage site (SEQ ID NO: 61), TPA-Prototype 4 tandem minigene (SEQ ID NO: 52); bGH polyadenylation site (SEQ ID NO: 67), and 5’ TcB ITR1 (SEQ ID NO: 68).

[0351] Schematics of additional illustrative expression constructs are shown in FIG. 3, FIG. 5 and FIG. 6.

[0352] TABLE 10: illustrative cleavable linkers.

[0353] TABLE 11: Illustrative nucleic acid sequences.WSGR Docket No. 57428-711.601WSGR Docket No. 57428-711.601

[0354] TABLE 12: Illustrative expression construct.WSGR Docket No. 57428-711.601WSGR Docket No. 57428-711.601EXAMPLE 4: Introduction of construct into cells

[0355] Anonymized healthy donor blood is obtained from commercial vendors. This material is provided as direct venous blood, apheresis material, or mononuclear cell waste material from plateletpheresis. Blood is processed either using Ficoll-Paque or ACK lysing buffer to reduce erythrocyte content. The yb T cell subset is purified from PBMC using positive and / or negative magnetic selection techniques then cryopreserved.

[0356] For example, isolation of yb T cells is performed using EasySep Human Gamma / delta T cell isolation kit from StemCell Technologies following the manufacturer’s protocol. Isolated T cells are then cryopreserved and stored frozen.

[0357] T cells are thawed, then activated with anti-TCRy / b antibody in the presence of anti- CD28 for 36-48 h. Tc Buster (TcB) and hyperactive TcBuster (TcB-M) mRNA are obtained from Bio-Techne (Minneapolis, MN). Transfection of TcB transposase mRNA and BAFF-CAR transposon plasmid are achieved using electroporation performed with Neon or Xenon electroporator. Electroporation is performed with the Neon NXT Electroporation system (ThermoFisher Scientific, Waltham, MA, USA).

[0358] T cells are centrifuged to remove the culture medium and resuspended in R buffer with RNase inhibitor. For stable integration, 5 pg of transposon DNA is combined with 5 pg of transposase RNA (BioTechne) per 10e6 cells. After electroporation, T cells are then cultured in CTS OpTmizer T-cell expansion SFM (ThermoFisher) supplemented with 10% human AB serum, 2 mM 1-glutamine, 2 ZU / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-15. Cells are maintained in gas-permeable rapid expansion (G-Rex) culture vessels (Wilson Wolf). All T cells are cultured at 37 °C and 5% CO2.

[0359] The engineering process can be refined for each CAR construct. This can involve testing different nucleofection apparatus (e.g., Maxcyte, Neon, Amaxa) and electroporation parameters (e.g., voltage, pulse width, number of pulses). These will be used to improve transfection efficiency and post-electroporation viability if necessary, in tandem with a titration of the amount of transposon donor plasmid and transposase. Cells will be expanded as described and assessed every 3-4 days for cell numbers, viability, and CAR expression. CARs will be evaluated on their integration efficiency, overall numbers at harvest, and activation state.EXAMPLE 5: Flow cytometry

[0360] Engineered cells are characterized on a CytoFLEX S flow cytometer (Beckman Coulter). Cells are stained with the following antibodies: Brilliant Violet 605™ anti-human TCR Vb2 Antibody clone B6 (BioLegend), Brilliant Violet 510™ anti-human TCR a / p Antibody clone IP26 (BioLegend), Alexa Fluor® 700 anti -human CD3 Antibody clone UCHT1WSGR Docket No. 57428-711.601(BioLegend), Brilliant Violet 421™ anti-human CD25 Antibody Clone BC96 (BioLegend), Brilliant Violet 650™ anti-human CD69 Antibody clone FN50 (BioLegend), FITC TCR V delta 1 Monoclonal Antibody clone TS8.2 (ThermoFisher), R-Phycoerythrin AffiniPure™ F(ab')2 Fragment Goat Anti-Mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch Laboratories Inc). Antibodies against CD3, TCR aP, TCR V81, and TCR V62 are used to characterize immune cell subtypes. Engineered cells are identified through anti F(ab')2 labeling of the extracellular anti mesothelin scFv fragment of the CAR. Antibodies against CD25 and CD69 are used for detection of T cell activation.EXAMPLE 6: Construct integration and cell expansion

[0361] An illustrative expression construct (POC6 in FIG. 5) was transfected into 2 million gamma delta T cells with TcB transposase mRNA via electroporation. After culturing, integration of the expression construct was measured via flow cytometry, with anti F(ab')2 labeling of the extracellular anti mesothelin scFv fragment of the CAR. 22% of cells were positive for CAR expression.

[0362] The engineered cells were expanded for approximately 13 days with two cycles of stimulation / restimulation. Cell numbers were determined before and after the expansion cycles as shown in TABLE 13.EXAMPLE 7: Antigen presentation assay

[0363] Antigens included in the TMG immunizing polypeptide that have known HLA binding epitopes and known TCR binders are characterized by measuring HLA display and TCR activation. One example is the mutation TP53 R175H, displayed on HLA-A*2 complexes, which can be recognized by TCRa TRAV12-1 / TRAJ13 and TCRp TRBV6-1 / TRBJ2-7 clone. Interactions between displayed peptide TAAs and TCRs are measured using the CD8+ T-Cell Activation Bioassay (TCRaP-KO), Propagation Model (Promega).

[0364] Engineered antigen expressing cells including the TP53 R175H minigene (SEQ ID NO: 32) are cocultured with modified immortalized T cells expressing this specific transgenic TCR and a luciferase reporter gene. Promega’s assay platform utilizes an engineered T-cell lineWSGR Docket No. 57428-711.601(Jurkat) in which the endogenous TCR gene has been knocked out, allowing greater assay sensitivity. Upon ligation, these cells express luciferase, which will be developed, read, and quantified using a Biotek Synergy LX multimode plate reader.

[0365] TCR binding to HLA-restricted antigens is evaluated with each transgenic TCR- expressing cell line by tetramer staining using antigen-matched HLA tetramers generated using the easYmer® MHC Tetramers Kit (Eagle Biosciences) and a synthetic peptide corresponding to the minigene antigen. Transgenic TCR-expressing cells are co-cultured with unmatched engineered APCs to measure and control for non-specific T-cell activation. Coculture is performed using effector Target (E:T) ratios ranging from 10: 1 to 1 : 10 and with biological replicates using y6 T cells derived from at least three independent donors.EXAMPLE 8: Neoantigen presentation via immunopeptidomics

[0366] Gamma delta T cells were engineered to express an immunizing polypeptide comprising colorectal cancer antigens (expression construct POC9 in FIG. 5). MHC-I presented neoantigen was detected via liquid chromatography tandem mass spectrometry using the sample preparation method described in Purcell et al. (2019) Mass spectrometry-based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 14, 1687-1707.

[0367] The cells were expanded, lysed, and a pulldown of peptide-MHC complexes conducted with an anti-MHC antibody. Peptide cargo was extracted and separated into fractions by HPLC, and the peptides in these fractions identified using nUPLC-MS / MS.

[0368] The KRAS G12V neoepitope VVVGAVGVGK (SEQ ID NO: 120) was detected, demonstrating MHC presentation of a T cell epitopes from the immunizing polypeptide by engineered gamma delta T cells (TABLE 14, FIG. 7, and FIG. 8).

[0369] TABLE 14: detection of KRAS G12V via immunopeptidomicsEXAMPLE 9: Activation of T cells specific for immunizing polypeptide antigen

[0370] T cell activation assays were conducted to measure activation of T cells with TCR specificity for an antigen included in an immunizing polypeptide. Engineered gamma delta T cells were generated that express an immunizing polypeptide that includes a TP53 R175H mutation. A peptide containing the mutation is displayed on HLA-A*2 complexes and can beWSGR Docket No. 57428-711.601 recognized by reporter T cells expressing TP53 R175H-specific transgenic TCRs, with a luciferase reporter gene downstream to indicate TCR signaling (FIG. 10). Details of the antigen and TCRs are provided in TABLES 15 and 16.

[0371] TABLE 15: TP53 R175H antigen details.

[0372] TABLE 16: details of TCRs specific for TP53 R175H

[0373] The engineered gamma delta T cells were cocultured with the reporter cell line, and luciferase expression measured to determine relative activation of TCR signaling. Signaling was observed for both reporter lines with TP53 R175H-specific TCRs in response to co-culture with gamma delta T cells expressing the TP53 R175H peptide, whereas no or low signaling was observed in control conditions (FIG. 11).

[0374] The reporter cell line was also analyzed via flow cytometry following co-culture. Increased CD69 expression was observed for reporter T cells co-cultured with engineered gamma delta T cells expressing the immunizing polypeptide with TP53 R175H (“y5 POC9” in FIG. 12)

[0375] These results demonstrate that tumor-specific antigens presented by engineered gamma delta T cells are recognized by TCRs and induce T cell activation.EXAMPLE 10: In vivo study

[0376] Studies are performed in humanized mice to demonstrate the cooperative effects of the APC and CAR functions of engineered cells using the CAR design. Study groups can include APC-only single-action (e.g., both TMG and li-CLIP replacement versions), CAR-only single-action, DACART using immunizing polypeptides disclosed herein (e.g., with CD4+ and / or CD8+ T cell epitopes), and pulsed control. Variations of APC single-action and DACART cells can be included to test at least 2-3 different li-CLIP replacement peptides (e.g.,WSGR Docket No. 57428-711.601 based upon which peptide antigens are best displayed as measured by tandem mass spectrometry experiments).

[0377] Tumor-killing by engineered cells can be evaluated in humanized hu-PBMC-NSG™ mice (The Jackson Laboratory). A pilot study determines the appropriate tumor size and treatment timeline. Groups of four mice are orthotopically injected with 0, 1 * 10A6, 5* 10A6, or 10>< 10A6 luciferase-expressing SW480 cells (or suitable cell lines, e.g., from TABLES 17 & 18).

[0378] TABLE 17: Illustrative colorectal cancer cell lines that can be used in in vitro and in vivo studies.

[0379] TABLE 18: Illustrative NSCLC cell lines that can be used in in vitro and in vivo studies.

[0380] A second pilot is conducted to evaluate CAR homing to tumor sites. Tumors are orthotopically established in mice. One group can receive, e.g., engineered cells expressing immunizing polypeptides comprising CD8+ T cell epitopes after 14 days, and another group can receive, e.g., engineered cells expressing immunizing polypeptides comprising CD4+ T cell epitopes after 14 days. Cells are intravenously injected. After another 7 days, tumors are dissociated, and colocalization of tumor and CAR T cells will be evaluated by flow cytometry.

[0381] Tumor sizes and timelines can be based on pilot findings. Bioluminescence imaging (BLI) is used to show established tumors 15 days after orthotopic inoculation. On Day 0, mice are injected intravenously with 2* 10A6 engineered cells. Tumor volumes are assessed by BLI on Day 0 and every 7 days thereafter. Peripheral blood is withdrawn on Day 1 and every 7 days thereafter to characterize engineered cells, endogenous T cells, and blood cytokine levels. T cells are assessed for viability, MSLN CAR, V51, V62, tEGFR, MHC-I, MHC-II, CD3, TIM-3,WSGR Docket No. 57428-711.601LAG-3, PD-1, T-bet, CD27, CD45RA, CD62L, IFN-y, CD69, and CD25. Weights and clinical observations (posture, activity, diet) are collected daily or weekly. On Day 29 or upon tumor clearance, remaining mice are rechallenged with 2* 10A6 tumor cells. Mice are sacrificed according to humane treatment guidelines or after 90 days. After sacrifice, tumors are harvested, weighed, and dissociated. Assays are conducted to asses apoptosis (e.g., cleaved caspase-3), engineered T-cell accumulation (e.g., MSLN CAR+, tEGFR+), and endogenous CD4+ and CD8+ T-cell accumulation by flow cytometry.

[0382] The pilot and main mouse studies are repeated using a lung cancer model (e.g., NCI- 141573 cells) and engineered cells containing an NSCLC-specific immunizing polypeptides.X. ADDITIONAL SEQUENCES

[0383] TABLE 19 provides illustrative sequences that can be used in compositions and methods provided herein.WSGR Docket No. 57428-711.601WSGR Docket No. 57428-711.601WSGR Docket No. 57428-711.601

[0384] A polypeptide or domain thereof disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of the sequences disclosed in this application.

[0385] A polypeptide or domain thereof disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 50 consecutive residues of any one of the sequences disclosed in this application.

[0386] A polypeptide or domain thereof disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least aboutWSGR Docket No. 57428-711.60197.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 100 consecutive residues of any one of the sequences disclosed in this application.

[0387] A polypeptide or domain thereof disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 250 consecutive residues of any one of the sequences disclosed in this application.

[0388] A polypeptide or domain thereof disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at most about 70%, at most about 71%, at most about 72%, at most about 73%, at most about 74%, at most about 75%, at most about 76%, at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about 88%, at most about 89%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 95.5%, at most about 96%, at most about 96.5%, at most about 97%, at most about 97.5%, at most about 98%, at most about 98.5%, at most about 99%, or at most about 99.5% sequence identity or sequence similarity to any one of the sequences disclosed in this application.

[0389] In some embodiments, a polypeptide or domain thereof comprises, consists essentially of, or consists of an amino acid sequence with about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, 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%, about95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, or about 99.5% or about 100% sequence identity or sequence similarity to any one of the sequences disclosed in this application.WSGR Docket No. 57428-711.601

[0390] In some embodiments, the polypeptide or domain thereof comprises, consists essentially of, or consists of the amino acid sequence of any one of the sequences disclosed in this application.

[0391] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with one or more insertions, deletions, and / or substitutions relative to any one of the sequences disclosed in this application.

[0392] For example, the polypeptide or domain thereof can comprise an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of the sequences disclosed in this application.

[0393] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid insertions relative to any one of the sequences disclosed in this application.

[0394] In some embodiments, the polypeptide or domain thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertions relative to any one of the sequences disclosed in this application.

[0395] The one or more insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be contiguous, non-contiguous, or a combination thereof.

[0396] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid deletions relative to any one of the sequences disclosed in this application.

[0397] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid deletions relative to any one of the sequences disclosed in this application.WSGR Docket No. 57428-711.601

[0398] In some embodiments, the polypeptide or domain thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to any one of the sequences disclosed in this application.

[0399] The one or more deletions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0400] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of the sequences disclosed in this application.

[0401] In some embodiments, the polypeptide or domain thereof comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions relative to any one of the sequences disclosed in this application.

[0402] In some embodiments, the polypeptide or domain thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of the sequences disclosed in this application.

[0403] The one or more substitutions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions can be contiguous, non-contiguous, or a combination thereof

[0404] A nucleic acid molecule disclosed herein can comprise, consist essentially of, or consist of a nucleotide sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity to any one of the nucleic acid sequences disclosed herein.

[0405] A nucleic acid molecule disclosed herein can comprise, consist essentially of, or consist of a nucleotide sequence with at most about 70%, at most about 71%, at most aboutWSGR Docket No. 57428-711.60172%, at most about 73%, at most about 74%, at most about 75%, at most about 76%, at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about 88%, at most about 89%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 95.5%, at most about 96%, at most about 96.5%, at most about 97%, at most about 97.5%, at most about 98%, at most about 98.5%, at most about 99%, or at most about 99.5% sequence identity to any one of the nucleic acid sequences disclosed herein.

[0406] In some embodiments, a nucleic acid molecule comprises, consists essentially of, or consists of a nucleotide sequence with about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, 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 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, or about 99.5% or about 100% sequence identity to any one of the nucleic acid sequences disclosed herein.

[0407] In some embodiments, the nucleic acid molecule comprises, consists essentially of, or consists of the nucleotide sequence of any one of the nucleic acid sequences disclosed herein.

[0408] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence with one or more insertions, deletions, and / or substitutions relative to any one of the nucleic acid sequences disclosed herein.

[0409] For example, the nucleic acid molecule can comprise a nucleotide sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 nucleotide insertions, deletions, and / or substitutions relative to any one of the nucleic acid sequences disclosed herein.

[0410] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 nucleotide insertions, deletions, and / or substitutions relative to any one of the nucleic acid sequences disclosed herein.

[0411] In some embodiments, the nucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotide insertions, deletions, and / or substitutions relative to any one of the nucleic acid sequences disclosed herein.WSGR Docket No. 57428-711.601

[0412] The one or more insertions, deletions, and / or substitutions can be at the 5' end, the 3' end, within the nucleotide sequence, or a combination thereof, the one or more insertions can be contiguous, non-contiguous, or a combination thereof.

[0413] The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs designed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm. A global alignment algorithm, such as a Needleman and Wunsch algorithm, can be used to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Default settings can be used.

[0414] To generate similarity scores for two amino acid sequences, scoring matrices can be used that assign positive scores for some non-identical amino acids (e.g., amino acids with similar physio-chemical properties and / or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs), Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0415] Amino acids can include genetically encoded and non-genetically encoded occurring amino acids. Amino acids can include naturally occurring and non-naturally occurring amino acids. Amino acids can be L forms or D forms. Substitutions disclosed herein can include conservative and / or non-conservative amino acid substitutions. A conservative amino acid substitution can be a substitution of one amino acid for another amino acid of similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be a substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gin (Q), Asp (D), Lys (K) and Arg (R). A conservative amino acid change can be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids can include He (I), Phe (F), Vai (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change can be an amino acid change from one acidic amino acid to another acidic amino acid. Acidic amino acids can include Glu (E) and Asp (D). A conservative amino acid change can be an amino acid change from one basic amino acid to another basic amino acid. BasicWSGR Docket No. 57428-711.601 amino acids can include His (H), Arg (R) and Lys (K). A conservative amino acid change can be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids can include Asn (N), Gin (Q), Ser (S) and Thr (T). A conservative amino acid change can be an amino acid change from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino acids can include Leu (L), Val(V), He (I), Met (M), Gly (G) and Ala (A). A conservative amino acid change can be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids can include Phe (F), Tyr (Y) and Trp (W). A conservative amino acid change can be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids can include Ala (A), Vai (V), Leu (L) and He (I). In some embodiments, a conservative amino acid substitution is an amino acid change from one amino acid to another amino acid within one of the following groups: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group IL Cys, Ser, Tyr, Thr; Group III: Vai, He, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0416] A protein or polypeptide disclosed herein can comprise an N-terminal methionine. A protein or polypeptide disclosed herein can lack an N-terminal methionine.

[0417] Polypeptides disclosed herein can comprise chemical modifications, such as glycosylation, fucosylation, sialylation, and / or pegylation.

[0418] A polynucleotide encoding a polypeptide of the disclosure can encode a signal peptide. In some cases, a polypeptide of the disclosure comprises a signal peptide. A signal peptide can be cleaved off during processing of the protein, thus in some cases a mature polypeptide disclosed herein does not contain a signal peptide.

[0419] In some instances, a value is “about” a recited value if the value is within ±10% of the recited value. In some specific embodiments, a value is “about” a recited value if the value is within ±5% of the recited value. It is also understood that any disclosure provided herein to “about” a number, also includes disclosure of that number itself (i.e., without “about”).

[0420] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 57428-711.601CLAIMSWHAT IS CLAIMED IS:

1. An engineered cell comprising a heterologous nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope, wherein the engineered cell is a gamma delta T cell.

2. The engineered cell of claim 1, further comprising a second heterologous nucleic acid that encodes a chimeric antigen receptor (CAR).

3. The engineered cell of claim 1, wherein upon administration of the engineered cell to a subject, the engineered cell induces activation of an endogenous T cell in the subject.

4. The engineered cell of claim 1, wherein upon administration of the engineered cell to a subject, the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject.

5. The engineered cell of claim 1, wherein the engineered cell is capable of inducing activation of an endogenous T cell of a subject upon administration of the engineered cell to the subject.

6. The engineered cell of claim 1, wherein the engineered cell is capable of inducing an anti-cancer immune response mediated by endogenous T cells in the subject upon administration of the engineered cell to the subject.

7. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a CD4+ T cell epitope.

8. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a CD8+ T cell epitope.

9. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a CD4+ T cell epitope and a CD8+ T cell epitope.

10. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a tumor- associated antigen (TAA).WSGR Docket No. 57428-711.60111. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a solid tumor antigen.

12. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a neoantigen.

13. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a neoepitope.

14. The engineered cell of claim 1, wherein the immunizing polypeptide comprises at least four T cell epitopes.

15. The engineered cell of claim 1, wherein the immunizing polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

16. The engineered cell of claim 1, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 1-36, 115, and 120.

17. The engineered cell of claim 1, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-36, 115, and 120.

18. The engineered cell of claim 1, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 37-42 and 52-54.

19. The engineered cell of claim 1, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 137-42 and 52-54.

20. The engineered cell of claim 1, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class I.

21. The engineered cell of claim 1, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class II.

22. The engineered cell of claim 1, wherein the immunizing polypeptide comprises a targeting sequence to promote HLA presentation of the T cell epitope.

23. The engineered cell of claim 22, wherein the targeting sequence comprises a human tissue plasminogen activator signal peptide.WSGR Docket No. 57428-711.60124. The engineered cell of claim 22, wherein the targeting sequence comprises a li-CLIP- replacement peptide.

25. The engineered cell of claim 22, wherein the targeting sequence comprises a lysosome targeting sequence.

26. The engineered cell of claim 22, wherein the targeting sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 43-51.

27. The engineered cell of claim 22, wherein the targeting sequence comprises the amino acid sequence of any one of SEQ ID NOs: 43-51.

28. The engineered cell of claim 1, wherein the nucleic acid molecule that encodes the immunizing polypeptide is DNA.

29. The engineered cell of claim 1, wherein the nucleic acid molecule that encodes the immunizing polypeptide is a tandem minigene.

30. The engineered cell of claim 1, wherein the nucleic acid molecule that encodes the immunizing polypeptide is genomically integrated.

31. The engineered cell of claim 1, wherein the nucleic acid molecule that encodes the immunizing polypeptide is not genomically integrated.

32. The engineered cell of claim 1, wherein the nucleic acid molecule that encodes the immunizing polypeptide is RNA.

33. The engineered cell of claim 2, wherein the CAR comprises an extracellular binding domain that binds to a target associated with a solid tumor.

34. The engineered cell of claim 2, wherein the CAR comprises an extracellular binding domain that binds to a target associated with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer.

35. The engineered cell of claim 2, wherein the CAR comprises an extracellular binding domain that binds to mesothelin.WSGR Docket No. 57428-711.60136. The engineered cell of claim 2, wherein the CAR comprises an extracellular binding domain that binds to carcinoembryonic antigen (CEA), CD70, CSPG4, B7-H3, CD133, EGFR, or MUCl.

37. The engineered cell of claim 2, wherein an extracellular binding domain of the CAR does not bind to the immunizing polypeptide or the T cell epitope.

38. The engineered cell of claim 2, wherein the CAR is a split CAR or dual CAR.

39. The engineered cell of claim 2, wherein the CAR comprises an FcsRIy cytoplasmic signaling domain.

40. The engineered cell of claim 2, wherein the CAR is a second, third, fourth, or fifth generation CAR.

41. The engineered cell of claim 2, wherein the CAR comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 59-60.

42. The engineered cell of claim 2, wherein CAR comprises the amino acid sequence of any one of SEQ ID NOs: 59-60.

43. The engineered cell of claim 1, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

44. The engineered cell of claim 1, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

45. The engineered cell of claim 1, wherein the engineered cell further comprises a safety switch.

46. The engineered cell of claim 1, wherein the safety switch comprises a truncated EGFR.WSGR Docket No. 57428-711.60147. A population of cells comprising a plurality of the engineered cell of claim 1, wherein the population of cells comprises: (i) at least 10% Vdl+ cells and at least 10% Vd2+ cells, (ii) at least 1% Vdl- Vd2- gamma delta T cells, or (iii) at least 11% Vd3+ cells.

48. A method of treating a subject in need thereof, the method comprising administering to the subject the engineered cell of claim 1.

49. The method of claim 48, wherein the method treats cancer in the subject.

50. The method of claim 49, wherein the cancer is a solid tumor.

51. The method of claim 49, wherein the cancer is colorectal cancer (CRC).

52. The method of claim 49, wherein the cancer is non-small cell lung cancer (NSCLC).

53. The method of claim 49, wherein the cancer is lung adenocarcinoma.

54. The method of claim 49, wherein the cancer is prostate cancer.

55. The method of claim 49, wherein the cancer is breast cancer.

56. The method of claim 48, wherein the engineered cell is allogeneic to the subject.

57. The method of claim 56, wherein the engineered cell is matched to the subject for at least one HLA allele.

58. The method of claim 48, wherein the engineered cell is autologous to the subject.

59. The method of claim 49, wherein the engineered cell induces activation of an endogenous T cell in the subject.

60. The method of claim 49, wherein the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject.

61. The method of claim 49, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.WSGR Docket No. 57428-711.60162. The method of claim 49, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

63. An engineered cell comprising: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope.

64. The engineered cell of claim 63, wherein upon administration of the engineered cell to a subject, the engineered cell induces activation of an endogenous T cell in the subject.

65. The engineered cell of claim 63, wherein upon administration of the engineered cell to a subject, the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject.

66. The engineered cell of claim 63, wherein the engineered cell is capable of inducing activation of an endogenous T cell of a subject upon administration of the engineered cell to the subject.

67. The engineered cell of claim 63, wherein the engineered cell is capable of inducing an anti-cancer immune response mediated by endogenous T cells in the subject upon administration of the engineered cell to the subject.

68. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a CD4+ T cell epitope.

69. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a CD8+ T cell epitope.

70. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a CD4+ T cell epitope and a CD8+ T cell epitope.

71. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a tumor-associated antigen (TAA).WSGR Docket No. 57428-711.60172. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a solid tumor antigen.

73. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a neoantigen.

74. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a neoepitope.

75. The engineered cell of claim 63, wherein the immunizing polypeptide comprises at least four T cell epitopes.

76. The engineered cell of claim 63, wherein the immunizing polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

77. The engineered cell of claim 63, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 1-36, 115, and 120.

78. The engineered cell of claim 63, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-36, 115, and 120.

79. The engineered cell of claim 63, wherein the immunizing polypeptide comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 37-42 and 52-54.

80. The engineered cell of claim 63, wherein the immunizing polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 137-42 and 52-54.

81. The engineered cell of claim 63, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class I.

82. The engineered cell of claim 63, wherein the engineered cell expresses the immunizing polypeptide and presents a peptide comprising the T cell epitope on HLA class II.

83. The engineered cell of claim 63, wherein the immunizing polypeptide comprises a targeting sequence to promote HLA presentation of the T cell epitope.WSGR Docket No. 57428-711.60184. The engineered cell of claim 83, wherein the targeting sequence comprises a human tissue plasminogen activator signal peptide.

85. The engineered cell of claim 83, wherein the targeting sequence comprises a li-CLIP- replacement peptide.

86. The engineered cell of claim 83, wherein the targeting sequence comprises a lysosome targeting sequence.

87. The engineered cell of claim 83, wherein the targeting sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 43-51.

88. The engineered cell of claim 83, wherein the targeting sequence comprises the amino acid sequence of any one of SEQ ID NOs: 43-51.

89. The engineered cell of claim 63, wherein the nucleic acid molecule that encodes the immunizing polypeptide is DNA.

90. The engineered cell of claim 63, wherein the nucleic acid molecule that encodes the immunizing polypeptide is a tandem minigene.

91. The engineered cell of claim 63, wherein the nucleic acid molecule that encodes the immunizing polypeptide is genomically integrated.

92. The engineered cell of claim 63, wherein the nucleic acid molecule that encodes the immunizing polypeptide is not genomically integrated.

93. The engineered cell of claim 63, wherein the nucleic acid molecule that encodes the immunizing polypeptide is RNA.

94. The engineered cell of claim 63, wherein the CAR comprises an extracellular binding domain that binds to a target associated with a solid tumor.

95. The engineered cell of claim 63, wherein the CAR comprises an extracellular binding domain that binds to a target associated with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, prostate cancer, or breast cancer.WSGR Docket No. 57428-711.60196. The engineered cell of claim 63, wherein the CAR comprises an extracellular binding domain that binds to mesothelin.

97. The engineered cell of claim 63, wherein the CAR comprises an extracellular binding domain that binds to carcinoembryonic antigen (CEA), CD70, CSPG4, B7-H3, CD133, EGFR, or MUCl.

98. The engineered cell of claim 63, wherein an extracellular binding domain of the CAR does not bind to the immunizing polypeptide or the T cell epitope.

99. The engineered cell of claim 63, wherein the CAR is a split CAR or dual CAR.

100. The engineered cell of claim 63, wherein the CAR comprises an FcsRIy cytoplasmic signaling domain.

101. The engineered cell of claim 63, wherein the CAR is a second, third, fourth, or fifth generation CAR.

102. The engineered cell of claim 63, wherein the CAR comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 59-60.

103. The engineered cell of claim 63, wherein CAR comprises the amino acid sequence of any one of SEQ ID NOs: 59-60.

104. The engineered cell of claim 63, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

105. The engineered cell of claim 63, wherein upon administration of the engineered cell to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

106. The engineered cell of claim 63, wherein the engineered cell further comprises a safety switch.WSGR Docket No. 57428-711.601107. The engineered cell of claim 63, wherein the safety switch comprises a truncated EGFR.

108. A population of cells comprising a plurality of the engineered cell of claim 63, wherein the population of cells comprises: (i) at least 10% Vdl+ cells and at least 10% Vd2+ cells, (ii) at least 1% Vdl- Vd2- gamma delta T cells, or (iii) at least 11% Vd3+ cells.

109. A method of treating a subject in need thereof, the method comprising administering to the subject the engineered cell of claim 63.

110. The method of claim 109, wherein the method treats cancer in the subject.

111. The method of claim 110, wherein the cancer is a solid tumor.

112. The method of claim 110, wherein the cancer is colorectal cancer (CRC).

113. The method of claim 110, wherein the cancer is non-small cell lung cancer (NSCLC).

114. The method of claim 110, wherein the cancer is lung adenocarcinoma.

115. The method of claim 110, wherein the cancer is prostate cancer.

116. The method of claim 110, wherein the cancer is breast cancer.

117. The method of claim 109, wherein the engineered cell is allogeneic to the subject.

118. The method of claim 117, wherein the engineered cell is matched to the subject for at least one HLA allele.

119. The method of claim 109, wherein the engineered cell is autologous to the subject.

120. The method of claim 109, wherein the engineered cell induces activation of an endogenous T cell in the subject.

121. The method of claim 110, wherein the engineered cell induces an anti-cancer immune response in the subject mediated by endogenous T cells of the subject.

122. The method of claim 110, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell reduces average tumor volume at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the controlWSGR Docket No. 57428-711.601 engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

123. The method of claim 110, wherein if the engineered cell is administered to a cohort of subjects, the engineered cell improves average survival time at least 5% more than a control engineered cell administered to a control cohort of subjects, wherein the control engineered cell lacks the nucleic acid molecule that encodes the immunizing polypeptide.

124. A method of making an engineered cell, comprising introducing into the cell: (a) a first nucleic acid molecule that encodes a chimeric antigen receptor (CAR), and (b) a second nucleic acid molecule that encodes an immunizing polypeptide, wherein the immunizing polypeptide comprises a T cell epitope.

125. The method of claim 124, wherein the cell is a gamma delta T cell.

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