Compositions and methods for reducing the immunogenicity of cell therapy

Abstract

This application provides, in part, methods and compositions for reducing the immunogenicity of cell therapies (e.g., CAR-T cell therapies) using transporter associated with antigen processing inhibitors (TAPi) and oligonucleotides that reduce the expression of immunogenic proteins (e.g., MHC class I and class II).

Description

[Technical field]

[0001] [Related Applications] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 331,773, filed April 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] [Government support] This invention was made with Government support under Grant No. 5R01CA238268-03 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Adoptive cell therapies, such as chimeric antigen receptor (CAR) CAR-T cell therapy, have revolutionized cancer treatment. However, clinical trials have demonstrated that some patients develop humoral and cellular anti-CAR immune responses against the non-self components of the CAR, limiting the persistence and success of multi-dose administration of CAR-T cells. The potential for CAR-T cell rejection is even higher when allogeneic immune effector cell products are used. Summary of the Invention

[0004] This application discloses methods and compositions for reducing a subject's immune response to adoptive cell therapy. In some aspects, the disclosure relates to the discovery that a subject's immune response to adoptive cell therapy (e.g., CAR-T cells) can be reduced by modifying the cells of the adoptive cell therapy to express a transporter associated with antigen processing inhibitor (TAPi), which reduces expression of MHC class I. The disclosure further relates to the discovery that a subject's immune response can additionally or alternatively be reduced by reducing expression of MHC class II (e.g., using RNAi targeting MHC class II transactivator protein). The methods and compositions of the disclosure based on these discoveries do not require deep host immune suppression or complex gene editing, thus avoiding the drawbacks associated with previous methods that rely on such host immune suppression and / or gene editing. Furthermore, in some embodiments, CAR-T cells expressing TAPi and RNAi targeting MHC class II do not exhibit increased susceptibility of therapeutic immune effector cells (IECs) to NK cell-mediated rejection (compared to previous methods), a risk associated with current β2M knockout methods.

[0005] In some aspects, the application discloses a cell comprising: (i) a transporter associated with antigen processing inhibitor (TAPi) or a variant thereof; and (ii) an oligonucleotide complementary to a polynucleotide encoding an MHC class II transactivator protein or a variant thereof, the oligonucleotide being selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide.

[0006] In some aspects, the application discloses a cell comprising an oligonucleotide complementary to a polynucleotide encoding (i) a chimeric antigen receptor (CAR); and (ii) a transporter inhibitor associated with antigen processing (TAPi) or variant thereof; and / or (iii) an MHC class II transactivator protein or variant thereof, the oligonucleotide being selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some embodiments, the oligonucleotide is complementary to any one of SEQ ID NOs: 7-12. In some embodiments, the oligonucleotide is complementary to SEQ ID NO: 7.

[0007] In some embodiments, the TAPi or variant thereof reduces MHC class I expression. In some embodiments, the TAPi is a viral TAPi. In some embodiments, the TAPi is a herpes virus TAPi. In some embodiments, the TAPi is selected from the group consisting of herpes simplex virus (HSV) TAPi, human cytomegalovirus (HCMV) TAPi, or Epstein-Barr virus (EBV) TAPi. In some embodiments, the TAPi is selected from the group consisting of herpes simplex virus (HSV) ICP47 TAPi, human cytomegalovirus (HCMV) US6 TAPi, or Epstein-Barr virus (EBV) BNLF2a TAPi. In some embodiments, the TAPi comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1-3. In some embodiments, the TAPi comprises an amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the RNAi oligonucleotide is selected from the group consisting of siRNA, miRNA, or shRNA. In some embodiments, the RNAi oligonucleotide is an shRNA. In some embodiments, the shRNA comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the shRNA comprises the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell.

[0008] In some embodiments, the cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises (i) an extracellular target binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the extracellular target binding domain binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain. In some embodiments, the extracellular target binding domain binds to CD19. In some embodiments, the extracellular target binding domain is not derived from a human polypeptide sequence. In some embodiments, the extracellular target binding domain is derived from a mouse polypeptide sequence. In some embodiments, the extracellular target binding domain comprises a VH amino acid sequence having at least 85% identity to SEQ ID NO:39 and a VL amino acid sequence having at least 85% identity to SEQ ID NO:40.In some embodiments, the transmembrane domain is selected from the group consisting of the alpha chain of the T cell receptor, the beta chain of the T cell receptor, or the zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the intracellular signaling domain is selected from the group consisting of CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0009] In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 41 and a nucleic acid sequence having at least 85% identity to SEQ ID NO: 17 or 18. In some aspects, the application discloses a polynucleotide comprising a nucleic acid sequence encoding (i) a TAPi or a variant thereof and (ii) an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein. In some embodiments, the TAPi is a viral TAPi. In some embodiments, the TAPi or a variant thereof reduces expression of MHC class I. In some embodiments, the TAPi is a herpes simplex virus (HSV) TAPi. In some embodiments, the TAPi is selected from the group consisting of a herpes simplex virus (HSV) TAPi, a human cytomegalovirus (HCMV) TAPi, or an Epstein-Barr virus (EBV) TAPi. In some embodiments, the TAPi is selected from the group consisting of Herpes Simplex Virus (HSV) ICP47 TAPi, Human Cytomegalovirus (HCMV) US6 TAPi, or Epstein-Barr Virus (EBV) BNLF2a TAPi. In some embodiments, the TAPi comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-3. In some embodiments, the TAPi comprises an amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the oligonucleotide is complementary to any one of SEQ ID NOs: 7-12 or variants thereof. In some embodiments, the oligonucleotide is complementary to SEQ ID NO: 7 or variants thereof.

[0010] In some embodiments, the oligonucleotide is selected from the group consisting of an RNAi oligonucleotide or a CRISPR interference guide RNA. In some embodiments, the RNAi oligonucleotide is selected from the group consisting of an siRNA, an miRNA, or an shRNA. In some embodiments, the RNAi oligonucleotide is an shRNA. In some embodiments, the shRNA is encoded by a nucleic acid sequence comprising SEQ ID NO:13.

[0011] In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises (i) an extracellular target binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the extracellular target binding domain binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain. In some embodiments, the extracellular target binding domain binds to CD19. In some embodiments, the extracellular target binding domain is not derived from a human polypeptide sequence. In some embodiments, the extracellular target binding domain is derived from a mouse polypeptide sequence.

[0012] In some embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0013] In some embodiments, the intracellular signaling domain is selected from the group consisting of CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0014] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO: 17-18. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19 and the nucleic acid sequence of SEQ ID NO: 20, 22, or 24.

[0015] In some embodiments, the polynucleotide is a vector, optionally a lentiviral vector. In some aspects, this application discloses a polynucleotide comprising an shRNA of SEQ ID NO: 13. In some aspects, this application discloses a cell comprising a polynucleotide described herein. In some aspects, the cell comprises a polynucleotide described herein.

[0016] In some aspects, the application discloses a method of modifying the immunogenicity of a cell, comprising introducing into the cell an oligonucleotide complementary to a polynucleotide encoding a MHC class II complex subunit of any one of SEQ ID NOs: 7-12, wherein the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some aspects, the application discloses a method of reducing a subject's immune response to a cell therapy, comprising introducing into a cell of the cell therapy an oligonucleotide complementary to a polynucleotide encoding a class II MHC transactivator complex protein of any one of SEQ ID NOs: 7-12, wherein the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide.

[0017] In some embodiments, the method further comprises introducing a virus-derived transporter associated with antigen processing inhibitor (TAPi) or variants thereof to a cell of the cell therapy. In some embodiments, the method comprises introducing a polynucleotide described herein to a cell of the cell therapy. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the cell is allogeneic to the subject. In some embodiments, the cell therapy is CAR-T cell therapy. In some embodiments, the CAR-T cell therapy comprises anti-CD19 CAR-T cells. In some embodiments, the subject is a human subject. In some embodiments, the method reduces natural killer cell activation. In some aspects, this application relates to a method of treating cancer in a subject, comprising administering to the subject a cell described herein. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is selected from the group consisting of leukemia, lymphoma, and myeloma. In some embodiments, the hematological cancer is selected from the group consisting of acute lymphoblastic leukemia or mantle cell lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, brain cancer, liver cancer, renal cancer, lung cancer, breast cancer, prostate cancer, pharyngeal cancer, thyroid cancer, colon cancer, testicular cancer, and skin cancer. In some embodiments, the cancer expresses CD19. [Brief description of the drawings]

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0019] [Figure 1A]Figures 1A-1E show that lentiviral transduction of viral TAP inhibitors reduces cell surface expression of MHC class I and allogeneic responses in human primary T cells while avoiding overt NK cell and pre-existing antiviral T cell responses. Figure 1A shows the design of lentiviral constructs expressing viral TAPi or CRISPR guide RNA for β2M. [Figure 1B] Figure 1B shows reduced MHC class I cell surface expression, and NK cell cytotoxicity and degranulation were found by flow cytometric analysis of TAPi-transduced human primary T cells (n=3 donors) after co-incubation with NK cells. [Fig. 1C-1D] Figure 1C shows that T cell allogeneic and autologous responses to human primary TAPi-expressing or β2M KO T cells, as measured by proliferation of responder T cells in MLR reactions by flow cytometric analysis of CellTrace-labeled responder cells, were found to be severely reduced by clearance of MHC I cell surface levels. Figure 1D shows that co-expression of viral TAPi and HCMV pp65 in primary T cells strongly reduces NLV antigen presentation, as assessed by IFNγ secretion by NLV-specific CD8+ T cells upon co-incubation measured by IFNγ ELISA. [Figure 1E] Figure 1E shows that coincubation of viral TAPi-expressing primary T cells with autologous T cells from donors with pre-existing antiviral cellular immunity does not induce T cell responses as measured by IFNγ ELISPOT (asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). [Figure 2A-2B]Figures 2A-2G show that lentiviral transduction of shRNA targeting CIITA reduced MHC class II cell surface expression and allogeneic responses in human primary T cells, and combined with expression of EBV TAPi reduced both MHC class I and II, allowing escape of allogeneic T cell responses. Figure 2A shows the design of lentiviral constructs expressing shRNA targeting CIITA or CRISPR guides of CIITA. Figures 2B-2C show that flow cytometry analysis of human primary T cells expressing shRNA targeting CIITA found reduced MHC class II cell surface expression, but T cell transduction with shRNA CIITA3 maintained similar proliferation compared to UTD (n=3 donors). [Fig. 2C-2D] Figure 2C (above). Figure 2D shows that T cell allogeneic and autologous responses to human primary T cells expressing shRNA targeting CIITA were measured by proliferation of responder T cells in MLR reactions by flow cytometric analysis of CellTrace labeled responder cells, and that proliferation was found to be severely impaired by clearance of MHCII cell surface levels. [Fig. 2E-2F] Figure 2E is a schematic diagram of the various utilized lentiviral constructs combining EBV TAPi and shRNA CIITA3, and Figure 2F shows the expression of MHC class I and II in primary human T cells expressing EBV TAPi and / or shRNA targeting CIITA3 analyzed by flow cytometry analysis (n=3 donors). [Figure 2G] Figure 2G shows that T cell allogeneic and autologous responses to primary human T cells expressing EBV TAPi and / or shRNA CIITA3 were assessed by responder cell proliferation in MLR assays (asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). [Figure 3A]Figures 3A-3F show that stealth modifications to αCD19 CAR T cells do not alter tumor elimination efficacy and CAR T cell proliferation, while stealth αCD19 CAR T cells are able to escape CAR-mediated immune recognition by T cells in patients who received one or two infusions of αCD19 CAR T cells. Figure 3A is a schematic diagram of various lentiviral constructs based on αCD19 CAR without the combination of EBV TAPi and shRNA CIITA3. [Figure 3B] Figure 3B shows MHC class I and II expression analyzed in αCD19 CAR T cells without stealth technology, NK cell cytotoxicity after co-incubation with NK cells, and T cell proliferation. [Figure 3C] Figure 3C shows luciferase cytotoxicity assays of αCD19 CAR T cells with or without stealth technology performed on the ALL cell line NALM-6 and the Mantle cell line JeKo-1, showing similar tumor clearance in vitro. [Figure 3D] Figures 3D-3E show that NSG mice were implanted with NALM6 cells and treated with αCD19 CAR T cells with or without stealth technology or left untreated. At days 7 and 14 post-treatment, blood was drawn to assess CAR T cell proliferation and BLI images were taken to assess tumor burden. [Figure 3E] (the above) [Figure 3F] Figure 3F shows the results of IFNγ ELISpot assays performed on T cells from patients receiving FMC63-based αCD19 CAR T cell (Yescarta or Kymriah) products and autologous αCD19 CAR T cells with or without stealth technology to evaluate CAR-mediated T cell immunity, showing efficient evasion of CAR-mediated T cell immunity by stealth technology (asterisks indicate statistical significance compared to UTD - *: P ≦ 0.05; **: P ≦ 0.01; ***: P ≦ 0.001; ****: P ≦ 0.0001). [Figure 4A]Figures 4A-4C show eGFP expression in TAPi-expressing primary T cells, and evaluation of CD25 and CD69 expression of responder cells in MLR assays evaluating allogeneic and autologous responses to TAPi-expressing T cells. Figure 4A shows flow cytometry analysis of eGFP expression in TAPi-expressing T cells. [Figure 4B-4C] Figures 4B-4C show that flow cytometry analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using TAPi-expressing T cells and β2M KO T cells indicates reduced immune activation and allogeneic responses. [Figure 5A] Figures 5A-5F show eGFP expression in primary T cells expressing shRNAs targeting CIITA and / or EBV TAPi, and evaluation of CD25 and CD69 expression in responder cells in MLR assays evaluating allogeneic and autologous responses to T cells expressing shRNAs targeting CIITA and / or EBV TAPi. Figure 5A shows flow cytometry analysis of eGFP expression in T cells expressing shRNAs targeting CIITA. [Figure 5B-5C] Figures 5B-5C show that flow cytometry analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using T cells expressing shRNA targeting CIITA or CIITA KO T cells shows reduced immune activation and allogeneic responses. [Figure 5D] FIG. 5D shows flow cytometry analysis of eGFP expression in T cells expressing shRNAs targeting CIITA and / or EBV TAPi. [Fig. 5E-5F] Figures 5E-5F show that flow cytometry analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using T cells expressing shRNA targeting EBV TAPi and / or CIITA indicates reduced immune activation and allogeneic responses. [Figure 6A]Figures 6A-6E show that lentiviral transduction of viral TAP inhibitors reduces cell surface expression of MHC class I and allogeneic responses in human primary T cells while avoiding overt NK cell and pre-existing antiviral T cell responses. Figure 6A shows a schematic of the MHC class I pathway and the design of lentiviral constructs expressing CRISPR guides for viral TAPi or β2M. [Figure 6B] Figures 6B-6D show reduced MHC class I cell surface expression, and NK cell cytotoxicity and degranulation were found by flow cytometry analysis of TAPi-transduced human primary T cells (n=3 donors) after co-incubation with NK cells. [Figure 6C] (the above) [Figure 6D] (the above) [Figure 6E] Figure 6E shows that T cell allogeneic and autologous responses to human primary TAPi-expressing T cells or β2M KO T cells were measured by proliferation of responder T cells in MLR reactions by flow cytometric analysis of CellTrace-labeled responder cells, and that proliferation was found to be severely reduced by clearance of MHC I cell surface levels. [Figure 7A] Figures 7A-7D show that lentiviral transduction of shRNA targeting CIITA reduces MHC class II cell surface expression and allogeneic responses in human primary T cells. Figure 7A shows a schematic of the MHC class II pathway and the design of lentiviral constructs expressing shRNA targeting CIITA or CRISPR guides for CIITA. [Figure 7B] Figures 7B-7C show that flow cytometry analysis of human primary T cells expressing shRNA targeting CIITA found reduced MHC class II cell surface expression, but T cells transduced with shRNA CIITA3 maintained similar proliferation compared to UTD (n=3 donors). [Figure 7C] (the above) [Figure 7D]Figure 7D shows that T cell allogeneic and autologous responses to human primary T cells expressing shRNA targeting CIITA, as measured by proliferation of responder T cells in MLR reactions by flow cytometric analysis of CellTrace-labeled responder cells, were found to be severely reduced by clearance of MHC II cell surface levels (asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). [Figure 8A-8B] Figures 8A-8D show that lentiviral transduction of a combination of EBV TAPi and shRNA targeting CIITA reduces both MHC class I and II and avoids allogeneic T cell responses. Figure 8A shows a schematic diagram of the various utilized lentiviral constructs combining EBV TAPi and shRNA CIITA3. Figures 8B-8C show that MHC class I and II expression was analyzed by flow cytometry analysis (n=3 donors) in human primary T cells expressing EBV TAPi and / or shRNA targeting CIITA. [Figure 8C] (the above) [Figure 8D] Figure 8D shows that T cell allogeneic and autologous responses to primary human T cells expressing EBV TAPi and / or shRNA CIITA3 were assessed by responder cell proliferation in MLR assays. (Asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001) [Figure 9A-9B]Figures 9A-9F show that stealth modifications to αCD19 CAR T cells do not alter the in vitro properties of CAR T cells. Figure 9A shows a schematic diagram of various lentiviral constructs based on αCD19 CAR without the combination of EBV TAPi and shRNA CIITA3. Figure 9B shows the expression of MHC class I, MHC class II, EBV TAPi, and CIITA, Figure 9C shows the cytotoxicity of NK cells after co-incubation with NK cells, and Figure 9D shows that T cell proliferation was analyzed in αCD19 CAR T cells without stealth technology. [Fig. 9C-9D] (the above) [Figure 9E] Figure 9E shows flow cytometry analysis of CAR-T cell CD4:CD8 ratio and memory phenotype by CD45RA and CCR7 expression. [Figure 9F] Luciferase cytotoxicity assays of αCD19 CAR T cells, with or without stealth technology, were performed in the ALL cell line NALM-6 and the mantle cell line JeKo-1 and showed similar tumor clearance in vitro (asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). [Figure 10A-10B] Figures 10A-10H show that stealth modifications to αCD19 CAR T cells do not alter in vivo tumor clearance efficacy and CAR T cell proliferation. In Figures 10A-10C and 10E-10G, NSG mice were implanted with NALM6 or JeKo-1 cells and treated with αCD19 CAR T cells with or without stealth technology or left untreated. 14 days after treatment, blood was drawn to assess CAR T cell proliferation and BLI images were taken to assess tumor burden. [Fig. 10C-10D] (Figure 10C above) Figures 10D and 10H show survival rates shown by Kaplan-Meier curves (asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001) [Fig. 10E-10F] (the above) [Fig. 10G-10H] (the above) [Figure 11A-11B] Figures 11A-11E show that stealth modifications to αCD19 CAR T cells enable CAR T cells to evade CAR-mediated immune recognition by T cells from patients who received one or two infusions of αCD19 CAR T cells. To evaluate CAR-mediated T cell immunity, IFNγ ELISpot assays were performed with T cells from patients who received FMC63-based αCD19 CAR T cell (Yescarta or Kymriah) products and autologous αCD19 CAR T cells with or without stealth technology, demonstrating efficient evasion of CAR-mediated T cell immunity by stealth technology. Figure 11A shows swimmer plots of selected patient populations. Figure 11B shows a schematic diagram depicting the predicted results of the ELISpot assay. [Fig. 11C-11D] 11C-11D show heat maps and representative wells of the ELISpot assay. [Figure 11E] Figure 11E shows a histogram showing eGFP expression levels after sorting and a graph showing CAR-mediated T cell activation and anti-CAR responses from ELISpot assays (asterisks indicate statistical significance compared to UTD - *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). [Figure 12A] Figures 12A-12H show that stealth modifications to αCD19 CAR T cells enable evasion of allogeneic responses in vitro and lead to increased CAR T cell proliferation in an allogeneic in vivo model. Figure 12A shows that stealth technology prevents induction of allogeneic responses following co-incubation with allogeneic T cells, as measured by IFNγ ELIspot and flow-based cytotoxicity assays. [Figure 12B]In Figures 12B-12C, NSG mice were engrafted with αCD3 / αCD28 expanded allogeneic T cells (UTD ND2), inoculated with NALM6, and treated with αCD19 CAR T cells (ND1) with or without stealth technology or left untreated. Blood was taken 14 days after treatment to assess CAR T cell proliferation and tumor burden. [Figure 12C] (the above) [Figure 12D] FIG. 12D shows BLI images taken to assess tumor burden. [Fig. 12E-12F] In Figure 12E, NSG mice were transplanted with allogeneic T cells (UTD ND2) pulsed twice with irradiated PBMCs from a CAR T cell donor (ND1), expanded with the REP protocol, inoculated with NALM6, and treated with αCD19 CAR T cells (ND1) with or without stealth technology or left untreated. Figure 12F shows weekly blood draws (days 7 to 28) and CAR T cell proliferation assessed by flow cytometry. [Fig. 12G-12H] In Figure 12G, tumor burden was quantified by BLI and total release was graphed. Figure 12H shows survival plotted in Kaplan-Meier curves, indicating that models with allogeneic T cells expanded in REP can be followed longer before causing severe GVhD. (Asterisks indicate statistical significance compared to UTD - *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). [Figure 13A-13B] Figures 13A-13B show evaluation of CD25 and CD69 expression of responder cells in MLR assays evaluating allogeneic and autologous responses to TAPi-expressing T cells. Figures 13A-13B show flow cytometric analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using TAPi-expressing T cells and β2M KO T cells shows reduced immune activation and allogeneic responses. (Asterisks indicate statistical significance compared to UTD-*: P≦0.05; **: P≦0.01; ***: P≦0.001; ****: P≦0.0001). [Figure 14A-14B] Figures 14A-14B show evaluation of CD25 and CD69 expression of responder cells in MLR assays evaluating allogeneic and autologous responses to T cells expressing shRNA targeting CIITA. Figures 14A-14B show flow cytometric analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using T cells expressing shRNA targeting CIITA or CIITA KO T cells shows reduced immune activation and allogeneic responses. (Asterisks indicate statistical significance compared to UTD-*: P≦0.05; **: P≦0.01; ***: P≦0.001; ****: P≦0.0001). [Figure 15A-15B] Figures 15A-15B show evaluation of CD25 and CD69 expression of responder cells in MLR assays evaluating allogeneic and autologous responses to T cells expressing shRNAs targeting EBV TAPi and CIITA. Figures 15A-15B show flow cytometric analysis of CD25 and CD69 of allogeneic and autologous responder cells in MLR assays using T cells expressing shRNAs targeting EBV TAPi and CIITA showing reduced immune activation and allogeneic responses. (Asterisks indicate statistical significance compared to UTD-*: P≦0.05; **: P≦0.01; ***: P≦0.001; ****: P≦0.0001). [Figure 16] Figure 16 shows the survival curves of mice in the allogeneic in vivo model. NSG mice were transplanted with αCD3 / αCD28 expanded allogeneic T cells (Allo T cells), inoculated with NALM6, and treated with αCD19 CAR T cells with or without stealth technology or left untreated. Survival is shown by Kaplan-Meier curves. Mice died early due to graft-versus-host disease observed by hair loss and sclerosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] [Cell composition] In some aspects, the application discloses a cell comprising (i) a transporter inhibitor associated with antigen processing (TAPi) or a variant thereof; and (ii) an oligonucleotide complementary to a gene encoding a subunit of MHC class II (e.g., SEQ ID NO: 6) or an MHC class II transactivator protein gene (e.g., SEQ ID NO: 7-12), the oligonucleotide being selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some aspects, the application discloses a cell comprising (i) a transporter inhibitor associated with antigen processing (TAPi) or a variant thereof; and (ii) an oligonucleotide complementary to a polynucleotide encoding an MHC class II transactivator protein (e.g., SEQ ID NO: 7-12), the oligonucleotide being selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide.

[0021] As used herein, an MHC class II transactivator protein refers to a protein that regulates MHC class II transcription or a protein present in a protein complex that regulates MHC class II transcription. MHC class II transactivator proteins include, but are not limited to, CIITA (SEQ ID NO:7), RFX (SEQ ID NO:8), RFXANK (SEQ ID NO:9), CREB (SEQ ID NO:10), NFYA (SEQ ID NO:11), and / or NFYC (SEQ ID NO:12).

[0022] A "variant" or "variants thereof" as referred to herein is a sequence (e.g., a polypeptide or polynucleotide) that is substantially homologous to a native or reference sequence, but has a sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide encompasses sequences that encode variant proteins or fragments thereof that contain one or more additions, deletions, or substitutions of nucleotides when compared to the native or reference DNA sequence, but that retain the activity of the non-variant polypeptide. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by one of skill in the art.

[0023] A variant amino acid or DNA sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native sequence and a variant sequence can be determined, for example, by comparing the two sequences using computer programs (e.g., BLASTp or BLASTn with default settings) that are freely available for this purpose and are commonly used on the World Wide Web.

[0024] [Transporter associated with antigen processing inhibitors (TAPi)] Transporters associated with antigen processing (TAPs) are proteins involved in translocating antigenic peptides and loading angiogenic peptides into MHC class I (e.g., HLA A, B, and C) for antigen presentation to the immune system, as described, for example, in Lehnert, Elisa, and Robert Tampe. Frontiers in immunology (2017):10, which is incorporated by reference in its entirety. The term "transporter associated with antigen processing inhibitors (TAPi)" refers to molecules that inhibit the activity, expression, or function of TAPs, as described, for example, in Matschulla et al., Scientific Reports 7.1 (2017):1-13, which is incorporated by reference in its entirety. In some embodiments, TAPi inhibits the activity expression or function of MHC class I. In some embodiments, the TAPi reduces intracellular MHC class I expression by at least 30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%). In some embodiments, the TAPi reduces intracellular MHC class I expression by 50-90%, 50-95%, or 50-99%.

[0025] In some embodiments, the TAPi is a viral TAPi. In some embodiments, the TAPi is a herpes virus TAPi. In some embodiments, the TAPi is selected from the group consisting of herpes simplex virus (HSV) TAPi, human cytomegalovirus (HCMV) TAPi, Epstein-Barr virus (EBV) TAPi, Varicellovirus (TAPi), or poxvirus TAPi, e.g., as described in Matschulla et al., Scientific Reports 7.1 (2017): 1-13. In some embodiments, the TAPi is selected from the group consisting of ICP47 (herpes simplex virus type 1, HSV-1), US6 (human cytomegalovirus, HCMV), BNLF2a (Epstein-Barr virus, EBV), UL49.5 (Varicellovirus), and CPXV12 (poxvirus). In some embodiments, the TAPi is selected from the group consisting of Herpes Simplex Virus (HSV) ICP47 TAPi, Human Cytomegalovirus (HCMV) US6 TAPi, or Epstein-Barr Virus (EBV) BNLF2a TAPi. In some embodiments, the TAPi is BNFL2a (EBV). In some embodiments, the TAPi comprises an amino acid sequence that is at least 85% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical) to any one of SEQ ID NOs: 1-3. In some embodiments, the TAPi comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 or a variant thereof. In some embodiments, the TAPi consists of an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-3.

[0026] [Cells containing alternative methods for reducing MHC class I expression] In some embodiments, the cell comprises an oligonucleotide (e.g., an RNAi oligonucleotide) comprising a sequence complementary to TAP. In some embodiments, the cell comprises an oligonucleotide comprising a sequence complementary to a gene encoding a subunit of MHC class I (e.g., a beta2-microglobin sequence (SEQ ID NO:5) or a variant thereof, or an HLA-B sequence (SEQ ID NO:4) or a variant thereof). In some embodiments, the cell comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides, each comprising a sequence complementary to a gene encoding a subunit of MHC class I. In some embodiments, the oligonucleotide is an RNAi (e.g., an siRNA, miRNA, or shRNA), an ASO, or a CRISPR sequence (e.g., a CRISPR guide RNA sequence), as are well known in the art and described below.

[0027] [Oligonucleotides complementary to MHC class II] In some aspects, the cells comprise a nucleic acid sequence encoding an MHC class II (e.g., HLA DR / DP / DQ) or a variant thereof, or an oligonucleotide complementary to a nucleic acid sequence encoding an MHC class II transactivator protein, hi some aspects, the cells comprise an oligonucleotide complementary to a nucleic acid sequence encoding an MHC class II transactivator protein.

[0028] The term "complementary" as used herein refers to the degree of Watson-Crick base pairing between two polynucleotides (e.g., an shRNA and a target mRNA). For example, two polynucleotides can be 90% complementary if 9 / 10 nucleotides of each polynucleotide form Watson-Crick base pairs. In some embodiments, complementary refers to at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nucleotides of a first polynucleotide form Watson-Crick base pairs with a second polynucleotide. In some embodiments, an oligonucleotide can be sufficiently complementary to a target gene to reduce expression of the target gene. One of skill in the art will understand that oligonucleotides (e.g., RNAi oligonucleotides) used to reduce gene expression can include a first sequence designed to be complementary to a target gene sequence (e.g., an mRNA) and other sequences (e.g., sequences for processing) that are not complementary to the target gene sequence. Thus, when an oligonucleotide complementary to a gene (e.g., a gene encoding a subunit of MHC class II) is disclosed, complementarity refers to the region of the oligonucleotide that is designed to be complementary to that gene.

[0029] In some aspects, the cells comprising the TAPi comprise an oligonucleotide complementary to a nucleic acid sequence encoding MHC class II (e.g., HLA DR / DP / DQ). In some embodiments, the MHC class II is a mammalian MHC class II. In some embodiments, the MHC class II is a human MHC class II. In some embodiments, the MHC class II is a mouse MHC class II.

[0030] In some embodiments, the cell comprises an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein (e.g., any one of CIITA (SEQ ID NO: 7), RFX (SEQ ID NO: 8), RFXANK (SEQ ID NO: 9), NYFA (SEQ ID NO: 10), NYFC (SEQ ID NO: 11), NF-gamma, and CREB (SEQ ID NO: 12)) or a variant thereof. In some embodiments, the cell comprises an oligonucleotide complementary to any one of SEQ ID NOs: 7-12 or a variant thereof. In some embodiments, the cell comprises an oligonucleotide complementary to CIITA or a variant thereof. In some embodiments, the cell comprises an oligonucleotide complementary to SEQ ID NO: 7 or a variant thereof. In some embodiments, the cell comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more oligonucleotides, each of which comprises a sequence complementary to a gene encoding a subunit of MHC class II and / or an MHC class II transactivator protein (e.g., SEQ ID NOs: 7-12). In some embodiments, the cells contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides each comprising a sequence complementary to the gene encoding CIITA (SEQ ID NO:7).

[0031] In some embodiments, the oligonucleotide is an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference oligonucleotide. In some embodiments, administration of the oligonucleotide reduces expression of MHC class II in cells.

[0032] In some embodiments, the RNAi oligonucleotide is selected from the group consisting of siRNA, miRNA, shRNA, or any other suitable RNAi oligonucleotide.The method of constructing and using siRNA, miRNA, and shRNA oligonucleotides to reduce the expression of genes (e.g., MHC class I, MHC class II, or MHC class II transactivator protein) is well known in the art, for example, as described in Agrawal et al., Microbiology and Molecular Biology Reviews 67.4(2003):657-685, and Taxman et al., RNA Therapeutics.Humana Press, 2010.139-156, both of which are incorporated by reference in their entirety.

[0033] In some embodiments, the RNAi oligonucleotide is an shRNA. In some embodiments, the cell comprises an shRNA comprising a sequence complementary to any one of SEQ ID NOs: 7-12 or variants thereof. In some embodiments, the cell comprises an shRNA comprising a sequence complementary to a gene encoding CIITA (SEQ ID NO: 7) or a variant thereof. In some embodiments, the shRNA is encoded by a nucleic acid sequence comprising SEQ ID NO: 13 or a variant thereof. In some embodiments, the shRNA is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0034] In some embodiments, the oligonucleotide complementary to a nucleic acid sequence encoding an MHC class II or MHC class II transactivator protein is an antisense oligonucleotide (ASO). ASOs are well known in the art, for example, as described in Quemener, Anais M. et al. Wiley Interdisciplinary Reviews: RNA 11.5 (2020): e1594, which is incorporated by reference in its entirety. In some embodiments, the ASO is a DNA sequence. In some embodiments, the ASO DNA sequence is modified. In some embodiments, the ASO sequence comprises one or more modifications selected from the group consisting of phosphorothioate (PS) oligodeoxynucleotides, 2' methoxyethyl (2'-MOE), 2' constrained ethyl (2'cEt) modifications, 2'-MOE conjugated with N-acetylgalactosamine (GalNAc) and 2'cEt PS ASO.

[0035] In some embodiments, the oligonucleotide complementary to the nucleic acid sequence encoding the MHC class II or MHC class II transactivator protein is a CRISPR gRNA sequence (e.g., a CRISPR interference guide RNA sequence). Methods for using CRISPR interference and designing CRISPR interference guide RNA sequences are well known in the art, as described in Mohr, Stephanie E. et al. The FEBS Journal 283.17 (2016): 3232-3238, which is incorporated by reference in its entirety. In some embodiments, the oligonucleotide complementary to the nucleic acid sequence encoding the MHC class II or MHC class II transactivator protein is a CRISPR oligonucleotide. In some embodiments, CRISPR can be used to mutate the MHC class II or MHC class II transactivator protein. In some embodiments, the mutation is a loss-of-function mutation (e.g., a frameshift mutation or an early stop codon mutation). In some embodiments, the oligonucleotide complementary to a nucleic acid sequence encoding an MHC class II or MHC class II transactivator protein is a base editor oligonucleotide. In some embodiments, the base editor is an adenosine base editor or a cytosine base editor. In some embodiments, the base editor mutates the gene encoding the MHC class II or MHC class II transactivator protein. In some embodiments, the mutation is a loss-of-function mutation (e.g., a frameshift mutation or an early stop codon mutation).

[0036] [Chimeric Antigen Receptor (CAR)] In some embodiments, the cells comprising a TAPi and an oligonucleotide (e.g., an RNAi oligonucleotide) complementary to a nucleic acid sequence encoding an MHC class II transactivator protein described herein (e.g., SEQ ID NOs:7-12) or a variant thereof, further comprise a chimeric antigen receptor (CAR).

[0037] As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered T cell receptor that transfers ligand or antigen specificity into a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0038] CARs place a chimeric extracellular antigen binding domain that specifically binds to a target, e.g., a polypeptide, expressed on the surface of a cell (e.g., a T cell) targeted for an immune cell response on a construct that includes a transmembrane domain and an intracellular domain of a T cell receptor molecule. In some embodiments, the chimeric extracellular antigen binding domain comprises an antigen binding domain of an antibody reagent that specifically binds to an antigen expressed on a cell targeted for a T cell response. In some embodiments, the chimeric extracellular antigen binding domain comprises a ligand that specifically binds to an antigen expressed on a cell targeted for a T cell response.

[0039] As used herein, "CART cell," "CAR-T cell," or "CAR T cell" refers to a T cell expressing a CAR. When expressed in a T cell, a CAR has the ability to redirect the specificity and reactivity of the T cell toward a selected target in an MHC-unrestricted manner, utilizing the antigen-binding properties of a monoclonal antibody. MHC-unrestricted antigen recognition endows the CAR-expressing T cell with the ability to recognize antigens independent of antigen processing, bypassing a major mechanism of tumor escape.

[0040] In some embodiments, the CAR polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the CAR polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 17. In some embodiments, the CAR polypeptide consists of the amino acid sequence of any one of SEQ ID NO: 17. As can be determined by one of skill in the art, various functionally similar or equivalent components of these CARs can be exchanged or substituted for each other and for other similar or functionally equivalent components known in the art or listed herein.

[0041] [Extracellular antigen-binding domain] As used herein, the term "extracellular antigen binding domain" refers to a polypeptide found outside of a cell sufficient to facilitate binding to a target. An extracellular target binding domain specifically binds to its binding partner, i.e., the target. As non-limiting examples, an extracellular antigen binding domain may include the antigen binding domain of an antibody or antibody reagent, or a ligand that recognizes and binds to a cognate binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Cognate binding partners of a ligand useful in the methods and compositions described herein may generally be found on the surface of a cell. Ligand:cognate partner binding may result in a change in the receptor bearing the ligand or may activate a physiological response, such as activation of a signal transduction pathway. In some embodiments, the ligand may be non-native to the genome. In some embodiments, the ligand has a function that is conserved across at least two species.

[0042] Any cell surface moiety can be targeted by the CAR. Often the target is a cell surface polypeptide that can be differentially or preferentially expressed on the cells to which one wishes to target a T cell response. In some embodiments, the extracellular target binding domain can be any of the polypeptides described in, for example, PCT / US2020 / 065733, PCT / US2020 / 036108, PCT / US2018 / 013215, PCT / US2018 / 013213, PCT / US2018 / 027783, PCT / US2018 / 013221, PCT / US2018 / 022974, PCT / US2019 / 042268, PCT / US2019 / 038518, PCT / US2019 / 066357, PCT / US2019 / 066357, PCT / US2019 / 066358, PCT / US2019 / 066359 ... As described in US2019 / 013103, PCT / US2019 / 017727, PCT / US2020 / 051018, and / or PCT / US2018 / 013095, the antibody binds to any one of CD19, CD37, CD70, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain. In some embodiments, the extracellular target binding domain is non-human. In some embodiments, the extracellular target binding domain is murine. In some embodiments, the extracellular target binding domain binds to CD19. In some embodiments, the CD19 antibody is FMC63 (VH: SEQ ID NO: 39 or VL: SEQ ID NO: 40) or a variant thereof. In some embodiments, the extracellular target binding domain comprises a VH amino acid sequence that is at least 85% identical to SEQ ID NO:39 and a VL amino acid sequence that is at least 85% identical to SEQ ID NO:40.

[0043] In various embodiments, the CAR described herein comprises an antibody reagent or an antigen-binding domain thereof as an extracellular target binding domain. As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. In some embodiments, the antibody reagent may comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, the antibody reagent may comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, the antibody may comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. In some embodiments, the antibody reagent is a bispecific antibody reagent.

[0044] The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur. J. Immunol. 26(3):629-639, 1996, which is incorporated herein by reference in its entirety)) as well as complete antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as combinations of subtypes thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primates) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. In some embodiments, a CAR comprises an antibody reagent. In some embodiments, a therapeutic agent comprises an antibody reagent.

[0045] Fully human antibody binding domains can be selected, for example, from phage display libraries using methods known to those of skill in the art. Additionally, antibody reagents include single domain antibodies, such as camelid antibodies.

[0046] The VH and VL regions can be further divided into regions of hypervariability called "complementarity determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The extent of the framework regions and CDRs has been precisely defined (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. Mol. Biol. 196:901-917, 1987; each of which is incorporated herein by reference in its entirety). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0047] In some embodiments, the antibody or antibody reagent is not a human antibody or antibody reagent (i.e., the antibody or antibody reagent is murine), but is humanized. By "humanized antibody or antibody reagent" is meant a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to the antibody or antibody reagent variants that are naturally produced in humans. One approach to humanizing an antibody employs grafting murine or other non-human CDRs onto a human antibody framework.

[0048] In some embodiments, the extracellular target binding domain of the CAR comprises or consists essentially of a single chain Fv (scFv) fragment generated by fusing the VH and VL domains of an antibody, typically a monoclonal antibody, via a flexible linker peptide. In various embodiments, the scFv is fused to a transmembrane domain and a T cell receptor intracellular signaling domain, e.g., an engineered intracellular signaling domain as described herein. In another embodiment, the extracellular target binding domain of the CAR comprises a camelid antibody.

[0049] In some embodiments, the antibody reagent binds to a tumor associated antigen. Non-limiting examples of additional tumor antigens, tumor associated antigens, or other antigens of interest include activated fibroblast markers, CD19, CD37, BCMA (Tumor necrosis factor receptor superfamily member 17 (TNFRSF17); NCBI Gene ID: 608; NCBI Reference Sequence: NP001183.2 and mRNA (e.g., NCBI Reference Sequence: NM_001192.2)), CEA, immature laminin receptor, TAG-72, HPV E6 and E7, BING-4, calcium activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, 15her2 / neu, telomerase, EGFR, EGFRviii These include SAP-1, survivin, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-NMART-1, gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, BRCA1 / 2, CDK4, MART-2, p53, Ras, MUC1, TGF-BetaRII, IL-15, IL-13Ra2, and CSF1R. In some embodiments, the activated fibroblast marker comprises any one of αSMA (ACTA2), fibroblast activation protein (FAP), platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB), fibroblast-specific protein 1 (FSP1 / S100A4), endoglin (ENG), transgelin (TAGLN), tenascin-C (TNC), periostin (POSTN), chondroitin sulfate proteoglycan 4 or neuron-glial antigen 2 (CSPG4 / NG2), podoplanin (PDPN), or osteopontin (SPP1).

[0050] [Hinge and transmembrane domains] Each CAR described herein comprises a transmembrane domain, e.g., a hinge / transmembrane domain, that connects the extracellular antigen binding domain to the intracellular signaling domain. The binding domain of the CAR is optionally followed by one or more "hinge domains," which serve to position the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. The CAR optionally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8 (e.g., CD8 alpha), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be modified.

[0051] In some embodiments, the hinge region is derived from an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain comprises the CD8a hinge region.

[0052] As used herein, a "transmembrane domain" (TM domain) refers to a portion of a CAR that fuses the extracellular binding portion, optionally via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and the intracellular signaling domain), anchoring the CAR to the cell membrane of an immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR that passes through the cell membrane of the cell. The TM domain can be a transmembrane region or fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Although specific examples are provided herein and used in the examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternative embodiments of the technology. It is preferred that the selected transmembrane region or fragment thereof does not interfere with the intended function of the CAR.

[0053] When used in reference to a transmembrane domain of a protein or polypeptide, "fragment thereof" means a portion of the transmembrane domain sufficient to anchor or attach the protein to a cell surface.

[0054] In some embodiments, the transmembrane domain of a CAR described herein or a fragment thereof is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT In some embodiments, the transmembrane domain is selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0055] As used herein, "hinge / transmembrane domain" refers to a domain that includes both the hinge domain and the transmembrane domain. For example, the hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of a CAR or a fragment thereof is derived from or includes the hinge / transmembrane domain of CD8. CD8 is an antigen found preferentially on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a co-receptor for T cells. CD8 consists of alpha (CD8 alpha or CD8a) and beta (CD813 or CD8b) chains. CD8a sequences are known for many species, for example, human CD8a (NCBI Gene ID: 925) polypeptide (e.g., NCBI Reference Sequence NP_001139345.1) and mRNA (e.g., NCBI Reference Sequence NM_000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to, e.g., canine, feline, bovine, equine, porcine, etc. CD8.

[0056] Homologs and / or orthologs of human CD8 are readily identified for a species by one of skill in the art using, for example, the NCBI ortholog search function, or by searching available sequence data for a given species for sequences similar to the reference CD8 sequence.

[0057] In some embodiments, the hinge and transmembrane sequences correspond to the amino acid sequence of SEQ ID NO:25; or comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:25, or an amino acid sequence having <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions relative to any thereof.

[0058] [Costimulatory domain] Each CAR described herein optionally includes an intracellular domain or costimulatory domain of one or more costimulatory molecules. As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal required for efficient activation and function of T lymphocytes. The costimulatory domain can be, for example, a costimulatory domain of 4-1BB, CD27, CD28, or OX40. In one example, the 4-1BB intracellular domain (ICD) can be used (see, for example, below and SEQ ID NO: 26, or a variant thereof). Additional illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-induced costimulatory molecule and a key regulator of the immune response.

[0059] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. 4-1BB sequences are known for many species, for example human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 Number: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB may refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, for example, in veterinary applications, 4-1BB may refer to 4-1BB, for example, canine, feline, bovine, equine, porcine, etc. Homologs and / or orthologs of human 4-1BB are easily identified for such species by those skilled in the art, for example, using the NCBI ortholog search function or by searching available sequence data of a given species for sequences similar to the reference 4-1BB sequence.

[0060] In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. In some embodiments, the 4-1BB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO:26; or comprises a sequence selected from SEQ ID NO:26; or comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:26, or <1, ​​<2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions relative to SEQ ID NO:26.

[0061] [Intracellular signaling domain] The properties of the intracellular signaling domain of the CAR can vary, as known in the art and as disclosed herein, but the chimeric target / antigen binding domain sensitizes the receptor to signaling activation upon binding of the chimeric target / antigen binding domain to a target / antigen on the surface of a target cell.

[0062] With respect to intracellular signaling domains, so-called "first generation" CARs include those that provide only a CD3 zeta signal upon antigen binding. So-called "second generation" CARs include those that provide both costimulatory (e.g., CD28 or CD137) and activation (CD3 zeta) domains, and so-called "third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) and activation domains (e.g., CD3 zeta). In various embodiments, CARs are selected to have high affinity or avidity for a target / antigen - for example, target or antigen binding domains derived from antibodies generally have higher affinity and / or avidity for target antigens than naturally occurring T cell receptors. This property, combined with the high specificity that can be selected for antibodies, results in highly specific T cell targeting by CAR T cells.

[0063] The CAR described herein comprises an intracellular signaling domain. By "intracellular signaling domain" is meant the portion of a CAR polypeptide that is involved in transmitting the message of effective CAR binding to a target antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, such as release of cytotoxic factors to a target cell bound to the CAR, or other cellular responses elicited after antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is derived from CD3 zeta (see, e.g., below). Further non-limiting examples of intracellular signaling domains that contain immunoreceptor tyrosine-based activation motifs (ITAMs) that are particularly useful in the art include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 sigma, CD3 eta, CD3 epsilon, CD3 zeta; CD22, CD79a, CD79b, and CD66d.

[0064] CD3 is a T cell coreceptor that promotes T lymphocyte activation when engaged simultaneously with appropriate costimulation (e.g., binding of costimulatory molecules). The CD3 complex consists of four different chains; mammalian CD3 consists of the CD3 gamma chain, the CD3 delta chain, and two CD3 epsilon chains.

[0065] These chains associate with a molecule known as the T cell receptor (TCR) and CD3 zeta to generate an activation signal in T lymphocytes. The complete TCR complex includes the TCR, CD3 zeta, and the complete CD3 complex.

[0066] In some embodiments of any of the aspects, the CAR polypeptide described herein comprises an intracellular signaling domain that comprises an immunoreceptor tyrosine-based activation motif or ITAM from CD3ζ, including variants of CD3ζ, such as, for example, ITAM-mutated CD3ζ, CD3η, or CD3θ. In some embodiments of any of the aspects, the ITAM comprises an ITAM triad of CD3ζ (ITAM3). In some embodiments of any of the aspects, the ITAM triad of CD3ζ is not mutated and thus comprises a native or wild-type sequence. In some embodiments, the CD3ζ sequence comprises a CD3ζ sequence set forth in the sequences provided herein, e.g., SEQ ID NO:27, or a variant thereof.

[0067] For example, a CAR polypeptide described herein comprises an intracellular signaling domain of CD3zeta. In some embodiments, the CD3zeta intracellular signaling domain corresponds to the amino acid sequence of SEQ ID NO:27; or comprises a selected sequence of SEQ ID NO:27; or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:27, or an amino acid sequence having <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions relative to SEQ ID NO:27.

[0068] In some embodiments, the intracellular signaling domain comprises a 4-1BB intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB intracellular signaling domain and a CD3-zeta intracellular signaling domain. In some embodiments, the 4-1BB intracellular signaling domain corresponds to the amino acid sequence of SEQ ID NO:26; or comprises a selected sequence of SEQ ID NO:26; or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:26, or an amino acid sequence having <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions to SEQ ID NO:26.

[0069] The individual CAR and other construct components described herein can be used together with each other and substituted in the various constructs described herein, as can be determined by one of skill in the art. Each of these components can comprise or consist of any of the corresponding sequences described herein, or variants thereof.

[0070] A more detailed description of CARs and CAR T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated herein by reference in its entirety.

[0071] [Signal peptide] In some embodiments, the CAR polypeptides described herein include a signal peptide. The signal peptide can be from any protein that has an extracellular domain or is secreted. The CAR polypeptides described herein can include any signal peptide known in the art. In some embodiments, the CAR polypeptides include a CD8 signal peptide, for example, an amino acid sequence that corresponds to or includes the amino acid sequence of SEQ ID NO:28, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:28, or has <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions relative to SEQ ID NO:28. In some embodiments, the CAR polypeptide comprises an IgK signal peptide, e.g., an IgK signal peptide comprising an amino acid sequence that corresponds to or comprises the amino acid sequence of SEQ ID NO:29, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:29, or has <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 substitutions relative to SEQ ID NO:29.

[0072] In further embodiments, the CAR polypeptides described herein can optionally exclude one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO:28 or the IgK signal peptide of SEQ ID NO:29.

[0073] [Linker domain] In some embodiments, the CAR further comprises a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region of about 2 to 100 amino acids in length that links together any of the domains / regions of the CAR described herein. In some embodiments, the linker can comprise or consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. Linker sequences useful in the present invention can be 2 to 100 amino acids in length, 5 to 50 amino acids in length, 10 to 15 amino acids in length, 15 to 20 amino acids in length, or 18 to 20 amino acids in length, and include any suitable linker known in the art. For example, linker sequences useful in the present invention include, but are not limited to, glycine / serine linkers, such as GGGSGGGSGGGS (SEQ ID NO:31) and Gly4Ser(G4S) (SEQ ID NO:30); linkers such as (G4S)3(GGGGSGGGGSGGGGS (SEQ ID NO:32)) and (G4S)4(GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:33)); the linker sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:34) described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, the contents of which are incorporated herein by reference in their entirety; linker sequences of GGSSRSSSSGGGGSGGGG (SEQ ID NO:35) as described by Harb. Protoc. 2011(9), 2011; as well as linker sequences with added functionality, such as coding sequences containing Cre-Lox recombination sites or epitope tags, as described by Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000, the contents of which are incorporated herein by reference in their entirety. Longer linkers can be used when it is desirable to prevent two adjacent domains from sterically interfering with each other.

[0074] Further, the linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A and T2A (SEQ ID NO:36)), 2A-like linkers or functional equivalents thereof and combinations thereof.

[0075] For example, the P2A linker sequence may correspond to the amino acid sequence of SEQ ID NO:37. In various examples, linkers having sequences described herein or variants thereof are used. It should be understood that the indication of a particular linker in a construct at a particular position does not mean that only that linker can be used therein. Rather, different linker sequences (e.g., P2A and T2A) can be exchanged for one another (e.g., in the context of the constructs of the present invention) as can be determined by one of skill in the art. In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmIFV2A. Linkers such as these can be used in the context of polyproteins such as those described below. For example, they can be used to separate the CAR component of a polyprotein from an oligonucleotide that includes a sequence complementary to a gene encoding TAPi and / or MHC class II transactivator protein (e.g., shRNA complementary to CIITA).

[0076] [Reporter molecule] In some embodiments, the CARs described herein further comprise a reporter molecule, in some cases, for example, to allow for non-invasive imaging (e.g., positron emission tomography PET scans). In bispecific CARs that include a reporter molecule, the first and second extracellular binding domains can include different or the same reporter molecule. In bispecific CAR cells, the first and second CARs can express different or the same reporter molecule. In another embodiment, the CARs described herein further comprise a reporter molecule (e.g., hygromycin phosphotransferase (hph)) that can be imaged alone or in combination with a substrate or chemical (e.g., 9-[4-[18F]fluoro-3]-(hydroxymethyl)butyl]guanine ([18F]FHBG)). In another embodiment, the CARs described herein further comprise a nanoparticle that can be easily imaged using a non-invasive technique (e.g., gold nanoparticles (GNPs) functionalized with 64Cu2+). Labeling of CART cells for non-invasive imaging is reviewed, for example, in Bhatnagar et al., Integr. Biol. (Camb). 5(1):231-238, 2013, and Keu et al., Sci. Transl. Med. 18;9(373), 2017, which are incorporated herein by reference in their entireties.

[0077] In some embodiments, GFP and mCherry can be used as fluorescent tags to image CARs expressed on T cells (e.g., CART cells). It is expected that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, it is not necessary for the CAR to include a fluorescent tag or fluorescent protein. Thus, in each of the specific construct examples provided herein, any markers present in the construct can be removed. The present invention includes constructs with or without markers. Thus, when a specific construct is referred to herein, it can be considered to be included in the present disclosure with or without a marker or tag (e.g., including a histidine tag such as the histidine tag of HHHHHH (SEQ ID NO: 38)).

[0078] In some embodiments, the CAR comprises a CD8 leader sequence, an anti-CD19 antibody, a CD8 hinge / transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3-zeta intracellular signaling domain and a T2A peptide domain. In some embodiments, the CAR comprises a CD8 leader sequence, an FMC63 heavy and light chains, a CD8 hinge / transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3-zeta intracellular signaling domain and a T2A peptide domain. In some embodiments, the CAR comprises a CD8 leader sequence, an FMC63 heavy chain, a linker, an FMC63 light chain, a CD8 hinge / transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3-zeta intracellular signaling domain and a T2A peptide domain.

[0079] [Cell type] In some embodiments, the cell comprising (i) a transporter inhibitor associated with antigen processing (TAPi) or a variant thereof and (ii) an oligonucleotide complementary to a polynucleotide encoding an MHC class II or MHC class II transactivator protein is a eukaryotic cell. In some embodiments, the cell comprising (i) a transporter inhibitor associated with antigen processing (TAPi) or a variant thereof and (ii) an oligonucleotide complementary to a polynucleotide encoding an MHC class II transactivator protein is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cells are T cells, NK cells, NKT cells, lymphocytes such as B cells and T cells, and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cells are T cells.

[0080] In some embodiments, immune cells are obtained from a subject having or diagnosed as having cancer, a plasma cell disorder, or an autoimmune disease, modified as described herein (e.g., to include a TAPi, an anti-MHC class II oligonucleotide, and a CAR), and then administered to the subject.

[0081] In some embodiments, the cells are allogeneic cells. The term allogeneic cells refers to cells that are not derived from or extracted from the subject being treated (e.g., the cells are extracted or derived from another). In some embodiments, the allogeneic cells are derived from embryonic stem cells or induced pluripotent stem cells. In some embodiments, the allogeneic cells are extracted from a healthy subject. Because allogeneic stem cells are derived from another, the immunogenicity of the stem cells in the subject may be increased. Thus, the introduction of TAPi and / or anti-MHC class II oligonucleotides may reduce the immunogenicity of the allogeneic cells in the subject being treated.

[0082] In some embodiments, immune cells, e.g., T cells, can be modified to include any of the TAPi or MHC class I-complementary oligonucleotides described herein, and / or MHC class II-complementary oligonucleotides. In some embodiments, immune cells, e.g., T cells, can be modified to include any of the CAR polypeptides described herein or known in the art. For example, T cells can be isolated from peripheral blood taken from a donor or patient. T cells can be isolated from a mammal. Preferably, T cells are isolated from a human.

[0083] [Polynucleotides encoding TAPi and oligonucleotides] In some embodiments, the application discloses a polynucleotide comprising a first nucleic acid sequence encoding a TAPi, as described herein, and a second nucleic acid sequence encoding an oligonucleotide complementary to MHC class II (e.g., MHC class II shRNA). In some embodiments, the application discloses a polynucleotide comprising a first nucleic acid sequence encoding a TAPi, as described herein, and a second nucleic acid sequence encoding an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein (e.g., CIITA). In some embodiments, the polynucleotide further comprises a third nucleic acid sequence encoding a CAR, as described herein.

[0084] In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are each operably linked to a promoter. In some embodiments, the first nucleic acid sequence is operably linked to a first promoter, and the second nucleic acid sequence is operably linked to a second promoter. In some such embodiments, the third nucleic acid sequence is operably linked to a first promoter, a second promoter, or a third promoter. The promoter can be a constitutively expressed promoter (e.g., EF1a promoter) or an inducibly expressed promoter (e.g., NFAT promoter). In some embodiments, the promoter is induced by CAR activity or T cell receptor (TCR) activity.

[0085] In some embodiments, the expression of TAPi and CAR is driven by the same promoter, for example, a constitutively expressed promoter (e.g., EF1a promoter). In other embodiments, the expression of TAPi and CAR is driven by different promoters. The polynucleotide sequence encoding CAR can be located upstream of the polynucleotide sequence encoding TAPi, or the polynucleotide sequence encoding TAPi can be located upstream of the polynucleotide sequence encoding CAR. In some embodiments, the expression of an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein (e.g., CIITA) is driven by a promoter (e.g., U6 promoter) different from the expression of TAPi or CAR. In some embodiments, the oligonucleotide complementary to a gene encoding an MHC class II transactivator protein is located upstream of TAPi and CAR. In some embodiments, the oligonucleotide complementary to a gene encoding an MHC class II transactivator protein is located downstream of TAPi and CAR.

[0086] In some embodiments, the nucleic acid sequence encoding the TAPi, the nucleic acid sequence encoding an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein, and the nucleic acid sequence encoding the CAR are encoded within the same vector. In some embodiments, the nucleic acid sequence encoding the TAPi, the nucleic acid sequence encoding an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein, and the nucleic acid sequence encoding the CAR are encoded on two or three vectors.

[0087] In various examples, the vector is a retroviral vector. Retroviruses, such as lentiviruses, provide a convenient platform for delivering nucleic acid sequences encoding genes of interest or chimeric genes. A selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak, Pharmacological Reviews 2000 52:493-512, which are incorporated herein by reference in their entirety. A lentiviral system for efficient DNA delivery can be purchased from OriGene (Rockville, MD). In various embodiments, the protein is expressed in the T cell by transfection or electroporation of an expression vector comprising a nucleic acid encoding the protein, using vectors and methods known in the art. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector), an adenoviral vector, or an adeno-associated viral vector.

[0088] In some embodiments, the cells (e.g., CAR-T cells) described herein comprise any one of the polynucleotides described above.

[0089] Vectors and nucleic acid transfection or electroporation techniques are known in the art.

[0090] Efficient expression of TAPi and oligonucleotides comprising sequences complementary to genes encoding MHC class II transactivator proteins (e.g., shRNA complementary to CIITA) and / or CAR can be assessed using standard assays that detect mRNA, DNA, or gene products of nucleic acids encoding TAPi, oligonucleotides and / or CAR (and optional antibody reagents or cytokines), such as RT-PCR, FAGS, Northern blotting, Western blotting, ELISA, or immunohistochemistry.

[0091] [How to use] In some embodiments, the application discloses a method of modifying a cell, comprising introducing into the cell an oligonucleotide complementary to a polynucleotide encoding an MHC class II or MHC class II transactivator protein (e.g., CIITA, RFX, RFXANK, NYFA, NYFC, NF-γ, and CREB), wherein the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide.

[0092] In some embodiments, the application discloses a method of modifying the immunogenicity of a cell, comprising introducing into the cell an oligonucleotide complementary to a polynucleotide encoding an MHC class II or MHC class II transactivator protein (e.g., CIITA, RFX, RFXANK, NYFA, NYFC, NF-γ, and CREB), wherein the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some embodiments, the application discloses a method of modifying the immunogenicity of a cell, comprising introducing into the cell an oligonucleotide complementary to a polynucleotide encoding an MHC class II transactivator protein CIITA (SEQ ID NO: 7), wherein the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some embodiments, the application discloses a method for modifying the immunogenicity of a cell, the method comprising introducing into the cell an oligonucleotide complementary to a polynucleotide encoding the MHC class II transactivator protein CIITA (SEQ ID NO: 7), wherein the oligonucleotide is an RNA interference (RNAi) oligonucleotide (e.g., shRNA).

[0093] In some embodiments, the application discloses a method of reducing a subject's immune response to a cell therapy (e.g., an allogeneic cell therapy). In some embodiments, the method includes introducing (e.g., via electroporation) an oligonucleotide complementary to a polynucleotide encoding an MHC class II or MHC class II transactivator protein (e.g., CIITA, RFX, RFXANK, NYFA, NYFC, NF-γ, and CREB) into cells of the cell therapy prior to administering the therapy to the subject, where the oligonucleotide is selected from the group consisting of an RNA interference (RNAi) oligonucleotide, an antisense oligonucleotide (ASO), or a CRISPR interference (CRISPRi) oligonucleotide. In some embodiments, introducing the oligonucleotide includes introducing a vector encoding the oligonucleotide (e.g., a vector encoding an shRNA). In some embodiments, introducing the oligonucleotide includes directly introducing the oligonucleotide into the cell (e.g., transfecting an shRNA). In some embodiments, the oligonucleotide is any one of the oligonucleotides comprising a sequence complementary to MHC class II described herein. In some embodiments, the oligonucleotide is an shRNA. In some embodiments, the oligonucleotide is complementary to CIITA (SEQ ID NO:7). In some embodiments, the oligonucleotide comprises the sequence of SEQ ID NO:8.

[0094] In some embodiments, the method further comprises introducing a TAPi or variant thereof described herein into cells of the cell therapy prior to administration to the subject, hi some embodiments, the TAPi is an EBV TAPi (e.g., SEQ ID NO:3) or a variant thereof.

[0095] In some embodiments, the cell therapy is an immune cell therapy described herein. In some embodiments, the cell therapy is an allogeneic cell therapy described herein. In some embodiments, the cell therapy is an allogeneic immune cell therapy described herein.

[0096] In some embodiments, the cell therapy is a CAR-T cell therapy. In some embodiments, the cell therapy is an allogeneic CAR-T cell therapy. In some embodiments, the cell therapy includes any known CAR, including any CAR described herein. In some embodiments, the cell therapy includes an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR includes an amino acid sequence at least 85% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical) to SEQ ID NO:41. In some embodiments, the anti-CD19 CAR includes the amino acid sequence of SEQ ID NO:41. In some embodiments, the anti-CD19 CAR includes FMC63 VH and VL (e.g., SEQ ID NOs:39-40), or variants thereof.

[0097] In some embodiments, the subject is a human subject. In some embodiments, the method reduces natural killer cell activation.

[0098] In some aspects, the application discloses a method of treating a subject (e.g., a subject diagnosed with cancer) comprising administering a cell therapy comprising an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein described herein (e.g., shRNA targeting CIITA) and / or a TAPi. In some aspects, the application discloses a method of treating a subject (e.g., a subject diagnosed with cancer) comprising administering a cell therapy comprising an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein described herein (e.g., shRNA targeting CIITA) and a TAPi. In some aspects, the application discloses a method of treating a subject (e.g., a subject diagnosed with cancer) comprising administering a cell therapy comprising an oligonucleotide complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA) and an oligonucleotide complementary to a subunit of MHC class I (e.g., shRNA complementary to beta2-microglobulin).

[0099] In some embodiments, the subject is diagnosed with cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is selected from the group consisting of leukemia, lymphoma, and myeloma. In some embodiments, the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma (many subtypes), chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the blood cancer is selected from the group consisting of acute lymphoblastic leukemia or mantle cell lymphoma. In some embodiments, the hematological cancer is B-cell acute lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia / lymphoma (ALL / LBL), or B-cell lymphoma.

[0100] In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor cancer is selected from the group consisting of ovarian cancer, mesothelioma, brain cancer, liver cancer, kidney cancer, lung cancer, breast cancer, prostate cancer, pharyngeal cancer, thyroid cancer, colon cancer, testicular cancer, and skin cancer.

[0101] In some embodiments, the cancer is characterized by cells that express CD19.

[0102] [subject] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, such as house cats, canine species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Mammals other than humans may be advantageously used as subjects that represent animal models of disease, such as cancer. The subject may be male or female.

[0103] The subject may have previously been diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, or mesothelioma, among others), or one or more complications associated with such a condition, and in some cases may have already been treated for the condition or one or more complications associated with the condition.

[0104] Alternatively, the subject may also not have been previously diagnosed with such a condition or an associated complication, for example, the subject may be one who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or one who exhibits no risk factors.

[0105] A "subject in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed as having the condition, or is at risk for developing the condition.

[0106] Pharmaceutical Compositions As used herein, the term "pharmaceutical composition" means an active agent (e.g., a cell therapy described herein) in combination with a pharma- ceutical acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry.

[0107] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, the pharma- ceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, the pharma-ceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, the pharma-ceutically acceptable carrier can be an artificial or modified carrier, e.g., a carrier in which the active ingredient is not found naturally occurring.

[0108] In one aspect of the technology, the technology described herein relates to a pharmaceutical composition comprising an activated CART cell comprising a TAPi as described herein and an oligonucleotide comprising a sequence complementary to a gene encoding a MHC class II transactivator protein (e.g., shRNA complementary to CIITA), and optionally a pharma- ceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least an activated CART cell (e.g., comprising a CD19 CAR) comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding a MHC class II transactivator protein (e.g., shRNA complementary to CIITA) as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of an activated CART cell (e.g., comprising a CD19 CAR) comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding a MHC class II transactivator protein (e.g., shRNA complementary to CIITA) as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of activated CAR T cells comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA), as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffers, Ringer's solution, and serum components such as serum albumin, HDL, and LDL. Terms such as "additives," "carriers," "pharmaceutical acceptable carriers," "pharmaceutical acceptable additives," and the like, are used interchangeably herein.

[0109] In some embodiments, a pharmaceutical composition comprising activated CAR T cells comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA) as described herein can be in a parenteral dosage form. Because administration of a parenteral dosage form typically circumvents the patient's natural defenses against contaminants, components other than the CART cells themselves are preferably sterile or can be sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products dissolved or suspended in a pharma- ceutically acceptable vehicle for injection, suspensions for injection, and emulsions. Any of these can be added to the activated CAR T cell preparation prior to administration. Suitable vehicles that can be used to provide a parenteral dosage form of the disclosed activated CAR T cells are well known to those skilled in the art. Examples include, but are not limited to, saline; glucose solution; aqueous vehicles including, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0110] [Dosage] As the term is used herein, "unit dosage form" refers to a dosage amount suitable for single administration. By way of example, a unit dosage form can be a quantity of a therapeutic agent disposed in a delivery device, such as a syringe or an intravenous infusion bag. In some embodiments, a unit dosage form is administered in a single administration. In other embodiments, more than one unit dosage form can be administered simultaneously.

[0111] In some embodiments, the activated CAR T cells comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., an shRNA complementary to CIITA) described herein are administered as a monotherapy, i.e., no other treatment for the condition is administered concomitantly to the subject. The pharmaceutical compositions comprising the T cells described herein are generally administered within 10 4 From 10 9 Cells / kg body weight, in some cases 10 5 From 10 6 The T cell composition can be administered in a dosage of cells / kg body weight, including all integer values ​​within these ranges. If necessary, the T cell composition can be administered multiple times at these dosages. The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. Med. 319:1676, 1988).

[0112] In certain embodiments, it may be desirable to administer to a subject activated CART cells comprising a TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA), then subsequently redraw blood (or perform an apheresis), activate T cells therefrom as described herein, and reinfuse the patient with these activated and expanded T cells. This method may be performed multiple times, every few weeks. In certain embodiments, T cells may be activated from a blood draw of 35 10cc to 400cc. In certain embodiments, T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.

[0113] [Administration] In some embodiments, the methods described herein involve treating a subject having or diagnosed as having cancer, a plasma cell dyscrasia or disorder, or an autoimmune disease or disorder with any of the CAR polypeptides described herein, or a mammalian cell comprising a nucleic acid encoding any of the CAR polypeptides described herein. A CART cell comprising a TAPi described herein and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., an shRNA complementary to CIITA) includes a TAPi described herein and any of the oligonucleotides comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., an shRNA complementary to CIITA) and any of the CAR polypeptides described herein (and optional antibody reagents or cytokines) known in the art, or a mammalian cell comprising a TAPi and a gene encoding an MHC class II transactivator protein (e.g., an shRNA complementary to CIITA) or a nucleic acid encoding any of the CAR polypeptides described herein.

[0114] Subjects with a condition can be identified by a physician using this method of diagnosing the condition. Symptoms and / or complications of the condition that characterize and aid in diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that can help diagnose a condition include, but are not limited to, blood tests and bone marrow tests, which are known in the art for a given condition. Family history of a condition, or exposure to risk factors for a condition, can also help determine whether a subject is likely to have the condition or to make a diagnosis of the condition.

[0115] The compositions described herein may be administered to a subject having or diagnosed with a condition. In some embodiments, the methods described herein include administering to a subject an effective amount of activated CART cells comprising a TAPi described herein and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA) to alleviate symptoms of the condition. As used herein, "alleviating symptoms of a condition" refers to ameliorating any condition or symptoms associated with a condition. Such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique, as compared to an equivalent untreated control. Various means for administering the compositions described herein to a subject are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered to the site of a tumor.

[0116] The term "effective amount" as used herein refers to the amount of the cell therapy described herein (e.g., activated CAR T cells comprising TAPi and an oligonucleotide (e.g., shRNA complementary to CIITA) comprising a sequence complementary to a gene encoding an MHC class II transactivator protein) necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a cell preparation or composition to produce a desired effect. Thus, the term "therapeutically effective amount" refers to an amount of the cell therapy described herein that is sufficient to produce an effect against a particular condition when administered to a typical subject. An effective amount as used herein may also include, in various circumstances, an amount sufficient to slow the progression of a disease symptom, alter the course of a disease symptom (e.g., but not limited to, slow the progression of a condition), or reverse a condition symptom. Thus, it is generally not practical to specify an exact "effective amount." However, in any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0117] Effective doses, toxicity, and therapeutic effects can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals. Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. Therapeutically effective doses can be estimated initially from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a cell therapy (e.g., activated CART cells) comprising a TAPi and an oligonucleotide (e.g., shRNA complementary to CIITA) comprising a sequence complementary to a gene encoding an MHC class II transactivator protein, as described herein, that achieves half-maximal inhibition of symptoms) determined in cell culture or a suitable animal model. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay, for example, an assay for bone marrow biopsy, among others. Dosages will be determined by the physician and may be adjusted as necessary to meet observed therapeutic effects.

[0118] [Administration method] Methods of administration of the cell therapies described herein can include, for example, intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient intraarterially, intratumorally, intranodal, intraperitoneally, or intramedullary. In some embodiments, the compositions of T cells can be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0119] In some embodiments, the subject may undergo leukapheresis, where leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate cells of interest, e.g., T cells. In some embodiments, T cells may be extracted from a healthy subject, e.g., by leukapheresis, or differentiated in vitro (e.g., using iPSCs or embryonic stem cells). Any of these T cell isolates may be expanded by contact with an artificial APC, e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs, and processed to allow for the introduction of one or more polynucleotides of the technology (e.g., TAPi, an oligonucleotide complementary to the gene encoding CIITA, and a polynucleotide comprising a CAR), thereby generating CAR T cells. The subject in need thereof may then undergo standard treatment with high-dose chemotherapy, followed by a peripheral blood stem cell transplant. After or simultaneously with the transplant, the subject may receive an infusion of the expanded CAR T cells. In some embodiments, the expanded cells are administered before or after surgery. In some embodiments, lymphodepletion is performed on the subject prior to administration of one or more CART cells described herein. In such embodiments, lymphodepletion may include administering one or more of melphalan, 40 urbibi, cyclophosphamide, and fludarabine. The dosages of the above therapeutic agents administered to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration may be performed according to art-accepted practices.

[0120] In some embodiments, a single treatment regimen is required. In other embodiments, one or more subsequent doses or treatment regimen administrations may be performed. For example, after a treatment is administered every two weeks for three months, the treatment may be repeated once a month for six months or a year or more. In some embodiments, no additional treatment is administered after the first treatment.

[0121] The dosage of the compositions described herein will be determined by a physician and may be adjusted, if necessary, to the observed effects of the treatment. With regard to duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine whether the treatment is providing a therapeutic effect and decide whether to administer more cells, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so high as to cause side effects such as cytokine release syndrome. In general, dosage will vary according to the age, condition, and sex of the patient and may be determined by one of skill in the art. Dosage may also be adjusted by an individual physician if complications arise.

[0122] [Effectiveness] The efficacy of the cell therapy described herein (e.g., activated CART cells comprising TAPi and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA)) in, for example, treating a condition described herein or in inducing a response described herein (e.g., reduction in cancer cells) can be determined by a skilled clinician. However, if, following treatment according to the methods described herein, one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically acceptable symptoms are improved or even alleviated, or a desired response is induced, for example, by at least 10%, then the treatment is considered to be an "effective treatment" as that term is used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a condition treated according to the methods described herein, or any other measurable parameter as appropriate.

[0123] Treatment with the methods described herein can reduce the levels of a marker or symptom of a condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0124] Efficacy can also be measured by the individual not getting worse, as assessed by the need for hospitalization or medical intervention (i.e., progression of the disease is halted). Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes (1) suppressing the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., inducing regression of symptoms. An amount effective for treating a disease means an amount sufficient to effect effective treatment, as that term is defined herein, for that disease when administered to a subject in need thereof. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response. It is within the ability of one of skill in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. The efficacy of a given approach can be evaluated in animal models of the conditions described herein. When using experimental animal models, efficacy of treatment is demonstrated when a statistically significant change in the marker is observed.

[0125] [Cell therapy] One aspect of the technology described herein is a method of treating cancer, plasma cell disorder, or autoimmune disease in a subject in need of treatment, comprising modifying a T cell to include any of the TAPi described herein and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II protein or an MHC class II transactivator protein (e.g., shRNA complementary to CIITA) on the surface of the T cell, and any CAR polypeptide described herein (e.g., CD19 CAR) or known in the art; administering the modified T cell to the subject. In the case of cancer, the method can be for treating a diagnosed cancer, preventing recurrence of cancer, or for use in an adjuvant or neoadjuvant setting. In some embodiments, the method includes providing a T cell modified to include any of the CAR polypeptides described herein or known in the art on the surface of the T cell; modifying the T cell to include any of the TAPi described herein and an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., shRNA complementary to CIITA); administering the modified T cell to the subject.

[0126] One aspect of the technology described herein relates to a method of treating cancer, a plasma cell disorder, or an autoimmune disease in a subject in need of such treatment, comprising administering any of the TAPi described herein, an oligonucleotide comprising a sequence complementary to a gene encoding an MHC class II transactivator protein (e.g., an shRNA complementary to CIITA), and a mammalian cell comprising any of the CAR polypeptides described herein or known in the art. In some embodiments of any of the aspects, the engineered CAR-T cells are stimulated and / or activated prior to administration to the subject.

[0127] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior art or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0128] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order, or may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide still further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, considerations of biological function equivalence allow for some modifications to protein structures without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0129] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0130] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. TIFF2025513894000002.tif244170TIFF2025513894000003.tif251170TIFF2025513894000004.tif251170TIFF2025513894000005.tif251170TIFF202 5513894000006.tif252170TIFF2025513894000007.tif252170TIFF2025513 894000008.tif251170TIFF2025513894000009.tif252170TIFF20255138940 00010.tif251170TIFF2025513894000011.tif252170TIFF2025513894000012.tif252170TIFF2025513894000013.tif251170TIFF2025513894000014.t if251170TIFF2025513894000015.tif250170TIFF2025513894000016.tif252170TIFF2025513894000017.tif247170TIFF2025513894000018.tif176170

[0131] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior art or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0132] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order, or may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide still further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, considerations of biological function equivalence allow for some modifications to protein structures without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0133] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0134] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. EXAMPLES

[0135] Example 1. Expression of a viral TAP inhibitor in primary T cells results in a reduction in cell surface levels of MHC class I Herpesviruses are a class of chronic viruses that infect a variety of human cells and somehow evade T cell immunity. They express TAP, a protein required for the transport of cytoplasmic peptides across the endoplasmic reticulum and loading them for presentation on MHC class I molecules at the cell surface. 13 Cells that naturally or experimentally lack expression of a functional TAP complex exhibit dramatically reduced levels of surface MHC I and are CD8 + Greatly reduces their susceptibility to T cells 14This disclosure is directed, in part, to the discovery that forced expression of a viral TAP inhibitor (TAPi) reduces MHC I expression in genetically modified cells, thereby preventing cell-mediated immune responses to foreign transgenes. To test whether expression of herpesvirus TAPi reduced surface MHC I expression in primary T cells, bicistronic lentiviral constructs were generated to express herpes simplex virus (HSV) ICP47, human cytomegalovirus (HCMV) US6, or Epstein-Barr virus (EBV) BNLF2a TAPi along with the fluorescent reporter eGFP as a transduction marker (Figure 1A). Lentiviral constructs expressing sgRNA for β-2-microglobulin (β2M) with or without electroporation with Cas9 mRNA were used as positive (β2M KO) or negative (β2M-) controls. Primary human T cells consistently expressed eGFP upon transduction with lentiviral vectors ( Fig. 4A ), and TAPi-transduced cells displayed reduced levels of surface MHC I without affecting the upregulation of MHC class II upon activation ( Fig. 1B ).

[0136] MHC I expression inhibits targeting by NK cells 15 We investigated the effect of MHC I downregulation on susceptibility to NK cell killing. 12Similarly, β2M KO T cells were susceptible to autologous NK cell lysis and induced NK cell degranulation, as measured by CD107a expression. Importantly, T cells expressing viral TAPi did not significantly induce NK cell lysis or degranulation compared to untransduced (UTD) T cells (Figure 1B). Similarly, MHC I expression mediates allogeneic T cell responses due to mismatches between MHC and TCR. To measure the allogeneic response of TAPi-expressing T cells, a mixed lymphocyte reaction (MLR) was performed. Transduced T cells were incubated with autologous or allogeneic labeled responder T cells in the presence or absence of MHC I and II blocking antibodies. Activation of responder T cells was measured by proliferation (Figure 1C) and changes in CD69 and CD25 expression (Figures 4B-4C). T cells transduced with viral TAPi, especially EBV BNLF2a, induced less activation of allogeneic responder T cells, which was further reduced by MHC I and / or MHC II blockade.

[0137] We next tested the functional capacity of TAPi-expressing T cells to present cytoplasmic antigens by assessing presentation of peptides derived from the highly immunogenic HCMV pp65 protein. * Presented on the 02:01 allele, NLV-specific CD8 + Triggering T cells to secrete IFNγ 16 NLV-specific "responder T cell" lines were cloned into HLA-A immunized mice, which had evidence of CMV-specific memory responses. *TAPi T cells were first generated by serial stimulation of PBMCs from a healthy donor at 0201. A panel of “stimulator T cells” from the same healthy donor was then generated, which were either left untransduced (UTD) or transduced with constructs as indicated, including three different viral TAPi. Co-culture of “stimulator” and “responder” cells demonstrated that viral TAPi expression, specifically from HSV or EBV, inhibited antigen presentation, as shown by reduced IFNγ secretion in “responder T cells” (Figure 1D). Despite reduced antigen presentation, the use of viral proteins to knock down MHC I increases the likelihood of an immune response to the sequence. To measure the immunogenicity of viral TAPi transduction in T cells, normal donors with pre-existing cellular immunity to the respective TAPi viruses were identified. PBMCs from normal donors were screened in an IFNγ ELISpot assay with peptides known to be immunogenic and derived from HCMV, EBV, or HSV. T cells from normal donors with detectable cellular responses to those viruses were then transduced with viral TAPi from the same viruses and expressed autologous CD8 + CD8 T cell activation was measured by IFNγ ELISpot (Figure 1E). T cells from HCMV-responsive donors were activated in response to transduction with HCMV pp65, but not with CMV TAPi. Similarly, HSV and EBV-responsive donors did not produce IFNγ in response to HSV or EBV TAPi, indicating that these viral TAP inhibitors do not induce T cell responses, despite their demonstrated response to other known immunogenic sequences of the same virus.

[0138] Taken together, the results indicate that primary T cells expressing HSV, EBV, or HCMV TAPi efficiently prevented cell surface expression of MHC I molecules, thereby limiting killing by NK cells and mitigating alloresponses. The few remaining MHC I molecules on the cell surface were not sufficient to initiate T cell activation in response to immunogenic peptides or peptides derived from the viral TAP inhibitor itself.

[0139] [Expression of shRNA targeting CIITA reduces cell surface levels of MHC class II, and it can be coexpressed with EBV TAPi to reduce both cell surface MHC class I and II] Activated human T cells express high levels of MHC class II molecules and may mediate rejection of genetically modified cells and antigen cross-presentation 4,17 MHC class II expression was previously reduced by targeting CIITA, the master regulator of MHC II gene transcription. 18 .

[0140] To avoid the use of gene editing and double-strand breaks, we encoded shRNAs targeting CIITA into lentiviral vectors (Figure 2A) and used a panel of shRNA sequences to compare shRNA vectors with CRISPR / Cas9-mediated gene knockout of CIITA. Transduction efficiency was measured based on eGFP expression (Figure 5A); primary human T cells transduced with shRNAs targeting CIITA showed reduced cell surface expression of MHC II to the same extent as CIITA KO, without affecting MHC I expression (Figure 2B). However, only shRNA CIITA3 reduced MHC II expression without compromising T cell proliferation (Figure 2C). Measurement of proliferation of responder allogeneic or autologous T cells in a mixed lymphocyte reaction (MLR) demonstrated that shRNA-mediated knockdown of CIITA reduced proliferation of responder T cells (Figure 2D, Figures 5B-5C). We next combined the MHC I and II downregulation strategies by including both EBV TAPi and shRNA CIITA3 in one lentiviral vector (Figure 2E). The combined EBV-TAPi / shRNA-CIITA3 vector, when transduced into primary human T cells, reduced MHC I and II expression (Figure 2F) and reduced the proliferative response in MLRs (Figure 2G; Figure 5D-E). These results demonstrated that genetically modified primary T cells could successfully evade cellular immune responses by the MHC class I and II downregulation strategy as proposed here, generating "stealth" T cells.

[0141] [Stealth-enabled αCD19 CAR T cells are functional and can evade CAR-mediated immune recognition by T cells in patients who received one or two infusions of αCD19 CAR T cells] Autologous CAR T cells based on FMC63-based αCD19 single-chain scFv can induce T cell responses against the murine scFv fraction of the patient's CAR 3,4Therefore, the stealth strategy proposed here was tested in the context of these CARs to confirm the retention of antitumor efficacy and evasion of cellular immunity. Two stealth FMC63-based αCD19 CARs were generated with alternating sequence positions of EBV TAPi and eGFP markers (Figure 3A). Both stealth αCD19 CAR-T cells had reduced MHC I and II molecules on their cell surface compared to αCD19 CAR alone. Interestingly, this reduction did not increase NK cell cytotoxicity and CAR-T cell proliferation was unchanged compared to non-transduced T cells (Figure 3B).

[0142] Stealth αCD19 CAR-T cells also maintained their ability to target tumor cells in vitro and in vivo. When co-incubated with luciferase-expressing acute lymphoblastic leukemia (ALL) NALM6 cells or mantle cell lymphoma JeKo-1 cells, stealth αCD19 CAR-T cells reduced tumor cell viability to the same extent as αCD19 CAR-T cells (Figure 3C). Due to its slight advantage in MHC I downregulation, the stealth 2 αCD19 CAR-T cell construct was chosen to be further studied in an in vivo NSG mouse model containing ALL NALM6 cells. After tumor engraftment, mice were left untreated or injected with αCD19 CAR-T cells with or without stealth technology and assessed for CAR-T cell proliferation by blood sampling and tumor clearance by bioluminescence (BLI) (Figure 3D). Both αCD19 CAR-T cells and stealth αCD19 CAR-T cells expanded similarly in the blood as observed by flow cytometric assessment of GFP+CD3+ cells on days 7 and 14. Tumor cells, GFP+CD3-NALM6 cells, were found to be absent at both time points, whereas significant proliferation was found in the untreated group. This was further confirmed by BLI imaging. Treatment of engrafted NALM6 with αCD19 CAR-T cells and stealth αCD19 CAR-T cells showed comparable tumor clearance, whereas luciferase-expressing NALM6 cells expanded significantly in untreated mice. Kaplan-Meier survival curves demonstrated no difference in survival of mice treated with αCD19 CAR-T cells with or without the additional stealth sequence in the vector (Figure 3E). In summary, stealth αCD19 CAR-T retained its ability to recognize and eliminate CD19-expressing cells in both in vitro and in vivo tumor models.

[0143] Finally, we tested the ability of the proposed stealth technology to avoid antigen presentation of immunogenic CAR sequences. We identified 11 patients who received one or two doses of autologous FMC63-based CAR and generated fresh αCD19 CAR-T cells with or without stealth technology from PBMCs 3 months after their infusion. Four of the patients had an initial response of their tumor to the CAR T cell product, four had tumors that did not respond to the CAR T cells, and three received a second infusion of CAR T cells due to tumor progression after the first infusion. To evaluate whether T cells from these patients could be activated by their autologous T cells expressing the FMC63-based αCD19 CAR, freshly generated αCD19 CAR-T cells with or without stealth technology were used as “stimulators” and co-cultured with autologous “responder” untransduced T cells in an IFNγ ELISpot assay (Figure 3F). Responder T cells were activated in the presence of FMC63-based αCD19 CAR-T cell products, but not UTD cells or stealth αCD19 CAR-T cells. Activation of responder T cells was particularly high in subjects who received two infusions of FMC63-based CAR T cells and in three of four non-responders. These data provide evidence that multiple infusions can increase anti-CAR immunity in patients and that a proportion of non-responders have a strong rejection response to their autologous αCD19 CAR-T cells.

[0144] [Consideration] In summary, the results demonstrate that combined expression of shRNAs targeting EBV TAPi BNLF2a and CIITA effectively reduces MHC expression and antigen presentation, and that incorporating these sequences into lentiviral vectors has potential applications in circumventing autologous and allogeneic cell immunity. Avoidance of endogenous T cell-mediated rejection may be particularly beneficial for αCD19 CAR-T cell therapy, where early expansion and persistence are associated with durable remission. 19,20 αCD19 CAR-T cells efficiently eliminate the B cell lineage, limiting humoral immunity to αCD19 CAR-T cell therapy, further enhancing the impact of evading T cell immunity in this setting.4,21 More generally, this stealth technology can be applied in any setting using genetically modified cells, where either the transgene, binding sequence, or cell type is not autologous, and avoiding early rejection can enhance the desired therapeutic effect. See, e.g., references 4, 6, and 22.

[0145] Furthermore, the results demonstrated that stealth CAR-T cells avoided the anti-CAR response derived from FMC63-based αCD19 CAR while avoiding NK cell activation due to the loss of cell surface MHC I. Moreover, an increase in CAR-reactive T cell responses was found in patients who received multiple FMC63-based αCD19 CAR-T cell infusions.

[0146] [method] [Mice and cell lines] NSG mice were purchased from the Jackson Laboratory and housed in a pathogen-free environment at the Center for Comparative Medicine at MGH. All experiments were performed according to protocols approved by the Massachusetts General Hospital Animal Care and Use Committee. Where indicated, cell lines were transduced and propagated to express Click Beetle Green (CBG) luciferase and enhanced GFP after clonal selection. HEKT cells, NALM-6 (ALL), JeKo-1 (MCL), and K562 (CML), were purchased from the American Type Culture Collection and maintained under conditions outlined by the supplier.

[0147] [(Stealth) CAR T cell production] Human T cells were purified under an Institutional Review Board-exempt protocol from healthy donor leukapheresis products (Stem Cell Technologies) purchased from the MGH blood bank. T cells from patients treated with axicabtagene ciloleucel or tisagenleucel at MGH were collected under an IRB-approved protocol with written informed consent; PMBCs from one subject who received two infusions of autologous FMC63-based CAR T cells at Seattle Cancer Care Alliance were provided and collected by Dr. Turtle with written informed consent. Cells were transduced with lentiviruses corresponding to various second-generation CAR-T cell constructs. Briefly, bulk human T cells were activated on day 0 using CD3 / CD28 Dynabeads (Life Technologies) and then incubated with 10% FBS and 20 IU ml -1 T cells were cultured in RPMI 1640 medium with GlutaMAX and HEPES supplemented with 1000 mL of recombinant human IL-2. Lentiviral transduction of cells was performed on day 1, CD3 / CD28 Dynabeads were removed on day 5, and T cells were electroporated with Cas9 mRNA, if applicable. If T cells needed to be sorted, they were sorted for purity on day 8 using the eGFP marker and expanded until day 14, after which they were transferred to storage in liquid nitrogen. When unsorted CAR T cells were used, CAR-T cells were normalized for transduction efficiency using untransduced but cultured and activated T cells from the same donor and expansion.

[0148] [Cytotoxicity assay] To assess the cytotoxicity of CAR T cells against target cells, CAR T cells were incubated with luciferase-expressing tumor targets at the indicated E / T ratios for 24 h. Residual luciferase activity was then measured with a Synergy Neo2 Luminescence Microplate Reader (Biotek). To assess the cytotoxicity of NK cells against stealth or CAR T cells, NK cells were purified from blood or frozen PBMCs (Stem Cell Technologies) and primed with 20 IU / ml recombinant human IL-2 before co-incubation with their respective target cells stained with CFSE (Life Technologies). After 3 h of co-incubation, αCD107a antibody was added and the assay was incubated for an additional hour. After a total of 4 h, cells were centrifuged and resuspended with the viability marker SYTOXred (Life Technologies) to assess target cell viability and NK cell degranulation on a flow cytometer.

[0149] [ELISpot assay] Plates with Immobilon-P membranes (Millipore) were activated with 35% ethanol for 30 seconds, washed with PBS, and incubated overnight with anti-human IFNγ antibody (Clone NIB42, Biolegend) in PBS. The next day, plates were blocked with 1% BSA in PBS and incubated with 5 × 10 5 PBMCs or 2 x 10 5 T cells were co-incubated with the respective peptide, antigen, or stimulator. After 24 h, the plates were washed with PBS containing 0.05% Tween-20 and incubated overnight with PBS containing biotinylated anti-human IFNγ antibody (Clone 4S.B3, Biolegend) as detection antibody. After washing with PBS containing 0.05% Tween-20, the plates were incubated with avidin-HRP (Biolegend) for 2 h, developed using BD Elispot AEC substrate set, and analyzed on an ImmunoSpot Reader system. All antibodies were used according to the manufacturer's recommendations.

[0150] [ELISA] Interferon-γ was measured from supernatants after overnight coincubation of NLV responder T cells and target cells at an E:T ratio of 1:5 using a human DuoSet ELISA kit (R&D systems).

[0151] [Flow cytometry] Generally, cells were stained for 30 min at 4°C in the dark and washed twice with RPMI before analysis. SYTOX Red or SYTOX Blue (Life Technologies) were added as viability markers, and singlet discrimination was performed with both FSC and SSC detectors. The following antibodies targeting their respective antigens were used in combination with their respective isotype controls according to the manufacturer's recommendations: CD4 (SK3, Biolegend), CD8 (SK1, Biolegend), CD3 (OKT3, Biolegend), CD25 (BC96, Biolegend), CD69 (FN50, Biolegend), HLA-A / B / C (W6 / 32, Biolegend), HLA-DR / DP / DQ (Tu39, Biolegend), CD107a (H4A3, Biolegend), mouse red blood cells (TER-119, Biolegend), mouse Ly6G / 6C (RB6-8C5, Biolegend), mouse CD11b (M1 / 70, Biolegend), and mouse NK1.1 (PK136, Biolegend). Analysis was performed with FlowJo software (BD Biosciences).

[0152] [Mixed lymphocyte reaction (MLR) assay] Stealth or CAR T cells were stained with CFSE (Life Technologies), and autologous or allogeneic T cells were stained with CellTrace Violet (Life Technologies), and then 20 IU ml -1T cells were co-incubated at a 4:1 ratio in the presence of recombinant human IL-2 and isotype or blocking antibodies for MHC I (W6 / 32, Biolegend) or MHC II (Tu39, Biolegend) or both MHC I and II. Fresh IL-2 was added every other day and T cells were pulsed with new stealth T cells and blocking antibodies on days 7 and 14. On day 16, T responder cells were stained and assessed for cell division and activation markers CD69 and CD25 by FCM.

[0153] [In vivo studies] Luciferase-treated NALM-6 were harvested during logarithmic growth phase, washed twice with PBS, counted, and then inoculated into the tail vein of NSG mice with these tumor cells (1 × 10 per mouse). 6 The presence of tumors was confirmed by bioluminescence 3 days later, at which point mice were injected with 2 × 10 6 Mice were treated with injections of CAR T cells. Tumor progression was then assessed longitudinally by bioluminescence emission using an Ami HT optical imaging system (Spectral Instruments) after intraperitoneal matrix injection. On days 7 and 14, blood from mice was collected by cheek punch and analyzed by FCM for the presence of NALM-6 and CAR T cells per microliter of blood.

[0154] [Stealth CAR Design] DNA constructs were synthesized and cloned into second generation lentiviral backbones under the control of the human EF-1α promoter for protein translation and / or the human U6 promoter for RNA transcription. Sequences of EBV BNLF2a, HSV ICP47 and HCMV US6TAPi were synthesized and combined with eGFP via the 2A self-cleaving peptide. shRNAs targeting CIITA were designed using software from Dharmacon and Whitehead Laboratories and combined into a plasmid expressing eGFP via the EF-1α promoter. Similarly, vectors containing CRISPR / Cas9 guides for β2M and CIITA and eGFP expression were constructed. Lentiviral vectors expressing a combination of shRNA CIITA3, EBV BNLF2a, and eGFP were also constructed. For CAR construction, a plasmid expressing an FMC63-based anti-CD19 CAR was constructed in combination with the expression of shRNAs targeting EBV BNLF2a and CIITA.

[0155] [Statistical method] All statistical analyses were performed using GraphPad Prism 9 software. Data are presented as mean ± sem with statistically significant differences determined by tests as indicated in the figure legends.

[0156] [References in Example 1] 1. Locke, FL et al. Axicabtagene Ciloleucel as Second-Line Therapy for Large B-Cell Lymphoma.N Engl J Med(2021). 2. Bishop, MR et al. Second-Line Tisagenlecleucel or Standard Care in Aggressive B-Cell Lymphoma.N Engl J Med (2021). 3. Turtle, C.J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 126, 2123-2138 (2016). 4. Wagner, D.L. et al. Immunogenicity of CAR T cells in cancer therapy. Nat Rev Clin Oncol 18, 379-393 (2021). 5. Jensen, M.C. et al. Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20 / CD19-specific chimeric antigen receptor redirected T cells in humans. Biol Blood Marrow Transplant 16, 1245-1256 (2010). 6. Lamers, C.H. et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood 117, 72-82 (2011). 7. Depil, S., Duchateau, P., Grupp, S.A., Mufti, G. & Poirot, L. ’Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 19, 185-199 (2020). 8. Tuladhar, R. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat Commun 10, 4056 (2019). 9. Allogene. Allogene Therapeutics Reports FDA Clinical Hold. (2021). 10. Choi, B.D. et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for immunotherapy of cancer 7, 304 (2019). 11. Biosciences, P. (2021). 12. Kagoya, Y. et al. Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy. Cancer Immunol Res 8, 926 - 936 (2020). 13. Verweij, M.C. et al. Viral inhibition of the transporter associated with antigen processing (TAP): a striking example of functional convergent evolution. PLoS Pathog 11, e1004743 (2015). 14. Goldsmith, K., Chen, W., Johnson, D.C. & Hendricks, R.L. Infected cell protein (ICP) 47 enhances herpes simplex virus neurovirulence by blocking the CD8+ T cell response. J Exp Med 187, 341 - 348 (1998). 15. Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat Immunol 9, 503 - 510 (2008). 16. Khan, N., Cobbold, M., Keenan, R. & Moss, P. A. Comparative analysis of CD8+ T cell responses against human cytomegalovirus proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, and phenotype. J Infect Dis 185, 1025 - 1034 (2002). 17. Costantino, C. M., Spooner, E., Ploegh, H. L. & Hafler, D. A. Class II MHC self - antigen presentation in human B and T lymphocytes. PLoS One 7, e29805 (2012). 18. Holling, T. M., van der Stoep, N., Quinten, E. & van den Elsen, P. J. Activated human T cells accomplish MHC class II expression through T cell - specific occupation of class II transactivator promoter III. J Immunol 168, 763 - 770 (2002). 19. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371, 1507 - 1517 (2014). 20. Porter, DL et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 7,303ra139 (2015). 21. Elahi, R., Heidary, AH, Hadiloo, K. & Esmaeilzadeh, A. Chimeric Antigen Receptor-Engineered Natural Killer (CAR NK) Cells in Cancer Treatment; Recent Advances and Future Prospects. Stem Cell Rev Rep 17,2081-2106(2021). 22. Jan, M. et al. Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.Sci Transl Med 13(2021).

[0157] Example 2. Further results demonstrating that expression of a viral TAP inhibitor in primary T cells results in a reduction in cell surface levels of MHC class I Herpesviruses express TAP, a protein required for transporting cytoplasmic peptides across the endoplasmic reticulum and loading them for presentation to MHC class I molecules on the cell surface. 29 Cells that naturally or experimentally lack expression of a functional TAP complex exhibit dramatically reduced levels of surface MHC I and are CD8 + Greatly reduces their susceptibility to T cells 30We hypothesized that forced expression of a viral TAP inhibitor (TAPi) would reduce MHC I expression in genetically modified cells, thereby preventing cell-mediated immune responses to foreign transgenes. To test whether expression of herpesvirus TAPi reduced surface MHC I expression in primary T cells, we generated bicistronic lentiviral constructs to express herpes simplex virus (HSV) ICP47, human cytomegalovirus (HCMV) US6, or Epstein-Barr virus (EBV) BNLF2a TAPi together with the fluorescent reporter eGFP as a transduction marker (Figure 6A). Lentiviral constructs expressing sgRNA for β-2-microglobulin (β2M) without electroporation with Cas9 mRNA were used as positive (β2M KO) or negative (β2M-) controls. With similar transduction efficiency, TAPi-transduced cells reduced surface MHC I levels without affecting MHC class II upregulation upon activation (Fig. 6B). Viral TAP inhibitors reduced total surface MHC I levels by at least one log-fold, which was maintained upon further stimulation with IFNγ or αCD3 antibodies (Fig. 6C).

[0158] MHC I expression inhibits targeting by NK cells 31 We investigated the effect of MHC I downregulation on susceptibility to NK cell killing. 26,27Similarly, β2M KO T cells were susceptible to autologous NK cell lysis and induced NK cell degranulation as measured by CD107a expression. Compared to β2M KO T cells, T cells expressing EBV viral TAPi caused significantly reduced NK cell lysis or degranulation (Figure 6D). Similarly, MHC I expression mediates allogeneic T cell responses due to mismatch between MHC and TCR. To measure the allogeneic response of TAPi-expressing T cells, a mixed lymphocyte reaction (MLR) was performed. Transduced T cells were incubated with autologous or allogeneic labeled responder T cells in the presence or absence of MHC I and II blocking antibodies. Activation of responder T cells was measured by proliferation (Figure 6E) and changes in CD69 and CD25 expression (Figures 13A-13B). T cells transduced with viral TAPi, particularly EBV BNLF2a, induced less activation of allogeneic responder T cells than did MHC I and / or MHC II blockade.

[0159] We next tested the ability of TAPi-expressing T cells to present cytoplasmic antigens by assessing presentation of peptides derived from the highly immunogenic HCMV pp65 protein. * Presented on the 02:01 allele, NLV-specific CD8 + Triggering T cells to secrete IFNγ 32 NLV-specific "responder T cell" lines were cloned into HLA-A immunized mice, which had evidence of CMV-specific memory responses. *TAPi T cells were first generated by serial stimulation of PBMCs derived from a healthy donor at 02:01. A panel of “stimulator T cells” derived from the same healthy donor was then generated, which were either left untransduced (UTD) or transduced with constructs as indicated, including three different viral TAPi (Figure 6A). Co-culture of “stimulator cells” with “responder cells” demonstrated that viral TAPi expression, specifically from HSV or EBV, reduced antigen presentation based on reduced IFNγ secretion in “responder T cells” (Figure 1D). Despite reduced antigen presentation, the use of viral proteins to knock down MHC I increases the likelihood of an immune response to the sequence. To measure the immunogenicity of viral TAPi transduction in T cells, normal donors with pre-existing cellular immunity to the respective TAPi viruses were identified. PBMCs from normal donors were screened in an IFNγ ELISpot assay with peptides known to be immunogenic and derived from HCMV, EBV, or HSV. 33-36 Then, T cells from normal donors with detectable cellular responses to the virus were transduced with viral TAPi from the same virus and expressed autologous CD8 + CD8 T cell activation was measured by IFNγ ELISpot (Figure 1E). T cells from HCMV-responsive donors were activated in response to transduction with HCMV pp65, but they did not respond to transduction with CMV TAPi. Similarly, HSV- and EBV-responsive donors did not produce IFNγ in response to HSV or EBV TAPi, indicating that these viral TAP inhibitors do not induce T cell responses, despite being responsive to other known immunogenic sequences from the same viruses.

[0160] [Expression of shRNA targeting CIITA reduces cell surface levels of MHC class II] Activated human T cells express high levels of MHC class II molecules. In genetically modified cells, high MHC II could induce rejection through antigen cross-presentation of the genetic modification. 3,37 As with MHC class I, direct targeting of MHC class II expression by DNA editing techniques is highly complex and potentially patient-specific, as these genes are highly polymorphic and harbor significant allelic variation. 38 Reduction of MHC class II expression was examined by targeting CIITA, a key regulator of MHCII gene transcription. 39 To avoid the use of gene editing and double-strand breaks, a panel of shRNA sequences was used to encode shRNAs targeting CIITA into lentiviral vectors (Figure 7A). The shRNA vectors were also compared to CRISPR / Cas9-mediated gene knockout of CIITA. Primary human T cells transduced with shRNAs targeting CIITA showed reduced cell surface expression of MHC II to the same extent as CIITA KO, without affecting MHC I expression (Figure 7B). Both CRISPR / Cas9 and shRNA CIITA targeting strategies resulted in T cells with fewer than 20,000 MHC class II molecules on their surface, which was unaffected by boosting with IFNγ or αCD3 antibodies (Figure 7C). However, only shRNA CIITA3 reduced MHC II expression without compromising T cell proliferation (Figure 2C). In mixed lymphocyte reactions (MLRs) using allogeneic or autologous responder T cells, shRNA-mediated knockdown of CIITA reduced proliferation of responder T cells (FIG. 7D; FIGS. 14A-14B).

[0161] [Expression of the viral TAP inhibitor EBV BNLF2a and shRNA targeting CIITA can be combined in primary T cells to reduce cell surface levels of both MHC class I and class II.] Although both strategies to downregulate cell surface expression of MHC I or II were effective individually, the question remained as to whether these strategies could be combined. TAPi EBV BNLF2a was chosen to combine with shRNA CIITA3. This TAPi sufficiently reduced cell surface MHC I to suppress antigen presentation, while remaining MHC I on the cell surface could potentially suppress NK cell activation. These MHC I and II downregulation strategies were combined by including both EBV TAPi and shRNA CIITA3 in one lentiviral vector (Figure 8A). The combined EBV-TAPi / shRNA-CIITA3 vector, when transduced into primary human T cells, reduced MHC I and II expression (Figures 8B-8C) and reduced proliferative responses in MLRs (Figure 8D, Figures 15A-15B). This demonstrates that genetically modified primary T cells can successfully evade cellular immune responses by the MHC class I and II downregulation strategy presented here, generating "stealth" T cells.

[0162] [Stealth-enabled αCD19 CAR T cells are functional in vitro and in vivo] The murine scFv FMC63, which recognizes CD19 and is used in four of the six FDA-approved CAR-T cell products, has been reported to induce autologous T cell responses in patients. 3,6Therefore, stealth strategies were tested in the context of FMC63 CARs to verify that they retain functionality and avoid eliciting cellular immunity. Stealth FMC63-based αCD19 CARs were generated by incorporating both EBV TAPi and shRNA CIITA3 (Figure 9A). Stealth αCD19 CAR-T cells had reduced MHC I and II molecules on their cell surface compared to T cells transduced with only αCD19 CAR, and showed strong expression of EBV TAPi and reduced expression of CIITA mRNA compared to αCD19 CAR alone by qPCR (Figure 9B). Interestingly, this reduction of MHC I molecules on the cell surface did not increase NK cell cytotoxicity, and CAR-T cell proliferation was unchanged compared to non-transduced T cells (Figure 9C-9D). In addition, phenotypic analysis by CD4, CD8, CCR7, and CD45RA further demonstrated no difference in CD4 / CD8 ratio and memory phenotype comparing αCD19 CAR-T cells with or without stealth technology (Figure 9E). Stealth αCD19 CAR-T cells also maintained their ability to target tumor cells in vitro. When co-incubated with luciferase-expressing acute lymphoblastic leukemia (ALL) NALM6 cells or mantle cell lymphoma JeKo-1 cells, stealth αCD19 CAR-T cells reduced tumor cell viability to the same extent as αCD19 CAR-T cells (Figure 9F).

[0163] In vivo functionality was also investigated. After tumor engraftment with NALM6 or JeKo-1 cells, mice were left untreated or injected with αCD19 CAR-T cells with or without stealth technology. CAR-T cell proliferation in the blood was assessed by flow cytometry and tumor clearance was measured by bioluminescence imaging (BLI) (Figure 10A). Mice treated with αCD19 CAR-T cells or stealth αCD19 CAR-T cells showed comparable tumor clearance, whereas tumors grew significantly in untreated mice by BLI (Figures 10B and 10F). Both αCD19 CAR-T cells and stealth αCD19 CAR-T cells proliferated similarly in the blood, as observed by the presence of GFP+CD3+ cells at day 14 (Figures 10C and 10G). There were no or very few tumor cells (GFP+CD3-NALM6 cells) in the blood of mice treated with CAR-T cells, whereas in the untreated group, significant proliferation was found, similar to BLI imaging. Kaplan-Meier survival curves demonstrated no difference in survival rates of mice treated with αCD19 CAR-T cells with or without additional stealth technology (Figures 10D and 10H). In summary, stealth αCD19 CAR-T cells retained their ability to recognize and eliminate CD19-expressing cells both in vitro and in vivo.

[0164] [Stealth-enabled αCD19 CAR T cells evade CAR-mediated immune recognition by T cells in patients who have received one or two infusions of αCD19 CAR T cells.] Next, we tested the ability of stealth technology to avoid antigen presentation of immunogenic CAR sequences. We identified 11 patients who received autologous FMC63-based CAR once or twice. Four of these patients showed an initial response of their tumors to the CAR T cell product, while four had tumors that did not respond to CAR T cells. Three patients received a second infusion of CAR T cells because their tumors progressed after the first infusion (Figure 11A). To evaluate whether the T cells of these patients could be activated by their autologous T cells expressing FMC63-based αCD19 CAR, fresh αCD19 CAR-T cells were generated from their T cells (collected 3 months after infusion, no CAR) as "stimulators" and co-cultured with autologous non-transduced T cells as "responders" with or without stealth technology in an IFNγ ELISpot assay (Figures 11B-11E). Responder T cells were activated in the presence of FMC63-based αCD19 CAR-T cell products, but not UTD cells or stealth αCD19 CAR-T cells. Activation of responder T cells was particularly high in subjects who received two infusions of FMC63-based CAR T cells and in three of the four nonresponders. These data suggest that multiple infusions increase anti-CAR immunity in patients, and that some nonresponders may potently reject their autologous αCD19 CAR-T cells upon reinfusion. However, larger numbers of patients will be needed to establish a correlation between lack of response and CAR T cell rejection.

[0165] [Stealth αCD19 CAR T cells reduce allogeneic responses in vitro and in vivo] Finally, we investigated the mechanism of evasion of stealth αCD19 CAR-T cells against allogeneic T cells. We co-incubated αCD19 CAR-T cells or stealth αCD19 CAR-T cells with in vitro expanded allogeneic T cells (expanded on αCD3 / αCD28 beads), and found that the stealth technology reduced both IFNγ secretion and cytotoxicity against CAR-T cells (Figure 12A). We also performed a previously reported in vitro mouse model.40 In this study, αCD3 / αCD28 expanded allogeneic T cells were injected prior to NALM6 inoculation and subsequently treated with CAR-T cells (Figure 12B). Stealth CAR-T cells expanded significantly more in the blood compared to αCD19 CAR-T cells at day 14 (by flow cytometry) despite the presence of similar levels of allogeneic T cells and tumor burden (Figures 12C-12D). However, because the stealth system did not remove large numbers of untransduced activated T cells, the incidence and severity of xeno-GvHD (as indicated by hair loss and sclerosis) was earlier and higher, with no change in survival (Figure 16). Therefore, a second allogeneic model was performed to boost the allogeneic response using allogeneic T cells primed by pulsing twice with irradiated PBMCs from the CAR T cell donor. These primed cells were then subjected to a rapid expansion protocol prior to injection into mice. 28 (Figure 12E). In this model, stealth αCD19 CAR-T cells proliferated more robustly over 4 weeks compared to αCD19 CAR-T cells (Figure 12F) and showed comparable antitumor activity (Figures 12G-12H). Primed allogeneic T cells allowed mice to be monitored for a longer period before the onset of severe xenogeneic GvHD. Importantly, stealth αCD19 CAR-T cells proliferated more robustly than αCD19 CAR T cells.

[0166] [Consideration] CD19-targeting CAR-T cells have frequently induced complete remissions in patients with previously incurable hematologic disorders, but certain hurdles remain in both the autologous and allogeneic settings. 15,16 Current clinical trials using allogeneic CAR T cells have attempted to circumvent one of these hurdles, the host immune response, by reducing MHC I and / or class II cell surface presentation. 3,4Here, we show that the inclusion of stealth transgenes, EBV TAPi BNLF2a, and shRNA targeting CIITA, effectively reduces MHC cell surface molecules and avoids autologous and allogeneic T cell responses. These stealth transgenes were incorporated within a CAR transduction vector to develop a one-shot transduction to generate CAR-T cells with T cell evasion properties. This simplified approach is particularly valuable as it does not rely on CRISPR / Cas9 gene editing technology to remove MHC I / II from the cell surface. 4,41 CRISPR / Cas9 can introduce off-target effects via INDELs that promote aberrant mRNA or protein products, which increases with the introduction of multiple targets. 20,42 CRISPR / Cas9 is also being investigated for a variety of other targets in CAR-T cells, including targets that enhance CAR T cell fitness and persistence. 22,41 Alternatively, alternative solutions to reduce HLA from the surface of CAR-T cells would allow CRISPR / Cas9 to continue to be used for these purposes.

[0167] Evasion of T cell immunity may be particularly useful in αCD19 CAR T cell therapy, which efficiently eliminates normal B cells in addition to the intended tumor cells, thereby naturally limiting humoral immune responses to the non-self CAR components, as anti-CAR or donor-specific antibodies and their potential interference with αCD19 CAR T cell therapy are highly limited. 3,42 Therefore, equipping αCD19 CAR T cells or αCD19 NK cells with mechanisms to prevent T cell immunity could have a major impact. Clinical trials using autologous CAR-T cells have shown that patients treated with CAR-T cells develop CAR-reactive T cell responses. 3,6,11 Here, we demonstrate that stealth CAR-T cells evade the anti-CAR response derived from FMC63-based αCD19 CAR and show increased proliferation in an allogeneic model. Furthermore, we found increased CAR-reactive T-cell responses in patients who received multiple FMC63-based αCD19 CAR-T cell infusions.

[0168] We have not exhaustively compared all the methods that can be used to avoid immunogenicity. Indeed, CRISPR / Cas9 and TALEN gene knockout are frequently used to eliminate T cell receptors and / or B2M in allogeneic T cell products. Also, shRNAs for B2M have been used. 43 , using base editing technology to mutate B2M 44 It may also be possible. The advantage of the present disclosure is that it can be easily combined with other gene editing strategies such as CRISPR / Cas9, while reducing the number of double-strand breaks or the possibility of translocation events. Furthermore, the incorporation of stealth transgenes into the self-product of "simple" lentiviral transduction may be performed quickly without the need to develop exhaustive sequencing-based strategies or additional release assays to measure off-target gene editing effects.

[0169] In addition to the potential of stealth genes in CAR-T cell therapy, this stealth technology may be useful in additional settings with genetically modified cells, where either the transgene, binding sequence, or cell type are not autologous, thus enhancing the desired therapeutic effect by avoiding early rejection. 3、8、45 .

[0170] [method] [Mice and cell lines] NSG mice were purchased from the Jackson Laboratory and housed in a pathogen-free environment at the Center for Comparative Medicine at MGH. Experiments were performed according to protocols approved by the Massachusetts General Hospital Animal Care and Use Committee. HEKT cells, NALM-6 (ALL), JeKo-1 (MCL), and K562 (CML), were purchased from the American Type Culture Collection, maintained under conditions outlined by the supplier, and, where indicated, transduced to express click beetle green luciferase and enhanced GFP. Cell lines were routinely authenticated by STR profiling and routinely tested to exclude mycoplasma infection.

[0171] [(Stealth) CAR T cell production] Human T cells were purified under an Institutional Review Board-exempt protocol from healthy donor leukapheresis products purchased from the MGH blood bank (Stem Cell Technologies). T cells from patients treated with axicabtagene ciloleucel or tisagenlecleucel at MGH were collected under an IRB-approved protocol (16-206) with written informed consent; PMBCs from one subject who received two infusions of autologous FMC63-based CAR T cells at Seattle Cancer Care Alliance were provided by Dr. Turtle and collected with written informed consent. Briefly, bulk human T cells were activated on day 0 using CD3 / CD28 Dynabeads (Life Technologies) and cultured in RPMI 1640 medium containing GlutaMAX and HEPES supplemented with 10% FBS and 20 IU / ml recombinant human IL-2. Lentiviral transduction was performed on day 1, and CD3 / CD28 Dynabeads were removed on day 5. Where applicable, T cells were electroporated with Cas9 mRNA on day 5. For flow-based sorting, T cells were sorted on day 8 using the eGFP marker and expanded until day 14 followed by cryopreservation. When unsorted CAR T cells were used, CAR-T cells were normalized for transduction efficiency using untransduced activated T cells from the same donor and expansion.

[0172] [Cytotoxicity assay] To assess the cytotoxicity of CAR T cells against target cells, CAR T cells were incubated with luciferase-expressing tumor targets at the indicated E / T ratios for 24 h. Residual luciferase activity was then measured with a Synergy Neo2 Luminescence Microplate Reader (Biotek). To assess the cytotoxicity of NK cells against stealth or CAR T cells, NK cells were purified from blood or frozen PBMCs (Stem Cell Technologies) and primed with 20 IU / ml recombinant human IL-2 before co-incubation with their respective target cells stained with CFSE (Life Technologies). After 3 h of co-incubation, αCD107a antibody was added and the assay was incubated for an additional hour. After a total of 4 h, cells were centrifuged and resuspended with the viability marker SYTOXred (Life Technologies) to assess target cell viability and NK cell degranulation on a flow cytometer.

[0173] [ELISpot assay] Plates with Immobilon-P membranes (Millipore) were activated with 35% ethanol for 30 seconds, washed with PBS, and incubated overnight with anti-human IFNγ antibody (Clone NIB42, Biolegend) in PBS. The next day, plates were blocked with 1% BSA in PBS and incubated with 5 × 10 5 PBMCs or 2 x 10 5 T cells were co-incubated with the respective peptide, antigen, or stimulator. After 24 h, the plates were washed with PBS containing 0.05% Tween-20 and incubated overnight with PBS containing biotinylated anti-human IFNγ antibody (Clone 4S.B3, Biolegend) as detection antibody. After washing with PBS containing 0.05% Tween-20, the plates were incubated with avidin-HRP (Biolegend) for 2 h, developed using BD Elispot AEC substrate set, and analyzed on an ImmunoSpot Reader system. All antibodies were used according to the manufacturer's recommendations.

[0174] [ELISA] Interferon-γ was measured from supernatants after overnight coincubation of NLV responder T cells and target cells at an E:T ratio of 1:5 using a human DuoSet ELISA kit (R&D systems).

[0175] [Flow cytometry] Generally, cells were stained for 30 min at 4°C in the dark and washed twice with RPMI before analysis. SYTOX Red or SYTOX Blue (Life Technologies) were added as viability markers, and singlet discrimination was performed with both FSC and SSC detectors. The following antibodies targeting their respective antigens were used in combination with their respective isotype controls according to the manufacturer's recommendations: CD4 (SK3, Biolegend), CD8 (SK1, Biolegend), CD3 (OKT3, Biolegend), CD25 (BC96, Biolegend), CD69 (FN50, Biolegend), HLA-A / B / C (W6 / 32, Biolegend), HLA-DR / DP / DQ (Tu39, Biolegend), CD107a (H4A3, Biolegend), mouse red blood cells (TER-119, Biolegend), mouse Ly6G / 6C (RB6-8C5, Biolegend), mouse CD11b (M1 / 70, Biolegend), and mouse NK1.1 (PK136, Biolegend). Where specified, antibody binding capacity was measured using Quantum Simply Cellar beads (Bangs laboratories). Analysis was performed with FlowJo software (BD Biosciences).

[0176] [Mixed lymphocyte reaction (MLR) assay] Stealth or CAR T cells were stained with CFSE (Life Technologies), and autologous or allogeneic cells were stained with CellTrace Violet (Life Technologies), then co-incubated with 20 IU / ml recombinant human IL-2 in the presence of isotype or blocking antibodies for MHC I (W6 / 32, Biolegend) or MHC II (Tu39, Biolegend) or both MHC I and II at a 4:1 ratio. Fresh IL-2 was added every other day, and T cells were pulsed with new stealth T cells and blocking antibodies on days 7 and 14. On day 16, T responder cells were stained with SYTOXRed (viability) and cell division was assessed by FCM. Cell allogeneity was assessed by PCR (American Red Cross), with at least five out of six mismatches (HLA-A / B / C / DP / DQ / DR) selected.

[0177] [In vivo studies] Luciferase-treated NALM-6 or JeKo-1 cells were collected, washed with PBS, counted, and then inoculated into the tail vein of NSG mice with these tumor cells (1 × 10 per mouse). 6 Tumor growth was confirmed by bioluminescence 3 days later, at which point mice were injected with 2 × 10 6 Mice were treated with injections of CAR T cells. Tumor progression was then assessed longitudinally by bioluminescence emission using an Ami HT optical imaging system (Spectral Instruments) after intraperitoneal matrix injection. On day 14 (or the designated day), mice's blood was collected by cheek punch and analyzed by FCM for the presence of NALM-6 and CAR T cells per microliter of blood. In the allogeneic T cell mouse model, "activated" allogeneic T cells were activated with CD3 / CD28 beads and mice were cultured at 7 × 10 per mouse. 6 The "primed" allogeneic T cells were pulsed twice with irradiated (100 Gy) PBMCs from the CAR T cell donor, followed by a rapid expansion protocol. 28 Each mouse was cultured at 4 × 10 6Allogeneic T cells were injected into the tail vein of NSG mice 1 day before NALM-6 tumor cell injection.

[0178] [Stealth CAR Design] DNA constructs were synthesized and cloned into second generation lentiviral backbones under the control of the human EF-1α promoter for protein translation and / or the human U6 promoter for RNA transcription. Sequences of EBV BNLF2a, HSV ICP47 and HCMV US6TAPi were synthesized and combined with eGFP via the 2A self-cleaving peptide. shRNAs targeting CIITA were designed using software from Dharmacon and Whitehead Laboratories and combined into a plasmid expressing eGFP via the EF-1α promoter. Similarly, vectors containing CRISPR / Cas9 guides for β2M and CIITA and eGFP expression were constructed. Lentiviral vectors expressing a combination of shRNA CIITA3, EBV BNLF2a, and eGFP were also constructed. For CAR construction, a plasmid expressing an FMC63-based anti-CD19 CAR was constructed in combination with the expression of shRNAs targeting EBV BNLF2a and CIITA.

[0179] [Statistical method] All statistical analyses were performed using GraphPad Prism 9 software. Data are presented as mean ± SEM with statistically significant differences determined by tests as indicated in the figure legends.

[0180] [References in Example 2] 1. Bishop MR, Dickinson M, Purtill D, et al. Second-Line Tisagenlecleucel or Standard Care in Aggressive B-Cell Lymphoma.N Engl J Med.2021. 2. Locke FL, Miklos DB, Jacobson CA, et al. Axicabtagene Ciloleucel as Second-Line Therapy for Large B-Cell Lymphoma. N Engl J Med. 2021. 3. Wagner DL, Fritsche E, Pulsipher MA, et al. Immunogenicity of CAR T cells in cancer therapy. Nat Rev Clin Oncol. 2021;18(6):379-393. 4. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ’Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19(3):185-199. 5. Young RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-Generation CAR T-cell Therapies. Cancer Discov. 2022:OF1-OF14. 6. Turtle CJ, Hanafi LA, Berger C, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest. 2016;126(6):2123-2138. 7. Jensen MC, Popplewell L, Cooper LJ, et al. Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20 / CD19-specific chimeric antigen receptor redirected T cells in humans. Biol Blood Marrow Transplant. 2010;16(9):1245-1256. 8. Lamers CH, Willemsen R, van Elzakker P, et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood. 2011;117(1):72-82. 9. Shah NN, Lee DW, Yates B, et al. Long-Term Follow-Up of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL. J Clin Oncol. 2021;39(15):1650-1659. 10. Xu X, Sun Q, Liang X, et al. Mechanisms of Relapse After CD19 CAR T-Cell Therapy for Acute Lymphoblastic Leukemia and Its Prevention and Treatment Strategies. Front Immunol. 2019;10:2664. 11. Gauthier J, Bezerra ED, Hirayama AV, et al. Factors associated with outcomes after a second CD19-targeted CAR T-cell infusion for refractory B-cell malignancies. Blood. 2021;137(3):323-335. 12. Nie Y, Lu W, Chen D, et al. Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies. Biomark Res. 2020;8:18. 13. Li X, Liu MJ, Mou N, et al. Efficacy and safety of humanized CD19 CAR-T as a salvage therapy for recurrent CNSL of B-ALL following murine CD19 CAR-T cell therapy. Oncol Lett. 2021;22(5):788。 14. Caldwell KJ, Gottschalk S, Talleur AC. Allogeneic CAR Cell Therapy - More Than a Pipe Dream. Front Immunol. 2020;11:618427。 15. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):439-448。 16. Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med. 2017;377(26):2531-2544。 17. Porter DL, Hwang WT, Frey NV, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7(303):303ra139。 18. Ghorashian S, Kramer AM, Onuoha S, et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med. 2019;25(9):1408-1414. 19. Lowe KL, Mackall CL, Norry E, Amado R, Jakobsen BK, Binder G. Fludarabine and neurotoxicity in engineered T-cell therapy. Gene Ther. 2018;25(3):176-191. 20. Tuladhar R, Yeu Y, Tyler Piazza J, et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat Commun. 2019;10(1):4056. 21. Allogene. Allogene Therapeutics Reports FDA Clinical Hold; 2021. 22. Dimitri A, Herbst F, Fraietta JA. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol Cancer. 2022;21(1):78. 23. Rezalotfi A, Fritz L, Forster R, Bosnjak B. Challenges of CRISPR-Based Gene Editing in Primary T Cells. Int J Mol Sci. 2022;23(3). 24. Choi BD, Yu X, Castano AP, et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. J Immunother Cancer. 2019;7(1):304. 25. Biosciences P. 2021. 26. Kagoya Y, Guo T, Yeung B, et al. Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy. Cancer Immunol Res. 2020;8(7):926-936. 27. Lee J, Sheen JH, Lim O, et al. Abrogation of HLA surface expression using CRISPR / Cas9 genome editing: a step toward universal T cell therapy. Sci Rep. 2020;10(1):17753. 28. Smith C, Okern G, Rehan S, et al. Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement. Clin Transl Immunology. 2015;4(1):e31. 29. Verweij MC, Horst D, Griffin BD, et al. Viral inhibition of the transporter associated with antigen processing (TAP): a striking example of functional convergent evolution. PLoS Pathog. 2015;11(4):e1004743. 30. Goldsmith K, Chen W, Johnson DC, Hendricks RL. Infected cell protein (ICP)47 enhances herpes simplex virus neurovirulence by blocking the CD8+ T cell response. J Exp Med. 1998;187(3):341 - 348. 31. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9(5):503 - 510. 32. Khan N, Cobbold M, Keenan R, Moss PA. Comparative analysis of CD8+ T cell responses against human cytomegalovirus proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, and phenotype. J Infect Dis. 2002;185(8):1025 - 1034. 33. Lubke M, Spalt S, Kowalewski DJ, et al. Identification of HCMV - derived T cell epitopes in seropositive individuals through viral deletion models. J Exp Med. 2020;217(3). 34. Dervillez X, Qureshi H, Chentoufi AA, et al. Asymptomatic HLA-A*02:01-restricted epitopes from herpes simplex virus glycoprotein B preferentially recall polyfunctional CD8+ T cells from seropositive asymptomatic individuals and protect HLA transgenic mice against ocular herpes. J Immunol. 2013;191(10):5124-5138。 35. Duraiswamy J, Burrows JM, Bharadwaj M, et al. Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions. J Virol. 2003;77(13):7401-7410。 36. Sukdolak C, Tischer S, Dieks D, et al. CMV-, EBV- and ADV-specific T cell immunity: screening and monitoring of potential third-party donors to improve post-transplantation outcome. Biol Blood Marrow Transplant. 2013;19(10):1480-1492。 37. Costantino CM, Spooner E, Ploegh HL, Hafler DA. Class II MHC self-antigen presentation in human B and T lymphocytes. PLoS One. 2012;7(1):e29805。 38. Wieczorek M, Abualrous ET, Sticht J, et al. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation. Front Immunol. 2017;8:292. 39. Holling TM, van der Stoep N, Quinten E, van den Elsen PJ. Activated human T cells accomplish MHC class II expression through T cell-specific occupation of class II transactivator promoter III. J Immunol. 2002;168(2):763-770. 40. Mo F, Watanabe N, McKenna MK, et al. Engineered off-the-shelf therapeutic T cells resist host immune rejection. Nat Biotechnol. 2021;39(1):56-63. 41. Razeghian E, Nasution MKM, Rahman HS, et al. A deep insight into CRISPR / Cas9 application in CAR-T cell-based tumor immunotherapies. Stem Cell Res Ther. 2021;12(1):428. 42. Elahi R, Heidary AH, Hadiloo K, Esmaeilzadeh A. Chimeric Antigen Receptor-Engineered Natural Killer (CAR NK) Cells in Cancer Treatment; Recent Advances and Future Prospects. Stem Cell Rev Rep. 2021;17(6):2081-2106. 43. Ramos CA, Courtney AN, Robinson SN, et al. Allogeneic NKT Cells Expressing a CD19-Specific CAR in Patients with Relapsed or Refractory B-Cell Malignancies: An Interim Analysis. Blood. 2021;138(23 November 2021):2819. 44. Webber BR, Lonetree CL, Kluesner MG, et al. Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nat Commun. 2019;10(1):5222. 45. Jan M, Scarfo I, Larson RC, et al. Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci Transl Med. 2021;13(575).

Claims

1. (i) Antigen-related transporter inhibitors (TAPi) or variants thereof; and (ii) Oligonucleotides complementary to a polynucleotide encoding an MHC class II transactivator protein or a variant thereof, selected from the group consisting of RNA interference (RNAi) oligonucleotides, antisense oligonucleotides (ASOs), or CRISPR interference (CRISPRi) oligonucleotides. Cells containing this substance.

2. (i) Chimeric antigen receptors (CARs); and (ii) Antigen-related transporter inhibitors (TAPi) or variants thereof; and / or (iii) Oligonucleotides complementary to a polynucleotide encoding an MHC class II transactivator protein or a variant thereof, selected from the group consisting of RNA interference (RNAi) oligonucleotides, antisense oligonucleotides (ASOs), or CRISPR interference (CRISPRi) oligonucleotides. Cells containing this substance.

3. The cell according to claim 1 or claim 2, wherein the oligonucleotide is complementary to any one of sequence numbers 7 to 12.

4. The cell according to claim 1 or claim 2, wherein the oligonucleotide is complementary to SEQ ID NO:

7.

5. The cell according to claim 1 or claim 2, wherein TAPi or a variant thereof reduces the expression of MHC class I.

6. The cell according to claim 1 or claim 2, wherein the TAPi is a viral TAPi.

7. The cell according to claim 1 or claim 2, wherein the TAPi is a herpesvirus TAPi.

8. The cell according to claim 1 or claim 2, wherein the TAPi is selected from the group consisting of herpes simplex virus (HSV) TAPi, human cytomegalovirus (HCMV) TAPi, or Epstein-Barr virus (EBV) TAPi.

9. The cell according to claim 1 or claim 2, wherein the TAPi is selected from the group consisting of herpes simplex virus (HSV) ICP47 TAPi, human cytomegalovirus (HCMV) US6 TAPi, or Epstein-Barr virus (EBV) BNLF2a TAPi.

10. The cell according to claim 1 or claim 2, wherein TAPi comprises an amino acid sequence that is at least 85% identical to any one of sequence numbers 1 to 3.

11. The cell according to claim 1 or claim 2, wherein TAPi contains any one amino acid sequence of sequence numbers 1 to 3.

12. The cell according to claim 1 or claim 2, wherein the RNAi oligonucleotide is selected from the group consisting of siRNA, miRNA, or shRNA.

13. The cell according to claim 12, wherein the RNAi oligonucleotide is shRNA.

14. The cell according to claim 13, wherein the shRNA comprises the nucleic acid sequence of SEQ ID NO:

3.

15. The cell according to claim 12, wherein the shRNA comprises the nucleic acid sequence of SEQ ID NO:

13.

16. A cell according to claim 1 or claim 2, which is a eukaryotic cell.

17. A cell according to claim 1 or claim 2, which is an immune cell.

18. The cell according to claim 17, wherein the immune cell is a T cell.

19. The cell according to claim 1, further comprising a chimeric antigen receptor (CAR).

20. CAR, (i) Extracellular target binding domain; (ii) Transmembrane domains; and (iii) Intracellular signaling domain The cell according to claim 2, comprising:

21. The cell according to claim 20, wherein the extracellular target binding domain binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, or claudin 6, or binds to any pair of CD19 / CD79b or BCMA / TACI, or is a TriPRIL antigen binding domain.

22. The cell according to claim 21, wherein the extracellular target binding domain binds to CD19.

23. The cell according to claim 20, wherein the extracellular target binding domain is not derived from a human polypeptide sequence.

24. The cell according to claim 20, wherein the extracellular target binding domain is derived from a mouse polypeptide sequence.

25. The cell according to claim 20, wherein the extracellular target binding domain comprises a VH amino acid sequence having at least 85% identity with SEQ ID NO: 39 and a VL amino acid sequence having at least 85% identity with SEQ ID NO:

40.

26. The transmembrane domain is the alpha chain of the T cell receptor, the beta chain of the T cell receptor, or the zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF) I) CD160, CD19, IL2R Beta, IL2R Gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Cells according to claim 20, selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

27. The cell according to claim 20, wherein the intracellular signaling domain is selected from the group consisting of CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

28. The cell according to any one of claims 20 to 27, wherein CAR comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 41 and a nucleic acid sequence having at least 85% identity with SEQ ID NO: 17 or 18.

29. A polynucleotide comprising a nucleic acid sequence encoding (i) TAPi or a variant thereof and (ii) an oligonucleotide complementary to the gene encoding an MHC class II transactivator protein.

30. The polynucleotide according to claim 29, wherein the TAPi is a viral TAPi.

31. The polynucleotide according to claim 29, wherein TAPi or a variant thereof reduces the expression of MHC class I.

32. The polynucleotide according to claim 29, wherein the TAPi is herpes simplex virus (HSV) TAPi.

33. The polynucleotide according to claim 29, wherein the TAPi is selected from the group consisting of herpes simplex virus (HSV) TAPi, human cytomegalovirus (HCMV) TAPi, or Epstein-Barr virus (EBV) TAPi.

34. The polynucleotide according to claim 29, wherein the TAPi is selected from the group consisting of herpes simplex virus (HSV) ICP47 TAPi, human cytomegalovirus (HCMV) US6 TAPi, or Epstein-Barr virus (EBV) BNLF2a TAPi.

35. The polynucleotide according to claim 29, wherein TAPi comprises an amino acid sequence that is at least 85% identical to any one of sequence numbers 1 to 3.

36. The polynucleotide according to claim 29, wherein TAPi comprises any one amino acid sequence of sequence numbers 1 to 3.

37. The polynucleotide according to claim 29, wherein the oligonucleotide is complementary to any one of sequence numbers 7 to 12 or a variant thereof.

38. The polynucleotide according to claim 29, wherein the oligonucleotide is complementary to SEQ ID NO: 7 or a variant thereof.

39. The polynucleotide according to claim 29, wherein the oligonucleotide is selected from the group consisting of RNAi oligonucleotides or CRISPR interference guide RNA.

40. The polynucleotide according to claim 39, wherein the RNAi oligonucleotide is selected from the group consisting of siRNA, miRNA, or shRNA.

41. The polynucleotide according to claim 39, wherein the RNAi oligonucleotide is shRNA.

42. The polynucleotide according to claim 41, wherein the shRNA is encoded by a nucleic acid sequence including sequence number 13.

43. The polynucleotide according to claim 29, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

44. CAR, (i) Extracellular target binding domain; (ii) Transmembrane domains; and (iii) Intracellular signaling domain The polynucleotide according to claim 43, comprising:

45. The polynucleotide according to claim 44, wherein the extracellular target binding domain binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, or claudin 6, or binds to any pair of CD19 / CD79b or BCMA / TACI, or is a TriPRIL antigen binding domain.

46. The polynucleotide according to claim 44, wherein the extracellular target binding domain binds to CD19.

47. The polynucleotide according to claim 44, wherein the extracellular target binding domain is not derived from a human polypeptide sequence.

48. The polynucleotide according to claim 44, wherein the extracellular target binding domain is derived from a mouse polypeptide sequence.

49. The transmembrane domain is the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R Beta, IL2R Gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The polynucleotide according to claim 44, selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

50. The polynucleotide according to claim 44, wherein the intracellular signaling domain is selected from the group consisting of CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

51. The polynucleotide according to claim 44, comprising a nucleic acid sequence having at least 85% identity with sequence numbers 17-18.

52. The polynucleotide according to claim 44, comprising the nucleic acid sequence of sequence number 19 and the nucleic acid sequences of sequence numbers 20, 22, or 24.

53. The polynucleotide according to claim 29, wherein the vector is optionally a lentiviral vector.

54. A polynucleotide containing shRNA with sequence number 13.

55. A cell comprising a polynucleotide according to any one of claims 29 to 54.

56. A cell according to claim 1 or claim 2, comprising a polynucleotide according to any one of claims 29 to 54.

57. A method for modifying the immunogenicity of a cell, comprising introducing an oligonucleotide complementary to a polynucleotide encoding any one of the MHC class II complex subunits of Sequence ID No. 7 to 12 into the cell, wherein the oligonucleotide is selected from the group consisting of RNA interference (RNAi) oligonucleotides, antisense oligonucleotides (ASOs), or CRISPR interference (CRISPRi) oligonucleotides.

58. A method for reducing the target immune response to cell therapy, comprising introducing an oligonucleotide complementary to a polynucleotide encoding any one of the class II MHC transactivator complex proteins of Sequence ID No. 7 to 12 into cells for cell therapy, wherein the oligonucleotide is selected from the group consisting of RNA interference (RNAi) oligonucleotides, antisense oligonucleotides (ASOs), or CRISPR interference (CRISPRi) oligonucleotides.

59. The method according to claim 57 or claim 58, further comprising introducing a virus-derived antigen-processing-related transporter inhibitor (TAPi) or a variant thereof into cells for cell therapy.

60. The method according to claim 57 or claim 58, comprising introducing a polynucleotide according to any one of claims 29 to 54 into cells for cell therapy.

61. The method according to claim 57 or claim 58, wherein the cells are eukaryotic cells.

62. The method according to claim 57 or claim 58, wherein the cells are immune cells.

63. The method according to claim 62, wherein the immune cell is a T cell.

64. The method according to claim 57 or claim 58, wherein the cells are of the same species and allosystem as the target cells.

65. The method according to claim 57 or claim 58, wherein the cell therapy is CAR-T cell therapy.

66. The method according to claim 65, wherein the CAR-T cell therapy comprises anti-CD19 CAR-T cells.

67. The method according to claim 58, wherein the subject is a human subject.

68. The method according to claim 58, which reduces natural killer cell activation.

69. A pharmaceutical product for treating the target cancer, (i) The cell according to claim 1 or claim 2; (ii) A cell comprising a polynucleotide as described in any one of claims 29 to 54; or (iii) A cell according to claim 1 or claim 2, comprising the polynucleotide according to any one of claims 29 to 54. Includes, (i), (ii), or (iii) above are pharmaceuticals administered to the subject.

70. The pharmaceutical product according to claim 69, wherein the cancer is a blood cancer.

71. The pharmaceutical product according to claim 70, wherein the blood cancer is selected from the group consisting of leukemia, lymphoma, and myeloma.

72. The pharmaceutical product according to claim 70, wherein the blood cancer is selected from the group consisting of acute lymphoblastic leukemia or mantle cell lymphoma.

73. The pharmaceutical product according to claim 69, wherein the cancer is a solid tumor.

74. The pharmaceutical product according to claim 73, wherein the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, brain cancer, liver cancer, kidney cancer, lung cancer, breast cancer, prostate cancer, pharyngeal cancer, thyroid cancer, colon cancer, testicular cancer, and skin cancer.

75. The pharmaceutical agent according to claim 69, wherein cancer expresses CD19.

76. A cell according to claim 1 or 2, or a polynucleotide according to any one of claims 29 to 54, wherein the MHC class II transactivator protein is class II MHC transactivator 3.

Images