Improving the efficacy and safety of T cell-mediated immunotherapy

Abstract

This document relates to engineered immune cells comprising differentially expressed tumor-CAR and FAPscFv-cytokine fusion proteins, the use of the engineered immune cells in the treatment of tumors expressing FAP, and methods and materials for the preparation of the engineered immune cells.

Description

[Technical Field]

[0001] This document relates generally to the field of cancer, and in particular to cell therapy and immunotherapy for the treatment of solid tumors or hematological cancers characterized by the presence of FAPs in the patient's tumor microenvironment. [Background technology]

[0002] Adoptive cell therapy, also known as cellular immunotherapy, is a form of treatment that uses immune system cells to eliminate diseased cells, such as infected or malignant cells. Some of these approaches involve directly isolating an individual's own immune cells to simply expand their numbers, while others involve genetically engineering immune cells from the patient (autologous approaches) or donors (allogeneic approaches) to enhance and / or redirect immune cells toward specific target tissues. In the case of cancer, immune cells, particularly cytolytic and helper T lymphocytes, natural killer cells, and macrophages, are particularly potent against cancer due to their ability to bind to markers known as antigens on the surface of cancer cells. Cellular immunotherapy harnesses this natural ability and can be deployed in a variety of ways, including tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) cell therapy, chimeric antigen receptor ("CAR") immune cell therapy, and natural killer (NK) cell therapy.

[0003] Chimeric antigen receptor-expressing immune cells are cells that have been genetically engineered to express a chimeric antigen receptor (CAR) and are often designed to recognize specific tumor antigens and kill cancer cells expressing the tumor antigen(s). Chimeric antigen receptor-expressing immune cells are typically CAR-expressing T cells ("CAR-T cells"), CAR-expressing natural killer cells ("CAR-NK cells"), or CAR-expressing macrophages.

[0004] CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single or multiple fusion molecules. Generally, the binding moiety of a CAR can comprise the antigen-binding domain of a single-chain antibody ("scFv"), which comprises variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation CARs is derived from the cytoplasmic region of the CD3 zeta (i.e., CD3ζ) chain or the Fc receptor gamma chain. First-generation CARs have been shown to successfully redirect T cell cytotoxicity, but failed to produce long-term expansion and antitumor activity in vivo. Addition of signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137), alone (second generation) or in combination (third generation), has enhanced the survival and proliferation of CAR-modified T cells. CARs have been successfully used to redirect T cells against antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors (Jena, Dotti et al., Blood (2010) 116(7):1035-44).

[0005] Adoptive immunotherapy, which involves the transfer of ex vivo generated autologous or allogeneic antigen-specific T cells, is a promising strategy for treating viral infections and cancer, as confirmed by the increasing number of clinical trials using CAR-T cells.

[0006] To date, only autologous CAR T cells have been approved by the U.S. Food and Drug Administration (FDA) (e.g., the anti-CD19 CAR-T tisagenlecleucel (Kymriah™) by Novartis for the treatment of precursor B-cell acute lymphoblastic leukemia, the anti-CD19 CAR-T axicabtagene ciloleucel (Yescarta™) by Kite Pharma for certain types of large B-cell lymphoma in adult patients who express CD19 as a marker, the anti-BCMA CAR-T idecbutagen biculeucel (Abecma™) for the treatment of adult patients with relapsed or refractory multiple myeloma, and the anti-CD19 CAR-T idecabtagene biculeucel (Abecma™) for the treatment of adult patients with relapsed or refractory large B-cell lymphoma). CAR-T lysocabtagene malareucel (Breyanzi™), anti-CD19 CAR-T brexocabtagene autolucel (Tecartus™) for patients with relapsed or refractory mantle cell lymphoma. Allogeneic approaches are more challenging due to the alloreactivity of the cells to the patient's own immune cells. State-of-the-art programs use specific rare-cutting endonucleases, such as TALE nucleases, to reduce the alloreactivity of the cells before administering them to patients, as reported by Poirot et al. (Cancer. Res. (2015) 75(18):3853-3864) and Qasim et al. (Science Translational (2017) 9(374)). Alternatively, inactivation of the TCR (e.g., TRAC and / or TRBC) in primary T cells can be combined with inactivation of MHC components such as beta-2-microglobulin (B2M) and / or inactivation of genes encoding checkpoint proteins, e.g., as described in WO 2014 / 184744.

[0007] T cell-mediated antitumor cytotoxicity is a promising immunotherapy strategy for both leukemia and solid tumors. However, the efficacy of tumor antigen-targeted CAR-T therapy for solid tumors is limited due to several factors, including a lack of tumor-infiltrating lymphocytes (TILs) and an immunosuppressive tumor microenvironment (TME) (Stern et al. (2020) Cancer Treat Res. 180:297-326).

[0008] The microenvironment of most solid tumors is characterized by the presence of activated fibroblasts called cancer-associated fibroblasts (CAFs), which express unique surface proteins such as FAPs (Kalluri R. Nat Rev Cancer (2016) 16:582-98). CAFs can inhibit TILs and promote immunosuppression (Wang et al. (2014) Cancer Immunol Res. 2:154-66).

[0009] Fourth-generation CAR-T cells have been described that aim to induce a pro-inflammatory environment by engineering CAR-T cells to release transgenic cytokines upon CAR signaling in targeted tumor tissues (Chmieswcki et al. (2020) Adv Cell Gene Ther. 3:e84; Sachdeva et al. Nature Communications (2019) 10:5100).

[0010] Waldhauer et al. (MABS (2021)) investigate the effect of immune cytokines in enhancing the efficacy of different immunotherapies.

[0011] Despite recent technological advances, the treatment of cancer, particularly cancers characterized by solid tumors, remains a major medical challenge. What is needed are new compositions and treatments that are effective against solid tumors and safe in patients, particularly those that are efficient at targeting tumor tissue and evading the immunosuppressive tumor microenvironment while exhibiting reduced overall side effects.

[0012] This background information is presented for informational purposes only. No admission is necessarily intended, nor should it be construed, that such information constitutes prior art against the present invention. Summary of the Invention

[0013] This document provides methods and materials for treating cancer. For example, this document provides cells (e.g., immune cells such as T cells or NK cells, or iPSCs that can be further differentiated into immune cells) that are engineered to express a CAR capable of binding to a tumor antigen and secrete stimulatory cytokines to produce potential effects on both the engineered immune cells and the patient's immune cells when and where they are most needed to treat the patient.

[0014] It is to be understood that both the foregoing general description and the following detailed description of the embodiments are exemplary and are therefore not limiting of the scope of the embodiments.

[0015] The methods and materials provided herein are particularly suitable for treating cancers characterized by the presence of FAPs in the tumor microenvironment. The methods and materials provided herein are also particularly suitable for achieving "universal" treatments in which components of the treatment can be used in many unrelated patients.

[0016] In general, one aspect of this document is: a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen, placed under the transcriptional control of an exogenous or endogenous constitutive promoter; b) an exogenous nucleic acid sequence encoding a secreted fusion protein comprising a fibroblast activation protein (FAP) binding domain and a stimulatory cytokine, placed under the transcriptional control of an endogenous inducible promoter; 1. An engineered cell comprising: the exogenous nucleic acid sequences of a) and b) are integrated into the genome of the cell; The engineered cells are featured, wherein the inducible promoter is inducible upon cellular activation.

[0017] As a result, expression of the fusion protein is induced upon activation of the engineered cells.

[0018] In some cases, the FAP binding domain can comprise VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody.

[0019] In some cases, the fusion protein does not include an antibody fragment crystallizable (Fc).

[0020] In some cases, the tumor antigen targeted by the tumor-CAR is not a FAP.

[0021] In some cases, the fusion protein may include a signal peptide that is removed during or after secretion of the fusion protein outside the cell.

[0022] In some cases, the engineered cell can be an immune cell, such as a T cell, an NK cell, or a macrophage.

[0023] In some cases, the engineered cells can be engineered T cells or engineered NK cells.

[0024] In some cases, the engineered cells may be iPSCs, which may be intermediates in the production of engineered immune cells such as T cells, NK cells, or macrophages, as described herein. In some cases, the engineered cells may be engineered immune cells derived from iPSCs after the engineered iPSCs have been subjected to one or more differentiation steps.

[0025] Although various aspects described herein are applicable to situations where the cell is an immune cell such as a T cell, these various aspects also apply to, and are therefore included herein, NK cells and macrophages.

[0026] In some cases, the engineered cells described herein may be T cells that have been genetically modified to suppress or abrogate expression of a T cell receptor (TCR) (e.g., an endogenous TCR) by inactivation of a gene (e.g., the TRAC gene and / or the TRBC gene) encoding a component of the TCR, optionally genetically modified to suppress or abrogate expression of at least one gene that controls MHC complex surface presentation, such as B2M and class II major histocompatibility complex transactivator (CIITA), optionally genetically modified to suppress or abrogate expression of CD52, and optionally genetically modified to suppress or abrogate expression of at least one immune checkpoint or receptor for an immune checkpoint.

[0027] In another aspect, this document provides: a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) (“tumor-CAR”) that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding (i) a fibroblast activation protein (FAP)-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. An engineered T cell comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The invention features engineered T cells, wherein the inducible promoter is inducible upon activation of the T cell.

[0028] In some cases, the exogenous nucleic acid sequence of a) is placed under the control of an exogenous constitutive promoter after integration into the genome of the cell.

[0029] In some cases, the exogenous nucleic acid sequence of a) is placed under the control of an endogenous constitutive promoter after integration into the genome of the cell.

[0030] In some cases, the fusion protein comprises: a) a signal peptide that directs the secretion of a fusion protein, the signal peptide being removed after secretion of the fusion protein; b) a FAP-binding domain, for example, a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody; c) at least one stimulatory cytokine selected from the group consisting of interleukin-2 or a variant thereof such as IL-2v, interleukin-7 or a variant thereof, interleukin-12 or a variant thereof, interleukin-15 or a variant thereof, interleukin-15 in complex with its high affinity receptor IL-15RA, interleukin-18 or a variant thereof, interleukin-23 or a variant thereof; Includes.

[0031] Optionally, the fusion protein does not include an antibody fragment crystallizable (Fc).

[0032] Optionally, the fusion protein does not include a complete monoclonal anti-FAP antibody.

[0033] In some cases, tumor-CARs a) an extracellular tumor antigen-binding domain, for example, an extracellular tumor antigen-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-tumor antigen antibody; b) a hinge selected from an FcγRIII hinge, a CD8α hinge, and an IgG1 hinge; c) a transmembrane domain comprising a CD8α transmembrane domain or a CD28 transmembrane domain; d) a cytoplasmic domain containing a CD3 zeta signaling domain and a costimulatory domain derived from 4-1BB or CD28; may include:

[0034] In some cases, the constitutive promoter may be selected from the group consisting of EF1A, CD52, GAPDH, CMV, hPGK, UBC, SV40, PGK, CAGG, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, RPBSA, CD4, and CD8 promoters, and / or the inducible promoter may be selected from the group consisting of PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, GOS2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 promoters.

[0035] In some cases, the constitutive promoter can be selected from the group consisting of EF1A, TRAC, B2M, CD52, CS1, CD45, CD5, and GAPDH promoters. For example, the constitutive promoter can be an EF1A promoter, a TRAC promoter, a CD52 promoter, or a B2M promoter. For example, the constitutive promoter can be an EF1A promoter.

[0036] In some cases, the inducible promoter can be selected from the group consisting of PDCD1, CD25, GM-CSF, TIM3, and TIGIT promoters. For example, the inducible promoter can be a PDCD1 promoter.

[0037] In some cases, the constitutive promoter can be the endogenous TRAC promoter or the exogenous EF1A promoter, and the inducible promoter can be the endogenous PDCD1 promoter.

[0038] In some cases, the constitutive promoter can be an exogenous EF1A promoter and the inducible promoter can be an endogenous PDCD1 promoter.

[0039] In some cases, the fusion protein may comprise a FAP-binding domain comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, or SEQ ID NO:36, and / or the tumor-CAR may comprise an extracellular tumor antigen-binding domain comprising the amino acid sequence of SEQ ID NO:45, SEQ ID NO:53, or SEQ ID NO:61.

[0040] In some cases, the fusion protein may comprise a FAP-binding domain comprising the amino acid sequence of SEQ ID NO: 9, and the tumor-CAR may comprise an extracellular tumor antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 61.

[0041] In some cases, an engineered T cell described herein may be a T cell that has been genetically modified to suppress or silence expression of a T cell receptor (TCR) (e.g., an endogenous TCR) by inactivation of a gene (e.g., TRAC and / or TRBC) encoding a component of the TCR, optionally genetically modified to suppress or silence expression of at least one gene that controls MHC complex surface presentation, such as B2M and CIITA, optionally genetically modified to suppress or silence expression of CD52, and optionally genetically modified to suppress or silence expression of at least one gene encoding an immune checkpoint or a receptor for an immune checkpoint.

[0042] In some cases, the engineered T cells described herein may be T cells that have been genetically modified to (i) suppress or inhibit expression of the T cell receptor (TCR) by inactivation of genes encoding components of the TCR (e.g., TRAC and / or TRBC), (ii) suppress or inhibit expression of PD1 or a receptor for PD1, optionally (iii) suppress or inhibit expression of B2M, and optionally (iv) suppress or inhibit expression of CD52 in the T cell.

[0043] In other embodiments of the present disclosure, the engineered immune cell(s) are NK cells. Thus, another embodiment is, for example, a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding (i) a fibroblast activation protein (FAP)-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. An engineered NK cell comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The invention relates to engineered NK cells, wherein the inducible promoter is inducible upon activation of the NK cells.

[0044] In some cases, the engineered NK cells described herein have been genetically modified to suppress or downregulate expression of at least one gene that controls MHC complex surface presentation, such as B2M and class II major histocompatibility complex transactivator (CIITA), in NK cells, and optionally, to suppress or downregulate expression of CD52, and optionally, to suppress or downregulate expression of at least one immune checkpoint or its receptor.

[0045] In other embodiments of the present disclosure, the engineered cell(s) are iPSCs. a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding (i) a fibroblast activation protein (FAP)-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. An engineered iPSC comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The invention relates to engineered iPSCs, wherein the inducible promoter is inducible upon activation of an immune cell into which the engineered iPSCs may further differentiate.

[0046] In some cases, the engineered iPSCs described herein have been genetically modified in the cells to suppress or silence expression of the T cell receptor (TCR) by inactivation of a gene encoding a T cell receptor (TCR) component (e.g., TRAC and / or TRBC), to suppress or silence expression of at least one gene that controls MHC complex surface presentation, such as B2M and class II major histocompatibility complex transactivator (CIITA), and optionally to suppress or silence expression of CD52, and optionally to suppress or silence expression of at least one immune checkpoint or its receptor.

[0047] In some cases, the tumor-CAR can be constitutively expressed in engineered immune cells (e.g., engineered T cells) by lentiviral integration or by nuclease-mediated cDNA insertion at one or more constitutively expressed gene loci, such as one or more of the TRAC, B2M, or CD52 gene loci.

[0048] In some cases, the TRAC and / or B2M loci can be disrupted, for example, by TALE nucleases, to increase the persistence of engineered immune cells (e.g., the persistence of engineered T cells) in an allogeneic environment to inhibit graft-versus-host disease (GvHD).

[0049] In some cases, fusion protein expression is inducible upon activation of an engineered immune cell (e.g., an engineered T cell), and the fusion protein is encoded by an exogenous nucleic acid sequence that can be integrated into the genome of the cell by nuclease-mediated cDNA insertion at one or more inducible gene loci.

[0050] In some cases, the fusion protein may be encoded by an exogenous nucleic acid sequence that is integrated into the genome of the cell by nuclease-mediated cDNA insertion at one or more inducible loci selected from the group consisting of the PDCD1, CD25, GM-CSF, TIM3, and TIGIT loci, e.g., at a PDCD1-inducible locus.

[0051] In some cases, the tumor antigen targeted by the tumor-CAR may be selected from the group consisting of mesothelin (e.g., human mesothelin), MUC1 (e.g., human MUC1), EGFR (e.g., human EGFR), VEGF (e.g., human VEGF), and Trop2 (e.g., human Trop2).

[0052] In some cases, the tumor antigen targeted by the tumor-CAR can be mesothelin (e.g., human mesothelin) or MUC1 (e.g., human MUC1).

[0053] In another aspect, this document features a method of treating a cancer characterized by the presence of a FAP in the tumor microenvironment, comprising administering a therapeutically effective amount of an engineered immune cell (e.g., an engineered T cell) described herein that includes (a) an exogenous nucleic acid sequence encoding a tumor-CAR placed under the transcriptional control of an exogenous or endogenous constitutive promoter, and (b) an exogenous nucleic acid sequence encoding a secreted fusion protein comprising a FAP-binding domain and a stimulatory cytokine placed under the transcriptional control of an endogenous inducible promoter.

[0054] In another aspect, this document features a pharmaceutical composition comprising a therapeutically effective amount of an engineered immune cell (e.g., an engineered T cell) described herein.

[0055] In another aspect, this document features a composition comprising a therapeutically effective amount of an engineered immune cell (e.g., an engineered T cell) described herein for use in treating a cancer characterized by the presence of a FAP in the tumor microenvironment. In another aspect, this document provides a method for producing a cell population comprising engineered immune cells (e.g., engineered T cells), comprising: (i) providing donor-derived immune cells or induced pluripotent stem cells (iPSCs); (ii) optionally, inhibiting or suppressing expression of a T cell receptor (TCR) (e.g., an endogenous TCR) in a cell or presentation of a TCR on the surface of said cell; (iii) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a tumor-CAR described herein placed under the transcriptional control of an exogenous or endogenous constitutive promoter; (iv) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, as described herein, placed under the transcriptional control of an endogenous inducible promoter; (v) optionally isolating the engineered cells that do not express a TCR (e.g., an endogenous TCR) on the cell surface; Including, The method features, wherein the inducible promoter is inducible upon activation of the engineered immune cell.

[0056] In yet another aspect, the document provides: (1)(a) at least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a tumor-CAR described herein placed under the transcriptional control of a constitutive promoter; or (1)(b) at least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a tumor-CAR described herein, wherein the nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to an endogenous constitutive locus in the cell; and (2) At least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a FAP-binding domain and a stimulatory cytokine, as described herein, wherein the exogenous nucleic acid sequence is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to an endogenous inducible locus in the cell. The present invention features a set of vectors, including:

[0057] This document also: (1)(a) at least one vector comprising a nucleic acid sequence comprising a constitutive promoter and a nucleic acid sequence encoding a tumor-CAR operably linked to the promoter; and / or (1)(b)(i) at least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a tumor-CAR, wherein the nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to an endogenous constitutive locus in a cell; and (ii) at least one sequence-specific endonuclease that targets the endogenous constitutive locus; and (2) (i) at least one vector comprising a nucleic acid sequence encoding a fusion protein comprising a FAP-binding domain and a stimulatory cytokine, the nucleic acid sequence being positioned between a left homology region and a right homology region, the left homology region and the right homology region being homologous to an endogenous inducible locus in the cell; and (ii) at least one sequence-specific endonuclease that targets the inducible locus. The present invention relates to a kit comprising:

[0058] As used herein, endogenous constitutive loci and endogenous inducible loci in a cell refer to loci in a cell that can be transduced with a set of vectors as described, said vectors constituting a means for integrating a defined cassette, as described, into the genome of the cell.

[0059] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0060] Those skilled in the art will understand that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0061] [Figure 1]A) Diagram of a strategy to improve the safety and efficacy of CAR-T-based therapy using a constitutively expressed tumor-targeting CAR and an immunocytokine (e.g., FAPscFv-IL2v) expressed under the control of an inducible promoter. B) Diagram of a different approach to deliver a tumor-CAR to the genome of a cell to express the tumor-CAR under the control of a constitutive promoter. C) Diagram of an approach to deliver an immunocytokine (e.g., FAPscFv-IL2v) to the genome of a cell to express the immunocytokine under the control of an inducible promoter. [Figure 2] A) Diagram of strategies for improving the safety and efficacy of CAR-T-based therapy, as described in the Examples section. B) Meso-CAR constructs are integrated into the genome of cells at the TRAC locus (TRAC-targeted integration), with expression of the Meso-CAR construct under the control of the endogenous TCR promoter, or randomly integrated into the genome of cells using rLV, with expression of the Meso-CAR construct under the control of the exogenous EF1 alpha promoter. C) FAPscFv-IL2v constructs are integrated into the genome of cells at the PDCD1 locus (PDCD1-targeted integration). [Figure 3A] Flow cytometry results showing TCR alpha / beta (Y-axis) and Meso-CAR staining (X-axis) of mock-electroporated T cells (Mock), T cells with Meso-CAR-targeted integration at the TRAC locus and PDCD1 knockout (TRACMesoCARPDCD1KO), T cells with TRAC knockout and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACKOPDCD1FAPscFv-IL2v), or T cells with Meso-CAR-targeted integration at the TRAC locus and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACMesoCARPDCD1FAPscFv-IL2v). [Figure 3B] Post-activation flow cytometry results showing PD-1 staining (X-axis) of the indicated T cells after activation with PMA and ionomycin. [Figure 3C]1 shows the results of a His-tag ELISA measuring FAPscFv-IL2v production and secretion after activation of the indicated T cells with recombinant mesothelin protein. [Figure 4] A) Schematic depicting the experimental design for investigating the anti-tumor activity of engineered TRACCAR T cells in a xenograft mouse model. B) Growth kinetics of mouse tumor xenografts treated with mock-electroporated T cells (Mock), T cells with Meso-CAR-targeted integration at the TRAC locus and PDCD1 knockout (TRACMesoCARPDCD1KO), T cells with TRAC knockout and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACKOPDCD1FAPscFv-IL2v), or T cells with Meso-CAR-targeted integration at the TRAC locus and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACMesoCARPDCD1FAPscFv-IL2v). [Figure 5A] Flow cytometry results showing TCR alpha / beta (X-axis) staining of T cells with TRAC knockout and PDCD1 knockout (TRACKOPDCD1KO), T cells with random Meso-CAR integration and double knockout of TRAC and PDCD1 (rLv-MesoCAR;TRACKOPDCD1KO), T cells with TRAC knockout and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACKOPDCD1FAPscFv-IL2v), or T cells with random Meso-CAR integration, TRAC knockout, and FAPscFv-IL2v-targeted integration at the PDCD1 locus (rLv-MesoCAR;TRACKOPDCD1FAPscFv-IL2v). [Figure 5B] Flow cytometry results showing Meso-CAR staining (X-axis) for the indicated T cells. [Figure 5C] Percentage of targeted integration of FAPscFv-IL2v at the PDCD1 locus as determined by ddPCR. [Figure 5D]1 shows the results of a His-tag ELISA demonstrating FAPscFv-IL2v production and secretion following activation of the indicated T cells with recombinant mesothelin protein. [Figure 6] A) Flow cytometry results showing GFP expression (Y-axis) and human FAP staining (X-axis) of tumor cell lines NCI-H226 and NCI-H226-FAP. B) Quantitative representation of flow cytometry analyzing dose-dependent binding of recombinant FAPscFv-IL2v protein to NCI-H226-FAP cells, normalized to NCI-H226 control cells. [Figure 7A] FIG. 1 is a schematic depicting the experimental design of a sequential killing assay to measure the anti-tumor cytotoxicity of engineered T cells. [Figure 7B] 1 is a graphical representation of tumor cell survival as a unit of time following co-incubation of tumor cells with T cells with TRAC knockout and PDCD1 knockout (TRACKOPDCD1KO), T cells with Meso-CAR random integration and double knockout of TRAC and PDCD1 (rLv-MesoCAR;TRACKOPDCD1KO), T cells with TRAC knockout and FAPscFv-IL2v-targeted integration at the PDCD1 locus (TRACKOPDCD1FAPscFv-IL2v), or T cells with Meso-CAR random integration and TRAC knockout and FAPscFv-IL2v-targeted integration at the PDCD1 locus (rLv-MesoCAR;TRACKOPDCD1FAPscFv-IL2v) in a sequential killing assay. [Figure 7C] Images of viable NCI-H226 and NCI-H226-FAP cells expressing the GFP reporter at 72 hours of a sequential killing assay after incubation with the indicated T cells. [Figure 7D] Graphical representation of the killing rates of NCI-H226 (left panel) and NCI-H226-FAP (right panel) by the indicated T cells over the 48-72 hour period of the serial killing assay. [Figure 7E]Graphical representation of IFN-g released by the indicated T cells in co-incubation supernatants with NCI-H226 (left panel) and NCI-H226-FAP (right panel) during the course of a sequential killing assay. [Figure 8] RNAseq data for CS1-CAR-T cells (unactivated) at t=0 h or 24 h after activation with recombinant CS1 protein (open symbols). Filled symbols represent genes selected based on expression levels (<100 TPM at 0 h, >50 TPM at 24 h, and fold change >5 at 24 h). DETAILED DESCRIPTION OF THE INVENTION

[0062] [Detailed explanation] This document provides methods and materials that can be used to exploit spatial features of the tumor microenvironment ("TME") to locally amplify the anti-tumor activity of immunotherapies. For example, in some cases, this document provides engineered CAR-T, as well as engineered T cells expressing tumor-targeting CARs that can be used as triggers to enhance the tumoricidal activity of a patient's immune cells when and where it is needed, thereby enhancing both the therapeutic efficacy and safety of targeted cellular immunotherapies in patients.

[0063] In interpreting this specification, the following definitions shall apply, and wherever appropriate, terms used in the singular shall include the plural, and vice versa. If any definition set forth below conflicts with the usage of that term in other documents, including documents incorporated herein by reference, the definition set forth below shall always control for the purposes of interpreting this specification and the claims associated therewith, unless a contrary meaning is clearly intended (e.g., in the document in which the term is first used). The use of "or" means "and / or" unless otherwise indicated. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures of cells. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Furthermore, when the term "comprising" is used in describing one or more embodiments, those skilled in the art will understand that in some specific instances, one or more embodiments may alternatively be described using the language "consisting essentially of" and / or "consisting of."

[0064] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is used.

[0065] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0066] The practice of the present invention will employ, unless otherwise indicated, techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, gene editing, and immunology within the knowledge of those skilled in the art, and such techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. Ausubel, 2000, Wiley and Sons Inc, Library of Congress, USA), Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Oligonucleotide Synthesis (M.J. Gait, ed., 1984), Mullis et al., U.S. Pat. No. 4,683,195, Nucleic Acid Hybridization (B.D. Harries & S.J. Higgins, eds., 1984), Transcription And Translation (B.D. Hames & S.J. Higgins, eds., 1984), Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987), Immobilized Cells And Enzymes (I.R.L. Press, 1986), B. Perbal, A Practical Guide To Molecular Biology (Frederick M. Ausubel, 2000, Wiley and Sons Inc, Library of Congress, USA), Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Oligonucleotide Synthesis (M.J. Gait, ed., 1984), Mullis et al., U.S. Pat. No. 4,683,195, Nucleic Acid Hybridization (B.D. Harries & S.J. Higgins, eds., 1984), Transcription And Translation (B.D. Hames & S.J. Higgins, eds., 1984), Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987), Immobilized Cells And Enzymes (I.R.L. Press, 1986), B Cloning (1984), in the series Methods In ENZYMOLOGY (J. Abelson and M. Simon, editors-in-chief, Academic Press, Inc., New York), in particular volumes 154 and 155 (Wu et al., eds.) and volume 185, "Gene Expression Technology" (D. Goeddel, ed.), Gene Transfer Vectors For Mammalian Cells (J.H. Miller and MPSee Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986), and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0067] Unless otherwise defined herein, all technical and scientific terms used have the same meaning as those generally understood by those skilled in the art of gene therapy, biochemistry, genetics, immunology, cancer, molecular biology and gene editing.The definitions of common terms in molecular biology can be found, for example, in Benjamin Lewin, Genes VII, Oxford University Press, 2000 (ISBN 019879276X), Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, Blackwell Publishers, 1994 (ISBN 0632021829), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).

[0068] As used herein, a "recipient" is a patient receiving a transplant, such as a transplant comprising a population of engineered immune cells, e.g., T cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0069] As used herein, a "donor" is a mammal (e.g., a human) from which one or more cells are isolated prior to administration of the cells or cell progeny to a recipient. The one or more cells may be, for example, a population of immune cells or hematopoietic stem cells that are manipulated, expanded, enriched, or maintained according to the methods described herein prior to administration of the cells or cell progeny to a recipient. In the allogeneic context contemplated herein, the "donor" is not the patient being treated.

[0070] "Expansion," in the context of cells, refers to an increase in the number of a particular cell type or types of cells from an initial population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells produced from the expansion.

[0071] A "cell population" includes eukaryotic cells, such as mammalian cells, e.g., human cells, isolated from a biological source, e.g., a blood product or tissue. A cell population can be derived from more than one cell.

[0072] As used herein, the term "pharmaceutical composition" refers to an active ingredient in combination with a pharmaceutically acceptable carrier and / or excipient, such as a carrier and / or excipient commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the bounds of sound medical knowledge, suitable for use in contact with the tissues of mammals, such as humans, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0073] As used herein, the term "administering" refers to introducing a compound, cell, or cell population disclosed herein into a subject by a method or route that results in at least partial delivery of the agent at the desired site. Pharmaceutical compositions containing the compounds or cells disclosed herein can be administered by any suitable route that results in effective treatment of the patient. Patients that can be treated with the materials and methods disclosed herein can be mammals, including humans and non-human primates.

[0074] As used herein, "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of phosphodiester bonds. Nucleic acids can be single-stranded or double-stranded.

[0075] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0076] As used herein, the terms "treat," "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects caused by the disease. As used herein, "treatment" includes any treatment of disease in a mammal (e.g., a human), including (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., preventing the onset of the disease, and (c) palliating the disease, e.g., causing a remission of the disease, e.g., completely or partially eliminating the symptoms of the disease.

[0077] As used herein, the term "subject" or "patient" includes mammals, including non-human primates and humans.

[0078] An "effective amount" or "therapeutically effective amount" refers to the amount of a composition described herein that, when administered to a subject (e.g., a human), is sufficient to help treat a disease. The amount of a composition that constitutes a "therapeutically effective amount" varies depending on the cell preparation, the condition and its severity, the mode of administration, and the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art having regard to their own knowledge and this disclosure. When referring to individual active ingredients or compositions administered alone, the therapeutically effective dose refers to that individual active ingredient or composition alone. When referring to a combination, the therapeutically effective dose refers to the combined amounts of the active ingredients, compositions, or both that result in the therapeutic effect, whether administered concomitantly, simultaneously, or sequentially.

[0079] "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. "Vector" may include, but is not limited to, viral vectors, plasmids, oligonucleotides, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Preferred vectors are vectors capable of autonomous replication (episomal vectors) and / or vectors capable of expressing a nucleic acid to which it has been linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses (AAV)), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, Examples of viruses include double-stranded DNA viruses, including rabies, flu, flu-like viruses (e.g., flu-like viruses, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma virus, mammalian type C viruses, type B viruses, type D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, J.M., Retroviridae: The viruses and their replication, In Fundamental Virology, 3rd ed., B.N. Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0080] As used herein, the term "locus" refers to the specific physical location of a DNA sequence (e.g., a gene) in a genome. The term "locus" may also refer to the specific physical location of a rare-cutting endonuclease target sequence in a chromosome. Such a locus may include a target sequence that is recognized and / or cleaved by a sequence-specific endonuclease described herein. It is understood that a locus of interest can identify not only a nucleic acid sequence present in the body of a cell's genetic material (i.e., a chromosome), but also a portion of genetic material that may exist independently of the body of genetic material, such as a plasmid, episome, virus, transposon, or an organelle such as, by way of non-limiting example, a mitochondria.

[0081] As used herein, a nucleic acid sequence is said to be "under the transcriptional control of a promoter" if the nucleic acid sequence is operably linked to the promoter, such that the nucleic acid sequence follows the promoter or is at the 3' end of the promoter, such that its transcription is controlled by the promoter.

[0082] The term "cleavage" when used in reference to nucleic acids refers to the disruption of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-strand and double-strand breaks are possible, and double-strand breaks can occur as a result of two different single-strand break events. Cleavage of double-stranded DNA, RNA, or DNA-RNA hybrids can result in the generation of either blunt ends or staggered ends.

[0083] "Sequence identity" refers to the identity between two nucleic acid molecules or polypeptides. Sequence identity refers to residues that are the same in two sequences when the two sequences are aligned for maximum correspondence. If a position in the compared sequences is occupied by the same base (or amino acid), the molecules are identical at that position. The degree of identity between nucleic acid sequences (or amino acid sequences) corresponds to the number of identical or matching nucleotides (or amino acids) at positions shared by the aligned nucleic acid sequences (or amino acid sequences). Various alignment algorithms and / or alignment programs, including FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), can be used to calculate identity between two sequences, for example, using default settings. For example, polypeptides and polynucleotides encoding such polypeptides that have at least 70%, 85%, 90%, 95%, 98%, or 99% identity to the specific polypeptides described herein and exhibit substantially the same function, are contemplated.

[0084] "Fibroblast activation protein" ("FAP") is also commonly referred to as prolyl endopeptidase FAP, or fibroblast activation protein alpha (NCBI Gene ID: 2191). In some cases, the FAP polypeptide can be a human FAP polypeptide. Examples of FAP polypeptides that can be targeted by the FAP binding domains described herein include, but are not limited to, human FAP polypeptides having the amino acid sequence set forth in NCBI Reference Sequence: NP_004451.2.

[0085] In one aspect, this document: a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding a secreted fusion protein comprising a fibroblast activation protein (FAP) binding domain and a stimulatory cytokine; 1. An engineered cell comprising: the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); An engineered cell is provided, wherein the inducible promoter is inducible upon activation of the cell.

[0086] In certain embodiments, the fusion protein does not comprise an antibody fragment crystallizable (Fc).

[0087] In certain embodiments, the fusion protein does not include an entire anti-FAP antibody.

[0088] In certain embodiments, the fibroblast activation protein binding domain comprises VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody.

[0089] The engineered cell can be any suitable cell. For example, the cell comprising the exogenous nucleic acid sequences of a) and b) can be an immune cell such as a T cell, an NK cell, or a macrophage. In some cases, the cell comprising the exogenous nucleic acid sequences of a) and b) can be an iPSC, which can be an intermediate in the production of engineered immune cells such as T cells, NK cells, or macrophages, as described herein.

[0090] In some cases, the engineered cells provided herein may be T cells that have been genetically modified to suppress or inhibit expression of a T cell receptor (TCR) (e.g., an endogenous TCR) on the surface of the T cell, and optionally, to suppress or inhibit expression of at least one gene that controls MHC complex surface presentation, such as the B2M gene encoding a β2m polypeptide and / or the CIITA gene encoding a CIITA polypeptide, and optionally, to suppress or inhibit expression of a gene encoding a CD52 polypeptide on the surface of the T cell.

[0091] In some cases, the engineered cells provided herein may be NK cells that have been genetically modified to suppress or repress the expression of at least one gene that controls MHC complex surface presentation, such as the B2M gene encoding the β2m polypeptide and / or the CIITA gene encoding the CIITA polypeptide, and optionally, to suppress or repress the expression of a gene encoding a CD52 polypeptide on the surface of the NK cell.

[0092] In some cases, the β2m polypeptide can be a human β2m polypeptide. Examples of B2M genes encoding β2m polypeptides whose expression can be silenced or inhibited as described herein include, but are not limited to, the human B2M gene encoding a β2m polypeptide having the amino acid sequence set forth in GeneBank Accession No. AAA51811 (e.g., NCBI Gene ID 567).

[0093] In some cases, the CIITA polypeptide may be a human CIITA polypeptide. Examples of CIITA genes encoding CIITA polypeptides whose expression can be suppressed or silenced as described herein include, but are not limited to, human CIITA genes encoding CIITA polypeptides having the amino acid sequence set forth in GeneBank Accession Nos. P33076.3 or AAU06586 (e.g., NCBI Gene ID 4261).

[0094] In some cases, the CD52 polypeptide can be a human CD52 polypeptide. Examples of CD52 genes (e.g., NCBI gene ID 1043) encoding CD52 polypeptides whose expression can be silenced or inhibited as described herein include, but are not limited to, the human CD52 polypeptide having the amino acid sequence set forth in GeneBank Accession No. AJC19276.

[0095] In some cases, the engineered T cells described herein may be designed such that the CD52 gene, the B2M gene, or both the CD52 and B2M genes are inactivated.

[0096] In some cases, the engineered T cells described herein can be designed (e.g., genetically modified) to suppress or silence expression of at least one immune checkpoint protein or a receptor for an immune checkpoint protein. For example, the engineered T cells described herein can be designed to inactivate the programmed cell death 1 (PDCD1) gene, the CTLA4 gene, or both the PDCD1 gene and the cytotoxic T lymphocyte-associated protein 4 (CTLA4) gene.

[0097] In some cases, the PD1 polypeptide can be a human PD1 polypeptide. Examples of PDCD1 genes encoding PD1 polypeptides whose expression can be silenced or inhibited as described herein include, but are not limited to, the human PDCD1 gene encoding a polypeptide having the amino acid sequence set forth in GeneBank Accession No. UMM61402.1 (e.g., NCBI Gene ID 5133 or ENSG00000188389).

[0098] In some cases, the CTLA4 polypeptide can be a human CTLA4 polypeptide. Examples of CTLA4 genes encoding CTLA4 polypeptides whose expression can be silenced or inhibited as described herein include, but are not limited to, the human CTLA4 gene encoding the CTLA4 polypeptide having the amino acid sequence of GeneBank Accession No. AAL07473 (e.g., NCBI Gene ID 1493).

[0099] In some cases, the engineered T cells described herein may be designed such that at least one gene encoding a TCR component and the PDCD1 gene are inactivated.

[0100] In another aspect, this document provides a pharmaceutical composition comprising the engineered immune cells described herein.

[0101] For example, this document: a) an exogenous nucleic acid sequence encoding a tumor-CAR; and b) an exogenous nucleic acid sequence encoding (i) a FAP-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. A pharmaceutical composition comprising an engineered immune cell (e.g., an engineered T cell) comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is placed under the transcriptional control of an endogenous or exogenous constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The pharmaceutical composition is provided, wherein the inducible promoter is inducible upon activation of an immune cell.

[0102] In some cases, the pharmaceutical compositions described herein may be for use in treating cancers characterized by the presence of FAPs in the tumor microenvironment.

[0103] In another aspect, this document provides: a) an exogenous nucleic acid sequence encoding a tumor-CAR; and b) an exogenous nucleic acid sequence encoding (i) a FAP-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; administering to a patient in need thereof a therapeutically effective amount of engineered immune cells (e.g., engineered T cells), comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The present invention provides a method for treating a cancer characterized by the presence of a FAP in the tumor microenvironment, the method comprising the step of: wherein the inducible promoter is inducible upon activation of an immune cell.

[0104] The engineered cells and methods described herein can be part of an autologous therapy or part of an allogeneic therapy. Autologous means that the cells used to treat a patient are derived from the patient. Allogeneic means that the cells or cell populations used to treat a patient are not derived from the patient, but are derived from a donor or cell line.

[0105] In some cases, the engineered cells described herein can be administered to a patient (e.g., a human) undergoing immunosuppressive therapy. In some cases, the administered cells can be cells that have been made resistant to at least one immunosuppressant. In some cases, the immunosuppressive therapy can aid in the selection and expansion of engineered immune cells (e.g., engineered T cells) in the patient.

[0106] The cells described herein can be administered to a patient using any suitable route of administration, including aerosol inhalation, injection, ingestion, infusion, implantation, and / or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In some cases, the cell compositions described herein can be administered to a patient by intravenous injection, so long as the cells are capable of migrating to the desired site of action.

[0107] While individual needs vary, determining the optimal range of effective amounts of a particular cell type for a particular disease or condition is within the skill of one of ordinary skill in the art. By effective amount is meant an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect. In some cases, administration of cells or cell populations is at a dose of about 10 cells / kg body weight. 4 ~10 9 In some cases, administration of about 10 cells 5 ~10 6 per kg body weight, or approximately 10 cells 5 ~5×10 6 Cells / kg body weight may be administered. All integer values ​​of cell numbers within these ranges are contemplated.

[0108] The cells may be administered in one or more doses. In some cases, an effective amount of cells may be administered as a single dose. In some cases, an effective amount of cells may be administered as multiple doses over a period of time. The timing of administration is within the discretion of the attending physician and is determined by the patient's clinical condition.

[0109] In some cases, administering engineered immune cells (e.g., T cells) may include treating the patient with a myeloablative and / or immunosuppressive regimen to deplete host bone marrow stem cells and prevent rejection. In some cases, the patient may be administered chemotherapy and / or radiation therapy. In some cases, the patient may be administered a dose-reduced chemotherapy regimen. In some cases, a dose-reduced chemotherapy regimen using busulfan at 25% of the standard dose may be sufficient to achieve significant engraftment of the modified cells while reducing conditioning-related toxicity (Aiuti A. et al. (2013), Science 23;341(6148)). More intensive chemotherapy regimens may be based on the administration of both busulfan and fludarabine as depletors of endogenous HSCs. In some cases, the doses of busulfan and fludarabine may be approximately 50% and 30% of the doses used in standard allogeneic transplants. In some cases, the cells may be administered after B-cell depleting therapy, such as an agent that reacts with CD20, e.g., Rituxan. In some cases, the patient may be administered chemotherapy such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 against CD3 or alemtuzumab (Campath®, Lemtrada®) against CD52. In some cases, the patient may be administered fludarabine and cyclophosphamide, and optionally alemtuzumab.

[0110] In certain cases, engineered immune cells (e.g., T cells) may be administered to a subject as a combination therapy with an immunosuppressant. Exemplary immunosuppressants include sirolimus, tacrolimus, cyclosporine, mycophenolic acid, antithymocyte globulin, corticosteroids, calcineurin inhibitors, antimetabolites such as methotrexate, post-transplant cyclophosphamide, or any combination thereof. In some cases, a subject may be pretreated with sirolimus or tacrolimus alone as prophylaxis against GVHD. In some cases, the cells may be administered to a subject before an immunosuppressant. In some cases, the cells may be administered to a subject after an immunosuppressant. In some cases, the cells may be administered to a subject simultaneously with an immunosuppressant. In some cases, the cells may be administered to a subject without an immunosuppressant. In some cases, a patient receiving genetically modified cells may receive an immunosuppressant for less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, less than 1 month, less than 3 weeks, less than 2 weeks, or less than 1 week.

[0111] 1. Engineered immune cells containing a tumor-CAR and a fusion protein, such as a FAPscFv-cytokine fusion protein The cell type for engineered cells expressing a) a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen, whose expression is constitutive, and b) a secreted fusion protein comprising (i) a fibroblast activation protein binding domain and (ii) a stimulatory cytokine, whose expression is inducible upon cell activation, is not particularly limited.

[0112] 1.1.Cell Types The engineered cells described herein can be immune cells, including T cells, NK cells, and macrophages.

[0113] The engineered cells described herein may be induced pluripotent stem cells ("iPSCs"), which can be subsequently differentiated into the immune cells described herein. The engineered iPSCs described herein are therefore intermediates in the production of engineered immune cells in accordance with the present disclosure.

[0114] In some cases, the engineered cells described herein may be any differentiated cells that may be subsequently dedifferentiated into iPSCs, which may subsequently be differentiated into the immune cells described herein. The engineered differentiated cells described herein are thus intermediates in the production of engineered immune cells in accordance with the present disclosure. The genetic manipulations described herein may be performed on differentiated cells, dedifferentiated cells, or iPSCs.

[0115] Methods for producing iPSCs from differentiated cells are well known to those skilled in the art and include methods based on nuclear transfer, the use of cell extracts and synthetic molecules, the forced expression of defined genes, and cytoplasmic level modifications (Telpalo-Carpio et al. (2013) J Stem Cells Regen Med. 9(1):2-8). Methods for producing immune cells from iPSCs are also well known to those skilled in the art and include, for example, the serum-free and feeder-free in vitro differentiation protocol to T cells disclosed by Themeli et al. (Nature Biotechnology (2013) 31:928-933) and the differentiation protocol to NK cells under completely chemically defined conditions described by Matsubara et al. (Biochem Biophys Res Commun. (2019) 515(1):1-8).

[0116] Thus, one aspect is a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding (i) a fibroblast activation protein (FAP)-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. An engineered immune cell comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The invention relates to engineered immune cells, wherein the inducible promoter is inducible upon activation of the immune cell.

[0117] In some cases, the immune cell can be a T cell.

[0118] In some cases, the immune cell can be an NK cell.

[0119] In some cases, the immune cell can be a macrophage.

[0120] Another aspect is a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding (i) a fibroblast activation protein (FAP)-binding domain, e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv), and (ii) a secretable fusion protein comprising a stimulatory cytokine; 1. An engineered iPSC comprising: Optionally, the fusion protein does not comprise an antibody fragment crystallizable (Fc); the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); The invention relates to engineered iPSCs, wherein the inducible promoter is inducible upon activation of the iPSCs or upon activation of an immune cell into which the engineered iPSCs may further differentiate.

[0121] In some cases, the engineered iPSCs described herein may be intermediate products in the production of the engineered immune cells described herein.

[0122] Tumor-CAR "Chimeric antigen receptor" or "CAR" generally refers to a synthetic receptor containing a targeting moiety (also called a "binding moiety") associated with one or more signaling domains as a single fusion molecule. As defined herein, the term "chimeric antigen receptor" includes single-chain CARs and multi-chain CARs. In some cases, the binding moiety of a CAR may comprise the antigen-binding domain of a single-chain antibody (scFv), which comprises variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation CARs is derived from the cytoplasmic region of the CD3 zeta chain or the Fc receptor gamma chain. First-generation CARs have been shown to successfully redirect T cell cytotoxicity. However, first-generation CARs have failed to provide long-term expansion and antitumor activity in vivo. The addition of signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), and 4-1BB (CD137), either alone (second generation) or in combination (third generation), has been shown to enhance the survival and proliferation of CAR-modified T cells. CARs are not necessarily single-chain polypeptides; multi-chain CARs are also possible. For example, in multi-chain CAR structures, such as those described in WO 2014 / 039523, the signaling and costimulatory domains are located on different polypeptide chains. Such multi-chain CARs can be derived from FcεRI by replacing the high-affinity IgE-binding domain of the FcεRI alpha chain with an extracellular ligand-binding domain, such as an scFv, while the N- and / or C-terminal tails of the FcεRI beta and / or gamma chains are fused to the signaling and costimulatory domains, respectively. The extracellular ligand-binding domain serves to redirect the specificity of immune cells (e.g., T cells) toward cellular targets, while the signaling domain activates the immune cell response. CARs are generally expressed in effector immune cells and redirect the immune activity of effector immune cells against antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors.A component of a CAR is any functional subunit of a CAR that is encoded by an exogenous polynucleotide sequence introduced into a cell. For example, the component may aid in the interaction with a target antigen, the stability of the CAR, or the localization of the CAR within the cell.

[0123] While the CARs of the present disclosure useful in the methods herein are not limited to a specific CAR structure, nucleic acids that can be used to engineer immune cells generally encode CARs comprising an extracellular antigen-binding domain that binds to a tumor antigen, a hinge, a transmembrane domain, and an intracellular domain comprising a stimulatory domain and / or a primary signaling domain. Typically, the extracellular antigen-binding domain is an scFv comprising the variable heavy chain (VH) and variable light chain (VL) of an antibody that binds to a tumor antigen, connected via a linker. The extracellular antigen-binding domain can also be derived from a single-domain antibody (e.g., a nanobody) or an ankyrin repeat domain. Thus, the extracellular antigen-binding domain can comprise one variable heavy chain without a variable light chain. Walser et al. (Viruses (2022):14, 2242) describes ankyrin repeat domains. The transmembrane domain can be, for example, a CD8α transmembrane domain, a CD28 transmembrane domain, or a 4-1BB transmembrane domain. The costimulatory domain can be, for example, a 4-1BB costimulatory domain or a CD28 costimulatory domain. The primary signaling domain can be, for example, a CD3ζ signaling domain.

[0124] The CARs described herein also generally include a signal peptide that directs the nascent protein to the endoplasmic reticulum for subsequent expression on the surface of the engineered cell. The signal peptide is cleaved after targeting the CAR to the cell surface. The signal peptide included in the CARs described herein can be a CD8α signal peptide, such as one having an amino acid sequence at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 76, or one having an amino acid sequence at least 80%, at least 90%, at least 95%, or at least 99% identical to the alternative signal peptide of SEQ ID NO: 77.

[0125] As used herein, in a broad sense, a tumor-CAR can also be a recombinant TCR that recognizes a tumor-expressed peptide / MHC complex. Thus, in some cases, the tumor-CARs described herein can be recombinant TCRα / β that include an extracellular ligand-binding domain and a transmembrane domain without a stimulatory and / or costimulatory domain. Indeed, typically, recombinant TCRα / β do not contain an activation or costimulatory domain; they rely on the endogenous CD3 domain for activation and on endogenous CD28 for costimulation.

[0126] [Table 1]

[0127] A tumor-CAR comprises an extracellular ligand-binding domain (or extracellular antigen-binding domain) that recognizes a tumor antigen. Thus, the tumor-CARs described herein comprise an extracellular tumor antigen-binding domain.

[0128] As used herein, the term "extracellular antigen-binding domain" or "extracellular ligand-binding domain" generally refers to an oligopeptide or polypeptide capable of binding to a specific antigen, such as a tumor antigen. In some cases, this domain can interact with a cell surface molecule, such as a ligand. For example, in some cases, the extracellular antigen-binding domain can be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some cases, the extracellular antigen-binding domain can comprise a single-chain antibody fragment (scFv) comprising fragments of the variable heavy chain (VH) and variable light chain (VL) of a target antigen-specific monoclonal antibody linked by a flexible linker. The antigen-binding domain of a CAR expressed on the cell surface of an engineered cell described herein can be any domain that binds to a target antigen and is derived from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and functional fragments thereof.

[0129] As used herein, the term "tumor antigen" is intended to include "tumor-specific antigens" and "tumor-associated antigens." Tumor-specific antigens (TSAs) are generally present only in tumor cells and not in other cells, whereas tumor-associated antigens (TAAs) are present in some tumor cells and some normal cells. As intended herein, "tumor antigen" also refers to a mutated form of a protein in which the non-mutated form is observed in non-tumor tissue but only appears in the mutated form in tumors.

[0130] The tumor antigen may be an antigen specific to or associated with a solid tumor. The tumor antigen is not limited. In some cases, the tumor antigen is selected from the group consisting of CEA, ERBB2, EGFR, GD2, mesothelin, MUC1, PSMA, GD2, PSMA1, LAP3, ANXA3, tumor-associated glycoprotein 72 (TAG72), MUC16, 5T4, FRα, MUC28z, NKG2D, HRG1β, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), carboxyanhydrase-IX (CA-IX), Trop2, claudin 18.2, folate receptor 1 (FOLR1), CXC R2, B7-H3, CD133, CD24, receptor tyrosine kinase-like orphan receptor 1 specific (ROR1), EGFR, EGFRvIII, VEGF, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), DLL3, glypican-3, epithelial cell adhesion molecule (EpCAM), GUCY2C (guanylate cyclase 2C), doublecortin-like kinase 1 (DCLK1), HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b , STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, and ERBB3. See, e.g., Marofi et al., Stem Cell Res Ther (2021) 12, 81, which is incorporated herein by reference.

[0131] In some cases, the tumor antigen is selected from the group consisting of mesothelin, Trop2, MUC1, EGFR, and VEGF. In some cases, the antigen is selected from the group consisting of mesothelin, MUC1, and Trop2.

[0132] In some cases, the tumor antigen is not a FAP. Thus, in some cases, the FAP is not the antigen targeted by the tumor-CAR.

[0133] Tumor antigens can also be antigens specific to or associated with hematological cancers characterized by the presence of FAPs in the tumor microenvironment, such as myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

[0134] In some cases, the tumor antigen associated with a hematological cancer is selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD123, CD70, CD33, CD135, CD44, CD276, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD37, CD79, CD79a, CD80, CD138, CD47, CRLF2, CD38, CLL-1, NKG2D, CALR, IL1RAP, ILT3, TIM3, CD96, VISTA, CS1, TACI, APRIL, GPRC5D, and CD44v6.

[0135] In some cases, the tumor antigen associated with a hematological cancer is selected from the group consisting of BCMA, CD19, CD123, CD20, CD22, CS1, CD138, CD80, CD2, CD3, CD4, CD5, CD7, and CD8.

[0136] In some cases, the tumor antigen associated with the hematological cancer is selected from the group consisting of BCMA, CD19, CD123, CD20, CD22, and CS1.

[0137] [Table 2]

[0138] [Table 3]

[0139] The tumor-CARs described herein are (a) an extracellular ligand-binding domain comprising the amino acid sequences of VH and VL of a monoclonal anti-tumor antigen antibody; (b) a hinge selected from an FcγRIII hinge, a CD8α hinge, and an IgG1 hinge; (c) a transmembrane domain comprising a CD8α transmembrane domain or a CD28 transmembrane domain; (d) a cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain of 4-1BB or CD28; may include:

[0140] In some cases, the tumor-CAR a) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, and the L-CDRs of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, and optionally the amino acid sequence SEQ ID NO: 45; b) amino acid sequences having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and the L-CDRs of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, and optionally the amino acid sequence SEQ ID NO: 53; c) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, and the L-CDRs of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, and optionally the amino acid sequence of SEQ ID NO: 61; or d) H-CDR and L-CDR contained in the amino acid sequence of SEQ ID NO: 63, and optionally an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 63. The extracellular binding domain may comprise:

[0141] In some cases, the tumor-CAR may be specific for mesothelin (Meso-CAR) and may have the amino acid sequence of SEQ ID NO: 62. In some cases, the Meso-CAR may comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62, and may optionally include the CDRs of SEQ ID NOs: 55 through 60.

[0142] In some cases, a nucleic acid sequence encoding a Meso-CAR described herein can comprise the nucleic acid sequence of SEQ ID NO: 100. In some cases, a nucleic acid sequence encoding a Meso-CAR described herein can comprise a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 100, and can encode a Meso-CAR comprising the amino acid sequence of SEQ ID NO: 62.

[0143] In some cases, the tumor-CAR may be specific for Trop2 (Trop2-CAR) and may have the amino acid sequence of SEQ ID NO: 46. In some cases, the Trop2-CAR may comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46, and may optionally comprise the CDRs of SEQ ID NOs: 39 to 44.

[0144] In some cases, a nucleic acid sequence encoding a Trop2-CAR described herein may comprise the nucleic acid sequence of SEQ ID NO: 102. In some cases, a nucleic acid sequence encoding a Trop2-CAR described herein may comprise a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 102, and may encode a Trop2-CAR comprising the amino acid sequence of SEQ ID NO:46.

[0145] In some cases, the tumor-CAR may be specific for mucin 1 (MUC1-CAR) and may have the amino acid sequence of SEQ ID NO: 54. In some cases, the MUC1-CAR may comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 54, and may optionally comprise the CDRs of SEQ ID NOs: 47 through 52.

[0146] In some cases, a nucleic acid sequence encoding a MUC1-CAR described herein may comprise the nucleic acid sequence of SEQ ID NO: 101. In some cases, a nucleic acid sequence encoding a MUC1-CAR described herein may comprise a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 101, and may encode a MUC1-CAR comprising the amino acid sequence of SEQ ID NO:54.

[0147] In some cases, the tumor-CAR may be specific for CS1 (CS1-CAR) and may have the amino acid sequence of SEQ ID NO: 88. In some cases, the CS1-CAR may comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88, and may optionally comprise the CDRs included in SEQ ID NO: 88.

[0148] 1.3 Fusion proteins The engineered immune cells described herein are capable of expressing and secreting a fusion protein comprising (i) a FAP-binding domain and (ii) a stimulatory cytokine.

[0149] In some cases, the FAP-binding domain may comprise a single-chain antibody fragment (scFv) comprising a heavy chain variable fragment (VH) and a light chain variable fragment (VL) of a FAP-specific monoclonal antibody linked by a flexible linker to form a "FAPscFv." The FAP-binding domain of the fusion proteins described herein binds to a FAP and can be any domain derived from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and functional fragments thereof. In some cases, the antigen-binding domain (e.g., a FAP-binding domain) may comprise the VH and VL fragments of an antibody, but does not include the antibody fragment crystallizable (Fc) domain.

[0150] It is also contemplated that the FAP-binding domain may be derived from a single-domain antibody (e.g., a nanobody) or an ankyrin repeat domain. Thus, the FAP-binding domain may comprise no variable light chains but one variable heavy chain. Thus, in some cases, the fusion protein may comprise: (i) optionally a signal peptide; (ii) a FAP-binding domain, for example, a FAP-binding domain (e.g., FAPscFv) comprising a VH amino acid sequence and a VL amino acid sequence derived from a monoclonal anti-FAP antibody; (iii) at least one stimulatory cytokine selected from the group consisting of interleukin-2 or a variant thereof such as IL-2v, interleukin-7 or a variant thereof, interleukin-12 or a variant thereof, interleukin-15 or a variant thereof, interleukin-15 in complex with the high affinity receptor for interleukin-15, IL-15RA, interleukin-18 or a variant thereof, and interleukin-23 or a variant thereof; may include:

[0151] In certain embodiments, the fusion protein does not comprise an antibody crystallizable fragment (Fc).

[0152] The fusion proteins described herein also generally contain a signal peptide, in their immature form, that directs the nascent protein to the endoplasmic reticulum and allows for subsequent secretion outside the cell. The signal peptide is cleaved after secretion of the fusion protein into the extracellular medium. The signal peptide included in the protein fusions described herein can be, for example, an IL2 signal sequence (e.g., SEQ ID NO: 90), an IgE signal sequence (e.g., SEQ ID NO: 91), a CTLA4 Ig signal sequence (e.g., SEQ ID NO: 92), or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 90, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 91, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 92.

[0153] [Table 4]

[0154] In some cases, the FAP binding domain (e.g., FAPscFv) comprises: a) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the L-CDRs of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and optionally the amino acid sequence SEQ ID NO:9; b) amino acid sequences having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the L-CDRs of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and optionally the amino acid sequence SEQ ID NO: 18; c) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and the L-CDRs of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, and optionally the amino acid sequence SEQ ID NO: 27; or d) amino acid sequences having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and the L-CDRs of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and optionally the amino acid sequence SEQ ID NO: 36. Includes.

[0155] In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 8. In some cases, the FAP-binding domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 7 and an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 8. In some cases, the FAP-binding domain comprises an amino acid sequence comprising SEQ ID NO: 7 and a complementarity-determining region (CDR) comprised in SEQ ID NO: 8. In some cases, the H-CDR comprised in SEQ ID NO: 7 comprises the amino acid sequence of SEQ ID NO: 1-3. In some cases, the L-CDR included in SEQ ID NO: 8 comprises the amino acid sequence of SEQ ID NO: 4 to SEQ ID NO: 6. In some cases, the FAP-binding domain comprises (i) the CDRs included in SEQ ID NOs: 7 and 8, and (ii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 7, and (iii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region of SEQ ID NO: 8.

[0156] In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises a VH region comprising SEQ ID NO: 16 and a VL region comprising SEQ ID NO: 17. In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 16 and an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 17. In some cases, the FAP-binding domain comprises an amino acid sequence comprising SEQ ID NO: 16 and a complementarity-determining region (CDR) comprised in SEQ ID NO: 17. In some cases, the H-CDR comprised in SEQ ID NO: 16 comprises the amino acid sequence of SEQ ID NO: 10-12. In some cases, the L-CDR included in SEQ ID NO: 17 comprises the amino acid sequences of SEQ ID NOs: 13 to 15. In some cases, the FAP-binding domain comprises (i) the CDRs included in SEQ ID NOs: 16 and 17, and (ii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 16, and (iii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 17.

[0157] In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises a VH region comprising SEQ ID NO: 25 and a VL region comprising SEQ ID NO: 26. In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 25 and an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 26. In some cases, the FAP-binding domain comprises an amino acid sequence comprising SEQ ID NO: 25 and a complementarity-determining region (CDR) comprised in SEQ ID NO: 26. In some cases, the CDR comprised in SEQ ID NO: 25 comprises the amino acid sequence of SEQ ID NO: 23-25. In some cases, the CDRs included in SEQ ID NO: 26 comprise the amino acid sequences of SEQ ID NOs: 26-28. In some cases, the FAP-binding domain comprises (i) the CDRs included in SEQ ID NOs: 25 and 26, and (ii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 25, and (iii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 26.

[0158] In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises a VH region comprising SEQ ID NO: 34 and a VL region comprising SEQ ID NO: 35. In some cases, the FAP-binding domain (e.g., a FAPscFv) comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 34 and an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 35. In some cases, the FAP-binding domain comprises an amino acid sequence comprising SEQ ID NO: 34 and a complementarity-determining region (CDR) comprised in SEQ ID NO: 35. In some cases, the CDR comprised in SEQ ID NO: 34 comprises the amino acid sequence of SEQ ID NO: 28-30. In some cases, the CDRs included in SEQ ID NO: 35 comprise the amino acid sequences of SEQ ID NOs: 31-33. In some cases, the FAP binding domain comprises (i) the CDRs included in SEQ ID NOs: 34 and 35, and (ii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH region comprising SEQ ID NO: 34, and (iii) an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VL region comprising SEQ ID NO: 35.

[0159] [Table 5]

[0160] In some cases, the amino acid sequence comprising the VH region and the amino acid sequence comprising the VL region are separated by one or more linker amino acid residues. The number of amino acids constituting the linker is not necessarily limited, but in some cases, the linker is at least about 5 amino acids in length, for example, at least about 10 amino acids in length. In some cases, the linker is about 10 to 25 amino acids in length. In some cases, the linker sequence is selected from any one of SEQ ID NOs: 37 to 38.

[0161] In some cases, a FAP-binding domain comprising a VH region and a VL region from a monoclonal anti-FAP antibody may comprise a sequence selected from any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, and SEQ ID NO: 36. In some cases, a FAP-binding domain may comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, and SEQ ID NO: 36, and optionally the CDRs of each of SEQ ID NOs: 1-6, 10-15, 19-24, and 28-33.

[0162] Stimulatory cytokines, as defined herein, are cytokines that affect the maturation, distribution, growth, survival, and function of specific cell populations, particularly CD8+ T cells, CD4+ T cells, macrophages, and NK cells.

[0163] Stimulatory cytokines according to the present disclosure include interleukin-2 or variants thereof, such as IL-2v, interleukin-7 or variants thereof, interleukin-12 or variants thereof, interleukin-15 or variants thereof, interleukin-15 complexed with its high-affinity receptor IL-15RA, interleukin-18 or variants thereof, and interleukin-23 or variants thereof. Variants of interleukin-2 include variants that preferentially or exclusively bind to the intermediate-affinity IL-2Rβγ but do not bind to CD25. According to the present disclosure, the stimulatory cytokine can be a variant of IL-2 with high affinity for IL-2Rβγ and low binding to IL-2Rα, such as the variant disclosed in WO 2012 / 10747. In some cases, the variant of IL-2 is IL-2v, which has the amino acid sequence of SEQ ID NO:93. IL-2v has been shown to activate NK cells and CD4+ / CD8+ T cells without preferentially activating Tregs (Waldhauer et al. (2021) MAbs. 13(1):1913791).

[0164] As used herein, the term "variant" as applied to cytokines, particularly interleukins, includes any mutant form of a naturally occurring cytokine, particularly an interleukin, which differs from the naturally occurring protein by at least one amino acid mutation that affects the activity of the naturally occurring protein, for example, the interaction of naturally occurring IL-2 with CD25. This mutation can include substitution, deletion, or modification of an amino acid residue normally located at the corresponding position in the full-length naturally occurring protein, as well as the addition of one or more amino acid residues, the deletion of one or more amino acid residues, or a fragment of the full-length naturally occurring protein. Reference native proteins can be human interleukin-2 (IL-2) (Swiss Prot Reference P01585), human interleukin-7 (Swiss Prot Reference P13232), human interleukin-12 (Swiss Prot References P29460 and P29459), human interleukin-15 (Swiss Prot Reference P40933), human interleukin-18 (Swiss Prot Reference Q14116), and human interleukin-23 (Swiss Prot Reference Q9NPF7).

[0165] As discussed above, the fusion proteins and tumor-CARs expressed by the engineered immune cells described herein are differentially expressed as a result of having the exogenous nucleic acid sequence encoding the tumor-CAR under the transcriptional control of a constitutive promoter and the exogenous nucleic acid sequence encoding the fusion protein under the transcriptional control of an inducible promoter.

[0166] A "constitutive promoter" generally refers to a promoter that is active under all circumstances in a particular cell or cell type that contains the promoter. A constitutive promoter drives transcription of an associated gene continuously in a cell. The level of transcription of a gene associated with a constitutive promoter may vary, but the transcript, and therefore the product of the gene (if present), remains detectable. Examples of constitutive promoters include the human elongation factor 1 alpha (EF1A) promoter, the cluster of differentiation 52 (CD52) promoter, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the human cytomegalovirus (CMV) promoter, the human phosphoglycerate promoter (hPGK) promoter, the RPBSA promoter, the human ubiquitin C (UBC) promoter, the simian virus 40 (SV40) early promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-actin promoter (CAGG) associated with the CMV early enhancer, the T cell receptor activator (TCR) promoter, and the T cell receptor activator (TCR). Constitutive promoters useful herein include the T cell receptor beta constant region (TRAC or TCRA) promoter, T cell receptor beta constant region 1 (TRBC or TCRB) promoter, T cell receptor gamma constant region 1 or 2 (TRGC1 or TCRG1, TRGC2 or TCRG2) promoter, T cell receptor delta constant region (TRDC or TCRD) promoter, beta-2-microglobulin (B2M) promoter, cluster of differentiation 5 (CD5) promoter, CS1 (also known as CD319, CRACC, and SLAMF7) promoter, cluster of differentiation 45 (CD45) promoter, cluster of differentiation 4 (CD4) promoter, and cluster of differentiation 8 (CD8) promoter. Constitutive promoters useful herein can be identical to promoters already present in the genome of the cell (i.e., without genetic manipulation as described herein). This is the case, for example, with the EF1A promoter, CD52 promoter, GAPDH promoter, TRAC promoter, TRBC promoter, TRGC promoter, TRDC promoter, B2M promoter, and CD5 promoter.A constitutive promoter useful herein may not be present in the genome of a cell prior to its introduction into the cell by genetic engineering, such as in the case of the synthetic RPBSA promoter (i.e., a synthetic promoter composed of a fragment of the RPL13a promoter fused to a region of the RPL41 gene), the CMV promoter, the mouse PGK promoter, the SV40 promoter, or the CAGG promoter. A constitutive promoter may be added to the genome of a cell as an exogenous polynucleotide, or it may be an endogenous polynucleotide that is already present in the genome of a cell independent of genetic engineering of the cell as described herein, i.e., without adding an exogenous polynucleotide corresponding to the constitutive promoter to the cell.

[0167] The terms "inducible locus" and "inducible promoter" generally refer to a locus and a promoter contained therein that are activated in a cell containing the locus or promoter only in response to a specific stimulus. Thus, an inducible locus / promoter is active only under certain circumstances. Unless it receives a stimulus, the inducible promoter contained in the inducible locus remains inactive, and a gene associated with the inducible promoter in the "off" state is generally not transcribed or is only weakly transcribed. An inducible promoter enters the "on" state when a specific stimulus is present and an activator protein binds to the inducible promoter, activating it and initiating transcription. Transcription of a gene associated with the inducible promoter increases when the inducible promoter transitions to the "on" state in response to a specific stimulus. Expression of a gene controlled by an inducible promoter is tightly regulated, and its expression decreases rapidly when the activation signal is removed. In the present disclosure, the inducible promoter responds to cell activation (e.g., activation of immune cells such as T cells) as defined herein. For example, a promoter that is inducible upon in vitro CAR-T cell activation (e.g., as described in Example 8) meets the criterion of a fold change between the mean expression at 0 hours and the mean expression at 24 hours of more than 3, e.g., more than 5.

[0168] "Cell activation" generally refers to the process by which changes occur in a cell in response to an "activation signal." An "activation signal" refers to a signal or stimulus that can directly or indirectly activate a cell. In the present disclosure, "cell activation," as applied to an engineered cell comprising a CAR described herein, refers primarily to changes that occur in the engineered cell after an activation signal is generated in the cell upon binding or recognition of an epitope of a tumor antigen by a tumor-CAR expressed by the engineered cell.

[0169] At the molecular level, activation of a cell also corresponds to activation of an inducible promoter. Indeed, an inducible promoter contains one or more regulatory elements that respond to one or more signaling pathways in the cell, such as NFAT-regulated signaling.

[0170] In some cases, the inducible promoter may be responsive to CD3 zeta signaling. Examples of inducible promoters useful herein include the programmed cell death protein 1 (PDCD1) gene, the cluster of differentiation 25 (CD25) gene, the T-cell immunoglobulin and mucin domain-containing 3 (TIM3) gene, the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) gene, the CC motif chemokine ligand 1 (CCL1) gene, the nuclear receptor subfamily 4 group A member 3 (NR4A3) gene, the early growth response 3 (EGR3) gene, the G0 / G1 switch 2 (G0S2) gene, the interleukin-22 (IL22) gene, and the regulator of G-protein signaling (G-protein signaling). Inducible promoters include promoters for gene 16 (RGS16), Fas ligand (FASLG), retinol dehydrogenase 10 (RDH10), colony-stimulating factor 1 (CSF1), colony-stimulating factor 2 (CSF2, also known as GM-CSF), lymphocyte activation 3 (LAG3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CD152), interleukin-10 (IL10), nuclear receptor subfamily 4 group A member 1 (NR4A1 or NUR77), and forkhead box P3 (FOXP3). Inducible promoters useful herein can be identical to promoters already present in the genome of a cell (i.e., without genetic manipulation as described herein). This is the case, for example, for the promoter of PDCD1 or the promoter of GM-CSF. Inducible promoters useful herein do not have to be present in the genome of a cell prior to their introduction into the cell by genetic manipulation. An inducible promoter may be added to the genome of a cell as an exogenous polynucleotide, or it may be an endogenous polynucleotide that is already present in the genome of a cell independent of genetic engineering of the cell as described herein, i.e., without adding an exogenous polynucleotide corresponding to the inducible promoter to the cell.

[0171] In some cases, after an activation signal in an engineered cell described herein, the abundance of cells expressing the fusion protein in a cell population comprising the engineered immune cell is at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0172] In some cases, expression of the fusion protein induced by an activation signal in the engineered cells returns to initial basal levels or is undetectable after removal, disappearance, or reduction of the activation signal in the engineered immune cells.

[0173] 1.3. Further Features of Engineered Cells In some cases, engineered immune cells, e.g., T cells, that have been modified to express a fusion protein comprising a FAP-binding domain and a stimulatory cytokine in addition to a CAR directed against a tumor antigen as described herein may have one or more additional modifications.

[0174] Additional genetic traits can be imparted by gene editing in immune cells to improve their therapeutic efficacy.

[0175] In some cases, the engineered cells can be further modified to improve persistence or longevity in patients by inactivating genes encoding MHC-I component(s), such as HLA or B2M, as described, for example, in WO 2015 / 136001 or by Liu et al. (2017, Cell Res 27:154-157).

[0176] Beta-2 microglobulin, also known as β2m, is the light chain of the MHC class I molecule and is therefore an integral part of the major histocompatibility complex. In humans, β2m is encoded by the B2M gene located on chromosome 15, rather than the other MHC genes, which are located in a gene cluster on chromosome 6. The human protein consists of 119 amino acids and has a molecular weight of 11,800 daltons.

[0177] In some cases, inhibition of B2M expression is achieved by genome modification, such as by expression in the cell of a rare-cutting endonuclease that can selectively inactivate a gene encoding β2m, such as the human B2M gene (NCBI Reference Sequence: NG_012920.1), by DNA cleavage. Such a rare-cutting endonuclease can be a TALE nuclease, a meganuclease, a zinc finger nuclease (ZFN), or an RNA-guided endonuclease (e.g., Cas9).

[0178] In some cases, inhibition of expression of B2M can be achieved by inhibiting gene transcription and / or translation by using (e.g., introducing into T cells) a nucleic acid molecule that specifically hybridizes (e.g., binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding β2m. In some cases, inhibition of expression of B2M is achieved by using (e.g., introducing into T cells) an antisense oligonucleotide molecule, a ribozyme molecule, or an interfering RNA (RNAi) molecule. In some cases, such a nucleic acid molecule can comprise at least 10 consecutive nucleotides of the complement of mRNA encoding human β2m.

[0179] In some cases, immune cells (e.g., T cells) or progenitor cells are provided that express a rare-cutting endonuclease that can selectively inactivate the gene encoding β2m by DNA cleavage. For example, such cells contain an exogenous nucleic acid molecule comprising a nucleotide sequence encoding the rare-cutting endonuclease, which can be a TALE nuclease, meganuclease, zinc finger nuclease (ZFN), or RNA-guided endonuclease. Thus, to provide immune cells (e.g., T cells) with reduced alloreactivity, the methods described herein can further include inactivating or mutating the β2m gene.

[0180] In some cases, engineered immune cells, such as T cells, have been modified to suppress or silence HLA expression in the cells. The class I HLA gene cluster in humans contains three major loci, B, C, and A, as well as several minor loci. The class II HLA cluster also contains three major loci, DP, DQ, and DR, and both the class I and class II gene clusters are polymorphic in that several different alleles of both class I and class II genes exist within the population. Several accessory proteins also exist that are involved in HLA function. The Tap1 and Tap2 subunits are part of the TAP transporter complex, which is essential for loading peptide antigens onto the class I HLA complex, and the LMP2 and LMP7 proteosome subunits are involved in the proteolysis of antigens into peptides for presentation by HLA. Reduction of LMP7 has been shown to reduce the amount of MHC class I on the cell surface, likely due to a lack of stabilization (Fehling et al. (1999) Science 265:1234-1237). In addition to TAP and LMP, there is the tapasin gene, whose product forms a bridge between the TAP complex and HLA class I chains, enhancing peptide loading. Reduction of tapasin results in impaired MHC class I assembly and reduced cell surface expression of MHC class I, resulting in cells with impaired immune responses (Grandea et al. (2000) Immunity 13:213-222 and Garbi et al. (2000) Nat. Immunol. 1:234-238). Any of the above genes may be inactivated as part of this document, for example, as disclosed in WO 2012 / 012667.

[0181] In some cases, the engineered immune cells, e.g., T cells, are modified to suppress or silence expression of CIITA in the cells, which is a gene encoding a class II major histocompatibility complex transactivator protein.

[0182] In some cases, the engineered immune cells, e.g., T cells, are inactivated in at least one gene selected from the group consisting of RFXANK, RFX5, RFXAP, TAP1, TAP2, ZXDA, ZXDB, and ZXDC. Inactivation can be achieved using genomic modification, such as by expression in the cells of a rare-cutting endonuclease that can selectively inactivate a gene selected from the group consisting of RFXANK, RFX5, RFXAP, TAP1, TAP2, ZXDA, ZXDB, and ZXDC by DNA cleavage. Such modification can allow for reduced alloreactivity of the engineered immune cells when infused into a patient.

[0183] Thus, in one embodiment, the engineered cells described herein may be genetically modified to suppress or silence expression of at least one gene that controls MHC complex surface presentation. Genes that control MHC complex surface presentation, as defined herein, include B2M, CIITA, HLA, RFXANK, RFX5, RFXAP, TAP1, TAP2, ZXDA, ZXDB, and ZXDC. In some cases, the engineered immune cells, e.g., T cells or NK cells, are genetically modified to suppress or silence expression in the cells of an immune checkpoint protein and / or a receptor for an immune checkpoint protein, such as PDCD1 or CTLA4, as described in WO 2014 / 184744.

[0184] As will be understood by those skilled in the art, the term "immune checkpoint" refers to a group of molecules expressed by T cells, NK cells, and antigen-presenting cells. These molecules effectively act as "brakes" to downregulate or inhibit immune responses. Immune checkpoint molecules include programmed death 1 (PD-1, also known as PDCD1 or CD279, e.g., human PD-1: Accession No. NM_005018), cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152, e.g., human CTLA-4: GenBank Accession No. AF414120.1), LAG3 (also known as CD223, e.g., human LAG3: Accession No. NM_002286.5), Tim3 (also known as HAVCR2, e.g., human Tim3: GenBank Accession No. JX049979.1), BTLA (also known as CD272, e.g., human BTLA: Accession No. NM_181780.3), and BY55, which directly inhibit immune cells. (also known as CD160, e.g., human BY55; GenBank accession number CR541888.1), TIGIT (also known as IVSTM3, e.g., human TIGIT; accession number NM_173799), LAIR1 (also known as CD305, e.g., human LAIR1; GenBank accession number CR542051.1), SIGLEC10 (e.g., human SIGLEC10; GenBank accession number AY358337.1), 2B4 (also known as CD244, e.g., human 2B4; accession number NM_001166664.1), PPP2CA (also known as NEDLBA, PP2Ac, PP2C alpha, RP-C; e.g., human PPP2CA; NCBI gene ID 5515), PPP2CB (also known as PP2A beta, e.g., human PPP2CB: NCBI Gene ID 5516), PTPN6 (also known as HCP, HCPH, HPTP1C, PTP-1C, SH-PTP1, SHP-1, SHP-1L, SHP1, e.g., human PTPN6: NCBI Gene ID 5777), PTPN22 (NCBI Gene ID 26191), CD96 (NCBI Gene ID 10225), CRTAM (NCBI Gene ID 56253), SIGLEC7 (NCBI Gene ID 27036), SIGLEC9 (NCBI Gene ID27180), TNFRSF10B (NCBI Gene ID 8795), TNFRSF10A (NCBI Gene ID 8797), CASP8 (NCBI Gene ID 841), CASP10 (NCBI Gene ID 843), CASP3 (NCBI Gene ID 836), CASP6 (NCBI Gene ID 839), CASP7 (NCBI Gene ID 840), FADD (NCBI Gene ID 8772), FAS (NCBI Gene ID 355), TGFBRII (NCBI Gene ID 7048), TGFRBRI (NCBI Gene ID 7046), SMAD2 (NCBI Gene ID 4087), SMAD3 (NCBI Gene ID 4088), SMAD4 (NCBI Gene ID 4089), SMAD10, SKI (NCBI Gene ID 6497), SKIL (NCBI Gene ID 6498), TGIF1 (NCBI Gene ID 7050), IL10RA (NCBI Gene ID 3587), IL10RB (NCBI Gene ID 3588), HMOX2 (NCBI Gene ID 3163), IL6R (NCBI Gene ID 3570), IL6ST (NCBI Gene ID 3572), EIF2AK4 (NCBI Gene ID 440275), CSK (NCBI Gene ID 1445), PAG1 (NCBI Gene ID 55824), SIT1 (NCBI Gene ID 27240), FOXP3 (NCBI Gene ID 50943), PRDM1 (NCBI Gene ID 639), BATF (NCBI Gene ID 10538), GUCY1A2 (NCBI Gene ID 2977), GUCY1A3 (NCBI Gene ID Examples of genes that may be used include, but are not limited to, CTLA-4 (NCBI gene ID 2977), GUCY1B2 (NCBI gene ID 2974), and GUCY1B3 (NCBI gene ID 2975). For example, CTLA-4 is a cell surface protein expressed on certain CD4 T cells and CD8 T cells, which, when engaged by its ligands (B7-1 and B7-2) on antigen-presenting cells, inhibits T cell activation and effector function. In some cases, the engineered T cells are further genetically modified by inactivating at least one gene encoding a protein involved in an immune checkpoint, such as PD1 and / or CTLA-4, or any of the immune checkpoint proteins mentioned herein.

[0185] In some cases, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRI, SM At least two genes encoding immune checkpoint proteins selected from the group consisting of AD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 are inactivated.

[0186] In some cases, the engineered immune cells, e.g., T cells, may be modified to confer resistance to at least one immunosuppressive or chemotherapeutic agent, and optionally to contain a suicide gene.

[0187] In some cases, the engineered immune cells, e.g., T cells, may be further modified to confer resistance to at least one immunosuppressant drug, such as by inactivating CD52, the target of anti-CD52 antibodies (e.g., alemtuzumab), as described, for example, in WO 2013 / 176915.

[0188] To improve cancer therapy and selective engraftment of allogeneic immune cells, engineered immune cells can be conferred drug resistance to protect them from the toxic side effects of chemotherapeutic or immunosuppressive agents. In some cases, engineered immune cells can be further modified to confer resistance to chemotherapeutic agents, such as purine analog drugs, by inactivating DCK, as described in WO 2015 / 75195, for example.

[0189] Because immune cells expressing drug resistance genes survive and proliferate compared to drug-sensitive cells, drug resistance of immune cells also allows for the enrichment of immune cells in vivo or ex vivo. In some cases, the methods further include a method for engineering allogeneic drug-resistant immune cells for immunotherapy, comprising the steps of: (a) providing immune cells, e.g., T cells; (b) selecting at least one drug; (c) modifying the cells to confer drug resistance to the cells; and (d) expanding the engineered cells in the presence of the drug. When the immune cells are T cells, the preceding steps may be combined with the steps of modifying the T cells by inactivating at least one gene encoding a T cell receptor (TCR) component, followed by selecting transformed T cells that do not express a TCR on their cell surface.

[0190] Therefore, engineered immune cells can be further modified to confer resistance to drugs, such as chemotherapeutic agents. Resistance to drugs can be conferred to immune cells by expressing a drug resistance gene. Variant alleles of several genes, such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), have been identified to confer drug resistance to cells. In some cases, a drug resistance gene can be expressed in a cell by introducing a transgene encoding the gene into the cell or by integrating the drug resistance gene into the cell's genome by homologous recombination.

[0191] Resistance to drugs can be conferred to immune cells by inactivating one or more genes (drug-sensitizing genes) that cause cell sensitivity to drugs, such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Genbank: M26434.1). For example, inactivating HPRT in engineered immune cells can confer resistance to the cytostatic metabolite 6-thioguanine (6TG). 6TG is converted by HPRT to the cytotoxic thioguanine nucleotide, which is currently used to treat patients with cancer, particularly leukemia (Hacke et al. (2013) Transplantation Proceedings, 45(5):2040-2044). Another example is the inactivation of CD3, which is normally expressed on the surface of T cells, which can confer resistance to anti-CD3 antibodies such as teplizumab.

[0192] Alternatively, drug resistance can be conferred to immune cells (e.g., T cells) by the expression of at least one drug resistance gene. A drug resistance gene refers to a nucleic acid sequence that encodes "resistance" to a drug, such as a chemotherapeutic agent (e.g., methotrexate). In other words, expression of a drug resistance gene in a cell allows the cell to grow in the presence of the drug to a greater extent than the growth of corresponding cells that do not have the drug resistance gene. The drug resistance gene can encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, analogs or derivatives thereof, and the like.

[0193] Several drug resistance genes have been identified that could potentially be used to confer drug resistance to target cells (Takebe et al. (2001) Mol. Ther. 3(1):88-96; Sugimoto et al. (2003) Mol. Cancer Ther. 2:105-112; Zielske et al. (2003) J. Clin. Invest. 112(10):1561-70; Nivens et al. (2004) Cancer Chemother Pharmacol 53(2):107-15; Bardenheuer et al. (2005) Leukemia 19(12):2281-8; Kushman et al. (2007) Carcinogenesis 28(1):207-14).

[0194] Another example of a drug resistance gene may be a mutant or modified form of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in cells and is essential for DNA synthesis. Folic acid analogs, such as methotrexate (MTX), inhibit DHFR and are therefore used clinically as anti-neoplastic agents. Various mutant forms of DHFR have been described that have increased resistance to inhibition by folate antagonists used in therapy. In some cases, the drug resistance gene may be a nucleic acid sequence encoding a mutant form of human wild-type DHFR (GenBank: AAH71996.1) that contains at least one mutation that confers resistance to folate antagonist treatment, such as methotrexate. In some cases, the mutant form of DHFR contains at least one mutant amino acid at position G15, L22, F31, or F34, e.g., at position L22 or F31 (Schweitzer, Dicker et al. 1990; WO 94 / 24277; U.S. Pat. No. 6,642,043).

[0195] As used herein, "antifolates" or "folate analogs" refer to molecules intended to interfere with the folate metabolic pathway at some level. Examples of antifolates include, for example, methotrexate (MTX), aminopterin, trimetrexate (Neutrexin™), edatrexate, N10-propargyl-5,8-dideazafolate (CB3717), ZD1694 (Tumodex), 5,8-dideazaisofolate (IAHQ), 5,10-dideazatetrahydrofolate (DDATHF), 5-deazaisofolate (IAHQ), 5,10-dideazatetrahydrofolate (DDATHF), 5-deazaisofolate (IAHQ), 5,10-dideazatetrahydrofolate (DDATHF), 5-deazaisofolate (IAHQ), 5,10-dideazaisofolate (IAHQ), 5,10-dideazaisofolate (DDATHF), 5-de ... These include folic acid, PT523 (Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin (DDATHF, Iomatrexol), piritrexim, 10-EDAM, ZD1694, GW1843, pemetrexate, and PDX (10-propargyl-10-deazaaminopterin).

[0196] Another example of a drug resistance gene may be a mutant or modified form of ionosine-5'-monophosphate dehydrogenase II (IMPDH2), the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or modified form of IMPDH2 is an IMPDH inhibitor resistance gene. The IMPDH inhibitor may be mycophenolic acid (MPA) or its prodrug, mycophenolate mofetil (MMF). The mutant IMPDH2 may contain at least one, for example, two, mutations in the MAP binding site of wild-type human IMPDH2 (NP_000875.2) that confer significantly increased resistance to IMPDH inhibitors. These mutations may be at positions T333 and / or S351 (Yam et al. (2006) Mol. Ther. 14(2):236-44; Jonnalagadda et al. (2013) PLoS One 8(6):e65519). In some cases, the threonine residue at position 333 may be replaced with an isoleucine residue, and the serine residue at position 351 may be replaced with a tyrosine residue.

[0197] Another drug resistance gene is a mutant form of calcineurin. Calcineurin (PP2B) is a ubiquitously expressed serine / threonine protein phosphatase involved in many biological processes and central to T cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA, three isoforms) and a regulatory subunit (CnB, two isoforms). After association with the T cell receptor, calcineurin dephosphorylates the transcription factor NFAT, translocating it to the nucleus and key target genes, such as 1L2, where it is activated. FK506 in complex with FKBP12 or cyclosporine A (CsA) in complex with CyPA blocks NFAT access to the active site of calcineurin, preventing its dephosphorylation and thereby inhibiting T cell activation (Brewin et al. (2009) Blood 114(23):4792-803). The drug resistance gene can be a nucleic acid sequence encoding a mutant form of calcineurin that confers resistance to calcineurin inhibitors, such as FK506 and / or CsA. In some cases, the mutant form can include at least one mutant amino acid of the wild-type calcineurin heterodimer at positions V314, Y341, M347, T351, W352, L354, or K360, e.g., a double mutation at positions T351 and L354 or V314 and Y341. The correspondence of amino acid positions described herein is often expressed in terms of the amino acid positions in the form of the wild-type human calcineurin heterodimer (GenBank: ACX34092.1).

[0198] In some cases, the variant may comprise at least one mutated amino acid of wild-type calcineurin heterodimer b at positions V120, N123, L124, or K125, e.g., a double mutation at positions L124 and K125. The correspondence of amino acid positions described herein is often expressed in terms of the amino acid position in the form of the wild-type human calcineurin heterodimer b polypeptide (GenBank: ACX34095.1).

[0199] Another drug resistance gene encodes human alkylguanine transferase (hAGT). 6 The enzyme that encodes AGT is 6-methylguanine methyltransferase (MGMT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an AGT inhibitor that enhances the toxicity of nitrosoureas and is coadministered with TMZ to enhance the cytotoxic effects of TMZ. Some mutant forms of MGMT, which encode variants of AGT, are highly resistant to inactivation by 6-BG but retain the ability to repair DNA damage (Maze, Kurpad et al., 1999). In some cases, AGT mutants may contain a variant amino acid at position P140 (UniProtKB:P16455) of wild-type AGT.

[0200] Another drug resistance gene may be the multidrug resistance protein 1 (MDR1) gene. The MDR1 gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic by-products across the cell membrane. The P-GP protein exhibits broad specificity for multiple structurally unrelated chemotherapeutic agents. Therefore, expression of a nucleic acid sequence encoding MDR-1 (NP_000918) can confer drug resistance to cells.

[0201] The drug resistance gene can also be a cytotoxic antibiotic, such as the ble gene or mcrA gene, whose ectopic expression in immune cells confers a selective advantage upon exposure to the chemotherapeutic agents bleomycin or mitomycin C, respectively.

[0202] With respect to immunosuppressants, this document describes the following optional steps: (a) providing immune cells, such as T cells from cell culture or a blood sample, or induced pluripotent stem cells (iPSCs), (b) selecting a gene in the cells that expresses a target of the immunosuppressant, (c) introducing into the cells an endonuclease that can selectively inactivate the gene encoding the target of the immunosuppressant by DNA cleavage, e.g., by double-strand breaks, and (d) optionally expanding the cells in the presence of the immunosuppressant. In some cases, the method includes the further step of inactivating a component of the T cell receptor (TCR).

[0203] Immunosuppressants are drugs that suppress immune function through one of several mechanisms of action. In other words, immunosuppressants are compounds that can reduce the magnitude and / or voracity of an immune response. By way of non-limiting example, immunosuppressants can be calcineurin inhibitors, targets of rapamycin, interleukin-2 α-chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, or immunosuppressive antimetabolites. Traditional cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act by inactivating T cells or inhibiting helper cell activation. The methods described herein enable immune cells (e.g., T cells) to be resistant to immunosuppression for immunotherapy by inactivating the target of the immunosuppressant in the cells. By way of non-limiting example, the target of the immunosuppressant can be a receptor for the immunosuppressant, such as CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.

[0204] In immunocompetent hosts, allogeneic cells are typically rapidly rejected by the host immune system. It has been shown that allogeneic leukocytes present in non-irradiated blood products do not persist for more than 5–6 days (Boni et al. (2008) Blood 112(12):4746–54). Therefore, to prevent allogeneic cell rejection, effective suppression of the host immune system is necessary. Glucocorticoid steroids are widely used therapeutically for immunosuppression (Coutinho and Chapman (2011) Mol. Cell Endocrinol. 335(1):2–13). This class of steroid hormones binds to the glucocorticoid receptor (GR) present in the cytosol of T cells, leading to nuclear translocation and binding of specific DNA motifs that regulate the expression of multiple genes involved in immunological processes. Treatment of T cells with glucocorticoid steroids results in reduced cytokine production, leading to T cell anergy and disruption of T cell activation. Alemtuzumab, also known as CAMPATH1-H, is a humanized monoclonal antibody that targets CD52, a 12-amino acid glycosylphosphatidyl-inositol (GPI)-linked glycoprotein (Waldmann and Hale (2005) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360:1701-1701). CD52 is expressed at high levels on T and B lymphocytes, at lower levels on monocytes, and absent from granulocytes and myeloid precursors. Treatment with alemtuzumab, a humanized monoclonal antibody against CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes. Alemtuzumab is frequently used in the treatment of T-cell lymphoma and, in certain cases, as part of conditioning regimens for transplantation. However, in adoptive immunotherapy, the use of immunosuppressive drugs also has adverse effects on the therapeutic immune cells (e.g., T cells) being transferred, and therefore, for adoptive immunotherapy approaches to be effective in these conditions, the transferred cells must be resistant to immunosuppressive treatment.

[0205] In some cases, the gene specific for immunosuppressive therapy is CD52, and the immunosuppressive therapy comprises a humanized antibody targeting the CD52 antigen. In some cases, the gene specific for immunosuppressive therapy is glucocorticoid receptor (GR), and the immunosuppressive therapy comprises a corticosteroid, such as dexamethasone. In some cases, the gene specific for immunosuppressive therapy is an FKBP family gene member or a variant thereof, and the immunosuppressive therapy comprises FK506, also known as tacrolimus or fujimycin. In some cases, the gene specific for immunosuppressive therapy is an FKBP family gene member or a variant thereof, such as FKBP12. In some cases, the gene specific for immunosuppressive therapy is a cyclophilin family gene member or a variant thereof, and the immunosuppressive therapy comprises cyclosporine.

[0206] Cytokine release syndrome (CRS) is the most common adverse event of CAR-T cell therapy. CRS is defined as a clinical syndrome that can occur after cell therapy due to the release of cytokines (substances secreted by immune cells) into the body's bloodstream. It has been shown that inactivation of granulocyte-macrophage colony-stimulating factor (GM-CSF) can prevent monocyte-dependent release of a key mediator of cytokine release syndrome (Sachdeva et al. (2019) J. Biol. Chem. 294(14)5430-5437). Therefore, in a further embodiment, the engineered immune cells described herein are genetically modified to suppress expression or cell surface presentation of GM-CSF.

[0207] In some cases, the engineered immune cells described herein are one or more of TCR-negative, B2M-negative, CIITA-negative, PDCD1-negative, GM-CSF-negative, CD52-negative, e.g., at least TCR-negative, or at least TCR-negative, B2M-negative and CD52-negative.

[0208] In some cases, to reduce fratricide effects, the engineered immune cells described herein do not present on their cell surface the antigen targeted by the tumor-CAR. For example, the engineered immune cells described herein may have the CD4 or CD8 gene inactivated or expression inhibited if the tumor-CAR targets CD4 or CD8, respectively.

[0209] 2. Methods for Producing the Engineered Cells Described Herein Another aspect is a method of producing a cell population comprising the engineered immune cells described herein, comprising: (i) providing donor-derived immune cells or induced pluripotent stem cells (iPSCs); (ii) optionally inhibiting or suppressing T cell receptor (TCR) expression in the cell or presentation of the TCR on the cell surface; (iii) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets a tumor antigen ("tumor-CAR"), wherein the exogenous nucleic acid sequence, after integration, is placed under the transcriptional control of a constitutive promoter; (iv) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a fusion protein comprising a signal peptide, a FAP-binding domain (e.g., a FAP-binding domain comprising VH and VL amino acid sequences from a monoclonal anti-FAP antibody (e.g., a FAPscFv)), and a stimulatory cytokine, wherein the exogenous nucleic acid sequence, after integration, is placed under the transcriptional control of an endogenous inducible promoter, and optionally the fusion protein does not comprise an antibody crystallizable fragment (Fc); (v) optionally isolating the engineered cells that do not express a TCR on their surface; Including, The method is provided wherein the inducible promoter is inducible upon activation of the engineered immune cell.

[0210] The origin of the cells provided in step (i), i.e., the cells to be manipulated, is not particularly limited. In some cases, the cells in step (i) may be immune cells derived from a donor or immune cells resulting from differentiation of iPSCs into immune cells. The cells in step (i) may also be iPSCs that can be differentiated into immune cells after any one of the genetic manipulation steps (ii) to (iv) disclosed above.

[0211] By "immune cell" is meant a cell derived from the hematopoietic system that is functionally involved in the initiation and / or execution of innate and / or adaptive immune responses, typically a CD45-positive cell, a CD3-positive cell, a CD8-positive cell, or a CD4-positive cell. Immune cells include dendritic cells, killer dendritic cells, mast cells, macrophages, natural killer cells (NK cells), cytokine-induced killer cells (CIK cells), B cells, or cytotoxic T lymphocytes, or T cells selected from the group consisting of helper T lymphocytes, gamma delta T cells, and natural killer T cells (NKT cells).

[0212] In some cases, the engineered immune cells (e.g., T cells) are derived from primary cells, and by "primary cell(s)" is intended cells taken directly from biological tissue (e.g., biopsy material) and established to grow in vitro for a limited time; i.e., primary cells are capable of undergoing a limited number of population doublings. Primary cells are distinct from tumorigenic continuous cell lines or artificially immortalized cell lines. Non-limiting examples of such cell lines include CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, and Molt 4 cells.

[0213] Primary immune cells can be obtained from multiple sources, including, but not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors (e.g., tumor-infiltrating lymphocytes). In some cases, the immune cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In some cases, the cells are part of a mixed population of immune cells that exhibit different phenotypic characteristics, such as including CD4-positive cells, CD8-positive cells, and CD56-positive cells. Primary immune cells are prepared from a donor or patient via various methods known in the art, such as the leukapheresis technique outlined by Schwartz J. et al. (Guidelines on the use of therapeutic apheresis in clinical practice - evidence-based approach from the Writing Committee of the American Society for Apheresis: 6th Special Issue (2013) J Clin Apher. 28(3):145-284).

[0214] In this document, primary immune cells also refer to stem cell-derived immune cells, such as those derived from induced pluripotent stem cells (iPSCs) (Yamanaka, K. et al. (2008) Science. 322(5903):949-53). Lentiviral expression of reprogramming factors has been used to induce pluripotent cells from human peripheral blood cells (Staerk et al. (2010) Cell stem cell. 7(1):20-4; Loh et al. (2010) Cell stem cell. 7(1):15-9).

[0215] In some cases, immune cells can be derived from human embryonic stem cells by techniques well known in the art that do not involve the destruction of the human embryo (Chung et al. (2008) Cell Stem Cell 2(2):113-117).

[0216] In some cases, T cells may be derived from cytotoxic T lymphocytes or helper T lymphocytes.

[0217] In some cases, immune cells, such as T cells or NK cells, can be derived from stem cells. The stem cells can be adult stem cells, embryonic stem cells, such as non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. A representative human cell is a CD34+ cell.

[0218] In some cases, the immune cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to cell expansion and genetic modification, cells can be obtained from a subject by a variety of non-limiting methods. T cells can be obtained from multiple sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain cases, any number of T cell lines available and known to those skilled in the art can be used. In some cases, the cells can be derived from a healthy donor or a patient diagnosed with cancer. In some cases, the cells are part of a mixed population of cells exhibiting different phenotypic characteristics. The scope of the present disclosure also includes cell lines obtained from transformed T cells according to the aforementioned methods. Modified cells that are resistant to immunosuppressive treatment and obtainable by the aforementioned methods are also disclosed herein.

[0219] In some cases, the engineered immune cells (e.g., T cells or NK cells) are allogeneic. "Allogeneic" means that the cells are derived from a donor, derived from a cell line, or generated and / or differentiated from stem cells, with the intention that they will be infused into a patient with a different haplotype. Such immune cells are generally engineered to have low alloreactivity and / or high persistence to the patient host. More specifically, methods for engineering allogeneic cells can include reducing or inactivating TCR expression in T cells or in stem cells that will give rise to T cells. This can be achieved by various sequence-specific reagents, such as, but not limited to, gene silencing or gene editing techniques using nucleases, base editing techniques, shRNA, and RNAi.

[0220] In some cases, the engineered immune cells, such as T cells or NK cells, may be derived from a human, where the human is the donor and not the patient.

[0221] In some cases, engineered T cells can include an inactivated T cell receptor (TCR), which may be modified by inactivating at least one component of the TCR, for example, by using a sequence-specific endonuclease, such as an RNA-guided endonuclease associated with a specific guide RNA, or by using other gene editing techniques such as TALE nuclease. T cell receptors (TCRs) are cell surface receptors involved in T cell activation in response to antigen presentation. TCRs are generally made up of two chains, alpha and beta, which assemble to form a heterodimer and associate with the CD3 transduction subunit to form a T cell receptor complex present on the cell surface. Each of the alpha and beta chains of the TCR consists of an immunoglobulin-like variable (V) and constant (C) region at the N-terminus, a hydrophobic transmembrane domain, and a short cytoplasmic region. Regarding immunoglobulin molecules, the variable regions of the alpha and beta chains are generated by V(D)J recombination, which generates a large diversity of antigen specificities in T cell populations. However, unlike immunoglobulins that recognize intact antigens, T cells are activated by processed peptide fragments associated with MHC molecules, introducing an additional characteristic, known as MHC restriction, into antigen recognition by T cells. Recognition of MHC differences between donor and recipient by the T cell receptor leads to T cell proliferation and the potential development of GvHD. Normal surface expression of the TCR has been shown to depend on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner (1990) Annu. Rev. Immunol. 8:139-67). Inactivation of TRAC (encoding the TCR alpha constant domain) or TRBC (encoding the TCR beta constant domain) can eliminate the TCR from the surface of T cells, preventing recognition of alloantigens and thus preventing GVHD. However, disruption of the TCR generally results in the loss of the CD3 signaling component, altering the means for further T cell expansion.

[0222] In some cases, at least 50%, at least 70%, at least 90%, or at least 95% of the engineered T cells in a population are mutated in a TRAC allele, a TRBC allele, and / or a CD3 allele.

[0223] In some cases, TCRs can be inactivated using specific TALE nucleases, better known by the trade name TALEN® (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS). This method uses RNA transfection as part of a platform that allows for the mass production of allogeneic T cells and has proven highly efficient in primary cells. See, e.g., International Publication No. WO 2013 / 176915, incorporated herein by reference in its entirety.

[0224] In some cases, the TCR can be inactivated using an RNA-guided endonuclease associated with a specific guide RNA. U.S. Patent No. 10,870,864 describes a method for inactivating a TCR in a cell using such a method and is incorporated herein by reference. Engraftment of allogeneic T cells is possible by inactivating at least one gene encoding a TCR component. In some cases, the TCR is rendered non-functional in the cell by inactivating the TRAC gene and / or the TCRB gene. TCR inactivation in allogeneic T cells aims to prevent or reduce GvHD.

[0225] In some cases, the TCR gene can be inactivated by inserting at least one exogenous polynucleotide encoding a tumor-CAR into the TRAC locus of the cell's genome, the polynucleotide comprising: (a) an extracellular tumor antigen-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-tumor antigen antibody; (b) a hinge selected from an FcγRIII hinge, a CD8α hinge, and an IgG1 hinge; (c) a CD8α transmembrane domain or a CD28 transmembrane domain; (d) The cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain from 4-1BB or CD28.

[0226] Inactivating a gene means that the gene of interest is not expressed in the form of a functional protein. In some cases, genetic modification of cells relies on expressing an endonuclease in the cell prepared for manipulation, allowing the endonuclease to catalyze the cleavage of a target gene, thereby inactivating the target gene. Nucleic acid strand breaks caused by endonucleases are usually repaired by different mechanisms: homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes in the DNA sequence at the break site. The mechanism involves rejoining the remaining portions of the two DNA ends by direct religation (Critchlow and Jackson (1998) Trends Biochem Sci. 23(10):394-8) or via so-called microhomology-mediated end joining (Betts et al. (2003) J. Immunol. Methods 281(1-2):65-78; Ma et al. (2003) Mol Cell Biol 23(23):8820-8). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used to generate specific gene knockouts. The modification can be a substitution, deletion, or addition of at least one nucleotide. Cells that have undergone a break-induced mutagenesis event, i.e., a mutagenesis event subsequent to an NHEJ event, can be identified and / or selected by methods well known in the art.

[0227] Because the engineered immune cells described herein may be derived from the differentiation of the engineered iPSCs described herein into said immune cells, another aspect described herein is a method of producing a cell population comprising the engineered iPSCs described herein, comprising: (i) providing induced pluripotent stem cells (iPSCs); (ii) optionally inactivating latent expression of a T cell receptor (TCR) in the cell or presentation of a TCR on the surface of the cell; (iii) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets a tumor antigen ("tumor-CAR"), wherein the exogenous nucleic acid sequence, after integration, is placed under the transcriptional control of a constitutive promoter; (iv) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a fusion protein comprising a signal peptide, a FAP-binding domain, and a stimulatory cytokine, wherein the exogenous nucleic acid sequence, after integration, is placed under the transcriptional control of an endogenous inducible promoter; Including, Optionally, the fusion protein does not comprise an antibody crystallizable fragment (Fc); Optionally, the FAP binding domain comprises VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody (e.g., a FAPscFv); and wherein the inducible promoter is inducible upon activation of an immune cell into which the engineered iPSCs may further differentiate.

[0228] Accordingly, another aspect includes a method of producing a cell population comprising engineered immune cells described herein, comprising the steps of: (i) producing a cell population comprising engineered iPSCs as described above; and (ii) differentiating the engineered iPSCs into immune cells.

[0229] Manipulation and Gene Editing Methods that can be used herein to engineer or gene-edit cells are not particularly limited. In some cases, cells can be modified (e.g., engineered or gene-edited) by contacting them with sequence-specific reagents.

[0230] By "sequence-specific reagent" is meant an active molecule capable of specifically recognizing a selected polynucleotide sequence at a genomic locus, termed a "target sequence," which is generally at least 12 bp, at least 15 bp, or at least 30 bp or 35 bp in length, in view of altering the expression of said genomic locus. The expression can be altered by mutations, deletions, or insertions in coding or regulatory polynucleotide sequences, by epigenetic changes such as methylation or histone modifications, or by interfering at the transcriptional level by interacting with transcription factors or polymerases.

[0231] Examples of sequence-specific reagents are endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, end-processing enzymes, e.g., exonucleases, and more particularly cytidine deaminases, such as those coupled to the CRISPR / cas9 system to effect base editing (i.e., nucleotide substitution) without necessarily relying on nuclease cleavage, as described by Hess et al. (Mol Cell. (2017) 68(1):26-43) and Rees et al. (Nat. Rev. Genet. (2018) 19, 770-788).

[0232] According to one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population express a short hairpin RNA (shRNA) or small interfering RNA (siRNA) against a polynucleotide sequence encoding a component of a TCR.

[0233] In one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population expresses a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding β2M.

[0234] According to one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population expresses a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding CD52.

[0235] According to one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population express a short hairpin RNA (shRNA) or small interfering molecule (siRNA) directed against a polynucleotide sequence encoding PDCD1. According to one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population express a short hairpin RNA (shRNA) or small interfering molecule (siRNA) directed against a polynucleotide sequence encoding LAG3.

[0236] In one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population expresses a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding TIM3.

[0237] In one embodiment, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population expresses a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding GM-CSF.

[0238] According to another aspect, at least 50%, at least 70%, at least 90%, or at least 95% of the cell population encodes a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding a component of a TCR, as well as a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding β2M and / or a short hairpin RNA (shRNA) or small interfering molecule (siRNA) against a polynucleotide sequence encoding CD3.

[0239] In some cases, the sequence-specific reagent can be a sequence-specific nuclease reagent, such as a sequence-specific endonuclease, such as a rare-cutting endonuclease, such as a TALE nuclease, or an RNA guide coupled to a guide endonuclease, such as a CRISPR.

[0240] The term "sequence-specific nuclease reagent" includes reagents with nickase or endonuclease activity. A sequence-specific nuclease reagent can be a chimeric polypeptide comprising a DNA-binding domain and another domain that exhibits catalytic activity. Such catalytic activity can be, for example, a nuclease for gene inactivation, or a nickase or double nickase for preferential gene insertion by creating cohesive ends to facilitate gene integration by homologous recombination.

[0241] The term "endonuclease" generally refers to a wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of DNA or RNA molecules, or internucleic acid bonds in DNA molecules. Endonucleases (and thus sequence-specific endonucleases) do not cleave DNA or RNA molecules regardless of their sequence, but rather recognize and cleave DNA or RNA molecules at specific polynucleotide sequences, hereafter referred to as "target sequences" or "target sites." Endonucleases can be classified as rare-cutting endonucleases if they have polynucleotide recognition sites that are typically greater than 10 base pairs (bp) in length or 14-55 bp in length. Rare-cutting endonucleases significantly increase homologous recombination by inducing DNA double-strand breaks (DSBs) at defined loci, thereby enabling gene repair or gene insertion therapy (Pingoud and Silva (2007) Nat. Biotechnol. 25(7):743-4).

[0242] In some cases, the sequence-specific reagent can be a base editor capable of performing base editing, for example, as described by Komor et al. (Nature (2019) 533 (7603), 420-424) and Mok et al. (Nature (2020) 583:631-637).

[0243] The term "base editor," as used herein, refers to a catalytic domain that can make modifications to bases (e.g., A, T, C, G, or U) within a nucleic acid sequence by converting one base to another (e.g., A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, or T to G). Base editors can include cytidine deaminases that convert targeted C / G to T / A, and adenine base editors that convert targeted A / T to G / C. The adenosine deaminase can be, for example, TadA or its variant TadA7.10, as described by Jeong et al. (Nat Biotechnol (2021) 39, 1426-1433). Various members of the apolipoprotein B mRNA editing enzyme (APOBEC) family, such as mouse rAPOBEC1 and human APOBEC3G developed by Lee et al. (Science Advances (2020) 6(29)), can be used to convert cytidine to thymidine.

[0244] In some cases, a base editor catalytic domain can convert a C to a T (cytidine deaminase), catalyzing the chemical reaction "cytosine + HO → uracil + NH" or "5-methyl-cytosine + HO → thymine + NH." As can be seen from the reaction equation, such a chemical reaction results in a nucleobase change from C to U / T. In the context of a gene, such a nucleotide change or mutation can result in an amino acid change in the protein, e.g., a loss-of-function or gain-of-function, which can affect the function of the protein.

[0245] The sequence-specific reagents defined herein include TALE-base editors (BEs), which can be generated by fusing a transcription activator-like effector array protein (TALE) with a base editor catalytic domain. The base editor catalytic domain can be a double-stranded DNA deaminase ("DddA") that precisely generates nucleotide changes and / or corrects pathogenic mutations rather than disrupting DNA by double-strand breaks (DSBs). For example, Mok et al. (Nature (2020) 583:631-637) recently developed a TALE base editor by splitting the bacterial cytidine deaminase toxin DddAtox from Burkholderia cenocepacia into two non-toxic halves, each fused to the C-terminus of a pair of (left and right) TALE-binding domains to form a heterodimeric TALE base editor. In this situation, the deaminase DddAtox becomes active when the two halves linked to their respective TALE-binding domains co-localize at a predetermined genomic locus. The split "DddA-N half" and "DddA-C half" can be obtained by cleaving the complete DddAtox protein (SEQ ID NO: 87) at positions 1333 or 1397.

[0246] In some cases, such TALE-base editors may also include a domain that inhibits uracil glycosylase, termed "UGI," and / or a nuclear localization signal. The term "uracil glycosylase inhibitor" or "UGI," as used herein, refers to a protein that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some cases, the UGI domain may include wild-type UGI or standard UGI. In some cases, the UGI protein may include fragments of UGI and proteins homologous to UGI or UGI fragments, which are useful for improving the specificity of base edits performed at a predetermined locus.

[0247] The methods and materials provided herein aim to improve the therapeutic potential of immune cells through gene editing techniques, particularly by gene-targeted integration.

[0248] After integration into the genome of a cell, the exogenous nucleic acid sequence encoding the tumor-CAR is placed under the transcriptional control of an exogenous or endogenous constitutive promoter. After integration into the genome of a cell, the exogenous nucleic acid sequence encoding the fusion protein comprising the FAP-binding domain and a stimulatory cytokine is placed under the transcriptional control of an endogenous inducible promoter.

[0249] An exogenous nucleic acid sequence encoding a tumor-CAR described herein can be integrated into the genome of a cell by random integration (e.g., by integration of a lentiviral vector) or by gene-targeted integration (e.g., by sequence-specific endonuclease-mediated cDNA insertion at a targeted locus in the genome of the cell).

[0250] An exogenous nucleic acid sequence encoding a fusion protein described herein can be integrated into the genome of a cell by gene-targeted integration (e.g., by sequence-specific endonuclease-mediated cDNA insertion at a targeted locus in the genome of the cell).

[0251] In one example, an exogenous nucleic acid sequence encoding a tumor-CAR described herein is integrated into the genome of a cell by random integration (e.g., by lentiviral vector integration), and an exogenous nucleic acid sequence encoding a fusion protein described herein is integrated into the genome of a cell by gene-targeted integration (e.g., by sequence-specific endonuclease-mediated cDNA insertion at a targeted locus in the genome of the cell).

[0252] In other examples, an exogenous nucleic acid sequence encoding a tumor-CAR described herein is integrated into the genome of the cell by gene-targeted integration (e.g., by sequence-specific endonuclease-mediated cDNA insertion at a targeted locus in the genome of the cell), and an exogenous nucleic acid sequence encoding a fusion protein described herein is integrated into the genome of the cell by gene-targeted integration (e.g., by sequence-specific endonuclease-mediated cDNA insertion at a targeted locus in the genome of the cell).

[0253] "Gene-targeted integration" refers to any known site-specific method that allows for the insertion, replacement, or modification of a genomic coding sequence in a living cell.

[0254] In some cases, gene-targeted integration involves inserting a target gene or replacing a target gene with at least one exogenous nucleotide sequence, such as a sequence of nucleotides (i.e., polynucleotides), e.g., a coding sequence, as a result of homologous recombination at the locus of the target gene.

[0255] By "DNA target," "DNA target sequence," "target DNA sequence," "nucleic acid target sequence," "target sequence," or "processing site" is intended a polynucleotide sequence that can be targeted and processed by the sequence-specific nuclease reagents described herein. These terms refer to a specific DNA location, such as a location within the genome of a cell, but also to a portion of genetic material that can exist independently of the body of genetic material, such as a plasmid, episome, virus, transposon, or a portion of genetic material that can reside in an organelle, such as, but not limited to, a mitochondria. A non-limiting example of an RNA-guided target sequence is a genomic sequence to which a guide RNA can hybridize that directs an RNA-guided endonuclease to a desired locus.

[0256] A "rare-cutting endonuclease" is any sequence-specific endonuclease reagent so long as the recognition sequence is in the range of 10-50 contiguous base pairs overall, e.g., 12-30 bp or 14-20 bp.

[0257] In some cases, the sequence-specific endonuclease reagent can be a nucleic acid encoding an "engineered" or "programmable" rare-cutting endonuclease, such as a homing endonuclease described by Arnould et al. (WO 2004067736), a zinc finger nuclease (ZFN) described by Urnov et al. (Nature (2005) 435:646-651), a TALE nuclease described by Mussolino et al. (Nucl. Acids Res. (2011) 39(21):9283-9293), or a megaTAL nuclease described by Boissel et al. (Nucleic Acids Research (2013) 42(4):2591-2601).

[0258] In some cases, the endonuclease reagent may be an RNA guide used in conjunction with an RNA-guided endonuclease such as Cas9 or Cpfl, as taught by Doudna and Charpentier (Science (2014) 346(6213):1077), which is incorporated herein by reference, among others.

[0259] In some cases, the endonuclease reagent can be transiently expressed in the cell, i.e., the reagent is not intended to be integrated into the genome or persist for long periods of time, as is the case with RNA such as mRNA, proteins, or mixed protein and nucleic acid complexes (e.g., ribonucleoproteins).

[0260] The endonuclease in mRNA form can be synthesized with a cap to enhance its stability according to techniques well known in the art, for example, as described by Kore et al. (J Am Chem Soc. (2009) 131(18):6364-5).

[0261] The nucleases described herein, polynucleotides encoding these nucleases, donor polynucleotides, and compositions comprising proteins and / or polynucleotides for genetically modifying cells can be delivered in vivo or ex vivo by any suitable means.

[0262] In some cases, a polypeptide can be synthesized in situ within a cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. In some cases, a polypeptide can be produced extracellularly and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art and include, but are not limited to, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and viral-mediated methods. In some cases, a polynucleotide can be introduced into a cell via a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, or the like. For example, transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. A polynucleotide can be contained in a vector, such as a plasmid or virus, to be expressed within the cell.

[0263] In some cases, a nucleic acid encoding an endonuclease reagent can be transfected into a cell, and in some cases, 80% of the endonuclease reagent is degraded by 30 hours, e.g., by 24 hours or 20 hours, after transfection.

[0264] In some cases, the nucleases and / or donor constructs described herein may be delivered using vectors that include sequences encoding one or more of a CRISPR / Cas system(s), zinc finger, or TALEN protein(s).

[0265] Any vector system may be used, including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors. See also U.S. Patent Nos. 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, which are incorporated herein by reference in their entireties. Furthermore, it will be apparent that any of these vectors may contain one or more sequences required for therapy. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nuclease and / or donor polynucleotide may be carried in the same or different vectors. When multiple vectors are used, each vector can contain sequences encoding one or more nucleases and / or donor constructs.

[0266] Any suitable viral and non-viral gene transfer method can be used to introduce nucleic acids encoding nucleases and donor constructs into cells (e.g., mammalian cells) and target tissues.

[0267] Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to the cell. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., Current Topics in See Microbiology and Immunology, Doerfler and Bohm (eds.) (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0268] In some cases, methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation:nucleic acid conjugates or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and pharmaceutical enhancement of DNA uptake. Sonoporation, for example, using the Sonitron 2000 system (Rich-Mar), can also be used for delivery of nucleic acids.

[0269] In general, the electroporation step used to transfect primary immune cells such as PBMCs is typically carried out in a closed chamber with parallel plate electrodes that generate a pulsed electric field between the parallel plate electrodes of greater than 100 volts / cm and less than 5,000 volts / cm that is substantially uniform throughout the treatment volume, as described in WO 2004 / 083379, particularly page 23, line 25 to page 29, line 11, which is incorporated herein by reference. One such electroporation chamber is one in which the square of the electrode gap (cm) is proportional to the chamber volume (cm). 3 ) divided by the quotient (cm -1 ), which has a view factor of 0.1 cm -1 The cells and sequence-specific reagents are suspended in a medium adjusted to have a conductivity ranging from 0.01 to 1.0 millisiemens. Typically, the cell suspension is subjected to one or more pulsed electric fields. This method allows for scalable throughput and substantially uniform cell processing time within the chamber.

[0270] In some cases, different transgenes or multiple copies of a transgene can be included in a single vector. The vector can include a nucleic acid sequence encoding a ribosomal skipping sequence, such as a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes the ribosome to "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by these codons (see Donnelly et al., J. of General Virology 82:1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78:13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13):4227-4239 (2008); Atkins et al., RNA 13:803-810 (2007)).

[0271] "Codon" refers to three nucleotides in an mRNA (or the sense strand of a DNA molecule) that are translated into one amino acid residue by a ribosome. Thus, two polypeptides can be synthesized from a single, continuous open reading frame in an mRNA if they are separated by an in-frame 2A oligopeptide sequence. Such ribosomal skipping mechanisms are well known in the art and are known to be used by some vectors for the expression of several proteins encoded by a single messenger RNA.

[0272] In some cases, the polynucleotide encoding the sequence-specific reagent can be mRNA, which is directly introduced into cells, for example, by electroporation. In some cases, cells can be electroporated using cytoPulse technology, which uses pulsed electric fields to transiently permeabilize live cells and allow substances to be delivered into the cells. This technology, based on the use of electroporation waveforms from PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, Mass. 01746, USA), allows precise control of pulse duration, intensity, and the interval between pulses (see U.S. Pat. No. 6,010,613 and published international application WO 2004 / 083379). All of these parameters can be modified to achieve optimal conditions for high transfection efficiency with minimal mortality. A first high-field pulse allows for pore formation, and a subsequent low-field pulse allows for the movement of the polynucleotide into the cells.

[0273] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam and Lipofectin). Suitable cationic and neutral lipids for efficient receptor-recognition lipofection of polynucleotides include those described by Felgner, WO 91 / 17424, WO 91 / 16024.

[0274] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992), U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0275] In some cases, the donor sequence and / or sequence-specific reagents can be encoded by a viral vector. In some cases, an adenovirus-based system can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained with such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy treatments (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in several publications, including U.S. Pat. No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0276] Recombinant adeno-associated viral vectors (rAAV) are a promising alternative gene delivery system based on the defective, nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that contain only the AAV 145-bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery by integration into the genome of transduced cells are key features of this vector system (Wagner et al., Lancet 351:9117 1702-3 (1998); Kearns et al., Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including, but not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6, may also be used in accordance with the present disclosure.

[0277] In some cases, the cells may be administered with an effective amount of one or more caspase inhibitors in combination with the AAV vector.

[0278] In some cases, the donor sequence and / or the sequence-specific reagent may be encoded by a recombinant lentiviral vector (rLV).

[0279] Integrase-deficient lentivirus (IDLV) may also be used herein. IDLV is derived from a typical integrating lentivirus but contains a loss-of-function mutation in its lentiviral integrase protein that prevents proviral DNA from inserting into the genome of transduced cells.

[0280] The nuclease-encoding sequence and donor construct can be delivered using the same system or different systems, for example, the donor polynucleotide can be carried in a viral vector and the nuclease(s) can be delivered as an mRNA composition.

[0281] In some cases, one or more reagents can be delivered to cells using nanoparticles. In some cases, the nanoparticles are coated with a ligand, such as an antibody, that has specific affinity for an HSC surface protein, such as CD105 (Uniprot number P17813). In some cases, the nanoparticles are biodegradable polymer nanoparticles in which a sequence-specific reagent in the form of a polynucleotide is conjugated to a polymer of polybeta-amino ester and coated with polyglutamic acid (PGA).

[0282] Due to their high specificity, TALE nucleases have proven to be particularly suitable sequence-specific nuclease reagents for therapeutic applications, especially in heterodimeric form, i.e., functioning as a pair of a "right" monomer (also referred to as "5'" or "forward") and a "left" monomer (also referred to as "3'" or "reverse") as reported, for example, by Mussolino et al. (Nucl. Acids Res. (2014) 42(10):6762-6773).

[0283] As mentioned above, the sequence-specific reagent can be in the form of a nucleic acid, such as a DNA or RNA form, encoding a rare-cutting endonuclease or a subunit thereof, but can also be part of a conjugate comprising a polynucleotide(s) and a polypeptide(s), such as a so-called "ribonucleoprotein." Such conjugates can be formed with reagents such as Cas9 or Cpf1 (RNA-guided endonucleases) as described by Zetsche et al. (Cell (2015) 163(3):759-771), respectively, that comprise an RNA guide or a DNA guide that can be complexed with the respective nuclease.

[0284] An "exogenous sequence" refers to a nucleotide or nucleic acid sequence that was not originally present at a selected locus. This sequence may be homologous to a genomic sequence, or may be a copy of a genomic sequence, or may be a foreign sequence introduced into a cell. In contrast, an "endogenous sequence" refers to a cellular genomic sequence that is originally present at a locus.

[0285] As used herein, a "donor construct" or "donor polynucleotide" includes an exogenous nucleotide sequence that is randomly inserted into the genome of a cell at any locus, or that is inserted into or replaces a targeted locus. A donor construct can include a nucleotide sequence encoding a CAR or fusion protein described herein, and can optionally include a promoter that controls transcription of the CAR or fusion protein.

[0286] In some cases, the donor construct may be a vector comprising a constitutive exogenous promoter and an exogenous nucleic acid sequence encoding a tumor-CAR operably linked to said promoter, as described herein. In this case, the donor construct may be randomly integrated into the genome of the cell at any locus, and transcription of the tumor-CAR is controlled by said constitutive exogenous promoter.

[0287] In some cases, the donor construct can be a vector comprising an exogenous nucleic acid sequence encoding a tumor-CAR flanked by left and right homology arms (or "left and right homology regions") having homology to a targeted locus (also referred to as "5' and 3' homology arms (or 5' and 3' homology regions)," respectively), whose expression is constitutive as described herein. In some cases, the vector does not comprise a promoter sequence, and the donor construct can be integrated into the genome of the cell by homologous recombination at the targeted constitutively expressed locus, such that transcription of the tumor-CAR is controlled by the constitutive (endogenous) promoter of the targeted locus. In some cases, the vector further comprises a constitutive exogenous promoter sequence, and a "cassette" comprising the promoter sequence and the exogenous nucleic acid sequence encoding the tumor-CAR is flanked by left and right homology arms having homology to the targeted locus. In these latter cases, the donor construct can be integrated into the cell's genome by homologous recombination at the targeted locus, while transcription of the tumor-CAR is controlled by a constitutive exogenous promoter provided by the vector.

[0288] In some cases, the donor construct can be a vector that includes an exogenous nucleic acid sequence encoding a fusion protein described herein flanked by left and right homology arms that have homology to a targeted locus that is an inducible locus. Generally, such vectors do not include a promoter sequence, and the donor construct can be integrated into the genome of a cell by homologous recombination at the targeted inducible locus such that transcription of the fusion protein is controlled by the inducible (endogenous) promoter of the targeted locus.

[0289] If the donor construct does not contain a promoter, the donor construct may contain, in addition to the CAR coding sequence or fusion protein coding sequence, an internal ribosome entry site (IRES) or a "self-cleaving" 2A peptide such as T2A, P2A, E2A, or F2A to enable production of a functional CAR or fusion protein, respectively.

[0290] Stable expression of proteins in the above-mentioned immune cells, such as T cells, particularly the tumor-CAR and fusion proteins described herein, can be achieved using, for example, viral vectors (e.g., lentiviral vectors, retroviral vectors, adeno-associated viral (AAV) vectors), or transposon / transposase systems, or integration of plasmids or PCR products. Other approaches include direct mRNA electroporation.

[0291] Non-limiting examples of TALE nucleases that target endogenous genes expressing PDCD1, TRAC, CD52, and B2M are shown in Table 6. The present invention can be practiced as described herein using such polynucleotides or polypeptides that have at least 70%, such as at least 80%, at least 90%, or at least 95% or 99% identity to the sequences referred to in Table 6.

[0292] [Table 6] TIFF2025536578000007.tif216149 TIFF2025536578000008.tif216149 TIFF2025536578000009.tif216149 TIFF2025536578000010.tif216149 TIFF2025536578000011.tif216149 TIFF2025536578000012.tif216149

[0293] In some cases, integration of the donor construct by homologous recombination at the targeted locus results in reduced or suppressed production of the product of the targeted gene.

[0294] Thus, in some cases, any of the donor constructs described herein can be integrated at loci encoding TCR, HLA, B2M, PDCD1, CTLA4, TIM3, LAG3, CD69, IL2Ra, GM-CSF, and / or CD52. As a result, in these cases, expression of the targeted gene is reduced or suppressed.

[0295] For example, in some cases, a polynucleotide encoding a tumor-CAR described herein is integrated at the endogenous TRAC locus, B2M locus, or CD52 locus in the genome of the engineered immune cell, e.g., a T cell. In some cases, a polynucleotide encoding a fusion protein described herein is integrated at the endogenous PDCD1 locus, CD25 locus, GM-CSF locus, TIM3 locus, or TIGIT locus in the genome of the engineered immune cell (e.g., a T cell).

[0296] Gene-targeted insertion of sequences encoding CAR or fusion proteins and / or other exogenous gene sequences can be performed using AAV vectors, particularly vectors from the AAV6 family, or the chimeric vector AAV2 / 6 previously described by Sharma et al. (Brain Research Bulletin. (2010) 81(2-3):273-278).

[0297] Thus, one aspect relates to transducing human primary immune cells, such as primary T cells, with such AAV vectors encoding the tumor-CAR or fusion proteins described herein in conjunction with expression of a sequence-specific endonuclease reagent, such as a TALE endonuclease, to increase gene integration at the aforementioned loci.

[0298] Another aspect relates to inactivating the aforementioned genes (e.g., TRAC, TRBC, CD3, HLA, B2M, PDCD1, CTLA4, TIM3, LAG3, CD69, IL2Ra, GM-CSF and / or CD52) by transduction of human primary immune cells, particularly primary T cells, with a recombinant lentiviral vector (rLV) encoding a CAR, such as a tumor-CAR, described herein, which can be done before or after introduction of a sequence-specific endonuclease reagent, such as a TALE endonuclease.

[0299] In some cases, the sequence-specific endonuclease reagent may be introduced into the cell by transfection, such as by electroporation, of mRNA encoding said sequence-specific endonuclease reagent.

[0300] Accordingly, there is provided a method for inserting an exogenous nucleic acid sequence encoding a tumor-CAR or fusion protein described herein into the genome of a cell at one of the aforementioned loci, the method comprising: transducing the cells with an AAV vector comprising an exogenous nucleic acid sequence encoding a tumor-CAR or fusion protein described herein and a sequence homologous to an endogenous DNA sequence to be targeted; and expressing a sequence-specific endonuclease reagent that cleaves the endogenous sequence at the site of insertion. It includes at least one of the following:

[0301] The insertion of the exogenous nucleic acid sequence achieved can result in the introduction of genetic material and replacement of the endogenous sequence, thus resulting in the inactivation of the endogenous locus.

[0302] Another object relates to AAV vectors used in the subject methods, which may include an exogenous coding sequence that is "promoterless," the coding sequence being any of those referred to herein.

[0303] Many other vectors known in the art, such as plasmids, episomal vectors, linear DNA substrates, etc., can also be used to effect gene insertion at these loci by following the teachings of the present application.

[0304] The DNA vectors used for the random integration described herein may comprise (i) a constitutive promoter and an exogenous nucleic acid to be inserted, the exogenous nucleic acid comprising a sequence encoding a tumor-CAR described herein, operably linked to the promoter.

[0305] As derived from the meaning of the term "exogenous sequence" provided herein, it should be understood that sequences contained in an integrated DNA vector are necessarily "exogenous" because they are intended to be added to the genome of the cell; therefore, the adjective "exogenous" may be omitted in this context.

[0306] In another embodiment, when the CAR is a multi-chain CAR, the nucleic acid of (1) further comprises an internal ribosome entry site (IRES) or a "self-cleaving" 2A peptide, such as T2A, P2A, E2A, or F2A, such that the inserted exogenous coding sequence is polycistronic. The IRES or 2A peptide can precede or follow the exogenous coding sequence.

[0307] The exogenous polynucleotide sequence encoding the tumor-CAR and / or fusion protein described herein can also be introduced into immune cells, e.g., T cells or NK cells, or into iPSCs, by using a viral vector, such as a lentiviral vector. Thus, the present disclosure provides viral vectors encoding the tumor-CAR and / or fusion protein described herein.

[0308] In some cases, the lentiviral or AAV vectors contemplated herein may comprise a CAR-encoding sequence separated by a T2A or P2A sequence as forming a single transcription unit. In a lentiviral vector, the tumor-CAR-encoding sequence described herein may form an expression cassette that is transcribed under the control of a constitutive exogenous promoter, such as the EF1 alpha promoter derived from the human EF1A1 gene.

[0309] In some cases, engineered cells are generated by a process that involves random integration in the genome of the cell of a lentiviral vector that includes a constitutive promoter sequence and a polynucleotide encoding a tumor-CAR described herein.

[0310] In some cases, the engineered cells are generated by a process that includes targeted integration of an exogenous sequence encoding a tumor-CAR.

[0311] In some cases, engineered cells are produced by a process that includes targeted integration of exogenous sequences encoding the fusion proteins described herein.

[0312] Thus, in some cases, engineered cells are produced by a process comprising targeted integration of a polynucleotide encoding a tumor-CAR described herein into the genome of the cell by sequence-specific endonuclease-mediated cDNA insertion at a constitutively expressed locus in the genome of the cell. In these cases, the cDNA comprises a polynucleotide encoding a tumor-CAR described herein, and the constitutively expressed locus is a locus controlled by an endogenous constitutive promoter as defined herein. In these cases, expression of the tumor-CAR is controlled by the endogenous constitutive promoter.

[0313] In some cases, engineered cells are generated by a process comprising targeted integration into the genome of a cell of a polynucleotide comprising an exogenous constitutive promoter and an exogenous nucleic acid sequence encoding a tumor-CAR described herein operably linked to the promoter via sequence-specific endonuclease-mediated insertion at any targeted locus in the genome of the cell. In these cases, expression of the tumor-CAR is controlled by the exogenous constitutive promoter.

[0314] In some cases, engineered cells are produced by a process that includes targeted integration of a polynucleotide encoding a fusion protein described herein into the genome of the cell via sequence-specific endonuclease-mediated cDNA insertion at an inducible locus in the genome of the cell. In these cases, the cDNA includes a polynucleotide encoding a fusion protein described herein, and the inducible locus is controlled by an inducible promoter as defined herein. In these cases, expression of the fusion protein is controlled by the endogenous inducible promoter.

[0315] Another aspect relates to a set of vectors or kits for producing the engineered immune cells or iPSCs described herein.

[0316] In one aspect, a set of vectors is provided that includes at least one vector comprising a nucleic acid sequence encoding a tumor-CAR described herein that allows for integration into the genome of a cell under the transcriptional control of an endogenous or exogenous constitutive promoter, and at least one vector comprising a nucleic acid sequence encoding a fusion protein described herein that allows for integration into the genome of a cell under the transcriptional control of an inducible endogenous promoter.

[0317] In one aspect, (1)(a) at least one vector comprising an exogenous nucleic acid sequence comprising a constitutive promoter and a nucleic acid sequence encoding a tumor-CAR operably linked to the promoter; or (1)(b) at least one vector comprising an expression cassette comprising an exogenous nucleic acid sequence encoding a tumor-CAR, wherein the exogenous nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to an endogenous constitutive locus in the cell; and (2) At least one vector comprising an expression cassette comprising an exogenous nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, optionally wherein the fusion protein does not comprise an antibody crystallizable fragment (Fc), and the exogenous nucleic acid sequence encoding the fusion protein is positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to an endogenous inducible locus in the cell. A set of vectors is provided, comprising:

[0318] As used herein, endogenous constitutive loci and endogenous inducible loci in a cell refer to loci present in the genome of a cell that can be transduced with a defined set of vectors, said vectors constituting the means for integrating said defined cassettes into the genome of said described cell.

[0319] In another aspect, (a) at least one vector comprising a nucleic acid sequence comprising an exogenous constitutive promoter and a nucleic acid sequence encoding a tumor-CAR operably linked to the promoter; (b) at least one vector comprising a nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, optionally wherein the fusion protein does not comprise an antibody crystallizable fragment (Fc), and wherein the nucleic acid sequence encoding the fusion protein is positioned between a left homology region and a right homology region, the left homology region and the right homology region being homologous to a locus targeted by the endonuclease of (c); (c) at least one sequence-specific endonuclease that targets one endogenous inducible locus; A kit is provided comprising:

[0320] In another aspect, (a) at least one vector comprising an expression cassette comprising an exogenous nucleic acid sequence encoding a tumor-CAR, wherein the exogenous nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (b); (b) at least one sequence-specific endonuclease that targets an endogenous constitutive locus; and (c) at least one vector comprising a nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, optionally wherein the fusion protein does not comprise an antibody crystallizable fragment (Fc), and wherein the nucleic acid sequence encoding the fusion protein is positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (d); (d) at least one sequence-specific endonuclease that targets one endogenous inducible locus; A kit is provided comprising:

[0321] In some of the kits described herein, the exogenous constitutive promoter is selected from the group consisting of EF1A promoter, CD52 promoter, GAPDH promoter, CMV promoter, hPGK promoter, UBC promoter, SV40 promoter, PGK promoter, CAGG promoter, TRAC promoter, TRBC promoter, TRGC promoter, TRDC promoter, B2M promoter, CD5 promoter, CS1 promoter, CD45 promoter, RPBSA promoter, CD4 promoter, and CD8 promoter; and / or the endogenous constitutive locus is selected from the group consisting of EF1A, CD52, GAPDH, hPGK, UBC, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, CD4, and CD8 loci.

[0322] In some of the kits described herein, the inducible locus is selected from the group consisting of the PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 loci.

[0323] In some of the kits, the sequence-specific endonuclease targeting one constitutive locus is a TALE nuclease. In some cases, the TALE nuclease can target one endogenous constitutively expressed locus selected from the group consisting of EF1A, CD52, GAPDH, hPGK, UBC, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, CD4, and CD8 loci. In some cases, the TALE nuclease can target one endogenous constitutively expressed locus selected from the group consisting of EF1A, TRAC, B2M, CD52, CS1, CD45, CD5, and GAPDH loci. In some cases, the TALE nuclease can target one endogenous constitutively expressed locus selected from the group consisting of EF1A, TRAC, B2M, and CD52 loci.

[0324] In some of the kits, the sequence-specific endonuclease targeting one inducible locus is a TALE nuclease. In some cases, the TALE nuclease may target one inducible locus selected from the group consisting of PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, GOS2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 loci. In some cases, the TALE nuclease may target one inducible locus selected from the group consisting of PDCD1, CD25, GM-CSF, TIM3, and TIGIT loci. In some cases, the TALE may target one inducible locus that is the PDCD1 locus.

[0325] In some cases, if the vector of the kit does not include a promoter that controls transcription of the exogenous nucleic acid sequence contained in the vector, transcription of the exogenous nucleic acid sequence is controlled by an endogenous promoter of the targeted endogenous locus after integration in the genome of the cell.

[0326] In some cases, if the vector of the kit contains a promoter that controls transcription of the exogenous nucleic acid sequence contained in the vector, transcription of the exogenous nucleic acid sequence is controlled by the exogenous promoter after integration into the genome of the cell.

[0327] In another aspect, (a) at least one vector comprising a nucleic acid sequence comprising a constitutive promoter and a nucleic acid sequence encoding a tumor-CAR described herein operably linked to the promoter; (b) at least one vector comprising a nucleic acid sequence encoding a fusion protein described herein positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (c); (c) at least one sequence-specific endonuclease that targets one inducible locus selected from the group consisting of the PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 loci. A kit is provided comprising:

[0328] In another aspect, (a) at least one vector comprising a nucleic acid sequence encoding a tumor-CAR described herein positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (b); (b) at least one sequence-specific endonuclease targeting one constitutively expressed locus selected from the group consisting of EF1A, CD52, GAPDH, hPGK, UBC, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, CD4, and CD8 loci; and (c) at least one vector comprising a nucleic acid sequence encoding a fusion protein described herein positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (d); (d) at least one sequence-specific endonuclease targeting one inducible locus selected from the group consisting of the PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 loci; A kit is provided comprising:

[0329] In another embodiment, (a) at least one vector comprising a constitutive promoter and a cassette comprising a nucleic acid sequence encoding a tumor-CAR described herein operably linked to the promoter, wherein the constitutive promoter is selected from the group consisting of EF1A, CD52, GAPDH, hPGK, UBC, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, RPBSA, CD4, and CD8, and wherein the cassette of (a) is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (b); (b) at least one sequence-specific endonuclease that targets the locus; and (c) at least one vector comprising a nucleic acid sequence encoding a fusion protein described herein positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to a locus targeted by the endonuclease of (d); (d) at least one sequence-specific endonuclease targeting one inducible locus selected from the group consisting of the PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 loci; A kit is provided comprising:

[0330] In this latter embodiment, since the tumor-CAR is under the control of a specified exogenous promoter, there is no limitation regarding the targeted locus in (b). Thus, in this latter embodiment, the targeted locus in (b) may be inducible or constitutively expressed.

[0331] Activation and expansion of immune cells Whether before or after genetic modification, the immune cells described herein can be activated or expanded, even if they can be activated or expanded independently of the antigen binding mechanism. For example, T cells can be activated and expanded using methods described in, e.g., U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and 7,572,631. T cells can be expanded in vitro or in vivo. T cells are generally expanded by contacting them with agents that stimulate the CD3 TCR complex and costimulatory molecules on the surface of the T cells, thereby generating an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for T cells.

[0332] As non-limiting examples, a population of T cells can be stimulated in vitro by contact with an anti-CD3 antibody, or an antigen-binding fragment of an anti-CD3 antibody, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. Co-stimulation of accessory molecules on the surface of T cells uses a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Suitable conditions for culturing T cells include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo 5 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human fetal serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-, or other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokine(s) sufficient for T cell growth and expansion, and may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1, and X-Vivo 20, OptTmizer, supplemented with amino acids, sodium pyruvate, and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air and 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics.

[0333] In some cases, the cells may be expanded by co-culturing with tissue or cells. The cells may be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0334] For example, any biological activity exhibited by engineered immune cells expressing a CAR can be determined, including cytokine production and secretion, degranulation, proliferation, or any combination thereof.

[0335] In some cases, the biological activity determined in step (iii) is cytokine secretion, cell proliferation, or both.

[0336] Biological activity can be measured by methods well known to those skilled in the art, for example by in vitro and / or ex vivo methods.

[0337] The secretion of any cytokine can be measured, for example, the secretion of IFNγ, TNFα can be determined. Standard methods for determining cytokine secretion include ELISA and flow cytometry. These methods are described, for example, by Sachdeva et al. (Front Biosci, 2007, 12:4682-95) and Pike et al. (2016) (Methods in Molecular Biology, Vol. 1458, Humana Press, New York, NY).

[0338] The level of cytokine secretion can be measured, for example, as the maximum level of cytokine (e.g., IFNγ) secreted per CAR-expressing immune cell (e.g., CAR-T cell), e.g., the maximum amount of IFNγ secreted per CAR-T cell.

[0339] Standard methods can be used to assess "degranulation," including, for example, CD107a degranulation assays or measurement of secreted granzyme B or perforin (e.g., as described in Lorenzo-Herrero et al., Methods Mol Biol (2019) 1884:119-130; Betts et al., Methods in Cell Biology (2004) 75:497-512).

[0340] To assess "proliferative" activity, standard methods can be performed, mainly based on methods including measurement of DNA synthesis, detection of proliferation-specific markers, measurement of continuous cell division by using cell membrane-binding dyes, measurement of cellular DNA content, and measurement of cellular metabolism.

[0341] In some cases, the methods described herein allow for the production of engineered T cells within a limited time frame of about 15-30 days, e.g., 15-20 days, or 18-20 days, such that the full immunotherapeutic potential of the cells, particularly with regard to their cytotoxic activity, is preserved.

[0342] These cells may be derived from or may be members of a cell population that may be derived from a single donor or patient. In some cases, these cell populations may be expanded in closed culture recipients to comply with best manufacturing practice requirements and frozen prior to infusion into patients, providing an "off-the-shelf" or "ready to use" therapeutic composition.

[0343] In some cases, a significant number of cells can be obtained from the same leukapheresis, which can be important for obtaining a sufficient dose to treat a patient. Although differences can be observed between cell populations derived from various donors, the number of immune cells procured by leukapheresis is generally around 10 PBMC cell counts. 8 ~10 10PBMCs contain several types of cells, such as granulocytes, monocytes, and lymphocytes, of which 30-60% are T cells, which is typically 10 cells from one donor. 8 ~10 9 This corresponds to primary T cells.

[0344] In some cases, the methods described herein generally involve approximately 10 8 more than 10 T cells, more commonly about 10 9 More than 10 T cells, and more commonly about 10 10 More than 10 T cells, usually 10 11 This typically results in a population of engineered cells amounting to more than 100 T cells. In some cases, the T cells are gene edited at at least two different loci.

[0345] Thus, such compositions of engineered cells or populations of engineered cells can be used as therapeutics, particularly to treat any of the cancers herein, for example, for the treatment of solid tumors in a patient, such as melanoma, neuroblastoma, glioma, or carcinomas such as lung, breast, colon, prostate, or ovarian tumors, in a patient in need of such treatment.

[0346] Also encompassed herein are therapeutically effective immune cell populations that comprise at least 30%, at least 50%, or at least 80% of the engineered cells described herein.

[0347] This document discloses a population of primary TCR-negative immune cells, e.g., T cells, derived from a single donor, wherein at least 20%, at least 30%, at least 50%, at least 90%, at least 95%, at least 96%, or at least 97% of the cells in the population have been genetically modified using sequence-specific reagents to be TCR-negative.

[0348] "TCR-negative immune cells" refer to immune cells, such as T cells or NK cells, in which TCR expression is undetectable by standard antibody-based methods, such as flow cytometry, Western blot, or ELISA. TCR-negative immune cells include immune cells in which the two endogenous alleles encoding components of the T cell receptor have been genetically modified (e.g., disrupted) such that the presence of a TCR on the cell surface of the engineered cell is reduced and / or undetectable. TCR-negative immune cells also include immune cells that do not normally express TCR genes in their natural, unengineered state, such as NK cells.

[0349] "CD52-negative immune cells" refer to immune cells, such as T cells or NK cells, in which CD52 expression is undetectable by standard antibody-based methods such as flow cytometry, Western blot, ELISA, etc. CD52-negative immune cells include immune cells in which the two endogenous alleles encoding CD52 have been genetically modified (e.g., disrupted) resulting in reduced and / or undetectable presence of CD52 on the cell surface of the engineered cells.

[0350] "B2M-negative immune cells" refer to immune cells, such as T cells or NK cells, in which β2M expression is undetectable by standard antibody-based methods such as flow cytometry, Western blot, ELISA, etc. B2M-negative immune cells include immune cells in which two endogenous alleles encoding β2M have been genetically modified (e.g., disrupted) resulting in reduced and / or undetectable presence of β2M on the cell surface of the engineered cells.

[0351] "PDCD1-negative immune cells" refer to immune cells, such as T cells or NK cells, in which PD1 expression is undetectable by standard antibody-based methods such as flow cytometry, Western blot, ELISA, etc. PDCD1-negative immune cells include immune cells in which the two endogenous alleles encoding PD1 have been genetically modified (e.g., disrupted) resulting in reduced and / or undetectable presence of PD1 on the cell surface of the engineered cells.

[0352] "GM-CSF-negative immune cells" refer to immune cells, such as T cells or NK cells, in which expression of GM-CSF is undetectable by standard antibody-based methods, such as flow cytometry, Western blot, ELISA, etc. GM-CSF-negative immune cells include immune cells in which two endogenous alleles encoding GM-CSF have been genetically modified (e.g., disrupted) resulting in reduced and / or undetectable presence of GM-CSF on the cell surface of the engineered cells.

[0353] Methods of Treatment and Products for Use in Immunotherapy Certain aspects relate to pharmaceutical compositions comprising a therapeutically effective amount of the immune cells described herein.

[0354] Also described herein are compositions comprising a therapeutically effective amount of the immune cells described herein for use in treating cancer, such as cancers characterized by the presence of FAPs in the tumor microenvironment.

[0355] Also contemplated herein are methods of treating cancer, such as cancers characterized by the presence of FAPs in the tumor microenvironment, comprising administering a therapeutically effective amount of the engineered immune cells described herein.

[0356] Cancers that can be treated by the compositions, cells, or methods of treatment described herein are not limited.

[0357] The cancer may be a solid tumor or a hematological cancer.

[0358] The cancer that expresses a solid tumor antigen may be selected from any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, testicular cancer, mesothelioma, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, or liver cancer.

[0359] Examples of such cancers that express solid tumor antigens include breast cancer (e.g., triple-negative breast cancer), pancreatic cancer, and lung cancer (e.g., malignant pleural mesothelioma).

[0360] The hematological cancer characterized by the presence of a FAP in the tumor microenvironment may be selected from the group consisting of myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

[0361] All of the above cancers may be treated with the engineered immune cells or pharmaceutical compositions described herein.

[0362] Cancers that are advantageously treated by the engineered immune cells or pharmaceutical compositions described herein are those in which the targeted tumor antigen is also present in normal, healthy tissue.

[0363] In some cases, the cancer is ovarian cancer and the tumor antigen is selected from one or more of mesothelin, glycoprotein 72 (TAG72), MUC16, Her2, 5T4, and FRα.

[0364] In some cases, the cancer is breast cancer and the tumor antigen is selected from one or more of MUC28z, NKG2D, HRG1β, and HER2.

[0365] In some cases, the cancer is prostate cancer and the tumor antigen is selected from one or more of prostate stem cell antigen (PSCA) and prostate-specific membrane antigen (PSMA).

[0366] In some cases, the cancer is renal cancer and the tumor antigen is carboxyanhydrase-IX (CA-IX).

[0367] In some cases, the cancer is gastric cancer and the tumor antigen is selected from one or more of Trop2, claudin 18.2, NKG2D, folate receptor 1 (FOLR1), and HER2.

[0368] In some cases, the cancer is pancreatic cancer and the tumor antigen is selected from one or more of mesothelin, MUC1, CXCR2, B7-H3, CD133, CD24, PSCA, CEA, and Her-2.

[0369] In some cases, the cancer is lung cancer and the tumor antigen is selected from one or more of mesothelin, receptor tyrosine kinase-like orphan receptor 1-specific (ROR1), EGFRvIII, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), PSCA, MUC1, and DLL3.

[0370] In some cases, the cancer is liver cancer and the tumor antigen is selected from one or more of MUC1, CEA, glypican-3, and epithelial cell adhesion molecule (EPCAM).

[0371] In some cases, the cancer is colorectal cancer and the tumor antigen is selected from one or more of MUC1, NKG2D, CD133, GUCY2C (guanylate cyclase 2C), TAG-72 doublecortin-like kinase 1 (DCLK1), and CEA.

[0372] In some cases, the hematological cancer is myelofibrosis and the tumor antigen is CALR.

[0373] In some cases, the hematological cancer is myelodysplastic syndrome and the tumor antigen is selected from one or more of CD123, CD33, and NKG2D.

[0374] In some cases, the hematological cancer is acute myeloid leukemia and the tumor antigen is selected from one or more of CD123, CLL-1, IL1RAP, CD33, CD135, CD70, CD44, CD276, ILT3, CD7, CD47, TIM3, CD96, and VISTA.

[0375] In some cases, the hematological cancer is acute lymphocytic leukemia and the tumor antigen is selected from one or more of CD19, CD22, CD79a, CD10, CD2, CD3, CD4, CD5, CD7, CD8, CRLF2, and CD38.

[0376] In some cases, the hematological cancer is non-Hodgkin's lymphoma and the tumor antigen is selected from one or more of CD19, CD20, CD22, CD80, CD37, CD79, CD30, CD70, and CD38.

[0377] In some cases, the hematological cancer is multiple myeloma and the tumor antigen is selected from one or more of BCMA, CD19, CD138, CS1, CD38, TACI, APRIL, GPRC5D, and CD44v6.

[0378] Treatment using the engineered primary immune cells described herein can be ameliorative, curative, or preventative.

[0379] In some cases, patients may undergo preliminary lymphodepletion (temporary depletion of the immune system) prior to administration of the engineered T cells. In some cases, lymphodepletion results in only partial depletion of the patient's immune system, but not complete depletion. In some cases, the combination of IL-2 treatment with preliminary lymphodepletion may enhance the persistence of the cellular therapy.

[0380] In some cases, the engineered immune cells, such as T cells, described herein are administered in a dose of about 10 cells, with or without a course of lymphodepletion, e.g., by administering cyclophosphamide and / or fludarabine, and / or alemtuzumab. 6 ~109 It may be administered in an amount of 1 / kg.

[0381] In some cases, the cells or cell populations comprising engineered immune cells, such as T cells, described herein are at a concentration of about 10 cells per kg of body weight. 4 ~10 9 pieces, about 10 cells 5 ~5×10 6 per kg body weight, or approximately 10 cells 5 ~10 6 The CAR-T cell therapy dosage is, for example, 10 cells / kg body weight (including all integer values ​​of the number of cells within these ranges). 5 Pieces or 10 6 ~10 9 The method may include administration of 100 mg / kg of leukemia-causing agent with or without a course of lymphodepletion, for example with fludarabine, cyclophosphamide, or alemtuzumab, or any combination thereof.

[0382] The cells or cell populations may be administered in one or more doses. In some cases, an effective amount of cells is administered as a single dose. In some cases, an effective amount of cells is administered as multiple doses over a period of time. The timing of administration is within the discretion of the attending physician and is determined by the patient's clinical condition. The cells or cell populations may be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of effective amounts of a particular cell type for a particular disease or condition is within the skill of one of ordinary skill in the art.

[0383] An effective amount of engineered immune cells, such as CAR-T cells, means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0384] The engineered immune cell treatment described herein may further be performed in combination with one or more therapies for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0385] For example, treatment with the engineered immune cells described herein may be performed in combination with the administration of an immune checkpoint antagonist, which may be administered intravenously in an amount of about 200 mg to 400 mg (including all integer values ​​within these ranges).

[0386] What is described herein about engineered T cells that contain an inactivated TCR, constitutively express a tumor-CAR, and express a fusion protein comprising a FAP-binding domain and a stimulatory cytokine upon activation of the T cell can be equally applied to engineered natural killer cells that constitutively express a tumor-CAR and express a fusion protein comprising a FAP-binding domain and a stimulatory cytokine upon activation of the natural killer cells.

[0387] Such engineered NK cells are necessarily TCR-negative. The NK cells described herein can be derived from a donor or from a cell line, such as the NK92 cell line. In some cases, the engineered NK cells are derived from engineered iPSCs described herein that have been differentiated into NK cells.

[0388] Optionally, the engineered NK cells have reduced expression of the β2M gene mediated by gene inactivation, and / or gene silencing, and / or insertion of at least one exogenous polynucleotide encoding a CAR as defined herein into the β2M locus of the genome of the NK cells.

[0389] The engineered NK cells can have reduced expression of the CD52 gene mediated by gene inactivation, and / or gene silencing, and / or insertion of at least one exogenous polynucleotide encoding a CAR as defined herein into the CD52 locus of the genome of the NK cells.

[0390] In some cases, the engineered NK cells contain either an inactivated CD52 gene or an inactivated β2M gene.

[0391] therefore, a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; b) an exogenous nucleic acid sequence encoding a secretable fusion protein comprising (i) a fibroblast activation protein (FAP) binding domain (e.g., a FAPscFv) comprising VH and VL amino acid sequences derived from a monoclonal anti-fibroblast activation protein (FAP) antibody, and (ii) a stimulatory cytokine, wherein the fusion protein optionally does not comprise an antibody crystallizable fragment (Fc); 1. An engineered NK cell comprising: the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is placed under the transcriptional control of an inducible promoter of the endogenous inducible locus; expression of the fusion protein is inducible upon activation of the NK cell; Also provided herein are engineered NK cells, optionally wherein the NK cells have been genetically modified to suppress or inhibit expression of at least one gene that controls MHC complex surface presentation, such as B2M or CIITA, in the NK cells.

[0392] Tumor-CARs and fusion proteins similar to those described herein can be expressed in the NK cells to produce engineered tumor-CAR / fusion protein-expressing NK cells, which can be used in methods of treating cancers characterized by the presence of FAPs in the tumor microenvironment, e.g., solid tumors and hematological cancers, as described herein.

[0393] Thus, also described herein are pharmaceutical compositions comprising engineered NK cells comprising (i) an exogenous nucleic acid sequence encoding a tumor-CAR placed under the transcriptional control of an exogenous or endogenous constitutive promoter, (ii) an exogenous nucleic acid sequence encoding a fusion protein comprising a FAP-binding domain and a stimulatory cytokine placed under the transcriptional control of an endogenous inducible promoter, and optionally (iii) an inactivated β2M gene. Optionally, the fusion protein that can be expressed and secreted by the engineered NK cells does not comprise an antibody crystallizable fragment (Fc).

[0394] Yet another embodiment described herein is a pharmaceutical composition as described above for use in the treatment of cancers characterized by the presence of FAPs in the tumor microenvironment, such as solid tumors and hematological cancers, wherein said exogenous nucleic acid sequences a) and b) are integrated into the genome of a cell, and expression of the fusion protein is inducible upon activation of NK cells.

[0395] The foregoing description is intended to enable those skilled in the art to make and use the invention by providing means and processes for making and using the invention, this enablement being provided with particular reference to the subject matter of the appended claims, which form a part of the original description.

[0396] Where a numerical limit or range is stated herein, the endpoints are included, and all values ​​and subranges within the numerical limit or range are specifically included as if they were expressly written out.

[0397] Having generally described the invention, a further understanding can be obtained by reference to certain specific examples, which are presented herein for illustrative purposes only and are not intended to limit the scope of the invention as claimed. [Example]

[0398] Example 1: Materials TALE nucleases targeting TRAC and PDCD1 TALEN-mRNAs targeting TRAC (SEQ ID NO: 66 and SEQ ID NO: 67) and TALEN-mRNAs targeting PDCD1 (SEQ ID NO: 64 and SEQ ID NO: 65) were produced by Trilink.

[0399] AAV constructs The Meso-CAR construct was inserted into an AAV vector in frame with the TRAC locus and peptide 2A (SEQ ID NO: 103). The TRAC-FAP-CAR donor construct consists of 300 bp TRAC left and right homology arms and the self-cleaving 2A peptide, which allows expression of Meso-CAR with amino acid sequence SEQ ID NO: 62.

[0400] The FAPscFv-IL2v construct was inserted into an AAV vector in frame with the PDCD1 locus and peptide 2A (SEQ ID NO: 104). The PDCD1-FAPscFv-IL2v donor construct consists of 300-bp PDCD1 left and right homology arms and a self-cleaving 2A peptide that allows expression of FAPscFv-IL2v fused to a C-terminal (His)6 tag. Furthermore, FAPscFv-IL2v is followed by another 2A peptide and the truncated surface protein DLNGFR. DLNGFR expression is used as a reporter for matrix insertion at the PDCD1 locus. This construct also contains a nucleic acid encoding an IL2 signal sequence (SEQ ID NO: 90) that directs secretion of the fusion protein outside the cell.

[0401] AAV vectors were produced by Vigene.

[0402] rLV constructs A recombinant lentiviral vector containing the Meso-CAR coding sequence (SEQ ID NO: 100) under the control of the EF1A promoter (SEQ ID NO: 105) was used to randomly insert the Meso-CAR expression cassette into the genome. Lentiviral particles were produced by Flash Therapeutics.

[0403] Example 2: Generation of CAR-T cells with constitutive expression of Meso-CAR and inducible expression of FAPscFv-IL2v protein by AAV transduction This example describes the generation of universal CAR-T cells with constitutive expression of Meso-CAR and inducible expression of FAPscFv-IL2v protein. The Meso-CAR construct was inserted into the endogenous TRAC locus, while the FAPscFv-IL2v construct was inserted into the endogenous PDCD1 locus. Expression of Meso-CAR and FAPscFv-IL2v was driven by the endogenous TRAC promoter and PDCD1 promoter, respectively (Figure 2A, B, and C).

[0404] Engineered CAR-T cells Cryopreserved PBMCs were thawed at 37°C, washed, resuspended in OpTmizer medium supplemented with 5% AB human serum, and incubated overnight at 37°C in a 5% CO2 incubator. Cells were then activated with Transact in OpTmizer medium (culture medium) supplemented with 5% AB human serum and recombinant human interleukin-2 (rhIL-2, 350 IU / mL) in a CO2 incubator. Three days after activation, T cells were electroporated with 5 μg each of Talen® arm mRNA specific for TRAC (SEQ ID NOs: 66 and 67) or Talen® arm mRNA specific for PDCD1 (SEQ ID NOs: 64 and 65). Transfection was performed in a 0.4 cm gap cuvette using the Pulse Agile technique in Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts) by applying two 0.1 mS pulses at 800 V followed by four 0.2 mS pulses at 130 V. Electroporated cells were then immediately transferred to prewarmed Optmizer serum-free medium and incubated at 37°C for 15 minutes. Cells were then concentrated and incubated in the presence of TRAC-Meso-CAR AAV particles (MOI = 1.1E5 vg / cell) and PDCD1-FAPscFv-IL2v AAV particles (MOI = 1.1E5 vg / cell) containing the donor constructs shown in Figure 2B and C, respectively. After 2 hours of incubation at 30°C, OpTmizer medium supplemented with 10% AB serum and IL-2 was added to the cell suspension, and the mixture was incubated for 16 hours under the same incubation conditions. The cells were then cultured at 37°C in the presence of 5% CO2. The cells were then cultured at 37°C in the presence of 5% CO2 and analyzed for TRAC, PD1, FAP-CAR, and MESO-CAR expression. After 24 hours of activation with PMA (20 μM) / ionomycin (800 ng / ml), the cells were analyzed by flow cytometry for TRAC knockout, Meso-CAR expression (Figure 3A), and PDCD1 knockout (Figure 3B).More than 90% TRAC knockout was achieved, with approximately 30% of the edited T cells expressing Meso-CAR (Figure 3A). Furthermore, PDCD1-positive cells were reduced from 70% (in mock controls) to 15-20% (Figure 3B). To determine the expression and secretion of FAPscFv-IL2v upon MesoCAR activation, cells were incubated for 48 hours on mesothelin protein (2 μg / ml; Lake Pharma)-coated 24-well plates. Secreted FAPscFv-IL2v protein in the cell culture supernatant was detected using a His-tag ELISA detection kit (Genscript, catalog no. L00436). FAPscFv-IL2v protein expressed TRAC. MesoCAR PDCD1 FAPscFv-IL2v It was detected in the cell supernatant at a concentration of 18 ng / mL (Fig. 3C).

[0405] Example 3: Improved cytotoxic activity of CAR-T cells with constitutive expression of MESO-CAR and inducible secretion of FAPscFv-IL2v in vivo To demonstrate the increased antitumor activity of CAR-T upon FAPscFv-IL2v expression in vivo using this strategy, 8-week-old female NSG mice were inoculated with 3 × 10 6 3 x 10 cells mixed with human triple-negative breast tumor-derived cancer-associated fibroblasts 6 Human triple-negative breast cancer cell line HCC70-NanoLuc-GFP was orthotopically implanted. 24 days after tumor implantation, tumor-bearing mice were injected with 5 × 10 6 moc-transfected T cells, TRAC MesoCAR PDCD1 KO T cells, TRAC MesoCAR PDCD1 FAPscFv-IL2v T cells, or TRAC KO PDCD1 FAPscFv-IL2v T cells were injected intravenously. Mice were monitored for tumor growth for 3 weeks after CAR-T injection (Figure 4A). MesoCAR Inducible expression of FAPscFv-IL2v in T cells was observed in TRAC MesoCARCompared with T cells alone, PDCD1 significantly reduced tumor growth (Fig. 4B). FAPscFv-IL2v Insert TRAC MesoCAR There was no effect on tumor progression in the absence of α-glucan, demonstrating the stringency of the induction (Fig. 4B).

[0406] Overall, our results demonstrate the clear advantages of combining FAPscFv-IL2v immune stimulation with antitumor CAR activity to achieve maximal tumor regression.

[0407] Example 4: Generation of CAR-T cells with constitutive expression of Meso-CAR by lentiviral transduction and inducible expression of FAPscFv-IL2v by AAV-mediated targeted integration T cells were electroporated with TALENs to knock out the TRAC and PDCD1 genes, transduced with lentivirus to constitutively express a CAR directed against the mesothelin protein, and with AAV for targeted integration of FAPscFv-IL2v at the PDCD1 locus.

[0408] To express Meso-CAR on the surface of primary T cells, cryopreserved PBMCs were thawed at 37°C, washed, resuspended in OpTmizer medium supplemented with 5% AB human serum, and incubated overnight at 37°C in a 5% CO2 incubator. Cells were then activated with Transfectant in OpTmizer medium (culture medium) supplemented with 5% AB human serum and recombinant human interleukin-2 (rhIL-2, 350 IU / ml) in a CO2 incubator. On the same day as activation, T cells were transduced at an MOI of 10 with lentiviral particles containing the anti-mesothelin CAR coding sequence (SEQ ID NO: 100) expressed under the control of the EF1A promoter.

[0409] Four days after transduction, Meso-CAR-T cells were electroporated with 5 μg each of Talen® arm mRNA specific for TRAC (SEQ ID NO: 66 and SEQ ID NO: 67) or Talen® arm mRNA specific for PDCD1 (SEQ ID NO: 64 and SEQ ID NO: 65). Transfection was performed in a 0.4 cm gap cuvette in cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts) using Pulse Agile technology by applying two 0.1 mS pulses at 800 V followed by four 0.2 mS pulses at 130 V. Electroporated cells were then immediately transferred to prewarmed Optmizer serum-free medium and incubated at 37°C for 15 minutes. Cells were then concentrated and incubated in the presence of PDCD1-FAPscFv-IL2v AAV particles (MOI = 1.1E5 vg / cell) containing the donor construct shown in Figure 2C. After 2 hours of incubation at 30°C, OpTmizer medium supplemented with 10% AB serum and IL-2 was added to the cell suspension, and the mixture was incubated under the same conditions for 16 hours. Then, the cells were cultured at 37°C in the presence of 5% CO2.

[0410] These cells were analyzed by flow cytometry for TRAC knockout and Meso-CAR expression. Greater than 90% TRAC knockout was achieved (Figure 5A), and greater than 30% of edited T cells expressed Meso-CAR (Figure 5B). Furthermore, FAPscFv-IL2v integration at the PDCD1 locus was assessed using digital droplet PCR (ddPCR), and FAPscFv-IL2v expression and secretion upon MesoCAR activation were determined by incubating cells on mesothelin protein (2 μg / ml; Lake Pharma)-coated 24-well plates for 48 hours. Secreted FAPscFv-IL2v protein in cell culture supernatants was detected using a His-tag ELISA detection kit (Genscript, catalog no. L00436). The FAPscFv-IL2v expression matrix was integrated at the PDCD1 locus with an allele frequency of approximately 8% (Figure 5C), and the FAPscFv-IL2v protein was expressed in the rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v It was detected only in the cell supernatant at a concentration of 3–4 ng / mL ( Fig. 5D ).

[0411] Example 5: Evaluation of binding of FAPscFv-IL2v to FAP-positive tumor cells To confirm the binding and activity of the FAPscFv-IL2v immunocytokine, we generated a human FAP-expressing cell line by transducing mesothelioma tumor cells NCI-H226 (ATCC, CRL5826), which express GFP and luciferase reporter genes, with lentiviral particles containing the human FAP gene expressed under the control of a CMV promoter. The NCI-H226-FAP cell line expressed human FAP in more than 40% of the cell population as assessed by flow cytometry (Figure 6A), recapitulating the heterogeneity of FAP expression in solid tumors. This cell line was further used to determine the FAP-binding ability of FAPscFv-IL2v. (His)6-tagged recombinant FAPscFv-IL2v protein synthesized by Lake Pharma was incubated with NCI-H226-FAP or control NCI-H226 cells at different concentrations. The fraction of cells that bound FAPscFv-IL2v was then detected using an APC fluorescent-conjugated anti-His antibody and subsequently analyzed by flow cytometry.

[0412] As shown in Figure 6B, FAPscFv-IL2v bound to human FAP-expressing NCI-H226 cells in a dose-dependent manner, with saturation reached at 100 ng / ml. This result confirms that the recombinant FAPscFv-IL2v fusion protein can successfully bind to human FAP protein in a specific manner.

[0413] Example 6. Antitumor cytotoxic activity of CAR-T cells with constitutive expression of Meso-CAR and inducible expression of FAPscFv-IL2v This example demonstrates that combining constitutive expression of Meso-CAR with inducible expression of the immunocytokine FAPscFv-IL2v enhances specific killing of tumor cells. The engineered T cells generated in Example 5 were used.

[0414] Sequential killing assay procedure To assess the antitumor activity of the engineered T cells, a sequential killing assay was performed as outlined in Figure 7A. The adherent tumor cell lines NCI-H226 or NCI-H226-FAP, expressing the reporter genes GFP and luciferase, were cultured at 5 × 10 per well in a total volume of 0.5 ml of DMEM medium supplemented with 10% FBS. 4 Cells were plated at a concentration of 0 cells per 100 cells (day 0). After 24 hours, TRAC KO PDCD1 KO ,TRAC KO PDCD1 FAPscFv-IL2v , rLv-MesoCAR;TRAC KO PDCD1 KO , or rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v engineered T cells to tumor cells in a 2:1 ratio with effector CAR + T cells were added at a T cell:tumor cell ratio. The mixture was incubated for 24 hours in an Incucyte ZOOM live cell analyzer, which measured live tumor cell GFP signals every 2 hours. Subsequently, suspended T cells were collected from the well supernatant, spun down, resuspended in 0.1 ml DM1M + 10% FBS medium, and added to a fresh well of tumor cells plated as described above. Simultaneously, T cell cytotoxicity during the first 24 hours was determined by measuring the luciferase activity of surviving tumor cells in the day 0 plate. This protocol was repeated for up to 5 days.

[0415] rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v Cytotoxic activity of T cells against NCI-H226 and NCI-H226-FAP tumor cells The highest antitumor cytotoxicity upon repeated challenge of engineered T cells with tumor cells was observed with rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v This was observed for T cells, which secreted FAPscFv-IL2v immunocytokines upon tumor cell exposure (Figure 7B). KO PDCD1 KOCompared with T cells, rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v Higher antitumor cytotoxicity of T cells was observed as early as 24 hours after tumor cell administration and persisted throughout the course of the assay, in contrast to rLv-MesoCAR;TRAC KO PDCD1 KO T cells exhibited lower cytotoxicity and ultimately suboptimal tumor cell clearance.

[0416] Furthermore, rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v The antitumor cytotoxicity of FAPscFv-IL2v was further enhanced when the target NCI-H266 expressed human FAP (NCI-H226-FAP), demonstrating the superiority of FAP anchoring for mediating immune cell stimulation (Figure 7B).

[0417] Because the maximum cytotoxic activity was recorded between 48 and 72 hours, we analyzed the killing kinetics of the engineered T cells during this period by assessing the time course of GFP signal changes as measured by Incucyte ZOOM. As shown in Figure 7C and Figure 7D, FAPscFv-IL2v-expressing rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v T cells specifically target NCI-H226-FAP tumor cells, whereas control rLv-MesoCAR;TRAC KO PCDC1 KO rLv-MesoCAR;TRAC induced faster tumor cell clearance than T cells (Figure 7D). This was due to the fact that rLv-MesoCAR;TRAC induced faster tumor cell clearance than T cells during the course of the assay as determined by ELISA. KO PDCD1 FAPscFv-IL2v This was further confirmed by increased and sustained IFNg secretion by T cells (Fig. 7E).

[0418] Overall, our results demonstrate a clear functional advantage of FAPscFv-IL2v secretion compared to MesoCAR T cells alone for boosting antitumor cytotoxicity and sustained activity of MesoCAR T cells, especially when FAP is expressed in the microenvironment.

[0419] Example 7. In vivo safety measurements To demonstrate the increased antitumor activity and reduced systemic toxicity of this strategy compared with IL-2 cytokine treatment in vivo, 8-week-old female NSG mice were treated with 3 × 10 6 3 x 10 cells mixed with human triple-negative breast tumor-derived cancer-associated fibroblasts 6 Human triple-negative breast cancer cell line HCC70-NanoLuc-GFP were orthotopically implanted in the left inguinal mammary fat pad. Twenty-one days after tumor implantation, tumor-bearing mice were treated with 8 × 10 6 Cars + rLv-MesoCAR;TRAC KO PDCD1 KO or rLv-MesoCAR;TRAC KO PDCD1 FAPscFv-IL2v of engineered T cells will be injected intravenously (IV). As a control, 8 x 10 6 TRAC KO PDCD1 KO or TRAC KO PDCD1 FAPscFv-IL2v T cells will be injected in two additional cohorts. For systemic toxicity comparison, one tumor-bearing mouse cohort will be injected with 8 x 10 T cells. 6 Cars + rLv-MesoCAR;TRAC KO PDCD1 KO T cells are injected IV and recombinant human IL-2 (0.4 mg / kg qdX3 by intraperitoneal injection ip, 3 cycles every 9 days). Mouse weights are closely monitored throughout the course of the study. Tumor volumes are measured weekly, and 40 days after the start of treatment, animals are euthanized, lungs and livers are removed, and analyzed for vascular leak syndrome (VLS) by Evans blue dye staining.

[0420] Administration of recombinant IL-2 boosts CAR-T activity but also results in systemic toxicity, as evidenced by rapid weight loss and VLS in peripheral organs. KO PDCD1 FAPscFv-IL2v Two levels of control were programmed in this system: (1) tumor site-specific production of FAPscFv-IL2v and (2) rLv-MesoCAR;TRAC. KO PDCD1 FAPscFv-IL2v The FAP anchoring of FAPscFv-IL2v prevents systemic accumulation of FAP, boosting tumor clearance without inducing systemic toxicity.

[0421] Example 8: Identification of several promoters inducible upon CAR-T cell activation Anti-CS1-CART cells were produced using an 18-day process briefly described below.

[0422] Human PBMCs were thawed and activated using TransAct beads. Three days later, cells were electroporated with mRNA encoding TRAC-specific TALE nucleases (SEQ ID NO: 66 and SEQ ID NO: 67) and CS1-specific TALE nucleases (SEQ ID NO: 74 and SEQ ID NO: 75). Two days later, cells were transduced with a lentiviral vector driving the expression of a CS1-specific second-generation CAR (SEQ ID NO: 88), followed by an in vitro expansion step and magnetic depletion of remaining alpha / beta TCR-positive cells. At the end of the production process, CAR-T cells were filled into vials and cryopreserved.

[0423] A portion of the CS1-CAR-T cells was thawed and CAR+ T cells were sorted by flow-activated cell sorting (FACS) using CAR-specific reagents (the "deactivated cell sample").

[0424] In parallel, another portion of CS1-CAR-T cells was thawed and activated using plate-bound CS1 recombinant protein (SEQ ID NO: 89). 24 hours after activation, CAR+ T cells were sorted by FACS ("activated cell sample").

[0425] Inactivated and activated samples from two independent donors were analyzed by RNA-seq.

[0426] To identify genes induced by activation, The maximum expression level at time 0 is less than 100 TPM (transcripts per million kilobases), The minimum expression level at 24 hours is greater than 50 TPM, and A fold change between the mean expression at 0 hours and the mean expression at 24 hours of greater than 5 Genes that met these criteria were selected.

[0427] These criteria led to the identification of 159 genes (shown in Figure 8). From this literature-based list, we identified genes that adversely affect T cell proliferation, tumor invasion, or function, some of which are shown in Table 7.

[0428] [Table 7]

Claims

1. a) an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen; and b) an exogenous nucleic acid sequence encoding a secretable fusion protein comprising a fibroblast activation protein (FAP) binding domain and a stimulatory cytokine; 1. An engineered immune cell comprising: the exogenous nucleic acid sequence of a) is integrated into the genome of the cell and is under the transcriptional control of a constitutive promoter; the exogenous nucleic acid sequence of b) is integrated into the genome of the cell at an endogenous inducible locus and is under the transcriptional control of a promoter of the endogenous inducible locus (an "inducible promoter"); An engineered immune cell, wherein said inducible promoter is inducible upon activation of said immune cell.

2. The immune cell of claim 1 , wherein the FAP-binding domain comprises VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody.

3. The immune cell of claim 1 or 2, wherein the fusion protein does not contain an antibody crystallizable fragment (Fc).

4. The immune cell of claim 1 or 3, wherein the tumor antigen targeted by the tumor-CAR is not a fibroblast-associated protein.

5. The immune cell of any one of claims 1 to 4, wherein the constitutive promoter is an exogenous polynucleotide.

6. The immune cell of any one of claims 1 to 5, wherein the constitutive promoter is selected from the group consisting of EF1A promoter, CD52 promoter, GAPDH promoter, CMV promoter, hPGK promoter, UBC promoter, SV40 promoter, PGK promoter, CAGG promoter, TRAC promoter, TRBC promoter, TRGC promoter, TRDC promoter, B2M promoter, CD5 promoter, CS1 promoter, CD45 promoter, RPBSA promoter, CD4 promoter, and CD8 promoter.

7. The immune cell of any one of claims 1 to 6, wherein the inducible promoter is selected from the group consisting of PDCD1 promoter, CD25 promoter, TIM3 promoter, TIGIT promoter, CCL1 promoter, NR4A3 promoter, EGR3 promoter, G0S2 promoter, IL22 promoter, RGS16 promoter, FASLG promoter, RDH10 promoter, CSF1 promoter, GM-CSF promoter, LAG3 promoter, CTLA-4 promoter, IL10 promoter, NUR77 promoter, and FOXP3 promoter.

8. The immune cell of any one of claims 1 to 7, wherein the constitutive promoter is a promoter of EF1A, TRAC, B2M, CD52, CS1, CD45, CD5, or GAPDH, and the inducible promoter is a promoter of PDCD1, CD25, GM-CSF, TIM3, TIGIT, or CTLA4.

9. The immune cell of any one of claims 1 to 8, wherein the constitutive promoter is an EF1A promoter, a TRAC promoter, a B2M promoter, or a CD52 promoter, and the inducible promoter is a PDCD1 promoter or a GM-CSF promoter.

10. The immune cell according to any one of claims 1 to 9, which is a primary immune cell.

11. The immune cell of claim 10, wherein the primary immune cell is a macrophage, a natural killer cell, or a T cell.

12. The immune cell of claim 11 , wherein the T cell is a cytotoxic T lymphocyte or a helper T lymphocyte.

13. The immune cell of claim 11 , wherein the T cell is a cytotoxic T lymphocyte.

14. The immune cell according to any one of claims 10 to 13, wherein the primary immune cell is derived from a human.

15. The immune cell of any one of claims 1 to 14, which has been genetically modified to suppress or inhibit expression of at least one component of the T cell receptor (TCR).

16. The immune cell of claim 15, which has been genetically modified to suppress or inhibit expression of the TCRα gene.

17. The immune cell of claim 15, which has been genetically modified to suppress or inhibit expression of the TCRβ gene.

18. The immune cell of claim 15, which has been genetically modified to suppress or inhibit expression of the TCRα gene and the TCRβ gene.

19. The immune cell according to any one of claims 1 to 18, which has been genetically modified to suppress or inhibit the expression of at least one gene encoding an MHC-I protein selected from β2m and HLA.

20. The immune cell according to any one of claims 1 to 19, which has been genetically modified to suppress or inhibit the expression of a gene encoding an immune checkpoint protein and / or its receptor.

21. 21. The immune cell of any one of claims 1 to 20, which has been genetically modified to confer resistance to at least one immunosuppressive or chemotherapeutic agent, and optionally to contain a suicide gene.

22. The immune cell of any one of claims 1 to 21, which is one or more of TCR-negative, B2M-negative, PDCD1-negative, and CD52-negative.

23. The immune cell according to any one of claims 1 to 22, which is at least TCR negative.

24. The immune cell of any one of claims 1 to 23, which is at least TCR-negative and PDCD1-negative, or at least TCR-negative and B2M-negative.

25. The immune cell of any one of claims 1 to 24, wherein the tumor antigen targeted by the tumor-CAR is an antigen present in a solid tumor or a hematological cancer, and the tumor or cancer is characterized by the presence of FAP in the microenvironment of the tumor or cancer.

26. The immune cell of any one of claims 1 to 25, wherein the tumor antigen targeted by the tumor-CAR is an antigen present in both solid tumors and some normal healthy tissues.

27. The tumor antigen is selected from the group consisting of CEA, ERBB2, EGFR, GD2, mesothelin, MUC1, PSMA, GD2, PSMA1, LAP3, ANXA3, TAG72, MUC16, 5T4, FRα, MUC28z, NKG2D, HRG1β, PSCA, PSMA, CA-IX, Trop2, claudin 18.2, FOLR1, CXCR2, B7-H3, CD133, CD24, ROR1, EGFR, and EGFR.

27. The immune cell of claim 25 or 26, wherein the immune cell is selected from the group consisting of: VEGF, EphA2, DLL3, glypican-3, EpCAM, GUCY2C, DCLK1, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Ley, Lex, Leb, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, and ErbB3.

28. The immune cell of any one of claims 1 to 27, wherein the tumor antigen targeted by the tumor-CAR is selected from the group consisting of mesothelin, MUC1, Trop2, EGFR, and VEGF.

29. The immune cell of any one of claims 1 to 28, wherein the tumor antigen targeted by the tumor-CAR is mesothelin or MUC1.

30. the tumor-CAR is (a) an extracellular tumor antigen binding domain, such as an extracellular tumor antigen binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-tumor antigen antibody, wherein the tumor antigen is as defined in any one of claims 4 and 25-29; and (b) a hinge selected from an FcγRIII hinge, a CD8α hinge, and an IgG1 hinge; (c) a transmembrane domain comprising a CD8α transmembrane domain or a CD28 transmembrane domain; (d) a cytoplasmic domain comprising (i) a CD3 zeta signaling domain, (ii) and a costimulatory domain derived from 4-1BB or CD28; The immune cell of any one of claims 1 to 29, comprising:

31. The immune cell of claim 30, wherein the hinge is a CD8α hinge, the transmembrane domain is a CD8α transmembrane domain, and the cytoplasmic domain comprises a CD3 zeta signaling domain and a costimulatory domain derived from 4-1BB.

32. the tumor-CAR is a) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NOs: 39, 40, and 41, and the L-CDRs of SEQ ID NOs: 42, 43, and 44, and the amino acid sequence set forth in SEQ ID NO: 45; b) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NOs: 47, 48, and 49, and the L-CDRs of SEQ ID NOs: 50, 51, and 52, and the amino acid sequence set forth in SEQ ID NO: 53; c) an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the H-CDRs of SEQ ID NOs: 55, 56, and 57, and the L-CDRs of SEQ ID NOs: 58, 59, and 60, and the amino acid sequence set forth in SEQ ID NO: 61; or d) H-CDR and L-CDR contained in the amino acid sequence of SEQ ID NO: 63, and an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

63. The immune cell of any one of claims 1 to 31, comprising an extracellular tumor antigen-binding domain comprising:

33. the FAP-binding domain (1) A variable heavy chain (VH) comprising H-CDRs of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO: 25; and a variable light chain (VL) comprising L-CDRs of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO: 26; or (2) A variable heavy chain (VH) comprising H-CDRs of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO:7, and a variable light chain (VL) comprising L-CDRs of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO:8; or (3) A variable heavy chain (VH) comprising H-CDRs of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO: 16; and a variable light chain (VL) comprising L-CDRs of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence SEQ ID NO: 17; or (4) A variable heavy chain (VH) comprising H-CDRs of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence SEQ ID NO: 34; and a variable light chain (VL) comprising L-CDRs of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence SEQ ID NO:

35. The immune cell of any one of claims 1 to 32, comprising:

34. The immune cell of any one of claims 1 to 33, wherein the FAP-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, or SEQ ID NO: 36, for example, SEQ ID NO:

27.

35. 35. The immune cell of any one of claims 1 to 34, wherein the stimulatory cytokine is selected from the group consisting of interleukin-2 or a variant thereof such as IL-2v, interleukin-7 or a variant thereof, interleukin-12 or a variant thereof, interleukin-15 or a variant thereof, interleukin-15 in complex with its high affinity receptor IL-15RA, interleukin-18 or a variant thereof, and interleukin-23 or a variant thereof.

36. The immune cell of any one of claims 1 to 35, wherein the stimulatory cytokine is IL-2v having the amino acid sequence shown in SEQ ID NO:

93.

37. A therapeutically effective immune cell population comprising at least 30%, at least 50%, or at least 80% of the cells of any one of claims 1 to 36.

38. A pharmaceutical composition comprising a therapeutically effective amount of the immune cells according to any one of claims 1 to 36.

39. 37. A composition comprising a therapeutically effective amount of the immune cells of any one of claims 1 to 36 for use in the treatment of cancer characterized by the presence of FAP in the tumor microenvironment.

40. 40. The composition for use of claim 39, wherein the cancer is a solid tumor or a blood cancer.

41. 40. The composition for use of claim 39, wherein the cancer is a solid tumor or blood cancer selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, testicular cancer, mesothelioma, thyroid cancer, brain cancer, esophageal cancer, stomach cancer, pancreatic cancer, colorectal cancer, liver cancer, myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

42. 37. A method for treating cancer characterized by the presence of FAP in the tumor microenvironment, comprising administering a therapeutically effective amount of the immune cells of any one of claims 1 to 36.

43. A method for producing a cell population comprising the immune cells according to any one of claims 1 to 36, comprising: (i) providing donor-derived immune cells or induced pluripotent stem cells (iPSCs); (ii) optionally inhibiting or suppressing T cell receptor (TCR) expression in the cell or presentation of a TCR on the cell surface; (iii) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) ("tumor-CAR") that targets a tumor antigen, placed under the transcriptional control of a constitutive promoter; (iv) integrating into the genome of the cell an exogenous nucleic acid sequence encoding a fusion protein comprising a signal peptide, a FAP-binding domain (e.g., a FAP-binding domain comprising VH and VL amino acid sequences derived from a monoclonal anti-FAP antibody), and a stimulatory cytokine, wherein the exogenous nucleic acid sequence is under the transcriptional control of an endogenous inducible promoter, and optionally the fusion protein does not comprise an antibody crystallizable fragment (Fc); (v) optionally isolating the engineered cells that do not express the TCR on their surface; Including, The method, wherein the inducible promoter is inducible upon activation of the engineered immune cell.

44. 44. The method of claim 43, wherein the integration in (iii) is achieved by random integration (e.g., by lentiviral vector integration) or by gene-targeted integration (e.g., by nuclease-mediated cDNA insertion at a single targeted locus) in the genome of the cell.

45. 44. The method of claim 43, wherein the integration in (iv) is carried out by gene-targeted integration in the genome of the cell (e.g., by nuclease-mediated cDNA insertion at a single targeted locus).

46. 46. ​​The method of any one of claims 43 to 45, comprising inactivating at least one of the TRAC locus, the B2M locus, and the CD52 locus in the genome of the cell.

47. The method of any one of claims 43 to 46, wherein the cell in (i) is an immune cell that is a T cell.

48. 47. The method of any one of claims 43 to 46, wherein the cells in (i) are iPSCs, and the method comprises the further step of differentiating the engineered cells obtained after step (iv) or (v) into immune cells.

49. 49. The method of claim 48, wherein said differentiating step differentiates said engineered cells into immune cells that are T cells, NK cells, or macrophages.

50. 49. The method of claim 48, wherein said differentiating step differentiates said engineered cells into immune cells that are T cells.

51. (1) (a) at least one vector comprising a nucleic acid sequence comprising a constitutive promoter and a nucleic acid sequence encoding a tumor-CAR, operably linked to the promoter; or (1)(b) at least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a tumor-CAR, wherein the nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to an endogenous constitutive locus in a cell; and (2) At least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, optionally wherein the fusion protein does not comprise an antibody crystallizable fragment (Fc), and wherein the nucleic acid sequence encoding the fusion protein is positioned between a left homology region and a right homology region, and wherein the left homology region and the right homology region are homologous to an endogenous inducible locus in a cell. A set of vectors comprising: Optionally, the tumor-CAR is as claimed in any one of claims 30 to 32, and the secretable fusion protein comprises a FAP-binding domain according to claim 33 or 34 and a stimulatory cytokine according to claim 35 or 36.

52. (1) (a) at least one vector comprising a nucleic acid sequence comprising a constitutive promoter and a nucleic acid sequence encoding a tumor-CAR operably linked to the promoter; and / or (1)(b)(i) at least one vector comprising an expression cassette comprising a nucleic acid sequence encoding a tumor-CAR, wherein the nucleic acid sequence encoding the tumor-CAR is positioned between a left homology region and a right homology region, and the left homology region and the right homology region are homologous to an endogenous constitutive locus in a cell; and (ii) at least one sequence-specific endonuclease that targets the endogenous constitutive locus; and (2) (i) at least one vector comprising a nucleic acid sequence encoding a secretable fusion protein comprising a FAP-binding domain and a stimulatory cytokine, optionally wherein the fusion protein does not comprise an antibody crystallizable fragment (Fc), and wherein the nucleic acid sequence is positioned between a left homology region and a right homology region, wherein the left homology region and the right homology region are homologous to an endogenous inducible locus in a cell; and (ii) at least one sequence-specific endonuclease that targets the inducible locus. Includes a kit.

53. 53. The kit of claim 52, wherein the tumor-CAR is as claimed in any one of claims 30 to 32, and the fusion protein comprises a FAP-binding domain according to claim 33 or 34 and a stimulatory cytokine according to claim 35 or 36.

54. 54. The kit of claim 52 or 53, wherein the constitutive promoter in (1)(a) is selected from the group consisting of EF1A promoter, CD52 promoter, GAPDH promoter, CMV promoter, hPGK promoter, UBC promoter, SV40 promoter, PGK promoter, CAGG promoter, TRAC promoter, TRBC promoter, TRGC promoter, TRDC promoter, B2M promoter, CD5 promoter, CS1 promoter, CD45 promoter, RPBSA promoter, CD4 promoter, and CD8 promoter; and / or the endogenous constitutive locus in (1)(b) is selected from the group consisting of EF1A, CD52, GAPDH, hPGK, UBC, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, CD4, and CD8 gene loci.

55. 55. The kit of any one of claims 52 to 54, wherein the inducible locus is selected from the group consisting of PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3, EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1, GM-CSF, LAG3, CTLA-4, IL10, NUR77, and FOXP3 gene loci.

Images