Tumor-activated alloreactive and heteroreactive t cells and their use in cancer immunotherapy

Abstract

Tumor-activated heteroreactive or heteroreactive T cells that are active only at the tumor site and methods of producing the same are provided. Also provided are methods of treating tumors and cancers using these tumor-activated alloreactive or heteroreactive T cells. The allogeneic or heterologous reaction of these T cells at the tumor site results in killing of tumor cells and stromal cells that express mismatched HLA molecules. The lack of activity of these T cells at non-tumor locations prevents attack to normal tissue. Further related methods and products are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 17 / 574,483 filed on January 12, 2022, the disclosure of which is incorporated by reference in its entirety into this application for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted in ASCII format via EFS-Web and referenced in corresponding U.S. Patent Application No. 17 / 574,483, which is hereby incorporated by reference in its entirety into this application. Technical Field

[0005] The present disclosure relates to alloreactive and xenoreactive T cells and methods of limiting their alloreactivity or xenoreactivity to tumor or cancer sites in order to kill tumor cells and tumor stromal cells in cancer immunotherapy without causing significant damage to normal tissues. Background Art

[0006] In recent years, T cell-based cancer immunotherapy has made significant progress. Chimeric antigen receptor (CAR)-modified T cells have shown remarkable efficacy in the treatment of B cell cancers (1-5). The FDA approved the first CAR T cell drug for B cell lymphoma in 2017, which was an important milestone in the fight against cancer. This approach isolates autologous T cells from the patient and genetically modifies them to express CAR that recognizes the pan-B cell marker CD19. CAR T cells infused into the patient are able to recognize cancer cells through CAR-CD19 interactions. Signals from the CAR lead to T cell activation and cytotoxicity against cancer cells. However, there are still major challenges in developing safe and effective T cell immunotherapies for a wider range of cancers, especially for solid tumors and non-B cell blood cancers.

[0007] A major challenge for T cell therapy of solid tumors is the heterogeneity of tumor antigen expression. To date, the success of CAR T cell therapy has been limited primarily to the treatment of B cell cancers. Complete elimination of cancer cells is achievable because CD19 is expressed homogeneously on all cancerous B cells. In fact, normal B cells are also eliminated, but the loss of B cell function can be easily compensated by immunoglobulin infusion. In contrast, the expression of most solid tumor antigens is more heterogeneous both in terms of tissue type and in terms of expression levels on tumor cells. Many tumor antigens can be highly expressed in tumors but may also be expressed at subtumoral levels in certain normal tissues (6). Furthermore, tumor antigen expression within a tumor is rarely uniform, with only a certain percentage of tumor cells expressing a particular type of tumor antigen (7-10). Some progress has been made in minimizing the “on-target, but not tumor” targeting of normal tissues by designing CARs that mediate T cell responses only to cells that highly express tumor antigens (11) or that express multiple tumor antigens (12-14). However, heterogeneity in tumor antigen expression by tumor cells remains a major obstacle. This obstacle was highlighted in a recent clinical study targeting EGFRvIII (epidermal growth factor receptor variant III) in glioblastoma (10). CAR T cells invaded the tumor and killed EGFRvIII-positive tumor cells, but failed to prevent glioblastoma progression, most likely due to the expansion of EGFRvIII-negative tumor cells under the selective pressure of CAR T cells. Similar results were also observed in animal models of glioblastoma targeting IL13Rα2 (15).

[0008] The challenge of target antigen heterogeneity also exists in non-B cell blood cancers. The lack of cancer-specific antigens has hampered the development of CAR T-cell therapy for acute myeloid leukemia (AML) (16). Most of the targets discovered to date, such as CD33 and CD123, are also expressed on normal myeloid cells and hematopoietic stem cells (17, 18). Therefore, targeting these antigens often requires rescue hematopoietic stem cell transplantation. The heterogeneity of target antigens in cancer cells is one of the main reasons for relapse after CAR T-cell therapy. For example, in multiple myeloma biopsies, the expression of B-cell maturation antigen (BCMA) varies in both percentage and expression level (19).

[0009] Therefore, there is an urgent need for effective strategies that can expand the target range to include tumor antigen-negative tumor cells and stromal cells that support tumor growth to overcome antigen-negative immune escape. There is also a need for methods targeting alternative antigens to overcome obstacles in treating non-B cell blood cancers. The present disclosure addresses these needs. Summary of the invention

[0010] The present disclosure relates to alloreactive and xenoreactive T cells and methods of limiting their alloreactive or xenoreactive responses to tumor sites or sites rich in cancer cells so as to kill tumor cells, tumor stromal cells and cancer cells without causing significant damage to normal tissues outside these sites, for the purpose of immunotherapy of cancer.

[0011] The present disclosure provides a tumor-activated alloreactive or heteroreactive T cell. The T cell can be used to kill tumor cells, tumor stroma cells and cancer cells. The advantage is that the T cell should not cause significant damage to normal tissues. Therefore, a method for treating patients with malignant tumors (particularly solid tumors and non-B cell blood cancers) is also provided. In addition, a method for preparing tumor-activated T cells and a related kit are also provided.

[0012] The methods of treatment described herein and compositions for use in these methods may additionally be viewed as using genetically modified tumor-activated alloreactive or xenoreactive T cells to treat cancer in a patient in need of treatment, or for the preparation of a medicament for treating cancer. The disclosed use of genetically modified tumor-activated T cells may be applied to any of the methods and combinations described above and below.

[0013] Provided is a genetically modified T cell comprising: (i) genetic disruption of the expression of at least one endogenous gene encoding a molecule essential for TCR signaling and T cell activation, (ii) an exogenous nucleotide sequence encoding a tumor sensing receptor that releases or activates a transcriptional activator when it binds directly or indirectly to a molecule enriched in tumor cells, tumor microenvironment, or tissue where blood cancer cells accumulate, and (iii) an exogenous nucleotide sequence comprising an expression cassette that expresses a copy of the endogenous gene disrupted in (i) when the transcriptional activator in (ii) is released or activated.

[0014] In some aspects, at least one disrupted endogenous gene encoding a molecule necessary for TCR signaling and T cell activation encodes a transmembrane protein selected from one or more of, for example, CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148, and CD58. In some embodiments, the disrupted endogenous gene is selected from CD3ε, CD3ζ, CD3γ, and CD3δ. In one embodiment, the disrupted endogenous gene is CD3ε. In some aspects, at least one disrupted endogenous gene encoding a molecule necessary for TCR signaling and T cell activation encodes an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT, and PDK1.

[0015] In the context of genetically modified T cells, the tumor sensing receptor comprises: (i) an extracellular domain that directly or indirectly binds to a target molecule that is enriched in tumor cells, tumor microenvironment, and tissues where blood cancer cells accumulate; and (ii) an intracellular domain that activates or releases a transcriptional activator when the extracellular domain binds to the target molecule. The tumor sensing receptor can be a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, or a chimeric antigen receptor (CAR).

[0016] In certain aspects, the target molecule is enriched in tumor cells and / or the tumor microenvironment and is derived from, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothlin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-related Antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and one or more of vimentin. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin, MUC1, EpCAM, GD2 and CEA. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin and MUC1. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII and CD19. In one embodiment, the target molecule is Her2 / neu.

[0017] In some aspects, the target molecule is enriched in tissues where blood cancer cells accumulate, and the tissue is lymphoid tissue and / or bone marrow tissue. In certain embodiments, the target molecule can be selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1.

[0018] In certain aspects, the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed repeat anchor protein (DARPin), a nanobody, a TCR, an Fc receptor, a growth factor receptor, a chemokine receptor, or a hormone receptor.

[0019] In certain aspects, the tumor sensing receptor is a chimeric antigen receptor that activates downstream transcription factors through a signaling pathway activated by binding of the extracellular domain to a target molecule.

[0020] In certain embodiments, the expression cassette encoding the disrupted endogenous gene copy includes a transcriptional control element that drives the expression of the disrupted endogenous gene copy by binding to a transcriptional activator selected from Gal4-VP64, Notch, tet transcriptional activator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs, and eomesodermin (Eomes). In certain embodiments, the expression cassette is selected from Gal4-CD3-PGK-BFP, NR4A-CD3-PKG-BFP, and NFAT-CD3-PKG-BFP. In certain embodiments, the tumor sensing receptor and the expression cassette are selected from SynNotch and Gal4-CD3-PGK-BFP, CAR and NR4A-CD3-PKG-BFP, and CAR and NFAT-CD3-PKG-BFP.

[0021] In some aspects, the T cell sample is from a donor individual, and the donor individual has at least one HLA allele mismatch relative to the intended recipient, and at least one HLA allele mismatch is located at a site selected from HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1, thereby generating tumor-activated alloreactive T cells. In certain embodiments, at least one HLA allele mismatch is located at a site selected from HLA-A, HLA-B, HLA-C, and HLA-DRB1. In certain embodiments, the HLA allele mismatch includes four alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1, six alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1, or all eight alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1. In one embodiment, the HLA allele mismatch includes all eight alleles of HLA-A, HLA-B, HLA-C, and HLA-DRBl.

[0022] In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, and the tumor sensing receptor is SynNotch or CAR. In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is CAR, and the expression cassette is NFAT-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is CD19, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP.

[0023] Also provided is a method for preparing tumor-activated allogeneic reactive or xenogeneic reactive T cells. The method comprises: a) selecting a T cell sample from a donor individual or a donor animal; b) optionally stimulating the proliferation of the T cell sample; c) destroying at least one molecule necessary for TCR signaling and T cell activation in the T cell to make it lose expression or function, thereby making the T cell lose its activation ability; d) transforming the T cell to (i) express a recombinant receptor that specifically binds to a target molecule enriched in tumor cells, tumor microenvironment, and blood cancer cell accumulation tissue, and the receptor releases or activates a transcriptional activator when binding to the target molecule; (ii) introducing an expression cassette so that the transcriptional activator in (i) drives the expression of the molecule destroyed in c), thereby restoring the expression or function of the destroyed molecule, thereby restoring the ability of the T cell to be activated by TCR recognition of antigens, thereby generating tumor-activated allogeneic reactive or xenogeneic reactive T cells. In this method, step d) is performed before step c), step c) is performed before step d), or steps c) and d) are performed simultaneously.

[0024] In certain aspects of the method, the T cell sample is from a donor individual, and the donor individual has at least one HLA allele mismatch relative to the intended recipient, and at least one HLA allele mismatch is located at a site selected from HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1, thereby generating tumor-activated alloreactive T cells. In certain embodiments, at least one HLA allele mismatch is located at a site selected from HLA-A, HLA-B, HLA-C, and HLA-DRB1. In certain embodiments, the HLA allele mismatch includes four alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1, six alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1, or all eight alleles in HLA-A, HLA-B, HLA-C, and HLA-DRB1. In one embodiment, the HLA allele mismatch includes all eight alleles of HLA-A, HLA-B, HLA-C, and HLA-DRBl.

[0025] In certain aspects of the method, step b) comprises: (i) co-culturing the donor T cells with cells of the intended recipient; (ii) co-culturing the donor T cells with cells of a second donor who matches the intended recipient in at least one HLA allele and (ii) is mismatched with the T cell donor; (iii) co-culturing the donor T cells with a cell line that expresses at least one HLA allele of the intended recipient; (iv) co-culturing the donor T cells with an artificial surface coated with a protein encoded by at least one HLA allele of the intended recipient.

[0026] In certain embodiments of the method, the cells used to stimulate T cells in step b) include: (i) tumor cells isolated from a patient; (ii) PMBCs isolated from a patient; and / or (iii) mature monocyte-derived dendritic cells (MoDCs) generated from monocytes isolated from patient PBMCs. In one embodiment, MoDCs are pulsed with tumor cell lysates isolated from a patient, or with lysates of tumor cell lines of the same tumor from different individuals.

[0027] In certain embodiments of preparing T cells, at least one molecule necessary for TCR signaling and T cell activation is a cell surface molecule selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58, or an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, NFAT, SLP76, PKCθ, NFκB, AKT and PDK1. In certain embodiments, the disrupted endogenous gene is selected from CD3ε, CD3ζ, CD3γ and CD3δ. In one embodiment, the disrupted endogenous gene is CD3ε.

[0028] In certain embodiments of the method, the method used in step b) to disrupt molecules necessary for TCR signaling and T cell activation comprises (i) CRISPR-Cas; (ii) transcription activator-like effector nucleases (TALENs); (iii) megaTALS; (iv) zinc finger nucleases; and / or (v) homing endonucleases.

[0029] In certain aspects of the method, step d) comprises introducing a nucleic acid encoding a tumor-sensing receptor into a T cell, wherein the tumor-sensing receptor comprises (i) an extracellular domain capable of directly or indirectly binding to a target molecule enriched in tumor cells, tumor microenvironment, and tissues in which blood cancer cells accumulate; and (ii) an intracellular domain capable of activating or releasing a transcriptional activator after the extracellular domain binds to the target molecule.

[0030] In certain aspects of the method, step d) comprises introducing into a T cell a nucleic acid encoding an expression cassette comprising (i) a transcriptional control element (TCE) that can be bound by a transcriptional activator activated or released by a tumor sensing receptor; and (ii) a DNA sequence encoding a copy of the gene disrupted in step c), such that the disrupted gene can be expressed after the tumor sensing receptor binds to a target molecule that is enriched on or in tumor cells, tumor microenvironments, and tissues where blood cancer cells accumulate. The DNA sequence encoding the copy of the gene disrupted in step c) comprises one or more silent mutations that do not change the protein sequence encoded by the DNA but render it resistant to methods for disrupting endogenous genes.

[0031] In certain embodiments of the method, the DNA sequence encoding the tumor sensing receptor and the expression cassette is introduced into the T cells in step d) by: (i) a retroviral vector; (ii) a lentiviral vector; and / or (iii) a transposon vector, such as Sleeping Beauty (Addgene) and piggyBac (VectorBuilder).

[0032] In certain embodiments of making T cells, the target molecule is enriched on tumor cells and / or in the tumor microenvironment and is selected from, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen HMW-MAA, MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-acetylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin are selected, or the target molecule is enriched in the tissue where blood cancer cells accumulate, wherein the tissue is lymphoid tissue and / or bone marrow tissue, wherein the target molecule is selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin, MUCI, EpCAM, GD2 and CEA.In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin, and MUCI. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, and CD19. In one embodiment, the target molecule is Her2 / neu.

[0033] In certain embodiments, the expression cassette encoding the disrupted endogenous gene copy includes a transcriptional control element to drive transcription of the endogenous gene copy, which element binds a transcriptional activator selected from the group consisting of Gal4-VP64, Notch, tet transactivator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs and eomesodermin (Eomes). In certain embodiments, the expression cassette is selected from Gal4-CD3-PGK-BFP, NR4A-CD3-PKG-BFP and NFAT-CD3-PKG-BFP. In certain embodiments, the tumor sensing receptor and the expression cassette are selected from SynNotch and Gal4-CD3-PGK-BFP, CAR and NR4A-CD3-PKG-BFP, CAR and NFAT-CD3-PKG-BFP.

[0034] In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, and the tumor sensing receptor is SynNotch. In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, and the tumor sensing receptor is CAR. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is CAR, and the expression cassette is NFAT-CD3-PGK-BFP.

[0035] A method of treating cancer in a patient is also provided. The method comprises administering the T cells described above or herein, or T cells prepared by any method described above or herein, to a patient in need thereof. In some aspects, the T cells are tumor-activated alloreactive T cells, which are alloreactive to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various methods and compositions disclosed herein. However, the disclosure is not limited to the specific arrangements and instrumentalities of the embodiments depicted in the drawings.

[0037] Figure 1 Depicted are the four steps for generating tumor-activated alloreactive T cells disclosed herein.

[0038] Figures 2A-2E illustrate the generation and use of tumor-activated alloreactive T cells based on SynNotch tumor sensing receptors and CD3ε expression cassettes. Figure 2A depicts a schematic diagram of an alloreactive T cell, including a schematic diagram of a T cell receptor (TCR) / CD3 complex. TCR is a heterodimer composed of an α chain and a β chain. The TCR / CD3 complex includes two CD3 epsilon (ε) chains, two CD3 gamma (γ) chains, two CD3 delta (δ) chains, and two CD3 zeta (ζ) chains. Gray boxes represent ITAM domains in the CD3 chains. ITAM: immunoreceptor tyrosine-based activation motif. The alloreactive T cell is genetically modified to disrupt the expression of CD3ε (ε knockout) using technologies such as CRISPR-Cas9, thereby generating T cells that have lost their activation ability. Figure 2B depicts an ε knockout T cell, in which the TCR / CD3 complex includes two CD3γ chains, two CD3δ chains, and two CD3ζ chains. The T cells shown in FIG2B that have lost their activation capacity are genetically modified by introducing a tumor-associated antigen (TAA)-specific tumor sensing receptor (in this example, a SynNotch-based tumor sensing receptor) into the T cells and introducing a CD3ε expression cassette into the nucleus. TCE: transcriptional control element. ε gene: CD3ε gene. These steps produce the tumor-activated alloreactive T cells shown in FIG2C. FIG2D depicts the activity of the tumor-activated alloreactive T cells (shown in FIG2C) in the presence of tumor cells expressing tumor antigens recognized by the TAA-specific tumor sensing receptor. When the tumor antigen binds to the TAA-specific tumor sensing receptor, the transcription activator is cleaved from the receptor and activates transcription of the CD3ε gene in the expression cassette. The CD3ε polypeptide thus produced restores functional TCR / CD3 expression on the T cells, which can recognize and bind to the allogeneic peptide-HLA on the tumor cells. This results in activation of the T cells and killing of the tumor cells (first killing). TCR / CD3 expression on the T cells is expected to persist for some time, although the signal from the tumor sensing receptor will begin to decay after the tumor cells are killed. As shown in Figure 2E, T cells may therefore be able to kill other HLA +Tumor cells or stromal cells. The allogeneic response of T cells may be maintained by contact with other tumor cells expressing TAAs. After leaving the tumor, T cells will lose allogeneic reactivity due to attenuation of SynNotch signaling and loss of CD3ε expression. Figure 2A-C illustrates this in vitro, and Figure 2D and E illustrate the activity in vivo.

[0039] Figure 3 Depicted are data on cytotoxicity of alloreactive T cells against U266 myeloma cells. SK1: CD8-specific monoclonal antibody SK1. % Killing: Specific killing was calculated as [1-(sample activity) / (maximum activity)] × 100. Data are presented as mean ± SD (n = 3).

[0040] Figure 4 Depicted are the expression data of TCRβ and CD3ε on engineered D10 cells and wild-type D10 cells. Figure 4 Described are flow cytometry data for wild-type D10 cells and D10 cells (D10-ζ-KO) knocked out mouse CD3ζ (ζ) using a gRNA containing the crRNA sequence 5'-cuccugggaaccgcacgugg-3' (SEQ ID NO: 14). Cells were stained with antibodies against TCRβ and CD3ε and analyzed using flow cytometry.

[0041] Figure 5 HLA-I expression on wild-type MDA-MB-231 cells and β2m knockout MDA-MB-231 cells is depicted. MDA-MB-231: human breast cancer cell line. β2m KO: β2 microglobulin knockout. WT: wild-type. Cells were stained with FITC-labeled anti-HLA-I monoclonal antibody clone W6 / 32.

[0042] Figures 6A and 6B depict flow cytometry data for proliferation of T cells co-cultured with MDA-MB-231 cells (wild type in Figure 6A or β2 microglobulin knockout in Figure 6B). MDA-MB-231: human breast cancer cell line. β2m KO: β2 microglobulin knockout. WT: wild type. CFSE: carboxyfluorescein diacetate succinimidyl ester.

[0043] Figures 7A-7CDepicted are flow cytometry data for CD3ε and anti-CD19 SynNotch expression in primary human CD8+ T cells that have been genetically engineered to contain the anti-CD19 SynNotch receptor and CD3ε expression cassettes. The anti-CD19 SynNotch receptor contains a cleavable artificial transcriptional activator Gal4-VP64; expression of the anti-CD19 SynNotch receptor is driven by a constitutively active PGK-1 promoter. The CD3ε expression cassette includes a blue fluorescent protein (BFP) coding sequence driven by a constitutively active PGK-1 promoter. The genetically engineered T cells have been stimulated by co-culture with MDA-MB-231 cells; anti-human CD28 antibody was added to the co-culture with MDA-MB-231 cells. Anti-CD19 SynNotch expression was analyzed by staining with PE-labeled anti-MycTag (Cell Signaling Technology, clone 9B11) (X axis). The presence of the CD3ε expression cassette in T cells is marked by the expression of blue fluorescent protein (BFP) (Y axis). Figures 7A and 7B show data of genetically modified primary human CD8+ T cells stimulated with MDA-MB-231 cells. Figure 7C shows data of the modified cells shown in Figure 7B after CD3ε knockout.

[0044] Figures 8A-8C Flow cytometry data of CD3ε and TCRβ expression of human CD8+T cells are depicted. CD3ε was knocked out in primary human CD8+T cells using CRISPR-Cas9 technology, and cells of CD3ε knockout (CD3-KO) were purified by magnetic separation. Figure 8A depicts data of wild-type T cells, and Figure 8B depicts data of CD3-KO T cells three days after electroporation with CRISPR-Cas9 complex (hCD3εsgRNAHs.Cas9.CD3E.1.AC and Alt-RSp Cas9 Nuclease V3). Figure 8C depicts data of purified CD3-KO cells.

[0045] Fig. 9 Depicts flow cytometry time course data for the proliferation of T cells (CD3-KO cells) that were evaluated by CD3ε knockout. Primary human T cells were knocked out of CD3ε by CRISPR. After knockout, CD3+T cells (i.e., wild-type T cells) were not removed from the mixture. Mixed WT and CD3-KO T cells were cultured for 25 days, and CD3ε and TCRβ expression were regularly assessed.

[0046] Fig.10Depicted is the expression of CD19 on wild-type MDA-MB-231 cells (MDA-MB-231WT) and engineered MDA cells (MDA-MB-231-CD19).

[0047] Figures 11A and 11B depict flow cytometry data demonstrating that binding of anti-CD19 SynNotch restored CD3 expression on CD3KO-19SN-εCS T cells and enabled activation of the cells upon anti-CD3 antibody stimulation, as demonstrated by degranulation (CD107a) and IFNγ production (see Figures 11A and 11B, respectively). Cells are the BFP+ gated population.

[0048] Fig.12 Depicted are cytotoxicity data of alloreactive CD3KO-19SN-εCS T cells against CD19-expressing MDA-MB-231 cells. Alloreactive CD3KO-19SN-εCS T cells showed significantly higher cytotoxicity against CD19-expressing MDA-MB-231 cells (MDA-MB-231-CD19-luci) than wild-type MDA-MB-231 cells (MDA-MB-231-luci). Data are expressed as mean ± standard deviation (n=3). ***: P<0.001, Student's t test.

[0049] Fig.13 Depicted are the cytotoxicity data of alloreactive wild-type (WT) and CD3ε knockout (CD3KO) T cells against MDA-MB-231 cells. Data are expressed as mean ± SD (n = 3). ***: P < 0.001, Student's t test.

[0050] Fig.14 A CAR-based tumor sensing receptor is described that restores CD3ε expression in alloreactive CD3-KO T cells through activation of the signaling pathway and transcription factor NFAT. The transcriptional control element (TCE) in the CD3 expression cassette contains the NFAT binding sequence. CD3 expression cassettes containing TCEs that contain binding sequences for other activating transcription factors NR4A, NFκB, and AP-1 can also be used. Re-expression of CD3ε restores surface expression of the TCR / CD3 complex.

[0051] Fig.15Depicted is a CD3ε expression cassette containing a sequence for constitutive expression of BFP. The first part of the expression cassette (left side) drives inducible expression of CD3ε and contains multiple transactivator binding sites, followed by a minimal IL2 promoter and CD3ε coding sequence. The second part (right side) drives constitutive expression of BFP and contains a PGK-1 promoter followed by the BFP coding sequence. Consistent expression of BFP can serve as a marker for the presence of the entire expression cassette in the cell.

[0052] Fig.16 Depicted are flow cytometry data showing the phenotype of PBMCs, immature monocyte-derived dendritic cells (MoDCs), and mature MoDCs. PBMCs were cultured in AIM V medium containing 100 ng / ml GM-CSF and 100 ng / ml IL-4 for 6 days to generate immature MoDCs. The cells were then stimulated with the maturation cocktail for 2 days. Mature MoDCs showed increased expression levels of CD83 and CD86 compared to immature MoDCs. PBMCs: peripheral blood mononuclear cells. MoDCs: monocyte-derived dendritic cells.

[0053] Fig.17 Depicted are flow cytometry data showing T cell proliferation stimulated by HLA-mismatched monocyte-derived dendritic cells (MoDCs). MoDCs are composed of HLA-A2 + PBMCs were generated from donors, matured with maturation cocktail, and spiked with MDA-MB-231 tumor cell lysate. Matured MoDCs were incubated with HLA-A2 - Donor CFSE-labeled T cells were co-cultured at a ratio of 1:3 for 9 days. T cell proliferation was analyzed by flow cytometry. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells cultured alone served as a control.

[0054] Fig.18 Data depicting cytotoxicity of alloreactive T cells against tumor cells expanded from monocyte-derived dendritic cells (MoDC). - Donor T cells with HLA-A2 +Donor-derived MoDCs were expanded. MoDCs were matured with a maturation mixture and added to MDA-MB-231 cell lysate. The expanded T cells were co-cultured with MDA-MB-231 cells expressing luciferase (MDA-luci) or MDA-MB-231 cells with β2m knockout (resulting in loss of HLA-I surface expression) and expressing luciferase (MDA-β2m-KO-luci) at a ratio of 3:1 for 16 hours. MDA-luci and MDA-β2m-KO-luci cells cultured alone were used as controls. The luciferase activity of the remaining surviving MDA-MB-231 cells was determined using the Bright-Glo kit (Promega), and the percentage of killing was calculated as 100x[1-(sample luciferase activity / control luciferase activity)]. Data are expressed as mean ± standard deviation (n=3). **: p<0.01, Student's t test.

[0055] Fig.19 Delineation of human CD8 + Flow cytometry data of CD3ε and TCRβ expression in T cells 3 days after CD3γ, CDδ, or CD3ζ expression was knocked out using CRISPR-Cas9 electroporation. CD3γ-KO: CD3γ knockout. CDδ-KO: CDδ knockout. CD3ζ-KO: CD3ζ knockout. WT: wild type.

[0056] Fig. 20 Delineation of human CD8 + Flow cytometry data of CD8α and TCRβ expression in T cells 3 days after CD8α expression was knocked out using CRISPR-Cas9 electroporation. CD8α-KO: CD8α knockout.

[0057] Fig.21 Depicts the presence of CD19 + In the case of MDA-MB-231 cells, CD3KO-19SN-εCS T cells expressed CD19 -Trans-killing data of MDA-MB-231 cells. MDA-MB-231 cells or MDA-MB-231 cells expressing CD19 (MDA-MB-231-CD19) were mixed with MDA cells expressing luciferase (MDA-MB-231-luci) at a ratio of 1:1. CD3KO-19SN-εCS T cells were added at a ratio of 3:1 (T cells to total MDA cells) and incubated for 48 hours, and then the luciferase activity of the remaining viable MDA-MB-231-luci cells was determined. The mixture of MDA-MB-231 cells cultured alone without T cells was used to determine the maximum luciferase activity. The killing percentage was calculated as [1–(sample activity) / (maximum activity)]×100. Data are expressed as mean ± SD (n = 3). ***: P < 0.001.

[0058] Figures 22A and 22B depict flow cytometry data characterizing CD3 KO-Her2SN-εCS T cells, including expression of CD3ε and Her2-specific SynNotch and integration of the CD3ε expression cassette. Figure 22A depicts data on CD3ε expression on unsorted and sorted CD3 KO-Her2SN-εCS T cells. Sorted CD3 KO-Her2SN-εCS T cells showed significantly lower CD3ε expression levels than wild-type T cells. Figure 22B: Sorted CD3 KO-Her2SN-εCS T cells were stained with anti-MycTag to detect SynNotch expression. The presence of the CD3ε expression cassette is indicated by persistently expressed blue fluorescent protein (BFP).

[0059] Figures 23A-23CFlow cytometry data showing Her2 and HLA-I expression are depicted, including wild-type MDA-MB-231 cells, MDA-MB-231 cells transduced to express Her2 (MDA-Her2), MDA-MB-231 cells transduced to express luciferase and Her2 (MDA-luci-Her2), MDA-MB-231 cells with low-level endogenous Her2 expression knocked out using CRISPR / Cas9 (MDA-Her2KO), and MDA-Her2KO cells transduced to express luciferase (MDA-luci-Her2KO). Figure 23A: Wild-type MDA-MB-231 cells express low levels of Her2. Cells were stained with anti-CD340 / Her2 antibodies (Biolegend). Figure 23B: MDA-Her2 and MDA-luci-Her2 express high levels of Her2. MDA-Her2KO and MDA-luci-Her2KO do not express Her2. For stable expression of luciferase, cells were transduced with pLX313 lentiviral vectors encoding firefly luciferase and hygromycin resistance genes. For stable expression of luciferase and Her2, cells were transduced with pLVx lentiviral vectors encoding Her2 and puromycin resistance genes. Transduced cells were selected in the corresponding antibiotics to obtain stable expression cells. For Her2 KO, sgRNA Hs.Cas9.ERBB2.1.AA (IDT) with a crRNA sequence of 5'-CAACUACCUUUCUACGGACG-3' (SEQ ID NO: 21) was used. Figure 23C: Wild-type and all modified MDA-MB-231 cells express HLA-I. Cells were stained with anti-human HLA-A, B, C antibodies (Biolegend).

[0060] Figures 24A and 24B depict flow cytometry data demonstrating that binding of anti-Her2 SynNotch restored CD3 expression on CD3KO-Her2SN-εCS T cells and enabled the cells to be activated upon stimulation with anti-CD3 antibodies, as demonstrated by degranulation (CD107a) (Figure 24A) and IFNγ production (Figure 24B). + Gated groups.

[0061] Fig.25Depicted are the cytotoxicity data of alloreactive CD3KO-Her2SN-εCS T cells against Her2-expressing MDA-MB-231 cells. Alloreactive CD3KO-19SN-εCS T cells showed significantly higher cytotoxicity against Her2-expressing MDA-MB-231 cells (MDA-luci-Her2) than against Her2-negative MDA cells (MDA-luci-Her2KO). T cells and target cells were cultured at a ratio of 1:1 or 1.5:1 for 16 hours, and then luciferase activity in the remaining surviving cells was measured. Data are expressed as mean ± standard deviation (n=3). ***: P<0.001, Student's t test.

[0062] Fig.26 Depicted in the presence of Her2 + In the case of MDA-MB-231 cells, CD3KO-Her2SN-εCS T cells were positive for Her2 - Trans-killing of MDA-MB-231 cells. Her2 - MDA-Her2KO or Her2 + MDA-Her2 cells and Her2 expressing luciferase - MDA cells (MDA-luci-Her2KO) were mixed at a ratio of 1:1. CD3KO-Her2SN-εCS T cells were added at a ratio of 3:1 or 5:1 (T cells to total MDA cells) and incubated for 48 hours, and then the luciferase activity of the remaining viable MDA-luci-Her2KO cells was determined. The mixture of MDA cells cultured alone without T cells was used to determine the maximum luciferase activity. The killing percentage was calculated as [1–(sample activity) / (maximum activity)]×100. Data are expressed as mean ± SD (n=3). :P<0.01. *:P<0.001, Student's t test.

[0063] Figures 27A and 27B depict flow cytometry data characterizing CD3 KO-Her2 CAR-εCS T cells, including the expression of CD3 ε and Her2-specific CARs and the integration of CD3 ε expression cassettes. Figure 27A: Expression of CD3 ε on unsorted and sorted CD3 KO-Her2 CAR-εCS T cells. Figure 27B: Sorted CD3 KO-Her2 CAR-εCS T cells were first stained with recombinant human Her2 and then stained with anti-CD340 / Her2 to detect Her2-specific CAR expression. The presence of the CD3 ε expression cassette is indicated by continuously expressed blue fluorescent protein (BFP).

[0064] Figures 28A and 28B depict flow cytometry data demonstrating that binding of anti-Her2 CAR restored CD3 expression on CD3KO-Her2CAR-εCS T cells and enabled the cells to respond to anti-CD3 antibody stimulation as demonstrated by degranulation (CD107a) (A) and IFNγ production (B). + Gated groups. DETAILED DESCRIPTION

[0065] The present disclosure relates to a tumor-activated alloreactive or xenoreactive T cell, a method of making the tumor-activated alloreactive or xenoreactive T cell, and a method of using the tumor-activated alloreactive or xenoreactive T cell.

[0066] Definitions and Abbreviations

[0067] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0068] The following abbreviations / acronyms have the following meanings unless otherwise stated:

[0069] -cDNA: complementary DNA

[0070] -DNA: deoxyribonucleic acid

[0071] -EDTA: Ethylenediaminetetraacetic acid

[0072] -kDa: kilodaltons

[0073] -MW: Molecular weight

[0074] -NCBI: National Center for Biotechnology Information

[0075] -PEG: Polyvinyl alcohol

[0076] -pI: isoelectric point

[0077] -ppm: parts per million

[0078] -PVA: poly(vinyl alcohol)

[0079] -PVP: poly(vinylpyrrolidone)

[0080] -RNA: Ribonucleic acid

[0081] -SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0082] -w / v: weight / volume

[0083] -w / w: weight / weight

[0084] -v / v: volume / volume

[0085] -wt%: weight percentage

[0086] -℃: Celsius

[0087] -H2O: water

[0088] -dH2O or DI: Deionized water

[0089] -dIH2O: deionized water, Milli-Q filtered

[0090] -g or gm: grams

[0091] -μg: microgram

[0092] -mg: milligrams

[0093] -kg: kilogram

[0094] -μL and μl: microliter

[0095] -mL and ml: milliliters

[0096] -mm: millimeters

[0097] -μm: micrometer

[0098] -M: Moore

[0099] -mM: millimole

[0100] -μM: micromolar

[0101] -U: unit

[0102] -sec: seconds

[0103] -min(s): minutes

[0104] -hr(s): hours

[0105] -ETOH: ethanol

[0106] -eq.: equivalent

[0107] -N: equivalent concentration

[0108] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry and hybridization are those well known and commonly used in the art.

[0109] The synthesis of nucleic acids and peptides is carried out using standard techniques. These techniques and procedures are generally carried out according to conventional methods in the field and various general references (e.g., Sambrook and Russell, 2001, Molecular Cloning, Laboratory Methods, Cold Spring Harbor Press, Cold Spring Harbor, New York, and Ausubel et al., 2002, Current Protocols in Molecular Biology, John Wiley & Sons Press, New York), which are mentioned many times in this document.

[0110] The nomenclature used herein and the laboratory procedures utilized in analytical chemistry and organic syntheses described below are those well known and commonly used in the art. Chemical syntheses and chemical analyses were performed using standard techniques or modifications thereof.

[0111] As used herein, each of the following terms has the meaning associated with it in this section.

[0112] As used herein, the articles "a" and "an" refer to one or more (ie, at least one) of the grammatical objects. For example, "an element" means one element or a plurality of elements.

[0113] It must be noted that as used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0114] As used herein, "about" when referring to a measurable value (such as an amount, a time duration, etc.) is intended to include variations of ±20% or ±10%, more preferably ±5%, more preferably ±1%, and even more preferably ±0.1% of the specified value, as these variations are suitable for performing the disclosed methods.

[0115] The term "autologous" as used herein refers to any material derived from one individual and subsequently reintroduced into the same individual.

[0116] As used herein, "effective amount" refers to the amount of a therapeutic compound or combination thereof that can provide a therapeutic benefit in alleviating one or more manifestations of a malignant tumor when administered to a patient suffering from a malignant tumor. However, it should be understood that a complete therapeutic effect is not necessarily achieved by a single administration, and may only be achieved after a series of administrations. Therefore, an effective amount can be achieved by one or more administrations. In the case of treatment or prevention applications, the active dose administered to the subject will depend on the type and severity of the disease or condition, as well as the subject's characteristics, such as general health, age, sex, body weight, and tolerance to the drug. It will also depend on the degree, severity, and type of the disease or condition. A skilled technician will be able to determine the appropriate dosage based on these and other factors.

[0117] As used herein, the terms "individual", "patient" or "subject" (as the object of treatment) refer to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc. In one embodiment, the individual is a human. Generally, the terms "individual", "subject" and "patient" are used interchangeably herein when referring to human subjects.

[0118] As used herein, the term "donor" refers to the individual or animal from which the T cells to be manipulated and used as a therapeutic agent are obtained.

[0119] As used herein, "recipient" refers to an individual, patient or subject to whom the T cells of the present disclosure are intended to be administered and / or to receive the T cells of the present disclosure. A "recipient" can refer to a class of individuals, patients or subjects having common characteristics, such as a specific HLA repertoire.

[0120] The term "HLA mismatch" refers to the HLA alleles expressed in tissues of a first individual that are different from those in tissues of a second individual. The term specifically refers to alleles in the highly polymorphic loci HLA-A, HLA-B, HLA-C, DRB1, DPA1, DPB1, DQA1, and DQB1.

[0121] The term "allogeneic" means that the T cells used for modification and treatment originate from a different person than the patient.

[0122] The term "alloreactivity" refers to the ability of some of a person's T cells to respond to cells and tissues of another HLA-mismatched person through TCR recognition of mismatched MHC and antigens presented by mismatched MHC molecules.

[0123] The term "xenogeneic" refers to the fact that the T cells used for manipulation and therapy are derived from non-human animals.

[0124] The term "xenoreactivity" refers to the ability of certain T cells from an individual of one species to respond to cells and tissues from an individual of a different species by recognizing mismatched HLA and antigens presented by mismatched HLA molecules through the TCR.

[0125] As used herein, a "normal subject" or "control subject," depending on the context, refers to a subject that does not suffer from a malignancy.

[0126] As used herein, a "control sample" refers to a sample from a control subject or a sample representative of a population of control subjects.

[0127] As used herein, "treating" a disease refers to reducing the frequency or severity of at least one symptom or sign of the disease or disorder experienced by a subject. Treatment may include delaying further disease progression, or lessening the severity of symptoms that have occurred or are expected to occur, ameliorating existing symptoms, and preventing the onset of additional symptoms.

[0128] "Antibodies" (Ab) shall include, but are not limited to, immunoglobulins that specifically bind to an antigen and are composed of at least two heavy (H) chains and two light (L) chains connected by disulfide bonds, or antigen-binding portions thereof. Each H chain includes a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (referred to herein as VL) and a light chain constant region. The light chain constant region includes a constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interlaced with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. Antibodies can exist in a variety of forms, such as polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv), heavy chain antibodies (e.g., camelid antibodies), and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0129] The "antigen binding portion" (also referred to as an "antigen binding fragment") of an Ab or its antigen binding portion refers to one or more sequences of an Ab (a fragment of a full-length or full-length antibody) that retain the ability of the entire Ab to specifically bind to an antigen. Examples of antigen binding fragments include intrabodies, bispecific antibodies, Fab, F(ab')2, scFv (single-chain variable fragment), Fab', dsFv, sc(Fv)2, and scFv-Fc.

[0130] The term "nanoantibody" (Nb) used herein refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from a natural heavy chain antibody, which is well known to those skilled in the art. They are derived from antibodies containing only heavy chains, found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996). Immunoglobulins without light polypeptide chains have been found in the "Camelidae" animal family. "Camelidae" includes Old World camels (Bactrian camels and dromedaries) and New World camels (e.g., alpacas, llamas, guanacos and llamas). Single variable domain heavy chain antibodies are referred to herein as nanoantibodies or VHH antibodies.

[0131] As used herein, the term "variable domain" refers to the immunoglobulin variable domain as defined by Kabat et al. in Sequences of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, Washington, D.C. (1991). The numbering and positioning of CDR amino acid residues within the variable domain follow the well-known Kabat numbering convention. VH, "variable heavy chain" and "variable heavy chain domain" refer to the variable domain of the heavy chain. VL, "variable light chain" and "variable light chain domain" refer to the variable domain of the light chain.

[0132] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized antibody, some, most or all of the amino acids outside the CDR domain have been replaced with amino acids from human immunoglobulins, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small amino acid additions, deletions, insertions, substitutions or modifications are allowed as long as the ability of the antibody to bind to a specific antigen is not destroyed. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0133] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology, for example, an antibody expressed by a bacteriophage as described herein. The term should also be understood to include antibodies produced by synthesizing a DNA molecule encoding an antibody that expresses an antibody protein, or an amino acid sequence that specifies an antibody, wherein the DNA or amino acid sequence is obtained using existing and well-known synthetic DNA or amino acid sequence technology.

[0134] Reference to a wild-type protein includes the mature form of the protein. A "mature" polypeptide refers to a wild-type polypeptide sequence from which the signal sequence has been removed during polypeptide expression. A mature protein can be a fusion protein between a mature polypeptide and a signal sequence polypeptide.

[0135] The term "variant" with respect to a polypeptide refers to a polypeptide that is different from a specified wild-type, parent or reference polypeptide, including an intentional substitution, insertion or deletion at one or more amino acid positions. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that is different from a specified wild-type, parent or reference polynucleotide in nucleotide sequence. The identity of the wild-type, parent or reference polypeptide or polynucleotide will be apparent from the context.

[0136] The term "recombinant" when used to refer to a cell, nucleic acid, protein, or vector of interest means that the cell has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions that differ from those found in nature.

[0137] As used herein, the term "purified" refers to material (eg, an isolated polypeptide or polynucleotide) that is in a relatively pure state, for example, at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0138] As used herein, the term "amino acid sequence" is synonymous with "polypeptide", "protein" and "peptide" and is used interchangeably. When these amino acid sequences exhibit activity, they may be referred to as "enzymes". Amino acid sequences are presented in the standard amino-terminal to carboxyl-terminal direction (i.e., N→C) using the conventional single-letter or three-letter codes for amino acid residues.

[0139] The term "nucleic acid" includes DNA, RNA, hybrid double strands and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, multiple codons can be used to encode specific amino acids, and the compositions and methods of the present invention include nucleotide sequences that encode specific amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0140] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system, then the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand, when used as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.

[0141] As used herein, "hybridization" refers to the process by which a nucleic acid chain forms a double strand with a complementary strand, i.e., base pairing, such as occurs in blot hybridization techniques and PCR techniques. An example of stringent hybridization conditions is hybridization under the following conditions: 65°C and 0.1X SSC (where 1X SSC = 0.15M NaCl, 0.015M Na3 citrate, pH 7.0). The hybridized double-stranded nucleic acid is characterized by a melting point (Tm), at which temperature half of the hybridized nucleic acid is unmatched from the complementary strand. Mismatched nucleotides within the double strand will reduce Tm. Nucleic acids encoding variant alpha amylases may be 1°C–3°C lower than the Tm of a double strand formed by the nucleotides of SEQ ID NO: 2 and its completely complementary sequence or more.

[0142] As used herein, a "synthetic" molecule refers to a molecule that is produced by in vitro chemical or enzymatic synthesis rather than by an organism.

[0143] As used herein, the terms "transformed," "stably transformed," and "transgenic," when used in reference to a cell, mean that the cell contains a non-native (eg, heterologous) nucleic acid sequence integrated into its genome or carried by an episome that is maintained for multiple generations.

[0144] In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," which are terms known in the art.

[0145] The term "heterologous" when referring to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell.

[0146] The term "endogenous" when referring to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0147] The term "expression" used herein refers to the process of producing polypeptides based on nucleic acid sequences. This process includes transcription and translation.

[0148] "Selectable marker" or "selectable marker" refers to a gene that can be expressed in a host cell to facilitate selection of host cells carrying the gene. Examples of selectable markers include, but are not limited to, antimicrobials (such as hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage on the host cell, such as a nutritional advantage.

[0149] "Vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term is also interpreted to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassette constructs, and the like.

[0150] "Expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, the coding sequence being operably connected to an appropriate control sequence capable of realizing DNA expression in an appropriate host. These control sequences may include a promoter for realizing transcription, an optional operator sequence for controlling transcription, a sequence suitable for ribosome binding sites on the encoding mRNA, an enhancer, and sequences controlling transcription and translation termination. Control sequences are also referred to herein as transcription control elements ("TCEs"). Expression vectors include all vectors known in the art, such as cosmids, plasmids (such as naked plasmids or plasmids contained in liposomes) and viruses (such as slow viruses, retroviruses, adenoviruses, and adeno-associated viruses), all of which contain recombinant polynucleotides.

[0151] The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that the expression of the coding sequence is controlled by the regulatory sequence.

[0152] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all mutually degenerate versions of nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins or RNAs may also include introns, because in some versions, nucleotide sequences encoding proteins may contain introns.

[0153] The terms "linker," "spacer," or "spacer domain," used interchangeably herein, refer to an amino acid or amino acid sequence that can be selectively positioned between two amino acid sequences in a fusion protein.

[0154] A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0155] As used herein, "biological activity" refers to a sequence that has a particular biological activity, such as enzymatic activity.

[0156] "Percent sequence identity" means that a variant has at least a certain percentage of identical amino acid residues with the wild-type protein when compared using the BLAST algorithm with default parameters. For example, a variant of a 617 amino acid polypeptide has 5 amino acid substitutions or deletions, and the percent sequence identity relative to the mature polypeptide is 99% (612 / 617 identical residues x 100, rounded to the nearest integer). Such a variant would be included in the variants having "at least 99% sequence identity" with the mature polypeptide.

[0157] As used herein, a "fusion protein" or "fusion polypeptide" is a polypeptide consisting of at least two polypeptides, optionally further comprising a linker sequence, and operatively linked into a continuous protein. The two polypeptides linked in the fusion protein are usually derived from at least two independent sources (i.e., not from the same parent polypeptide), so the fusion protein includes at least two linked polypeptides that are not usually linked together in nature. Typically, at least two polypeptides can be operatively linked directly by a peptide bond, or can be linked by a linker (such as a spacer domain). An example of a fusion polypeptide is a polypeptide that serves as an antigen receptor, in which an antigen-binding polypeptide that forms an extracellular domain is fused to other polypeptides to form a "chimeric antigen receptor". Fusion proteins comprising 3, 4, 5, 6, 7, 8, 9 or 10 or more heterologous polypeptides are also contemplated herein.

[0158] As used herein, "eliminating gene expression" means disrupting the expression of a gene.

[0159] As used herein, "eliminating gene product function" means disrupting the function and activity of the gene product.

[0160] Range: Various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the disclosed range. Therefore, the description of a range should be considered to specifically disclose all possible subranges within the range as well as individual values. For example, describing a range of 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0161] As used herein, an "isolated" compound is one that is removed from at least one component with which it is naturally associated in nature.

[0162] Regarding the material compositions and methods disclosed herein, in one aspect, embodiments of the present invention include components and / or steps disclosed herein. In another aspect, embodiments of the present invention are substantially composed of components and / or steps disclosed herein. In yet another aspect, embodiments of the present invention are composed of components and / or steps disclosed herein.

[0163] describe

[0164] Embodiments of the present invention are described below. However, it should be clearly pointed out that the present invention is not limited to these embodiments, but is intended to also include modifications and equivalents obvious to those skilled in the art.

[0165] The present disclosure utilizes the alloreactivity or heteroreactivity of allogeneic or xenogeneic T cells to broadly target cancer cells and stromal cells in tumor or hematologic cancer cell accumulation tissues. Each T cell naturally expresses a unique type of T cell receptor (TCR) on the cell surface to recognize specific antigens on target cells. In humans, antigens are short peptides (about 10 amino acid residues) complexed with cell surface proteins called human leukocyte antigens (HLA). HLA class I (HLA-I) proteins consist of α heavy chains and β2 microglobulin (β2M). Only the α chain participates in peptide binding and TCR interactions. The α chain is encoded by three gene loci: HLA-A, B, and C, resulting in the expression of three gene products on the cell surface. All three gene loci are highly polymorphic, with hundreds to thousands of different alleles in the population. HLA-I molecules are expressed on almost all human cells, including many tumor cells. Peptide-HLA-I antigens are expressed by CD8 + TCRs are recognized by cytotoxic T cells that, upon activation, kill cells expressing the antigen. HLA class II (HLA-II) molecules are αβ heterodimers, with both chains involved in peptide presentation and TCR binding. HLA-II molecules are encoded by six gene loci (HLA-DRA1, DRB1, DPA1, DPB1, DQA1, and DQB1), all of which, except DRA1, are highly polymorphic. HLA class II is primarily expressed on B cells and professional antigen-presenting cells (APCs), but expression can also be induced on epithelial cells and various solid tumor cells (20). Peptide-HLA-II antigens activate CD4 + Helper T cells, which release inflammatory cytokines and help CD8 +T cell survival and function. Due to the polymorphism of each HLA gene locus in the human population, it is rare for any two individuals to express the same combination of HLA genes. A significant proportion (1-10%) of an individual's T cells can recognize and respond to cells from another individual through TCR recognition of mismatched HLA and antigens presented by mismatched HLA molecules (21,22). T cell alloresponses, that is, responses to cells and tissues from different individuals of the same species, play a key role in organ transplant rejection through host versus graft (HvG) activity (23). If the transplant recipient is immunocompromised and cannot eliminate T cells in the graft through HvG, the transplanted T cells may attack the recipient's tissues and cause severe, sometimes fatal graft-versus-host (GvH) disease (24). Therefore, close matching of HLA genes between organ donor and recipient is essential for successful transplantation. Matching of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci was found to be most critical for successful transplantation, indicating that they are more involved in alloreactivity than HLA-DP and HLA-DQ (25). Due to the diploid nature of the human genome, matching of all eight HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci (8 / 8 matches) has the greatest chance of success in transplantation. T cell responses to cells of a different species, i.e., xenoreactivity, are often more potent than alloreactivity (26) and are a major obstacle to the use of animals (e.g., pigs) as sources of organs for human transplantation. It should be noted that the term "allogeneic" refers to the source of T cells from an individual different from the recipient. "Alloreactivity" can refer to the reactivity of transplanted allogeneic T cells to recipient tissues (GvH) or the reactivity of tissue transplant recipient T cells to transplanted tissues (including transplanted allogeneic T cells) (HvG). Not all allogeneic T cells are alloreactive to recipient tissues. On the other hand, the alloreactive T cells used in the present disclosure for cancer treatment are allogeneic in nature. Since the T cells of the present disclosure are administered to cancer patients, the terms "recipient" and "patient" are used interchangeably depending on the context.

[0166] Alloreactive T cells have been used to treat cancer in special circumstances. Donor lymphocyte infusion (DLI) is an established treatment for patients with blood cancers who have residual disease after allogeneic stem cell transplantation. In this setting, patients receive intensive chemotherapy or radiation therapy to kill cancer cells. Stem cells from a partially HLA-matched donor are used to restore normal hematopoietic activity that was impaired by the radiation or chemotherapy. T cells from the same donor are then infused to eliminate the remaining cancer cells through an alloreactive response. Approximately 70% of these patients will develop GvH disease, which is associated with a lower risk of cancer recurrence (27,28). A unique factor in this setting is that the infused allogeneic T cells are less likely to be eliminated by the patient through HvG because the patient’s T cells develop from stem cells from the same donor and are therefore tolerant to the donor’s HLA.

[0167] Therefore, the use of alloreactive or xenoreactive T cells for cancer therapy must overcome two major obstacles. The first is to avoid or minimize the patient's HvG activity against allogeneic and xenogeneic T cells to allow these cells to survive and perform antitumor activities. In this regard, lymphocyte depletion caused by radiation or chemotherapy that cancer patients receive can impair the patient's immune system, potentially creating a window of reduced HvG activity for the use of allogeneic T cell therapy (29). Advances have also been made in rendering allogeneic T cells "invisible" to the recipient's immune system. Using gene editing techniques such as CRISPR-Cas9, HLA expression can be knocked out in allogeneic T cells, rendering them "invisible" to the recipient's T cells (30,31). These cells can be further engineered to express nonclassical HLA-I molecules, such as HLA-E and HLA-G, to protect them from attack by natural killer (NK) cells (32), which kill cells that do not express any HLA. The second hurdle is to restrict the destructive allogeneic, or xenogeneic, response of T cells to the tumor site to minimize damage to normal tissues and avoid severe GvH disease.

[0168] The present disclosure describes a method for generating genetically modified T cells whose alloreactivity or xenoreactivity is localized to the tumor site or the tissue where blood cancer cells accumulate. The method includes eliminating the ability of T cells to be activated by TCR and introducing a mechanism that restores this ability at the tumor site in response to molecular cues enriched in tumor cells or tumor microenvironment. The present disclosure further describes the prepared genetically modified T cells and their therapeutic uses.

[0169] I. Methods for Generating Tumor-Activated Alloreactive or Xenoreactive T Cells

[0170] The present disclosure describes methods for generating tumor-activated alloreactive or xenoreactive T cells and their use in treating cancer patients. The T cells are generated through four main steps ( Figure 1 , for simplicity only alloreactive T cells are described).

[0171] The first step is T cell collection. T cells are isolated from the blood of an HLA-mismatched donor. The degree of HLA mismatch is determined by comparing the alleles of the donor and recipient at the HLA-A, HLA-B, HLA-C, DRB1, DPA1, DPB1, DQA1, and DQB1 loci. A donor is selected that has at least one HLA allele mismatch with the recipient or patient. The mismatched allele is preferably located at one of the HLA-A, HLA-B, HLA-C, and DRB1 loci. Donors with more mismatches are better suited to produce a strong T cell allogeneic response. Xenogeneic T cells are isolated from animals (such as pigs). Total T cells (CD4 + and CD8 + T cell mixture), CD4 + T cells or CD8 + T cells.

[0172] The second step is stimulation to activate the T cells and drive their proliferation (i.e., drive the cells into the cell cycle). This step facilitates genetic manipulation of the T cells and expansion of the T cells in subsequent steps. T cells can be stimulated nonspecifically or specifically. For nonspecific stimulation, T cells can be co-cultured with anti-CD3 and anti-CD28 antibodies coated on beads or plastic surfaces to activate all T cells, regardless of whether they are alloreactive or xenoreactive. For specific stimulation, T cells can be co-cultured with cells from the patient, including peripheral blood mononuclear cells (PBMCs), cultured monocyte-derived dendritic cells (DCs), and cells isolated from resected tumors. Alternatively, T cells can be co-cultured with PBMCs or DCs from another donor who shares at least one HLA allele with the patient. Alloreactive T cells proliferate in response to PBMCs from HLA-mismatched individuals, and this proliferation response is the result of stimulation of the T cells by antigen presenting cells (APCs) in the PBMCs through TCR-HLA interactions. This is the basis of the mixed lymphocyte reaction (MLR), which has been used to determine the alloreactivity of T cells since 1964 (33). To expand the alloreactive T cells of the present disclosure, T cells from HLA-mismatched donors can be labeled with the cell division tracking fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with the patient's PBMCs. Prior to culture, the patient's PBMCs are irradiated (2,500 rad) or treated with chemotherapeutic drugs (such as mitomycin C) to eliminate their proliferation potential. Proliferated donor T cells with low CFSE levels are sorted using flow cytometry and used as a source of alloreactive T cells for downstream genetic manipulation. The use of DCs can improve the efficiency of alloreactive T cell expansion. As professional antigen presenting cells (APCs), DCs highly express HLA-I, HLA-II and a series of co-stimulatory molecules, such as CD40, CD80 and CD86. DCs may also strongly activate T cells by binding to the TCR and co-stimulatory receptors such as CD28. Monocyte-derived dendritic cells (MoDCs) have been tested for decades as immunotherapies for the treatment of cancer, autoimmunity, and other diseases. As of August 2019, there are 120 human clinical trials listed on ClinicalTrials.gov. MoDCs have been shown to effectively stimulate the expansion of alloreactive T cells (34-36). The generation of MoDCs involves culturing monocytes enriched from PBMCs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (34-42). The procedures for monocyte enrichment, generation, and cryopreservation of MoDCs are well established (43,44), and a number of commercial kits are available for the generation of clinical-grade MoDCs.To enhance the ability of T cells to recognize and kill tumor cells, T cells can be stimulated with MoDCs in the presence of lysates generated from the patient's tumor cells or from a tumor cell line of the same type from another individual (45-47).

[0173] T cells can also be expanded by co-culturing with a cell line expressing an HLA protein encoded by at least one HLA allele of the patient, or with an artificial surface (such as plastic) coated with an HLA protein encoded by at least one HLA allele of the patient. Specific stimulation results in the selective activation and expansion of T cells that are alloreactive or xenoreactive to the patient's HLA. The stimulated and expanded T cells can be cryopreserved, for example in an appropriate percentage of DMSO, such as 10% DMSO or 7.5% DMSO, and thawed later for downstream genetic manipulation. If the genetic manipulation in the subsequent steps can be achieved without activating the T cells, and it is preferred that the T cells are activated and expanded after infusion into the patient, the activation step can be omitted.

[0174] The third step is to eliminate the ability of T cells to be activated through TCR signaling, thereby rendering the T cells incapable of activation. This is achieved by disrupting the expression or function of at least one molecule necessary for TCR signaling and T cell activation ( Figure 1and Figures 2A, 2B). For example, gene editing technologies such as CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), megaTALS, zinc finger nucleases, or homing endonucleases can be used to disrupt the expression of genes encoding key proteins (48). Disruption occurs in both copies of the target gene in the cell, resulting in a complete lack of expression in the cell and its progeny. In recent years, CRISPR-cas9 has become a routine technique in research laboratories and has been used in a variety of clinical applications, including immunotherapy (49,50). A large number of commercial products are available for guide RNA (gRNA) design and synthesis, recombinant cas9 protein, and tools for delivering gRNA and cas9 into a variety of cells. For primary human T cells, electroporation transfection of gRNA-cas9 ribonucleoprotein (RNP) has become the preferred method for efficient gene knockout (51,52). Genes encoding polypeptides essential for TCR signaling and T cell activation can encode transmembrane proteins. For example, transmembrane proteins expressed on the plasma membrane include CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58. Genes encoding polypeptides necessary for TCR signaling and T cell activation can encode intracellular signaling molecules involved in TCR signaling and T cell activation. For example, intracellular signaling molecules include, but are not limited to, Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT, NcK and PDK1. The sequences of these exemplary molecules are readily available in public databases, such as the National Institutes of Health. (U.S. Department of Health and Human Services) and UniProt. There are many free online tools that can be used to design gRNA sequences to target any specific gene in the human genome. For example: URL: https: / / www.idtdna.com / site / order / designtool / index / CRISPR_CUSTOM, URL: https: / / zlab.bio / guide-design-resources, and URL: https: / / www.synthego.com / products / bioinformatics / crispr-design-tool. Pre-designed gRNAs for specific gene targets are also available from commercial sources, for example: URL: https: / / www.idtdna.com / site / order / designtool / index / CRISPR_PREDESIGN.

[0175] The fourth step is to generate tumor-activated alloreactive or xenoreactive T cells by endowing the inactivated T cells with the ability to restore activation at the tumor site ( Figure 1and Figure 2C). This is achieved by introducing a tumor sensing receptor and an expression cassette for the disrupted gene. The tumor sensing receptor includes an extracellular domain that binds a molecule enriched on tumor cells or in the tumor microenvironment and causes the release or activation of transcriptional activators from the intracellular domain. The extracellular domain can be, for example, a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a nanobody, a TCR, an Fc receptor, a growth factor receptor, a chemokine receptor, or a hormone receptor. The types of molecules recognized by these receptors include, for example, tumor-associated antigens (TAAs) expressed on tumor cells, including CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc. Cancer-associated antigens also include, for example, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tensin C, TGF beta 2. TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin.Tumor sensing receptors may also recognize soluble factors that are enriched in the tumor microenvironment, such as chemokines (53), growth factors, and growth hormones (54). In another embodiment, tumor sensing receptors recognize molecules that are enriched in tissues where blood cancer cells accumulate. For example, acute myeloid leukemia (AML) and myeloma cells tend to accumulate in the bone marrow and lymphoid organs. Tumor sensing receptors may recognize molecules expressed on hematopoietic cells, particularly lymphocytes, that are abundant in these tissues. These molecules include CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123, and CCL-1.

[0176] The expression cassette includes a promoter-like transcriptional control element (TCE) DNA sequence operably linked to a DNA sequence encoding a copy of the disrupted gene (see Fig.15 ). Binding of transcription activators to transcription control elements drives expression of the gene disrupted in step 3 and restores the ability of T cells to activate and respond to antigens. Specifically, the TCE contains multiple transcription activator binding sites and a minimal promoter. Downstream of the TCE is the coding sequence of the gene disrupted in step 3. The coding sequence should encode the polypeptide of the gene, but needs to be modified so that, for example, the gRNA used to disrupt the original gene by the CRISPR / Cas9 method no longer works. This can be achieved by creating multiple silent mutations in the sequence targeted by the gRNA, especially in the PAM sequence. The expression cassette can also contain a coding sequence for a fluorescent protein (such as BFP) driven by a continuously active promoter. The expression of the fluorescent protein serves as a marker for the presence of the expression cassette in the cell.

[0177] Two types of engineered receptors can be used as tumor sensing receptors. The first type releases a transcriptional activator from the intracellular domain upon ligand binding. The transcriptional activator then translocates to the nucleus where it activates the expression cassette. These receptors include, for example: synthetic Notch (SynNotch) (14, 55) and (U.S. Patent Publication US20160264665A1 and U.S. Patent No. 9834608B2), Modular Extracellular Sensor Architecture (MESA) (56) (U.S. Patent Publication US20140234851A1) and Tango (57). SynNotch receptors exploit the ability of Notch to cleave and release its intracellular domain upon ligand binding, which acts as a transcription factor after translocation to the nucleus. SynNotch receptors are constructed by replacing the extracellular recognition domain of Notch with a binding domain for a specific target and replacing the intracellular domain with a transcriptional activator that can bind to the TCE in the expression cassette and drive expression of the target gene. The patented DNA construct of the SynNotch receptor, which includes the Gal4-VP64 transcriptional activator and the extracellular binding domains for CD19 (Addgene Cat.#79125) and Her2 (Addgene Cat.#85424), can be easily modified to construct SynNotch receptors with other binding specificities and / or transcriptional activators. A DNA construct of the patented expression cassette with the Gal4-VP64 TCE is also available from Addgene (Cat.#79123) and can be used to express a gene of interest in response to SynNotch activation. A CD3ε expression cassette (Gal4-CD3-PKG-BFP), which contains an additional BFP coding sequence controlled by a constitutively active PGK-1 promoter, is shown in SEQ ID NO:1. Since SynNotch activation depends on surface-anchored ligands, it can be used to sense tumor antigens expressed on the surface of tumor cells, such as Her2.

[0178] MESA can be used to sense soluble factors in the tumor microenvironment, such as vascular endothelial growth factor (VEGF) or soluble tumor antigens shed from tumor cells. Target binding leads to receptor dimerization, which brings the protease on the intracellular domain of one monomer into proximity with the substrate sequence on the other monomer. Cleavage of the substrate sequence leads to the release of the transcriptional activator that is linked to the cytoplasmic domain of the second monomer through the substrate sequence.

[0179] Tango is similar to MESA, except that the protease is linked to an intracellular signaling molecule that is recruited to the intracellular domain of the receptor upon ligand binding. Thus, Tango can be used to sense a variety of soluble factors in the tumor microenvironment, including chemokines (53), growth factors, and growth hormones (54).

[0180] The second type of engineered tumor-sensing receptors activate endogenous transcription factors in T cells through signaling pathways. For example, typical CARs have intracellular immunoreceptor tyrosine-based activation motif (ITAM) domains that can initiate multiple signaling pathways after ligand binding, leading to the activation of transcription factors NFAT, AP-1, NFκB / Rel, or NR4A1 (Nur77) ( Fig.14 ). Expression cassettes that use TCE sequences containing these transcription factor binding sequences to drive luciferase or GFP expression have been widely used in T cell activation reporter systems. The expression cassettes NFAT-CD3-PKG-BFP (SEQ ID NO: 2), AP1-CD3-PKG-BFP (SEQ ID NO: 3), NFκB-CD3-PKG-BFP (SEQ ID NO: 4), and NR4A-CD3-PKG-BFP (SEQ ID NO: 5) can be generated by replacing the Gal4 binding site in the Gal4-CD3-PKG-BFP cassette with the binding sites of these transcription factors (Table 1). These expression cassettes can be used to drive the expression of downstream target genes in response to CAR recognition of ligands. The combination of CAR and transcription factor-driven expression cassettes has been used to develop TRUCK (universal redirected T cells for cytokine-mediated killing), which secrete proinflammatory cytokines in response to CAR signals to enhance CART cell function (58-60). TRUCK T cells have also entered clinical stage research (NCT02498912 and NCT03721068).

[0181] Table 1. Transcription factor binding sites in the expression cassettes of CAR-based tumor sensing receptors

[0182]

[0183]

[0184] DNA sequences for the tumor sensing receptor and expression cassette will be introduced into T cells using retroviral or lentiviral vectors, or transposon vectors such as Sleeping Beauty (Addgene) and piggyBac (VectorBuilder) to promote their stable integration into the T cell genome. In practice, steps 3 and 4 can be performed simultaneously or in reverse order. Nonspecifically or specifically stimulated T cells can be restimulated. T cells may be cryopreserved after stimulation / expansion, after step 3, or after step 4. Thus, tumor-activated alloreactive T cells can be generated in a variety of ways. Three examples are: (i) stimulation / expansion → SynNotch and expression cassette introduction → CD3ε knockout → (cryopreservation) → treatment; (ii) stimulation / expansion → cryopreservation → restimulation → SynNotch and expression cassette introduction → CD3ε knockout → (cryopreservation) → treatment; and (iii) stimulation / expansion → CD3ε knockout → cryopreservation → restimulation with PMA and ionomycin → SynNotch and expression cassette introduction → (cryopreservation) → treatment.

[0185] II. Tumor-activated alloreactive or xenoreactive T cells

[0186] The present disclosure provides a tumor-activated alloreactive or heteroreactive T cell. The T cell is derived from a healthy donor, and it is known that the genotype of at least one HLA-A, B, C and DRB1 site of the donor does not match the patient. The T cell is modified to lose the ability to activate. Specifically, the T cell is genetically modified to destroy the expression of at least one endogenous gene, which encodes a molecule that is critical to TCR signaling and T cell activation. The gene to be destroyed can encode a transmembrane protein expressed on the plasma membrane. Exemplary transmembrane proteins include, but are not limited to, CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58. The gene to be destroyed can encode an intracellular signaling molecule involved in TCR signaling and T cell activation. Exemplary intracellular signaling molecules include, but are not limited to, Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT, NcK, and PDK1.

[0187] The T cell that has lost the ability to activate also contains an expression cassette, which contains a copy of the gene encoding the destroyed molecule in the T cell. The expression vector contains a transcription control element (TCE) operably linked to the gene, and the binding of a specific transcription activator to the TCE leads to the expression of the gene encoding the destroyed molecule in the T cell. The T cell also contains an exogenous tumor sensing receptor. The binding of the tumor sensing receptor to its specific tumor antigen leads to the release or activation of a transcription activator. The tumor antigen used in this article can be a tumor cell surface molecule (such as Her2), or a soluble factor present in the tumor microenvironment (such as vascular endothelial growth factor (VEGF)), or a tumor antigen shed from a tumor cell. In another embodiment, the tumor sensing receptor recognizes molecules enriched in tissues where blood cancer cells accumulate. These include molecules expressed on hematopoietic cells abundant in the bone marrow and lymphoid organs.

[0188] Genetically modified T cells containing the above characteristics are expected to provide at least one of the following beneficial properties: alloreactivity localized to tumor sites or tissues where blood cancer cells accumulate, and the ability to target HLA-expressing tumor cells as well as tumor stromal cells.

[0189] In certain embodiments of tumor-activated alloreactive or xenoreactive T cells and methods for preparing the same, the disrupted endogenous gene is selected from CD3ε, CD3ζ, CD3γ, CD3δ, and CD8α. In one embodiment, the disrupted endogenous gene is CD3ε.

[0190] In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin, MUCI, EpCAM, GD2, and CEA. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, CD19, IL-13RR-a2, mesothelin, and MUCI. In certain embodiments, the target molecule is selected from Her2 / neu, EGFRvIII, and CD19. In one embodiment, the target molecule is Her2 / neu. In one embodiment, the target molecule is CD19.

[0191] In certain embodiments, the expression cassette is selected from Gal4-CD3-PGK-BFP, NR4A-CD3-PKG-BFP, and NFAT-CD3-PKG-BFP. In certain embodiments, the tumor sensing receptor and the expression cassette are selected from SynNotch and Gal4-CD3-PGK-BFP, CAR and NR4A-CD3-PKG-BFP, and CAR and NFAT-CD3-PKG-BFP.

[0192] In certain aspects, the T cell sample is from a donor individual, and the donor individual has at least one HLA allele mismatch compared to the intended recipient, and the HLA allele mismatch is located at one of the following groups: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, thereby generating tumor-activated alloreactive T cells. In certain embodiments, at least one HLA allele mismatch is located at one of the following groups: HLA-A, HLA-B, HLA-C and HLA-DRB1. In certain embodiments, the HLA allele mismatch includes four alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci, six alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci, or all eight alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In one embodiment, the HLA allele mismatch includes all eight alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[0193] In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, and the tumor sensing receptor is SynNotch.

[0194] In certain embodiments, the disrupted endogenous gene is CD3ε, the target molecule is selected from Her2 / neu, EGFRvIII and CD19, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is CD19, the tumor sensing receptor is SynNotch, and the expression cassette is Gal4-CD3-PGK-BFP. In one embodiment, the disrupted endogenous gene is CD3ε, the target molecule is Her2 / neu, the tumor sensing receptor is CAR, and the expression cassette is NFAT-CD3-PGK-BFP.

[0195] III. Methods of treatment and use

[0196] Tumor-activated alloreactive or xenoreactive T cells will be expanded in culture medium supplemented with cytokines such as IL-2 and IL-7. These T cells will be introduced into the patient or cryopreserved for later use. T cells from different donors or stimulated differently can be used in the same patient for optimal results.

[0197] Several additional steps can be taken to reduce HvG and enhance T cell survival in patients. First, patients can be given lymphocyte-depleting radiation or chemotherapy to suppress the immune system before infusion.

[0198] Second, T cells can be obtained from donors with partial HLA matches to reduce HvG. For example, T cells from a donor matched at HLA-A, B, C, and DRB1 5 / 8 loci can be used and stimulated with patient cells or a cell line expressing the patient's three mismatched loci alleles to expand T cells that are alloreactive to these alleles. Third, T cells can be additionally modified to make them "invisible" to the patient's immune system. For example, CRISPR-Cas9 can be used to disrupt the expression of HLA-A, B, or C on T cells. It should be noted that only the mismatched HLA genes need to be disrupted. Alternatively, the expression of all HLA-I molecules on the cell surface can be eliminated by knocking out the shared β2M component, which is required for HLA-I to reach the plasma membrane. The α chain of HLA-E or HLA-G fused to β2M is then introduced in the fourth step (25). HLA-E and HLA-G are non-classical HLA molecules that do not participate in alloreactivity but can inhibit attack by natural killer (NK) cells. Finally, for patients with blood cancer who have received an allogeneic hematopoietic stem cell transplant, T cells can be obtained from the stem cell donor. In this case, the patient's immune system will tolerate these T cells. Moreover, in this case, the restored alloreactive T cells will only target the patient's cells (including cancer cells), but avoid the hematopoietic cells that developed from the donor's stem cells.

[0199] In patients, tumor-activated alloreactive or xenoreactive T cells are unable to respond to HLA in normal tissues due to the absence of key components of TCR signaling and should not cause graft-versus-host (GvH) disease. However, after migrating to tissues where tumor or blood cancer cells accumulate, binding of tumor-sensing receptors to cue molecules enriched in the tumor or target tissue will restore the alloreactive or xenoreactive nature of the T cells, leading to T cell killing of HLA-expressing tumor cells and stromal cells (Figures 2D and 2E). The duration of responsiveness depends on two factors. The first is the decay rate of the signal generated by the tumor-sensing receptor, which determines how long the re-expression of the knocked-out molecule can last after the receptor dissociates from the cue molecule. For example, the half-life of expression driven by the SynNotch receptor is approximately 8 hours. The second is the distribution of tumor cue molecules within the tumor, which determines how frequently the tumor-sensing receptor can re-bind and activate. Figures 2A-2E illustrate the responsiveness of alloreactive T cells, in which CD3ε expression is controlled by TAA-specific tumor-sensing receptors. CD3ε is a member of the TCR / CD3 complex. Loss of CD3ε expression results in the retention of the other components in the endoplasmic reticulum (61). Here, tumor-activated alloreactive T cells enter a tumor in which only a subset of tumor cells express TAAs. The activation capacity of the T cell is compromised by one of the TAAs. + Tumor cell recovery occurs through interaction of the TAA with the tumor sensing receptor, which results in expression of CD3ε in the expression cassette, thereby restoring surface expression of the TCR / CD3 complex. Partial restoration of TCR / CD3 expression levels may be sufficient to restore T cell alloreactivity (62). Alloreactivity to the HLA on the tumor cell will activate the T cell and result in killing of the tumor cell (cis killing). Assuming that at least 50% CD3ε expression is required for the T cell to remain alloreactive, the T cell would be able to kill other TAA-negative tumor cells (trans killing) within 8 hours before losing killing activity. If the T cell encounters other TAAs before leaving the tumor, + tumor cells, it may regain alloreactivity. After leaving the tumor, the T cell will lose alloreactivity due to attenuation of tumor-sensing receptor signaling and CD3ε expression, although it may cause limited damage to surrounding normal tissue due to residual CD3ε expression. The above scenario is consistent with a study involving T cells expressing SynNotch that binds to CD19 and drives the expression of a CAR specific for the tumor antigen ROR1 (63). When these T cells were administered to immunodeficient mice with bone marrow-disseminated lymphoma cells expressing CD19 and ROR1, the T cells expressed the ROR1-specific CAR due to SynNotch-CD19 binding and killed the lymphoma cells in vivo (cis-killing). However, these T cells also killed CD19- / ROR + In another example of SynNotch-mediated trans-killing, T cells expressing EGFRvIII-specific SynNotch (driving expression of an EphA2-specific CAR) were shown to kill EGFRvIII in mice. + EphA2 + and EGFRvIII - EphA2 + of tumor cells (64).

[0200] The potency and specificity of tumor-activated alloreactive or xenoreactive T cells can be optimized in several ways. First, the potency of the cells can be controlled by selecting the degree of HLA mismatch between the donor and the patient. For example, a mismatch at all six HLA-I loci is expected to elicit a stronger alloreactive response than a mismatch at only one locus.

[0201] Second, the ability of T cells to selectively target tumors and avoid normal tissues can be optimized by tuning the sensitivity of tumor-sensing receptors so that they only signal in response to high levels of tumor cue molecules. This can be achieved by adjusting the expression level, affinity, and extracellular linker length of the receptor.

[0202] Third, tumor selectivity of T cells can be further controlled by using two tumor-sensing receptors, one that releases the DNA binding domain of a transcriptional activator and the other that releases the activation domain of a transcriptional activator. In this way, only cells that express both tumor antigens can restore expression of key molecules and thus restore T cell responsiveness. Alternatively, tumor selectivity can be controlled by using multiple tumor-sensing receptors, each directing the expression of a different key component required for TCR signaling. For example, CD3ζ and CD3ε can be knocked out simultaneously, and two tumor-sensing receptors introduced: one that recognizes Her2 and directs the expression of CD3ζ; the other that recognizes a different tumor antigen, MUC1, and directs the expression of CD3ε. Since cell surface expression of the TCR / CD3 complex requires both CD3ζ and CD3ε, only cells that express both tumor antigens can activate T cells.

[0203] Fourth, the balance between tumor cell killing and normal tissue damage can be fine-tuned by adjusting the duration of alloreactivity. This can be achieved by manipulating the stability of the mRNA transcripts of re-expressed key molecules or controlling their protein degradation rate using degraders (65). This balance can also be adjusted by controlling the degree of lymphocyte depletion in the patient and, in turn, the HvG activity against allogeneic or xenogeneic T cells to enhance or reduce T cell survival. Finally, the potency and specificity of tumor-activated alloreactive or xenoreactive T cells can be controlled by using "universal" tumor-sensing receptors whose extracellular domains can bind peptides or chemical tags on soluble factors that bind to tumor antigens, or whose extracellular domains are derived from Fc receptors that can bind tumor antigen-specific IgA, IgG, or IgE antibodies. Tumor-sensing receptors will be activated by soluble factors or antibodies bound to tumor cells. The level of alloreactivity or xenoreactivity of T cells can be manipulated by controlling the type and dose of tumor-bound soluble factors or antibodies.

[0204] Tumor-activated alloreactive or xenoreactive T cells are thought to have several advantages over CAR T cells in terms of efficacy and cost. First, once activated at the tumor site, allogeneic T cells attack not only tumor cells expressing tumor antigens, but also other tumor cells expressing HLA-I. Although HLA-I downregulation is a common mechanism for tumor cells to evade immune surveillance, HLA-I expression is still well maintained in many cancers. For example, 68% in gastric cancer (66), 57% in esophageal cancer (67), 45% in osteosarcoma (68), 34% in breast cancer (69), and 30% in lung cancer (70).

[0205] Second, alloreactive or xenoreactive T cells also attack HLA-I-expressing stromal cells, such as cancer-associated fibroblasts, angiogenic vascular cells (71), and myeloid-derived suppressor cells (72), which play an important role in supporting tumor growth and creating an immunosuppressive tumor microenvironment. Even in cases where tumor cells are HLA-negative, stromal cells have been found to strongly and uniformly express HLA-I (73-75). These two features make it possible to treat tumors with a low proportion of tumor antigen-expressing cells, which may be considered unsuitable for CAR T cell therapy, with tumor-activated alloreactive or xenoreactive T cells. In this sense, cells expressing tumor antigens not only serve as targets, but also specify that the entire tumor be attacked by therapeutic T cells.

[0206] Third, because T cells are derived from healthy donors, tumor-activated alloreactive T cells that recognize certain common HLA alleles or haplotypes can be mass-produced, cryopreserved, and provided as off-the-shelf products. T cell products that are alloreactive to each HLA allele of the patient can be selected, combined, and administered for treatment. This will reduce costs and enable repeated dosing for better efficacy.

[0207] In summary, tumor-activated alloreactive or xenoreactive T cells have the potential to be more effective against a wider range of tumors compared with CAR T cells.

[0208] T cells are administered to a subject in need of treatment for a cell proliferative disease, including but not limited to cancer. T cells can be administered alone or as a pharmaceutical composition together with one or more pharmaceutically acceptable carriers, diluents or excipients and / or other ingredients (such as cytokines or other cell populations). These compositions may contain pharmaceutically acceptable buffers, such as neutral buffered saline, phosphate buffered saline, etc.; pharmaceutically acceptable carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; pharmaceutically acceptable antioxidants; pharmaceutically acceptable chelating agents, such as EDTA or glutathione; pharmaceutically acceptable adjuvants (such as aluminum hydroxide); and pharmaceutically acceptable preservatives. The composition is preferably formulated in a form suitable for intravenous injection.

[0209] Cancers that can be treated or prevented according to the present disclosure include a wide range of tumor types, including but not limited to: ovarian cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, lung cancer, kidney cancer, colorectal cancer, skin cancer, brain cancer, and tumors that may be caused by hematological malignancies, such as leukemia, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia and chronic lymphocytic leukemia. More specifically, the compounds, compositions and methods of the present disclosure can treat cancers including but not limited to the following: cardiac cancer, lung cancer, gastrointestinal cancer, urogenital tract cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer and adrenal cancer.

[0210] Cancer may include solid tumors that may or may not metastasize. Cancer may also occur in the form of diffuse tissues. Therefore, the term "tumor cell" provided herein includes cells affected by any of the above diseases.

[0211] T cells or their pharmaceutical compositions can be administered by a route that can effectively deliver an effective amount of cells into the patient's body to produce a pharmacological effect. Administration is usually non-oral. Intravenous administration is the preferred route, using infusion techniques commonly used in immunotherapy (see, for example, Rosenberg et al., New Eng. J. Med. 319: 1676, 1988). The number of T cells and the frequency of administration are determined by factors such as the patient's condition, the type and severity of the disease, although the appropriate dose can be determined through clinical trials. The "effective amount" is determined by the doctor taking into account individual differences in age, weight, disease state, and severity of the disease. Typically, the number of T cells administered in a single dose will be about 10 6 to 10 9 The range of cells / kg body weight includes all integer values ​​within these ranges. T cells can be given at these doses multiple times. The optimal dose and treatment regimen for a particular patient can be easily determined by a professional in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0212] The composition comprising T cells may be provided in a pharmaceutical package or pharmaceutical kit, which includes one or more containers or compartments containing one or more compositions. Together with these containers, there may also be a notice in a form prescribed by a governmental agency that is responsible for regulating the production, use or sale of drugs or biological products, which reflects the agency's approval for human use. Also included with these containers are instructions for performing the disclosed methods.

[0213] The instructional material may include a publication, recording, chart, or any other medium of expression that can be used to convey the usefulness of the method. The package insert may include text in any physical medium, such as paper, cardboard, film, or may be stored in an electronic medium, such as a disk, chip, memory stick, or other electronic storage form. The instructional material of the disclosed kit may, for example, be attached to a container containing the other contents of the kit or shipped with the container containing the kit. Alternatively, the instructional material may be shipped separately from the container, with the intent that the recipient use the instructional material in conjunction with the contents of the kit.

[0214] Exemplary embodiments

[0215] Embodiments provided herein include:

[0216] Embodiment 1. A method for producing tumor-activated alloreactive or xenoreactive T cells, the method comprising:

[0217] a) selecting T cell samples from HLA-mismatched donor individuals or donor animals;

[0218] b) optionally stimulating the T cell sample to proliferate;

[0219] c) eliminating the expression or function of at least one molecule necessary for TCR signaling and T cell activation in T cells, thereby rendering the T cells incapable of activation;

[0220] d) engineering T cells to (i) express a recombinant receptor molecule that specifically binds to a target molecule that is enriched in at least one tumor cell, tumor microenvironment, and tissue in which blood cancer cells accumulate, wherein binding of the recombinant receptor to the target molecule releases or activates a transcriptional activator; and (ii) introducing an expression cassette that enables the transcriptional activator in (i) to drive expression of the molecule eliminated in c),

[0221] wherein step c) is performed before step d), step d) is performed before step c), or steps c) and d) are performed simultaneously,

[0222] The binding of the recombinant receptor to the target molecule restores the expression or function of the eliminated molecule, thereby restoring the ability of T cells to activate through TCR recognition of antigens.

[0223] Tumor-activated alloreactive or xenoreactive T cells are thereby generated.

[0224] Embodiment 2. The method of embodiment 1, wherein step b) is not performed.

[0225] Embodiment 3. The method of embodiment 1, wherein step b) is performed.

[0226] Embodiment 4. The method of any one of embodiments 1 to 3, wherein step d) is performed before step c).

[0227] Embodiment 5. The method of any one of Embodiments 1 to 3, wherein step c) is performed before step d).

[0228] Embodiment 6. The method of any one of Embodiments 1 to 4, wherein steps c) and d) are performed simultaneously.

[0229] Embodiment 7. The method of any one of embodiments 4 to 6, wherein the T cell stimulation step comprises culturing donor T cells with specific antibodies against CD3 and CD28.

[0230] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the T cell sample is from a donor individual who has at least one HLA allele mismatch relative to the intended recipient, wherein the at least one HLA allele mismatch is located at a locus selected from the following group: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, thereby generating tumor-activated alloreactive T cells.

[0231] Embodiment 9. The method of any one of embodiments 1-8, wherein step b) comprises co-culturing the donor T cells with cells of the intended recipient.

[0232] Embodiment 10. The method of embodiment 9, wherein the cells from the intended recipient are peripheral blood mononuclear cells, dendritic cells, tumor cells, or a mixture thereof.

[0233] Embodiment 11. The method of embodiments 9 and 10, wherein the donor T cells are co-cultured with dendritic cells pulsed with lysates of tumor cells isolated from the patient, or pulsed with lysates of the same type of tumor cell line from a different individual.

[0234] Embodiment 12. The method of any one of embodiments 1-8, wherein step b) comprises co-culturing the donor T cells with cells from a second donor that (i) matches the intended recipient in at least one HLA allele and (ii) is mismatched with the T cell donor.

[0235] Embodiment 13. The method of embodiment 12, wherein the cells from the second donor are peripheral blood mononuclear cells, dendritic cells, tumor cells, or a mixture thereof.

[0236] Embodiment 14. The method of any one of embodiments 1-8, wherein step b) comprises co-culturing the donor T cells with a cell line expressing at least one HLA allele of the intended recipient.

[0237] Embodiment 15. The method of any one of embodiments 9 to 14, wherein the cells co-cultured with the donor T cells are irradiated or chemically treated to block cell proliferation.

[0238] Embodiment 16. The method of any one of embodiments 1-8, wherein step b) comprises co-culturing the donor T cells with the artificial surface.

[0239] Embodiment 17. The method of embodiment 16, wherein the artificial surface is a plastic coated with at least one protein encoded by at least one HLA allele of the intended recipient.

[0240] Embodiment 18. The method of any one of embodiments 1 to 17, wherein step c) comprises eliminating the expression of at least one protein that is critical for TCR signaling and T cell activation by disrupting the gene encoding the protein.

[0241] Embodiment 19. The method of embodiment 18, wherein the gene editing technology used to disrupt the gene is selected from CRISPR-Cas, transcription activator-like effector nuclease (TALEN), megaTALS, zinc finger nuclease or homing endonuclease.

[0242] Embodiment 20. The method of any one of embodiments 1 to 19, wherein at least one molecule essential for TCR signaling and T cell activation is a cell surface molecule selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58.

[0243] Embodiment 21. The method of any one of embodiments 1 to 19, wherein at least one molecule essential for TCR signaling and T cell activation is an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, NFAT, SLP76, PKCθ, NFκB, AKT and PDK1.

[0244] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the T cells that have lost the ability to activate in step c) are purified by first staining the cells using specific antibodies against surface molecules and then separating the cells lacking antibody binding by flow cytometry, magnetic beads and / or purification columns.

[0245] Embodiment 23. The method according to any one of embodiments 1 to 21, wherein the T cells that have lost the activation ability in step c) are purified by using a live cell-specific DNA imaging technique.

[0246] Embodiment 24. The method of embodiment 23, wherein the live cell specific DNA imaging technology is CRISPRLiveFish. Embodiment 25. The method of any one of embodiments 1 to 21, wherein the T cells that have lost the activation ability in step c) are purified by selecting T cells that cannot be activated and proliferated after further stimulation by TCR.

[0247] Embodiment 26. The method of any one of embodiments 1 to 25, wherein step d) comprises introducing a nucleic acid encoding a tumor sensing receptor into T cells using, for example, a lentiviral vector, a retroviral vector, or a transposon vector (such as Sleeping Beauty and piggyBac).

[0248] Embodiment 27. The method of embodiment 26, wherein the tumor sensing receptor comprises (i) an extracellular domain that directly or indirectly binds to at least one target molecule enriched in tumor cells, tumor microenvironment, and tissues where blood cancer cells accumulate; and (ii) an intracellular domain that activates or releases a transcriptional activator in response to the binding of the extracellular domain to the target molecule.

[0249] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the target molecule is enriched in tumor cells and / or tumor microenvironment and is selected from the following: CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight- Melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tensin C, TGF beta 2. TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin.

[0250] Embodiment 29. The method of any one of embodiments 1 to 27, wherein the target molecule is enriched in a tissue where blood cancer cells accumulate, which tissue is lymphoid tissue and / or bone marrow tissue.

[0251] Embodiment 30. The method of embodiment 29, wherein the target molecule is selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1.

[0252] Embodiment 31. The method of any one of embodiments 27 to 30, wherein the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a TCR, a nanobody, an Fc receptor, a growth factor receptor, a chemokine receptor, or a hormone receptor.

[0253] Embodiment 32. The method of any one of embodiments 27 to 30, wherein the tumor sensing receptor is a version of synthetic Notch (SynNotch), modular extracellular sensor architecture (MESA), or Tango technology, wherein the transcription activator is released from the intracellular domain of the receptor in response to binding of the extracellular domain to a target molecule.

[0254] Embodiment 33. The method of any one of embodiments 27 to 30, wherein the tumor sensing receptor is a chimeric antigen receptor, wherein the downstream transcription factor is activated through a signaling pathway activated in response to binding of the extracellular domain to the target molecule.

[0255] Embodiment 34. The method of any one of embodiments 26 to 33, wherein the nucleic acid comprises an expression cassette comprising a transcriptional control element operably linked to a DNA sequence encoding a functional copy of at least one molecule required for TCR signaling and T cell activation that was eliminated in step c), wherein binding of a transcriptional activator activated and / or released from a tumor sensing receptor to the transcriptional control element activates transcription of the functional copy of at least one molecule required for TCR signaling and T cell activation.

[0256] Embodiment 35. The method of embodiment 34, wherein the transcriptional control element is bound by a transcriptional activator selected from Gal4-VP64, Notch, tet transcriptional activator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs and eomesodermin (Eomes).

[0257] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the potency and specificity of tumor-activated alloreactive or xenoreactive T cells are modulated by controlling the affinity, specificity and expression level of tumor-sensing receptors or by controlling the mRNA or protein stability of key molecules for T cell activation.

[0258] Embodiment 37. The method of any one of embodiments 1 to 36, further comprising engineering the T cells to reduce the likelihood of being detected by the patient's immune system and eliminated by host-versus-graft (HvG).

[0259] Embodiment 38. The method of embodiment 37, wherein engineering the T cell comprises eliminating expression of an HLA allele that is mismatched between the T cell and the recipient by disrupting a gene encoding an HLA class I alpha heavy chain.

[0260] Embodiment 39. The method of embodiment 38, wherein disruption of the gene encoding the HLA class I alpha heavy chain uses a gene editing technology selected from CRISPR-Cas, transcription activator-like effector nuclease (TALEN), megaTALS, zinc finger nuclease or homing endonuclease.

[0261] Embodiment 40. The method of embodiment 37, wherein modifying the T cells comprises eliminating all HLA class I expression on the surface of the T cells by disrupting the gene encoding β2-microglobulin (β2M).

[0262] Embodiment 41. The method of embodiment 40, wherein the gene encoding β2-microglobulin is disrupted using a gene editing technology selected from CRISPR-Cas, transcription activator-like effector nuclease (TALEN), megaTALS, zinc finger nuclease or homing endonuclease, and further includes introducing HLA-G or HLA-E α chain fused to β2M using a non-viral or viral vector.

[0263] Embodiment 42. The method of any one of Embodiments 1 to 41, further comprising cryopreserving the tumor-activated T cells for later use.

[0264] Embodiment 43. A method of treating cancer in a patient by administering T cells prepared by the method of any one of Embodiments 1 to 42.

[0265] Embodiment 44. The method of embodiment 43, wherein T cells from different donors or generated using different stimuli are administered to the same patient.

[0266] Embodiment 45. The method of embodiment 43 or 44, wherein prior to administration of T cells, the patient is pretreated with IFNγ (interferon gamma) to upregulate the expression of HLA on tumor cells and stromal cells.

[0267] Embodiment 46. The method of embodiment 43 or 44, wherein prior to administration of the T cells, the patient receives lymphocyte-depleting radiation therapy or chemotherapeutic drug pretreatment to suppress the immune system.

[0268] Embodiment 47. A genetically modified T cell (or a population thereof), comprising:

[0269] (i) genetic disruption of at least one endogenous gene encoding a molecule essential for TCR signaling and T cell activation, (ii) an exogenous nucleotide sequence encoding a tumor sensing receptor that releases or activates a transcriptional activator in response to direct or indirect binding to a molecule enriched on tumor cells, in the tumor microenvironment, or in tissues where blood cancer cells accumulate, and (iii) an exogenous nucleotide sequence comprising an expression cassette that expresses a copy of the endogenous gene disrupted in (i) in response to the transcriptional activator released or activated in (ii).

[0270] Embodiment 48. The genetically modified T cells of embodiment 47, wherein at least one disrupted endogenous gene encoding a molecule necessary for TCR signaling and T cell activation encodes a transmembrane protein selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58, or encodes an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT and PDK1.

[0271] Embodiment 49. The genetically modified T cell of embodiment 47 or 48, wherein the tumor sensing receptor comprises (i) an extracellular domain that directly or indirectly binds to at least one target molecule enriched in tumor cells, tumor microenvironment, and tissues where blood cancer cells accumulate; and (ii) an intracellular domain that activates or releases a transcriptional activator in response to the binding of the extracellular domain to the target molecule.

[0272] Embodiment 50. The genetically modified T cell of embodiment 49, wherein the tumor sensing receptor is a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor or a Tango receptor.

[0273] Embodiment 51. The genetically modified T cells of embodiment 49 or 50, wherein the target molecule is enriched in tumor cells and / or tumor microenvironment, and is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanin Tumor-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, cross protein C, TGF beta 2. Select from TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin.

[0274] Embodiment 52. The genetically modified T cell of embodiment 49 or 50, wherein the target molecule is enriched in a tissue where blood cancer cells accumulate, and the tissue is lymphoid tissue and / or bone marrow tissue.

[0275] Embodiment 53. The genetically modified T cell of embodiment 52, wherein the target molecule is selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1.

[0276] Embodiment 54. The genetically modified T cell of any one of embodiments 49 to 53, wherein the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a TCR, a nanobody, an Fc receptor, a growth factor receptor, a chemokine receptor or a hormone receptor.

[0277] Embodiment 55. The genetically modified T cell of embodiment 47 or 49, wherein the tumor sensing receptor is a chimeric antigen receptor, wherein the downstream transcription factor is activated through a signaling pathway activated in response to binding of the extracellular domain to the target molecule.

[0278] Embodiment 56. The genetically modified T cell of any one of embodiments 47 to 55, wherein the transcriptional control element driving the expression of the disrupted endogenous gene copy is bound by a transcriptional activator selected from Gal4-VP64, Notch, tet transcriptional activator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs and eomesodermin (Eomes).

[0279] Embodiment 57. A pharmaceutical composition comprising an effective amount of the genetically modified T cells of any one of Embodiments 47 to 56 and a pharmaceutically acceptable carrier.

[0280] Embodiment 58. A kit comprising the genetically modified T cell of any one of embodiments 47 to 56 and instructional materials for using it in a method of treatment.

[0281] Embodiment 59. A method of treating cancer in a patient by administering the T cells of any one of embodiments 47 to 56 or the pharmaceutical composition of embodiment 56.

[0282] Embodiment 60. The method of embodiment 59, wherein T cells from different donors or generated using different stimuli are administered to the same patient.

[0283] Embodiment 61. The method of embodiment 59 or 60, wherein prior to administration of T cells, the patient is pretreated with INFγ to upregulate expression of HLA on tumor cells and stromal cells.

[0284] Embodiment 62. The method of embodiment 59 or 60, wherein prior to administration of the T cells, the patient receives lymphocyte-depleting radiation therapy or pretreatment with a chemotherapeutic drug to suppress the immune system.

[0285] Embodiment 63. Use of the genetically modified T cells of any one of embodiments 47 to 56 to treat cancer in a patient in need thereof. Embodiment 64. Use of the genetically modified T cells of any one of embodiments 47 to 56 to manufacture a medicament for treating cancer.

[0286] This article also provides the following implementation schemes:

[0287] Embodiment P1: A method for producing tumor-activated alloreactive or xenoreactive T cells, the method comprising: a. selecting a T cell sample from an HLA-mismatched donor individual or donor animal;

[0288] b. stimulating the T cells to drive the cells into the cell cycle (proliferation);

[0289] c. generating T cells that are unable to activate by eliminating the expression or function of at least one molecule that is critical for TCR signaling and T cell activation; and

[0290] d. Generate tumor-activated alloreactive or xenoreactive T cells by introducing a mechanism that recognizes molecules enriched on tumor cells or in the tumor microenvironment, and as a result of recognition, restore the expression or function of the molecules that were eliminated in the previous step, thereby restoring the ability of T cells to be activated by recognizing antigens through the TCR.

[0291] Embodiment P2: The method of Embodiment P1, wherein steps c) and d) are performed simultaneously or in reverse order.

[0292] Embodiment P3: The method of embodiment P1, wherein the T cell sample is from a donor individual whose HLA gene does not match the patient at a single site or multiple sites, and the sites are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 sites.

[0293] Embodiment P4: The method of embodiment P1, wherein the T cell stimulation step comprises culturing T cells with specific antibodies against CD3 and CD28.

[0294] Embodiment P5: The method of embodiment P1, wherein the T cell stimulation step comprises co-culturing the donor T cells with cells of the patient, wherein the patient cells comprise peripheral blood mononuclear cells, dendritic cells, tumor cells, or a mixture of these cells. Embodiment P6: The method of embodiment P1, wherein the T cell stimulation step comprises co-culturing the donor T cells with peripheral blood mononuclear cells, dendritic cells, or a mixture of these cells from another donor, wherein the other donor has at least one HLA allele that matches the patient but does not match the T cell donor.

[0295] Embodiment P7: The method of Embodiment P1, wherein the T cell stimulation step comprises co-culturing the donor T cells with a cell line expressing at least one patient HLA allele.

[0296] Embodiment P8: The method of embodiment P1, wherein the T cell stimulation step comprises co-culturing the donor T cells with an artificial surface, such as a plastic coated with a protein encoded by at least one of the patient's HLA alleles.

[0297] Embodiment P9: The method of embodiment P5, P6 or P7, wherein the stimulator cells (non-T cells) are irradiated or chemically treated to block cell proliferation.

[0298] Embodiment P10: The method of embodiment P1, wherein the step of generating inactivatable T cells comprises using gene editing technology (including CRISPR-Cas, transcription activator-like effector nucleases (TALENs), megaTALS, zinc finger nucleases and homing endonucleases) to destroy protein-encoding genes, thereby eliminating the expression of at least one protein that is critical for TCR signaling and T cell activation.

[0299] Embodiment P11: The method of embodiment P10, wherein proteins that are critical for TCR signaling and T cell activation include cell surface molecules CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58, and intracellular signaling molecules Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, NFAT, SLP76, PKCθ, NFκB, AKT and PDK1.

[0300] Embodiment P12: The method of embodiment P10, wherein T cells whose surface molecule expression has been eliminated are purified by staining the cells using specific antibodies against the surface molecules and then separating the antibody-free cells using techniques such as flow cytometry, magnetic beads and purification columns.

[0301] Embodiment P13: The method of embodiment P10, wherein live cell-specific DNA imaging technology including CRISPR LiveFISH is used to isolate T cells in which the expression of intracellular signaling molecules is eliminated.

[0302] Embodiment P14: The method of embodiment P10, wherein the purification of T cells having at least one molecule that is critical for TCR signaling and activation destroyed is based on the inability of the T cells to activate and proliferate after further stimulation through the TCR.

[0303] Embodiment P15: The method of embodiment P1, wherein the step of generating tumor-activated alloreactive or xenoreactive T cells comprises introducing nucleic acid of a tumor-sensing receptor into the T cells.

[0304] Embodiment P16: The method of embodiment P15, wherein the tumor sensing receptor comprises an extracellular domain that directly or indirectly binds to a molecule enriched on tumor cells or in the tumor microenvironment, and an intracellular domain that activates or releases a transcriptional activator in response to binding of the extracellular domain.

[0305] Embodiment P17: The method of embodiment P16, wherein the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a TCR, a nanobody, an Fc receptor, a growth factor receptor, a chemokine receptor or a hormone receptor.

[0306] Embodiment P18: The method of embodiment P15, wherein the tumor sensing receptor is based on synthetic Notch (SynNotch), modular extracellular sensor architecture (MESA) or Tango technology.

[0307] Embodiment P19: The method of embodiment P1, wherein the step of generating tumor-activated alloreactive or xenoreactive T cells comprises introducing into T cells a nucleic acid of an expression cassette, wherein the expression cassette comprises an operably linked transcriptional control element and a DNA sequence encoding the disrupted gene of embodiments P10 and P11, wherein binding of the transcriptional activator activated and / or released from the tumor sensing receptor in embodiment P16 to the transcriptional control element activates transcription of the disrupted gene.

[0308] Embodiment P20: The method of embodiment P1, wherein the potency and specificity of tumor-activated alloreactive or xenoreactive T cells are regulated by controlling the affinity, specificity and expression level of tumor-sensing receptors or controlling the mRNA or protein stability of key molecules for T cell activation.

[0309] Embodiment P21: The method of embodiment P1, wherein the tumor-activated alloreactive or xenoreactive T cells are further engineered to reduce the likelihood of being detected by the patient's immune system and eliminated by host versus graft (HvG).

[0310] Embodiment P22: A method of embodiment P21, wherein T cell expression of HLA alleles that do not match the patient is eliminated by destroying the gene encoding the HLA class I alpha heavy chain using gene editing technology (including CRISPR-Cas, transcription activator-like effector nucleases (TALENs), megaTALS, zinc finger nucleases, and homing endonucleases).

[0311] Embodiment P23: The method of embodiment P21, wherein the expression of all HLA class I on the cell surface is eliminated by destroying the gene encoding β2M using gene editing technology, and then introducing HLA-G or HLA-E α chain fused to β2M using a non-viral or viral vector.

[0312] Embodiment P24: The method of embodiment P1, wherein the tumor-activated alloreactive or xenoreactive T cells are cryopreserved for later use.

[0313] Embodiment P25: A method for treating cancer in a patient by administering T cells prepared according to the method of Embodiment 1.

[0314] Embodiment P26: The method of embodiment P25, wherein T cells from different donors or generated using different stimuli are administered to the same patient.

[0315] Embodiment P27: The method of Embodiment P25, wherein prior to administration of T cells, the patient receives INFγ pretreatment to upregulate expression of HLA on tumor cells and stromal cells.

[0316] Embodiment P28: The method of Embodiment P25, wherein prior to administration of T cells, the patient receives lymphocyte-depleting radiation therapy or chemotherapeutic drug pretreatment to suppress the immune system.

[0317] Embodiment P29: A genetically modified tumor-activated alloreactive or xenoreactive T cell.

[0318] Embodiment P30: The T cell of embodiment P29, comprising a genetic modification that disrupts the expression of at least one endogenous gene encoding a molecule that is critical for TCR signaling and T cell activation.

[0319] Embodiment P31: The T cell of embodiment P30, wherein at least one disrupted endogenous gene encodes a transmembrane protein or an intracellular signaling molecule.

[0320] Embodiment P32: The T cell of embodiment P31, wherein the destroyed endogenous gene is a transmembrane protein selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58.

[0321] Embodiment P33: The T cell of embodiment P31, wherein the disrupted endogenous gene is an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, NFAT, SLP76, PKCθ, NFκB, AKT, NcK and PDK1.

[0322] Embodiment P34: A pharmaceutical composition comprising an effective amount of the genetically modified tumor-activated T cells of Embodiment P29 and a pharmaceutically acceptable carrier.

[0323] Embodiment P35: A method of treating cancer in a patient by administering genetically modified tumor activated T cells of embodiment P29. Embodiment P36: The method of embodiment P35, wherein T cells from different donors or stimuli are administered to the same patient. Embodiment P37: The method of embodiment P35, wherein prior to administering the T cells, the patient receives INFγ pretreatment to upregulate HLA expression on tumor cells and stromal cells.

[0324] Embodiment P38: The method of Embodiment P35, wherein prior to administration of T cells, the patient receives lymphocyte-depleting radiation therapy or chemotherapy drug pretreatment to suppress the immune system.

[0325] Embodiment P39: A kit comprising the genetically modified tumor-activated T cells of embodiment P29 and instructional materials for using them in treatment methods.

[0326] Example

[0327] The present invention is further described in detail by the following experimental examples. These examples are for illustrative purposes only and should not be considered as limiting unless otherwise stated. Therefore, the present invention should not be interpreted as being limited to the following examples, but should be interpreted as covering any and all variations that appear due to the teachings provided herein.

[0328] Example 1: T cell alloreactivity to U266 myeloma cells

[0329] Alloreactive T cells were activated for 72 h with anti-CD3 / CD28 beads and expanded for another 5 days in medium supplemented with 100 U / ml recombinant human IL2. 5T cells and 2.5x10 4 U266 cells expressing luciferase were incubated at a 10:1 ratio in the absence or presence of 10 μg / ml CD8-specific monoclonal antibody SK1 for 16 h. TM The luciferase activity of the remaining viable U266-luciferase cells was determined using the luciferase assay system (Promega, Madison, WI). 4 The luciferase activity of each U266-luciferase cell was determined as the maximum activity. Specific killing was calculated as {1–(sample activity) / (maximum activity)}×100.

[0330] Data such as Figure 3 U266 is a cell line established from an IgE myeloma patient that expresses high levels of HLA class I (76). U266 cells were incubated with preactivated HLA class I-mismatched human CD8 + T cell co-culture resulted in significant killing of U266 cells. This killing was largely inhibited by the anti-CD8 antibody SK1, which has been shown to effectively block T cell activation through TCR-HLA class I interactions, in which CD8 plays a key role (77, 78).

[0331] Example 2: Reduced expression of the TCR / CD3 complex on mouse D10 T cells after knockout of CD3ζ using CRISPR-Cas9 The experiment was performed as follows. CD3ζ is required for the assembly and cell surface expression of the TCR / CD3 complex, which also includes the TCRα, TCRβ, CD3γ, and CD3ε chains. A guide RNA (gRNA) designed to target the extracellular domain of mouse CD3ζ was generated using GeneArt TM Precision gRNA synthesis kit (Invitrogen) was used to synthesize. gRNA and Cas9 ribonucleoproteins (RNPs) were introduced into D10 cells by electroporation-based Neon transfection system (Invitrogen). Cells were stained with specific antibodies against TCRβ and CD3ε and analyzed using flow cytometry.

[0332] Data such as Figure 4 CD3ζ was knocked out in more than 60% of D10 cells, as shown by the obvious reduction in the surface expression levels of TCRβ and CD3ε.

[0333] Example 3: Non-specific activation and expansion of primary T cells using anti-CD3 / CD28 beads. 500,000 purified human CD8 +T cells were cultured with human T activator CD3 / CD28 Dynabeads (Life Technologies) at a 1:1 cell to bead ratio in 48-well plates. Cells were cultured in OpTmizer medium (Thermo Fisher; CTSTM OpTmizerTM T cell expansion SFM, 6mM GlutaMAXTM, and 2% CTSTM Immune Cell SR) and supplemented with 10ng / ml recombinant human IL2 (rhIL-2; R&D Systems). Beads were removed after 3 days of stimulation.

[0334] T cells were expressed at 0.5x10 6 / ml to 2x10 6 The concentration of 100 μg / mL was maintained at 400 μg / mL, and the culture medium and rhIL2 were replaced every 1 to 2 days.

[0335] Example 4: Specific stimulation and expansion of alloreactive T cells using the human breast cancer cell line MDA-MB-231 The human breast cancer cell line MDA-MB-231 (ATCC) expresses relatively high levels of HLA-I (A02:17; A02:01; B41:01; B40:02; C17:01; C02:02) (79). Figure 5 , MDA-MB-231WT). To generate a negative control that does not express HLA-I, β2-microglobulin (β2m) was knocked out by transfecting cells with Streptococcus pyogenes (Sp) Cas9 nuclease V3 (IDT) and a single guide RNA (sgRNA; IDTHs.Cas9.B2M.1.AA; crRNA sequence: 5'-CGUGAGUAAACCUGAAUCUU-3' SEQ ID NO: 15). MDA-MB-231 cells with β2m knocked out showed extremely low levels of HLA-I expression ( Figure 5 , MDA-MB-231β2m KO).

[0336] To specifically stimulate and expand alloreactive T cells, 0.125 x 10 6 MDA-MB-231 cells were seeded in 48-well plates and cultured overnight. 0.75 x 10 6 Purified primary human CD8 T cells from HLA-I mismatched donors (A 34:02, A 68:02; B 07:02, B 57:03; C 07:01, C 07:02) were cultured in vitro and supplemented with 10 ng / ml recombinant human IL-2 and 1 μg / ml Ultra-LEAF TM OpTmizer for purification of anti-CD28 monoclonal antibody (Biolegend, clone CD28.2) TM(Thermo Fisher Scientific) medium. After 5 days of culture, T cells were transferred to flasks and cultured at 0.5x10 6 Up to 2x 10 6 The cells were cultured at a density of 10 cells / ml.

[0337] The data are shown in Figures 6A and 6B. CFSE (carboxyfluorescein diacetate) labeled T cells proliferate rapidly when co-cultured with WT MDA cells, but have limited proliferation when co-cultured with HLA-I knockout MDA cells. See Figures 6A and 6B, respectively. Example 5: Lentiviral transduction of primary human T cells to introduce anti-CD19 SynNotch and CD3ε expression cassettes The transfer vector pHR_PGK_antiCD19_synNotch_Gal4VP64 (Addgene Cat.#79125) was used to package the anti-CD19SynNotch lentiviral vector. This vector drives the expression of the anti-CD19 SynNotch receptor downstream of a constitutively active PGK-1 promoter. The anti-CD19 SynNotch receptor consists of an N-terminal MycTag followed by an extracellular single-chain variable fragment (scFv) specific for CD19, a Notch core sequence containing a transmembrane domain, and an intracellular domain containing the cleavable artificial transcription activator Gal4-VP64, which consists of the Gal4 DNA binding domain and the VP64 transcription activation domain (14, 55).

[0338] To prepare a lentiviral vector for the CD3ε expression cassette, the transfer vector pHR_Gal4UAS_IRES_mC_pGK_tBFP (Addgene #79123) (14, 55) was modified to insert the human CD3ε coding sequence downstream of the Gal4 binding site and remove the IRES-mCherry sequence. The human CD3ε coding sequence was codon-optimized so that the target-specific sequence of the gRNA used to knock out CD3ε could no longer recognize the CD3ε coding sequence in the expression cassette and allow Cas9 to cut it. The final transfer vector encodes a Gal4-CD3-PKG-BFP expression cassette (SEQ ID NO: 1), in which the expression of CD3ε is controlled by the Gal4 binding sequence and the expression of blue fluorescent protein (BFP) is driven by the constitutively active PGK-1 promoter ( Fig.15 ). Thus, expression of BFP serves as a marker for the presence of the expression cassette in the transduced cells.

[0339] Lentiviral vectors were packaged in Lenti-X 293T cells (Takara) and concentrated 100 times using lentiviral precipitation solution (ALSTEM). To transduce T cells, 50 μl of concentrated anti-CD19 SynNotch and Gal4-CD3-PKG-BFP expression cassette lentiviral vectors were added to T cells stimulated with anti-CD3 / CD28 beads or MDA-MB-231 cells on day 1 after stimulation. After incubation for 24 hours in the presence of 10 μg / ml protamine sulfate, the medium was replaced to dilute the protamine sulfate and 10 ng / ml rhIL-2 was added. When co-cultured with MDA-MB-231 cells, 1 μg / ml of anti-human CD28 antibody (Biolegend, clone CD28.2) was added. The expression of anti-CD19 synNotch was analyzed by staining with PE-labeled anti-MycTag antibody (Cell Signaling Technology, clone 9B11). The presence of the CD3ε expression cassette in T cells is marked by the expression of BFP.

[0340] As shown in Figures 7A and 7B, primary human CD8 + T cells, more than 50%, 20%, and 17% of the cells expressed Gal4-CD3-PKG-BFP, anti-CD19 SynNotch, and both at the same time, respectively. Example 6: Knockout of CD3ε using CRISPR-Cas9 and purification of CD3-KO cells by magnetic separation To knockout CD3ε expression, primary human CD8 + T cells were electroporated using the Neon transfection system (ThermoFisher). Specifically, 7.5 pmol of hCD3εAlt-R CRISPR-Cas9 single guide RNA (sgRNA, IDT#Hs.Cas9.CD3E.1.AC, crRNA sequence: 5'-agggcaugtcaauauuacug-3'; SEQ IDNO: 16) was incubated with 7.5 pmol of Alt-R Sp Cas9 nuclease V3 (IDT) in 5 μl buffer R for 15 minutes to form ribonucleoprotein (RNP). RNP was then mixed with 0.2x10 6T cells were mixed, placed in a 10 μl pointed tube, and electroporated using program #24 (1600v, 10ms, 3 pulses). As shown in Figures 8A and 8B, 3 days after electrotransfection, about 87% of T cells were negative for both CD3ε and TCRβ, indicating effective knockout of CD3ε and intracellular retention of the TCR / CD3 complex. In order to purify CD3-KO cells, CD3+T cells were removed using the EasySep Human CD3 Positive Selection Kit II (StemCell Technologies). After two rounds of removal using this kit, the purity of CD-KO cells reached more than 99% (Figure 8C). In order to determine whether the lack of CD3ε expression affects the proliferation potential of T cells, CD3ε was knocked out according to the above method, but CD3 was not removed. + T cells, mixed WT and CD3-KO T cells were cultured for 25 days.

[0341] like Fig. 9 As shown in Figure 7, the ratio of CD3-KO to WT T cells remained relatively stable over time, indicating that the proliferation potential of CD3-KO cells was similar to that of WT T cells. As shown in Figure 7C, CD3 knockout had no significant effect on the expression of SynNotch and the expression cassette in transduced T cells.

[0342] Example 7: CD3 knockout eliminates the allogeneic cytotoxic activity of T cells

[0343] To determine the cytotoxicity of alloreactive T cells and the effects of CD3 knockout, T cells from HLA mismatched donors (A29:02, A30:01; B35:01, B53:01; C04:01) were stimulated with MDA-MB-231 cells and electroporated to knock out CD3ε according to the method of Example 6. Wild-type (WT; complete CD3ε) or purified CD3-KO cells were co-cultured with MDA-MB-231 cells (MDA-MB-231-luci) expressing firefly luciferase at a ratio of 3:1 for 6 hours. MDA-MB-231-luci cells cultured alone were used as controls. The luciferase activity of the culture was determined using Bright-Glo reagent (Promega) and read on a Victor multi-well plate detector (PerkinElmer). The percentage of specific killing was calculated as 100x[1-(sample luciferase activity / control luciferase activity)].

[0344] like Fig.13 As shown, WT T cells exhibited more than 60% specific killing of MDA-MB-231 cells. In contrast, CD3-KO cells showed negative specific killing percentages. The negative values ​​were due to slightly higher luciferase activity in co-cultured cells than in control cells.

[0345] Example 8: Binding of anti-CD19 SynNotch restores CD3ε expression on CD3-KO T cells expressing anti-CD19 SynNotch and Gal4-CD3-PKG-BFP expression cassette and enables cells to respond to anti-CD3 stimulation To generate CD3-KO T cells expressing anti-CD19 SynNotch and Gal4-CD3-PKG-BFP expression cassette (CD3KO-19SN-εCS T cells), primary human CD8 + T cells were stimulated with MDA-MB-231 cells, transduced with a lentiviral vector encoding anti-CD19 SynNotch and Gal4-CD3-PKG-BFP expression cassettes, CD3ε was knocked out by electroporation, and CD3-negative T cells were purified as described in Examples 4, 5, and 6. To test the ability of cells to restore CD3 expression and respond to anti-CD3 stimulation, 200,000 CD3KO-19SN-εCS T cells were incubated with 80,000 WT MDA-MB-231 cells or MDA-MB-231 cells expressing human CD19 ( Fig.10 ) were stimulated for 16 hours in a 96-well tissue culture plate. T cells were then transferred to another well containing anti-CD3 / CD28 Dynabeads (Life Technologies) and incubated for 6 hours at a 1:1 beads to T cells ratio, with the addition of monensin (Biolegend) and FITC-labeled anti-human CD107a antibodies (Biolegend, clone H4A3). The beads were then removed, the cells were fixed in 4% formaldehyde, permeabilized with permeabilization buffer (0.5% BSA, 0.1% saponin in DPBS / sodium azide), stained with anti-human CD3 and anti-human IFNγ (Biolegend clone 4S.B3) antibodies, and analyzed using a NovoCyteQuanteon flow cytometer.

[0346] The data is shown in Figure 11, with a significant number of BFPs + CD3KO-19SN-εCS T cells restored CD3 expression in response to MDA-MB-231-CD19 cells, but not to WT MDA-MB-231 cells. Among T cells with restored CD3 expression, most cells degranulated, as indicated by positive CD107a staining (Figure 11A), and produced IFNγ (Figure 11B). The results indicate that the binding of anti-CD19 SynNotch on CD3KO-19SN-εCS T cells restored CD3 expression and the ability of the cells to be activated through the TCR / CD3 signaling pathway.

[0347] Example 9: CD3KO-19SN-εCS T cells against CD19 + Cis-allelochemical killing of target cells

[0348] To determine the cytotoxicity of alloreactive CD3KO-19SN-εCS T cells against target cells expressing CD19, primary CD8 + T cells were stimulated with MDA-MB-231 cells and transduced and electroporated to knock out CD3ε according to the methods of Examples 4, 5, and 6. The cells were sorted and CD3ε was collected. - BFP + MycTag + Cells were used as CD3KO-19SN-εCS T cells. T cells were co-cultured with MDA-MB-231 cells (MDA-MB-231-luci) expressing firefly luciferase or MDA-MB-231-CD19 cells (MDA-MB-231-CD19-luci) expressing firefly luciferase at a ratio of 1.5: 1 for 16 hours. MDA-MB-231-luci and MDA-MB-231-CD19-luci cells cultured alone were used as corresponding controls. The luciferase activity of the culture was determined using Bright-Glo reagent (Promega) and read on a Victor multi-well plate detector (PerkinElmer). The percentage of specific killing was calculated as 100x [1-(sample luciferase activity / control luciferase activity)].

[0349] like Fig.12 As shown, alloreactive CD3KO-19SN-εCS T cells exhibited significantly higher cytotoxicity against CD19-expressing MDA-MB-231 cells relative to WT cells. Combined with the data in Figure 11, the results indicate that alloreactive CD3KO-19SN-εCS T cells are able to restore TCR / CD3 expression in response to SynNotch binding and kill HLA-I mismatched target cells through alloreactivity.

[0350] Example 10: Stimulation and expansion of alloreactive T cells using patient PBMCs or MoDCs matured with TNF-α According to previously published protocols (80), peripheral blood mononuclear cells (PBMCs) were isolated from patients' leukocytes by density gradient centrifugation using Ficoll-Paque (MP Biomedicals, Aurora, OH, USA) and purified using an OpTmizer TMTo generate MoDCs, PBMCs from patients were cultured at a final concentration of 3 to 5 × 10 6 Cells / ml are resuspended in culture medium and incubated in standard tissue culture bottles at 37°C and 5% CO2 for 2 hours. Non-adherent cells are removed by strong blowing. The remaining adherent cells will be cultured in culture medium supplemented with 200ng / mL recombinant human GM-CSF (R&D Systems, Minneapolis, MN, USA) and 4ng / mL recombinant human interleukin IL-4 (R&D Systems). Fresh cytokines will be added every 2 to 3 days. In order to mature DCs, the culture medium will be replaced on the 6th day and 1100U / mL recombinant human TNF-α (R&D Systems) will be added for 24 hours. Before co-culturing with T cells, PBMCs and DCs will be irradiated (2500rad) using an RS2000 irradiator (Radsource). T cells will be purified from PBMCs of HLA-mismatched donors using the EasySep Human T Cell Enrichment Kit (StemCell Technologies). T cells will be labeled with 2.5 μM CFSE in labeling buffer (DPBS plus 5% FBS) for 5 minutes and washed three times with labeling buffer. To stimulate and expand alloreactive T cells, CFSE-labeled T cells will be mixed with PBMCs or mature DCs at a 1:1 ratio and cultured in the presence of 10 ng / ml rh IL2 (R&D Systems) for 4 days. Cells will be collected, stained with anti-CD3-APC, and stained for CD3 + CFSE 低 The cells are then further expanded in culture and used immediately for downstream genetic manipulations or cryopreserved for future use.

[0351] Example 11: Methods for in vivo functional studies of tumor-activated alloreactive T cells in the NSG mouse model To study the efficacy and toxicity of tumor-activated alloreactive T cells against tumors in vivo, MDA-MB-231 cells were used to culture in severely immunodeficient NSG mice. TM Mouse Subcutaneous tumors are formed. In order to simulate the heterogeneity of TAA expression in tumor tissue, tumors will be formed by mixing MDA-MB-231 cells expressing firefly luciferase and tumor antigen Her2 with MDA-MB-231 cells expressing only luciferase in different proportions. The development of the tumor will be monitored by imaging the luciferase activity of the tumor using IVIS Lumina LT imager (PerkinElmer). After the tumor becomes detectable, mice will be treated, including allogeneic reactive T cells activated by tumors (CD3KO-Her2SN-εCS T cells), traditional Her2-specific second-generation CAR T cells, T cells activated but not transformed with anti-CD3 / CD28 beads, or no treatment. Tumor-activated allogeneic reactive T cells will be prepared using primary human T cells from donors whose HLA-I does not match the HLA-I of MDA-MB-231 cells. T cells will be stimulated with MDA-MB-231 cells, transduced with lentiviral vectors encoding Her2-specific SynNotch and Gal4-CD3-PKG-BFP expression cassettes, and electroporated to knockout CD3ε as in Examples 4, 5, and 6. T cells will be administered intravenously (iv) and their efficacy in controlling tumor growth will be monitored by imaging tumor luciferase activity over time and comparing survival curves for each treatment. CD3KO-Her2SN-εCS T cells are expected to be the most effective in controlling tumor growth, followed by conventional Her2-specific second-generation CAR T cells, and finally anti-CD3 / CD28 bead-activated but unmodified T cells. To monitor GvH side effects caused by therapeutic T cells, T cell infiltration in non-tumor tissues such as skin, liver, lung, and heart will be examined by immunohistochemistry. GvH side effects will also be compared by treating tumor-free NSG mice with tumor-activated alloreactive T cells, conventional Her2-specific second-generation CAR T cells, anti-CD3 / CD28 bead-activated but unmodified T cells, or no treatment. Weight loss and survival will be monitored, and levels of serum inflammatory cytokines including INFγ, IL2, IL-12, IL-17, IL5, and TNF-α will be monitored and compared. It is expected that mice treated with CD3KO-Her2SN-εCS T cells will exhibit lower GvH levels than mice treated with conventional Her2-specific second-generation CAR T cells or anti-CD3 / CD28 bead-activated but unmodified T cells.

[0352] Example 12: Stimulation and expansion of alloreactive T cells using a cytokine cocktail to mature MoDCs pulsed with tumor cell lysate

[0353] To generate MoDCs, HLA-A2+ Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors by density gradient centrifugation using Ficoll-Paque (MP Biomedicals, Aurora, OH, USA) were cultured at 37°C in a humidified atmosphere of 5% CO2 for 10 min. 6 / cm 2 The cells were cultured at a density of 100 μg / mL in AIM V serum-free medium (Thermo Fisher) for 2 h. Non-adherent cells were removed by washing three times with warm DPBS without calcium chloride and magnesium chloride. Adherent cells were cultured in AIM V medium supplemented with 100 ng / mL recombinant human GM-CSF (Peprotech) and 100 ng / mL recombinant human IL-4 (Peprotech) for 3 days, and then the medium was replaced with fresh AIM V medium with the same concentrations of GM-CSF and IL-4. The cells were cultured for another 2 days, and loosely adherent or non-adherent immature DCs were harvested and transferred to another culture dish at 1 × 10 6 The concentration of cells / ml was cultured in AIM V medium supplemented with 100 ng / mL recombinant human GM-CSF and 100 ng / mL recombinant human IL-4. The next day, a maturation mixture was added to a final concentration of 10 ng / mL TNF-α (Peprotech), 10 ng / mL IL-1β (Peprotech), 10 ng / mL IL-6 (Peprotech) and 1 μg / mL PGE2 (Sigma). In addition, MDA-MB-231 tumor cell lysate was added to DCs at a ratio of 3:1. To prepare tumor cell lysate, MDA-MB-231 cells were heat shocked at 42°C for 25 minutes (45-47). The cells were collected, washed with phosphate-buffered saline (PBS), and in DPBS at 1×10 7 The cells were resuspended at a concentration of 10 cells / ml and then subjected to 5 cycles of freezing and thawing (using ethanol plus dry ice and a 56°C water bath). The solid residue was then removed by centrifugation at 1700 g for 5 minutes, and the supernatant was collected and used immediately as tumor cell lysate or stored at -80°C.

[0354] Two days after the addition of maturation cocktail and tumor cell lysate, mature MoDCs were analyzed by flow cytometry to detect the expression of surface markers CD14, CD83, and CD86. Fig.16 As shown, mature MoDCs showed increased expression levels of CD83 and CD86 compared with immature MoDCs.

[0355] Example 13: Expansion of alloreactive T cells by MoDCs and cytotoxicity of the expanded alloreactive T cells against tumor cells

[0356] To determine the ability of mature MoDCs pulsed with tumor lysate to expand alloreactive T cells, the MoDCs described in Example 12 were incubated with cells from HLA-A2 - Donors and T cells labeled with carboxyfluorescein diacetate (CFSE) were co-cultured at a ratio of 1:3 for 9 days. Significant T cell proliferation was observed ( Fig.17 ), CFSE - T cells represent expanded alloreactive T cells.

[0357] To determine whether the expanded alloreactive T cells kill HLA-A2 + To test the ability of MDA-MB-231 tumor cells, T cells were co-cultured with luciferase-expressing MDA-MB-231 cells (MDA-luci) or luciferase-expressing HLA-I-MDA-MB-231 cells (MDA-β2m-KO-luci) (β2m knockout, resulting in loss of HLA-I surface expression) at a ratio of 3:1 for 16 h. MDA-β2m-KO-luci cells were generated by transducing MDA-MB-231β2m KO cells with the pLX313 lentiviral vector encoding firefly luciferase ( Figure 5 ) and stable expressers were selected by hygromycin. The killing of MDA-MB-231 cells was determined by luciferase-based assay. Fig.18 Description.

[0358] like Fig.18 As shown, alloreactive T cells exhibited significant cytotoxicity against wild-type MDA-MB-231 cells, but had little effect on MDA-MB-231 cells that do not express HLA-I. These data demonstrate the alloreactive nature of the killing. Example 14: Knockout of CD3γ, CD3δ, CD3ζ, and CD8α using CRISPR-Cas9

[0359] To knock out the expression of CD3γ, CD3δ, CD3ζ, or CD8α in human T cells, primary human CD8 +T cells were stimulated with anti-CD3 / CD28 beads for 4 to 5 days and then electroporated using the Neon transfection system (ThermoFisher) to prepare four different cell populations, each with one knockout. Specifically, 7.5 pmol of single guide RNA (sgRNA) was incubated with 7.5 pmol of Alt-RSp Cas9 nuclease V3 (IDT) in 5 μl buffer R for 15 min to form ribonucleoprotein (RNP). The crRNA sequences of the sgRNA sequences used were: for CD3γ, Hs.Cas9.CD3G.1.AH (5'-GUAAUGCCAAGGACCCUCGA-3'; SEQ ID NO: 17); for CD3δ, Hs.Cas9.CD3D.1.AB (5'-CCCCUUCAAGAUACCUAUAG-3'; SEQ ID NO: 18); for CD3ζ, Hs.Cas9.CD247.1.AC (5'-GAUGGAAUCCUCUUCAUCUA-3'; SEQ ID NO: 19); for CD8α, Hs.Cas9.CD8A.1.AA (5'-GCUGCUGUCCAACCCGACGU-3'; SEQ ID NO: 20). RNPs were then mixed with 0.2x10 6 T cells were mixed, pipetted into a 10 μl tip tube, and electroporated using program #24 (1600 v, 10 ms, 3 pulses).

[0360] like Fig.19 As shown, more than 30% of T cells stained negative for both CD3ε and TCRβ 3 days after electroporation of CD3γKO, CD3δKO, and CD3ζKO. These data indicate effective knockout of CD3 and intracellular retention of the TCR / CD3 complex. Fig. 20 As shown, approximately 74% of T cells lost CD8 expression after CD8α KO, and as expected, the expression of TCR was not affected.

[0361] Example 15: CD3KO-19SN-εCS T cells against CD19 - Trans-allelochemical killing of target cells

[0362] To determine whether CD3KO-19SN-εCS T cells are activated in the presence of CD19 + Killing CD19 on target cells -To investigate the ability of target cells, CD3KO-19SN-εCS T cells were generated as described in Example 16, except that a lentiviral vector encoding CD19-specific SynNotch was used instead of a lentiviral vector encoding Her2-specific SynNotch. Briefly, primary CD8 + T cells were stimulated with MDA-MB-231 cells, transduced and electroporated to knockout CD3ε as described in Examples 4, 5, and 6. Cells were sorted and CD3ε was collected. - BFP + MycTag + To prepare target cells for trans-killing, MDA-MB-231 cells or MDA-MB-231 cells expressing CD19 (MDA-MB231-CD19) ( Fig.10 ) were mixed with luciferase-expressing MDA cells (MDA-MB-231-luci) at a 1:1 ratio. CD3KO-19SN-εCS T cells were added at a 3:1 ratio (T cells to total MDA cells) and co-cultured for 48 hours, and then the luciferase activity of the remaining viable MDA-MB-231-luci cells was measured. The mixture of MDA-MB-231 cells not co-cultured with T cells was used to determine the maximum luciferase activity.

[0363] like Fig.21 As shown, when mixed with MDA-MB-231-CD19 cells, significantly higher killing of MDA-MB-231-luci cells was observed than when mixed with MDA-MB-231 cells. Since MDA-MB-231-luci cells do not express CD19, the higher killing percentage is due to the alloreactivity restored by T cells through interaction with MDA-MB-231-CD19.

[0364] Example 16: Generation of Her2-specific SynNotch-based tumor-activated alloreactive T cells (CD3KO-Her2SN-εCS T cells)

[0365] The transfer vector pHR_PGK_antiHer24D5-8_synNotch_Gal4VP64 (Addgene Cat.#85425) was used as a lentiviral vector to package the Her2-specific SynNotch. The expression of the Her2-specific SynNotch receptor is driven by a constitutively active PGK-1 promoter. The Her2-specific SynNotch receptor consists of an N-terminal MycTag followed by a Her2-specific extracellular single-chain variable fragment (scFv), a Notch core sequence containing a transmembrane domain, and an intracellular domain containing the cleavable artificial transcriptional activator Gal4-VP64, which consists of the Gal4 DNA binding domain and the VP64 transcriptional activation domain (14, 55).

[0366] To prepare a lentiviral vector for the CD3ε expression cassette, the transfer vector pHR_Gal4UAS_IRES_mC_pGK_tBFP (Addgene #79123) (14, 55) was modified to insert the human CD3ε coding sequence downstream of the Gal4 binding site and remove the IRES-mCherry sequence. The human CD3ε coding sequence was codon-optimized so that the target-specific sequence of the gRNA used to knock out CD3ε could no longer recognize the CD3ε coding sequence in the expression cassette and allow Cas9 to cut it. The final transfer vector encodes a Gal4-CD3-PKG-BFP expression cassette (SEQ ID NO: 1), in which the expression of CD3ε is controlled by the Gal4 binding sequence and the expression of blue fluorescent protein (BFP) is driven by the constitutively active PGK-1 promoter ( Fig.15 ). Thus, the expression of BFP served as a marker for the presence of the expression cassette in the transduced cells. Lentiviral vectors for both the Her2-specific SynNotch and CD3ε expression cassettes were packaged in Lenti-X 293T cells (Takara) and concentrated 100-fold using a lentiviral precipitation solution (ALSTEM).

[0367] The following steps were used to generate Her-specific SynNotch-based tumor-activated alloreactive T cells. Step 1) T cell activation. On day 0, 0.75x10 T cells extracted from a healthy donor that was HLA-I mismatched with MDA-MB-231 cells were cultured. 6 Purification of primary human CD8 + T cells and 0.125x10 6 MDA-MB-231 cells were grown in 48-well plates using OpTmizer TM Culture medium supplemented with 10 ng / ml rhIL-2 and 1 μg / ml Ultra-LEAF TMPurify anti-CD28 monoclonal antibody (Biolegend, clone CD28.2). Step 2) Transduction. On day 1, 50 μl of concentrated lentiviral vectors expressing Her2-specific SynNotch and Gal4-CD3-PKG-BFP cassettes were added to the co-culture. Protamine sulfate was also added at a concentration of 10 μg / ml. On day 2, the medium was replaced with fresh medium containing 10 ng / ml rhIL-2 and 1 μg / ml anti-human CD28 antibody to dilute the protamine sulfate. On day 5, the expanded T cells were cultured at 10 6 / ml concentration transferred to a 6-well plate. Step 3) CD3ε knockout. On day 6, T cells were electroporated using the Neon transfection system (ThermoFisher) to introduce sgRNA and Cas9 against CD3ε, as described in Example 6. Step 4) Analysis and purification of CD3KO-Her2SN-εCS T cells. On day 9, cells were collected and stained with anti-CD3ε antibody to detect CD3 expression and with anti-MycTag (Cell Signaling Technology, clone 9B11) antibody to detect SynNotch expression. The presence of the CD3ε expression cassette in T cells is marked by the expression of BFP. CD3 - MycTag + BFP + The cells were sorted as CD3KO-Her2SN-εCS T cells.

[0368] As shown in Figures 22A and 22B, sorted CD3KO-Her2SN-εCS T cells showed negative or low expression of CD3ε, positive expression of MycTag, indicating Her2-specific SynNotch expression, and positive expression of BFP, indicating integration of the CD3ε expression cassette.

[0369] Example 17: Binding of anti-Her2 SynNotch restores CD3ε expression on CD3KO-Her2SN-εCS T cells and enables cells to respond to anti-CD3 stimulation

[0370] To test the ability of CD3KO-Her2SN-εCS T cells to restore CD3 expression and response to anti-CD3 stimulation, 200,000 CD3KO-Her2SN-εCS T cells generated as described in Example 16 were inoculated with 80,000 MDA-MB-231 cells knocked out for low-level endogenous Her2 expression (MDA-HerKO) or MDA-MB-231 cells transduced to express high levels of Her2 (MDA-Her2) ( Figures 23A-23C) were stimulated for 16 hours in 96-well tissue culture plates. MDA-HER2KO cells were generated by knocking out Her2 expression in MDA-MB-231 cells using CRISPR / Cas9 and sgRNA Hs.Cas9.ERBB2.1.AA(IDT) (crRNA sequence 5'-CAACUACCUUUCUACGGACG-3'; SEQ ID NO:21). MDA-Her2 cells were generated by transducing MDA-MB-231 cells with a pLVx lentiviral vector encoding Her2, and stable expressers were selected by hygromycin. T cells were then transferred to another well containing anti-CD3 / CD28 Dynabeads (Life Technologies), incubated at a 1:1 beads to T cell ratio for 6 hours, and monensin (Biolegend) and FITC-labeled anti-human CD107a antibody (Biolegend, clone H4A3) were added. The beads were then removed and the cells were fixed in 4% formaldehyde, permeabilized using permeabilization buffer (0.5% BSA, 0.1% saponin in DPBS / sodium azide), stained using anti-human CD3 and anti-human IFNγ (Biolegend clone 4S.B3) antibodies, and analyzed using a NovoCyte Quanteon flow cytometer.

[0371] The data are shown in Figures 24A and 24B. A significant number of BFP + CD3KO-Her2SN-εCS T cells restored CD3 expression in response to MDA-Her2 cells, but not to MDA-Her2KO cells. Among T cells with restored CD3 expression, most cells showed degranulation, as shown by positive CD107a staining (Figure 24A), and produced IFNγ (Figure 24B). The results indicate that CD3KO-Her2SN-εCS T cells are able to restore CD3 expression in response to Her2-specific SynNotch binding, thereby mediating T cell activation in response to anti-CD3 stimulation.

[0372] Example 18: CD3KO-Her2SN-εCS T cells against Her2 + To determine the alloreactive CD3KO-Her2SN-εCS T cells to Her2 +Cytotoxicity of target cells, CD3KO-Her2SN-εCS T cells generated according to the description of Example 16 were co-cultured with MDA-MB-231 cells (MDA-luci-Her2) (Figure 23) expressing firefly luciferase and Her2 at a T cell ratio of 1: 1 or 1.5: 1 for 16 hours. As a control, T cells were co-cultured with MDA-MB-231 cells (MDA-luci-Her2KO) (Figure 23) expressing luciferase and knocked out low-level endogenous Her2 expression. MDA-luci-Her2 cells and MDA-luci-Her2KO cells were generated by transducing MDA-Her2 and MDA-Her2KO cells with pLX313 lentiviral vectors encoding firefly luciferase, respectively, and stable expressers were selected by hygromycin. MDA-luci-Her2 and MDA-luci-Her2KO cells cultured alone were used as corresponding controls. The luciferase activity of the cultures was determined using Bright-Glo reagent (Promega) and read on a Victor multiwell plate reader (PerkinElmer). The percentage of specific killing was calculated as 100x[1-(sample luciferase activity / control luciferase activity)].

[0373] like Fig.25 As shown, the cytotoxicity of alloreactive CD3KO-Her2SN-εCS T cells to MDA-luci-Her2 cells was significantly higher than that to MDA-luci-Her2KO cells. Combined with the data of Figures 24A and 24B, the results indicate that alloreactive CD3KO-19SN-εCS T cells are able to restore TCR / CD3 expression in response to SynNotch binding and kill HLA-I mismatched target cells through alloreactive reactions.

[0374] Example 19: CD3KO-Her2SN-εCS T cells against Her2 - To determine the effect of CD3KO-Her2SN-εCS T cells in the presence of Her2 + Killing Her2 in target cells - Target cell capacity, using three types of MDA-MB-231 cells as target cells: Her2 - MDA-luci-Her2KO, Her2-MDA-Her2KO and Her2 +MDA-Her2 (Figures 23B and 23C). To prepare target cells for trans-killing, MDA-Her2-KO or MDA-Her2 was mixed with MDA-luci-Her2KO in a 1:1 ratio. CD3KO-Her2SN-εCS T cells generated as described in Example 16 were added in a ratio of 3:1 or 5:1 (T cells to total MDA-MB-231 cells) and incubated for 48 hours. The MDA-MB-231 cell mixture not co-cultured with T cells was used to determine the maximum luciferase activity. Fig.26 As shown, when mixed with MDA-Her2 cells, significantly higher killing of MDA-luci-Her2KO cells was observed than when mixed with MDA-Her2KO. Since MDA-luci-Her2KO cells do not express Her2, the higher killing percentage is due to the alloreactivity restored by T cells through interaction with MDA-Her2.

[0375] Example 20: Tumor-activated alloreactive T cells generated based on Her2-specific CAR and NFAT-driven CD3ε expression cassette (CD3KO-Her2CAR-εCS T cells)

[0376] The transfer vector z368-EF1a-4D5-2gCAR is used to package the lentiviral vector of Her2-specific CAR. The vector encodes a Her2-specific second-generation CAR (SEQ ID NO: 22) driven by the EF1α promoter. The Her2-specific CAR includes an N-terminal 4D5 single-chain variable fragment (scFv), followed by a CD8α linker, a transmembrane domain, a 4-1BB signaling domain, and a CD3ζ signaling domain containing three ITAMs.

[0377] To prepare a lentiviral vector of the NFAT-driven CD3ε expression cassette, the transfer vector pHR_Gal4UAS_IRES_mC_pGK_tBFP (Addgene #79123) (14, 55) was modified by inserting the human CD3ε coding sequence downstream of the Gal4 binding site, removing the IRES-mCherry sequence, and then replacing the five Gal4 binding sequences and the minimal promoter with four NFAT (4xNFAT) binding sequences and a synthetic minimal promoter. The human CD3ε coding sequence was codon-optimized so that the target-specific sequence of the gRNA used to knock out CD3ε could no longer function by recognizing the CD3ε coding sequence in the expression cassette and allowing Cas9 to cleave it. The final transfer vector encodes a 4xNFAT-CD3-PKG-BFP expression cassette (SEQ ID NO:23), in which the expression of CD3ε is controlled by the 4xNFAT binding sequence and the expression of blue fluorescent protein (BFP) is driven by a constitutively active PGK-1 promoter ( Fig.15 ). Therefore, the expression of BFP served as a marker for the presence of the expression cassette in the transduced cells. Both the Her2-specific CAR and the 4xNFAT-CD3-PKG-BFP expression cassette lentiviral vectors were packaged in Lenti-X 293T cells (Takara) and concentrated 100-fold using a lentiviral precipitation solution (ALSTEM).

[0378] The following steps are used to generate tumor-activated alloreactive T cells based on Her-specific CARs. Step 1) T cell activation. On day 0, 0.75x10 T cells from healthy donors that are HLA-I mismatched with MDA-MB-231 cells were cultured. 6 Purification of primary human CD8 + T cells and 0.125x10 6 MDA-MB-231 cells were grown in 48-well plates using OpTmizer TM Culture medium supplemented with 10 ng / ml rhIL-2 and 1 μg / ml Ultra-LEAF TM Purify anti-CD28 monoclonal antibody (Biolegend, clone CD28.2). Step 2) Transduction. On day 1, 50 μl of concentrated lentiviral vectors of Her2-specific CAR and 4xNFAT-CD3-PKG-BFP expression cassette were added to the co-culture. Protamine sulfate was also added at a concentration of 10 μg / ml. On day 2, the culture medium was replaced with fresh medium containing 10 ng / ml rhIL-2 and 1 μg / ml anti-human CD28 antibody to dilute the protamine sulfate. On day 5, the expanded T cells were cultured at 10 6 / ml concentration was transferred to a 6-well plate. Step 3) CD3ε knockout. On day 6, T cells were electroporated using the Neon transfection system (ThermoFisher) to introduce sgRNA and Cas9 against CD3ε, as described in Example 6. Step 4) Analysis and purification of CD3KO-Her2CAR-εCS T cells. On day 9, cells were collected and stained with anti-CD3ε antibody to detect CD3 expression. The cells were also stained with recombinant human Her2 protein and then stained with anti-Her2 / CD340 antibody (Biolegend) to detect the expression of Her2-specific CAR. The presence of the NFAT-driven CD3ε expression cassette in T cells is marked by the expression of BFP. CD3 - CAR + BFP + The cells were sorted as CD3KO-Her2CAR-εCS T cells.

[0379] As shown in Figures 27A and 27B, the sorted CD3KO-Her2CAR-εCS T cells showed negative or low expression of CD3ε, positive expression of Her2-specific CAR, and positive expression of BFP, indicating the integration of the NFAT-driven CD3ε expression cassette.

[0380] Example 21: Binding of anti-Her2 CAR restores CD3ε expression on CD3KO-Her2CAR-εCS T cells and enables them to respond to anti-CD3 stimulation To test the ability of CD3KO-Her2CAR-εCS T cells to restore CD3 expression and respond to anti-CD3 stimulation, 200,000 CD3KO-Her2CAR-εCS T cells generated as described in Example 20 were stimulated for 16 hours in 96-well tissue culture plates with 80,000 MDA-MB-231 cells with low levels of endogenous Her2 expression knocked out (MDA-HerKO) or MDA-MB-231 cells expressing Her2 at high levels (MDA-Her2) (Figure 23). The T cells were then transferred to another well containing anti-CD3 / CD28 Dynabeads (Life Technologies), incubated for 6 hours at a 1:1 ratio of beads to T cells, and monensin (Biolegend) and FITC-labeled anti-human CD107a antibodies (Biolegend, clone H4A3) were added. The beads were then removed, and the cells were fixed in 4% formaldehyde, permeabilized with permeabilization buffer (0.5% BSA, 0.1% saponin in DPBS / sodium azide), stained with anti-human CD3 and anti-human IFNγ (Biolegend clone 4S.B3) antibodies, and analyzed using a NovoCyte Quanteon flow cytometer. The results showed that CD3KO-Her2CAR-εCS T cells stimulated with MDA-Her2 had 50% more CD3ε compared to CD3KO-Her2CAR-εCS T cells stimulated with MDA-Her2KO. + cells, 4.4 times more CD3ε + CD107a + T cells and 4.1-fold more CD3ε + INFγ + Cells. See Figures 28A and 28B. These data indicate that, in response to Her2CAR binding, CD3KO-Her2CAR-εCS T cells are able to restore CD3 expression, thereby mediating T cell activation in response to anti-CD3 stimulation.

[0381] Example 22: In vivo functional studies of tumor-activated alloreactive T cells in the NSG-HLA-A2 / HHD mouse model A xenograft mouse tumor model will be used to determine the ability of tumor-activated alloreactive T cells to inhibit tumor growth, including tumors with homogeneous and heterogeneous tumor antigen expression. To evaluate the impact of tumor-activated alloreactive T cells on potential normal tissue damage, we will establish a model in the NSG-HLA-A2 / HHD mouse (Jax, #014570), a highly immunodeficient NSG mouse that expresses human HLA-A02 in addition to mouse MHC class I and class II molecules (81, 82). MDA-MB-231 cells form rapidly growing tumors in NSG mice (83), and xenograft models have been widely used to test T cell therapies against cancer (84-86). Studies have shown that expression of HLA-A02 enhances the expression of HLA-A02 by transplanted human HLA-A02 - T cell-induced GvHD is due to the superposition of alloreactivity and xenoreactivity (87). Therefore, these models serve as a sensitive platform for studying the normal tissue damage and GvHD that may be caused by tumor-activated alloreactive T cells, which are derived from HLA-A02 - Donor T cells generated, targeting HLA-A02 + MDA-MB-231 cells. CD3KO-Her2CAR-εCS and CD3KO-Her2SN-εCS tumor-activated alloreactive T cells generated by MDA-MB-231 stimulation or MoDC stimulation will be tested as described in Examples 16 and 20.

[0382] 1) Determine the ability of tumor-activated alloreactive T cells to inhibit the growth of homologous tumor antigen-expressing tumors. To establish the xenograft mouse model, a group of NSG-HLA-A2 / HHD mice were injected subcutaneously on the right back with 5x10 6 Her2 + MDA-luci-Her2 cells. On day 7 after injection, 1x10 6 Tumor-activated alloreactive T cells. As a negative control, mice were treated with T cells from the same donor activated with anti-CD3 / CD28 beads (ThermoFisher). To confirm that tumor suppression was due to alloreactivity of tumor-activated alloreactive T cells, mice were treated with Her2 + HLA -Mice with tumors established by MDA-β2m-KO-luci cells will be treated. Tumor progression will be assessed weekly by measuring tumor size using calipers and measuring luciferase emission from tumors using the Lumina LT IVIS in vivo imaging system (Perkin Elmer) after intraperitoneal injection of d-luciferin (GoldBio). The survival of mice will be recorded over time for Kaplan-Meier survival analysis.

[0383] 2) Determine the ability of tumor-activated alloreactive T cells to inhibit the growth of tumors expressing heterogeneous tumor antigens and compare the efficacy of tumor-activated alloreactive T cells with CAR T cells. + MDA-MB-231 cells and Her2 - MDA-luci-Her2KO cells were mixed at a ratio of 10:1, 1:1, and 1:10 to establish tumors. Two groups of mice were treated with tumor-activated alloreactive T cells or Her2-specific CAR T cells, and tumor progression and mouse survival were analyzed.

[0384] 3) Determine the extent of normal tissue damage and GvHD in mice treated with tumor-activated alloreactive T cells. + NSG-HLA-A2 / HHD mice bearing MDA-MB-231 tumors will be treated with tumor-activated alloreactive T cells as described above and monitored for GvHD for 6 months. As positive controls, mice will be treated with expanded but unmodified alloreactive T cells or expanded alloreactive T cells expressing Her2-specific CARs. All tumor-activated alloreactive T cells, unmodified alloreactive T cells, and CAR T cells were obtained from the same HLA-A02 -T cell generation from donors. The severity of GvHD will be assessed three times a week using a scoring system that combines five clinical parameters: weight loss greater than 10% of initial body weight, hunched posture, skin damage, dull hair, and diarrhea (88). Each of the five parameters will be scored as 0 (if missing) or 1 (if present). If weight loss exceeds 30% of initial body weight or reaches the maximum clinical grade (i.e., 5 / 5), the mice will be euthanized. In addition, histological analysis will be performed on small intestine, large intestine, liver, and skin samples of dead or euthanized mice. The small intestine and large intestine will be scored for six parameters, and the liver will be scored for seven parameters, as described by Cooke et al. (89). The skin will be scored for three parameters as described by Ferrara et al. (90). The presence of tumor-activated alloreactive T cells or CAR T cells in these tissues will also be recorded separately based on BFP expression and anti-CD3 staining. The disclosures of each patent, patent application, and publication cited herein are fully incorporated herein by reference.

[0385] Although the present embodiment has been described in detail and with reference to the above examples, it should be understood that various modifications can be made without departing from the spirit of these embodiments, and these modifications will be familiar to those skilled in the art. The embodiments of the appendix are intended to be interpreted as including all such embodiments and equivalent variations.

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[0487]

Claims

1. A genetically modified alloreactive or xenoreactive T cell comprising: (i) disruption of the expression of at least one endogenous gene encoding a molecule essential for T cell receptor (TCR) signaling and T cell activation by genetic means, (ii) an exogenous nucleotide sequence encoding a tumor-sensing receptor that releases or activates a transcriptional activator upon direct or indirect binding to a molecule that is enriched on tumor cells, present in the tumor microenvironment, or present in tissues where blood cancer cells accumulate, and (iii) an exogenous nucleotide sequence comprising an expression cassette which, in response to the transcriptional activator released or activated in (ii), expresses a copy of the endogenous gene disrupted in (i), The genetically modified T cells are alloreactive or xenoreactive.

2. The genetically modified T cells according to claim 1, wherein at least one destroyed endogenous gene encoding a molecule essential for TCR signaling and T cell activation encodes a transmembrane protein selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58, or encodes an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT and PDK1.

3. The genetically modified T cell of claim 1, wherein the tumor sensing receptor comprises (i) an extracellular domain that directly or indirectly binds to a target molecule that is enriched on at least one tumor cell, in the tumor microenvironment, and in tissues where blood cancer cells accumulate; and (ii) an intracellular domain that activates or releases a transcriptional activator in response to binding of the extracellular domain to the target molecule.

4. The genetically modified T cell of claim 3, wherein the tumor sensing receptor is a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, or a Tango receptor, whose intracellular transcription activator is released from the receptor in response to binding of the extracellular domain to a target molecule.

5. The genetically modified T cell of claim 3, wherein the tumor sensing receptor is a chimeric antigen receptor (CAR) with an intracellular ITAM domain, wherein an endogenous transcription factor is activated through a signaling pathway in response to the extracellular domain binding to a target molecule.

6. The genetically modified T cell of claim 1, wherein the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a TCR, a nanobody, an Fc receptor, a growth factor receptor, a chemokine receptor or a hormone receptor.

7. The genetically modified T cells according to claim 3, wherein the target molecule is enriched on tumor cells and / or in the tumor microenvironment, and is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanin, Tumor-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tensin C, TGF beta 2. Choose from TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin, or, The target molecule is enriched in tissues where blood cancer cells accumulate, which are lymphoid tissues and / or bone marrow tissues, and the target molecule is selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1.

8. The genetically modified T cell according to claim 1, wherein The expression cassette comprises a transcriptional control element operably linked to a copy of the disrupted gene of claim 1(i), wherein expression of the disrupted gene is driven by binding of a transcriptional activator selected from the group consisting of Gal4-VP64, Notch, tet transcriptional activator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs and eomesodermin (Eomes) to the transcriptional control element.

9. A method for producing tumor-activated alloreactive or xenoreactive T cells, the method comprising: a) selecting a T cell sample from a donor individual or a donor animal; b) optionally stimulating the T cell sample to proliferate; c) eliminating the expression or function of at least one molecule necessary for TCR signal transduction and T cell activation in T cells, thereby rendering the T cells incapable of activation; and d) engineering T cells to (i) express a recombinant receptor that specifically binds to a target molecule that is enriched on at least one tumor cell, in a tumor microenvironment, or in a tissue where blood cancer cells accumulate, wherein binding of the recombinant receptor to the target molecule releases or activates a transcriptional activator; and (ii) introducing an expression cassette such that the transcriptional activator in (i) drives the expression of the molecule eliminated in step c), thereby restoring the expression or function of the eliminated molecule, thereby restoring the ability of the T cell to be activated by recognizing an antigen through the TCR, thereby generating tumor-activated alloreactive or xenoreactive T cells.

10. The method according to claim 9, wherein step d) is performed before step c), step c) is performed before step d), or steps c) and d) are performed simultaneously.

11. The method of claim 9, wherein the T cell sample is from a donor individual and the donor individual has at least one HLA allele mismatch compared to the intended recipient, and at least one HLA allele mismatch is located at a locus selected from the following group: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1.

12. The method according to claim 9, wherein step b) comprises: (i) co-culturing donor T cells with cells of the intended recipient; (ii) co-culturing donor T cells with cells of a second donor that (1) has at least one HLA allele that matches the intended recipient and (2) is mismatched with the T cell donor; (iii) co-culturing donor T cells with at least one cell line that expresses at least one HLA allele of the intended recipient; (iv) co-culturing donor T cells with an artificial surface coated with at least one protein encoded by at least one HLA allele of the intended recipient.

13. The method of claim 9, wherein at least one molecule necessary for TCR signaling and T cell activation is a cell surface molecule selected from CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD8α, CD8β, LAT, TRIM, CD45, CD28, LFA-1, CD2, CD54, CD52, CD148 and CD58, or an intracellular signaling molecule selected from Lck, Zap70, calcineurin, PI3K, Fyn, PLCγ, SLP76, PKCθ, AKT and PDK1.

14. The method of claim 9, wherein step d) comprises introducing a nucleic acid encoding a tumor-sensing receptor into a T cell, wherein the tumor-sensing receptor comprises (i) an extracellular domain that directly or indirectly binds to a target molecule enriched on tumor cells, in a tumor microenvironment, or in a tissue where blood cancer cells accumulate; and (ii) an intracellular domain that activates or releases a transcriptional activator in response to the binding of the extracellular domain to the target molecule.

15. The method of claim 14, wherein the tumor sensing receptor is a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, or a Tango receptor, and in response to the extracellular domain binding to a target molecule, the intracellular transcription activator is released from the receptor.

16. The method of claim 14, wherein the tumor sensing receptor is a chimeric antigen receptor (CAR) with an intracellular ITAM domain, wherein the endogenous transcription factor is activated through a signaling pathway activated in response to binding of the extracellular domain to a target molecule.

17. The method of claim 9, wherein the extracellular domain of the tumor sensing receptor is a single chain variable fragment (scFv), a Fab fragment, a designed ankyrin repeat protein (DARPin), a nanobody, a TCR, an Fc receptor, a growth factor receptor, a chemokine receptor or a hormone receptor.

18. The method according to claim 9, wherein the target molecule is enriched on tumor cells and / or in the tumor microenvironment and is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-related Antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycosylneuraminic acid, NPC-IC, PDGF-Ra, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tensin C, TGF beta 2. Choose from TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, mesothelin, NKG2D, CD147, NKR2, B7H3 and vimentin, or, The target molecule is enriched in tissues where blood cancer cells accumulate, which are lymphoid tissues and / or bone marrow tissues, and the target molecule is selected from CD45, CD19, CD20, CD4, CD8, CD2, CCR4, CD58, CD28, CD23, CD69, CD25, CD33, CD123 and CCL-1.

19. The method of claim 9, wherein step d) comprises introducing an expression cassette comprising a transcriptional control element operably linked to a copy of the disrupted gene of claim 9(c), wherein expression of the disrupted gene is driven by binding of a transcriptional activator selected from the group consisting of Gal4-VP64, Notch, tet transcriptional activator, NFAT, AP-1, NFκB / Rel, NR4A1 (Nur77), T-Bet, IRF4, STATs and eomesodermin (Eomes) to the transcriptional control element.

20. A method of treating cancer in a patient by administering the T cells of claim 1.

21. The method of claim 20, wherein the T cells are tumor-activated alloreactive T cells prepared by the method of claim 9.

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