Immune cells lacking SOCS1

Abstract

The present invention relates to engineered immune cells that are defective in SOCS1. Preferably, the engineered immune cells further comprise an engineered antigen receptor that specifically binds to a target antigen. The present invention also relates to a method for obtaining engineered immune cells, comprising a step consisting of inhibiting the expression and / or activity of SOCS1 in immune cells; optionally, further comprising a step consisting of introducing into the immune cells an engineered antigen receptor that specifically binds to the target antigen. The present invention also encompasses engineered immune cells for their use in adoptive therapy, particularly for the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to the field of adoption therapy. This invention provides SOCS1-deficient immune cells that have enhanced growth, survival, and functionality in vivo. [Background technology]

[0002] Introduction Adoptive T-cell therapy (ATCT), which includes T cells modified using recombinant T-cell receptors (TCRs), chimeric antigen receptors (CARs), or tumor-infiltrating lymphocytes (TILs), is emerging as a promising cancer therapy.

[0003] In vitro manufacturing processes allow for the genetic reprogramming of heterogeneous mixtures of CD4 and CD8 T cell live drugs, which exhibit complex sensing-response behaviors (Lim and June 2017). While CD8 or CD4 T cells alone can produce considerable therapeutic effects (Freitas and Rocha 2000), co-injection of both subsets is often a critical requirement for optimal and sustained antitumor activity (Linnemann, Schumacher, and Bendle 2011; Sadelain 2015; Borst et al. 2018). CD4 T cells exhibit multifaceted effects and plasticity, and can boost antitumor immune responses through both helper cells (Corthay et al. 2005; Bos and Sherman 2010; Z. Zhu et al. 2015) and cytotoxic functions (Xie et al. 2010; Quezada et al. 2010; Kitano et al. 2013; Sledzinska et al. 2020a).

[0004] However, activated CD4 and CD8 T cells differ in their ability to proliferate and persist in vivo. CD8 T cells undergo broad and autonomous clonal growth, while CD4 T cells require repeated antigen induction, exhibit growth arrest in early division, and result in a growth of approximately 1 / 10 to 1 / 20 (Homann, Teyton, and Oldstone 2001; Foulds et al. 2002; Seder and Ahmed 2003; Ravkov and Williams 2009).

[0005] The differences in the magnitude and duration of CD4 and CD8 T cell proliferation are not attributable to external signals or resource competition (see references above). Instead, by comparing the entry of naive and antigen-experienced (Ag-exp) CD4 T cells into the immune response after antigen stimulation, several studies have reported that Ag-exp CD4 T cells specifically reduce their own proliferation and exhibit decreased IL-2 production (Foulds et al. 2002; Merica et al. 2000; MacLeod, Kappler, and Marrack 2010; Helpt et al. 2008). We have previously developed an in vivo model that reproduces the increasing dysfunction of Ag-exp CD4 T cells during the ongoing immune response. In this physiologically relevant model, generalizable to several CD4 TCR transgenic (Tg) T cells, Ag-exp CD4 T cell proliferation essentially disappears, while naive CD4 T cell proliferation is maintained, demonstrating that the absence of Ag-exp CD4 proliferation is not related to insufficient priming (Helft et al. 2008). The strong inhibition they previously reported is Ag-specific, begins on day 2 (long before Ag disappearance), and is not attributable to exogenous factors such as the lack of regulatory T cells (Tregs) or antigen-presenting cell (APC) education, nor to Ag competition (Helft et al. 2008). Instead, they showed that Ag-exp CD4 T cell proliferation is halted by an intrinsic, active, and dominant phenomenon that cannot be overcome by providing new, Ag-loaded DCs.

[0006] In adoptive T-cell therapy (ATCT), where T cells are activated in vitro before the manipulation process, Ag-exp CD4 T cells can become a limited subset under in vivo recall conditions, impairing an efficient protective immune response (Homann, Teyton, and Oldstone 2001). The underlying molecular mechanisms for this limited increase are unclear, but the low dose of T cells injected into the patient may interfere with the efficacy of ATCT.

[0007] Therefore, there remains a need for engineered immune cells, particularly engineered T cells, that exhibit enhanced growth potential and survival after adoption. There is also still a need for engineered T cells with improved functional efficacy, especially enhanced cytotoxicity, which would support efficient and broad-spectrum cancer treatment.

[0008] Furthermore, the production of autologous T cells is time-consuming, costly, and often inefficient. To ensure the stable establishment of ATC therapy as a drug, well-characterized, stored, and pre-produced therapeutic cells from healthy donors would address these limitations. Therefore, efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system fulfill a critical clinical need.

[0009] Therefore, the inventors are investigating the intrinsic resistance of T cells to host immune elimination with the aim of creating universal cell therapies from healthy donors. Although the use of autologous CAR T cells has yielded outstanding clinical data to date (Neelapu et al. 2017; Maude et al. 2018), it has certain well-known drawbacks.

[0010] Firstly, complex personalized manufacturing reduces their scalability (Graham et al. 2018). In addition, the current manufacturing process takes approximately three weeks (Kohl et al. 2018), which limits their availability, especially for patients with highly proliferative diseases (Depil et al. 2020). Finally, the efficacy of autologous T cells can be negatively affected by immunosuppression resulting from previous therapies or the tumor microenvironment (Thommen and Schumacher 2018).

[0011] Conversely, the use of allogeneic T cell products (HLA mismatched) derived from healthy donors allows for immediate access to standardized batches of T cells, improves their potency (multiple cell modifications, target combinations), and reduces their cost and industrialization process (Lin et al. 2019), but nevertheless it presents two major challenges. First, the transfer of allogeneic T cells can cause graft-versus-host disease (GVHD), a life-threatening disease induced by donor T lymphocytes. One strategy to prevent this is genetic inactivation of the TCRα stationary (TRAC) gene. Second, TCR-negative allogeneic T cells can still be recognized as non-self HLA by the host immune system and rapidly eliminated, which would limit their antitumor activity. In this regard, it has been suggested that lymphocyte depletion using chemotherapy or radiation prior to universal CAR-T cell infusion delays rejection until the recipient's immune system recovers (Gattinoni et al. 2005), but these are accompanied by considerable toxicity and problematic viral reactivation (Chakrabarti, Hale, and Waldmann 2004).

[0012] Since HLA-I molecules are the primary mediators of immune rejection, another proposed strategy has been the genetic disruption of β2-microglobulin, which is essential for the formation of functional HLA class I molecules on the cell surface (Poirot et al. 2015; D. Wang et al. 2015; Torikai et al. 2013). However, these cells can become targets for NK cells, which are sensitive to reduced HLA expression (missing-self mechanism) (Bern et al. 2019). Solutions to block NK-mediated rejection may depend on the overexpression of HLA-E molecules (Gornalusse et al. 2017), which are ligands for the inhibitory complex CD94 / NGK2A (Braud et al. 1998), or HLA-G molecules (Rajagopalan and Long 1999; Pazmany et al. 1996; Gonen-Gross et al. 2010), which are normally expressed by the cytotrophoblast and bind to the inhibitory receptor KIR2DL4 / IT2.

[0013] Finally, the use of inducible pluripotent stems (iPSCs) of low immunogenic cells, combined with CAR technology, may also provide a promising and unrestricted source of lymphocytes independent of antigen specificity and HLA constraints (Themeli et al. 2013). However, challenges remain, particularly regarding differentiation methods that do not currently comply with good manufacturing practice (GMP) standards for pharmaceuticals, as they involve the presence of serum and mouse-derived feeder cells. Furthermore, because the differentiation process involves multiple steps, developmental transitions can occur with varying efficiencies. [Prior art documents] [Patent Documents]

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[0016] Therefore, the production of mature single-positive T cells for clinical application, particularly for allotransfer, remains a challenge (Nianias and Themeli 2019). [Means for solving the problem]

[0017] The inventors have developed a strategy to genetically engineer primary T cells at a genome-wide (GW) level using CRISPR technology. This innovative approach enables rapid, systematic, and unbiased identification of functionally non-redundant, T-cell-specific limiting factors in vivo (13, 14). First, the inventors investigated the specific factors that limit reloaded CD4 T cell enlargement in vivo. Their screening identified cytokine signaling suppressor 1 (SOCS1) as a CD4 limiting factor. + They were identified as non-redundant and unique inhibitors of T cell proliferation and survival. SOCS1 was found to be a CD4 inhibitor. + We demonstrated that it is a critical node for integrating cytokine signals (IFN-γ and IL-2) that actively limit T cell function. The inventors also demonstrated mouse and human CD4 + and CD8 + The function of SOCS1 was investigated in both antitumor adoptive cell therapies. SOCS1 inactivation was associated with CD4 + It promotes T helper-1 (Th1) cell enlargement and restores cytotoxic function, while CD8 + In T cells, this greatly boosted their cytotoxic potential.

[0018] Next, using a similar genome-wide screening strategy in vivo and transferring a pool of genomically mutated Marilyn T from C57BL6 mice (H2-Kb) into fully immunocompetent BALB / c (H2-Kd) MHC-mismatched mice, the inventors re-identified β2m, an empirical target whose decreased expression occurs concomitantly with immune evasion in nature (Lanza, Russell, and Nagy 2019), particularly in cancer (Koopman et al. 2000; He et al. 2017). Further and completely unexpectedly, the inventors identified Fas (CD95, Tnfrsf6), which they now validate, as a major target for improving the survival of allogeneic T cells in vivo. These results represent a major advance for the development of allogeneic (universal) cell-based therapies (such as immune cell therapies) based on allotransplantation.

[0019] The inventors further provide results supporting that the combination of SOCS1 and FAS inactivation provides a universal T cell product with "fratricide / sibling death resistance".

[0020] Therefore, the present invention relates to modified or engineered immune cells, particularly modified T cells, in which SOCS-1 is inactivated. In some embodiments, the immune cells are also defective in FAS and / or Suv39h1.

[0021] Typically, the engineered immune cells of the present application are T cells or NK cells. More particularly, the T cells are CD4+ or CD8+ T cells. Preferred cells can be selected from naive T cells (T N cells), stem memory T cells (TSC M cells), memory T cells (TC M cells), tumor infiltrating lymphocytes (TIL), or effector memory T cells (TE M cells), and combinations thereof.

[0022] Also typically, the engineered immune cells are isolated from a subject. Preferably, the subject has cancer or is at risk of developing cancer.

[0023] The target antigen to which the genetically engineered antigen receptor specifically binds is preferably an antigen expressed on cancer cells and / or a common tumor antigen.

[0024] Genetically modified antigen receptors can be chimeric antigen receptors (CARs) that contain an extracellular antigen-recognition domain that specifically binds to the target antigen. Genetically modified antigen receptors can also be T cell receptors (TCRs).

[0025] Preferably, the activity and / or expression of SOCS-1, and in some embodiments also of FAS and / or Suv39h1, are selectively inhibited or blocked in the engineered immune cells. In one embodiment, the engineered immune cells express SOCS-1, FAS, or Suv39h1 nucleic acids that encode non-functional SOCS-1, FAS, or Suv39h1 proteins, respectively.

[0026] This application also relates to a method for producing genetically modified immune cells, particularly universal immune cells (usable in allogeneic transplantation, especially in allogeneic adoptive cell therapy), comprising the steps of: inhibiting the expression and / or activity of SOCS-1 and / or FAS, and in some embodiments further inhibiting the expression and / or activity of β2m and / or Suvh39h1 in immune cells; and optionally, introducing genetically modified antigen receptors that specifically bind to a target antigen into immune cells.

[0027] In some embodiments, inhibition of SOCS-1, FAS, Suv39h1, or β2m activity and / or expression comprises contacting or bringing into contact a cell with at least one active agent that inhibits the expression and / or activity of the SOCS-1, FAS, Suv39h1, or β2m protein and / or disrupts the FAS, β2m, SOCS-1, and / or Suv39h1 genes. The active agent may be selected from small molecule inhibitors; antibody derivatives, aptamers, nucleic acid molecules that block transcription or translation, or gene editing agents that target the SOCS1, FAS, Suv39h1, or B2N genes, respectively.

[0028] The present invention also relates to engineered immune cells as described herein, or compositions comprising engineered immune cells, for use in adoptive cell therapy, particularly in adoptive cell therapy for cancer. [Brief explanation of the drawing]

[0029] [Figure 1]This figure shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identified SOCS1 as a non-redundant inhibitor of antigen-experienced (Ag-exp) CD4 T cell proliferation during an ongoing immune response. (A) Two cohort experimental designs used in B-D to assess naive and Ag-exp CD4 T cell proliferation over the course of an ongoing immune response. (B) Flow plot and percentage of 106 proliferative Marilyn CD4 T cells, either naive or in vitro Ag-exp, during an ongoing immune response in C57BL / 6 mice (highlighted percentages are from singlet-vivo CD45.1+ CD4 T cells). 106 cells were injected intravenously into mice and primed in vivo by injection of 106 Dby peptide-loaded LPS-mature DCs into the paw. (C, D) Survival and IL2 production of CD45.1 Ag-exp CD4 T cells compared with naive CD45.1 Marilyn CD4 T cells during the recall response in vivo. (E) CD44 / CD62L phenotype and lentiviral library transduction efficiency (BFP+) of Ag-exp Cas9-Marilyn CD4 T cells before puromycin selection and in vivo injection. (F) Scatter plot comparing sgRNA normalized read counts in the original plasmid DNA library and transduced T cells 4 days after puromycin selection (5 μg / mL). (G) Representative flow plots and quantification of proliferative CD45.1 library transduced Cas9-Marilyn CD4 T cells compared with CD45.1 mock transduced Cas9-Marilyn CD4 T cells. (H) Enrichment of hits in the CFSElo subset of CD45.1 library transduced CD4 T cells compared to the CFSEhi subset in the ongoing immune response in vivo. (I) Representative plots and percentages of proliferative Ag-exp mock marilyn or sgSOCS1 marilyn cells during the recall response at day 14 (gated with singlet-live CD45.1+ CD4 T cells).Mice were injected IV with 2,106 CD4 T cells and primed with 106 peptide-pulsed LPS-mature DCs on day 0 and day 7. (G, H) The data shown are from two independent primary GW screenings. (H) p-values ​​correspond to the gene-enriched p-values, and log2-fold change (LFC) corresponds to the median LFC of all sgRNAs supporting the enriched RRA score. Targets with FDR < 0.5 are highlighted in black. Each dot is an individual mouse, and the white-outlined symbols are replicas from independent experiments (FP: paw pad, DC: dendritic cell, pept: peptide, Ag-exp: antigen-experienced). [Figure 2]This figure shows that SOCS1 is a node that integrates several cytokine signals that actively silence polycytokine release. (A) SORTing strategies for CFSElo (green) and CFSEhi (red) naive or Ag-exp Marilyn cells from the ongoing immune response. (B) Heatmap showing the expression of a selected list of cytokine receptors by proliferative or inhibited Marilyn cells (first 7 receptors p<0.01, FDR<0.5). (C) Representative flow plots and quantification of 106 Marilyn naive IFNγ-R+ / - or Marilyn Ag-exp IFNγ-R+ / - or Ag-exp IFNγ-R- / - increases in vivo after cell transfer and foot-plantar vaccination on day 14 with or without cohort 1 increases (highlighted percentages are from singlet live CD45.1+ CD4 T cells). (D) Representative flow plots of 106 Marilin Ag-exp increases in vivo during recall response in the presence of isotype, antibody IL2Rβ, and anti-IFNγRα, and quantification of blocking antibody (200 μg) administered intraperitoneally on days 7, 9, and 11 (highlighted percentages are from singlet live CD45.1+ CD4 T cells). (E) Flow cytometry evaluation of CD69, CD25, and IRF4 expression in sgSOCS1 Ag-exp Marilin compared to mock cells after overnight co-culture with peptide pulsed LPS mature DCs in vitro. (F) Flow plots and percentages of mock or sgSOCS1 Marilin producing IFN-γ, TNFα, and IL-2. Values ​​are shown as mean or mean ± SD. Each point is an individual mouse, and the white sign indicates a replica from an independent experiment analyzed by Mann-Whitney U test or two-way ANOVA (E). [Figure 3-1]Figure 3A shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (A) Schematic of Marilyn CD4 T cells (ACT) in C57BL / 6 female mice with male DBY-expressing bladder tumor line MB49. Figure 3B shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (B) Tumor-free survival after ACT, log-rank (Mantel-Cox) test. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=4-6 mice / group. Figure 3C shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (C) Growth curves of MB49 tumors in C57BL6 mice after adoptive transfer of various ACT:PBS controls, 106 mock Ag-exp Marilyn cells, or 106 sgSOCS1 Ag-exp Marilyn Cas9 cells in mice treated with anti-CD8a and anti-AsialoGM1 (anti-GM1) depletion antibodies. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=4-6 mice / group. Figure 3D shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (D) Representative flow plots and quantification of mock or sgSOCS1 Marilyn cells in tumor draining lymph nodes (TdLNs), tumors, and unrelated lymph node LNs (irr-LNs) at 7 days post-ACT. The data are presented as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments, n=4–6 mice / group. [Figure 3-2]Figure 3E shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (E) Representative flow plots and percentages of mock and sgSOCS1 Marilyn cell proliferation in TdLN at 7 days post-ACT. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=4–6 mice / group. Figure 3F shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (F) Gene set enrichment analysis (GSEA) for selected characteristic transcriptional signatures (MSigDB) with FDR values ​​< 0.05 in Ag-exp sgSOCS1 vs. Ag-exp mock marilyn T cells in TdLN (n=3 replicas from 2 pooled mice). Figure 3G shows that Ag-exp sgSocs1 marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (G) Differentially expressed genes in tumor dissipation lymph node (TdLN) infiltrating CD45.1 marilyn sgSOCS1 cells compared with marilyn mock cells. Transcripts with FDR values ​​< 0.05 are highlighted in light green. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments, n=4–6 mice / group. Figure 3H shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (H) Representative flow plots and quantification of mock or sgSOCS1 Marilyn CD4 T cells producing IFNγ+ IL2+ and IFNγ+ TNFα+ in TdLN at 7 days post-transfer. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=4–6 mice / group. [Figure 3-3]Figure 3I shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (I) Representative flow plot of MHC-II molecules expressed by MB49 tumors. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments, n=4–6 mice / group. Figure 3J shows that Ag-exp sgSocs1 Marilyn CD4 T cells acquire a pluripotent Th cell-toxic phenotype and enhance rejection of male bladder MB49 tumors. (J) Flow plot and quantification of granzyme B (GZMB) expressed by tumor-infiltrating sgSOCS1 Marilyn CD4 T cells at day 7. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments, n=4–6 mice / group. [Figure 4]Improved ACT-based B16-OVA tumor rejection: This figure shows that Socs1 gene inactivation restores OT2 cell proliferation and enhances OT1 cell survival and cytotoxicity. (A) Schematic of OT1 CD8 and OT2 CD4 adoptive T cell therapy (ACT) in C57BL / 6 mice with B16-OVA melanoma tumors. (B) Growth curves of B16-OVA tumors in C57BL6 mice after adoptive transfer using OT1 (2,106 mock or 2,106 sgSOCS1) and OT2 cells (2,106 mock or 2,106 sgSOCS1). (C) Kaplan-Meier survival analysis and log-rank (Mantel-Cox) test in mice with B16-OVA after ACT. (D) Representative plots and quantifications of mock or sgSOCS1 OT1 and OT2 cells in tumor dissipation lymph nodes (TdLNs), tumors, or unrelated lymph nodes (Irr-LNs) at 7 days post-ACT, gated with singlet-derived Vα2+ T cells. (E) Representative flow plots and percentages of mock or sgSOCS1 OT1 and OT2 cells proliferating in TdLNs at 7 days post-ACT. (F, G) Representative flow plots and quantifications of mock or sgSOCS1 OT2 and OT1 tumor-infiltrating cells producing IFN-γ and granzyme B molecules at 7 days post-transplantation. Data are shown as mean, analyzed by the Mann-Whitney U test from two independent experiments, n=5–8 mice / group. [Figure 5-1]Figure 5A shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (A) Schematic of adoptive T cell therapy (ATCT) using CAR-T cell manipulation and 2,106 CD4 CAR (CAR4) and 2,106 CD8 CAR (CAR8) T cells from mice with NALM6-Luc. Figure 5B shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (B) CAR expression assessed using CD19 / Fc fusion protein and central memory phenotype before NSG injection. Data are presented as mean, analyzed by Mann-Whitney U test, from two independent experiments (n=5-6 mice / group). Figure 5C shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (C) Representative flow plots and quantifications of bone marrow infiltration using CAR4 and CAR8 mocks and sgSOCS1 in NSG mice with NALM6-Luc at 7 and 28 days post-transfer, gated with singlet live HLA-I+, CD45.2- mouse cells. Data are presented as mean, analyzed by Mann-Whitney U test from two independent experiments (n=5-6 mice / group). Figure 5D shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (D) Representative flow plots and quantification of bone marrow infiltration using CAR4 and CAR8 mocks and sgSOCS1 in NSG mice with NALM6-Luc at 7 and 28 days post-transfer, gated with singlet live HLA-I+, CD45.2- mouse cells. Data are presented as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (n=5-6 mice / group). [Figure 5-2]Figure 5E shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (E) Heatmap of selected differentially expressed genes (FDR<0.05) between mock and sgSOCS1 CAR T cells, associated with activation (red), proliferation / survival (blue), and effector function (green) at 7 days post-transfer. Data are presented as mean, analyzed by Mann-Whitney U test from two independent experiments (n=5-6 mice / group). Figure 5F shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (F) Gene set enrichment analysis for transcriptional signatures from characteristic signatures in CAR4 / 8 sgSOCS1 vs CAR4 / 8 mock (n=6 mice). [Figure 5-3]Figure 5G shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (G) Representative flow plot and quantification of effector molecules produced by CAR T cells from infiltrated BM at day 28. Data are presented as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (n=5-6 mice / group). Figure 5H shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (H) Representative flow plot and quantification of effector molecules produced by CAR T cells from infiltrated BM at day 28. Data are presented as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (n=5-6 mice / group). Figure 5I shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (I) NALM6-LUC tumor growth after ATCT using 2,106 CAR4 / 8 mocks or 2,106 CAR4 sgSOCS1 / 8 mocks or 2,106 CAR4 mocks / 8 sgSOCS1 or 2,106 CAR4 / 8 sgSOCS1, as detailed in Figure 5A. Data are presented as mean, analyzed by Mann-Whitney U test from two independent experiments (n=5-6 mice / group). [Figure 5-4]Figure 5J shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (J) Kaplan-Meier analysis of survival in NSG mice, log-rank (Mantel-Cox) test. Data are presented as mean analyzed by Mann-Whitney U test from two independent experiments (n=5-6 mice / group). Figure 5K shows that SOCS1 inactivation restores CAR4 T cell proliferation in vivo and boosts CAR8 T cell efficacy in controlling B-ALL disease. (K) Mice with NALM6-LUC were treated with 4,106 CAR-T cells. Tumor volume, shown as quantified bioluminescence signal per animal, over a 35-day period, n=5 mice / group. Data are presented as mean analyzed by Mann-Whitney U test from two independent experiments (n=5-6 mice / group). [Figure 6-1]Figure 6A shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (A) Representative flow plots and absolute numbers of live CD45.1(H2-Kb) Marilyn CD4 T cells in the spleen of well-immunized C57BL6 (syngene) and BALB / c (allogeneic) mice 4 days after intravenous (IV) injection. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6B shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (B) Representative flow plots and absolute numbers of live CD45.1(H2-Kb) Marilyn CD4 T cells in the spleen of well-immunized C57BL6 (synthetic) and BALB / c (allogeneic) mice 4 days after intravenous (IV) injection. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6C shows that in vivo genome-wide (18,400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (C) Schematic of the in vivo genome-wide CRISPR screening design. [Figure 6-2]Figure 6D shows that in vivo genome-wide (18,400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (D) Representative flow plots and absolute numbers of live CD45.1 mock or library mutant Marilyn CD4 T cells in the spleen of well-immunized C57BL6 and BALB / c mice 4 days after IV injection of 107 CD4 T cells. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6E shows that in vivo genome-wide (18,400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (E) Representative flow plots and absolute numbers of live CD45.1 mock or library mutant Marilyn CD4 T cells in the spleen of well-immunized C57BL6 and BALB / c mice 4 days after IV injection of 107 CD4 T cells. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. [Figure 6-3]Figure 6F shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (F) Meta-analysis of in vivo genome-wide CRISPR pooling screening of library mutant Marilyn cell survival in BALB / c mice compared to diversity from C57BL6-infiltrating mice using MAGECK analysis. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments, n=3–6 mice / group. Figure 6G shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (G) Gene-level representation of significant individual sgRNA distributions across each experiment, showing enriched genes (Fas, B2m) in BALB / c mice compared to C57BL6 mice. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6H shows that in vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (H) Representative flow plots and quantifications of spleens infiltrated by co-injected mock (expressing congenic marker CD45.1 / 2) and Fas-inactivated Marilyn CD4 T cells (sgFas, CD45.1 / 1) in C57BL6 mice (syngene) or BALB / c mice (allogeneic) 4 days after IV injection. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments, n=3–6 mice / group. [Figure 6-4]Figure 6I shows that in vivo genome-wide (18,400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (I) Percentage of Fas-negative Marilyn cells in C57BL6 and BALB / c mice. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6J shows that in vivo genome-wide (18,400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (J) Representative flow plots and quantifications of spleens infiltrated by co-injected mock (expressing congenic marker CD45.1 / 2) and B2m-inactivated Marilyn CD4 T cells (sgB2m, CD45.1 / 1) in C57BL6 or BALB / c mice 4 days after IV injection. Data are shown as mean values ​​analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. Figure 6K shows that in vivo genome-wide (18400 genes) CRISPR pooled screening identifies Fas and B2m as non-redundant targets enabling T cell survival in MHC mismatched hosts. (K) Percentage of B2m-negative Marilyn cells in C57BL6 and BALB / c mice. Data are shown as mean values ​​analyzed by Mann-Whitney U test from two independent experiments, n=3–6 mice / group. [Figure 7-1]Figure 7A shows that Fas targeting enhances resistance to both T cell and NK-mediated allogeneic rejection and can be enhanced in vivo by Socs1 inactivation. (A) Schematic of experimental design (screening model) for complete MHC mismatched rejection of C57BL6 T cells in BALB / c mice. Figure 7B shows that Fas targeting enhances resistance to both T cell and NK-mediated allogeneic rejection and can be enhanced in vivo by Socs1 inactivation. (B) Representative flow plots and absolute numbers of live CD45.1 polyclonal (CD4 and CD8) T cells in the spleen of well-immunized C57BL6 and BALB / c mice 4 days after IV injection of 2,106 T cells in mice treated with either IgG or anti-CD8a (per 200 ug / day) or anti-asialoGM1 (per 30 ug / day) depletion antibodies. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7C shows that Fas targeting improves resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (C) Representative flow plots and absolute numbers of live CD45.1 polyclonal (CD4 and CD8) T cells in the spleen of well-immunized C57BL6 and BALB / c mice 4 days after IV injection of 2,106 T cells in mice treated with either IgG or anti-CD8a (200 ug / day) or anti-asialoGM1 (anti-GM1, 30 ug / day) depletion antibodies. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. [Figure 7-2]Figure 7D shows that Fas targeting enhances resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (D) Pre-injection expression of Fas in polyclonal CD45.1 T cells (H2-Kb) 4 days after electroporation with sgRNA and HIFI-Cas9. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments (G, H), n=3–6 mice / group. Figure 7E shows that Fas targeting enhances resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (E) Percentage of indels in polyclonal CD45.1 T cells (H2-Kb) electroporated with sgSOCS1, using tide analysis. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7F shows that Fas targeting improves resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (F) Percentage of indels in polyclonal CD45.1 T cells (H2-Kb) electroporated with sgSIOCS1 and Fas, using tide analysis. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7G shows that Fas targeting improves resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (G) Representative flow plots and absolute numbers of live polyclonal CD45.1 T cells (H2-Kb) in the spleens of well-immunized C57BL6 and BALB / c mice 4 days after IV injection of 2,106 T cells. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments (G, H), n=3–6 mice / group.Figure 7H shows that Fas targeting enhances resistance to both T cell and NK-mediated allogeneic rejection and can be enhanced in vivo by Socs1 inactivation. (H) Double survival of inactivated polyclonal CD45.1 T cells compared to mock cells in syngeneic and allogeneic mice. Data are shown as mean, analyzed by Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7I shows that Fas targeting enhances resistance to both T cell and NK-mediated allogeneic rejection and can be enhanced in vivo by Socs1 inactivation. (I) Schematic of experimental design for semi-allogeneic rejection of F1 T cells (C57BL6×BALB / c) in C57BL6 mice. [Figure 7-3]Figure 7J shows that Fas targeting enhances resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (J) Number of F1 Marilyn cells in the spleen of C57BL6 mice 4 days after IV injection of 2,106 cells in mice treated with either IgG or anti-CD8a (per 200 ug / day) or anti-NK1.1 (per 30 ug / day) depletion antibodies. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments (G, H), n=3–6 mice / group. Figure 7K shows that Fas targeting enhances resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (K) Expression of H2-Kb and (et)H2-Kd in CD45.1 F1 Marilyn CD4 T cells from C57BL6 mice treated with anti-NK1.1. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7L shows that Fas targeting improves resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (L)2. Representative flow plot and absolute number of F1 Marilyn CD4 T cell viability in C57BL6 mice 4 days after IV injection of 106 F1 cells. Data are shown as mean values ​​analyzed by the Mann-Whitney U test from two independent experiments (G, H), n=3–6 mice / group. Figure 7M shows that Fas targeting improves resistance to both T cell and NK-mediated allore rejection and can be enhanced in vivo by Socs1 inactivation. (M) Representative flow plots and absolute numbers of F1 Marilyn CD4 T cell survival in C57BL6 mice 4 days after IV injection of 2,106 F1 cells. Data are shown as mean, analyzed by Mann-Whitney U test, from two independent experiments (G, H), n=3–6 mice / group. [Figure 8-1]Figure 8A shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (A) Schematic of an immunocompetent mouse model for assessing CD4 or CD8 tumor-specific T cell functionality across the MHC barrier. Figure 8B shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (B) Representative flow plot and absolute number of CD45.1 F1 OT1 cells infiltrating the spleen of C57BL6 mice with B16-OVA 15 days after IV injection with 2,106 F1 OT1 cells. Data are shown as mean, n=3-5 mice / group. Figure 8C shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (C) Representative flow plot and absolute number of (GZB+)CD45.1 F1 OT1 cells infiltrating tumors and expressing granzyme B in C57BL6 mice with B16-OVA, 15 days after IV injection with 2.106 F1 OT1 cells. Data are shown as averages, n=3-5 mice / group. [Figure 8-2]Figure 8D shows that dual inactivation of Fas and SOCS1 protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (D) Schematic of experimental design using human CAR-T cells. Figure 8E shows that dual inactivation of Fas and SOCS1 protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (E) Pre-injection CAR-T cell phenotype showing CD4 and CD8 T cell composition and CD19-CARbbz and TCRb expression after electroporation with sgTRAC. Data are shown as mean, n=3-5 mice / group. Figure 8F shows that dual inactivation of Fas and SOCS1 protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (F) Fas expression in engineered CAR-T cells by flow 4 days after electroporation. Data are shown as mean, n=3-5 mice / group. Figure 8G shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (G) Relative expression of SOCS1 mRNA assessed by RT-qPCR in TRAC / FAS / SOCS1 inactivated A2-CAR-T cells compared with TRAC inactivated A2-CAR-T cells 4 days after electroporation. Data are shown as mean, n=3-5 mice / group. [Figure 8-3]Figure 8H shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (H) Representative flow plot of HLA, A, B, C+ cells in the bone marrow (BM) of NSG mice 15 days after IV injection with 2,106 TRAC-inactivated A2-CAR-T cells. Data are shown as mean, n=3-5 mice / group. Figure 8I shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (I) Number of A2-CAR-T cells infiltrating the bone marrow of NSG mice 15 days after CAR-T cell injection. Data are shown as mean, n=3-5 mice / group. Figure 8J shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (J) Number of Nalm6-sgB2m cells infiltrating NSG mouse bone marrow 15 days after CAR-T cell injection. Data are shown as mean values, n=3-5 mice / group. Figure 8K shows that Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. (K) Number of A2+ T cells infiltrating NSG mouse bone marrow 15 days after CAR-T cell injection. Data are shown as mean values, n=3-5 mice / group. [Modes for carrying out the invention]

[0030] Detailed explanation definition The term “antibody” as used herein is used in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies, as well as single-chain antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG(rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding to antigens, single-chain variable fragments (scFv), and functional (antigen-binding) antibody fragments, including single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also includes intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, as well as genetically engineered and / or otherwise modified forms of immunoglobulins, such as multispecific, bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv, etc. Unless otherwise stated, the term “antibody” should be understood to encompass its functional antibody fragments. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0031] An "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions; single-chain antibody molecules such as scFv; and single-domain VH monoantibodies; as well as multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0032] A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody.

[0033] As used herein, “repression” of gene expression refers to the elimination or reduction of the expression of one or more gene products encoded by the gene of interest in a cell, compared to the level of expression of the gene product in the absence of repression. Exemplary gene products include mRNA and protein products encoded by the gene. In some cases, repression is transient or reversible, and in other cases, permanent. In some cases, repression is of a functional or full-length protein or mRNA, regardless of the fact that a cleaved or non-functional product may be produced. In some embodiments herein, gene activity or function is repressed as opposed to expression. Gene repression is generally induced by artificial means, i.e., by the addition or introduction of compounds, molecules, complexes, or compositions, and / or by the disruption of a gene or a gene-related nucleic acid, such as at the DNA level. Exemplary methods for gene repression include gene editing, gene silencing, knockdown, knockout, and / or gene disruption techniques. Examples include antisense technologies such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in a transient decrease in expression, and gene editing techniques that result in the inactivation or destruction of a target gene, for example, by inducing rupture and / or homologous recombination.

[0034] As used herein, gene “disruption” refers to a change in the sequence of a gene at the DNA level. Examples include insertions, mutations, and deletions. Disruption typically results in the suppression and / or complete absence of the expression of the normal or “wild-type” product encoded by the gene. Examples of such gene disruption include insertions, frameshifts, and missense mutations, deletion of a gene or part of a gene, including whole gene deletions, knock-ins, and knockouts. Such disruption may occur in the coding region, for example, in one or more exons, by insertion of a stop codon, and result in the inability to produce the full-length product, a functional product, or any product. Such disruption can also occur by disruption in the promoter or enhancer or other regions that affect transcriptional activation, thereby blocking gene transcription. Gene disruption includes gene targeting, including homologous recombination to inactivate the target gene.

[0035] The cells of the present invention The cells used in accordance with the present invention are typically eukaryotic cells such as mammalian cells (also referred to as animal cells in this invention), for example, human cells.

[0036] More specifically, the cells of the present invention are cells of the immune system (i.e., immune cells), such as cells of innate or adaptive immunity, including myeloid cells or lymphoid cells, such as lymphocytes, typically T cells and / or NK cells, derived from blood, bone marrow, lymphatic fluid, or lymphoid organs (particularly the thymus).

[0037] Preferably, according to the present invention, the cells are lymphocytes, including T cells, B cells, and NK cells in particular.

[0038] Cells according to the present invention may be immune cell precursors such as lymphoid precursors, and more preferably T cell precursors.

[0039] T cell precursors typically express a set of consensus markers including CD44, CD117, CD135, and Sca-1. See also Petrie HT, Kincade PW, Many roads, one destination for T cell progenitors. The Journal of Experimental Medicine. 2005;202(1):11-13.

[0040] The cells are typically primary cells, such as those directly isolated from the subject and / or those isolated from the subject and frozen.

[0041] With respect to the target to be treated, the cells of the present invention may be homogeneous and / or autologous.

[0042] In some embodiments, the cells include a whole T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, or one or more subsets of T cells or other cell types, defined by function, activation state, maturity, differentiation potential, growth, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation.

[0043] Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells, there are naive T(T) N ) cells, effector T cells (T EFF ), memory T cells, and stem cell memory T (TSC) M ), Central Memory T (TC M ), Effector Memory T (T EM), or its subtypes include terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, helper T cells such as follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. Preferably, cells according to the present invention have stem / memory characteristics and higher reconstitution ability due to inhibition of Suv39h1. EFF Cells, and T N cells, TSC M , TC M , TE M Cells, and combinations thereof.

[0044] In some embodiments, one or more T cell populations are enriched or depleted with cells that are positive for or express at high levels of one or more specific markers, such as surface markers, or cells that are negative for or express at relatively low levels of one or more markers. In some cases, such markers are absent or expressed at relatively low levels in certain T cell populations (e.g., non-memory cells), but present or expressed at relatively higher levels in certain other T cell populations (e.g., memory cells). In one embodiment, cells (CD8 + Cells or T cells, for example, CD3 +Cells (e.g., cells) are enriched (i.e., positively selected) with cells that are positive for or express high surface levels of CD117, CD135, CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or depleted (i.e., negatively selected) with cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched or depleted with cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra(CD127). In some examples, CD8+ T cells are enriched with cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L.

[0045] For example, according to this application, cells are a population of CD4+ T cells and / or a subpopulation of CD8+ T cells, for example, central memory (T CM ) may include enriched subpopulations of cells. Alternatively, the cells may be other types of lymphocytes, including natural killer (NK) cells, MAIT cells, innate lymphoid cells (ILCs), and B cells.

[0046] Cells and cell-containing compositions for operations according to the present invention are isolated, for example, from samples obtained from or derived from a subject, particularly from biological samples. Typically, the subject requires and / or will undergo cell therapy (adoptive cell therapy). The subject is preferably a mammal, particularly a human. In one embodiment of this application, the subject has cancer.

[0047] Samples include tissues, body fluids, and other samples taken directly from subjects, as well as samples resulting from one or more processing steps such as separation, centrifugation, genetic manipulation (e.g., transduction using viral vectors), washing, and / or incubation. Therefore, biological samples may be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, including processed samples derived therefrom. Preferably, samples from which cells are derived or isolated are blood or blood-derived samples, or apheresis or leukocyte apheresis products or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, and / or cells derived therefrom. The samples include autologous and allogeneic source samples in the context of cell therapy (typically adoptive cell therapy).

[0048] In some embodiments, the cells are derived from a cell line, such as a T cell line. Cells can also be obtained from heterologous sources such as mice, rats, non-human primates, or pigs. Preferably, the cells are human cells.

[0049] Cells lacking SOCS-1 This disclosure includes cells that are deficient in SOCS1, more specifically, immune cells. In some embodiments, SOCS1-deficient cells may be further deficient in FAS, β2m, SUV39h1, or a combination thereof.

[0050] As used herein, the terms “SOCS-1” or “cytokine signaling suppressor 1” have their general meaning in the art and refer to a subset of SOCS family proteins that form part of a classical negative feedback system that modulates cytokine signaling. There are eight SOCS proteins encoded in the human genome, SOCS1-7 and CIS. All eight are characterized by the presence of SOCS box 1 in the SH2 domain and short C-terminal domain. The SOCS box of all SOCS proteins is found bound to elongin B and C in the adapter complex. This linkage enables the guidance of the E3 ubiquitin ligase scaffold (Cullin 5), which catalyzes the ubiquitination of signal transduction intermediates guided by their SH2 domains (Kamizono S et al., "The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2", J Biol Chem. April 20, 2001; 276(16): 12530-8).

[0051] In addition to their ubiquitin ligase activity, SOCS1 and SOCS3 are unique in their ability to directly inhibit the kinase activity of JAK (Janus kinase). This activity depends on a short motif located immediately upstream of the SH2 domain, known as the KIR (kinase inhibitory region). The KIR of SOCS1 is a highly evolved inhibitor of JAK, and mutations in any residue within this motif, including histidine residues that mimic the substrate tyrosine, lead to a significant decrease in affinity. SOCS1 is a particularly direct, potent, and selective inhibitor of the catalytic activity of JAK1, JAK2, and TYK2, and is therefore typically involved in the negative regulation of the signaling of numerous cytokines, including interleukin-4 (IL-4), IL-6, IL-2, interferon (IFN)-alpha, interferon (IFN)-gamma, prolactin, growth hormone, and erythropoietin, through the JAK / STAT3 pathway. (For details on SOCS1 activity in particular, see Sharma J, Larkin J 3rd. "Therapeutic Implications of SOCS1 Modulation in the Treatment of Autoimmunity and Cancer", Front Pharmacol. 2019;10:324; Liau NPD, Laktyushin A, Lucet IS et al. "The molecular basis of JAK / STAT inhibition by SOCS1", Nat Commun. 2018;9(1):1558;Sporri B, Kovanen PE, Sasaki A, Yoshimura A, Leonard WJ. “JAB / SOCS1 / SSI-1 is an interleukin-2-induced inhibitor of IL-2 signaling”, Blood.2001;97(1):221-226; Alexander WS, Starr R, Fenner JE et al. "SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine", Cell. 1999;98(5):597-608, and Kamizono S, Hanada T, Yasukawa H et al. "The SOCS box of "SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2", J Biol Chem. 2001;276(16):12530~12538; and Frantsve J, Schwaller J, Sternberg DW, Kutok J, Gilliland DG. "Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation”, Mol Cell See Biol. 2001;21(10):3547-3557). This protein is also known as JAK-binding protein (JAB), STAT-inducible STAT inhibitor 1 (SSI-1), or Tec-interacting protein 3 (TIP-3). The human SOCS-1 protein is referred to as O15524 in UNIPROT, encoded by the SOCS-1 gene located on chromosome 16 (11,254,408-11,256,204 reverse strand), and referred to as ENSG00000185338 in the Ensembl database. The term SOCS-1 also encompasses all SOCS-1 orthologues. In some embodiments, the protein SOCS-1 according to the present invention is that of Sequence ID No. 1:

[0052] [ka]

[0053] As used herein, the expression “SOCS1-deficient” in accordance with the present invention refers to inhibition or blockade of SOCS-1 activity, such as, for example, blockade of SOCS1 binding to JAK and / or blockade of the guidance of the E3 ubiquitin ligase scaffold (Culin 5) via elongin BC. In some embodiments, inhibition of SOCS1 can be achieved by blocking the binding of SOCS1 to JAK (including JAK1 / 2 and / or TYK2) and / or by blocking the binding of the SOCS1 box to elongin C, a key intermediate of the E3 complex guidance.

[0054] Furthermore, as used herein, the terms "Suv39h1" or "H3K9 histone methyltransferase Suv39h1" have their general meanings in the art and refer to the histone methyltransferase "suppressor of variegation 3-9 homolog 1 (Drosophila)" that specifically trimethylates the Lys-9 residue of histone H3 using monomethylated H3-Lys-9 as a substrate (Aagaard L, Laible G, Selenko P, Schmid M, Dorn R, Schotta G, Kuhfittig S, Wolf A, Lebersorger A, Singh PB, Reuter G, Jenuwein T (June 1999) "Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component See also M3 1, EMBO J 1 8(7):1923-38). Histone methyltransferase is also known as MG44, KMT1A, SUV39H, SUV39H1, histone-lysine N-methyltransferase SUV39H1, H3-K9-HMTase 1, OTTHUMP00000024298, Su(var)3-9 homolog 1, lysine N-methyltransferase 1A, histone H3-K9 methyltransferase 1, position-effect variegation 3-9 homolog, histone-lysine N-methyltransferase, or H3-lysine-9 specific 1. Human Suv39h1 methyltransferase is referred to as O43463 in UNIPROT and is encoded by the gene Suv39h1 (gene ID: 6839 in NCBI) located on the X chromosome. In accordance with this invention, the term Suv39h1 also encompasses all orthologs of SUV39H1, such as SU(VAR)3-9.In some embodiments, the protein SUV39H1 according to the present invention is the one designated as SEQ ID NO: 2 or 3.

[0055] Sequence ID 2:

[0056] [ka]

[0057] Sequence ID 3:

[0058] [ka]

[0059] As used herein, the terms “Fas” or “Fas cell surface death receptor” have their general meanings in the art and refer to the receptor for TNFSF6 / FASLG. Also known as the Fas receptor (FasR), apoptosis antigen 1 (APO-1 or APT), differentiation antigen group 95 (CD95), or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), Fas is a protein encoded in humans by the FAS gene. FAS is a cell surface death receptor that leads to programmed cell death (apoptosis) when it binds to its ligand, Fas ligand (FasL), thus forming a cell death induction signaling complex (DISC) and inducing subsequent caspase 8 activation via the adapter molecule FADD. It is one of two apoptotic pathways, the other being the mitochondrial pathway. Human Fas is referred to as P25445(TNR6_HUMAN) in UNIPROT, encoded by the gene FAS located on chromosome 10 (88,990,531~89,017,059 in the forward chain), and referred to as ENSG00000026103 in the Ensembl database. The term FAS also encompasses all FAS1 orthologues.

[0060] In some embodiments, the protein FAS intended herein is that of SEQ ID NO: 4.

[0061] Sequence ID 4:

[0062] [ka]

[0063] Beta-2-microglobulin (β2m) is a component of the class I major histocompatibility complex (MHC). It is involved in the presentation of peptide antigens to the immune system. Human β2m is encoded by the B2M gene located at chromosome position 15q21.1 (chromosome 15: 44,711,487~44,718,851 forward) and is referred to as B2M ENSG00000166710 (or HGNC ID: HGNC:914) in the Ensembl database.

[0064] The β2m precursor is typically the one represented by Sequence ID No. 5, which is further processed into its mature form.

[0065] [ka]

[0066] As used herein, the expressions “SOCS1-defective,” “Suv39h1-defective,” “FAS-defective,” or “β2m-defective” as used in this application refer to the inhibition or blockade of SOCS1 and / or Suv39h1 and / or FAS activity, and / or β2m activity, as detailed above, in cells.

[0067] In this application, “inhibition of SOCS1 activity,” “inhibition of Suv39h1 activity,” “inhibition of FAS activity,” or “inhibition of β2m activity” refers to a reduction in SOCS1 activity, Suv39h1 activity, FAS activity, or β2m activity by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, compared to the activity or level of SOCS1, Suv39h1, or FAS protein that is not inhibited in the corresponding wild-type cell. Preferably, inhibition of SOCS1 activity, Suv39h1 activity, or FAS activity leads to the absence of substantially detectable activity of SOCS1, Suv39h1, or FAS, respectively, in the cell.

[0068] It should be noted that cells lacking SOCS1 and / or Suv39h1 and / or FAS and / or β2m can be acquired by repression or disruption of the SOCS1 and / or Suv39h1 and / or FAS and / or B2M genes, respectively, but also at the post-transcriptional level (SOCS1 mRNA and / or Suv39h1 and / or FAS mRNA and / or β2m mRNA), as well as at the post-translational or protein level of SOCS1 and / or FAS and / or Suv39h1 and / or β2m.

[0069] Therefore, inhibition of SOCS1 and / or FAS and / or Suv39h1 and / or β2m activity can also be achieved through the suppression of SOCS1 and / or FAS and / or Suv39h1 and / or β2m gene expression, or through SOCS1 and / or FAS and / or Suv39h1 and / or B2M gene disruption. According to the present invention, suppression reduces the expression of SOCS1 and / or FAS and / or Suv39h1 and / or β2m in cells, particularly immune cells of the present invention, by at least 50, 60, 70, 80, 90, or 95% of the expression of the same cells (i.e., corresponding cells) produced by the method in the absence of suppression or in corresponding wild-type cells (as illustrated in the results contained herein). Gene disruption may also lead to decreased expression of SOCS1 and / or FAS and / or Suv39h1 and / or β2m proteins, or to the expression of non-functional SOCS1 and / or non-functional FAS and / or non-functional Suv39h1 and / or non-functional β2m proteins.

[0070] "Non-functional SOCS1 protein," "Non-functional FAS protein," "Non-functional Suv39h1 protein," or "Non-functional β2m protein" are, in this specification, intended to be proteins having reduced activity or lack of detectable activity as described above.

[0071] In some embodiments, the inhibitor of SOCS1 activity in cells according to the present invention may be selected from any natural or non-natural compound or active substance having the ability to block the binding of SOCS1 to JAK and / or elongin C or to inhibit SOCS1 gene expression. The inhibitor of SOCS1 activity in cells according to the present invention may be selected from any natural or non-natural compound or active substance having the ability to inhibit SOCS1 activity or to inhibit SOCS1 gene expression, in particular as mentioned above.

[0072] In some embodiments, the peptide mimetic or autophosphorylation site pJAK2(1001-1013) of SOCS1 described in Lilian W Waiboci, Howard M Johnson, James P Martin, and Chulbul M Ahmed, J Immunol April 1, 2007, 178(Supplement 1)S170; or Waiboci LW, Ahmed CM, Mujtaba MG et al., J Immunol. 2007;178(8):5058-5068 may be used.

[0073] In some embodiments, the inhibitor of FAS activity in cells according to the present invention may be selected from any natural or non-natural compound or active substance having the ability to block FAS receptor activation or inhibit FAS gene expression.

[0074] In some embodiments, the inhibitor of Suv39h1 activity in cells according to the present invention may be selected from any natural or non-natural compound or active substance having the ability to inhibit the methylation of Lys-9 of histone H3 by H3K9-histone methyltransferase or to inhibit the expression of the H3K9-histone methyltransferase SUV39H1 gene. The inhibitor of Suv39h1 activity in cells according to the present invention may be selected from any natural or non-natural compound or active substance having the ability to inhibit the methylation of Lys-9 of histone H3 by H3K9-histone methyltransferase or to inhibit the expression of the H3K9-histone methyltransferase SUV39H1 gene.

[0075] Inhibition of SOCS1 and / or FAS and / or Suv39h1 and / or β2m in immune cells according to this application (at the gene and / or protein level) may be permanent and irreversible, transient or reversible. However, preferably, SOCS1 inhibition and / or FAS inhibition and / or Suv39h1 inhibition are permanent and irreversible. Inhibition of SOCS1 and / or FAS and / or Suv39h1 in cells may be achieved before or after injection of cells in a target patient, as described below.

[0076] Genetically modified cells according to the present invention In some embodiments, the cells include one or more genetically modified nucleic acids that encode one or more antigen receptors.

[0077] Typically, nucleic acids are heterogeneous (i.e., not normally found in the cells being manipulated and / or in the organism from which such cells originate). In some embodiments, nucleic acids do not exist in nature, including chimeric combinations of nucleic acids encoding various domains derived from multiple different cell types.

[0078] Antigen receptors according to the present invention include genetically modified T cell receptors (TCRs) and their components, as well as functional non-TCR antigen receptors such as chimeric antigen receptors (CARs).

[0079] Chimeric antigen receptor (CAR) In some embodiments, the manipulated antigen receptors include chimeric antigen receptors (CARs), including activating or stimulating CARs, co-stimulating CARs (see WO2014 / 055668), and / or inhibitory CARs (see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)).

[0080] Chimeric antigen receptors (CARs), also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors, are engineered receptors that graft arbitrary specificity onto immune effector cells (T cells). Typically, these receptors are used to graft the specificity of monoclonal antibodies onto T cells using the transfer of their coding sequences, facilitated by retroviral vectors.

[0081] In some embodiments, CARs typically comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components via a linker and / or transmembrane domain. Such molecules typically mimic or approximate signaling through innate antigen receptors, signaling through such receptors in combination with costimulatory receptors, and / or signaling through costimulatory receptors alone.

[0082] In some embodiments, CARs are constructed to have specificity for specific antigens (or markers or ligands), such as cancer markers or antigens expressed in specific cell types targeted by adoptive therapy. Therefore, CARs typically contain, in their extracellular portion, one or more antigen-binding fragments, domains, or parts, or one or more antigen-binding molecules, such as one or more antibody-variable domains and / or antibody molecules.

[0083] The portion used to bind to the antigen falls into three general categories: single-chain antibody fragments (scFv) derived from antibodies, Fab' selected from a library, or innate ligands involved in their homologous receptors (for first-generation CARs). Successful examples in each of these categories are reported, in particular, in Sadelain M, Brentjens R, and Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013;3(4):388-398 (see Table 1 in particular), and are included in this application. scFv derived from mouse immunoglobulins are frequently used because they are readily obtainable from well-characterized monoclonal antibodies.

[0084] Typically, a CAR contains one or more antigen-binding moieties of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0085] In some embodiments, the CAR includes an antibody heavy chain domain that specifically binds to a target cell, such as tumor cells or cancer cells, or to an antigen such as a cancer marker or cell surface antigen of a disease, such as any of the target antigens described herein or known in the Art.

[0086] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.

[0087] In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g., scFv), that specifically recognizes intracellular antigens such as tumor-associated antigens presented on the cell surface as MHC-peptide complexes. In some embodiments, an antibody that recognizes an MHC-peptide complex or an antigen-binding portion thereof can be expressed on the cell surface as part of a recombinant receptor such as an antigen receptor. Among antigen receptors, there are functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against a peptide-MHC complex can also be referred to as a TCR-like CAR.

[0088] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0089] The transmembrane domain in some embodiments is derived from either a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least their transmembrane regions). The transmembrane domain can also be synthetic.

[0090] In some embodiments, a short oligo or polypeptide linker, e.g., a linker of 2 to 10 amino acids in length, is present between and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0091] CARs generally include at least one or more intracellular signaling components. First-generation CARs typically had an intracellular domain derived from the CS3ζ chain, which is the main transducer of signals from the endogenous TCR. Second-generation CARs typically further include an intracellular signaling domain derived from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) in the cytoplasmic tail of the CAR to provide additional signals to T cells. Preclinical studies have shown that the second generation improves the anti-tumor activity of T cells. More recently, third-generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to enhance efficacy.

[0092] For example, the CAR can include an intracellular component of the TCR complex, such as the CD3 zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. The CAR can also further include a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16.

[0093] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the corresponding unmanipulated immune cells (typically T cells). For example, the CAR can induce functions of T cells, such as cytolytic activity or T helper activity, secretion of cytokines or other factors.

[0094] In some embodiments, the intracellular signaling domain includes the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also a co-receptor that acts in coordination with such receptors in a native context to initiate signal transduction after antigen receptor involvement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capacity.

[0095] In some embodiments, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, the CAR comprises one or both of such signaling components.

[0096] In some embodiments, the CAR includes a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex either stimulatingly or inhibitorily. Primary cytoplasmic signaling sequences that act stimulatingly may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0097] The CAR may also include the signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some embodiments, the same CAR may contain both activating and costimulatory components; alternatively, the activating domain may be provided by one CAR, while the costimulatory component may be provided by another CAR that recognizes a different antigen.

[0098] In some embodiments, the CAR is a CD19 BBz CAR typically known in the literature. Typically, such a CAR comprises the following constructs: scFv anti-CD19(FMC63)-CD8 hinge and transmembrane-CD3z intracellular. Optionally, the construct comprises the following CD8 signal peptide: CD8 signal peptide-scFv anti-CD19(FMC63)-CD8 hinge and transmembrane-CD3z intracellular.

[0099] CARs or other antigen receptors may also be inhibitory CARs (e.g., iCARs) and contain intracellular components that weaken or suppress responses such as immune responses. Examples of such intracellular signaling components are found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and EP2 / 4 adenosine receptors including A2AR. In some embodiments, engineered cells contain inhibitory CARs containing signaling domains of or derived from such inhibitory molecules, thereby functioning to weaken responses. Such CARs can be used, for example, to reduce the potential for off-target effects when activating receptors, such as antigens recognized by the CAR, are expressed or could be expressed on the surface of normal cells.

[0100] TCR In some embodiments, the genetically engineered antigen receptor includes recombinant T cell receptors (TCRs) and / or TCRs cloned from naturally occurring T cells.

[0101] A “T cell receptor” or “TCR” refers to a molecule containing variable α and β chains (also known as TCRa and TCRp, respectively) or variable γ and δ chains (also known as TCRy and TCR5, respectively) that can specifically bind to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR exists in the αβ form. Typically, TCRs existing in the αβ and γδ forms are generally structurally similar, but the T cells expressing them may have distinct anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), and they are generally involved in recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, Part 3, Current Biology Publications, pp. 4:33, 1997). For example, in some embodiments, each chain of the TCR may possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is bound to an invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass its functional TCR fragment. The term also encompasses intact or full-length TCRs, including TCRs in αβ or γδ morphology.

[0102] Therefore, for the purposes of this specification, references to TCR include any TCR or functional fragment, such as the antigen-binding portion of a TCR that binds to a specific antigen peptide bound to an MHC molecule, i.e., an MHC-peptide complex. The “antigen-binding portion” or “antigen-binding fragment” of a TCR, which may be used interchangeably, refers to a molecule that contains a portion of the structural domain of a TCR but binds to an antigen (e.g., an MHC-peptide complex) to which a complete TCR binds. In some cases, the antigen-binding portion contains variable domains of the TCR, such as the variable a-chain and variable β-chain of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as when each chain generally contains three complementarity-determining regions.

[0103] In some embodiments, the variable domains of the TCR chain associate to form loops, or complementarity-determining regions (CDRs) similar to those of immunoglobulins, which determine peptide specificity by conferring antigen recognition and forming binding sites for the TCR molecule. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Pwc. Nat'lAcad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR involved in recognizing the processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigen peptide, while CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain may contain an additional hypervariability (HV4) region.

[0104] In some embodiments, the TCR chain contains a constant domain. For example, like immunoglobulins, the extracellular portion of a TCR chain (e.g., a chain, β chain) may contain two immunoglobulin domains, a variable domain at the N-terminus (e.g., Va or Vp; typically amino acids 1-116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, Part 5), and one constant domain close to the cell membrane (e.g., the a chain constant domain or Ca, typically amino acids 117-259 based on Kabat, the β chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains a short connecting sequence in which cysteine ​​residues form disulfide bonds, creating a link between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues on each of the a and β chains, thereby the TCR containing two disulfide bonds in its constant domain.

[0105] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to bind to other molecules such as CD3. For example, a TCR containing a constant domain along with a transmembrane region may immobilize the protein on the cell membrane and bind to the invariant subunit of the CD3 signaling apparatus or complex.

[0106] Generally, CD3 is a polyprotein complex that can possess three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex may contain a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of the CD3ζ chain. The CD3y, CD35, and CD3s chains are highly closely related cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, a feature that allows these chains to bind to positively charged T cell receptor chains. Each of the intracellular tails of the CD3y, CD35, and CD3s chains contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while each CD3ζ chain has three. Generally, ITAMs are involved in the signaling activity of the TCR complex. These auxiliary molecules possess negatively charged transmembrane domains and play a role in transmitting signals from the TCR into the cell. The CD3 chain and ζ chain, together with the TCR, form what is known as the T cell receptor complex.

[0107] In some embodiments, the TCR may be a heterodimer of two chains a and β (or optionally γ and δ), or it may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer containing two distinct chains (a and β chains or γ and δ chains) linked by one or more disulfide bonds, etc.

[0108] A recombinant HLA-independent (or non-HLA-restricted) T cell receptor (referred to as "HI-TCR") that binds to an antigen of interest in an HLA-independent manner is described in International Application WO2019 / 157454. Such an HI-TCR comprises an antigen-binding chain including (a) an antigen-binding domain that binds to the antigen in an HLA-independent manner, e.g., an antigen-binding fragment of an immunoglobulin variable region; and (b) a constant domain that can bind to (and is consequently activated by) a CD3ζ polypeptide. Typically, since TCRs bind to antigens in an HLA-dependent manner, the antigen-binding domain that binds in an HLA-independent manner must be heterogeneous. Preferably, the antigen-binding domain or fragment thereof includes (i) the heavy chain variable region (VH) of an antibody, and / or (ii) the light chain variable region (VL) of an antibody. The constant domain of the TCR is, for example, a natural or modified TRAC polypeptide, or a natural or modified TRBC polypeptide. The constant domain of a TCR is, for example, the native TCR constant domain (alpha or beta) or a fragment thereof. Unlike chimeric antigen receptors, which typically contain intracellular signaling domains themselves, HI-TCRs do not directly produce an activation signal; instead, the antigen-binding chain binds to the CD3ζ polypeptide and is activated as a result. Immune cells containing recombinant TCRs exhibit superior activity when the antigen has a low density on the cell surface, less than approximately 10,000 molecules per cell.

[0109] The CD3ζ polypeptide is, for example, a naturally occurring CD3ζ polypeptide or a modified CD3ζ polypeptide. The CD3ζ polypeptide is optionally fused to the intracellular domain of a co-stimulatory molecule or a fragment thereof. Alternatively, the antigen-binding domain optionally includes a co-stimulatory region, such as an intracellular domain, that can stimulate immune-responsive cells upon binding of an antigen-binding chain to an antigen. Examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, fragments thereof, or combinations thereof. In some embodiments, the recombinant HI-TCR is expressed by a transgene incorporated into endogenous loci of immune-responsive cells, such as the CD3δ, CD3ε, CD247, B2M, TRAC, TRBC, TRDC, and / or TRGC loci. In most embodiments, the expression of the recombinant HI-TCR is driven from the endogenous TRAC or TRBC loci. In some embodiments, a transgene encoding a portion of the recombinant HI-TCR is incorporated into the endogenous TRAC and / or TRBC loci in a manner that disrupts or eliminates the endogenous expression of the TCR, including the native TCRα and / or native TCRβ chains. This disruption prevents or eliminates mispairing between the recombinant TCR and the native TCRα and / or native TCRβ chains in immune-responsive cells. The endogenous loci may also include modified transcriptional terminator regions, such as the TK transcriptional terminator, GCSF transcriptional terminator, TCRA transcriptional terminator, HBB transcriptional terminator, bovine growth hormone transcriptional terminator, SV40 transcriptional terminator, and P2A element.

[0110] Recombinant HI-TCRs can be further combined with other characteristics of the immune cells of the present invention. For example, the immune cells are cells in which the antigen-specific receptor is a modified TCR comprising a heterologous antigen-binding domain and a native TCR constant domain or fragment thereof, and the antigen-specific receptor can activate a CD3 zeta polypeptide. For example, the immune cells may further include at least one chimeric costimulatory receptor (CCR) and / or at least one chimeric antigen receptor.

[0111] Furthermore, in immune cells, nucleic acids encoding the antigen-binding domain of the HI-TCR can be inserted into the endogenous TRAC locus and / or TRBC locus of immune cells. Insertions or other smaller mutations of the HI-TCR nucleic acid sequence can disrupt or eliminate the endogenous expression of the TCR, including the native TCR alpha chain and / or native TCR beta chain. Insertions or mutations can reduce endogenous TCR expression by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. Because a single gene encodes the alpha chain (TRAC), rather than two genes encoding the beta chain, the TRAC locus is a typical target for reducing TCRαβ receptor expression. Therefore, nucleic acids encoding antigen-specific receptors (e.g., CAR or TCR) can be incorporated into the TRAC locus, preferably in the 5' region of the first exon, at a position that significantly reduces the expression of the functional TCR alpha chain. See, for example, Jantz et al., WO2017 / 062451; Sadelain et al., WO2017 / 180989; Torikai et al., Blood, 119(2): pp. 5697-705 (2012); Eyquem et al., Nature, March 2, 2017; 543(7643): pp. 113-117. The expression of endogenous TCR-alpha can be reduced by at least approximately 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In such embodiments, the expression of nucleic acids encoding antigen-specific receptors is optionally controlled by an endogenous TCR-alpha or endogenous TCR-beta promoter.

[0112] Optionally, the immune cell may also include a modified CD3 having a single active ITAM domain, and optionally, the CD3 may further include one or more or two or more co-stimulatory domains. In some embodiments, the CD3 includes two co-stimulatory domains, optionally CD28 and 4-1BB. The modified CD3 having a single active ITAM domain may include, for example, a modified CD3 zeta intracellular signaling domain in which ITAM2 and ITAM3 are inactivated or ITAM1 and ITAM2 are inactivated. In some embodiments, the modified CD3 zeta polypeptide retains only ITAM1, and the remaining CD3ζ domain is deleted (residues 90-164). As another example, ITAM1 is replaced with the amino acid sequence of ITAM3, and the remaining CD3ζ domain is deleted (residues 90-164).

[0113] Thus, the modified immune cells of the present invention may further include two or more or three or more or four or more combinations of the foregoing aspects.

[0114] For example, the modified immune cell is (a) an immune cell in which the antigen-specific receptor is a modified TCR comprising a heterologous antigen-binding domain and a native TCR constant domain or a fragment thereof, the antigen-specific receptor can activate a CD3 zeta polypeptide, and / or the antigen-specific receptor is a CAR, and optionally (b) the immune cell includes a modified CD3 having a single active ITAM domain, for example, in which ITAM2 and ITAM3 are inactivated, and optionally (c) the TCR is under the control of an endogenous TRAC and / or TRBC promoter, and optionally (d) the expression of the native TCR-alpha chain and / or the native TCR-beta chain is disrupted or eliminated. In a further embodiment, the cell may include at least one chimeric co-stimulatory receptor (CCR).

[0115] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for manipulating cells and introducing receptors into cells, are described, for example, in International Patent Applications Publications WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, and U.S. Patent Applications Publications US2002131960, US2013287748, US20130 As described in Patent No. 149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer This includes those described in Discov. April 2013; 3(4): 388-398; Davida et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5): 633-639; and Wu et al., Cancer, March 2012, 18(2): 160-175. In some embodiments, genetically engineered antigen receptors include those described in U.S. Patent No. 7,446,190 and International Patent Application Publication No. WO / 2014055668 A1.

[0116] antigen Some antigens targeted by genetically engineered antigen receptors are expressed in the context of diseases, conditions, or cell types targeted by adoptive cell therapy. These diseases and conditions include proliferative, neoplastic, and malignant diseases and conditions, and more particularly cancers; therefore, in some embodiments, one or more antigens are selected from tumor antigens (e.g., those expressed by tumor cells, and more specifically by cancer cells).

[0117] Cancer can be a solid tumor, or a “liquid tumor” that affects the blood, bone marrow, and lymphatic system, also known as a tumor of hematopoietic and lymphoid tissue, including leukemia and lymphoma in particular. Liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphoblastic leukemia (CLL) (including various lymphomas such as mantle cell lymphoma and non-Hodgkin lymphoma (NHL), adenoma, squamous cell carcinoma, pharyngeal carcinoma, gallbladder and bile duct cancer, and retinal cancers such as retinoblastoma). Includes.

[0118] Solid tumors include, in particular, cancers affecting one of the organs selected from the group consisting of the colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma), uterus, bone (osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma, etc.), liver, kidney, esophagus, stomach, bladder, pancreas, neck, brain (meningioma, glioblastoma, mild astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain tumor, etc.), ovaries, breast, head and neck region, testes, prostate, and thyroid.

[0119] Preferably, the cancers according to the present invention are those that affect the blood, bone marrow, and lymphatic systems, as described above. Typically, the cancers are multiple myeloma or related thereto.

[0120] Diseases covered by the present invention include, but are not limited to, infectious diseases or conditions caused by viruses, retroviruses, bacteria, and protozoa, immunodeficiencies, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc.; and autoimmune or inflammatory diseases or conditions, such as arthritis, e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or diseases or conditions associated with transplantation.

[0121] In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or pathological cells, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on manipulated cells. In some such embodiments, specificity and / or efficacy are enhanced using multi-targeting and / or gene disruption techniques provided herein.

[0122] In some embodiments, the antigen is expressed in cancer cells and / or is a universal tumor antigen. The term “universal tumor antigen” refers to immunogenic molecules such as proteins that are generally expressed at higher levels in tumor cells than in non-tumor cells and in tumors of various origins. In some embodiments, universal tumor antigens are expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% of human cancers. In some embodiments, universal tumor antigens are expressed in at least three, at least four, at least five, at least six, at least seven, at least eight, or more different types of tumors. In some cases, universal tumor antigens may be expressed in non-tumor cells, such as normal cells, but at lower levels than they are expressed in tumor cells. In some cases, universal tumor antigens are not expressed at all in non-tumor cells, such as not being expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double microchromosome 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms oncogene 1 (WT1), livin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin (D1). Peptide epitopes of tumor antigens, including universal tumor antigens, are known in the art and, in some embodiments, can be used to create MHC-restricted antigen receptors such as TCRs or TCR-like CARs (see, for example, published PCT applications WO2011009173 or WO2012135854 and published US application US20140065708).

[0123] In some embodiments, antigens such as CD38, CD138, and / or CS-1 are expressed in multiple myeloma. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, and / or CD44. Antibodies or antigen-binding fragments directed to such antigens are known and include, for example, those described in U.S. Patent Nos. 8,153,765; 8,603477, 8,008,450; U.S. Publication Application No. US20120189622; and published International PCT Applications No. WO2006099875, WO2009080829, or WO2012092612. In some embodiments, such antibodies or their antigen-binding fragments (e.g., scFv) can be used to produce CARs.

[0124] In some embodiments, antigens may be expressed or upregulated on cancer or tumor cells, but may also be expressed on immune cells such as resting or activated T cells. For example, in some cases, the expression of hTERT, survivin, and other universal tumor antigens has been reported to be present on lymphocytes, including activated T lymphocytes (see, e.g., Weng et al. (1996) J Exp. Med., 183:2471-2479; Hathcock et al. (1998) J Immunol, 160:5702-5706; Liu et al. (1999) Proc. Natl Acad Sci., 96:5147-5152; Turksma et al. (2013) Journal of Translational Medicine, 11:152). Similarly, in some cases, CD38 and other tumor antigens may also be expressed on immune cells such as T cells, such as being upregulated on activated T cells. For example, in some embodiments, CD38 is a known T cell activation marker.

[0125] In some embodiments provided herein, immune cells such as T cells may be manipulated to suppress or destroy the genes encoding antigens in the immune cells, thereby preventing the expressed genetically modified antigen receptors from specifically binding to the antigen in the background of their expression on the immune cells themselves. Therefore, in some embodiments, this can avoid off-target effects, such as the binding of the modified immune cells to themselves, which may reduce the efficacy of the modified immune cells, for example in relation to adoptive cell therapy.

[0126] In some embodiments, such as in the case of inhibitory CARs, the target is an off-target marker, such as an antigen expressed on normal or non-infected cells, which is not expressed on infected cells or target cells, but also expresses disease-specific targets that are targeted by activating or stimulating receptors in the same manipulated cells. Exemplary such antigens are MHC molecules, such as MHC class I molecules, in relation to treating diseases or conditions in which such molecules are downregulated but still expressed in non-target cells.

[0127] In some embodiments, the manipulated immune cells may contain antigens that target one or more other antigens. In some embodiments, one or more other antigens are tumor antigens or cancer markers. Other antigens targeted by antigen receptors on the provided immune cells include, in some embodiments, orphan tyrosine kinase receptors ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigens, antifolate receptors, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine (acethycholine) e receptor, GD2, GD3, H It may include MW-MAA, IL-22R-alpha, IL-13R-alpha-2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and cyclins such as MAGE A3, CE7, Wilms tumor 1 (WT-1), cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0128] For example, one or more antigens include pHER95, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD70, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, κ-light chain, KDR, LeY, and L1 cell adhesion. Tumor antigens may be selected from a group including molecules, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB.

[0129] In some embodiments, CARs bind to pathogen-specific antigens. In some embodiments, CARs are specific to viral antigens (such as HIV, HCV, and HBV), bacterial antigens, and / or parasitic antigens.

[0130] In some embodiments, the CAR comprises one or more 4-1BB costimulatory domains and binds to the CD19 antigen (also known as the 19BBz CAR in the literature).

[0131] In some embodiments, the cells of the present invention are genetically engineered to express two or more genetically engineered receptors on the cell, each recognizing a different antigen and typically containing different intracellular signaling components. Such multi-targeting strategies are described, for example, in International Patent Application Publication WO 2014055668 A1 (which describes, for example, a combination of activating and co-stimulating CARs that target two different antigens that are off-target, e.g., individually present on normal cells but present together only on cells of the disease or pathology being treated), and in Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013) (which describes cells expressing activating and inhibitory CARs, such as an activating CAR that binds to one antigen expressed on both normal or unaffected cells and cells of the disease or pathology being treated, and an inhibitory CAR that binds to another antigen expressed only on normal cells or cells that are not to be treated).

[0132] In some contexts, overexpression of stimulating factors (e.g., lymphokines or cytokines) can be toxic to the subject. Therefore, in some contexts, engineered cells contain gene segments that make the cells more susceptible to negative selection in vivo, such as during administration in adoptive immunotherapy. For example, in some embodiments, cells are engineered so that they can be eliminated as a result of changes in the in vivo pathology of the patient to whom they are administered. Negatively selectable phenotypes can arise from the insertion of genes that confer sensitivity to the administered active agent, such as a compound. Genes that can be negatively selected include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers ganciclovir sensitivity (Wigler et al., Cell II:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene; the cellular adenine phosphoribosyltransferase (APRT) gene; and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0133] In other embodiments of the present invention, cells, such as T cells, are not engineered to express recombinant receptors, but rather contain naturally occurring antigen receptors specific to a desired antigen, such as T cells cultured in vitro or ex vivo during an incubation step, to promote the growth of tumor-infiltrating lymphocytes and / or cells having specific antigen specificity. For example, in some embodiments, cells are produced for adoptive cell therapy by isolating tumor-specific T cells, such as autologous tumor-infiltrating lymphocytes (TILs). Direct targeting of human tumors using autologous tumor-infiltrating lymphocytes may, in some cases, mediate tumor regression (see Rosenberg SA et al. (1988) N Engl J Med. 319:1676-1680). In some embodiments, lymphocytes are extracted from excised tumors. In some embodiments, such lymphocytes are grown in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (e.g., IL-2). In some embodiments, such lymphocytes mediate the specific lysis of autologous tumor cells, rather than allogeneic tumor cells or autologous normal cells.

[0134] Additional nucleic acids, such as genes for introduction, may enhance the efficacy of the therapy by promoting the viability and / or function of the introduced cells; genes that provide genetic markers for cell selection and / or evaluation, such as for assessing in vivo survival or localization; genes that improve safety, for example, by making cells more susceptible to negative selection in vivo, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the PCT / US91 / 08442 and PCT / US94 / 05601 publications by Lupton et al., describing the use of bifunctional selectable fusion genes obtained by fusing a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0135] Method for obtaining cells according to the present invention The present invention also relates to a method for producing modified or manipulated immune cells, comprising the step of inhibiting the expression and / or activity of SOCS1 and / or FAS and / or Suv39h1 in immune cells.

[0136] Preferably, a method for obtaining cells according to the present invention further comprises the step of introducing a genetically modified antigen receptor or T cell receptor that specifically binds to a target antigen into immune cells.

[0137] Inhibition of SOCS1 expression and / or activity (and, in some embodiments, additional inhibition of FAS and / or Suv39h1 expression and / or activity), and introduction of genetically engineered antigen receptors that specifically bind to target antigens in immune cells, can be carried out simultaneously or sequentially in any order.

[0138] Inhibition of SOCS1, FAS, Suv39h1, and / or β2m The methods described herein for inhibiting gene expression or protein activity are applicable to four genes / proteins of interest, namely SOCS1, FAS, Suv39h1, and optionally β2m. If cells are defective in addition to SOCS1, the same or different methods can be used to further deficiency the cells in FAS and / or Suv39h1. Thus, the embodiments described herein can be combined according to the knowledge of those skilled in the art.

[0139] According to the present invention, engineered immune cells may be contacted with at least one active agent that inhibits or blocks the expression and / or activity of SOCS1, and optionally, in some embodiments, with at least one additional active agent that inhibits or blocks the expression and / or activity of Suv39h1, FAS, and / or β2m. The present invention also provides embodiments in which Fas is inactivated in immune cells (in particular cells) in combination with Suv39h1 and / or β2m, optionally.

[0140] The active substance may be selected from small molecule inhibitors; antibody derivatives such as peptide inhibitors, intrabodies, nanobodies, or aphibodies; aptamers; nucleic acid molecules that block transcription or translation, such as SOCS1, FAS, or antisense molecules complementary to Suv39h1; RNA interferants such as small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwiRNA (piRNA); ribozymes, and combinations thereof.

[0141] At least one active agent may be an exogenous nucleic acid comprising a) one or more manipulated, non-naturally occurring clustered, regularly spaced short palindromic repeat (CRISPR) guide RNAs that hybridize with SOCS1, Suv39h1, FAS, or β2m genomic nucleic acid sequences, and / or b) a nucleotide sequence encoding a CRISPR protein (typically type II Cas9 protein), and the cell is optionally transgenic with respect to expressing the Cas9 protein. The active agent may also be a zinc finger protein (ZFN) or a TAL protein.

[0142] The term "small organic molecule" refers to molecules of a size comparable to those commonly used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size to approximately 5000 Da, more preferably 2000 Da, and most preferably approximately 1000 Da.

[0143] In some embodiments, the inhibitor of H3K9-histone methyltransferase SUV39H1 is described in: Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. "Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9", Nat Chem Biol. August 2005; 1(3): pp. 143-145; Weber, HP et al., "The molecular structure and absolute configuration of chaetocin", Acta Cryst, B28, pp. 2945-2951 (1972); Udagawa, S. et al., "The production of chaetoglobosins, sterigmatocystin, O-methylsterigmatocystin, and chaetocin by Chaetomium spp. and related fungi", Can. J. microbiol, 25, pp. 170-177 (1979); and Gardiner, DM et al., "The This is ketocin (chaetocin) (CAS 28097-03-2), described in "epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis," Microbiol, 151, pp. 1021-1032 (2005). For example, ketocin is commercially available from Sigma Aldrich.

[0144] Another inhibitor of Suv39h1 may be ETP69 (Rac-(3S,6S,7S,8aS)-6-(benzo[d][1,3]dioxol-5-yl)-2,3,7-trimethyl-1,4-dioxohexahydro-6H-3,8a-epidithiopyrrolo[1,2-a]pyrazine-7-carbonitride), a racemic analog of the epidithiodiketopiperazine alkaloid ketosin A (see WO2014066435, but see Baumann M, Dieskau AP, Loertscher BM et al., Tricyclic Analogues of Epidithiodioxopiperazine Alkaloids with Promising In Vitro and In Vivo Antitumor Activity. Chemical Science (Royal Society of Chemical Science)). See also Chemistry:2010), 2015;6:4451-4457, and Snigdha S, Prieto GA, Petrosyan A et al., H3K9me3 Inhibition Improves Memory, Promotes Spine Formation, and Increases BDNF Levels in the Aged Hippocampus. The Journal of Neuroscience. 2016;36(12):3611-3622).

[0145] The inhibitory activity of the compound can be determined using various methods described in Greiner D. et al., Nat Chem Biol. August 2005;1(3):143-145, or Eskeland, R. et al., Biochemistry 43, pp. 3740-3749 (2004).

[0146] Inhibition of SOCS1, FAS, Suv39h1, and / or β2m in cells can be achieved before or after injection in the target patient. In some embodiments, the previously defined inhibition is performed in vivo after administration of cells to the subject. For example, the Suv39h1 inhibitor defined herein may be included in a cell-containing composition. One or more SOCS1, FAS, Suv39h1, or β2m inhibitors may also be administered separately before, incidentally to, or after administration of cells to the subject.

[0147] Typically, inhibition of SOCS1, FAS, Suv39h1, and / or β2m according to this application can be achieved by incubation of cells according to the present invention with a composition containing at least one previously described pharmacological inhibitor. The inhibitor is included during the proliferation of antitumor T cells in vitro and therefore modifies their reconstitution, survival, and therapeutic efficacy after adoptive transfer.

[0148] Inhibition of SOCS1, FAS, Suv39h1, and / or β2m in cells according to the present invention can be achieved using intrabodies.Intrabodies are antibodies that are produced within the same cell and then bind to those antigens intracellularly (for a review, see, for example, Marschall AL, Dubel S, and Boldicke T, "Specific in vivo knockdown of protein function by intrabodies," MAbs. 2015;7(6):10 pp. 10-35; Van Impe K, Bethuyne J, Cool S, Impens F, Ruano-Gallego D, De Wever O, Vanloo B, Van Troys M, Lambein K, Boucherie C et al., "A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis," Breast Cancer Res 2013;15:R116; Hyland S, Beerli RR, Barbas CF, Hynes NE, Wels W., "Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor," Oncogene) 2003;22:1557~67; Lobato MN, Rabbitts TH. "Intracellular antibodies and challenges facing their use as therapeutic agents", Trends Mol Med 2003;9:390~6; and Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, Benvenuto E. "Engineering stable cytoplasmic intrabodies with designed J Mol Biol. 2003 July 4;330(2):323-32).

[0149] Intrabodies can be created by cloning their respective cDNAs from existing hybridoma clones, or, more conveniently, novel scFv / Fabs can be selected from in vitro display techniques such as phage display, which provide the necessary genes encoding the antibody from the outset and allow for more detailed pre-design of antibody fine specificity. In addition, bacterial, yeast, mammalian cell surface displays and ribosome displays can be employed. However, the most commonly used in vitro display system for the selection of specific antibodies is phage display. In a procedure called panning (affinity selection), recombinant antibody phages are selected by incubation of the antibody phage repertoire with antigens. This process is repeated several times, leading to an enriched antibody repertoire containing specific antigen-binding factors to almost any conceivable target. To date, recombinant human antibody libraries assembled in vitro have already produced thousands of novel recombinant antibody fragments. It should be noted that a prerequisite for specific proteins to be knocked down by cytoplasmic intrabodies is that the antigen is neutralized / inactivated by antibody binding. Five different methods have emerged for producing appropriate antibodies: 1) in vivo selection (antigen-dependent and independent) of functional intrabodies in eukaryotes such as yeast and prokaryotes such as Escherichia coli (E. coli); 2) production of antibody fusion proteins to improve cytoplasmic stability; 3) use of special frameworks to improve cytoplasmic stability (e.g., by grafting CDRs into a stable antibody framework or introducing synthetic CDRs); 4) use of single-domain antibodies to improve cytoplasmic stability; and 5) selection of stable intrabodies without disulfide bonds. These methods are described in particular in Marschall, A. L et al., mAbs 2015, mentioned above.

[0150] The most commonly used form for intrabodies is scFv, consisting of H and L chain variable antibody domains (VH and VL) linked by a short, flexible linker sequence (frequently (Gly4Ser)3) to avoid the need for separate expression and association of two antibody chains of a complete IgG or Fab molecule. Alternatively, the Fab form, which additionally includes the C1 domain of the heavy chain and the constant region of the light chain, is used. More recently, scFab, a new conceivable form for intrabodies, has been described. The scFab form promises easier subcloning of available Fab genes into intracellular expression vectors, but it has not yet been confirmed whether this offers any advantages compared to the well-established scFv form. In addition to scFv and Fab, bispecific forms have been used as intrabodies. A bispecific Tie-2×VEGFR-2 antibody targeting the ER showed an extended half-life compared to its monospecific antibody counterpart. Bispecific transmembrane intrabodies have been developed as a special form that simultaneously recognizes intracellular and extracellular epitopes of epidermal growth factor by combining the individual characteristics of related monospecific antibodies, namely inhibition of autophosphorylation and ligand binding.

[0151] Another intrabody form particularly suitable for cytoplasmic expression is the single-domain antibody (also called a nanobody), derived from camel or consisting of a single human VH domain or human VL domain. These single-domain antibodies often possess advantageous properties, such as high stability; good solubility; ease of library cloning and selection; and high expression yields in E. coli and yeast.

[0152] Intrabody genes can be expressed inside target cells after transfection with an expression plasmid or viral transduction using recombinant viruses. Typically, selection aims to provide optimal intrabody transfection and production levels. The success of transfection and subsequent intrabody production can be analyzed by immunoblotting detection of the produced antibodies, but for accurate assessment of the correct intrabody / antigen interaction, co-immunoprecipitation from HEK 293 cell extracts transiently co-transfected with the corresponding antigen and intrabody expression plasmids may be used.

[0153] Inhibition of SOCS1 and / or FAS and / or Suv39h1 in cells according to the present invention can also be achieved by using aptamers that inhibit or block SOCS1, FAS, or Suv39h1 expression or activity, respectively. An aptamer is a class of molecules that serve as an alternative to antibodies in terms of molecular recognition. An aptamer is an oligonucleotide (DNA or RNA) or oligopeptide sequence that has the ability to recognize virtually any class of target molecules with high affinity and specificity.

[0154] Oligonucleotide aptamers can be isolated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method of random sequence libraries, as described in Turek C. and Gold L., 1990. Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of its own sequence, ultimately chemically modified. Possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena SD, 1999.

[0155] Peptide aptamers consist of a conformationally constrained antibody variable region exhibited by platform proteins such as E. coli thioredoxin A, selected from a combinatorial library by a two-hybrid method (Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. "Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2", Nature. April 11, 1996; 380(6574): pp. 548-550).

[0156] Inhibition of SOCS1, FAS, Suv39h1, and β2m in cells according to the present invention can also be achieved using affibody molecules. Affibody molecules are small proteins engineered to bind with high affinity to a number of target proteins or peptides, mimicking monoclonal antibodies, and are therefore members of the antibody mimetic family (for a review, see Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. June 18, 2010; 584(12): pp. 2670-80). Affibody molecules are based on an engineered variant (Z domain) of the B domain in the immunoglobulin-binding region of staphylococcal protein A, which theoretically has specific binding to any given target. Affibody molecular libraries are typically constructed by randomizing combinations of 13 amino acid sites in helices 1 and 2, including the original Fc-binding surface of the Z domain. The library is typically presented on a phage, followed by biopanning to the desired target. The affinity of the first should be increased, and affinity maturation generally results in improved binding factors and can be achieved by either helix shuffling or sequence alignment combined with directed combinatorial mutagenesis. Newly identified molecules with their modified binding surfaces generally retain the original helix structure and high stability, although unique exceptions with interesting properties have been reported. Due to their small size and rapid folding characteristics, affibody molecules can be produced by chemical peptide synthesis.

[0157] In other embodiments of the present invention, inhibition of SOCS1 and / or FAS and / or Suv39h1 and / or β2m activity can be achieved by gene repression / suppression by gene knockdown using RNA or DNA, particularly recombinant DNA or RNA, typically dsRNA (double-stranded RNA), miRNA (microRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA or DNA, or ribozyme-coding sequences, etc. For the purposes of the present invention, the term “recombinant DNA or RNA” refers to nucleic acid sequences that have been altered, rearranged, or modified by genetic engineering. The term “recombinant” does not refer to alterations of nucleic acid sequences resulting from naturally occurring events such as spontaneous mutation, or from non-spontaneous mutagenesis followed by selective breeding.

[0158] As used herein, the term “RNA” refers to a molecule containing at least one ribonucleotide residue. “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of the a.beta-D-ribofuranose moiety. The term encompasses isolated RNA such as double-stranded RNA, single-stranded RNA, RNA having both double-stranded and single-stranded regions, partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, and modified RNA or analog RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material to the terminal or internal parts of the RNA molecule, for example, at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the subject disclosed herein may also include non-standard nucleotides, such as naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0159] siRNA technology includes RNAi-based techniques that utilize double-stranded RNA molecules having sequences homologous to and complementary to the nucleotide sequence of mRNA transcribed from a gene. siRNA can generally consist of multiple RNA molecules that are homologous / complementary to one region of mRNA transcribed from a gene, or homologous / complementary to different regions.

[0160] Antisense oligonucleotides, including antisense RNA and antisense DNA molecules, are thought to directly block the translation of SOCS1, FAS, H3K9-histone methyltransferase SUV39H1, or β2m, thus inhibiting protein translation or increasing mRNA degradation, and thus reducing the levels of SOCS1, FAS, H3K9-histone methyltransferase SUV39H1, or β2m, respectively, and therefore their activity in cells. For example, antisense oligonucleotides consisting of at least about 15 bases and complementary to a specific region of the mRNA transcript sequence encoding SOCS1, FAS, H3K9-histone methyltransferase SUV39H1, or β2m can be synthesized, for example, by conventional phosphodiester techniques and administered, for example, by intravenous injection or infusion. Methods for using antisense techniques to specifically inhibit the gene expression of genes whose sequences are publicly known are well known in the art (see, for example, U.S. Patents 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0161] As used herein, “RNA interferant” is defined as any active substance that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interferants include, but are not limited to, nucleic acid molecules or fragments thereof, including RNA molecules, short interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of a target nucleic acid by RNA interference (RNAi), all of which are homologous to the target gene of the present invention (e.g., Suv39h1).

[0162] Small inhibitory RNAs (siRNAs) may also function as expression inhibitors for use in accordance with this application. SOCS1 gene expression, FAS expression, H3K9-histone methyltransferase SUV39H1, and / or B2M gene expression may be reduced by contacting a target or cell with small double-stranded RNA (dsRNA), or a vector or construct that induces the production of small double-stranded RNA, thereby specifically inhibiting SOCS1 gene expression, FAS expression, H3K9-histone methyltransferase SUV39H1, or B2M gene expression (i.e., RNA interference or RNAi). Methods for selecting a suitable dsRNA or dsRNA coding vector are well known in the art with respect to genes whose sequences are known (see, for example, Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patents 6,573,099 and 6,506,559; and International Patent Publications WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836). All or part of the phosphodiester bonds of the siRNA of the present invention are advantageously protected. This protection is generally carried out via a chemical route using methods known in the art. The phosphodiester bonds may be protected, for example, by thiol or amine functional groups, or by phenyl groups. The 5' and / or 3' ends of the siRNA of the present invention are also advantageously protected, for example, by using the techniques described above with respect to protecting phosphodiester bonds. The siRNA sequence advantageously comprises at least 12 consecutive dinucleotides or derivatives thereof.

[0163] shRNA (short hairpin RNA) can also function as an expression inhibitor for use in the present invention. shRNA typically consists of a short (e.g., 19-25 nucleotides) antisense strand, followed by a 5-9 nucleotide loop, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure, followed by the antisense strand.

[0164] As used herein, the term “microRNA” (miRNA or RNA) refers to a single-stranded RNA molecule with 21 to 23 nucleotides, preferably 21 to 22 nucleotides, that can regulate gene expression. Each miRNA is processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non-protein-coding genes. Precursor miRNAs have two complementary regions that allow them to form stem-loop-like or folded-like structures. Processed miRNAs (also called “mature miRNAs”) become part of a larger complex that downregulates specific target genes.

[0165] In some embodiments, the recombinant DNA described herein is recombinant DNA encoding a ribozyme. Ribozymes can also function as expression inhibitors for use in the present invention. A ribozyme is an enzymatic RNA molecule capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by endonuclease cleavage. Thus, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze the endonuclease cleavage of the H3K9-histone methyltransferase SUV39H1 mRNA sequence are useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, typically including the following sequences: GUA, GUU, and GUC. Once identified, the short RNA sequence of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for its predicted structural characteristics, such as secondary structures that may impair the oligonucleotide sequence.

[0166] Both antisense oligonucleotides and ribozymes useful as expression inhibitors can be prepared by known methods. These include techniques for chemical synthesis, such as by solid-phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of a DNA sequence encoding an RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors incorporating a suitable RNA polymerase promoter, such as the T7 or SP6 polymerase promoter. Various modifications to the oligonucleotides of the present invention can be introduced as means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'-O-methyl rather than phosphodiesterase linkage within the oligonucleotide backbone.

[0167] The antisense oligonucleotides, siRNAs, shRNAs, and ribozymes of the present invention can be delivered in vivo, either alone or in combination with a vector. In its broadest sense, “vector” means any vehicle capable of facilitating the transfer of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into cells, preferably cells expressing SOCS1, preferably cells expressing SOCS1 and H3K9-histone methyltransferase SUV39H1. Preferably, the vector transports the nucleic acid into the cell with reduced degradation compared to the degree of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that are manipulated by the insertion or incorporation of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid sequence. Viral vectors are preferred types of vectors and include, but are not limited to, nucleic acid sequences derived from RA viruses such as: retroviruses including Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary cancer virus, and Rous sarcoma virus; adenoviruses and adeno-associated viruses; SV40 type viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and retroviruses. Other vectors known to the art but not specifically named can be readily employed.

[0168] Preferred viral vectors are based on non-cellular eukaryotic viruses in which non-essential genes are replaced with genes of interest. Non-cellular viruses include retroviruses (e.g., lentiviruses) whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral incorporation into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are replication-deficient retroviruses (i.e., those capable of directing the synthesis of the desired protein but lacking the ability to produce infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient gene transduction in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfection of the plasmid into a packaging cell line, production of recombinant retrovirus by the packaging cell line, recovery of viral particles from tissue culture medium, and infection of target cells with viral particles) are provided in Kriegler, 1990 and Murry, 1991.

[0169] Preferred viruses for certain applications are adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses already approved for human use in gene therapy. In fact, 12 different AAV serotypes (AAV1-12), each with different histotropies, are known (Wu, Z Mol Ther 2006;14:316-27). Recombinant AAVs are derived from AAV2-dependent parvovirus (Choi, VW J Virol 2005;79:6801-07). Adeno-associated viruses 1-12 can be engineered to be replication-deficient and can infect a wide range of cell types and species (Wu, Z Mol Ther 2006;14:316-27). They further have advantages such as thermal and lipid solvent stability; high transduction frequency in diverse cell lineages, including hematopoietic cells; and lack of co-infection inhibition, thus enabling multiple transduction sequences. Reports suggest that adeno-associated viruses can integrate into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertional mutations and variability in inserted gene expression characteristic of retroviral infections. In addition, wild-type adeno-associated virus infections have been tracked in tissue culture for more than 100 passages in the absence of selective pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in extrachromosomal forms.

[0170] Other vectors include plasmid vectors. Plasmid vectors are extensively described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989. In recent years, plasmid vectors have been used as DNA vaccines for delivering antigen-coding genes to cells in vivo. They are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids, which have promoters adapted to host cells, can express peptides from genes operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Additionally, plasmids can be custom-engineered using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by a variety of parenteral, mucosal, and local routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. They can also be administered intranasal spray or drops, rectal suppositories, and orally. It can also be administered to the epidermal or mucosal surface using a gene gun. Plasmids can be given in aqueous solution, dried on gold particles, or in combination with other DNA delivery systems, including but not limited to liposomes, dendrimers, cochleate delivery vehicles, and microencapsulation.

[0171] Antisense oligonucleotides, siRNAs, shRNAs, or ribozymes or nucleic acid sequences encoding ribozymes according to the present invention are generally under the control of heterologous regulatory regions, such as heterologous promoters. Promoters may be specific to Müller glial cells, microglia, endothelial cells, pericytes, and astrocytes. For example, specific expression in Müller glial cells can be achieved through the promoter of the glutamine synthetase gene. Promoters may be viral vectors such as the CMV promoter, or any synthetic promoter.

[0172] Repression or disruption of the SOCS1 and / or FAS and / or Suv39h1 and / or β2m gene. Inhibition of SOCS1, FAS, Suv39h1, and / or β2m in cells according to the present invention may also be brought about by suppression or disruption of the SOCS1, FAS, Suv39h1, or B2M genes, respectively, by deletion, e.g., deletion of the entire gene, exon, or region, and / or substitution with an exogenous sequence, and / or mutation, e.g., by intragene, typically intra-exonal frameshift or missense mutation. In some embodiments, the disruption results in an immature stop codon being incorporated into the gene, thereby preventing the expression or rendering of the SOCS1, FAS, Suv39h1, or β2m protein non-functional. The disruption is generally performed at the DNA level. The disruption is generally permanent, irreversible, or non-transient. In some embodiments, inducible and / or reversible gene inactivation of SOCS1 (and / or FAS and / or Suv39h1 and / or β2m) may be preferred.

[0173] A well-suited method for editing immune cells for cancer immunotherapy in accordance with this application is described, in particular, in Lucibello F, Menegatti S, Menger L, "Methods to edit T cells for cancer immunotherapy," Methods Enzymol. 2020;631:107-135.

[0174] In some embodiments, gene disruption or suppression is achieved using gene editing agents such as DNA-targeting molecules, including DNA-binding proteins or DNA-binding nucleic acids that specifically bind to or hybridize with genes, or complexes, compounds, or compositions containing them. In some embodiments, the DNA-targeting molecule includes DNA-binding domains, such as zinc finger protein (ZFP) DNA-binding domains, transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domains, clustered regularly spaced short palindromic repeat (CRISPR) DNA-binding domains, or meganuclease-derived DNA-binding domains.

[0175] Zinc fingers, TALEs, and CRISPR system-binding domains can be "engineered" to bind to a given nucleotide sequence.

[0176] In some embodiments, the DNA-targeting molecule, complex, or combination includes a DNA-binding molecule and one or more additional domains, such as effector domains, that promote gene repression or disruption. For example, in some embodiments, gene disruption is carried out by a fusion protein comprising a DNA-binding protein and heterologous regulatory domains or functional fragments thereof.

[0177] Typically, the additional domain is a nuclease domain. Therefore, in some embodiments, gene disruption is induced by gene or genome editing using a manipulated protein, such as a nuclease-containing complex or fusion protein, which consists of a nuclease and a sequence-specific DNA-binding domain fused to or compounded with a nonspecific DNA-cleaving molecule such as a nuclease.

[0178] These target chimeric nucleases or nuclease-containing complexes induce targeted double-strand or single-strand breaks and stimulate cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR), thereby enabling precise genetic modification. In some embodiments, the nuclease is an endonuclease such as a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), an RNA-guided endonuclease (RGEN) such as a CRISPR-related (Cas) protein, or a meganuclease. Such systems are well known in the art (e.g., U.S. Patent No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Publications 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One) See 6:el9722; Li et al. (2011) Nucl. Acids Res. 39:6315~6325; Zhang et al. (2011) Nat. Biotech. 29:149~153; Miller et al. (2011) Nat. Biotech. 29:143~148; Lin et al. (2014) Nucl. Acids Res. 42:e47). Such genetic strategies may use constitutive or inducible expression systems according to well-known methods in the art.

[0179] ZFP and ZFN; TAL, TALE, and TALEN In some embodiments, the DNA-targeting molecule includes one or more DNA-binding proteins, such as zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to effector proteins such as endonucleases. Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Frontiers in Immunology, 4(221), pp. 1-7 (2013).

[0180] In some embodiments, the DNA-targeting molecule comprises one or more zinc finger proteins (ZFPs) or their domains that bind to DNA in a sequence-specific manner. A ZFP or its domain is a domain within a protein or larger protein that binds to DNA in a sequence-specific manner through one or more zinc finger regions of amino acid sequences within the binding domain, whose structure is stabilized by the coordination of zinc ions. Generally, the sequence specificity of a ZFP can be modified by amino acid substitutions at four helical sites (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, in some embodiments, the ZFP or ZFP-containing molecule is engineered to bind to a target site that does not exist naturally, for example, a site of selection. For example, see Beerli et al. (2002) Nature Biotechnol. 20: pp. 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: pp. 313-340; Isalan et al. (2001) Nature Biotechnol. 19: pp. 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: pp. 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: pp. 411-416.

[0181] In some embodiments, the DNA-targeting molecule forms a zinc finger nuclease (ZFN) by being or containing a zinc finger DNA-binding domain fused to a DNA cleavage domain. In some embodiments, the fusion protein includes a cleavage domain (or cleavage half-domain) derived from at least one IIS-type restriction enzyme, and one or more zinc finger-binding domains, which may be manipulated or unmanipulated. In some embodiments, the cleavage domain is derived from the IIS-type restriction endonuclease Fok I. For example, see U.S. Patent Nos. 5,356,802; 5,436,150; and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89: pp. 4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90: pp. 2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91: pp. 883-887; Kim et al. (1994b) J. Biol. Chem. 269: 31, 978-31, 982.

[0182] In some embodiments, ZFNs efficiently induce double-strand breaks (DSBs) at predetermined sites in the coding region of a target gene (i.e., Suv39h1). Typical target gene regions include exons, the region encoding the N-terminal region, the first exon, the second exon, and the promoter or enhancer region. In some embodiments, transient expression of a ZFN promotes highly efficient and permanent disruption of the target gene in engineered cells. In particular, in some embodiments, ZFN delivery results in permanent gene disruption with an efficiency of over 50%. Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA), in collaboration with Sigma-Aldrich (St. Louis, MO, USA), has developed a platform for zinc finger construction (CompoZr) that allows researchers to completely bypass zinc finger construction and validation, providing specifically targeted zinc fingers for thousands of proteins. Gaj et al., Trends in Biotechnology, 2013, 31(7), pp. 397-405. In some embodiments, commercially available zinc fingers are used or custom-made. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).

[0183] In some embodiments, the DNA-targeting molecule includes a naturally occurring or engineered (not naturally occurring) activator-like protein (TAL) DNA-binding domain, such as that found in activator-like protein effector (TALE) proteins. See, for example, U.S. Patent Publication 20110301073. In some embodiments, the molecule is a DNA-binding endonuclease, such as TALE-nuclease (TALEN). In some embodiments, TALEN is a fusion protein comprising a DNA-binding domain derived from TALE and a nuclease catalytic domain that cleaves a nucleic acid target sequence. In some embodiments, the TALE DNA-binding domain is engineered to bind to a target sequence in a gene encoding a target antigen and / or an immunosuppressive molecule. For example, in some embodiments, the TALE DNA-binding domain may target adenosine receptors such as CD38 and / or A2AR.

[0184] In some embodiments, TALENs recognize and cleave target sequences in genes. In some embodiments, DNA cleavage results in double-strand breaks. In some embodiments, breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an incomplete repair process that often results in alterations to the DNA sequence at the site of the cleavage. In some embodiments, the repair mechanism involves the rejoining of the remaining two DNA ends by direct religation (Critchlow and Jackson, Trends Biochem Sci. October 1998; 23(10): pp. 394-398) or via so-called microhomology-mediated end joining. In some embodiments, NHEJ-mediated repair may result in small insertions or deletions that can be used to disrupt and thereby repress genes. In some embodiments, the alteration may be a substitution, deletion, or addition of at least one nucleotide. In some embodiments, cells undergoing a cleavage-induced mutagenesis event, i.e., a mutagenesis event following an NHEJ event, can be identified and / or selected by methods well known in the art.

[0185] TALE repeats can associate to specifically target the Suv39h1 gene (Gaj et al., Trends in Biotechnology, 2013, 31(7), pp. 397-405). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology, 31, pp. 251-258 (2013)). Custom TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Specifically, TALENs targeting CD38 are commercially available (see catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3, available worldwide on the Gencopoeia website at www.genecopoeia.com / product / search / detail.php?prt=26&cid=&key=HTN222870). Exemplary molecules are described, for example, in U.S. Patent Publications US2014 / 0120622 and 2013 / 0315884.

[0186] In some embodiments, the TALEN is introduced as a transgene encoded by one or more plasmid vectors. In some embodiments, the plasmid vector may contain a selection marker that provides identification and / or selection of the vector-receiving cells.

[0187] RGEN (CRISPR / Cas system) Gene repression can be achieved using one or more DNA-binding nucleic acids, such as disruption via RNA-guided endonucleases (RGENs) or other forms of repression by other RNA-guided effector molecules. For example, in some embodiments, gene repression can be achieved using clustered, regularly spaced short palindromic repeats (CRISPR) and CRISPR-related proteins. See Sander and Joung, Nature Biotechnology, 32(4):347–355.

[0188] Generally, the “CRISPR system” refers collectively to transcripts and other elements involved in the expression or direction of activity of CRISPR-related (“Cas”) genes, including the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (which, in the context of the endogenous CRISPR system, includes “direct repeats” and partial direct repeats processed by tracrRNA), the guide sequence (also referred to as “spacers” in the context of the endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0189] Typically, a CRISPR / Cas nuclease or CRISPR / Cas nuclease system comprises a non-coding RNA molecule (guide) that sequence-specifically binds to DNA, and a CRISPR protein having nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system, such as the Cas nuclease, may be derived from a type I, type II, or type III CRISPR system. Preferably, the CRISPR protein is a Cas enzyme such as Cas 9. Cas enzymes are well known in the art; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the Cas nuclease and gRNA are introduced into cells. In some embodiments, the CRISPR system induces a DSB at the target site, followed by the disruption discussed herein. In other embodiments, a Cas9 variant considered a "nickase" may be used to introduce a nick into the single strand at the target site. For example, to improve specificity, paired nickases can also be used, each directed by a different pair of gRNAs that target a sequence. In yet another embodiment, catalytically inactive Cas9 can be fused to heterologous effector domains, such as transcriptional repressors, to influence gene expression.

[0190] Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of a target sequence. Typically, in the context of CRISPR complex formation, the “target sequence” generally refers to a sequence designed to have complementarity with a guide sequence, and hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not necessarily required, provided that there is sufficient complementarity to induce hybridization and facilitate the formation of the CRISPR complex. The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. Generally, a sequence or template that can be used for recombination to a target locus containing the target sequence is referred to as an “editing template,” “edited polynucleotide,” or “edited sequence.” In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0191] It should be noted that in some embodiments, catalytically dead CAS9 (dCas9) can be used in conjunction with an activator or repressor domain to control gene expression.

[0192] In some embodiments, one or more vectors promoting the expression of one or more elements of the CRISPR system are introduced into cells, thereby directing the expression of CRISPR system elements to the formation of CRISPR complexes at one or more target sites. For example, the Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements into distinct vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some embodiments, the CRISPR system elements combined in a single vector can be organized in any suitable orientation. In some embodiments, the CRISPR enzyme, guide sequence, tracr-mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. In some embodiments, the vector includes a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.

[0193] In some embodiments, a CRISPR enzyme (optionally compounded with) a guide sequence is delivered to the cell. Typically, CRISPR / Cas9 technology can be used to knock down the gene expression of Suv39h1 in manipulated cells. For example, a Cas9 nuclease and a guide RNA specific to the Suv39h1 gene can be introduced into cells using a lentiviral delivery vector or one of several known delivery methods or vehicles for transfer into the cell, such as one of several known methods or vehicles for delivering the Cas9 molecule and the guide RNA (see also below).

[0194] In some embodiments, inducible gene repression systems, particularly inducible CRISPR gene inactivation, may be preferred, as described in Chylinski, K., Hubmann, M., Hanna, RE et al., CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat Commun 10, 5454 (2019), or in MacLeod, RS, Cawley, KM, Gubrij, I. et al., Effective CRISPR interference of an endogenous gene via a single transgene in mice. Sci Rep 9, 17312 (2019).

[0195] Delivery of nucleic acids encoding gene disruption molecules and complexes In some embodiments, nucleic acids encoding DNA-targeting molecules, complexes, or combinations are administered to or introduced into cells. Typically, viral and nonviral-based gene transfer methods can be used to introduce nucleic acids encoding components of CRISPR, ZFP, ZFN, TALE, and / or TALEN systems into cells under culture.

[0196] In some embodiments, the polypeptide is synthesized in situ within the cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. In some embodiments, the polypeptide may be produced outside the cell and then introduced therein.

[0197] Methods for introducing polynucleotide constructs into animal cells are known, and non-limiting examples include stable transformation methods in which the polynucleotide construct is integrated into the cell's genome, transient transformation methods in which the polynucleotide construct is not integrated into the cell's genome, and virus-mediated methods.

[0198] In some embodiments, polynucleotides can be introduced into cells by means of recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include microinjection, electroporation, or particle bombardment. Nucleic acids are administered in the form of expression vectors. Preferably, the expression vector is a retrovirus expression vector, an adenovirus expression vector, a DNA plasmid expression vector, or an AAV expression vector. It should be noted that in mammalian expression vectors, the promoter driving Cas9 expression can be constitutive or inductive. The U6 promoter is typically used for gRNA.

[0199] Methods of nonviral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistex, virosomes, liposomes, immunoliposomes, polycationic or lipid: nucleic acid conjugates, naked DNA, artificial virions, and the incorporation of active ingredient-enhancing DNA. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, Cas9 RNP (ribonucleoprotein) may be used. Cas9 RNP consists of purified Cas9 protein conjugated with gRNA. They can be assembled in vitro and delivered directly to cells using standard electroporation or transfection techniques. Cas9 RNP can cleave genomic targets with similar efficiency to plasmid-based expression of Cas9 / gRNA. Cas9 RNP is delivered as an intact complex, is detectable at high levels immediately after transfection, and is rapidly removed from cells via proteolytic pathways.Cas9 RNP delivery to target cells is typically carried out by lipid-mediated transfection or electroporation (for details, see Wang, Ming et al., "Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles," Proceedings of the National Academy of Sciences 113.11(2016): pp. 2868-2873; Liang, Xiquan et al., "Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection," Journal of biotechnology 208(2015): pp. 44-53; Zuris, John A. et al., "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo," Nature biotechnology 33.1(2015): pp. 73-80; or Kim, Sojung et al., "Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins," Genome Research). See pp. 1012-1019 (24.6(2014)).

[0200] RNA or DNA virus-based systems include retrovirus, lentivirus, adenovirus, adeno-associated, and herpes simplex virus vectors for gene transfer.

[0201] For reviews of gene therapy procedures, see Anderson, Science 256: pp. 808-813 (1992); Nabel & Feigner, TIBTECH 11: pp. 211-217 (1993); Mitani & Caskey, TIBTECH 11: pp. 162-166 (1993); Dillon, TIBTECH 11: pp. 167-175 (1993); Miller, Nature 357: pp. 455-460 (1992); Van Brunt, Biotechnology 6(10): pp. 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8: pp. 35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1): pp. 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: pp. 13-26 (1994).

[0202] Reporter genes, including but not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), can be introduced into cells to encode gene products that serve as markers for measuring changes or modifications in gene product expression.

[0203] Cell preparation Cell isolation involves one or more preparation and / or affinity-based cell separation steps in accordance with well-known techniques in the art. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents for, for example, removing unwanted components, enriching with desired components, or lysing or removing cells sensitive to specific reagents. In some examples, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to specific components.

[0204] In some embodiments, cell preparation includes a step of freezing the cells, for example, for cryopreservation, before or after isolation, incubation, and / or manipulation. Any of the various known cryopreservation solutions and parameters may be used in some embodiments.

[0205] Typically, cells are incubated before or together with genetic manipulation and / or SOCS1 (and / or Suv39h1 and / or FAS and / or β2m) inhibition.

[0206] The incubation process may include culturing, incubation, stimulation, activation, growth, and / or reproduction.

[0207] In some embodiments, inhibition of SOCS1 (and / or Suv39h1 and / or FAS and / or β2m in some embodiments) according to the present invention can also be achieved in vivo after injection of cells into the target patient. Typically, inhibition of SOCS1 can be carried out using pharmacological inhibitors as previously described.

[0208] In other embodiments, inhibition of SOCS1 (and / or Suv39h1 and / or FAS and / or β2m in some embodiments) according to the previously described methods may also be carried out during the stimulation, activation, and / or growth steps. For example, PBMCs or purified T cells or purified NK cells or purified lymphoid precursors are grown in vitro in the presence of pharmacological inhibitors of SOCS1 and / or FAS and / or Suv39h1 and / or β2m before adoptive transfer to a patient. In some embodiments, the composition or cells are incubated under stimulating conditions or in the presence of stimulants. Such conditions include those designed to prime cells to genetic engineering, such as those that induce cell proliferation, growth, activation, and / or survival in a population, mimic antigen exposure, and / or introduce genetically engineered antigen receptors.

[0209] Incubation conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, active ingredients, such as nutrients, amino acids, antibiotics, ions, and / or stimulants such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other active ingredients designed to activate cells.

[0210] In some embodiments, the stimulating conditions or stimulant include one or more activators, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some embodiments, the activators turn on or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such activators may include antibodies, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, such as those specific to TCR components and / or costimulatory receptors, bound to a solid support such as beads. Optionally, the augmentation method may further include the addition of anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15, e.g., at a concentration of at least about 10 units / mL of IL-2.

[0211] In some embodiments, incubation is carried out in accordance with techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al., J Immunother. 2012;35(9):651-660, Terakura et al., Blood. 2012;1:72-82, and / or Wang et al., J Immunother. 2012,35(9):689-701.

[0212] In some embodiments, T cells are increased by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to a culture-initiating composition (for example, so that the resulting cell population contains at least about 5, 10, 20, or 40, or more, PBMC feeder cells for each T lymphocyte in the initial population to be increased); and incubating the culture (for example, for a time sufficient to increase the number of T cells). In some embodiments, the non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to inhibit cell division. In some embodiments, the feeder cells are added to the culture medium before the addition of the T cell population.

[0213] In some embodiments, the stimulation conditions include temperatures suitable for human T lymphocyte growth, such as at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally 37 degrees Celsius or about 37 degrees Celsius. Optionally, the incubation may further include the step of adding non-dividing EBV-transformed lymphoblast-like cells (LCLs) as feeder cells. The LCLs may be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some embodiments, the LCL feeder cells are provided in any suitable amount, such as a ratio of at least about 10:1 LCL feeder cells to initial T lymphocytes.

[0214] In this embodiment, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are acquired by stimulating naive or antigen-specific T lymphocytes with the antigen. For example, by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen, an antigen-specific T cell line or clone can be produced in response to the cytomegalovirus antigen.

[0215] In some embodiments, the method includes a step of assessing the expression of one or more markers on the surface of the manipulated or manipulated cells. In one embodiment, the method includes a step of assessing the surface expression of one or more target antigens (e.g., antigens recognized by genetically modified antigen receptors) that are to be targeted by adoptive cell therapy, for example, by affinity-based detection methods such as flow cytometry.

[0216] Vectors and methods for cell gene manipulation In some embodiments, genetic manipulation involves the introduction of the component to be genetically modified, or nucleic acids encoding other components for introduction into cells, such as components encoding gene-disrupted proteins or nucleic acids.

[0217] Generally, manipulating immune cells (e.g., T cells) with CARs requires that the cells be cultured in a way that allows for transduction and proliferation. While transduction can be performed using various methods, stable gene transfer is necessary to enable continuous CAR expression in order to clonally proliferate and sustain the manipulated cells.

[0218] In some embodiments, gene transfer is carried out by first stimulating cell growth, such as T cell growth, proliferation, and / or activation, followed by transduction of the activated cells and subsequent growth in culture to a number sufficient for clinical application.

[0219] Various methods for introducing genetically modified components, such as antigen receptors, such as CARs, are well known and can be used in conjunction with the methods and compositions provided. Exemplary methods include those for introducing nucleic acids encoding receptors, including via viral transduction, such as retroviruses or lentivirals, transposons, and electroporation.

[0220] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as vectors derived from Simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy April 3, 2014; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucle Acids 2, e93; Park et al. Trends Biotechnol. November 2011; 29(11):550-557).

[0221] In some embodiments, retroviral vectors derived from retroviral vectors, such as Moloney's mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeat sequences (LTRs). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically bispecies-specific, meaning they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Several example retrovirus systems are described (for example, U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7: pp. 980-990; Miller, AD (1990) Human Gene Therapy 1: pp. 5-14; Scarpa et al. (1991) Virology 180: pp. 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: pp. 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: pp. 102-109).

[0222] Methods for lentiviral transduction are also known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): pp. 689-701; Cooper et al. (2003) Blood. 101: pp. 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: pp. 97-114; and Cavalieri et al. (2003) Blood. 102(2): pp. 497-505.

[0223] In some embodiments, recombinant nucleic acids are transferred to T cells by electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3):e60298, and Van Tedeloo et al. (2000) Gene Therapy 7(16):pp. 1431-1437). In some embodiments, recombinant nucleic acids are transferred to T cells by translocation (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):pp. 427-437; Sharma et al. (2013) Molec Ther Nucle Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:pp. 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), plasmofusion, cationic liposome-mediated transfection; tungsten particle-induced microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0224] Other methods and vectors for the importation of genetically modified nucleic acids encoding genetically modified products are described, for example, in International Patent Application Publication WO2014055668 and U.S. Patent No. 7,446,190.

[0225] Composition of the present invention The present invention also includes compositions containing cells produced by the methods described and / or provided herein. Typically, the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.

[0226] The pharmaceutical compositions of the present invention generally comprise at least one of the engineered immune cells of the present invention and a pharmaceutically acceptable carrier.

[0227] As used herein, the term "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for pharmaceutically effective administration. Supplementary active compounds may be further incorporated into the composition. In some embodiments, the selection of the carrier in the pharmaceutical composition is determined in part by a specific manipulated CAR or TCR, a vector or cell expressing a CAR or TCR, and a specific method used to administer the vector or host cell expressing a CAR. Thus, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001 to about 2% by mass of the total composition.

[0228] Pharmaceutical compositions are formulated to conform to their intended route of administration.

[0229] treatment method The present invention also relates to cells previously specified for use in adoptive therapy (particularly adoptive T-cell therapy), typically in the treatment of cancer in subjects requiring such therapy. In some embodiments, the cells disclosed herein may be used in allotransfer, particularly in the case of cells lacking SOCS1 and / or FAS, in combination with optional inactivation of Suv39h1 and / or β2m.

[0230] As used herein, “treatment” or “to treat” is defined as the application or administration of cells or compositions comprising cells according to the present invention to a patient in need, for the purpose of curing, resolving, alleviating, reducing, modifying, relieving, improving, enhancing, or influencing a disease, such as cancer or any symptom of a disease (e.g., cancer). In particular, the terms “to treat” or “to treat” refer to reducing or alleviating at least one adverse clinical symptom associated with a disease such as cancer, such as pain, swelling, or low blood cell count.

[0231] In relation to cancer treatment, the terms “to treat” or “to cure” also refer to slowing or reversing the uncontrolled cellular proliferation of a neoplasm, i.e., shrinking an existing tumor and / or halting tumor growth. The terms “to treat” or “to cure” also refer to inducing apoptosis in cancer or tumor cells in a subject.

[0232] The subjects of this invention (i.e., patients) are mammals, typically primates such as humans. In some embodiments, primates are monkeys or apes. Subjects may be male or female and may be of any appropriate age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, subjects are non-primate mammals such as rodents. In some examples, patients or subjects are animal models validated for disease, adoptive cell therapy, and / or assessment of toxic outcomes such as cytokine release syndrome (CRS). In some embodiments of this invention, subjects have cancer, are at risk of having cancer, or are in remission from cancer.

[0233] Cancer can be a solid tumor, or a “liquid tumor” that affects the blood, bone marrow, and lymphatic system, also known as a tumor of hematopoietic and lymphoid tissue, including leukemia and lymphoma in particular. Liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphoblastic leukemia (CLL) (including various lymphomas such as mantle cell lymphoma and non-Hodgkin lymphoma (NHL), adenoma, squamous cell carcinoma, pharyngeal carcinoma, gallbladder and bile duct cancer, and retinal cancers such as retinoblastoma).

[0234] Solid tumors include, in particular, cancers affecting one of the organs selected from the group consisting of the colon, rectum, skin, endometrium, lung (including non-small cell lung carcinoma), uterus, bone (osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma, etc.), liver, kidney, esophagus, stomach, bladder, pancreas, neck, brain (meningioma, glioblastoma, mild astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain tumor, etc.), ovaries, breast, head and neck region, testes, prostate, and thyroid.

[0235] In some embodiments, the subjects are suffering from or at risk of suffering from an infectious disease or condition, including but not limited to viral, retroviral, bacterial, and protozoan infections, immunodeficiencies, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or transplant-related diseases or conditions.

[0236] The present invention also relates to methods of treatment and, in particular, adoptive cell therapy, preferably adoptive T cell therapy, which include administering previously described compositions to subjects in need thereof.

[0237] In some embodiments, the cells or composition are administered to subjects such as those having or at risk of having cancer or any of the diseases mentioned above. In some embodiments, the method thereby treats a disease or condition such as cancer, for example, improving one or more of its symptoms, by reducing the tumor load in cancer expressing an antigen recognized by the manipulated cells.

[0238] Methods for administering cells for adoptive cell therapy are publicly known and may be used in conjunction with the methods and compositions provided. For example, methods for adoptive T cell therapy are described in U.S. Patent Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10): pp. 577-585. See also, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): pp. 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): pp. 84-89; and Davila et al. (2013) PLoS ONE 8(4): e61338.

[0239] In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is carried out by autotransfer, in which cells are isolated and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some embodiments, the cells originate from a subject in need of treatment, such as a patient, and the cells, after isolation and processing, are administered to the same subject.

[0240] In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allotransfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that should or will ultimately receive cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. In such embodiments, the use of cells deficient in SOCS1 and / or FAS, optionally in combination with SUV39h1 and / or β2m inactivation, is preferred.

[0241] Administration of at least one cell according to the present invention to a subject requiring it may be combined with one or more additional therapeutic agents or another therapeutic intervention, simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy at a sufficiently close time interval so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell population is administered before one or more additional therapeutic agents. In some embodiments, the cell population is administered after one or more additional therapeutic agents.

[0242] In relation to cancer treatment, combination cancer therapies may include, but are not limited to, chemotherapy agents, hormones, anti-angiogens, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and / or radiotherapy.

[0243] Immunotherapy includes, but is not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, and cancer vaccines.

[0244] Preferably, the administration of cells in adoptive T-cell therapy according to the present invention is combined with the administration of an immune checkpoint modulator, in particular a checkpoint inhibitor. Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, V-domain Ig suppressor (VISTA) inhibitors for T-cell activation, and CTLA-4 inhibitors and IDO inhibitors. Co-stimulatory antibodies deliver a positive signal through immunomodulatory receptors, including, but not limited to, ICOS, CD137, CD27, OX-40, and GITR. Most preferably, the immune checkpoint modulator includes a PD-1 inhibitor (e.g., anti-PD-1), a PDL1 inhibitor (e.g., anti-PDL1), and / or a CTLA4 inhibitor.

[0245] In addition to or as an alternative to checkpoint blocking, the immune cells (in particular, immune cell compositions) of this disclosure may also be genetically modified to make them resistant to immune checkpoints using gene editing techniques, including but not limited to TALENs and Crispr / Cas. Such methods are known in the art; see, for example, US20140120622. Gene editing techniques may be used to block the expression of immune checkpoints expressed by T cells, including but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA, CTLA-4, and combinations thereof. The immune cells discussed herein may be modified by any of these methods.

[0246] Immune cells conforming to this disclosure may also be genetically modified to express molecules that increase tumor homing, or to deliver inflammatory mediators, including but not limited to cytokines, soluble immunomodulatory receptors, and / or ligands, to the tumor microenvironment.

[0247] The present invention also relates to the use of compositions comprising manipulated immune cells as described herein for the manufacture of pharmaceuticals for the treatment of cancer, infectious diseases or conditions, autoimmune diseases or conditions, or inflammatory diseases or conditions in a subject.

[0248] The present invention also includes a method for producing universal immune cells, particularly universal T cells, usable in allogeneic adoptive therapy, for example, in the treatment of cancer, which includes a step of suppressing FAS and / or SOCS1 activity in T cells (at the gene, mRNA, or gene level, as previously described) in combination with optionally inactivating Suv39h1 and / or β2m.

[0249] The present invention - A process of obtaining at least one type of immune cell from the target. - A step of modifying at least one type of immune cell to inactivate Fas and / or SCO1. - The process of administering at least one type of immune cell, typically in the form of a pharmaceutical composition, to another subject that requires it. Methods for allogeneic adoption therapy, particularly allogeneic cancer adoption therapy, particularly allogeneic ATCT, Optionally, at least one type of immune cell is further modified to express one or more genetically modified antigen receptors as previously described; Optionally, at least one type of immune cell is further modified to inactivate Suv39h1 and / or β2m; Optionally, at least one cell type is a mixed population of CD4+ T cells or CD4+ / CD8+ T cells, as previously described. It also includes methods.

[0250] This method can also be combined with embodiments previously described. [Examples]

[0251] Materials and methods Cell lines and mice B16-OVA and MB49 cell lines, kindly provided by E. Piaggio and C. Thery, and the FFLuc-BFP NALM6 (NALM6) cell line, provided by O. Bernard, were maintained in RPMI-1640 supplemented with 10% FBS. HY male antigen-specific CD45.1 and CD45.2 female Marilyn TCR transgenic Rag2 cells were also maintained. - / - Mice were crossed with Rosa26-Cas9-EGFP knock-in mice (026179, Jackson lab). Thy1.1 and Thy1.2 OT-II TCR transgenic Rag2 - / - Mouse, OVA-specific, and female CD45.1 female OT-I TCR transgenic Rag2 - / - Mouse, male NOD-scid IL2Rg - / - (NSG) mice were also used in this study. Female C57BL / 6 mice were purchased from Charles River Laboratories (L'Arbresle, France). All experiments were conducted using 6-12 week old mice in an animal facility accredited by the French Veterinary Department, in accordance with ethical guidelines approved by the relevant ethics committee (AP AF1S#6030-20 16070817147969 v2, permit #XX DAP 2017-023).

[0252] Cell culture and adoptive transfer Naive CD4+ T cells were obtained from the peripheral lymph nodes of Marilyn or OT-II mice. Lymph nodes and splenocytes from CD45.1 Marilyn mice or Thy1.1 OT-II mice were primed with 10 nM Dby (NAGFN-SNRANSSRSS, Genscript) and 5 μM OVAII peptide (InvivoGen), respectively, to obtain antigen-experienced CD4+ T cells. +T cells were generated in vitro. IL-2 (10 ng / mL) and IL-7 (2 ng / mL) (Peprotech) were added to complete RPMI-1640 supplemented with 10% FBS and 0.55 mM β-mercaptoethanol, starting on day 4 and added every 3 days. Ag-exp OT-I cells derived from lymph nodes and spleen were cultured with 0.5 μM SIINFEKL (InvivoGen) and maintained every 2 days with IL-15 (50 ng / mL) (Peprotech). T cells were labeled with 5 μM CFSE (Invitrogen) in PBS at 37°C for 8 minutes. For in vivo GS screening, see 4.10. 6 Individual Naive CD45.2 Marilyn CD4 + T cells were transferred to the soles of the feet, and 4.10 6 Individual LPS-mature bone marrow-derived dendritic cells (BMDCs) loaded with Dby were vaccinated. 7 days later, 12.10 6 Individual library transduction or 12.10 6 Individual mock transduced CD45.1 Cas9-Marilyn cells were intravenously injected into mice at the same time as 4.10 6 Several Dby-loaded LPS-mature BMDCs were vaccinated in the soles of their feet. For the validation experiment, 10 6 The first cohort of naive CD45.2 Marilyn or Thy1.2 OT-II cells was divided into 10 6 Several peptide-loaded LPS-mature BMDCs were transferred to the soles of vaccinated CD45.2 B6 hosts. After 7 days, 10 6 Individual naive CD45.1 Marilyn, Thy1.1 OT-558 II cells, or 2.10 6 Mice were injected with a second cohort of either Ag-exp CD45.1 Marilyn or Thy1.1 OT-II CD4+ T cells, and then given 10 6LPS-mature BMDCs loaded with peptides were inoculated into the soles of the feet. BMDCs were produced by culturing them for 10 days in complete IMDM containing 20 ng / ml GM-CSF (Peprotech), maturation was induced by 20 hours of treatment with 1 ug / ml lipopolysaccharide (Sigma-Aldrich), and pulsed for 2 hours with 50 nM Dby or 20 μM OVAII peptide. Mice were treated intraperitoneally on days 7, 11, and 11 after ACT (10 mg / kg) with blocking antibodies from Bioxcell, including isotype control rat IgG2b (clone LTF2), IgG2a (clone 2A3), anti-mouse CD122 antibody (clone TM-Beta 1), and anti-mouse IFN-gR (clone GR-20). For adoptive cell therapy, female C57BL6 hosts were treated as described in 1.5.10. 6 Individual male bladder MB49 tumor cells or 4.10 5 One of the B16-OVA melanoma cells was implanted subcutaneously. On day 10 for the MB49 model and on day 7 for the B16-OVA model, 106 Marilyn CD4+ T cells or 2.10 6 Individual OT-I and 2.10 6 Individual OT-II cells were adopted into mice with tumors (n=4-6 / group). For the B16-OVA model, mice were sacrificed when their tumors exceeded 15 mm in diameter.

[0253] Peripheral blood mononuclear cells (PBMCs) derived from healthy donors were isolated by density gradient centrifugation. T lymphocytes were purified using a Pan T cell isolation kit (Miltenyi Biotech) and activated with Dynabeads Human T-Activator CD3 / CD28 (1:1 beads:cells) (ThermoFisher) at a density of 10⁶ cells / mL in X-vivo 15 medium (Lonza) supplemented with 5% human serum (Sigma) and 0.5 mM β-mercaptoethanol. 48 hours after activation, T cells were transduced with lentiviral supernatant of the anti-CD19(FMC63)-CD8tm-4IBB-CD3ζ CAR construct (rLV.EF1.19BBz, Flash Therapeutics) at an MOI of 10. Two days later, the CD3 / CD28 beads were magnetically removed, and CAR T cells were electroporated with Cas9-ribonucleoprotein (Cas9-RNP) and maintained in supplemented X-vivo with IL7 (5 ng / mL) and IL15 (5 ng / mL). Six days after electroporation, CD4 was used for mutagenesis quantification of gDNA and Western blot analysis of SOCS1 expression. + and CD8 + CAR T cells, CD8 + T cells were isolated using a T cell isolation kit (Miltenyi). Male or female 8-12 week old NSG mice were given 4.10 5 10 NALM6 cells were injected intravenously via tail vein injection. Three days later, 2.10 6 CAR T cells were administered intravenously via tail vein injection (Day 0). Tumor volume was measured by bioluminescence imaging using the Lumina IVIS Imaging System (PerkinElmer). Radiance >5.10 6 The mice were slaughtered when [p / s / cm ≤ / sr].

[0254] Cytotoxic assay The cytotoxicity of CAR-transduced T cells was determined by co-culturing CAR T cells (effector) and Nalm6 cells (target) in a triple culture in X-vivo medium at a total volume of 100 μl per well, using the indicated E / T ratio. Maximum luciferase expression (relative luminous units; RLUmax) was determined using target cells alone seeded at the same cell density. After 18 hours, 100 μl of luciferase substrate (Perkin Elmer) was added directly to each well. Luminescence was detected using a SpectraMax ID3 plate reader (VWR). Lysis was determined as (1 - (RLU sample) / (RLUmax)) × 100.

[0255] Antibody and flow cytometry analysis Lymph node cells, spleen cells, and tumor samples enriched with density gradient media (Histopaque, Sigma) were incubated with mouse antibodies (STAR ​​method). Human cultured cells, NSG mouse cell-derived bone marrow cells, and spleen cells were stained with the indicated Ab or human-specific soluble protein:fluorescent dye conjugate antibody (STAR ​​method). Intracellular staining was performed using either intracellular staining permeabilization wash buffer (BD Bioscience) or Foxp3 kit (eBioscience). CAR expression was assessed using 9269-CD-050 Recombinant Human CD19 Fc Chimera Protein (Bio Techne) at a 1 / 100 dilution at 4°C for 1 hour. Viability was evaluated using Fixable Viability Dye eFluor 780 (eBioscience) or Aqua Live dead (Thermo Fisher). Restimulation was performed using 20 ng / mL PMA (Sigma), 1 μM ionomycin (Sigma), and a BD Golgi plug at 37°C for 4 hours. Cell counts were quantified using Cell Sorting Set-up Beads (Life Technologies) and normalized between samples and experiments. Staining was performed in blocking solutions: 5% FCS and 2% anti-FcR 2.4G2. Samples were acquired using LSRII / Fortessa (BD) and analyzed using FlowJo software (V10, Tree Star). Cell sorting was performed using ARIAII (BD).

[0256] Western blot analysis T cells (2.10 6The cells were lysed using RIPA lysis buffer (Thermofisher) and 1× Protease Inhibitor Cocktail (Sigma). Cell debris was removed by centrifugation at 14,000 rpm for 15 minutes at 4°C, and 20-40 μg of protein was separated from the supernatant using SDS-PAGE and transferred to a PVDF membrane. SOCS1 and β-actin (loading control) were visualized using the Chemidoc Touch Imaging system (Biorad) with monoclonal antibody anti-SOCS1 (1 μg / mL) (ab62584; Abcam), anti-actin mouse (Millipore, clone C4), HRP anti-rabbit IgG1 (Cell Signaling Technology), and HRP anti-mouse IgG (Cell Signaling Technology). Signal intensity was quantified using ImageJ software.

[0257] Genome-wide CRISPR-Cas9 screening A lentiviral gRNA plasmid library (Mouse Improved Genome-wide Knockout CRISPR Library v2, Pooled Library #67988#) and mock vector (#67974) for genome-wide CRISPR-Cas9 screening were obtained from Addgene. The libraries were amplified according to the protocol provided by Addgene. Briefly, 4 × 25 μl of NEB 10-beta Electrocompetent E. coli (NEB, catalog number C3020K) was electroporated with 4 × 10 ng / μl, cultured in 4 × 500 mL of ampicillin-treated Luria Bertani (LB), and incubated overnight at 37°C with shaking. Plasmids were extracted using 12 columns of the EndoFree plasmid Maxi kit (Qiagen). To prepare the viral library, 293 T cells in 20 cm dishes (×15) with a low passage (<7) were transfected with 11 μg of gRNA library, 11 μg of psPAX2, and 2.5 μg of pVSV-G. 24 hours after transfection, the medium was replaced with DMEM-1% BSA, and the samples were collected at 48, 60, and 72 hours, then centrifuged, filtered through a 0.45 μM PVDF membrane (Millipore), concentrated using an Amicon Ultra 15 ml centrifuge filter (Merck), and used fresh. One day before T cell transduction, CD4 + T cells, MagniSort Mouse CD4 +Cells were enriched using a T cell enrichment kit (Thermofisher Scientific) and seeded at a density of 1.5106 cells / ml in fresh medium supplemented with IL-2 (10 ng / ml) and IL-7 (2 ng / ml). Cells were spin-fected at 32°C and 900 g for 90 minutes with 10 ug / ml protamine sulfate (Sigma) and 8 ug / ml DEAE-dextran (Sigma). The volume of lentivirus library used was required to achieve an optimal transduction efficiency of 0.3 MOI after 5 days of selection with 5 ug / ml puromycin (Sigma). (CFSEhi and CFSElo Cas9-CD45.1 Marilyn CD4) + T cells were sorted, and their gDNA was extracted using 10 μl of lysis buffer-AL (Qiagen - DNeasy blood and tissue kit) and 1 μl of proteinase K (Qiagen). This was followed by incubation at 56°C for 30 minutes, incubation at 95°C for 30 minutes, and resuspension in 20 μl of ddH2O on ice. The gRNAs were amplified by a two-step PCR method using Herculase II Fusion DNA Polymerase (Agilent). For the first step PCR, all extracted gDNA was used to perform a PCR reaction of approximately 30 × 50 μl with forward primer 50 bp-F and reverse primer 50 bp-R (STAR ​​method); the PCR program used consisted of 16 cycles of 94°C for 180 seconds, 94°C for 30 seconds, 60°C for 10 seconds, and 72°C for 25 seconds, followed by a final 2-minute extension at 68°C.

[0258] The products from the first step PCR were pooled, purified using Ampure XP (Agencourt), and quantified using a dsDNA HS assay kit. Three 50-μl PCR reactions were performed using one of the forward primer Index-F and one of the reverse primers (Index-R1~R6). The PCR program used consisted of 18 cycles of 94°C for 180 seconds, 94°C for 30 seconds, 54°C for 10 seconds, and 72°C for 18 seconds, followed by a final 2-minute extension at 68°C.

[0259] The product of the second step PCR reaction was purified and analyzed using a Caliper Labchip (HT DNA High Sensitivity LabChip Kit; Perkin Elmer) on the DNA sample before sequencing using a Miseq or HiSeq2500 instrument (Illumina) to determine the library representation. DNA quality was assessed and quantified using the Agilent DNA 1000 Series II assay and a Qubit fluorometer (Invitrogen).

[0260] Sequencing was performed using a 25-bp single-ended sequencing protocol preceded by 23 dark cycles marking repeating structures in the target region, with a 10% Phix control.

[0261] Bulk mRNA sequencing and analysis 10 4 ~3.10 4Mouse and human T cells were sorted from lymph nodes and tumors in TCL buffer (Qiagen) containing 1% β-mercaptoethanol. Total RNA was purified using the Single Cell RNA purification kit (Norgen) according to the manufacturer's instructions, including a DNAse treatment step (Qiagen). RNA integrity numbers were then evaluated using the Agilent RNA 6000 pico kit. cDNA synthesis and Illumina-compatible libraries were prepared from total RNA (0.25–10 ng) using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian on the Curie Institute's next-generation sequencing platform, according to the manufacturer's instructions. The libraries were then sequenced using Illumina NovaSeq-S1 in 100 bp paired-end mode (OR HiSeq-Rapid Run-PE100). FASTQ files were mapped to the reference genome hg19 (human) or mm10 (mouse) using Hisat2, and read count tables were created by counting featureCounts from the Subread R package. Read counts were then normalized using EdgeR, and genes with >0.5 cpm expression in at least three sets of replicas were retained for subsequent analysis. Differential gene expression was performed using the limma-voom R package. Enrichment scores were calculated using the fgsea R package. For Affymetrix analysis, gene expression was performed using the Mouse Clariom D chip (Thermo Fisher). RNA samples were amplified using the Ovation Pico WTA System v2 (Nugen) and labeled with the Encore biotin module (Nugen). The arrays were hybridized with 5 μg of labeled DNA and assayed using the GeneChip Scanner 3000 7G (Affymetrix).Raw data was generated and controlled using Expression Console (Affymetrix) at the Institut Curie Genomic facility.

[0262] Genome-wide data processing The FASTQ files obtained after sequencing were demultiplexed using HiSeq Analysis software (Illumina). Then, per-sgRNA read count tables were generated by matching single-ended reads with sgRNA sequences from the genome-wide sgRNA Yusa library (Koike-Yusa et al., 2014) using the MAGeCK (Li et al., 2014) count command. Before mapping, the library was first deselected to remove (i) all sgRNAs that did not map to the reference genome (mm10 in this specification), and (ii) all sgRNAs that mapped to multiple spots in the reference genome (multi-hits). Redundant sgRNAs were then consolidated. Next, normalization coefficients for each sample were calculated using the Trimmed Mean of M-value (TMM) method performed in the edgeR R package (Robinson and Oshlack, 2010). Normalized counts were filtered for low-expression sgRNAs (only sgRNAs with at least 4 counts per million in the three samples were retained) and converted to log2 counts per million using voom, which is run in the limma R package. Differential expression of each sgRNA was calculated using the lmFit function in limma, with high and low CFSE cell fractions from each screening.

[0263] For each sgRNA, enrichment and depletion p-values ​​were calculated using a paired one-sided Student's t-test. From these, a Robust Rang Aggregation (RRA) score (10.1093 / bioinformatics / btr709) was calculated for each gene among multiple sgRNAs (n=5) for each gene. Gene-level association p-values ​​and corresponding adjusted p-values ​​[False Discovery Rate (FDR)] were obtained using a sorting test with 1,000,000 replicates on a randomized gene set of the same size. Finally, the genes were graphed according to the median logarithmic change in sgRNAs supporting these enrichment p-values ​​and RRA scores.

[0264] Cas9-RNP Verification For mouse T cells, 1 μl Oligos crRNA (100 nM) and 1 μl tracrRNA (100 nM) were annealed at 95°C for 5 minutes, and for human T cells, 1 μl Oligos crRNA1 + 1 μl Oligos crRNA2 + 1 μl Oligos tracrRNA were incubated with 10 μg Sp Hifi Cas9 Nuclease V3 at room temperature for 10 minutes (STAR ​​method). 2.10 6Each T cell was resuspended in 20 μl of nucleofection solution containing 3 μl or 4 μl RNPs and transferred to a nucleofection cuvette strip (4D-Nucleofector X kit S; Lonza). Mouse T cells were electroporated using the DN110 program of the 4D nucleofector (4D-Nucleofector Core Unit: Lonza, AAF-1002B), and human CAR T cells were electroporated using the E0115 program. The T cells were then incubated at 32°C for 24–48 hours before resuspending in supplemented fresh medium to increase mutagenesis potency (Doyon et al., 2010). Mouse CD4+ T cells were maintained in complete RPMI containing IL2 (10 ng / mL) and IL-7 (2 ng / mL). Human T cells were maintained in X-Vivo containing 5% human serum and IL7 (5 ng / mL) and IL15 (5 ng / mL). Locus-specific PCR (STAR ​​method) was performed on genomic DNA, and the frequency of NHEJ mutations was assessed by sequencing (Eurofins, Mix2seq) and TIDE analysis (https: / / tide.deskgen.com).

[0265] statistical analysis One-way ANOVA, two-way ANOVA, or Mann-Whitney nonparametric tests were performed using Prism 8.0 software (GraphPad) with p<0.05. Multiple comparisons were corrected using Bonferroni coefficients, and Kaplan-Meier survival curves were compared using log-rank tests.

[0266] result 1) SOCS1 as a major intrinsic checkpoint for T cells, particularly CD4+ T cells. In vivo genome-wide screening uses antigen-experienced CD4 + SOCS1 was identified as a major non-redundant inhibitor of T cell proliferation. The inventors previously demonstrated that Ag-exp CD4+ transgenic T cell proliferation is inhibited during an ongoing immune response, while naive T cells of the same specificity can proliferate efficiently (Helft et al., 2008). + To elucidate the inhibitory mechanisms that control T cell proliferation, they used A b :Dby-specific Marilyn monoclonal CD4 + T cells (TCR-Tg Rag2 - / - Marilyn mice (Lantz et al., 2000) were used. Naive CD45.2 Marilyn CD4 in C57BL / 6 hosts. + Following intravenous (iv) adoptive transfer of T cells, they initiated an immune response by injecting dendritic cells (DCs) loaded with Dby peptide into the soles of their feet (Figure 1A). Such an ongoing immune response was then catapulted to newly induced Ag-specific CD4 + To track T cell fate, they proliferated a first cohort of primed Marilyn cells for one week, followed by IV injection of a second cohort of naive or in vitro activated CD45.1 Marilyn CD4+ T cells (Ag-exp) (Helft et al., 2008). In this monoclonal recall response, in vitro primed Ag-exp CD45.1 Marilyn CD4+ T cells exhibited reduced proliferation and IL-2 production ability compared to naive CD45.1 Marilyn T cells (Figures 1B-1D). This model expands the possibility of genetically engineering Ag-exp CD4+ T cells before analyzing their fate in vivo during the immune response.

[0267] To identify intrinsic negative regulators of the CD4+ T cell immune response, they conducted a positive genome-wide CRISPR screening to explore genes whose inactivation would restore Ag-exp CD4+ T cell proliferation during the immune response. They transduced a genome-wide knockout (GWKO) sgRNA lentiviral library (18,400 genes, 90K sgRNA) into in vitro-generated Ag-exp Marilyn-R26-Cas9 (Cas9) T cells (Tzelepis et al., 2016), achieving an efficiency of 20–25% (BFP+)114 (Figure 1E). After puromycin selection, 40% of the transduced T cells survived and showed a single-infection rate of 75% (Chen et al., 2015). Prior to injection into adoptive hosts, mock and library-transduced Ag-exp Marilyn-Cas9 T cells exhibited a central memory phenotype (CD62L+CD44+), allowing them to similarly hom to dLNs (Figure 1E). Analysis of sgRNA in transduced Marilyn-Cas9 T cells revealed that less than 0.5% of sgRNA was expressed lower compared to the original plasmid library (Figure 1F). They measured 12 × 10⁶ mice per C57BL / 6 mouse. 6 Two independent GWKO pooled screenings were performed using individual Ag-exp library transducers or mock transducer Marilyn-Cas9 T cells (Figure 1A). Seven days after transfusion and priming, proliferation of mock transducer Marilyn-Cas9 cells ceased. However, proliferation of library transducer Marilyn-Cas9 cells was higher compared to the ratio of mock transducer Marilyn-Cas9 cells. lo BFP in a subset + / BFP - Significant recovery was observed, as indicated by the higher ratio, and the release of proliferation blockade by some sgRNAs was shown (Figure 1G).

[0268] In the absence of the first cohort, library transduced Marilyn cells increased to some extent, demonstrating efficient priming (Figure 1G). Following CFSE-based cell sorting of CD45.1 Marilyn-Cas9 T cells, CFSE loThe amplified sgRNA sequence enriched in a subset is used in non-dividing T cells (CFSE). hi ) was compared with sgRNA from CFSE. lo The small amount of sgRNA expressed in the subset demonstrates the effectiveness of in vivo selection. CFSE from two independent screenings. lo Analysis of individual sgRNAs enriched in subsets is used in vivo with Ag-exp CD4 + Socs1 was identified as a major gene involved in the restoration of T cell proliferation (p<1.10). -6 The false detection rate (FDR) was <1% (Figure 1H), while other lower-ranked targets showed an FDR > 0.5. Interestingly, Socs1 sgRNA was found to be CFSE hi CFSE of transduced Marilyn cells from the library injected alone, compared to a subset. lo Significant enrichment was also observed in the subset, which is due to the mutual inhibition of Ag-exp CD4. + This is consistent with the capabilities of T cells. Overall, these data are relevant to T cell biology, particularly the still-unexplored CD4 + This supports the non-redundant and critical role of SOCS1 in T cells.

[0269] The inventors then described two different CD4 + In Marilyn and OT2 cells, which are TCR-Tg models (the latter expressing a TCR specific to MHC-II-restricted ovalbumin peptide), electroporation of individual sgRNA-Cas9 ribonucleoprotein complexes (RNPs) was used (Seki and Rutz, 2018) to expel Ag-exp CD4 + The effect of SOCS1 inactivation on T cell proliferation was assessed.

[0270] In short, a CD4 primed in vitro + RNP was electroporated into TCR-Tg cells. 2.10 6 Individual naive, Ag-exp mock, or Ag-exp sgSOCS1 CD4 +T cells were labeled with CFSE142 and subsequently injected into C57BL / 6 mice as a secondary response factor during the ongoing immune response. In both models, a large increase in naive CD4+ T cells indicated efficient priming, while Socs1 gene inactivation indicated mock Ag-exp CD4+ T cell proliferation. + We observed a braking effect on T cell proliferation (Figure 1I). These results highlight the role of SOCS1 as a key intrinsic regulator involved in Ag-exp CD4+ T cell arrest during the ongoing immune response. In particular, we observed no Treg conversion after Marilyn and OT2 cell transfer in vivo, suggesting that Ag-specific Tregs are not involved in their model, contrary to what was suggested in another report (Akkaya et al., 2019).

[0271] SOCS1 is CD4 + It is a critical node that integrates multiple cytokine signals that actively inhibit T cell function. CD4 + To mechanistically characterize SOCS1-mediated inhibition of T cells, the inventors searched for potential inducing factors, and subsequently, Ag-exp CD4 + The functional consequences of Socs1 inactivation on T cells were assessed. SOCS1 expression in mouse splenocytes is induced by both cytokine and TCR stimulation using various schedules and intensities (Sukka-Ganesh and Larkin, 2016). While basal levels of SOCS1 are present in untreated T cells, the increase in SOCS1 protein levels in response to cytokine stimulation occurs rapidly (6 hours), while its maximum expression occurs 48 hours after TCR stimulation (Sukka-Ganesh and Larkin, 2016). This is consistent with the inhibition timeframe in their model, which is initiated in vivo 2 days after priming (Helft et al., 2008). These results suggest that TCR involvement in the presence of cytokines is induced by Ag-exp CD4 + This suggests that it may be a reason for SOCS1 induction in T cells.

[0272] To assess whether differential sensitivity to cytokine signaling can account for the selective inhibitory activity between naive and antigen-experienced cells, the inventors compared the transcriptional expression of cytokine receptors between selected proliferating (*) and inhibited subsets (**) during an ongoing immune response (Figure 2A). They observed a significant increase in the expression of Il2ra (also called CD25, confirmed at the protein level), Ifngr1, and Ifngr2 in CFSE lo cells compared to CFSE hi cells (Figure 2B). This correlation between inhibited cells and cytokine receptor expression was confirmed at the protein level. Furthermore, naive and Ag-exp CD4 + T cells secrete IL-2, while only Ag-exp murine CD4 + T cells produced both IL-2 and IFN-γ.

[0273] Since SOCS1 is a known regulator of IFN-γ signaling (Alexander et al., 1999), they evaluated the proliferation of Ag-exp IFN-γR - / - murine cells during an ongoing immune response, but the absence of the receptor only slightly restored the expansion of these cells in vivo (Figure 2C). SOCS1 can also be induced by IL-2 in T cells and potently inhibits the IL-2-induced Stat5 function by binding to IL-2Rβ (Liau et al., 2018; Sporri et al., 2001). Using blocking antibodies in conjunction with Ag restimulation of Ag-exp CD4 + T cells in vivo, the inventors then assessed the role of IL-2 and IFN-γ alone and in combination in this inhibition. Blocking of IL-2 signaling using anti-mouse IL-2Rb, which inhibits the binding of IL-2 to the IL-2R, did not reverse the impaired proliferation of Ag-exp CD4 + T cells (Figure 2D). However, blocking of both IL-2 and IFN-γ signaling (anti-IFN-γRα and Ag-exp IFNγ-R - / -The use of Marilyn T significantly rescued the increase in restimulated Ag-exp Marilyn T cells (Figure 2D).

[0274] This indicates redundancy between two cytokine receptors upstream of SOCS1 that impairs Ag-exp CD4 + T cell expansion.

[0275] Next, the inventors estimated the functional consequences of Socs1 deletion on TCR-induced activation of Ag-exp CD4 + T cells, reflected by the expression of the early activation marker CD69, the late activation marker CD25, and the T cell receptor-responsive transcription factor interferon regulatory factor 4 (IRF4) (Figure 2E). After overnight stimulation with titrated peptide-pulsed DCs, both Ag-exp Socs1-inactivated Marilyn and OT2 cells exhibited similar sensitivity to Ag stimulation (Ag dose leading to 50% of the maximal response) compared to mock-treated cells. However, the inventors observed a marked increase in CD25 and IRF4 expression with an elevated "plateau" at higher Ag doses (Figure 2E). This suggests that SOCS1 does not directly regulate proximal signals induced by cognate peptide stimulation but rather inhibits downstream signaling events. This would suggest the release of a negative feedback loop related to the secretion of IL-2 and IFN-γ in the medium.

[0276] Since IRF4 is a central regulator of Th1 cytokine secretion in CD4+ T cells (Mahnke et al., 2016; Wu et al., 2017), they evaluated the ability of Socs1-inactivated CD4 + T cells to exhibit multifunctionality. Socs1-inactivated Marilyn and OT2 cells exhibited a higher percentage of Th1 polycytokine (IFN-γ, TNFα, and IL-2) production after restimulation (Figure 2F). Thus, by integrating several cytokine signals, SOCS1 actively impairs the multifunctionality of Ag-exp CD4 + T cells.

[0277] These findings suggest that SOCS1 is a node that can receive signals from several inputs (IFN-γ and IL2) and interfere with multiple signaling outputs, leading to the blockage of proliferative and effector functions.

[0278] Tumor-responsive Marilyn CD4 + Socs1 inactivation in T cells induces a multifunctional cytotoxic phenotype that enhances rejection of male bladder MB49 tumors. Socs1 inactivated Ag experience CD4 + Since the function of T cells was restored, the inventors have found that adoptively transferred antitumor CD4 + The therapeutic potential of Socs1 deletion on T cells was evaluated. The inventors loaded female C57BL / 6 mice with Dby(HY)-expressing MB49 male bladder cancer cells, and after 10 days, mock or sgSOCS1 Ag-exp Marilyn cells were intravenously transferred (Figure 3A).

[0279] In the absence of Marilyn cell transplantation, immunogenic but nevertheless invasive MB49 tumors grew smoothly through an endogenous immune response (Figure 3B, Figure 3C). Mock Ag-exp Marilyn cell transplantation was observed in MB49 tumors. + This led to T cell and NK cell-dependent rejection (Figure 3B, Figure 3C). However, Ag-exp sgSOCS1 Marilyn CD4 + T cell transfer induced tumor rejection that was partially maintained after antibody-induced depletion (Figures 3B and 3C).

[0280] To determine whether Marilyn sgSOCS1 T cells are helpers or "independent" effectors in ACT, researchers analyzed the number, phenotype, and transcriptome of transferred Marilyn T cells in tumor dissipation lymph nodes (TdLNs), tumors, and distant, unrelated LNs (irr-LNs) on day 7, prior to tumor rejection. Surprisingly, the inventors observed that Ag-exp sgSOCS1 Marilyn T cells infiltrated tumors nearly 10 times more efficiently than mock Marilyn cells (Figure 3D). This was associated with a higher percentage of proliferative Ag-exp sgSOCS1 Marilyn cells in TdLN infiltration compared to mock Marilyn cells, which exhibited dominant cessation of their proliferation (Figure 3E).

[0281] Accurately reflecting this increased proliferation, bulk RNA-seq analysis of Marilyn cells sorted from TdLN revealed upregulation of genes involved in the cell cycle and DNA replication (G2M checkpoint, E2F transcription factor, mitotic spindle), as well as IL2 / STAT5 signaling, in sgSOCS1 Marilyn cells (Figure 3F). This pathway, along with molecules such as Il12rb2, Il2rb, Tbx21, Cxcr3, Cxcr5, Ifng, and Ctla2b (Figure 3G), has recently been suggested to be involved in the differentiation program of cytotoxic CD4 T helper-1 (Th1) cells (Krueger et al. 2021; Sledzinska et al. 2020b) and multifunctional antitumor activity (Z.-C. Ding et al. 2020). Examination of Ag-exp sgSOCS1 protein expression in Marilyn T cells confirmed increased multifunctionality, which is enhanced by Th1 cytokine expression in TdLNs, and the ability to produce granzyme B at tumor sites (Figure 3H, Figure 3I).

[0282] Overall, these data suggest that, along with the strong expression of MHC-II molecules by MB49 tumors (Figure 3J), Ag-exp sgSOCS1 Marilyn T cells may be directly cytotoxic and tumor-killing, in addition to their role as helper T cells.

[0283] Therefore, Socs1 deletion enables Marilyn CD4 T cells to exhibit robust growth and persistence in vivo, infiltrate tumors, and elicit an antitumor response with a multifunctional molecular signature indicating Th1 cell-toxic properties (Figures 3B, 3C, 3D, 3G, 3H, 3I).

[0284] CD4 used in adoptive transfer for melanoma tumors + and CD8 + Differential effects of Socs1 inactivation on T cell characteristics CD4 response to antitumor response + and / or CD8 + To compare the biological effects of Socs1 deletion in T cells, we compared in vitro activated tumor-specific CD4 cells that either lack or do not lack SOCS1 as described above. + and CD8 + T cells were independently generated (Figure 4A). They recognized MHC-II and MHC-I-restricted ovalbumin peptides, respectively: CD90.1, OT2, and CD4. + and CD45.1 OT1 CD8 + Using T cells, B16-OVA melanoma cells were subcutaneously implanted as a tumor model without conditioning or cytokine supply (Figure 4A). Compared to the results presented in Figure 3, Socs1 inactivation in OT2 cells had a slight antitumor effect (Figures 4B, 4C). This suggests the use of a highly immunosuppressive B16 melanoma model or a large number of high-avidity antitumor-specific CD8 cells. + It may have been involved in one of the T cell co-transfers.

[0285] However, after adoptive transfer of sgSOCS1 OT1 T cells (Figures 4B and 4C), we observed significant and prolonged rejection of settled tumors compared to transfer of mock OT1 T cells (p<0.001, log-rank). T cell infiltration 7 days after transfer showed increased accumulation in TdLNs and tumors compared to the group receiving both sgSOCS1 OT1 and sgSOCS1 OT2 cells compared to the group receiving mock-transferred cells (Figure 4D).

[0286] Importantly, in TdLN, Socs1 inactivation is accompanied by a significant increase in fully divided CD4+ T cells, OT2 CD4 + It has a tremendous effect on T cell proliferation, while OT1 CD8 + The pattern of T cell proliferation was hardly affected, and SOCS1 was more affected than CD8 proliferation. + This suggested an effect on T cell survival (Figure 4E). Sixty days after transfer, the number of sgSOCS1 OT2 cells eventually decreased in the blood of B16-OVA-loaded mice, while the central memory sgSOCS1 OT1 cell population remained 15 times more abundant than mock OT1 cells.

[0287] These results suggest that SOCS1 reduces the survival of Ag-exp CD8+ T cells, or CD8 + This suggests that the creation of a long-lived subset of T cells is inhibited. The former hypothesis is more likely, as tumor-infiltrating sgSOCS1 OT1 cells analyzed 14 days after transfer expressed higher mRNA levels of molecules involved in T cell survival (Tnfaip3, Bcl2, Il2ra, Il2rb, Jak2) and cytotoxic / effector molecules (Gzmb, Ifngr, Irf1, Fasl, Srgn, Tbx21). Furthermore, feature analysis highlighted pathways in tumor-infiltrating sgSOCS1 OT1 cells associated with TNFα, IL-2, and IFN-γ responses (FDR<0.05). Interestingly, GSEA of Socs1-inactivated OT1 T cells shows that genes associated with effector function are expressed more than those involved in exhaustion.

[0288] Targeting Socs1 in both OT1 and OT2 cells is CD4 + and CD8 + Both T cells maintain cytokine production associated with effector function (Figure 4F, Figure 4G), while GzmB is associated with CD8 +Increased in T cells (Figure 4F). Overnight in vitro stimulation of sgSOCS1 OT1 cells with titrated SIINFEKL pulsed DCs led to increased IFN-γ and granzyme B production at high antigen doses after Socs1 inactivation, and Socs1 was found to be CD8 + We demonstrated that these cytokines are actively suppressed in T cells.

[0289] The conservation or increase in functionality associated with the increase in the number of both sgSOCS1 OT2 and OT1 cells led to a much higher number of effector cells at the tumor site (Figure 4F, Figure 4G), which likely explained the stronger antitumor effect of Socs1-inactivated T cells. Overall, these results suggest that SOCS1 is effective in controlling CD4 in vivo. + and CD8 + This demonstrates that T cells have unique and differential roles in their regulation.

[0290] Potential for immunotherapy using SOCS1-edited human CD4+ and CD8+ CAR T cells To investigate the therapeutic potential of SOCS1 for human T cell adoptive transfer, we inactivated the SOCS1 gene using Cas9 RNP in human peripheral blood lymphocytes (PBLs) that were activated and then transduced with a chimeric antigen receptor called 19BBz, which contains a CD19-targeting 4-1BB costimulatory domain (Figures 5A and 5B).

[0291] CD8 + This construct (Guedan et al., 2018), known to preferentially enhance the survival of CAR-T cells (CAR8), was found to be effective against CD4 cells, which have a limited in vivo lifespan. + This made it possible to investigate the effects of SOCS1 inactivation on CAR-T cells (CAR4) (Turtle et al., 2016; Yang et al., 2017b).

[0292] After overnight co-culture with acute lymphoblastic leukemia (ALL) FFLuc-BFP NALM6 cell line (NALM6), sgSOCS1 CAR4 and sgSOCS1 CAR8 produced higher levels of effector molecules TNFα, IFN-γ, and GzmB compared to mock CAR T cells in three healthy donors, consistent with twice the toxic activity.

[0293] Furthermore, the inventors have found that NOD-scid IL2Rg injected with NALM6 - / - (NSG) In mice, 4.10 6 Individual PBL mocks or sgSOCS1 processing (2.10 6 CAR4 and 2.10 6 We modeled in vivo CAR therapy by injecting individual CAR8 cells. Seven days after transplantation, the number of sgSOCS1 CAR T cells accumulated in the bone marrow (BM) was twice as high as that of mock CAR T cells (Figure 5C, Figure 5D).

[0294] Reflecting higher T cell infiltration and more efficient tumor control in the bone marrow, the transcriptome profiles of sgSOCS1 CAR4 and CAR8 cells demonstrated upregulation of activation-related molecules (FOS, JUND, CD69, SOCS3), longevity-related factors (IL7R, PIM1 (Knudson et al., 2017), TCF7 (Zhou and Xue, 2012), and KLF2 (Carlson et al., 2006)), resistance to apoptosis (BCL2L11 (Hildeman et al., 2002), NDFIP2 (O'Leary et al., 2016)), and key regulators of cytotoxic effector function (GMZB, interferon-inducible molecules GBP5 (Krapp et al., 2016), and IRF1, as well as killer-related NKG7 (Patil et al., 2018)) (Figure 5E).

[0295] As observed in several studies on CAR-T cell response kinetics (Guedan et al., 2018) and CD4 / 8 CAR T subset analysis in ALL patients (Turtle et al., 2016; Yang et al., 2017b), CAR8 preferentially increased compared to CAR4 in our model. Therefore, they examined the persistence of sgSOCS1 CAR T cells 28 days after transfer. While mock CAR4 decayed over time, sgSOCS1 CAR4 and sgSOCS1 CAR8 significantly accumulated in both the BM and spleen of NSG mice and correlated with NALM6 rejection. Most notably, sgSOCS1 CAR4 increased to the level of sgSOCS1 CAR8 (Figure 5C, Figure 5D). Therefore, compared to their mock CAR counterparts in bone marrow, both sgSOCS1 CAR4 and CAR8 expressed increased levels of cytotoxic / effector-related molecules, including IFNG, FCRL6 (Wilson et al., 2007), CTSB (Balaji et al., 2002), and TBX21, as well as known SOCS1 target / survival genes such as IL2RB, JAK3, BCL3, and CXCL13, consistent with the antitumor activity of sgSOCS1 CAR-T cells (Li et al., 2019).

[0296] Interestingly, the inventors observed subset-specific transcriptome patterns in SOCS1-inactivated CAR4 and CAR8 cells. On the one hand, CAR4 cells showed increased expression of genes associated with the proliferation signature represented by E2F targets (Figure 5F), as well as metabolic genes such as the insulin growth factor regulator HTRA1 (H. Ding and Wu 2018) and the AMPK-TORC1 metabolic checkpoint NUAK1 (Monteverde et al. 2018). On the other hand, CAR8 cells showed signs of enhanced cytotoxicity (GZMB, GZMH, TNFSF10 (TRAIL), Secreted and Transmembrane 1 (SECTM1) (T. Wang et al. 2012), killer cell lectin-like receptor D1 KLRD1 (H. Li et al. 2019)), some of which were confirmed by flow cytometry analysis (Figures 5G, 5H).

[0297] Furthermore, in contrast to sgSOCS1 CAR4 cells, sgSOCS1 CAR8 cells expressed lower levels of E2F targets in vivo (Figure 5F), downregulated genes involved in the cell cycle and DNA replication, and suggested that the higher number of cells found in BM were more related to survival than proliferation (Ren et al. 2002).

[0298] sgSOCS1 CAR cells are PD1 + LAG3 + Although it exhibited a phenotype suggesting increased levels of activation, the sgSOCS1 CAR8 transcription signature at day 28 and over time (from day 28 to day 7) was more similar to effector memory than to the exhausted phenotype (Wherry and Kurachi 2015).

[0299] Regarding sgSOCS1 CAR4 cells, GSEA analysis did not reveal a significant exhaustion phenotype, and at day 28, specific transcription factors, including BATF, TOX, EOMES, or BCL6, with the exception of PRDM1 (Blimp1), were not upregulated compared to mock CAR4 cells (Figure S5J). Overall, this suggests that there is no evidence of exhaustion, even if SOCS1 inhibition leads to hyperactivation in CAR T cells.

[0300] At this later stage, SOCS1 inactivation led not only to an increase in the number of CAR4 and CAR8 cells, but also to higher cytokine secretion and cytotoxic activity (Figure 5G, Figure 5H).

[0301] To elucidate the relative contribution of CAR4 vs. CAR8 T cells to tumor rejection and the importance of SOCS1 inactivation in each subset, we tracked the bioluminescence of NALM6 tumors treated with the following combinations: mock CAR4 mock CAR8, sgSOCS1 CAR4 sgSOCS1 CAR8, mock CAR4 sgSOCS1 CAR8, and sgSOCS1 CAR4 mock CAR8 in vivo.

[0302] The inventors observed a significant delay in tumor progression in both the CAR4 mock-CAR8 sgSOCS1 and CAR4 sgSOCS1-CAR8 mock groups; however, SOCS1 targeting in both subsets was essential to achieve tumor eradication (Figure 5J, Figure 5K). This suggests that the robust growth, persistence, and functionality of sgSOCS1 CAR T cells all explain their optimal synergistic antitumor effect.

[0303] Overall, SOCS1 deletion in both CAR4 and CAR8 is a primary target for improving the efficacy of ACT therapy for solid tumors and hematological malignancies.

[0304] Consideration CD4 during antigen response + In exploring the mechanisms involved in regulating T cell proliferation, we discovered SOCS1 as a non-redundant signaling node that leads to a negative feedback loop downstream of TCR and lymphokine signaling. SOCS1 appears to actively suppress T cell proliferation, survival, and effector function in vivo.

[0305] SOCS1 is CD4 + and CD8 + It demonstrated different inhibitory effects on T cells: CD4 + It can inhibit T cell proliferation, survival, and multifunctionality, while CD8 + This significantly reduces T cell survival and effector function. Current data further demonstrate the potent effect of Socs1 gene inactivation on CD4 T cell enlargement, which is particularly associated with improved CAR-T cell composition and efficacy.

[0306] In the case of a synchronous immune response that can be induced by systemic infection or intravenous injection of an antigen, all naive CD4 T cells are led in simultaneously. However, during a localized asynchronous immune response, new naive cells and recirculated Ag-experienced CD4 T cells continue to enter the LN.

[0307] A cohort system can distinguish between naive and Ag-exp CD4 T cells during an ongoing immune response and assess their inherent differences. Our previous data (Helft et al. 2008) and current work show that, when both naive and Ag-exp CD4 T cells are present, Ag-exp CD4 T cells are at a disadvantage in terms of proliferation, as often happens during recall responses.

[0308] This strong and highly reproducible inhibition of Ag-exp CD4 T cells, likely involved in CD4 T cell response diversity and polyclonality, is not related to Ag elimination, regulatory T cell suppression, or competition among responsive T cells for APCs.

[0309] Instead, the study supported the existence of direct TT interactions that lead to intrinsic, dominant, and preferential inhibition of effector / memory CD4 T cell proliferation (Helft et al. 2008). This also applies to SC-injected solid tumors where Ag-exp CD4 T cells interact with each other in TdLNs, rather than utilizing newly arriving naive CD4 T cells.

[0310] The inventors have now demonstrated that this inhibition is due to the TCR-induced expression of SOCS1 and cytokine receptors. Surprisingly, because the inventors used a constitutive CRISPR / Cas9 system, their in vivo genome-wide positive screening only identified Socs1, likely because genes necessary for in vitro growth and survival were missed.

[0311] In addition, the restoration of Ag-exp Marilyn cell proliferation by blocking both the IL2 and IFN-γ pathways in their model (Figure 2D) suggests genetic redundancy and compensation between inactivating receptors, which could not be revealed by their screening strategy. Although not demonstrated in the current work, the inventors hypothesized that during synaptic TT interactions, Ag-exp CD4 T cells expressing high levels of SOCS1 and cytokine receptors (initiated 2 days after TCR induction) are inhibited by IL2 / IFN-γ produced in the "cis" state and by IL2 produced by naive cells in the "trans" state, which then activates SOCS1.

[0312] Finally, the researchers compared two different CD4s that exhibited distinct avidity and used various types of antigen stimulation, such as DC-peptides or tumor loading. + In T cell models (Marilyn and OT2), SOCS1 was shown to be effective in in vivo Ag-exp CD4 + We demonstrated that it is a major, unique inhibitor of T cell proliferation.

[0313] Overall, this is all CD4 + We emphasize the generalizable aspects of the present invention that are effective on T cells.

[0314] The inventors' data show that cytokine sensing occurs after Ag re-exposure / chronic stimulation on CD4 + This suggests that it plays a role in impairing T cell immunity.

[0315] CD4 + This paradoxical cytokine-mediated suppression of T cells blocks chronic IFN-I signaling during persistent infection, which is linked to CD4 + It has already been described that this enhances T cell-dependent viral clearance (Teijaro et al. 2013; Wilson et al. 2013).

[0316] SOCS1 may be involved in so-called activation-induced cell death (AICD), and IL-2 (Lenardo 1991) or IFN-γ (Berner et al. 2007), which are provided prematurely after antigen stimulation, may affect CD4 + This can lead to apoptosis of T cells (Majri et al. 2018).

[0317] Therefore, the authors observed that SOCS1 inhibits the expression of genes involved in resistance to apoptosis, such as Bcl2, Bcl3, Tnfaip3, and Hopx (Albrecht et al. 2010).

[0318] SOCS1 is used in human and mouse CD4 + In both T cells, inhibiting the expression of E2F targets, which are the main regulators of cell cycle progression, leads to in vivo CD8 + Compared to T cells, CD4 + It also appears to selectively regulate T cell proliferation (JW Zhu et al. 2001).

[0319] Therefore, targeting SOCS1 makes them insensitive to unordered lymphokine-induced cell death, thereby targeting CD4 + It improves T cell survival and proliferation. This phenomenon is observed in human and mouse CD4 in vivo after pre-exposure to cytokines. + SOCS3, another member of the SOCS family involved in T cell damage, has been described (Sckisel et al. 2015). However, SOCS3 expression is associated with Th2 differentiation lineage commitment, while SOCS1 is involved in Th1 differentiation (Egwuagu et al. 2002).

[0320] SOCS1 produces several cytokines essential for antitumor immunity in vitro, including Ag-exp CD4. + Because it negatively regulates T cell capacity (Dobrzanski 2013) (Figure 2), the inventors have identified adoptively transferred antitumor CD4 +We investigated the effects of Socs1 deletion on T cells. Targeting SOCS1 is effective in in vivo with Ag-exp CD4 + It also increases T cell pluripotency and enhances their lymphokine secretion, particularly IFN-γ in TdLNs (Figure 3) and GZMB in tumor sites (Figures 3 and 5).

[0321] Therefore, SOCS1-targeted mice and human CD4 + Both types of T cells exhibit increased expression of the Th1 phenotype, which has cytotoxic characteristics at the tumor site (Figures 3 and 5).

[0322] CD4 + The acquisition of such multifunctional characteristics by T cells has recently been described as a two-step modular program involving IL2 / STAT5 / BLIMP1 (Sledzinska et al. 2020b) and IFN-γ / IL12 / ZEB2 (Krueger et al. 2021).

[0323] These molecules are sgSOCS1 CD4 + STAT5 is significantly upregulated in T cells, and constitutive activation of STAT5 is essential for promoting multifunctional antitumor activity (Z.-C. Ding et al. 2020).

[0324] This suggests that deleting SOCS1 is involved in inducing such a differentiation program, and that it is involved in the adoption of CD4 + This demonstrates that T cells enhance the anti-tumor immune response.

[0325] Without any effect on the OT1 CD8 T cell CFSE pattern (Figure 4E), downregulation of the KEGG pathway was associated with cell cycle / DNA replication at sgSOCS1 CAR8 on day 7 and E2F target / G2M checkpoint genes on day 28 (Figure 5F), suggesting that SOCS1 inactivation actually reduces Ag-dependent proliferation of CD8 T cells in vivo.

[0326] Therefore, the defective enlargement after Ag stimulation is due to SOCS1-deficient CD8 in vivo.+ This has been previously reported in T cells (Ramanathan et al. 2010).

[0327] However, SOCS1 targeting can still enhance cytokine-driven (ag-independent) proliferation of CD8 T cells in vitro in a TCR-dependent manner (Ramanathan et al. 2010; Shifrut et al. 2018), promote the survival of CD8 T cells that accumulate in tumor sites (Figure 4D) (Figure 5E), and robustly increase their cytolytic activity (Figure 4F, Figure 5G) (Shifrut et al. 2018; Wei et al. 2019; Zhou et al. 2014).

[0328] Finally, we demonstrate that SOCS1 inactivation induces differentiation in the effector-memory phenotype in both TCR-Tg and CAR CD8 T cells without significant signs of exhaustion.

[0329] Because of improved in vivo persistence, SOCS1-targeted CD4+ T cells are likely to be subjected to chronic stimulation that can lead to anergy and Treg conversion (Alonso et al. 2018).

[0330] However, SOCS1 is essential for maintaining Foxp3 expression and for inhibiting Treg in vivo (Takahashi et al. 2011;2017).

[0331] Therefore, Socs1 deactivated CD4 + In contrast to Treg genes, T cells exhibit enrichment of conventional T cell markers, and, at later stages, reduced gene expression of FOXP3 and IKZF2 in sgSOCS1 CAR4 compared to mock CAR4.

[0332] Overall, the authors believe that CD4 + We confirmed that targeting SOCS1 in T cells prevents them from converting to Treg cells.

[0333] CD8 +Forced expression of cytokine-coding genes or constructs containing JAK / STAT signaling domains in CAR-T cells enhances their persistence and antitumor effects in vivo, highlighting the importance of signal 3 (mediated by cytokines and initiated after CD3 signaling: signal 1 and co-stimulation: signal 2) for CAR-T cell function (Markley and Sadelain 2010; Quintarelli et al. 2007; Kagoya et al. 2018).

[0334] Here, the inventors believe that inactivating major inhibitors of cytokine signaling in CAR-T cells also enhances their therapeutic potential, and most importantly, CD4 + and CD8 + We will demonstrate that it selectively affects CAR-T cells.

[0335] This has significant implications for designing next-generation adoptive T-cell therapies for cancer and viral infections that have improved efficacy and optimized CD4 / CD8 composition.

[0336] The inventors of the present invention have identified CD4 + We elucidated the importance of signal 3 regulation in T cell biological function and identified key intracellular checkpoints critical to the magnitude, duration, and quality of the T cell immune response, which may demonstrate clinical efficacy.

[0337] 2) In vivo genome-wide CRISPR screening identifies targets to evade host immune rejection. In vivo genome-wide (18,400 genes) CRISPR pooling screening identifies Fas and B2m as non-redundant targets that enable T cell survival in MHC mismatched hosts. Next generation ATCT 1 To address the significant clinical need for such a design, the inventors developed a genome-wide (GS) CRISPR screening to identify factors that enable T cell resistance to allogeneic cell death.

[0338] The inventors established allogeneic rejection screening conditions by transferring activated Marilyn CD4 T cells (C57BL6, H2-Kb) into completely MHC-mismatched BALB / c mice (H2-Kd), demonstrating that most of the donor T cells were rejected from the spleen as early as 4 days after injection (Figures 6A and 6B), enabling them to perform screening using a target window.

[0339] To overcome the challenge of Cas9 delivery in primary T cells, the inventors first developed Rosa26-Cas9 knock-in mice (with broad Cas9 expression and eGFP) (41), and CD45.1 / 1 Marilyn (CD4) anti-Dby TCR transgenic (42) TCR transgenic Rag2 - / - They were bred with mice.

[0340] Genome-wide genetic inactivation of T cells was achieved by incorporating a specific single guide RNA (sgRNA) using the Mouse Improved Genome-wide Knockout CRISPR lentiviral Library v2 (Addgene #67988, BFP reporter), which consists of 90°230 sgRNAs targeting 18,400 mouse genes (Figure 6C).

[0341] This innovative approach enables the rapid, systematic, and unbiased identification of functionally non-redundant, T cell-specific limiting factors in vivo (Dong et al. 2019; Wei et al. 2019).

[0342] A BALB / c mouse with a completely MHC mismatch (H-2K) d ) to 10 7By transferring mock or library mutant CD45.1 Marilyn T cells (C57BL6, H2-Kb) into BALB / c mice, the inventors demonstrated that library mutant Marilyn T cells could survive significantly better than mock Marilyn T cells (Figure 6D, Figure 6E). The surviving library mutant Marilyn T cells were selected from harvested spleens on day 4, and their gDNA was analyzed by deep sequencing.

[0343] MAGECK analysis of gDNA-enriched sgRNAs from library mutant CD45.1 Marilyn T cells derived from BALB / c mice (W. Li et al. 2015), compared with sgRNA diversity from C57BL6 mice, highlighted Fas and β2m as potential targets for reducing T cell allogeneic rejection (p<10). -6 (FDR < 0.07) (Figures 6F, 6G).

[0344] For the verification experiments, the inventors used Marilyn T cells expressing various congenic markers in each mouse, which enabled precise control of the survival of Fas or B2m inactivated T cells (CD45.1 / 1) compared to mock Marilyn T cells (CD45.1 / 2).

[0345] To efficiently inactivate either the Fas or B2m gene in enlarged Marilyn T cells (60-70% inactivation, data not shown), the authors switched to a Cas9-ribonucleoprotein (RNP) strategy (Doench et al. 2016), demonstrating a significant improvement in the survival of Fas-inactivated T cells (H2-Kb) in BALB / c mice (H2-Kd) at day 4 (Figure 6H, Figure 6I). Similarly, B2m-inactivated Marilyn T cells survived better in BALB / c mice compared to mock Marilyn T cells, which serves as a benchmark for the success and technical rigor of the inventors' screening strategy (Figure 6J, Figure 6K).

[0346] Fas targeting enhances resistance to both CD8 T cell and NK cell-mediated allogeneic rejection and can be enhanced in vivo by Socs1 inactivation. It is well known that cellular immune rejection is mediated by activated host alloreactive T and NK cells (Elliott and Eisen 1988; Ciccone et al. 1992; Ruggeri et al. 2002).

[0347] In our initial model (C57BL6 T cells in BALB / c mice), Marilyn T cells that were inactivated for B2m and expressed H2-Kb were effectively eliminated from C57BL6 mice, while they remained alive in BALB / c mice on day 4 (Figure 6J, Figure 6K).

[0348] B2m inactivation in T cells leads to downregulation of MHC-I molecules, which typically triggers their destruction by the auto-loss reactivity of NK cells (Bix et al. 1991). If this mechanism is efficient in C57BL6 mice, then host cell-mediated rejection by BALB / c mice appears to be largely mediated by alloreactive T cells.

[0349] To elucidate the contribution of each subset to targeted inactivated CD45.1 polyclonal T cell (CD4 and CD8) resistance to allogeneic rejection in immune BALB / c mice, we used either NK cells or CD8 T cell depletion antibodies (anti-CD8a 2.43; anti-asialoGM1) in conjunction with the in vivo selection process (Figure 7A).

[0350] After depleting NK cells from BALB / c mice using an anti-GM1 antibody, we observed no significant difference in CD45.1 T cell (H2-Kb) splenic infiltration compared to BALB / c+IgG mice (Figure 7B, Figure 7C).

[0351] However, CD8 T cell depletion using anti-CD8a antibodies was sufficient to restore mock CD45.1 T cell survival to the level of Fas-inactivated CD45.1 T cells (Figures 7B and 7C).

[0352] Overall, this demonstrates that the enhanced survival of Fas-inactivated H2-Kb T cells in BALB / c mice is due to resistance to alloreactive CD8 T cell lysis.

[0353] The inventors further hypothesized that inactivation of the Fas gene could prevent rejection by NK cells and allo-T cells, as well as the killing of sibling CAR T cells.

[0354] In fact, it has recently been shown that CAR-T cells actively transport their target antigens through trogocytosis, thereby promoting sibling-killing T cells (Hamieh et al. 2019a).

[0355] In addition to its persistence, a key aspect of the clinical success of allogeneic CAR T-cell therapy is its robust and immediate antitumor response during the engraftment window.

[0356] The inventors previously discovered that deletion of SOCS1, a non-redundant inhibitory checkpoint in activated T cells, can improve CAR-T cell proliferation and effector function (Del Galy et al. 2021) (Figures 1-5).

[0357] Furthermore, SOCS1-deficient CAR T cells upregulate the TRAIL and FasL molecules (Figures 4 and 5), which is a known escape mechanism used by fetal trophoblast cells against maternal immune tolerance (Vacchio and Hodes 2005).

[0358] Therefore, the inventors hypothesized that in a weaponized graft-promoting system similar to an immune-privileged site in the human body (Forrester et al. 2008), dual inactivation of SOCS1 and FAS would enable allogeneic CAR T cells to accumulate robustly, become more functional, and be insensitive to sibling elimination (Hamieh et al. 2019b).

[0359] Finally, targeting SOCS1, a potent JAK / STAT inhibitor, also increased cytokine-dependent proliferation and survival of TRAC-inactivated CAR T cells prior to injection.

[0360] As shown in Figures 7D and 7E, we efficiently inactivated both the Fas and Socs1 genes in polyclonal T cells derived from C57BL6 donor mice expressing the congenic marker CD45.1. Four days after IV injection of mock or targeted inactivated CD45.1 T cells, splenic infiltration revealed a significantly higher number of viable Fas / Socs1 inactivated T cells compared to Fas-targeted T cells in BALB / c mice (Figure 7G). Double-change analysis showed that Fas-inactivated T cells survived 10 times better than mock T cells, demonstrating that Fas / Socs1 dual inactivation induces a 30-fold enhanced survival of allogeneic T cells in BALB / c mice (Figure 7H).

[0361] The inventors of this invention have identified OTI(H2 b ) or Marilyn (H2 b ) Either mouse and BALB / c H2 d Injectable F1 T cells (H2) produced by mating mice b / d We designed a model of semi-allogeneic transfer in vivo based on (Figure 7I).

[0362] In this model, depletion of either NK cells (using anti-NK1.1 antibody) or CD8 T cells (anti-CD8a 2.43) increased the survival of mock F1 T cells, suggesting that both subsets are alloreactive in this hemi-allogeneic transfer in C57BL6 recipients (Figure 7J). Furthermore, we observed a higher percentage of surviving H2-Kd-expressing F1 T cells after NK depletion compared to H2-Kb-expressing T cells after in vivo selection, implying a role of NK cells in the specific targeting of H2-Kd F1 T cells (Figure 7K).

[0363] Similar to previous models, we demonstrated that Fas-inactivated F1 T cells survive better than mock F1 T cells in C57BL6 recipients, and that Socs1 targeting can enhance the resistance of Fas-deficient T cells to allogeneic catalysis in vivo (Figures 7L and 7M).

[0364] Fas and SOCS1 dual inactivation protects mouse and human tumor-reactive T cells from alloimmune rejection in vivo. To assess the antitumor efficacy of identified hits across the major histocompatibility barrier, we have used F1 mice (H2) to whole-body irradiated (TBI) and reconstituted C57BL6 recipient mice with specific tumors. b / d We adopted a previously developed protocol (Boni et al. 2008) based on the transfer of TCR-Tg donor T cells derived from [the specified organism].

[0365] This type of haploidentical transfer using a conditioned regime is closer to a clinical setting than our in vivo screening strategy in completely mismatched hosts.

[0366] More importantly, it offers the potential to evaluate the long-term behavior and exogenous effects of synergistic targets in immunocompromised models, complementing functional validation using human CAR-T cells in NSG mice.

[0367] After crossing OTI(H2-Kb) or Marilyn(H2-Kb) mice with BALB / c H2-Kd mice, F1 generation T cells (H2b / d) were reconstituted into C57BL6(H2 b In mice irradiated with 7 Gy, the effect is thought to last for up to 24 days (Figure 8A), and is thought to regulate the growth of B16-OVA melanoma or male Dby-expressing bladder tumor MB49, respectively.

[0368] F1 OT-1 T cells (H2 b / d The cells were efficiently inactivated with respect to Socs1 / Fas and injected into irradiated and reconstituted C57BL6 recipient mice with B16-OVA tumors. On day 15, the inventors observed that the adoptive, allogeneic tumor-specific sgFas / sgSocs1 T cells persisted 10 times longer in the spleen (Figure 8B), infiltrated the tumor 100 times more extensively, and maintained cytotoxicity (Figure 8C) compared to their mock counterparts.

[0369] Adapting from an available protocol (Mo et al. 2020), the inventors evaluated the function of Fas and Fas / SOCS1 inactivated CAR T cells in an acute lymphoblastic leukemia model (human ALL, NAML6-luciferase cells), and CAR-T cells should resist immune rejection from allogeneic T cells while protecting NOD / SCID / IL2rγ null (NSG) mice from cancer progression (Figure 8D).

[0370] In short, NSG mice would undergo pretreatment cell ablation (cytoablation) (TBI) to promote robust growth of recipient T cells (A2+ T cells). Subsequently, to avoid nonspecific allogeneic rejection by A2+ T cells, HLA expression would be deleted from tumor cells (B2M inactivation in NALM6-luciferase cells, Naml6 sgB2m), and donor CAR-T cells (A2-) would be TCR inactivated to prevent the destruction of A2+ T cells.

[0371] CD4 and CD8 T cells derived from healthy donors were efficiently transduced with a CD19 CAR bbz construct (Figure 8E), followed by electroporation using sgRNA and HIFI Cas9, after which the cells were subjected to TRAC, FAS, and SOCS1 inactivation (Figures 8E, 8F, and 8G).

[0372] Bone marrow infiltration in NSG mice 15 days after A2-CAR-T cell injection revealed increased engraftment and persistence of A2+ T cells, leukemia eradication in all CAR-T cell-treated groups, and increased survival of FAS-inactivated and FAS / SOCS1-inactivated A2-CAR T cells (Figures 8H, 8I, 8J, and 8K).

[0373] This data indicates that both FAS and FAS / SOCS1 targeting enhance the persistence of CAR-T cells in the presence of allogeneic recipient T cells while preserving antitumor activity.

[0374] (References) TIFF0007886313000006.tif224170TIFF0007886313000007.tif235170TIFF0007886313000008.tif235170TIFF0007886313000009.tif235170 TIFF0007886313000010.tif235170TIFF0007886313000011.tif235170TIFF0007886313000012.tif235170TIFF0007886313000013.tif235170 TIFF0007886313000014.tif235170TIFF0007886313000015.tif235170TIFF0007886313000016.tif235170TIFF0007886313000017.tif235170 TIFF0007886313000018.tif235170TIFF0007886313000019.tif235170TIFF0007886313000020.tif235170TIFF0007886313000021.tif235170

Claims

1. Engineered T cells that are defective in SOCS-1 and FAS, and are CD4+ or CD8+ T cells.

2. The manipulated T cell according to claim 1, further having defects in Suv39h1.

3. The engineered T cell according to claim 1 or 2, further comprising a genetically engineered antigen receptor that specifically binds to a target antigen.

4. A manipulated T cell according to any one of claims 1 to 3, isolated from a subject.

5. The subject is the manipulated T cell according to claim 4, which is a person who has cancer or is at risk of developing cancer.

6. The engineered T cells according to any one of claims 1 to 5, wherein (i) the activity and / or expression of SOCS1 and FAS, or (ii) SOCS1, Suv39h1, and FAS are selectively inhibited or blocked in the engineered T cells.

7. - Gene disruption of the SOCS-1 gene caused by modification of the SOCS-1 nucleic acid such that the SOCS-1 protein has reduced activity or lacks detectable activity, and - Genetic disruption of the FAS gene caused by modifications in the FAS nucleic acid, resulting in the FAS protein having reduced activity or lacking detectable activity. The manipulated T cells according to any one of claims 1 to 5, comprising:

8. The engineered T cell according to claim 7, further comprising gene disruption of the Suv39h1 gene caused by modification in the Suv39h1 nucleic acid such that the Suv39h1 protein has reduced activity or lacks detectable activity.

9. The engineered T cell according to any one of claims 3 to 8, wherein the target antigen is expressed by cancer cells and / or is a universal tumor antigen.

10. The engineered T cell according to any one of claims 3 to 9, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or TCR comprising an extracellular antigen recognition domain that specifically binds to a target antigen.

11. The genetically engineered T cell according to any one of claims 3 to 10, wherein the genetically engineered antigen receptor is a T cell receptor (TCR).

12. - A step of inhibiting the expression and / or activity of SOCS1 in T cells; and - A step of inhibiting the expression and / or activity of FAS in T cells. A method for producing universally genetically engineered T cells, wherein the T cells are CD4+ or CD8+ T.

13. - A step of inhibiting the expression and / or activity of Suv39h1 in the T cells. The method according to claim 12, further comprising:

14. - A step of introducing genetically modified antigen receptors that specifically bind to a target antigen into the T cells. The method according to claim 12 or 13, further comprising:

15. Inhibition of SOCS1 and FAS expression and / or activity affects T cells, - At least one active substance that inhibits SOCS1 expression and / or activity and / or disrupts the SOCS1 gene, - At least one active agent that inhibits the expression and / or activity of FAS and / or disrupts the FAS gene. The method according to any one of claims 12 to 14, comprising the step of bringing into contact with, wherein the active substance is a gene editing agent.

16. The method according to claim 15, wherein the active substance is a gene editing agent comprising a CRISPR guide RNA and a Cas9 nuclease specific to the SOCS1 and FAS genes, respectively.

17. The method according to claim 16, further comprising the step of contacting the T cells with at least one active substance that inhibits the expression and / or activity of Suv39h1 and / or disrupts the Suv39h1 gene, wherein the active substance is a gene editing agent.

18. The method according to claim 17, wherein at least one active agent that inhibits the expression and / or activity of Suv39h1 is a gene editing agent comprising a CRISPR guide RNA and a Cas9 nuclease specific to the Suv39h1 gene.

19. A pharmaceutical composition comprising engineered T cells according to any one of claims 1 to 11, or engineered T cells obtained according to the method according to any one of claims 12 to 18, for use in adoptive cell therapy for cancer.

20. The pharmaceutical composition according to claim 19 for use in allogeneic cell therapy for cancer.

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