Car-t cells with interferon-gamma receptor loss of function

Abstract

This disclosure provides immune cells (e.g., CAR-T cells) comprising a chimeric antigen receptor and / or an engineered immune cell receptor, that are deficient in interferon y receptor (IFNyR) expression and / or signaling, and methods of use thereof, wherein the CAR-immune cell have increased persistence.

Description

[0001] CAR-T CELLS WITH INTERFERON- GAMMA RECEPTOR LOSS OF FUNCTION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 740,544, filed December 31, 2024, entitled “CAR-T Cells With Interferon-Gamma Receptor Loss of Function”, and U.S. Provisional Application No. 63 / 784,645, filed April 7, 2025, entitled “CAR-T Cells With Interferon-Gamma Receptor Loss of Function”, the entire contents of each of which are incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under CA249062 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (M105370056WO00-SEQ-ARM.xml; Size: 145,083 bytes; and Date of Creation: December 24, 2025) are herein incorporated by reference in their entirety.

[0008] BACKGROUND

[0009] Cell therapy has revolutionized the treatment of some cancers. Some cell therapies engineer immune cells (e.g., host immune cells) to attack cancer cells in the host. For example, immune cells can be engineered to express a chimeric antigen receptor (CAR) or an engineered immune cell receptor that target a cell surface antigen of the cancer, thus directing the immune cell to the cancer. Despite success, these engineered immune cells can become exhausted in the host, which can decrease cancer killing efficacy.

[0010] SUMMARY

[0011] This disclosure provides immune cells that are deficient in interferon y receptor (IFNyR) expression and / or signaling. Surprisingly, results described herein show that decreasing the expression and / or activity of IFNyR in CAR-T cells increases CAR-T cell persistence in liquid tumors and solid tumors in in vivo model systems.

[0012] In some embodiments, this disclosure provides an immune cell comprising a first polynucleotide encoding a chimeric antigen receptor and / or an engineered immune cell receptor, wherein the immune cell is deficient in interferon y receptor (IFNyR) expression and / or signaling.

[0013] #14645213vlIn some embodiments, the immune cell comprises a mutation in one or more IFNyR gene loci of the immune cell. In some embodiments, the immune cell comprises an insertion and / or deletion in one or more IFNyR genes of the immune cell. In some embodiments, the insertion and / or deletion results in a frameshift of the one or more IFNyR genes. In some embodiments, the immune cell comprises a loss of function mutation in one or more IFNyR genes of the immune cell. In some embodiments, the immune cell comprises a knockout of one or more IFNyR genes of the immune cell. In some embodiments, the immune cell comprises an insertion and / or deletion, a frameshift, or a knockout of each IFNyR gene of the immune cell.

[0014] In some embodiments, the immune cell comprises a loss of function mutation in each IFNyR gene of the immune cell. In some embodiments, the immune cell further comprises: (i) a functional RNA comprising a portion that is complementary to an IFNyR gene and / or an mRNA transcribed from an IFNyR gene; and / or (ii) a second polynucleotide encoding the functional RNA.

[0015] In some embodiments, the functional RNA is an siRNA, a micro-RNA, a shRNA, an antisense oligonucleotide, a ribozyme, or a guide RNA. In some embodiments, the functional RNA is a guide RNA comprising a homology region that is complementary to a strand of an IFNyR gene of the immune cell. In some embodiments, the guide RNA comprises a sequence of any one of SEQ ID NOs: 32-47. In some embodiments, the immune cell is deficient in TRAC expression. In some embodiments, the immune cell further comprises: (i) a TRAC-targeted guide RNA comprising a homology region that is complementary to a TRAC gene of the immune cell; and / or (ii) a third polynucleotide encoding the TRAC-targeted guide RNA.

[0016] In some embodiments, the immune cell further comprises ribonucleoprotein (RNP) comprising: (i) the guide RNA and / or the TRAC -targeted guide RNA; and (ii) a Cas protein. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the immune cell further comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a meganuclease that targets one or more IFNyR genes of the immune cell. In some embodiments, two or more of the first polynucleotide, the second polynucleotide, and the third polynucleotide are part of the same contiguous polynucleotide. In some embodiments, the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to a promoter. In some embodiments, the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to different promoters. In some embodiments, the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to the same promoter.

[0017] #14645213vlIn some embodiments, the CAR comprises: (i) an extracellular antigen-binding domain, (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single-chain antibody fragment (scFv) that binds a cell surface protein. In some embodiments, the extracellular antigen-binding domain binds CD19, BCMA, TACI, CD79b, CD22, CD30, CS1, Claudin 18.2, GPC3, GD2, GPCR, PSMA, mesothelin, MUC1, MUC16, EGFR, IL-13Ralpha2, EGFRvIII, CD20, CD79a, or combinations thereof. In some embodiments, the extracellular antigen-binding domain is a CD 19 binding extracellular domain.

[0018] In some embodiments, the CD 19 extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 24; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 25; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 26; and (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 27; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 28; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 29.

[0019] In some embodiments, the CD 19 extracellular antigen-binding domain comprises a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 50. In some embodiments, the extracellular antigen-binding domain is a mesothelin binding extracellular domain. In some embodiments, the mesothelin extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 4; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 5; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 7; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 8; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 9.

[0020] In some embodiments, the mesothelin extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 13; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 14; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable

[0021] #14645213vldomain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 16; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 17; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 18.

[0022] In some embodiments, the transmembrane domain is a transmembrane domain of an alpha chain of an immune cell receptor, beta chain of an immune cell receptor, zeta chain of an immune cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ey9 (CD229), CD160 (BY55), PSGE1, CD100 (SEMA4D), SEAMF6 (NTB-A, EylO8), SEAM (SEAMF1, CD150, IPO-3), BEAME (SEAMF8), SEEPEG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0023] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, in the intracellular signaling domain comprises an intracellular signaling domain from 4- IBB, CD27, CD28, 0X4, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain further comprises a TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain and a CD3-zeta intracellular signaling domain.

[0024] In some embodiments, the extracellular antigen-binding domain further comprises a leader. In some embodiments, the CAR comprises any amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the CAR comprises any amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the CAR comprises any amino acid sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the

[0025] #14645213vlimmune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, this disclosure provides a polynucleotide encoding: (i) a chimeric antigen receptor (CAR); and (ii) a guide RNA comprising a homology region that is complementary to IFNgR. In some embodiments, the polynucleotide further comprises a nucleic acid encoding a guide RNA that comprises a homology region that is complementary to TRAC.

[0026] In some embodiments, this disclosure provides a method of treating cancer in a subject, the method comprising administering an immune cell described herein. In some embodiments, the CAR comprises a CD28 intracellular signaling domain. In some embodiments, the cancer is a CD 19 expressing cancer, the CAR comprises an extracellular antigen binding domain that bind to CD 19. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is leukemia or lymphoma. In some embodiments, the cancer is a mesothelin expressing cancer, the CAR comprises an extracellular antigen binding domain that bind to mesothelin. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is pancreatic cancer. In some embodiments, this disclosure provides a method of increasing the persistence of a CAR-immune cell in a subject, the method comprising decreasing the expression of interferon y receptor (IFNyR) in the immune cell. In some embodiments, the method further comprises knocking out one or more interferon y receptor (IFNyR) genes in the immune cell. In some embodiments, knocking out one or more IFNyR genes comprises introducing a loss of function mutation into the one or more IFNyR genes. In some embodiments, knocking out one or more IFNyR genes comprises introducing an insertion, a deletion, and / or a frameshift into the one or more IFNyR genes. In some embodiments, decreasing the expression of IFNyR in the immune cell comprises decreasing the expression using CRISPR. In some embodiments, decreasing the expression using CRISPR, comprising introducing a loss of function mutation into one or more IFNyR genes of the immune cell using CRISPR. In some embodiments, decreasing the expression of IFNyR in the immune cell comprises decreasing the expression using RNA interference. In some embodiments, decreasing the expression of IFNyR in the immune cell comprises decreasing using an antisense oligonucleotide (ASO). In some embodiments, the CAR comprises a CD28 intracellular signaling domain. In some embodiments, this disclosure provide a method of increasing the persistence of a CAR-immune cell in a subject, the method comprising contacting the CAR-immune cell with an anti-IFNgR antibody. In some embodiments, this disclosure provide a method of increasing the persistence of a CAR-immune cell in a subject, the method comprising administering the CAR-immune cell and an anti-IFNgR antibody.

[0027] #14645213vlBRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIGs. 1A-1I show deletion of IFNg or IFNg receptor drives greater CD28 CAR T cell expansion in vitro. FIG. 1A shows constructs used to generate CAR T cells with a CD 19 scFv and CRISPR / Cas9 editing of T cell receptor A constant (TRAC; CARCDI ), TRAC and IFNg (IFNgKO CARCDW) or TRAC and IFNg receptor 1 (IFNgRKO CARCDW) from healthy donor T cells. FIGs. 1B-1C show expression of CD3 and IFNgR on resting CAR-T and donor-matched untransduced T cells (UTD) (FIG. IB) and IFNg following a six-hour activation with PMA and ionomycin (FIG. 1C) was assessed by flow cytometry; representative histograms and graphs, n=3. FIG. ID shows the functional loss of IFNgR was confirmed via phosphoSTATl signaling following exposure of CAR-T to recombinant human IFNg; representative histogram, n=3. FIG. IE shows CD4 and CD8 expression was determined using flow cytometry; n=6. FIG. IF shows CAR-T were activated overnight with CD 19 antigen and cytokines were observed by Euminex; averages shown from n=3 biological replicates. FIG. 1G shows expansion of CD28 CAR-T in response to Nalm6 cells (left) or JeKo-1 (right) cells was monitored using real-time Incucyte imaging and statistics were calculated at 24-, 48- and 72-hour timepoints; n=5 biological replicates. FIG. 1H shows constructs used to generate CAR T cells with a CD 19 scFv, 4- IBB costimulatory domain and CRISPR / Cas9 editing of T cell receptor A constant (TRAC;

[0030] CARCDW), TRAC and IFNg (IFNgKO CARCDW) or TRAC and IFNg receptor (IFNgRKO CARCDW) from healthy donor T cells. FIG. II shows expression of CD3 and IFNgR on resting CAR-T and donor-matched UTD T cells (FIG. II; left, middle) and IFNg following a six-hour activation with PMA and Ionomycin (FIG. II; right) was assessed by flow cytometry; representative histograms, n=3 biological replicates. FIG. 1J shows expansion of 4- IBB CAR T cells in response to Nalm6 cells was monitored using real-time Incucyte imaging; n=5 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ****<0.0001, ns=not significant

[0031] FIGs. 2A-2F show IFNgKO and IFNgRKO CARCDI9 T cells maintain antitumor activity against hematologic malignancies and display greater persistence in vivo. FIG. 2A shows CAR-T were mixed at a 1:1 ratio with Nalm6 cells and T cell cytolysis was observed by Incucyte. At 3 and 6 days, cells were collected, counted and CAR-T were re-plated at a 1 : 1 ratio with fresh Nalm6 cells before being returned to the Incucyte; n=3 biological replicates. FIGs. 2B-2F show

[0032] #14645213vlNSG mice bearing intravenously administered CBGGFP+JeKo-1 tumor cells (le6I.V.) were left untreated (tumor only) or treated with le6CARCDW, IFNgKO CARCDI or IFNgRKO CARCDI9 T cells (I.V.) (FIG. 2B). Tumor burden was assessed using weekly bioluminescent imaging and graphed by flux (FIGs. 2C-2D) and overall survival was monitored (FIG. 2E). Mice were bled 14 days post-CAR-T infusion and T cell persistence was determined using flow cytometry (FIG.

[0033] 2F). For FIGs. 2B-2F, n=4-5 mice / group for each healthy donor (2 donors total). Data are shown as mean ± s.e.m. with P values by One-way ANOVA (FIGs. 2D, 2F) or Kaplan-Meier survival curve (FIG. 2E). P: *<0.05, ****<0.0001, ns=not significant.

[0034] FIGs. 3A-3E show abrogation of IFNg signaling prompts minimal effects on CD 19 CAR-T differentiation and exhaustion in vitro. FIGs. 3A-3C show CARCDI9, IFNgKO CARCDI9 and IFNgRKO CARCDI9 were generated from healthy donor T cells. After expansion, CAR-T were activated overnight with plate-bound CD 19 antigen, RNA sequenced using NanoString and graphed as volcano plots; n=3. In volcano plots, genes involved in interferon signaling (FIGs.

[0035] 3A, 3B; <0 Log2 fold change), differentiation (FIG. 3C; >0 Log2 fold change) and exhaustion (FIG. 3C; <0 Log2 fold change) are highlighted. Three adjusted -value cutoffs are shown ranging from dotted line (bottom) to dashed line (top): <0.05, <0.01, <0.001. FIG. 3D shows expression of memory markers CD45RA and CD62L on CAR T cells was assessed by flow cytometry and graphed by representative FACS plots and summary pie charts showing naive (CD45RA+CD62L+), central memory (CM; CD45RA CD62L+), effector memory (EM;

[0036] CD45RA CD62L ) and effector (CD45RA+CD62L ); representative FACS plot and average pie charts from n=7. FIG. 3E shows CAR-T were mixed at a 1:1 ratio with irradiated JeKo-1 cells on days 0, 3 and 7. Expression of exhaustion markers by flow cytometry was determined prior to activation (0) and on days 3, 7 and 10; n=3 biological replicates.

[0037] FIGs. 4A-4H show deletion of IFNg or IFNgR protects CD 19 CAR T cells from cell death. FIG. 4A shows CAR-T were activated overnight with plate-bound CD 19 antigen, RNA sequenced using NanoString and graphed as volcano plots; n=3 biological replicates. In volcano plots, genes involved in proliferation (>0 Log2 fold change) and cell death (<0 Log2 fold change) are highlighted. Three adjusted -value cutoffs are shown ranging from dotted line (bottom) to dashed line (top): <0.05, <0.01, <0.001. FIG. 4B shows proliferation of CAR-T in response to CD19+Nalm6 cells was assessed using cell trace violet 6 days post-activation (n=3 biological replicates). FIGs. 4C-4D show Annexin V (FIG. 4C) and cleaved Caspase 3 (FIG. 4D) expression was determined by flow cytometry for CAR-T groups at resting (no activation; NA) and 48 hours post-activation with plate-bound CD19 antigen (CD19); n=3 biological replicates.

[0038] FIG. 4E shows annexin V expression on CAR-T groups following activation with plate-bound

[0039] #14645213vlCD19 antigen was assessed using Incucyte real-time analysis; n=5. FIGs.4F-4G show CAR-T were activated overnight with plate-bound CD 19 antigen in the absence or presence of exogenous recombinant human IFNg, and RNA sequenced using NanoString; n=3. Data is presented as bar graphs (FIG. 4F) and volcano plots (FIG. 4G). In the volcano plots, genes involved in interferon signaling (purple) and cell death (magenta) are highlighted. FIG. 4H shows wildtype CD19-specific CAR-T with no CRISPR / Cas9 editing were activated with platebound CD 19 antigen in the absence or presence of blocking antibodies to IFNg (alFNg) or IFNgR (alFNgR) and Annexin V expression was assessed by Incucyte; n=3 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0040] FIGs. 5A-5I shows loss of IFNg signaling drives greater survival in mesothelin- specific CAR T cells. FIG. 5A shows CAR-T with the anti-mesothelin SSI scFv, CD28 costimulatory domain and CRISPR / Cas9 editing of T cell receptor A constant (TRAC; CARMESO), TRAC and IFNg (IFNgKO CARMESO) or TRAC and IFNg receptor 1 (IFNgRKO CARMESO) were generated from healthy donor T cells. FIGs. 5B-5C show expression of CD3, IFNgR and IFNg (FIG. 5B; representative histograms) and CD4 / CD8 (FIG. 5C) was assessed by flow cytometry; n=3-6 biological replicates. FIG. 5D shows expansion of CAR-T in response to mesothelin antigen was monitored using real-time Incucyte imaging (left) and quantitative flow cytometry (right); n=3 biological replicates. FIG. 5E shows memory markers CD45RA and CD62L were assessed on resting CAR-T and shown by representative FACS plots and pie charts for overall averages; n=3 biological replicates. FIG. 5F shows CAR-T were activated with plate-bound mesothelin antigen and exhaustion markers were observed using flow cytometry with both pre- (PRE) and post-activation (POST) values graphed; n=3 biological replicates. FIG. 5G shows annexin V expression on CAR-T groups following activation with plate-bound mesothelin antigen was assessed using Incucyte real-time analysis and displayed as fold change of average green area compared to timepoint zero; n=5 biological replicates. FIGs. 5H-5I show proliferation of CAR-T in response to plate-bound mesothelin antigen was assessed using cell trace violet and is displayed as a representative donor and average pie chart (FIG. 5H) (each curve corresponding to the number of divisions is shown right to left, i.e., 0 divisions is the right-most curve)and collective averages (FIG. 51; from left to right for each division number: CARMESO, IFNyKO CARMESO, IFNyRKO CARMESO); n=3 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001.

[0041] FIGs. 6A-6G show IFNgRKO CARMESO T cells exhibit greater antitumor activity and survival against pancreatic cancer. FIG. 6A shows CAR-T and donor-matched untransduced T

[0042] #14645213vlcells (UTD) T cellswere activated with plate-bound mesothelin antigen overnight and assessed for Granzyme B and Perforin expression by flow cytometry; representative histograms from n=3 biological replicates. FIG. 6B shows CARMESO, IFNgKO CARMESO and IFNgRKO CARMESO T cells were mixed with mesothelin* AsPC-1, BxPC-3 or Capan2 cells at a 1:1 ratio and CAR-T cytolysis was assessed by Incucyte imaging; n=5 biological replicates. FIGs. 6C-6F show NSG mice bearing subcutaneous AsPC-1 tumor cells (1.5e6S.C.) were left untreated (tumor only; TO) or treated with 3e6CARMESO, IFNgKO CARMESO or IFNgRKO CARMESO T cells (I.V.) (FIG. 6C). Mice were bled at weekly through day 35 post-CAR-T infusion and T cell persistence was determined using flow cytometry (FIG. 6D, individual time points; FIG. 6E, summarized across time). Tumor burden was assessed by weekly caliper measurements (FIG.

[0043] 6F) and overall survival monitored (FIG. 6G). For FIGs. 6C-6G, n=5 mice / group for each healthy donor (2 donors total). Data are shown as mean ± s.e.m. with P values by One-way ANOVA (FIGs. 6B, 6F) and Kaplan-Meier survival curve (FIG. 6G). P: *<0.05, **<0.01.

[0044] FIGs. 7A-7L show pancreatic tumors from IFNgRKO CARMEso-treated mice exhibit greater interferon signaling and apoptosis. FIG. 7A shows NSG mice bearing subcutaneous AsPC-1 tumor cells (1.5e6S.C.) were left untreated (tumor only; TO) or treated with 3e6CARMESO, IFNgKO CARMESO or IFNgRKO CARMESO T cells (I.V.). Tumors were collected 14 days post-CAR-T infusion, processed and sorted into mCherry* CAR-T cells and CBGGFP* AsPC-1 tumor cells. FIG. 7B shows representative FACS plots of the tumor / CAR-T cells isolated from the tumors of AsPC-1 -bearing mice. FIGs. 7C-7L show isolated CAR-T (FIG. 7C) and tumor (FIG. 7D) cells were analyzed by NanoString and displayed as a heatmap of all genes using Z-scores. Normalized gene counts from CAR-T (FIGs. 7E-7H) and tumor cells (FIGs. 7I-7L) were plotted as heatmaps or scatter plots using normalized gene counts. In FIG.

[0045] 7C, FIG. 7G and FIG. 7H, both pre- (PRE) and post-infusion (POST) CAR T cells were graphed. For this figure, n=2 mice / group for each healthy donor (2 donors total). Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0046] FIGs. 8A-8J show EGFR-targeting IFNgRKO CAR T cells confer protection from tumor rechallenge. FIG. 8A shows CAR and IFNgRKO CAR T cells targeting EGFR were generated from healthy donors. FIG. 8B shows CAR-T were activated with plate-bound EGFR antigen and T cell expansion was tracked by Incucyte; n=3 biological replicates. FIG. 8C shows CAREGFR and IFNgRKO CAREGFR T cells were mixed at a 1:1 ratio with CBGGFP* AsPC-1 cells and T cell cytolysis was monitored by Incucyte; n=3 biological replicates. FIGs.

[0047] 8D-8J, NSG mice bearing subcutaneous AsPC-1 tumor cells (1.5e6S.C.) were left untreated

[0048] #14645213vl(tumor only; TO) or treated with 3e6CAREGFR or IFNgRKO CAREGFR T cells (I.V.) (FIG. 8D) and tumor burden was measured by caliper weekly (FIG. 8E). Mice were bled at days 7, 14, 21, 28 and 35 post-CAR-T infusion and T cell persistence was determined using flow cytometry (FIG. 8F). Seventy days post-CAR-T infusion, mice displaying curative responses (3 mice / group) were re-challenged with 1.5e6AsPC-1 tumor cells in the flank opposite of the initial tumor injection. Tumor burden was assessed using weekly caliper measurements (FIG. 8G, FIG. 81) and overall survival monitored (FIG. 8H, 8J). For FIGs. 8D-8J, n=5 mice / group with 3 mice / group for re-challenge. Data are shown as mean ± s.e.m. with P values by unpaired t-test (FIGs. 8B, 8F), One-way ANOVA (FIGs. 8E, 8G, 81) and Kaplan-Meier survival curve (FIG.

[0049] 8H, 8J). P: *<0.05, **<0.01, ***<0.001.

[0050] FIGs. 9A-9I show IFNy and IFNyR in human T cells can be targeted using CRISPR / Cas9 editing. FIG. 9A shows a schematic of IFNy production and uptake among CAR T cell groups; Created with BioRender.com. FIGs. 9B-9D shows CAR-T with CRISPR / Cas9 editing of T cell receptor A constant (TRAC; CARCDW), TRAC and IFNy (IFNyKO CARci ) or TRAC and IFNyR (IFNyRKO CARCDW) were generated from healthy donor T cells as depicted in FIG. 9B and expression of CAR, CD3 and IFNyR was assessed by flow cytometry prior to (FIG. 9C) and post- (FIG. 9D) CD3 isolation and graphed in relation to donor-matched untransduced T cells (UTD); representative histograms and FACS plots from n=3 biological replicates. FIGs. 9E-9F show summary graphs of CD3 (FIG. 9E) and IFNyR (FIG. 9F) expression by flow cytometry after expansion / selection protocol; n=3. FIGs. 9G-9H show post-CD3 isolation, CAR-T were left untreated or given recombinant human IFNy prior to flowbased staining of phosphoSTATl; data shown as representative histograms (FIG. 9G) and scatter plots (FIG. 9H) from n=3 biological replicates. FIG. 91 shows CAR T cells were activated with plate-bound CD 19 antigen overnight and assessed for activation by CD69 expression; representative histograms from n=3 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ****<0.0001.

[0051] FIGs. 10A-10C show loss of IFNy or IFNyR does not affect overall CARCD19 T cell function. CAR T cells were activated overnight with plate-bound CD 19 antigen and assessed for cytokines using Luminex (FIG. 10A) and flow cytometry (FIGs. 10B-10C); n=3 biological replicates. Data for FIG. 10B is shown as representative FACS plots and FIG. 10C depicts average percentage of cells are making 1-5 cytokines simultaneously. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, ***<0.001, ****<0.0001.

[0052] FIGs. 11A-11E show IFNyKO and IFNyRKO CARCD19 T cells exhibit increased expansion in an antigen- dependent manner. CAR-T with CRISPR / Cas9 editing of T cell

[0053] #14645213vlreceptor A constant (TRAC; CARCD19), TRAC and IFNy (IFNyKO CARCD19) or TRAC and IFNyR (IFNyRKO CARCD19) were generated from healthy donor T cells. FIG. 11A shows CAR-T expansion in response to CD19+Nalm6 leukemia cells (left) or CD19+JeKo-1 lymphoma cells (right) was assessed using counting beads for flow cytometry; n=3 biological replicates. FIG. 11B shows CAR-T activated with plate-bound CD 19 were monitored using Incucyte (left) and flow cytometry (right); n=3 biological replicates. FIG. 11C shows wildtype CAR-T (WT) were combined with Nalm6 target cells (left) or plate-bound CD 19 antigen (right) in the absence or presence of blocking antibodies to IFNy (ocIFNy) or IFNyR (ocIFNyR) and CAR-T expansion was monitored by Incucyte; n=3. FIGs. 11D-11E show CBGGFP+Nalm6 tumor cells were sorted into CD19low, CD19mdand CD 191""1' populations as shown by MFI (FIG. 11D) and combined with CAR T cells to observe T cell expansion by Incucyte (FIG. HE); n=5. FIGs. 11F-11G show CAR-T cells were plated in the presence or absence of IL-2 and assessed for viability (FIG. HF) and cell count (FIG. 11G); n=5 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0054] FIGs. 12A-12O show manipulation of IFNy signaling does not affect CARCD19 efficacy in vitro or in vivo. FIGs. 12A-12B show CAR-T were activated with plate-bound CD 19 antigen overnight and assessed for Granzyme B and Perforin expression by flow cytometry and displayed as representative histograms (FIG. 12A) and averages (FIG. 12B) from n=3 biological replicates. FIG. 12C shows CARCD19, IFNyKO CARCD19 and IFNyRKO CARCD19 T cells were mixed with CD19+CBGGFP+Nalm6 (left) or JeKo-1 (right) cells at a 1:1 ratio and CAR-T cytolysis was assessed by Incucyte imaging; n=5. FIGs. 12D-12H show manipulation of IFNy signaling does not affect CARCDI efficacy against leukemia. NSG mice bearing intravenously administered CBGGFP+Nalm6 tumor cells (le61.V.) were left untreated (tumor only; TO) or treated with le6CARCD19, IFNyKO CARCD19 or IFNyRKO CARCD19 T cells (I.V.) (FIG. 12D). Tumor burden was assessed using weekly bioluminescent imaging and graphed by flux (FIGs. 12E-12F) and overall survival was monitored (FIG. 12G). Mice were bled 2 days post-CAR-T infusion and IFNy levels in the serum were determined using Luminex (FIG. 12H). FIGs. 121-120 show NSG mice bearing intravenously administered CBGGFP+JeKo-1 tumor cells (le6I.V.) were left untreated (tumor only; TO) or treated with le6CARCD19, IFNyKO, CARCDI or IFNyRKO CARCDI9 T cells (I.V.) (FIG. 121). Tumor burden was assessed using weekly bioluminescent imaging and graphed by flux (FIGs. 12J, 12M) and overall survival was monitored (FIGs. 12K, 12N). Mice were bled 14 days post-CAR-T infusion and T cell persistence in the blood was determined using flow cytometry (FIGs. 12L,

[0055] #14645213vl120). For FIGs. 12D-12O, n=5 mice / group for each healthy donor (2 donors total). Data are shown as mean ± s.e.m. with P values by One-way ANOVA (FIGs. 12F, 12H, 12K, 12N) or Kaplan-Meier survival curve (FIGs. 12G, 12L). P: **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0056] FIGs. 13A-13B show alteration of IFNy signaling in CARCD19 T cells is detectable at the RNA level. CAR-T were left inactivated (no activation; NA) or activated with plate-bound CD 19 antigen and RNA sequenced using NanoString. FIG. 13A shows data is plotted as volcano plots with common highly expressed genes highlighted in gray; n=3. Three adjusted p-value cutoffs are shown ranging from above the dotted line (bottom) to above the dashed line top) <0.05, <0.01, <0.001 FIG. 13B shows bar graphs for normalized gene counts in cultures with or without CD19 activation; n=3 biological replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0057] FIGs. 14A-14D show differentiation of IFNyKO and IFNyRKO CARCD19 is comparable to control CAR. FIG. 14A shows memory of CAR-T groups was assessed by CD45RA and CD62L on resting cells using flow cytometry with gating for memory subsets shown on top; n=7 biological replicates. FIGs. 14B-14C show CAR-T were left inactivated or activated with plate-bound CD 19 antigen and RNA sequenced using NanoString. Data is shown by cell type profiling (FIG. 14B) and bar graphs for normalized gene counts (FIG. 14C); n=3.

[0058] FIG. 14D shows resting CAR T cells were stained for CCR4, CCR6 and CXCR3 by flow cytometry and graphed by subsets shown on top (for each subset, from left to right: CARCDI , IFNyKO CARCDI9, IFNyRKO CARCDW); n=3. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0059] FIGs. 15A-15D show loss of IFNy signaling does not significantly impact CARCD19 exhaustion in vitro. FIGs. 15A-15C show CAR-T were activated with plate-bound CD 19 (FIG.

[0060] 15A), JeKo-1 (FIG. 15B) or Nalm6 (FIG. 15C) cells overnight and assessed by flow cytometry; n=3. FIG. 15D shows CAR-T were serially restimulated with irradiated (IRR) Nalm6 cells and observed by flow cytometry on days 0 (pre- activation), 3, 7 and 10; n=3 biological replicates..

[0061] FIGs. 16A-16C shows deletion of IFNy or IFNyR does not affect CARCD19 proliferation in vitro. CAR-T were stained with cell trace violet and activated with plate-bound CD19 antigen (FIG. 16A; each curve corresponding to the number of divisions is shown right to left, i.e., 0 divisions is the right-most curve) (summarized in FIG. 16B; for each division number, from left to right: CARCDW, IFNyKO CARCDW, IFNyRKO CARCDW) or Nalm6 cells (FIG. 16C; for each division number, from left to right: CARCDI9, IFNyKO CARCDI9, IFNyRKO

[0062] #14645213vlCARCDW) to monitor proliferation; n=3. Data is displayed as a representative histogram with summary data shown as pie charts and a bar graph.

[0063] FIGs. 17A-17H show mesothelin-specific IFNyKO and IFNyRKO CAR-T maintain functionality. FIGs. 17A-17B show CAR-T with CRISPR / Cas9 editing of T cell receptor A constant (TRAC; CARMESO), TRAC and IFNy (IFNyKO CARMESO) or TRAC and IFNyR (IFNyRKO CARMESO) and donor-matched untransduced T cells (UTD) were generated from healthy donor T cells. Expression of CAR (FIG. 17A; pre-CD3 isolation), CD3 and IFNyR were assessed by flow cytometry and IFNy production in response to CD 19 antigen was determined by Luminex (FIG. 17B; post-CD3 isolation); representative histograms and FACS plots from n=3. FIG. 17C shows post-CD3 isolation, CAR-T were left untreated or given recombinant human IFNy prior to flow-based staining of phosphoSTATl; n=3 biological replicates. FIG. 17D shows CAR-T were left untreated or activated with plate-bound mesothelin antigen and assessed for CD69 expression by flow cytometry; representative histogram from n=3 biological replicates. FIGs. 17E-17H, CAR-T were activated with plate-bound mesothelin antigen and assessed for cytokine production by Luminex (FIG. 17E) and flow cytometry (FIGs. 17F-17H); n=3 donors biological replicates. Data is shown as average ng / ml for Luminex (FIG. 17E), representative FACS plots with summary data (FIG. 17F, 17G) and percent cells making 1-5 cytokines simultaneously (FIG. 17H). Data are shown as mean ± s.e.m. with P values by Oneway ANOVA. P: **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0064] FIGs. 18A-18F show IFNyRKO CARMESO have higher accumulation, but not proliferation, following mesothelin exposure. FIGs. 18A-18C show CARMESO, IFNyKO CARMESO and IFNyRKOMESO CAR T cells were combined at a 1:1 ratio with AsPC-1 (FIG. 18A), BxPC-3 (FIG. 18B) or Capan-2 (FIG. 18C) pancreatic tumor cells and CAR-T expansion was monitored by Incucyte (left) and quantitative flow cytometry (right); n=3 biological replicates. FIGs. 18D-18F show CAR-T were labeled with cell trace violet and combined at a 1:1 ratio with AsPC-1 (FIG. 18D; for each division number (bottom), from left to right: CARMESO, IFNyKO CARMESO, IFNyRKO CARMESO), BXPC-3 (FIG. 18E; for each division number (bottom), from left to right: CARMESO, IFNyKO CARMESO, IFNyRKO CARMESO) or Capan-2 (FIG. 18F; for each division number (bottom), from left to right:

[0065] CARMESO, IFNyKO CARMESO, IFNyRKO CARMESO) tumor cells and proliferation of CAR-T was assessed using flow cytometry; n=2 biological replicates. Data are displayed as representative histograms with summary data shown as pie charts and bar graphs. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05.

[0066] #14645213vlFIGs. 19A-19H show tumors from IFNyRKO CARMESO-treated mice display a heightened expression of tumor suppressor genes. FIGs. 19A-19G show, as depicted in FIGs 7A-7L, NSG mice bearing subcutaneous AsPC-1 tumor cells (1.5e6S.C.) were treated with 3e6CARMESO, IFNyKO CARMESO or IFNyRKO CARMESO T cells (I.V.). Tumors were collected 14 days post-CAR-T infusion, processed and sorted into mCherry+CAR-T cells and CBGGFP+AsPC-1 tumor cells. Average percentage of GFP+tumor cells and mCherry+CAR-T cells from the two donors (FIG. 19A). For CAR T cells, heatmaps for normalized gene counts were made for differentiation (FIG. 19B) and immune checkpoints (FIG. 19C), and cell type profiling using NanoString Advanced Analysis software is shown (FIG. 19D); samples from pre- (PRE) and post-infusion (POST) are included. For the tumor side, genes were categorized by immune checkpoints (FIG. 19E) and tumor progression (FIGs. 19F-19G); tumor only (TO) is included. FIG. 19H shows pancreatic cancer cell lines were assessed for expression of mesothelin and EGFR antigens by flow cytometry, representative ofn=3 technical replicates. Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: ****<0.0001.

[0067] FIGs. 20A-20C show IFNyRKO CAREGFR protect mice from a secondary tumor challenge. NSG mice bearing subcutaneous AsPC-1 tumor cells (1.5e6S.C.) were left untreated (tumor only; TO) or treated with 3e6CAREGFR or IFNyRKO CAREGFR T cells (I.V.) and tumor burden was measured by caliper weekly (FIG. 20A). Seventy days post-CAR-T infusion, mice displaying curative responses (3 mice / group) were re-challenged with 1.5e6AsPC-1 tumor cells in the flank opposite of the initial tumor injection. Naive NSG mice were used for the tumor only group and tumor burden was assessed using weekly caliper measurements (FIG. 20B) and overall survival monitored (FIG. 20C). For this figure, n=5 mice / group with 3 mice / group for re-challenge. Data are shown as mean ± s.e.m. with P values by One-way ANOVA (FIGs. 20A-20B) and Kaplan-Meier survival curve (FIG. 20C). P: *<0.05, **<0.01, ***<0.001, ns=not significant.

[0068] FIGs. 21A-21H show IFNgRKO CAR T cells display increased efficacy and survival in a syngeneic model of triple-negative breast cancer. FIG. 21A shows T cells were isolated from CD45.2+C57BE / 6 wildtype (WT) or Ifngr KO (IFNgRKO) mice and retrovirally transduced to express control (CARFITC) or B7H3-targeted (CARBVHS) CAR constructs prior to in vitro and in vivo assessment. FIGs. 21B-21C show transduction efficiency (FIG. 21B) and memory (FIG.

[0069] 21C) was assessed by flow cytometry. Memory was defined as naive (CD44 CD62E+), central memory (CM; CD44+CD62E+), effector memory (EM; CD44+CD62E ) and double negative (DN; CD44 CD62E ); data shown is n=3 technical replicates, representative ofn=3 biological replicates. FIGs. 21D-21G show 3e5triple-negative breast cancer cells overexpressing B7H3

[0070] #14645213vl(E0771 B7H3) were injected into the mammary fat pad of CD45.1+C57BL / 6 mice. Tumorbearing mice were conditioned with lOOmg / g cyclophosphamide prior to intravenous (I.V.) administration of 5e6CD8+CAR T cells and tumor size was monitored by caliper (FIGs. 21D-21F). On day 23, tumors were collected and processed for flow cytometry (FIG. 21G). For FIGs. 21D-21G, n=5 mice / group. FIG. 21H shows a summary schematic for data presented herein. Data are shown as mean ± s.e.m. with P values by Two-way ANOVA (FIG. 21D), Oneway ANOVA (FIG. 21F) and unpaired t-test (FIGs. 21B, 21C, 21G). P: *<0.05, **<0.01, ***<0.001, ****<0.0001, ns=not significant.

[0071] FIGs. 22A-22D show loss of IFNg or IFNgR does not affect CARCDW efficacy in a stress model of lymphoma. NSG mice bearing intravenously administered CBGGFP+JeKo-1 tumor cells (le6I.V.) were left untreated (tumor only; TO) or treated with 5e5CARCD19, IFNyKO CARCD19 or IFNyRKO CARCD19 T cells (I.V.) (FIG. 22A). Tumor burden was assessed using weekly bioluminescent imaging and graphed by flux (FIGs. 22B-22D); n=5 mice / group for each healthy donor (2 donors total). Data are shown as mean ± s.e.m. with P values by One-way ANOVA. P: *<0.05.

[0072] DETAILED DESCRIPTION

[0073] In some aspects, this disclosure provides an immune cell that is deficient in interferon y receptor (IFNyR) expression and / or signaling.

[0074] Interferon y receptor (IFNyR) Deficient Immune Cells

[0075] “Interferon y receptor (IFNyR)” includes a protein receptor that binds to interferon y (IFNy). In some embodiments, the IFNyR is a heterodimer of two chains, IFNyRl (e.g., UniProt P15260, accessed December 23, 2024) and IFNyR2 (e.g., UniProt P38484, accessed December 23, 2024). In some embodiments, IFNyRl comprises an amino acid sequence comprising at least 85% identity (e.g., at least 90% identity, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 51. In some embodiments, IFNyRl comprises a nucleic acid sequence comprising at least 85% identity (e.g., at least 90% identity, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 53. In some embodiments, IFNyR2 comprises an amino acid sequence comprising at least 85% identity (e.g., at least 90% identity, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 52. In some embodiments, IFNyR2 comprises a nucleic acid sequence comprising at least 85% identity (e.g., at least 90% identity, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 54. In some embodiments, IFNyRl comprises an amino acid sequence comprising SEQ ID NO: 51. In some embodiments, IFNyRl comprises a nucleic acid sequence comprising SEQ

[0076] #14645213vlID NO: 53. In some embodiments, IFNyR2 comprises an amino acid sequence comprising SEQ ID NO: 52. In some embodiments, IFNyR2 comprises a nucleic acid sequence comprising SEQ ID NO: 54. In some embodiments, the IFNyR is a mammalian IFNyR. In some embodiments, the IFNyR is a non-human primate IFNyR. In some embodiments, the IFNyR is a rodent IFNyR. In some embodiments, the IFNyR is a human IFNyR.

[0077] In some aspects, this disclosure provides an immune cell that is deficient in interferon y receptor (IFNyR) expression.

[0078] An immune cell that is deficient in interferon y receptor (IFNyR) expression (e.g., IFNyR 1 and / or IFNyR2 expression) refers to an immune cell that has less IFNyR mRNA, protein and / or cell surface protein than a corresponding control immune cell of the same type (e.g., an immune cell that has not been treated with an agent that decreases IFNyR expression). In some embodiments, an immune cell that is deficient in IFNyR expression refers to an immune cell that has at least 10% (e.g., at least 25%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99%) less IFNyR mRNA, protein and / or cell surface protein than a corresponding control immune cell of the same type. In some embodiments, an immune cell that is deficient in IFNyR expression comprises an IFNyR knockdown (e.g., via RNAi or CRISPR interference) of one or more alleles that encode IFNyR. In some embodiments, an immune cell that is deficient in IFNyR expression comprises an IFNyR knockout (e.g., via CRISPR or lox-cre) of one or more alleles that encode IFNyR. In some embodiments, an IFNyR knockout is a heterozygous knockout. In some embodiments, an IFNyR knockout is a homozygous knockout. In some embodiments, an immune cell that is deficient in IFNyR expression comprises a mutation in a gene loci of IFNyR. A mutation includes an insertion, a deletion, a single nucleotide polymorphism, a synonymous mutation, a non- synonymous mutation, and a frameshift mutation. In some embodiments, an IFNyR mutation is a frameshift mutation. In some embodiments, an IFNyR mutation is an insertion / deletion mutation. In some embodiments, an IFNyR mutation is a single nucleotide mutation. In some embodiments, a IFNyR mutation is a heterozygous mutation. In some embodiments, a IFNyR mutation is a homozygous mutation. In some embodiments, a IFNyR mutation is a loss of function mutation. In some embodiments, an IFNyR mutation is a IFNyR promoter mutation.

[0079] In some embodiments, an immune cell that is deficient in IFNyR expression comprises an agent that decreases IFNyR expression. In some embodiments, the agent is a functional RNA. A “functional RNA” refers to a non-coding RNA that can be used to decrease the expression and / or activity of a target gene (e.g., IFNyRl and / or IFNyR2). In some embodiments, the functional RNA is RNA interference (RNAi). In some embodiments, the

[0080] #14645213vlRNAi is siRNA, miRNA, shRNA or piwiRNA. In some embodiments, the functional RNA is a guide RNA. In some embodiments, the functional RNA comprises a portion (e.g., a series of nucleotides) that is complementary to a target gene and / or mRNA. For example, a guide RNA comprises a portion (i.e., a homology region) that is complementary to a target polynucleotide. In some embodiments, the agent is a CRISPR-Cas reagent. In some embodiments, the agent is an antisense oligonucleotide.

[0081] As used herein the term “clustered regularly interspaced short palindromic repeats” or “CRISPR” may refer to a gene editing system that comprises a guide RNA component and a CRISPR associated (Cas) protein component. The guide RNA polynucleotide may comprise a homology region that is complementary to a target gene (e.g., IFNyRl or IFNyR2) and a stem loop region that is capable of binding to a Cas protein. The Cas protein may comprise a guide RNA binding site and nuclease activity. The Cas protein and the guide RNA form a complex that is capable of binding to the target gene (based on the homology region), and cleaving the DNA (using the nuclease activity of the Cas protein). In some embodiments, the Cas protein guide RNA complex binds to sequence that is adjacent to and downstream of a protospacer adjacent motif (PAM). Cleavage results in a DNA strand break and repair of that strand break may introduce a mutation (e.g., single nucleotide polymorphism, insertion, or deletion). In some embodiments, the Cas protein is any suitable Cas protein for mutating and / or altering the expression of a target gene (a Cas protein may also be referred to as a CRISPR protein herein). In some embodiments, the Cas protein is selected from the group consisting of a Cas9 protein, a Cas 12 protein, or a Cas 13 protein. The skilled person will understand that Cas proteins (e.g., Cas9) may have many different orthologs (e.g., SpyoCas9, spCas9, spyCas9, and geoCas9). In some embodiments, the Cas protein is SpyoCas9. Cas proteins and orthologs thereof are well known in the art as discussed in Gasiunas, Giedrius, et al, Nature communications 11.1 (2020): 1-10; and Fancheng Y et al., Cell Biology and Toxicology 35.6 (2019): 489-492, each of which is incorporated by reference in its entirety. Methods for designing guide RNAs (e.g., selecting homology region sequences for targeting a specific gene) are also well known in the art as described in Liu, Guanqing L. et al., Computational and Structural Biotechnology Journal 18 (2020): 35-44. In some embodiments, gRNAs (encoded by gRNA polynucleotides) are designed using CRISPick (portals.broadinstitute.org / gppx / crispick / public), which performs as described in Doench et al., Nature Biotechnology, 34(2), 184-191 (2016) and Sanson et al., Nature Communications, 9(1), 5416 (2018).

[0082] #14645213vlIn some embodiments, the CRISPR-Cas reagent comprises a Cas protein (e.g., Cas9) and a guide RNA comprises a homology region that is complementary to IFNyR 1 (e.g., SEQ ID NOs: 33-40) and / or IFNyR2 (SEQ ID NOs: 41-48).

[0083] In some aspects, this disclosure provides an immune cell that is deficient in interferon y receptor (IFNyR) signaling. “IFNyR signaling” refers to signaling that includes IFNyR and downstream components of the IFNyR pathway. In some embodiments, an immune cell that is deficient in IFNyR signaling is an immune cell that has been contacted with a IFNyR inhibitor (e.g., an IFNyR antibody). In some embodiments, an immune cell that is deficient IFNyR signaling is deficient in expression of IFNyR and / or downstream components of the IFNyR pathway. Components of the IFNyR downstream pathway include, but are not limited to Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Signal transducer and activator of transcription 1 (STAT1), Suppressor of cytokine signaling 1 (SOCS-1), and Interferon regulatory factor 1 (IRF1).

[0084] In some embodiments, an immune cell that is deficient in IFNyR signaling is deficient JAK1 expression or signaling. JAK1 expression may be decreased in any suitable way including via JAK1 knockout and / or knockdown. JAK1 expression and / or signaling may be decreased via a mutation in the JAK1 gene (e.g., a frameshift mutation) or gene locus. In some embodiments, an immune cell deficient in JAK1 signaling comprises an immune cell that has been treated with a JAK1 inhibitor (e.g., Abrocitinib, Baricitinib, Delgocitinib, Peficitinib, Ruxolitinib, Tofacitinib, Filgotinib, Oclacitinib, and / or Upadacitinib).

[0085] In some embodiments, an immune cell that is deficient in IFNyR signaling is deficient JAK2 expression or signaling. JAK2 expression may be decreased in any suitable way including via JAK2 knockout and / or knockdown. JAK2 expression and / or signaling may be decreased via a mutation in the JAK2 gene (e.g., a frameshift mutation) or gene locus. In some embodiments, an immune cell deficient in JAK2 signaling comprises an immune cell that has been treated with a JAK2 inhibitor (e.g., Abrocitinib, Baricitinib, Delgocitinib, Peficitinib, Ruxolitinib, Tofacitinib, Fedratinib, and / or Pacritinib).

[0086] In some embodiments, an immune cell that is deficient in IFNyR signaling is deficient STAT1 expression or signaling. STAT1 expression may be decreased in any suitable way including via STAT1 knockout and / or knockdown. STAT1 expression and / or signaling may be decreased via a mutation in the STAT1 gene (e.g., a frameshift mutation) or gene locus. In some embodiments, an immune cell deficient in STAT1 signaling comprises an immune cell that has been treated with a STAT1 inhibitor (e.g., fludarabine and / or Nifuroxazide).

[0087] #14645213vlIn some embodiments, an immune cell that is deficient in IFNyR signaling has increased SOCS-1 expression or signaling. SOCS-1 expression may be increased in any suitable way including via plasmid expression of SOCS-1 or CRISPR activation of SOCS-1. In some embodiments, an immune cell deficient in SOCS-1 signaling comprises an immune cell that has been treated with a SOCS-1 mimetic.

[0088] In some embodiments, an immune cell that is deficient in IFNyR signaling is deficient in IRF1 expression and / or signaling. IRF1 expression may be decreased in any suitable way including via knockout and / or knockdown. IRF1 expression and / or signaling may be decreased via a mutation in the IRF1 gene (e.g., a frameshift mutation) or gene locus. In some embodiments, an immune cell deficient in IRF1 signaling comprises an immune cell that has been treated with a IRF1.

[0089] In some embodiments, an immune cell that is deficient in interferon y receptor (IFNyR) expression and / or signaling is a T cell. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogenic T cell. In some embodiments, an immune cell is deficient in interferon y receptor (IFNyR) expression and / or signaling is a natural killer (NK) cell. In some embodiments, the NK cell is an autologous NK cell. In some embodiments, the NK cell is an allogenic NK cell.

[0090] In some embodiments, a cell described herein (e.g., a T cell) is an isolated cell. As used herein, an “isolated” cell refers to a cell that has been extracted from a subject (e.g., via whole blood collection, an apheresis system, a leukapheresis system, a subject-connected closed loop system, etc.). In some embodiments an isolated cell (e.g., an isolated T cell) is a cell that has been extracted from the subject and engineered (e.g., engineered to express a CAR polypeptide provided herein).

[0091] In some embodiments, an immune cell that is deficient in interferon y receptor (IFNyR) expression and / or signaling is also deficient in T cell receptor alpha constant (TRAC) expression (e.g., compared to a control immune cell of the same type that does not comprise an agent that decreases TRAC expression or a TRAC knockout). In some embodiments, the immune cell comprises a TRAC loss of function mutation and / or knockout (e.g., induced via CRISPR).

[0092] Polynucleotides, Plasmids, and Vectors

[0093] In some embodiments, an immune cell comprises a polynucleotide encoding a chimeric antigen receptor and / or an engineered immune cell receptor, wherein the immune cell is deficient in IFNyR expression and / or signaling. In some embodiments, an immune cell

[0094] #14645213vlcomprises a first polynucleotide encoding a chimeric antigen receptor, wherein the immune cell is deficient in IFNyR expression and / or signaling. In some embodiments, an immune cell comprises a first polynucleotide encoding an engineered immune cell receptor, wherein the immune cell is deficient in IFNyR expression and / or signaling.

[0095] The term "polynucleotide" is used herein interchangeably with "nucleic acid molecule" to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double- stranded forms (and complements of each single- stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein the term, “heterologous nucleic acid molecule” refers to a nucleic acid molecule that does not naturally exist within a given cell or a nucleic acid sequence that has been engineered into a cell. For example, a heterologous nucleic acid molecule may be a nucleic acid molecule encoding a gene that is engineered into a cell (e.g., via a plasmid, vector or some other method). A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

[0096] In some embodiments, the polynucleotide further comprises a CRISPR guide polynucleotide (e.g., a polynucleotide that when expressed produces a guide RNA). In some embodiments, the guide RNA targets (e.g., comprises a homology region that is complementary to) an IFNyR gene (e.g., IFNyRl and / or IFNyR2). In some embodiments, the guide RNA target an IFNyR pathway component that is downstream of IFNyR (e.g., JAK1, JAK2, STAT1 or IRF1). In some embodiments, the polynucleotide comprises from 5’ to 3’ a nucleic acid encoding a CAR and a guide polynucleotide. In some embodiments, the polynucleotide comprises from 5’ to 3’ a guide polynucleotide and a nucleic acid encoding a CAR. In some embodiments, the polynucleotide further comprises a guide RNA that targets TRAC. Knockout of TRAC removes TRAC surface expression, which can be detected and used as a control for expression of the polynucleotide in the immune cell. In some embodiments, the polynucleotide

[0097] #14645213vlcomprises an IFNyR gene targeting guide RNA, a TRAC targeting guide RNA, and a nucleic acid encoding a CAR. In some embodiments, the polynucleotide comprises, from 5’ to 3’, an IFNyR gene targeting guide RNA, a TRAC targeting guide RNA, and a nucleic acid encoding a CAR. In some embodiments, the polynucleotide comprises, from 5’ to 3’, a TRAC targeting guide RNA, an IFNyR gene targeting guide RNA, and a nucleic acid encoding a CAR. In some embodiments, the polynucleotide comprises, from 5’ to 3’, a nucleic acid encoding a CAR, an IFNyR gene targeting guide RNA, and a TRAC targeting guide RNA. In some embodiments, the polynucleotide comprises, from 5’ to 3’, a nucleic acid encoding a CAR, a TRAC targeting guide RNA, and an IFNyR gene targeting guide RNA. In some embodiments, the polynucleotide comprises, from 5’ to 3’, an IFNyR gene targeting guide RNA, a nucleic acid encoding a CAR, and a TRAC targeting guide RNA. In some embodiments, the polynucleotide comprises, from 5’ to 3’, a TRAC targeting guide RNA, a nucleic acid encoding a CAR, and an IFNyR gene targeting guide RNA.

[0098] In some embodiments, the CAR is operably linked to a first promoter. In some embodiments, the IFNyR gene targeting guide RNA is operably linked to a second promoter. In some embodiments, the TRAC targeting guide RNA is operably linked to a third promoter. In some embodiments, the second promoter and the third promoter are selected from a hU6 or mU6 promoter. In some embodiments, the first promoter is a EFl -alpha promoter.

[0099] As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a CAR or a bispecific CAR, where the polynucleotide molecules are so arranged that the promoter can direct an RNA polymerase to transcribe the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0100] In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the U6 promoter is from a non-human species. In some embodiments, the promoter is selected from the group consisting of a CMV promoter, an EFla promoter, an EFla-short promoter, a CAG promoter, a PGK promoter, Hl promoter, or a U6 promoter. In some embodiments, the U6 promoter is from a human U6 promoter. In some embodiments, the U6 promoter is from cow, mice, rat, pig, yeast, dog, cat, drosophila, or C. elegans. In some embodiments, the promoter is a Hl promoter. In some embodiments, the promoter is a tissuespecific promoter (e.g., the HP1, CD14, CD43, CD45, C68, elastase, endoglin, fibronectin, Fit,

[0101] #14645213vlGFAP, GPIIb, ICAM-2, mIFN-beta, Mb, NphsI, OG-2, SP-B, SYN1, or WASP gene promoter). In some embodiments, the promoter is an inducible promoter (e.g., a tet or lac promoter).

[0102] In some embodiments, an immune cell that is deficient in IFNyR expression and / or signaling comprises a polynucleotide encoding a chimeric antigen receptor and / or an engineered immune cell receptor. In some embodiments, the polynucleotide is a vector. The term "vector," as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non- viral. The term "vector" encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

[0103] As used herein, the term "expression vector" may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

[0104] As used herein, the term "viral vector" may refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. In some embodiments, the viral vector is an adeno-associated viral, adenoviral, lentiviral, or a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a second generation lentiviral vector.

[0105] By "recombinant vector" may be a vector that includes a heterologous nucleic acid sequence or "transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable

[0106] #14645213vlepisomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0107] In some embodiments, a polypeptide, polynucleotide, plasmid and or / vector as described herein optionally further comprises a reporter molecule, e.g., to determine if the vector is properly expressed in a cell. In some embodiments, the reporter molecule may be a fluorescent protein (e.g., GFP, YFP, RF, mCherry), antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), or a radioisotope. In some embodiments, the reporter molecule is hygromycin phosphotransferase (hph) that can be imaged alone or in combination with a substrate or chemical (for example 9-[4-[18F]fhioro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

[0108] In some embodiments, GFP and mCherry may be used as fluorescent tags for imaging a bispecific CAR expressed on a T cell (e.g., a CAR-T cell). It is expected that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR need not include a fluorescent tag or fluorescent protein. In each instance of particular constructs provided herein, therefore, any markers present in the constructs can be removed. The invention includes the constructs with or without the markers. Accordingly, when a specific construct is referenced herein, it can be considered with or without any markers or tags (including, e.g., histidine tags) as being included within the invention.

[0109] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., a CAR) is comprised by a plasmid. The term “plasmid” may refer to a circular piece of DNA the comprises an origin of replication. In some embodiments, the plasmid comprises a prokaryotic origin of replication. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the plasmid comprises a eukaryotic origin of replication. In some embodiments, the plasmid comprises a mammalian origin of replication. In some embodiments, the plasmid comprises a prokaryotic and eukaryotic origin of replication. In some embodiments, the plasmid comprises an origin of replication that is active in a cell which the plasmid is located. In some embodiments, the plasmid is a lentiviral plasmid (e.g., a second generation lentiviral plasmid).

[0110] In some embodiments, this disclosure provide a cell comprising a polynucleotide described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is an NK cell.

[0111] In some embodiments, the disclosure provides a sequence that shares a certain percentage of identity with a reference sequence (e.g., a sequence having at least 90% identity to

[0112] #14645213vla reference sequence). In some embodiments, determining the percentage of identity of a sequence with a reference sequence comprises aligning the sequence and the reference sequence, e.g., using Emboss Needle as described in Madeira F et al. Nucleic Acids Res. 2024 Jul;52(Wl):W521-W525.

[0113] Chimeric Antigen Receptors (CARs)

[0114] The terms "chimeric antigen receptor" or "CAR" or "CARs", as used herein, refer to engineered T cell receptors, which graft a ligand or antigen specificity onto T cells (for example, naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.

[0115] In some embodiments, a CAR places a chimeric extracellular antigen-binding domain that specifically binds a target, e.g., a polypeptide, expressed on the surface of a cell to be targeted for a T cell response onto a construct including a transmembrane domain and intracellular domain(s) of a T cell receptor molecule. In some embodiments, the chimeric extracellular antigen-binding domain includes the antigen domain(s) of an antibody reagent that specifically binds an antigen expressed on a cell to be targeted for a T cell response. In some embodiments, the chimeric extracellular antigen-binding domain includes a ligand that specifically binds an antigen expressed on a cell to be targeted for a T cell response.

[0116] As used herein, a "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. In some embodiments, when expressed in a T cell, CARs have the ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0117] As used herein, a "CAR-NK cell" or "CAR-NK" refers to a natural killer (NK) cell that expresses a CAR.

[0118] In some embodiments, the CAR excludes a CD8 signal peptide as described herein. As can be determined by those of skill in the art, various functionally similar or equivalent components of these CARs can be swapped or substituted with one another, as well as other similar or functionally equivalent components known in the art or listed herein.

[0119] Any cell- surface moiety can be targeted by a CAR. Often, the target will be a cellsurface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. In some embodiments, the extracellular target binding

[0120] #14645213vldomain binds to any one of CD19, CD37, CD70, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL extracellular antigen-binding domain, e.g., as described in PCT / US2020 / 065733, PCT / US2020 / 036108, PCT / US2018 / 013215, PCT / US2018 / 013213, PCT / US2018 / 027783, PCT / US2018 / 013221, PCT / US2018 / 022974, PCT / US2019 / 042268, PCT / US2019 / 038518, PCT / US2019 / 066357, PCT / US2019 / 013103, PCT / US2019 / 017727, PCT / US2020 / 051018, and / or PCT / US2018 / 013095.

[0121] Extracellular antigen-binding domain

[0122] As used herein, the term "extracellular antigen-binding domain" refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding to a target. The extracellular target binding domain may specifically bind to its binding partner, i.e., the target. As nonlimiting examples, the extracellular antigen-binding domain can include an antigen domain of an antibody or antibody reagent, or a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule that binds specifically to a portion of a protein and / or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand:cognate partner binding can result in the alteration of the ligand-bearing receptor, or activate a physiological response, for example, the activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a conserved function across at least two species.

[0123] Any cell- surface moiety can be targeted by a CAR. In some embodiments, the target will be a cell-surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. To target Tregs, antibody reagents can be targeted against, e.g., Glycoprotein A Repetitions Predominant (GARP), latency-associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, 0X40, 4- IBB, and LAG-3.

[0124] In some embodiments, the CAR vector comprises a CAR polynucleotide encoding an extracellular antigen-binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL extracellular antigen-binding domain. In some embodiments, a CAR polypeptide comprises an extracellular antigen-binding domain that binds to B7H3.

[0125] In some embodiments, the extracellular antigen-binding domain comprises:

[0126] #14645213vl(a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 24; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 25; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 26; and

[0127] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 27; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 28; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 29.

[0128] In some embodiments, the extracellular antigen-binding domain comprises:

[0129] (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 24 or a conservatively modified variant thereof; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 25 or a conservatively modified variant thereof; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 26 or a conservatively modified variant thereof; and

[0130] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 27 or a conservatively modified variant thereof; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 28 or a conservatively modified variant thereof; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 29 or a conservatively modified variant thereof.

[0131] In some embodiments, the CD 19 extracellular antigen-binding domain comprises a VH of SEQ ID NO: 30 and a VL of SEQ ID NO: 31. In some embodiments, the CD 19 extracellular antigen-binding domain comprises a VH having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 30 and a VL having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 31.

[0132] In some embodiments, the CD 19 extracellular antigen-binding domain comprises a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 50. In some embodiments, the CD 19 extracellular antigen-binding domain comprises a VH having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 49 and a VL having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 50. In some embodiments, the CD19 extracellular antigen-binding domain comprises CD19 scFv FMC63 (e.g., as described in Seigner, J., Zajc, C.U., Dotsch, S. et al. Sci Rep 13, 23024 (2023)). In some embodiments, the CD 19 extracellular antigen-binding domain comprises an amino acid sequence of SEQ ID NO:

[0133] #14645213vl115 or an amino acid sequence having at least at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 115.

[0134] In some embodiments, the CD 19 extracellular antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 110 or an amino acid sequence having at least at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 110.

[0135] In some embodiments, the mesothelin extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 4; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 5; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 6; and

[0136] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 7; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 8; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 9.

[0137] In some embodiments, the mesothelin extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 4 or a conservatively modified variant thereof; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 5 or a conservatively modified variant thereof; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 6 or a conservatively modified variant thereof; and

[0138] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 7 or a conservatively modified variant thereof; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 8 or a conservatively modified variant thereof; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 9 or a conservatively modified variant thereof.

[0139] In some embodiments, a mesothelin extracellular antigen-binding domain comprises a VH of SEQ ID NO: 2 and a VL of SEQ ID NO: 3. In some embodiments, a mesothelin antigenbinding domain comprises a VH having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 2 and a VL having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 3.

[0140] In some embodiments, a mesothelin extracellular antigen-binding domain comprises:

[0141] #14645213vl(a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 13; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 14; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 15; and

[0142] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 16; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 17; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 18.

[0143] In some embodiments, a mesothelin extracellular antigen-binding domain comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 13 or a conservatively modified variant thereof; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 14 or a conservatively modified variant thereof; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 15 or a conservatively modified variant thereof; and

[0144] (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 16 or a conservatively modified variant thereof; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 17 or a conservatively modified variant thereof; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 18 or a conservatively modified variant thereof.

[0145] In some embodiments, a mesothelin extracellular antigen-binding domain comprises a VH of SEQ ID NO: 19 and a VL of SEQ ID NO: 20. In some embodiments, a mesothelin antigen-binding domain comprises a VH having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 19 and a VL having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 20.

[0146] In some embodiments, the mesothelin extracellular antigen-binding domain comprises an amino acid sequence of any one of SEQ ID NOs: 111-113 or an amino acid sequence having at least at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to any one of SEQ ID NOs: 111-113.

[0147] Hinge and Transmembrane Domains

[0148] In some embodiments, the CAR polypeptide further comprises a transmembrane domain, e.g., a hinge / transmembrane domain, which joins the extracellular antigen-binding domain to the

[0149] #14645213vlintracellular signaling domain. The binding domain of the CAR is, in some embodiments, followed by one or more "hinge domains," which plays a role in positioning the extracellular antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi- synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8alpha), CD4, CD28, 4- IBB, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, the CAR comprises polynucleotide encoding CD8alpha hinge / transmembrane domain. In some embodiments, the CAR comprises a polynucleotide encoding a 41BB intracellular domain.

[0150] In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain includes a CD8a hinge region.

[0151] As used herein, "transmembrane domain" (TM domain) refers to the portion of the CAR that fuses the extracellular binding portion, in some embodiments via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and intracellular signaling domain) and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR, which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used herein, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR.

[0152] As used in relation to a transmembrane domain of a protein or polypeptide, "fragment thereof" refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0153] In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8,

[0154] #14645213vlCD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0155] As used herein, a "hinge / transmembrane domain" refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4- IBB. In some embodiments, the hinge / transmembrane domain of a CAR or fragment thereof is derived from or includes the hinge / transmembrane domain of CD8 (e.g., any one of SEQ ID NOs: 1, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha or CD8a) and beta (CD813 or CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.

[0156] Homologs and / or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD8 sequence.

[0157] In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO: 114 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYC); or includes the sequence of SEQ ID NO: 1; or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 114.

[0158] #14645213vlCo-stimulatory Domains

[0159] Each CAR described herein optionally includes the intracellular domain of one or more co-stimulatory molecule or co-stimulatory domain. As used herein, the term "co-stimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fe receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4- IBB, CD27, CD28, or 0X40. In one example, a 4- IBB intracellular domain (ICD) can be used (see, e.g., below and SEQ ID NO: 100 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or variants thereof). In some embodiments, the CAR comprises a CD28 ICD. In some embodiments, the CAR comprises a CD28 ICD comprising an amino acid sequence of SEQ ID NO: 21 or having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 21.

[0160] Additional illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SEAMF1), CD152 (CTEA4), CD223 (EAG3), CD270 (HVEM), CD273 (PD-E2), CD274 (PD-E1), CD278 (ICOS), DAP10, EAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1 BB. 4- IBB (CD 137; TNFRS9) is an activation induced costimulatory molecule, and is an important regulator of immune responses.

[0161] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4- IBB is expressed on activated T lymphocytes. 4-1BB sequences are known for a number of species, e.g., human 4-1 BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI Reference Sequence:

[0162] NM_001561.5). 4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BB can refer to the 4-1BB of, e.g., dog, cat, cow, horse, pig, and the like.

[0163] Homologs and / or orthologs of human 4- IBB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4- IBB sequence.

[0164] Intracellular Signaling Domains

[0165] #14645213vlIn some embodiments, the CAR comprises a polynucleotide encoding a CD3zeta intracellular signaling domain.

[0166] The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target / extracellular antigen-binding domains(s) render the receptor sensitive to signaling activation when the chimeric target / extracellular antigen-binding domain binds the target / antigen on the surface of a targeted cell.

[0167] With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3-zeta signals upon antigen binding. So-called "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD 137) and activation (CD3-zeta;) domains, and so-called "third-generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3-zeta). In various embodiments, the CAR is selected to have high affinity or avidity for the target / antigen - for example, antibody-derived target or extracellular antigen-binding domains will generally have higher affinity and / or avidity for the target antigen than would a naturally occurring T cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific T cell targeting by CAR-T cells.

[0168] CARs as described herein include an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is from CD3-zeta; (see, e.g., below).

[0169] Additional non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling domains that are of particular use in the technology include those derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0170] CD3 is a T cell co-receptor that facilitates T lymphocyte activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co- stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammalian CD3 consists of a CD3-gamma chain, a CD3delta chain, and two CD3-epsilon chains.

[0171] These chains associate with a molecule known as the T cell receptor (TCR) and the CD3-zeta to generate an activation signal in T lymphocytes. A complete TCR complex includes a TCR, CD3-zeta, and the complete CD3 complex.

[0172] #14645213vlIn some embodiments of any aspect, a CAR polypeptide described herein includes an intracellular signaling domain that includes an Immunoreceptor Tyrosine-based Activation Motif or IT AM from CD3-zeta, including variants of CD3-zeta such as IT AM-mutated CD3-zeta, CD3-eta, or CD3-theta. In some embodiments of any aspect, the IT AM includes three motifs of IT AM of CD3-zeta (ITAM3). In some embodiments of any aspect, the three motifs of IT AM of CD3-zeta are not mutated and, therefore, include native or wild-type sequences. In some embodiments, the CD3-zeta sequence includes the sequence of a CD3-zeta as set forth in the sequences provided herein, e.g., a CD3-zeta sequence of SEQ ID NO: 99 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or variants thereof.

[0173] For example, a CAR polypeptide described herein includes the intracellular signaling domain of CD3-zeta. In some embodiments, the CD3-zeta intracellular signaling domain corresponds to an amino acid sequence of SEQ ID NO: 99 or includes a sequence of SEQ ID NOs: 99; or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence of SEQ ID NOs: 99.

[0174] In some embodiments, the intracellular domain is the intracellular domain of a 4- IBB. In some embodiments, the 4- IBB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO: 100; or includes a sequence selected from SEQ ID NO: 100; or includes at least 75%, at least 80%, at least 85%, 35 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 100.

[0175] Individual CAR and other construct components as described herein can be used with one another and swapped in and out of various constructs described herein, as can be determined by those of skill in the art. Each of these components can include or consist of any of the corresponding sequences set forth herein, or variants thereof.

[0176] A more detailed description of CARs and CAR-T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated by reference herein in its entirety.

[0177] Signal Peptide

[0178] #14645213vlIn some embodiments, a CAR polypeptide as described herein includes a signal peptide. Signal peptides can be derived from any protein that has an extracellular domain or is secreted. A CAR polypeptide as described herein may include any signal peptides known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, e.g., a CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO: 101 (MALPVTALLLPLALLLHAARP), or including the amino acid sequence of SEQ ID NO: 101, or including an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 101.

[0179] In further embodiments, a CAR polypeptide described herein may optionally exclude one of the signal peptides described herein, e.g., a CD8 signal peptide of SEQ ID NO: 101 or an IgK signal peptide of SEQ ID NO: 102 (METDTLLLWVLLLWVPGSTGD).

[0180] Linker Domain

[0181] In some embodiments, the CAR further includes a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains / regions of the CAR as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Linker sequences useful for the invention can be from 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids in length, and include any suitable linkers known in the art. For instance, linker sequences useful for the invention include, but are not limited to, glycine / serine linkers, e.g., GGGSGGGSGGGS (SEQ ID NO: 103) and linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO: 104)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 105)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 106) as described by Whitlow et al., Protein Eng.

[0182] 6(8) :989-95, 1993, the contents of which are incorporated herein by reference in its entirety; the linker sequence of GGSSRSSSSGGGGSGGGG (SEQ ID NO: 107) as described by Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011 (9), 2011, the contents of which are incorporated herein by reference in its entirety; as well as linker sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing Cre-Lox recombination site as described by Sblattero et al., Nat. Biotechnol. 18(l):75-80, 2000, the contents of which are incorporated herein by reference in its entirety. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.

[0183] #14645213vlFurthermore, linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 108, GSGATNFSLLKQAGDVEENPGP) and T2A (SGGGGEGRGSLLTCGDVEENPGPR, SEQ ID NO: 109), 2A-like linkers or functional equivalents thereof and combinations thereof.

[0184] For example, a P2A linker sequence can correspond to the amino acid sequence of SEQ ID NO: 108. In various examples, linkers having sequences as set forth herein, or variants thereof, are used. It is to be understood that the indication of a particular linker in a construct in a particular location does not mean that only that linker can be used there. Rather, different linker sequences (e.g., P2A and T2A) can be swapped with one another (e.g., in the context of the constructs of the present invention), as can be determined by those of skill in the art. In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. Linkers such as these can be used in the context of polyproteins, such as those described below. For example, they can be used to separate a CAR component of a polyprotein from a therapeutic agent (e.g., an antibody, such as a scFv, single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) component of a polyprotein (see below).

[0185] Full CARs

[0186] In some embodiments, the CAR is selected from a group consisting of (1) a CAR that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, (2) a CAR that binds to any pair of CD19 / CD79b, BCMA / TACI, or (3) is a TriPRIL extracellular antigen-binding domain. In some embodiments, the CAR comprises a CD28 ICD.

[0187] In some embodiments, the CAR polypeptide comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity of a sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the CAR polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98. In some embodiments, the CAR polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98.

[0188] #14645213vlIn some embodiments, the CAR polypeptide comprises an extracellular antigen binding domain (e.g., a CD 19 extracellular- antigen binding domain or a mesothelin binding extracellular-antigen binding domain), a CD8 transmembrane domain (e.g., SEQ ID NO: 114), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 21), and a CD3-zeta intracellular signaling domain (e.g., SEQ ID NO: 99). In some embodiments, the CAR polypeptide comprises a CD19 extracellular- antigen binding domain (e.g., comprising a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 50), a CD8 transmembrane domain (e.g., SEQ ID NO: 114), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 21), and a CD3-zeta intracellular signaling domain (e.g., SEQ ID NO: 99). In some embodiments, the CAR further comprises a CD8 signal peptide (e.g., SEQ ID NO: 101).

[0189] Sequences

[0190] Table 1: CARs and CAR parts

[0191]

[0192] #14645213vl

[0193]

[0194] #14645213vl

[0195]

[0196] #14645213vl

[0197]

[0198] #14645213vl

[0199]

[0200] #14645213vl

[0201]

[0202] IFNGR Guide RNA homology region sequences

[0203]

[0204] #14645213vl

[0205]

[0206] #14645213vl

[0207]

[0208] #14645213vl

[0209]

[0210] #14645213vlTable 3: Chimeric Antigen Receptors cont.

[0211]

[0212] #14645213vl

[0213]

[0214] #14645213vl

[0215]

[0216] #14645213vl

[0217]

[0218] #14645213vl

[0219]

[0220] #14645213vl

[0221]

[0222] #14645213vl

[0223]

[0224] #14645213vl

[0225]

[0226] #14645213vl

[0227]

[0228] #14645213vl

[0229]

[0230] #14645213vl

[0231]

[0232] #14645213vl

[0233]

[0234] #14645213vl

[0235]

[0236] #14645213vl

[0237]

[0238] #14645213vl

[0239]

[0240] Table 4: CAR Parts cont.

[0241]

[0242] Methods of Treatment

[0243] #14645213vlIn some aspects, this disclosure provide a method treating cancer in a subject, the method comprising administering an immune cell (e.g., a CAR-T cell) that is deficient in IFNyR expression and / or signaling to a subject. "Cancer" as used herein can refer to a hyperproliferation of cells whose unique trait, loss of normal cellular control, results in unregulated growth, lack of differentiation, local tissue invasion, and / orr metastasis. In some embodiments, the cancer is a CD 19 expressing cancer and the CAR-T cell comprises a CAR having an CD 19 binding extracellular antigen-binding domain. In some embodiments, the cancer is a mesothelin expressing cancer and the CAR-T cell comprises a CAR having an mesothelin binding extracellular antigen-binding domain. In some embodiments, the cancer expresses one or more of the following antigens: CD19, B7H3, BCMA, TACI, CD79b, CD22, CD30, CS1, Claudin 18.2, GPC3, GD2, GPCR, PSMA, mesothelin, MUC1, MUC16, EGFR, IL-13Ralpha2, EGFRvIII, CD20, CD79a, or combinations thereof, and the CAR comprises an extracellular antigen-binding domain that binds to one or more of these antigens.

[0244] In some embodiments, the method of treating a cancer in subject comprises treating a solid tumor. In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer is triple negative breast cancer (TNBC). TNBC includes breast cancer that is human epidermal growth factor receptor 2 (Her2) negative, estrogen receptor (ER) negative and progesterone receptor (PR) negative. In some embodiments, the triple negative breast cancer is B7H3 positive. In some embodiments, treating TNBC in a subject comprises administering an B7H3 binding CAR-T cell that is deficient in IFNyR expression to a subject, wherein TNBC cells of the subject are B7H3 positive. In some embodiments, the method of treating a cancer in subject comprises treating a liquid tumor. In some embodiments, the method of treating a cancer in subject comprises treating pancreatic cancer. In some embodiments, the method of treating a cancer in subject comprises treating ovarian cancer. In some embodiments, the method of treating a cancer in subject comprises treating ovarian cancer. In some embodiments, the method of treating a cancer in subject comprises treating a B-cell malignancy. In some embodiments, the method of treating a cancer in subject comprises treating lymphoma or leukemia. In some embodiments, the method of treating a cancer in subject comprises treating B cell lymphoma or B cell leukemia. In some embodiments, the method of treating a cancer in subject comprises treating B-cell acute lymphoblastic leukemia (B-ALL), B-cell chronic lymphocytic leukemia (B-CLL), B-cell prolymphocytic leukemia (B-PLL), Hairy cell leukemia (HCL), acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, juvenile myelonmonocytic leukemia, Juvenile Myelomonocytic Leukemia, Larger Granular Lymphocytic Leukemia, or Blastic plasmacytoid

[0245] #14645213vldendritic cell neoplasm. In some embodiments, the method of treating a cancer in subject comprises treating Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Waldenstrom macroglobulinemia, Primary intraocular lymphoma, or Primary mediastinal B-cell lymphoma (PMBCL). In some embodiments, the method of treating a cancer in subject comprises treating T cell lymphoma or T cell leukemia.

[0246] Subject

[0247] In some embodiments, methods described herein comprise treating a subject having cancer. As used herein, a “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female.

[0248] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., diagnosed with a mesothelin expressing cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.

[0249] Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0250] A “subject in need” of treatment for a particular condition (e.g., a cancer described herein) can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0251] Pharmaceutical Compositions

[0252] #14645213vlIn some embodiments, the methods described herein comprise administering to the subject a pharmaceutical composition comprising the immune cells (e.g., CAR-T cell deficient in IFNgR expression or signaling). As used herein, the term “pharmaceutical composition” refers to the active agent (e.g., a CAR-T cell expressing a CAR) in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry.

[0253] The phrase “pharmaceutically acceptable carrier” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found to occur in nature.

[0254] In one aspect of the technology, the technology described herein relates to a pharmaceutical composition including activated CAR-T cells comprising a CAR or a bispecific CAR described herein, and optionally a pharmaceutically acceptable carrier. In one aspect of the technology, the technology described herein relates to a pharmaceutical composition including activated CAR-NK cells comprising a CAR or a bispecific CAR described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for cell-based therapeutic formulation include saline and aqueous buffer solutions, Ringer’s solution, and serum component, such as serum albumin, HDL and LDL. The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient” or the like are used interchangeably herein.

[0255] In some embodiments, the pharmaceutical composition including immune cells described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient’s natural defenses against contaminants, the components apart from the immune cells themselves are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Any of these can be added to the immune cells in preparation prior to administration. Suitable vehicles that can be used to provide parenteral dosage forms of immune cells as disclosed within are known to those skilled in the art. Examples include, without limitation:

[0256] #14645213vlsaline solution; glucose solution; aqueous vehicles including but not limited to, sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer’s injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0257] Dosage

[0258] In some embodiments, the immune cells comprising a CAR and / oror engineered immune cell receptor described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject. A pharmaceutical composition including the immune cells described herein can generally be administered at a dosage of 104to 109cells / kg body weight. If necessary, immune cells compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 30 319:1676, 1988).

[0259] Administration

[0260] In some embodiments, the methods described herein include administering an effective amount of the immune cells described herein (e.g., activated CAR-T cells or NK cells that are deficient in IFNgR expression or signaling). As used herein, “treating a subject having a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of a tumor.

[0261] The term "effective amount" as includes the amount active immune cell administered to a subject. Described herein needed to treat at least one or more symptom of the cancer and relates to a sufficient amount of the cell preparation or composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of activated immune cells (e.g., activated CAR-T cells) described herein that is sufficient to provide a particular anti-

[0262] #14645213vlcondition effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom cancer, alter the course of the cancer (for example but not limited to, slowing the progression of the cancer), or reverse a symptom of a cancer. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0263] In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intravenous administration.

[0264] In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intravascular administration. In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intraperitoneal administration.

[0265] Modes of Administration

[0266] The immune cells described herein can be administered to a patient transarterially, intratumorally, intranodally, intraperitoneally, intrathecally, intramedullary, or orally. In some embodiments, the compositions of the immune cells (e.g., the CAR-T cells) may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0267] In some embodiments, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate the cells of interest, e.g., T cells. T cell isolates can be expanded by contact with an artificial APC (aAPC), e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs, and treated such that one or more CAR constructs or engineered immune cell receptor constructs of the technology may be introduced, thereby creating a CAR-T cell or a engineered immune cell receptor T cell. Subjects in need thereof can subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. Following or concurrent with the transplant, subjects can receive an infusion of the expanded CAR-T cells. In some embodiment, expanded cells are administered before or following surgery. In some embodiments, lymphodepletion is performed on a subject prior to administering one or more immune cell as described herein. In such embodiments, the lymphodepletion can include administering one or more of melphalan, survivin, cyclophosphamide, and fludarabine. The dosage of the above treatments to be administered to a

[0268] #14645213vlpatient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

[0269] In some embodiments, a single treatment regimen is required. In others, administration of one or more subsequent doses or treatment regimens can be performed. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. In some embodiments, no additional treatments are administered following the initial treatment.

[0270] Efficacy

[0271] The efficacy of immune cells described herein in, e.g., to treatment a cancer, or to induce a response as described herein (e.g., a reduction in cancer cells) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein is altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced, e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a mesothelin-expressing cancer treated according to the methods described herein or any other measurable parameter appropriate. Treatment according to the methods described herein can reduce levels of a marker or symptom of a mesothelin-expressing cancer, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0272] Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of cancer in an individual or an animal (some nonlimiting examples include a human or an animal) and includes: (1) inhibiting the cancer, e.g., preventing a worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the cancer, e.g., causing regression of symptoms. An effective amount for the treatment of a cancer means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for cancer. Efficacy of an agent can be determined by assessing physical indicators of mesothelin-expressing cancer or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination

[0273] #14645213vlof parameters. Efficacy of a given approach can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0274] Methods of Increasing Persistence

[0275] In some embodiments, this disclosure provides a method of increasing the persistence of a CAR-immune cell (e.g., a CAR-T cell or CAR-NK cell) in a subject, the method comprising contacting the CAR-immune cell with an anti-IFNgR antibody. In some embodiments, this disclosure provides a method of increasing the persistence of a CAR-immune cell in a subject, the method comprising administering the CAR-immune cell and an anti-IFNgR antibody.

[0276] “Persistence” as used in the context of the immune cells described herein refers to the duration with which an immune cell administered to a subject remains present and / or active in the subject. Persistence can be determined in any suitable way. In some embodiments, persistence is determined by measuring an amount of immune cells (that were administered to the subject (e.g., CAR-T cells) in a biological sample from the subject (e.g., a volume of blood from the subject). In some embodiments, persistence is determined by measuring exhaustion markers (e.g., CTEA-4, Eag-3, PD-1 and / or Tim-3) of the immune cells that were administered to the subject. In some embodiments, immune cells with increased persistence in a subject have increased persistence relative to a control (e.g., an immune cell that is deficient in IFNgR expression and / or signaling). For example, an immune cell that is deficient in IFNgR expression and / or signaling may have at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 95%, at least 98%, or at least 99%) increased persistence compared to a control immune cell that is not deficient in IFNgR expression and / or signaling.

[0277] EXAMPLES

[0278] Example 1. IFNg-resistant CAR T cells demonstrate increased survival, efficacy and durability in multiple tumor models.

[0279] Introduction

[0280] CAR T cells have transformed the landscape of treatment for patients with hematologic malignancies, with multiple recent approvals in B-cell lymphomas, B-cell leukemias, and

[0281] #14645213vlmultiple myeloma. One of the most clinically significant challenges in this setting is the incidence of relapse, which, broadly, can occur through either antigen escape of the tumor or insufficient engraftment and persistence of the CAR T cells. Current evidence suggests that both the peak expansion and duration of persistence of the CAR T cells correlates with durable responses in patients with B-ALL, chronic lymphocytic leukemia (CLL) and various other forms of B cell lymphoma (7-5). While data from patients with solid tumors who have received CAR T cells is limited, a higher CAR-T peak expansion was associated with response in patients with gastrointestinal tumors (6). This is further supported by the correlation of CAR-T persistence beyond 6 weeks with superior clinical outcomes in neuroblastoma patients (7). Collectively, these data highlight the importance of enhancing the survival and persistence of CAR-T cells post-infusion.

[0282] Interferon-gamma is a proinflammatory cytokine released primarily by T cells and NK cells that activates innate immune cells (8, 9), upregulates immune checkpoint proteins such as PD-L1 on cancer cells and immune cells (JO, 77) and can drive target cell death (72-74). Uptake of IFNg by T cells also increases their cytotoxicity (75), restricts memory formation in response to weak TCR activation (76), and drives activation-induced cell death (77). IFNg receptor signaling in tumor cells is thought to be important for the success of immune checkpoint blockades (ICB), such as pembrolizumab and nivolumab, based on retrospective studies identifying IFNG and IFNg-responsive genes to be predictive of ICB response in patients with metastatic melanoma (18) and non-small cell lunger cancer (79). However, IFNg can also lead to reduced efficacy of ICB due to immune cell death (20) and disruption of intratumoral stem-like T cells (27). Given the high levels of IFNg that are produced by CAR T cells after infusion into patients, it was sought to better understand the role of this cytokine and its receptor in mediating both toxicities and anti-tumor effects in hematologic malignancies and in solid tumors.

[0283] Previous studies have shown that pharmacologic blockade or genetic knockout of IFNg production by CAR T cells did not affect cytotoxic activity against hematologic malignancies and has the potential to mitigate cytokine release syndrome by reducing downstream monocyte and macrophage activation (22). IFNg knockout CAR T cells had reduced cytotoxicity against in solid tumor. A separate study indicated that engagement of the IFNgR signaling cascade leads solid tumor cells to upregulate adhesion molecules that are important for achieving sufficient avidity for CAR T cells to mediate successful cytotoxicity (23).

[0284] Here, it is demonstrated that IFNg limits CAR T cell responses to antigen, primarily via inducing T cell death. Using genetic and pharmacologic approaches, it is shown that blockade of IFNg or IFNgR protects CAR T cells from IFNg-mediated cell death. In models of hematologic

[0285] #14645213vlmalignancies, this resulted in greater CAR T cell persistence without compromising antitumor activity. It was discovered that while IFNg knockout (IFNgKO) CAR T cells had reduced cytotoxic activity toward solid tumors, IFNgR knockout (IFNgRKO) CAR-T had greater expansion and persistence, as well as improved anti-tumor activity in both xenograft and syngeneic models of solid tumors. RNA sequencing of these tumors suggests that lack of IFNg uptake by CAR T cells drives greater interferon signaling in tumor cells, resulting in increased tumor cell death. Further, IFNgRKO CAR T cells formed long term memory and protected mice from developing tumors on re-challenge with pancreatic tumor cells, whereas mice treated with control CAR-T had rapid tumor growth. Collectively, these data demonstrate that IFNg signaling limits CAR T cell survival, expansion, persistence, and memory formation and suggests that strategic manipulation of this pathway could enhance the efficacy of cell therapies for patients with hematologic or solid malignancies.

[0286] Results

[0287] Knockout of IFNg or IFNg receptor drives greater CAR T cell expansion in vitro.

[0288] Previous studies reported that the genetic deletion of IFNg in CD19-targeting CAR-T cells with either a CD28 or 4- IBB costimulatory domain limited potential toxicities without compromising antitumor efficacy in hematologic malignancies (22). Unexpectedly, these IFNgKO CAR-T exhibited greater expansion in response to the CD19+Nalm6 leukemia cell line in vitro, and the effect was particularly notable in CAR T cells bearing a CD28 costimulatory domain. To better study the effect of IFNg signaling on these events, CD19-directed, CD28-bearing CAR T cells were generated with either IFNg or IFNgR knocked out via CRISPR / Cas9. Similar to previous studies (22), the same vectors included guide RNA to knock out the T cell receptor alpha constant (TRAC) — either alone (referred to as CARci ) or in combination with the IFNg (IFNgKO CARCDW) or IFNgR (IFNgRKO CARci ) ; this approach enabled selection and confirmation of successful gene targeting (FIG. 1A). These constructs enabled investigation of the various aspects of IFNg signaling with an array of CAR T cells capable of (1) producing and responding to IFNg (CAR; control), (2) IFNg-deficient, which respond to but don’t produce IFNg (IFNgKO CAR) and (3) IFNg-resistant, which produce IFNg but do not respond to it (IFNgRKO CAR) (FIG. 9A).

[0289] Following expansion and CD3 selection, as depicted in FIGs. 9B-9D, the KO phenotype was verified through the absence of CD3, IFNgR, and / or IFNg on the CAR T cells compared to untransduced T cells (UTD; FIGs. IB, ID; FIGs. 9E-9F). The functional loss of IFNg receptor

[0290] #14645213vlwas confirmed through exposure to recombinant human IFNg, which resulted in the loss of STAT1 phosphorylation in IFNgRKO CARCDI (FIG. ID; FIGs. 9G-9H). Basic phenotyping revealed that IFNgKO CARCDW and, to a greater extent IFNgRKO CARCDI9, had a CD4:CD8 ratio closer to 1:1 compared to control CARCDI9, which was closer to 1:2 (FIG. IE). Exposure of the CAR T cells to CD 19 antigen resulted in a similar level of activation among groups, as measured by CD69 expression (FIG. 91). In agreement with these findings, all conditions exhibited similar cytokine production in response to CD 19 antigen, aside from IFNg in the IFNg-deficient CAR T cells, as expected (FIG. IF; FIGs. 10A-10C). Both IFNgKO and IFNgRKO CARCDI9 cells exhibited greater expansion in response to antigen exposure in the form of Nalm6 and JeKo-1 target cells (FIG. 1G; FIG. 11A), or plate-bound CD19 antigen (FIG.

[0291] 11B), suggesting that loss of IFNg signaling drives expansion of CAR T cells independent of tumor / T cell interactions.

[0292] These findings were confirmed with a pharmacologic approach by using blocking antibodies to either IFNg or IFNgR and observed greater expansion of non-CRISPR / Cas9 edited "wildtype" CAR-T in response to stimulation via Nalm6 target cells or CD19 antigen alone (FIG. 11C). The enhanced expansion of IFNgKO or IFNgRKO CARCDI9 T cells was also observed across varying levels of CD19 antigen density (FIGs. 11D-11E). Importantly, the increased expansion of IFNgKO and IFNgRKO CARCDW T cells was antigen-dependent as they did not survive or expand in the absence of antigen or IL-2 (FIGs. 11F-11G). To determine if these effects could be more broadly generalized to CAR T cells as a whole, versus only those with a CD28 costimulatory domain, IFNgKO and IFNgRKO CD19-targeted CAR T cells bearing a 4-1BB costimulatory domain were generated (FIGs. 1H-1I). In contrast to CD28 CAR T cells, it was discovered that the loss of IFNg or IFNgR on CAR T cells with a 4- IBB costimulatory domain did not drive increased T cell expansion in response to tumor cells (FIG.

[0293] 1J). These data suggest that IFNg signaling restricts the growth of CD28, but not 4- IBB, CAR T cells.

[0294] IFNgKO and IFNgRKO CARCDI9 T cells maintain antitumor activity against hematologic malignancies and display greater persistence in vivo.

[0295] To determine if the loss of IFNg signaling in CAR T cells impacts their cytotoxic activity, similar levels of granzyme B and perforin in control, IFNgKO and IFNgRKO CARCDI9 T cells following exposure to CD 19 antigen were first measured and confirmed (FIGs. 12A-12B). In real-time long-term cytotoxicity assays, it was observed that the loss of IFNg signaling did not compromise cytotoxicity against a one-time exposure to CD19+Nalm6 or JeKo-1 cell

[0296] #14645213vllines in vitro (FIG. 12C). Furthermore, IFNg and IFNgR knockout CARCDI maintained indistinguishable cytotoxic potential following repetitive exposure to Nalm6 cells in vitro (FIG.

[0297] 2A).

[0298] Next, the anti-tumor activity and persistence of control, IFNgKO and IFNgRKO CARCDW was measured in vivo using two different xenograft models. NSG mice bearing JeKo-1 tumors were treated with each of these CAR constructs and monitored for tumor burden, survival, and CAR T cell expansion. (FIG. 2B). It was found that while there was some donor variability (FIGs. 12I-12K, 12M-12N) IFNgKO and IFNgRKO CARCDI9 mediated similar antitumor effects and survival compared to control CARCDI9 (FIGS. 2C-2E). Furthermore, mice receiving IFNgKO, and to a greater extent IFNgRKO, CARCDI9 cells exhibited higher T cell expansion in the blood (FIG. 2F, FIGs.l2L, 120). The anti-tumor effects were also similar in a stress model of lymphoma (FIGs. 22A-22D), with a transient improvement in anti-tumor activity in the absence of IFNg signaling. The anti-tumor effects were confirmed in a Nalm6 xenograft model (FIGs. 12D-12G). While early serum collection confirmed the presence of human IFNg in mice that received control and IFNgRKO CARCDI9 T cell-treated mice, with corresponding absence of IFNg in IFNgKO CARcm9-treated mice (FIG. 12H), differences in CAR-T expansion and persistence could not be determined, as T cells were not detected at later timepoints, likely due to the aggressive nature of this particular stress model. Collectively, these data demonstrate neither IFNg deficiency nor resistance in CAR T cells compromised their antitumor efficacy in models of B-cell malignancies, and IFNg resistance appeared to increase the expansion of CAR T cells in vivo.

[0299] Abrogation of IFNg signaling prompts minimal effects on CD19 CAR-T differentiation and exhaustion in vitro.

[0300] To better understand the effects of IFNg on CAR T cells, RNA sequencing was performed on in vztra-activated CARCDI9, IFNgKO CARCDW, and IFNgRKO CARCDI9 cells. Briefly, CAR-T subsets were left untreated or activated with plate-bound CD 19 antigen overnight prior to transcriptional profiling. Initial observation revealed that all groups had a similar upregulation of highly expressed genes following exposure to CD19 antigen (CCL1, CCL3 / L1, CSF2, GZMB, IL3, NFKBIA, TNFRSF9), suggesting that IFNg does not change the overall biology of antigen activation (FIG. 13 A). It was next confirmed that the loss of IFNg signaling in IFNgKO and IFNgRKO CARCDW cells could be visualized at the RNA level as shown by reduced interferon-related gene expression (STAT1, TBX21, IL12RB2) following CD19 activation (FIG. 13B). Both IFNgKO (FIG. 3A) and IFNgRKO (FIG. 3B) CARCDI9 displayed a marked decrease in interferon signaling-related genes compared to control CARCDW.

[0301] #14645213vlIn agreement with the reported roles of IFNg in T cell differentiation (16), it was observed that IFNgKO and IFNgRKO CARCDI cells exhibited a less differentiated profile (lower CD45RO', higher LEF1, TCF7) compared to control CARCDI (FIG. 3C). To determine if these characteristics translated to a protein level, the memory phenotype of the different subsets was first assessed. While IFNgKO and IFNgRKO CARCDI9 cells trended towards a higher naive phenotype, donor variability was high and there were no significant differences in the differentiation of the CAR-T subsets using these metrics (FIG. 3D; FIG. 14A). Intriguingly, cell type profiling further revealed a possible skewing of IFNg-deficient and -resistant CARCDI9 T cells away from a type 1 phenotype (FIGs. 14B-14C). Given that IFNg can influence T cell subsets (24), it was predicted that IFNg and IFNgR knockout CARCDI9 might have a higher percentage of CD4+type 2 helper cells (Th2; CCR4+CCR6 CXCR3 ) compared to type 1 (Thl; CCR4 CCR6 CXCR3+). While slight skewing towards a Type 2 (Th2) phenotype in the knockout cells was observed, there were no significant differences in T cell composition between the groups (FIG. 14D).

[0302] Since IFNg is also known to influence immune checkpoint expression (77), the CTLA-4, Lag-3, PD-1 and Tim-3 levels were next assessed on all CAR-T subsets by flow cytometry following overnight activation with plate-bound CD19 antigen (FIG. 15A), JeKo-1 (FIG. 15B) or Nalm6 (FIG. 15C). Despite seeing changes at the RNA level at this time point, protein expression of these markers was not significantly different between groups. Given that these 'exhaustion' markers are also tied to T cell activation, these findings were not surprising. To better identify long-term exhaustion, CAR T cells were repeatedly exposed to irradiated JeKo-1 or Nalm6 cells and monitored over time (FIG. 3E; FIG. 15D). While IFNg plays a well-known role in the upregulation of immune checkpoint proteins, IFNgKO and IFNgRKO CARCDI9 did not exhibit a marked change in single positive or triple positive (Lag-3+PD-l+Tim-3+) populations. Collectively, these data suggest that while the loss of IFNg signaling in CAR-T could potentially yield a slightly less differentiated and exhausted population, these do not appear to be the dominant drivers of the observed increases in CAR-T cell expansion.

[0303] Knockout of IFNg or IFNgR protects CD19-directed CAR T cells from cell death.

[0304] Since the differentiation and exhaustion profile of IFNg-deficient and IFNg-resistant CARCDW cells were not significantly different from control CARCDI9, it was next sought to determine if they differed in proliferative potential. RNA sequencing revealed a potential proliferative advantage as

[0305]

[0306] all increased in IFNgKO (left) and IFNgRKO (right) CARCDI9 cells compared to control (FIG. 4A). Despite this upregulation, cell

[0307] #14645213vltrace violet assays revealed that all CAR groups exhibited similar proliferative capacity in response to CD19 antigen (FIGs. 16A-16B) and Nalm6 target cells (FIG. 4B; FIG. 16C).

[0308] It was next hypothesized that the persistence of IFNgKO and IFNgRKO CARCDW in response to antigen exposure could be due to protection from IFNg-mediated cell death, consistent with known IFNg biology in other settings (14, 17). A distinct upregulation of cell death-related genes (STAT1, XAF1, FASLG, CASP1, CASP3) was noted in control CARCDI cells compared to IFNgKO and IFNgRKO CARCDI9 after antigen stimulation (FIG. 4A). This was further validated through an increased expression of Annexin V and cleaved Caspase 3 in control CARCDW T cells compared to IFNgKO and IFNgRKO CARCDI9 following exposure to CD19 antigen (FIGs. 4C-4D). To further confirm cell death in the CAR T cell population following antigen exposure, live cell imaging was performed to visualize real-time Annexin V expression and observed a reduction in cell death in both IFNgKO and IFNgRKO CARCDW populations (FIG. 4E). Collectively, these data suggest that the enhanced persistence of IFNg-deficient and -resistant CAR T cells is likely due to reduced cell death rather than a proliferative advantage.

[0309] To confirm that IFNg signaling alone could trigger these changes, IFNgKO and IFNgRKO CARCDI9 T cells were stimulated with plate-bound CD 19 antigen overnight in the absence or presence of recombinant human IFNg (rhIFNg). The addition of rhIFNg was sufficient to restore STAT1 gene expression in IFNgKO, but not IFNgRKO, CARCDI9 (FIG. 4F). This was further confirmed as the addition of rhIFNg yielded an upregulation of interferon-related genes, such as IRF1, IRF9, TBX21 and JAK2 in IFNgKO CARCDI9 compared to IFNgRKO CARCDI9 (FIG. 4G; left). This also coincided with an upregulation of cell death-related genes (XAF1, ERAP2, PPARA) (FIG. 4G; right). These findings were confirmed with a pharmacologic approach using blocking antibodies to either IFNg or IFNgR, which also reduced apoptotic cell death as measured by Annexin V over time (FIG. 4H). Overall, these data demonstrate that IFNg limits CD28 CAR T cell persistence, in part, by promoting T cell death.

[0310] Loss of IFNg signaling drives greater survival of mesothelin-specific CAR T cells.

[0311] It was previously demonstrated that IFNg production by CAR-T and IFNgR signaling in the tumor cells is required for optimal cytotoxic activity against solid tumors (22, 23). Now, it was hypothesized that deletion of IFNgR in CAR T cells might enhance their survival and antitumor activity in solid tumors. A parallel array of control, IFNg-deficient, and IFNg-resistant constructs targeting mesothelin, CARMESO, IFNgKO CARMESO and IFNgRKO CARMESO, were generated and the loss of CD3, IFNg and / or IFNgR was confirmed by flow cytometry (FIGs.

[0312] #14645213vl5A-5B; FIGs. 17A-17B). The functional loss of IFNg signaling in mesothelin- specific CAR-T with targeted deletion of IFNgR was confirmed by the loss of pSTATl signaling following exposure to rhIFNg (FIG. 17C). Similar to CD19-targeted CAR-T, a similar activation (FIG. 17D) and functional profile (FIGs. 17E-17H) was observed between all groups despite a noticeable shift towards a more equal CD4:CD8 ratio in CAR-T lacking IFNg or IFNgR (FIG.

[0313] 5C).

[0314] Both IFNgKO and IFNgRKO CARMESO cells trended towards greater expansion following exposure to mesothelin antigen, as shown by live imaging analysis (left) and flow cytometry-based bead counts (right) (FIG. 5D). When challenged with a variety of mesothelin+pancreatic tumor cell lines AsPC-1, BxPC-3 and Capan-2 in vitro, IFNgRKO CARMESO cells exhibited a consistent increase in growth compared to control and IFNgKO CARMESO (FIGS. 18A-18C). In agreement with the CD19-targeting CAR-T, absence of IFNg signaling did not lead to differences in CAR T cell differentiation (FIG. 5E), expression of immune checkpoint proteins (FIG. 5F) or proliferation (FIG. 5H; FIGs. 18D-18F). Instead, the absence of IFNg signaling following antigen exposure resulted in reduced CAR T cell apoptotic death (FIG. 5G). Collectively, these data suggest that blocking IFNg signaling through deletion of IFNg, and to a greater extent IFNgR, results in improved CAR-T survival regardless of the target antigen specificity or histology of the antigen-expressing cell.

[0315] IFNgRKO CARMESO T cells mediate improved antitumor activity and survival in pancreatic cancer.

[0316] First, similar levels of Granzyme B and Perforin were measured and confirmed in CARMESO, IFNgKO CARMESO and IFNgRKO CARMESO after antigen exposure (FIG. 6A). When challenged with mesothelin+AsPC-1, BxPC-3 or Capan-2 tumor cells, it was observed that while IFNg-deficient CARMESO had diminished antitumor activity, IFNg-resistant CARMESO retained antitumor efficacy similar to control CARMESO (FIG. 6B). It was hypothesized that while IFNgKO CARMESO would be incapable of clearing tumors in mice, IFNgRKO CARMESO would retain antitumor activity and exhibit increased expansion compared to control CARMESO. To test this, NSG mice were injected with AsPC-1 tumor cells two weeks prior to treatment with CAR T cells (FIG. 6C). Both IFNgKO and IFNgRKO-treated mice displayed elevated levels of CAR T cells in the blood 28 days post-treatment, though this did not reach statistical significance (FIG. 6D). Despite increased expansion, mice receiving IFNgKO CARMESO demonstrated reduced tumor control and survival. (FIGs. 6E-6F). Intriguingly, mice receiving IFNgRKO CARMESO displayed significantly longer tumor control and survival compared to both

[0317] #14645213vlIFNgKO CARMESO and control CARMESO. Of note, several mice from the CARMESO group reached poor body conditions quickly following CAR-T treatment. Overall, these data show that while the production of IFNg by CAR T cells is important for antitumor efficacy, the uptake of IFNg by CAR T cells themselves is not required. . Notably, blocking IFNg uptake by CAR T cells slightly increased CAR-T expansion in the blood, enhanced antitumor efficacy and prolonged survival.

[0318] Pancreatic tumors from IFNgRKO CARMEso-treated mice exhibit greater interferon signaling and apoptosis.

[0319] Despite the absence of increased persistence of IFNgRKO CARMESO in the blood of tumor-bearing mice, the heightened antitumor response suggests that there could still be differences in CAR-T survival in the tumor. Given that injected CAR-T cells should be recruited to the tumor site, it was hypothesized that while T cell persistence in the blood was not significantly different between treatment groups, there could be increased accumulation of IFNgRKO CARMESO T cells at the tumor site. To address this and identify potential differences between the top two performers-control CARMESO and IFNgRKO CARMEso-tumor-bearing mice were treated with CAR T cells prior to tumor collection and processing two weeks post-CAR-T injection (FIG. 7A). Analysis of the tumor isolate revealed a 2-fold increase in the CAR-T population in conjunction with 50% less tumor cells in mice receiving IFNgRKO CARMESO (FIG. 7B; FIG. 19A).

[0320] To better understand what could be driving this increased CAR-T presence, FACS sorting was used to isolate mCherry+CAR-T and GFP+tumor cells from the tumor isolate and performed RNA sequencing. This analysis revealed that CAR-T from both groups had a similar resting profile prior to injection (FIG. 7C; PRE), while CAR-T retrieved from the tumors of mice given control CARMESO VS IFNgRKO CARMESO exhibited strikingly different transcriptional profiles (FIG. 7C; POST). Furthermore, tumor cells collected from mice receiving no treatment (tumor only; TO), control CARMESO, or IFNgRKO CARMESO displayed an overall difference in gene expression (FIG. 7D).

[0321] The CAR-T compartment was first explored, where some interferon-related genes were upregulated more in IFNgRKO CARMESO, while the majority of the interferon signature was higher in control CARMESO (FIG. 7E). There was a very subtle, but insignificant, trend towards less differentiation of IFNgRKO CARMESO in vitro (PRE), while IFNgRKO CARMESO retrieved from the tumor (POST) exhibited greater differentiation (higher CD45RO, GZMA, GZMB but interestingly retained expression of the sternness gene CTNNB1 (FIG. 19B). Although not

[0322] #14645213vlsignificant, there was a broader expression of immune checkpoint proteins, such as HAVCR2, NT5E, PDCD1, and TIGIT, on control CARMESO from the tumor, while IFNgRKO CARMESO exhibited higher CTLA4, ENTPD1, and LAG3 (FIG. 19C). Finally, cell profiling revealed a similar expression of Type 1 CD4+helper T cells (Thl), but less regulatory T cells (Treg) in the IFNgRKO CARMEso-treated mice (FIG. 19D; left). This further correlated with higher maintenance of CD8+T cells and cytotoxic cells in the tumors of mice receiving IFNgRKO CARMESO compared to control CARMESO (FIG. 19D; middle and right, respectively).

[0323] Collectively, these data identify subtle, but insignificant, differences in the differentiation, potential exhaustion, and cell types of CAR T cells with IFNg-resistance.

[0324] The expression of apoptosis and cell-death-related genes was next assessed in CAR-T collected from the tumors. In line with previous observations, control CARMESO exhibited a higher expression of pro-apoptotic genes (FIG. 7F). In contrast, IFNgRKO CARMESO displayed a marked upregulation of anti-apoptotic genes. Collectively, the upregulation of interferon-related genes in control CARMESO T cells (POST) correlated with a significant elevation in pro-apoptotic genes (FIGs. 7G-7H). Compared to resting CAR-T (PRE), control CARMESO from tumors had a markedly higher pro-apoptotic gene expression profile than that of IFNgRKO CARMESO. These findings confirm the in vitro findings and suggest that inhibiting the uptake of IFNg through deletion of IFNgR helps protect CAR-T from cell death.

[0325] The data herein show that CAR T cells consume and are affected by IFNg, suggesting that CAR-T themselves have the potential to compete with tumor cells for IFNg. It was hypothesized that deletion of IFNgR on CAR T cells would reduce competition for IFNg, thereby increasing the exposure of the tumor cells in the microenvironment. Consistent with this hypothesis, it was found that interferon-related genes were more highly expressed in the tumors from mice receiving IFNgRKO CARMESO compared to control CARMESO (FIG. 71). This correlated with a broader (but not significant) upregulation of immune checkpoint proteins, such as CTLA4, ENTPD1 and LAG3, on the tumor compartment (FIG. 19E). Interestingly, it was further discovered that while tumor driver genes, such as IER3 and UBB, were elevated in tumor only and control CARMESO groups, tumors from mice treated with IFNgRKO CARMESO exhibited a higher expression of tumor suppressor genes (FIGs. 19F-19G). In line with the previous data, the upregulation of interferon-related genes in tumors of mice treated with IFNgRKO CARMESO exhibited a significant increase in pro-apoptotic markers compared to tumor only and CARMEso-treated mice (FIGs. 7J-7L). Collectively, these data reveal that inhibiting the uptake of IFNg by CAR T cells drives greater interferon signaling, and subsequent

[0326] #14645213vlcell death, in tumor cells. Additionally, the skewing of interferon signaling to the tumor cells correlates with a greater expression of tumor suppressor genes.

[0327] EGFR-targeting IFNgRKO CAR T cells confer protection from tumor rechallenge.

[0328] It was next sought to determine if IFNg-resistant CAR T cells could persist and protect from tumor rechallenge in a different in vivo model. To test this, control CAREGFR and IFNgRKO CAREGFR cells were generated with an scFv to EGFR (FIGs. 8A-8B). Since EGFR antigen is more highly expressed on pancreatic tumor cell lines than mesothelin (FIG. 19H), EGFR-targeted CAR-T can produce more potent responses in this mouse model. Before testing these in vivo, it was first confirmed that IFNgRKO CAREGFR exhibit enhanced growth following antigen exposure (FIG. 8B) and cytotoxic activity against EGFR+AsPC-1 tumor cells (FIG. 8C).

[0329] Mice were treated with EGFR-specific CAR T cells two weeks after AsPC-1 injection (FIG. 8D). In agreement with mesothelin-targeted CAR T cells, IFNgRKO CAREGFR demonstrated more potent antitumor activity compared to CAREGFR (FIG. 81). Intriguingly, while overall survival was not impacted, an increased expansion of CAR T cells was detected in the blood of mice treated with IFNgRKO CAREGFR compared to control, though the difference did not meet statistical significance (FIG. 8F). Additionally several mice treated with CAREGFR or IFNgRKO CAREGFR exhibited complete responses, resulting in no measurable disease in 3 / 5 mice through day 70 post-CAR-T injection (FIG. 8E; FIG. 20A). On day 70 after initial CAR-T transfer, the cured mice from both the CAREGFR and IFNgRKO CAREGFR groups, plus 5 treatment-naive mice, were injected with AsPC-1 tumor cells and monitored for tumor growth. (FIG. 8D). Despite the initial curative response, mice who had originally received control CAREGFR were not protected against a secondary tumor challenge and exhibited tumor growth and survival that was only slightly delayed from the tumor only group (FIG. 8G; FIG. 20B). On the contrary, IFNgRKO CAREGFR-treated mice exhibited a small tumor relapse upon rechallenge but were able to rapidly clear it and yield long-term curative responses in all mice. This additionally led to significantly longer survival in IFNgRKO CAREGFR-treated mice compared to tumor only and control CAREGFR (FIG. 8H). Overall, these findings show that the deletion of IFNgR yields CAR-T that are capable of eliciting long-term antitumor activity and survival. Further, IFNgRKO CAR T demonstrate the ability to protect from tumor rechallenge where control CAR T cells fail. These data suggest that IFNg-resistant CAR-T cells have improved expansion and memory formation in vivo.

[0330] IFNgRKO CAR T cells display increased efficacy and survival in a syngeneic model of triple-negative breast cancer.

[0331] #14645213vlThis data demonstrates that IFNgRKO CAR T cells have greater expansion, antitumor activity and survival; however, interpretation of this data is limited due to the lack of an endogenous tumor microenvironment in NSG mice. Since IFNg can affect multiple cell subsets, including endothelial cells and innate immune cells, it was next sought to determine if these phenomena held true in a syngeneic mouse model. To do this, T cells were isolated from CD45.2+wildtype (WT) or IFNGRKO (IFNgRKO) C57BL / 6 mice and retrovirally transduced to express a non-specific control CAR (CARFITC) or a B7H3-targeting CAR (CARBVHS) (FIG.

[0332] 21 A; top). The transduction efficiency in CD4+and CD8+T cells was similar between WT and IFNgRKO T cells (FIG. 2 IB). Intriguingly, IFNgRKO CAR T cells displayed a significantly greater population of central memory T cells in both the CD4+and CD8+subsets compared to WT CAR T cells (FIG. 21C).

[0333] To test these CAR T cells in vivo, a triple-negative breast cancer cell line was first generated that over-expressed the tumor antigen B7H3 (E0771 B7H3) and injected it into the mammary fat pad of CD45.1+C57BL / 6 mice (FIG. 21 A; bottom). Tumor-bearing mice were conditioned with cyclophosphamide two days prior to the intravenous administration of 5e6CD8+CAR T cells. Of note, CD4+CAR T cells were not injected due to insufficient numbers. Control WT CARFITC and IFNgRKO CARFITC cells had no effect on tumor growth (FIGs. 21D-2 IF). In agreement with the xenograft tumor models, IFNgRKO CARBVHS displayed significantly greater antitumor activity compared to WT CARB7H3. Analysis of the tumor 25 days post-CAR T cell treatment revealed a higher percentage (left) and number (right) of IFNgRKO CARB7H3 cells compared to their wildtype counterpart (FIG. 21G). Collectively, these data support the previous findings and demonstrate that IFNgRKO CAR T cells display increased expansion, persistence, and antitumor activity in both immunodeficient / xenogeneic and immunocompetent models. As demonstrated in FIG. 21H, it is reported herein that IFNg uptake can limit CAR T cells by inducing apoptosis and restricting their expansion. Deletion of IFNgR conferred CAR T cells with protection from cell death and increased expansion, while the tumor compartment from IFNgRKO CAR-treated mice displayed greater interferon signaling and cell death.

[0334] Discussion

[0335] Presented herein is an investigation of the effects of IFNg biology on CAR T cells and demonstrated that IFNg resistance enhances CAR T cell engraftment, long-term persistence, and efficacy against hematologic and solid malignancies. Mechanistically, it was shown that genetic targeting of either IFNg or IFNgR can protect CAR T cells from IFNg-mediated cell death in

[0336] #14645213vlvitro and in vivo. Loss of either of these proteins increased persistence without compromising antitumor efficacy in models of leukemia and lymphoma. In models of xenograft solid tumors, knockout of IFNgR in CAR T cells resulted in greater tumor control and survival. Furthermore, deletion of IFNgR endowed CAR T cells with functional long-term memory. Finally, these results were confirmed in a syngeneic mouse model, demonstrating that IFNgRKO CAR T cells exhibit increased expansion in the tumor and anti-tumor efficacy in both immune-competent and -incompetent mouse models.

[0337] Because of the risk of tumor relapse, one goal in both hematologic and solid tumors is to generate long-lived memory T cells. Incorporation of costimulatory domains was the first significant modification that increased engraftment and persistence of engineered CAR T cells. While CAR T cells containing a CD28 costimulatory domain exhibit potent antitumor activity and rapid expansion, they tend to persist for shorter duration relative to CAR-T designed with the 4- IBB costimulatory domain (5). This shorter persistence and tendency to lead to T cell exhaustion (25) may limit its clinical efficacy in solid tumors (26), despite its more potent cytotoxic activity in vitro or in the short term (27). Previous studies have explored genetic editing of the CD28 costimulatory domain (28), signal strength (29) and signaling pathway (30) to increase the persistence of these T cells. As presented herein, it was discovered that uptake of IFNg by CAR T cells bearing the CD28 signaling domain, but not 4-1BB, limited their durability, in part, through the induction of apoptotic cell death.

[0338] The effects of IFNg signaling in T cells have been studied in the context of TCR signaling, and indicate that while IFNg can increase the motility and cytotoxicity of CD8+T cells (75), it also restricts the T cell memory precursor pool (16) and is required for activation-induced cell death (77). Recent studies have further demonstrated that IFNg signaling in tumorspecific T cells can dampen their antitumor activity through inducing cell death (20) and inhibiting the maintenance of stem-like T cells (27). In contrast to TCR-based studies, the effects of IFNg signaling in the specific context of CD28 CAR T cells is largely unknown. Due to the potent release of IFNg by CAR-T following antigen exposure, the question of how its uptake affects CAR-T function is raised. The data here demonstrate that while IFNg has minimal effect on the differentiation, exhaustion and proliferation of CD28 CAR T cells in the tested models, it plays a significant and direct role in the induction of cell death following antigen exposure. Notably, these effects were not observed in CAR T cells with a 4- IBB costimulatory domain, suggesting that response to IFNg could differ between clinical CAR T cell products.

[0339] While these findings demonstrate that IFNg limits CAR T cell responses, the importance of IFNg signaling in tumor cells for response to immunotherapies is becoming increasingly

[0340] #14645213vlrecognized and should be noted. Retrospective studies in patients with metastatic melanoma or non-small cell lung cancer have identified the correlation between IFNG and IFNg-responsive gene signatures with clinical response following treatment with the immune checkpoint inhibitors, pembrolizumab or nivolumab (18, 19). These findings are further supported by preclinical work demonstrating that loss of IFNgR in glioblastoma or pancreatic cancer cells reduces the avidity of CAR T cells, thereby conferring solid tumors with resistance to CAR-T killing (23). These data collectively suggest that while IFNg signaling in the tumor compartment is required for effective immunotherapy, its uptake by T cells might limit their activity. To this end, it was discovered that IFNg-resistant CAR-T, capable of producing but not internalizing IFNg, induced more potent, durable responses in both hematologic and solid malignancies.

[0341] In addition to the protection of CAR T cells from cell death via IFNgR loss, this approach could result in increased exposure of tumor to IFNg. Competition for cytokines (31) and nutrients (32, 33) occurs in the tumor microenvironment and can limit effective T cell responses. These data suggest that CAR-T could be serving as a sink for IFNg, thereby limiting the uptake of this cytokine by tumor cells and, potentially, surrounding tumor microenvironment. Greater interferon uptake in pancreatic tumor cells correlated with increased gene expression of cell death-related markers, as well as a higher expression of tumor suppressor genes. Given the reported potential of IFNg to skew innate immune cells to an antitumorigenic phenotype (9), removing CAR-T from competition for IFNg could not only increase tumor cell death but may potentiate a more favorable tumor microenvironment.

[0342] While the increase of interferon signaling in tumor cells reported here correlated with greater cell death gene expression, there was also a widespread upregulation of immune checkpoint proteins, such as CTLA4 and LAG3. Given the role of IFNg in inducing immune checkpoint proteins (10, 11), this finding is not surprising but highlights the potential for combination with immune checkpoint blockade therapies. As previously mentioned, IFNg is mechanistically required for effective anti-tumor responses to immune checkpoint therapies (18, 19), but can inhibit the maintenance of intratumoral stem-like T cells (21) and drive the cell death of tumor- specific T cells (20). Thus, IFNg-resistant CAR-T cells may be particularly well suited for combination with immune checkpoint blockades and other immunotherapies.

[0343] In summary, the data presented herein reveals that IFNg-resistant CAR T cells have enhanced anti-tumor efficacy via reduction in CAR-T cell death, enabling enhanced persistence, long-term memory formation, potentially increasing exposure of tumor cells to IFNg. These discoveries demonstrate that uptake of IFNg can limit CAR-T responses and that strategic

[0344] #14645213vlpharmacologic or genetic targeting of this pathway could generate more potent, persistent therapies for patients in the clinic.

[0345] Materials and Methods

[0346] Study design. The objective of the study was to define how IFNg signaling limits CAR T cells and leverage gene editing of this pathway to elicit potent, long-term efficacy of CAR-T in hematologic and solid malignancies. CRISPR / Cas9 editing was used to generate IFNg-deficient (IFNgKO) and IFNg-resistant (IFNgRKO) CAR T cells, and their phenotype, activation, functionality, cytotoxicity and proliferation was characterized. Direct effects of IFNg were further explored using pharmacologic blockade of IFNg or IFNgR, or introduction of exogenous recombinant human IFNg. In vitro replicates were variable and are indicated in the Brief Description of the Drawings. RNA sequencing of CAR T cells from both in vitro and in vivo models was used to define genetic changes stemming from loss of IFNg signaling.

[0347] Xenograft and syngeneic mouse models of leukemia, lymphoma and pancreatic cancer were evaluated by bioluminescence, caliper measurements, and serum collection for detection of IFNg and bleeds for CAR-T persistence. For in vivo experiments, tumor burden was used to randomize mice into treatment groups.

[0348] Blocking antibodies. IFNg signaling was pharmacologically blocked with 20mg / ml Purified NA / LE Mouse Anti-Human IFN-g Clone NIB42 (BD Biosciences) or anti-IFNgR (GIR-208; BD Biosciences). Blocking antibodies were refreshed every 24 hours as needed.

[0349] CAR design and generation.

[0350] Human. A CD19-specific CAR construct with a CD8 transmembrane domain, an intracellular CD28 costimulatory domain and a CD3z signaling domain was synthesized and cloned into a second-generation lenti viral backbone under the regulation of a human EF-la promoter. Additional constructs targeting CD 19 were designed to include guide RNA sequences for TRAC (AGAGTCTCTCAGCTGGTACA) (SEQ ID NO: 120) + / - IFNg (CCAGAGCATCCAAAAGAGTG) (SEQ ID NO: 121) or IFNg receptor 1 (IFNgR 1) (GGAGTACCAGATCATGCCAC) (SEQ ID NO: 40). Guides were chosen based on previous work (22, 23) and were further used to design anti-mesothelin (CARMESO, IFNgKO CARMESO, IFNgRKO CARMESO) or anti-EGFR (CAREGFR, IFNgRKO CAREGFR) constructs. Lentiviral vectors were prepared by transient transfection of 293 T cells with packaging and transfer plasmids. Supernatant was harvested at 24- and 48-hours post-transfection, filtered through 45mm filters and concentrated by ultracentrifugation (20000g for 2 hours at 4°C).

[0351] Murine. CAR constructs targeting FITC (control) or the tumor antigen B7H3 with a CD8 transmembrane domain, an intracellular CD28 costimulatory domain and a CD3z signaling

[0352] #14645213vldomain were incorporated into a retroviral vector (MSCV-B7H3-FITC or MSCV-B7H3-CAR). transient transfection of Platinum-E cells with packaging and transfer plasmids. Supernatant containing RVs was collected 72 hours post-transfection, filtered through 45mm filters and concentrated by ultracentrifugation (20000g for 2 hours at 4°C).

[0353] CAR-T production.

[0354] Human. Peripheral blood of anonymous healthy donor leukapheresis product was purchased from the MGH Blood Bank under an Institutional Review Board- approved protocol and T cells were isolated using the RosetteSep Human T Cell Enrichment Cocktail (STEMCELL Technologies). T cells were re-suspended in RIO media (RPMI 1640 + 10% FBS + Pen / Strep) supplemented with 20IU / ml IL-2 (PeproTech) and activated using anti-CD3 / CD28 Dynabeads (Life Technologies) at a 1T:3B ratio. Twenty-four hours post-activation, T cells were transduced with one of the lentiviral vectors encoding anti-CD19, -mesothelin or -EGFR CAR constructs described above. Cells were de-beaded five days post-activation and expanded in the presence of 20IU / ml IL-2. For knockout CAR-T, T cells were re-suspended at 5e6 / 100ml in Opti-Mem (Thermo Fisher Scientific) after de-beading on day 5 and electroporated with lOmg Cas9 mRNA (TriLink) at 360V x 001ms. Three to five days later, CD3" T cells were isolated by column purification (EasySep Human APC Positive Selection Kit II; STEMCELL Technologies) or flow-based cell sorting using the BD FACSAria. Transduction efficiency was determined using mCherry (PE-Texas Red) expression by flow cytometry. Deletion of CD3 (TRAC), IFNg and IFNgR was confirmed by flow cytometry. For all functional assays, CAR-T were normalized for transduction / isolation efficiency.

[0355] Murine. T cells were harvested from the spleen and lymph nodes of CD45.2+C57BL / 6 (Stock No. 000664) and Ifngr KO (Stock No. 003288) mice. CD4 and CD8 T cells were purified by bead-based selection (Miltenyi) and plated at 5e5cells / ml in 24- well plates pre-coated with CD3 (2mg / ml) and CD28 (2mg / ml) antibodies in T cell clone medium supplemented with mouse IL-2 (30 lU / ml, Miltenyi). T cell clone medium was made using DMEM, 10% FBS, penicillin / streptomycin (lOOU / ml), glutamine (2mM), sodium pyruvate (1.5mM), non-essential amino acids (lx, Lonza Cat. 13-114E), vitamins (lx, Thermo Fisher Scientific, Cat. 11120052), arginine (1.16mg / ml), asparagine (36mg / ml), folic acid (14mM), b-mercaptoethanol (57.2mM) and recombinant mouse IL-2 (30U / ml). 24 hours post activation, fresh concentrated RVs and 8mg / ml of polybrene were added to T cells and T cells were spinoculated at 2400rpm for 2 hours at 32°C. 12-14 hours later, fresh T cell clone medium was added to the T cells. 72 hours post-T cell activation, T cells were moved to uncoated plates for expansion in the presence of

[0356] #14645213vlmurine IL-2 (30 lU / ml). Transduction efficiency was determined using an anti-Myc-Tag (Cell Signaling).

[0357] Cell lines.

[0358] Human. The human JeKo-1, Nalm6, AsPC-1, BxPC-3 and Capan-2 cell lines were purchased from American Type Culture Collection (ATCC). Cell lines were engineered to constitutively express click beetle green luciferase (CBG) and enhanced GFP (GFP) prior to sorting on FACSAria (BD) to obtain a >99% CBG-GFP+population. JeKo-1, Nalm6, AsPC-1 and BxPC-3 cell lines were cultured in RIO media (RPMI1640 + 10%FBS + Penicillin / Streptomycin). Capan-2 cells were culture in DIO media (DMEM + 10%FBS + Penicillin / Streptomycin). All cell lines were routinely tested for mycoplasma.

[0359] Murine. The triple-negative breast cancer cell line E0771 was purchased from ATCC and transduced with a lentiviral construct encoding murine B7H3 cDNA and a blue fluorescence protein (BFP) reporter cDNA under the EFla promoter (EFla-mB7H3-p2a-BFP) to generate a cell line over-expressing mouse B7H3 (E0771 mB7H3 OE).

[0360] Flow cytometry. For extracellular staining, cells were stained in the dark for 20 minutes at room temperature in BD Horizon Brilliant Stain Buffer (BD Biosciences) and washed twice with FACS Buffer (PBS + 2% FBS) prior to acquisition. Cells to be saved for analysis at later time points were fixed using Fixation Buffer (BioLegend) according to protocol. For cytokine staining, cells were fixed with Fixation Buffer (BioLegend) and incubated in Intracellular Staining Permeabilization Wash Buffer (10X) (BioLegend) with flow antibodies for 30 minutes at room temperature prior to running on a Fortessa X-20 and analyzing using FlowJo software. The following human antibodies were purchased for flow cytometry from BioLegend: CCR6-PE / Cy7 (G034E3), CD3-APC (OKT3), CD39-BV510 (Al), CD4-FITC (CSK3), CD45RA-PerCPCy5.5 (HI100), CD45RO-APC (UCHL1), CD62L-FITC (DREG-56), CD69-BV785 (FN50), CTLA-4-APC (L3D10), CXCR3-BV421, Granzyme B-APC (CB9), IFNg-BV510 (B27), IL-2-BV785 (MQ1-17H12), Perforin-BV421 (B-D48), and TNFa-BV711 (MAbll); BD Biosciences: CD8-BUV395 (RPA-T8), Lag3-BV421 (T47-530), PD-1-BV605 (EH12.1), and Tim3-BV711 (7D3); R&D Systems: CCR4-AF488; Abeam: IFNgRl (EPR7866). Murine antibodies were purchased from BioLegend and included: CD4-Pe-Dazzle (RM4-5), CD44-APC-Fire750 (IM7), CD62L-BV785 (MEL14), CD8-PerCP / Cyanine5.5 (53-6-7).

[0361] Flow cytometry: Bead count assay. Cells were stained using the above protocol and then prepared for the bead count assay as follows. CountBright Plus Absolute Counting Beads (Thermo Fisher Scientific) were set out to come to room temperature, vortexed for 30 seconds, added to samples at 50ml beads / well and placed on a plate shaker. All samples were run on a

[0362] #14645213vlFortessa X-20, analyzed using FlowJo software and calculations were performed according to Thermo Fisher Scientific protocol.

[0363] Flow cytometry: Cell death assay. To measure viability, cells were left untreated or activated with plate-bound antigen and stained for flow cytometry using Cleaved Caspase 3 purchased from Cell Signaling Technology (Pacific Blue, D3E9) or Annexin V purchased from BioLegend (APC) in Annexin V binding buffer according to protocol (BioLegend). All samples were kept on ice throughout processing and run on a Fortessa X-20 prior to analysis with FlowJo software.

[0364] Flow cytometry: Cell trace violet. CAR T cells were collected (~4e6CAR+ / group), centrifuged and resuspended in 2ml CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Thermo Fisher Scientific; C34557) that had been diluted in pre-warmed sterile PBS according to protocol. Cells were incubated at 37 °C for 20 minutes and then quenched with 8ml RIO media for 5 minutes at RT. Cells were centrifuged, counted and added to wells with platebound antigen or tumor cells as described in the Brief Description of the Drawings, le5cells were saved and fixed for a day 0 control. Cells were taken down 6 days later, and all samples were run on a Fortessa X-20 before analyzing with FlowJo software.

[0365] Flow cytometry: Mouse blood. Following the collection of blood from mice, samples were processed for flow cytometry. APC-conjugated mouse dump antibodies from BioLogend (Ly-6G / Ly-6C (RB6-8C5), NK1.1 (PK136), CDllb (MI / 70), TER- 119 / Erythroid cell (TER-119) were added to a FACS tube at Iml / sample each. Iml / sample of anti-human CD4 (FITC; CSK3; BioLegend) and CD8 (BUV395; RPA-T8; BD Biosciences) antibodies were added to the same tube. The antibody tube was filled to 20ml / sample with PBS, vortexed and 20ul of mix was carefully added to the bottom of TruCount Tubes (BD Biosciences). TrueCount Tubes were vortexed and incubated in the dark for 10 minutes at room temperature (RT). 50ml of mouse blood was added to corresponding TrueCount Tubes, vortexed and incubated at RT in the dark for 10 minutes. BD FACS™ Lysing Solution 10X Concentrate (BD Biosciences; #349202) was diluted and 450ml was added to each TrueCount Tube. Tubes were vortexed and incubated at RT for 15 minutes prior to running on a Fortessa X-20 and analyzing using FlowJo software.

[0366] Flow cytometry: phosphoSTATl staining. For phosphoflow, cells were resuspended at le6 / ml and left untreated or given 20ng / ml recombinant human IFNg (BioLegend) for 20 minutes at 37°C. Cells were fixed at 1:1 ratio with BD Cytofix (BD Biosciences) for 15 minutes at room temperature. Following centrifugation, cells were resuspended in Perm Buffer III (BD Biosciences) at 2e6 / ml, vortexed vigorously and put on ice for 30 minutes. Cells were stained with PhosphoStatl Tyr701 (Clone 58D6; Cell Signaling Technology ) in 1% PBSA followed by

[0367] #14645213vlanti-Rabbit IgG (H+L), F(ab')2 Fragment AF647 (Cell Signaling Technology). All cells were run on a Fortessa X-20 (BD) and analyzed using FlowJo software.

[0368] Incucyte. Donor-derived CAR, IFNgKO CAR and IFNgRKO CAR-T targeting CD 19 were combined with JeKo-1 lymphoma or Nalm6 leukemia cells at a 1:1 effector-to-target ratio and tumor burden (GFP) and proliferation of mCherry+CAR T cells was assessed for three days on the Incucyte SX5 (Sartorius). Anti-mesothelin CAR-T were cultured at a 1:1 ratio with pancreatic cancer cell lines AsPC-1, BxPC-3 and Capan-2, and tumor burden (GFP) and CAR-T proliferation (mCherry) was monitored for 3-5 days. To measure real-time apoptosis of T cells in response to antigen stimulation, all CAR-T groups were added to plates coated with CD 19 or mesothelin antigen in the presence of Annexin V Green according to protocol (Sartorius).

[0369] Luminex. CAR-T were activated with plate-bound CD 19 or mesothelin antigen overnight and cytokines were measured using the Thl / Th2 Multiplex Panel according to protocol (Thermo Fisher Scientific) and run on the FLEXMAP 3D System (Luminex).

[0370] NanoString: in vitro. CAR-T cells were combined with plate-bound CD 19 antigen overnight in the presence or absence of lOng / ml recombinant human IFNg. All isolated CAR T cells were lysed using RLT buffer to obtain RNA and code set probes were hybridized with RNA by PCR for 18 hours at 65°C according to NanoString protocol. nCounter gene expression assays (NanoString Technologies) were performed using NanoString XT CAR-T Panel Standard + Customized PLUS panel. Hybridized RNA was loaded into nCounter MAX cartridges, run on the nCounter MAX and quantified using nSolver or nSolver Advanced software. For advanced analysis, donors as covariates was considered. Data is displayed as volcano plots and normalized gene counts.

[0371] NanoString: in vivo. 6-10-week-old NSG mice were subcutaneously injected with 1.5e6AsPC-1 CBG-GFP+cells (resuspended in 1:1 ratio of sterile PBS and Matrigel (Coming)) 14 days prior to IV administration of 3e6mesothelin-specific control or IFNgRKO CAR T cells. Mice were sacrificed at 14 days post-CAR-T and tumors were collected and processed as follows. Each tumor was cut into small pieces using razor blades, treated with digestion media that had 10ml warm RPMI (no FBS), 500ml Collagenase P (2mg / ml; Sigma-Aldrich) and 5ml DNase I (50mg / ml; Sigma-Aldrich) and placed in a shaking incubator at 37°C for two hours. Digested tumor samples were filtered through a 70mM filter, then resuspended in cold RIO media (RPMI 1640 + 10% FBS + Pen / Strep). GFP+tumor cells and mCherry+CAR-T were isolated from digested tumors using a BD FACSAria. Isolated cells were lysed using RLT buffer to obtain RNA and code set probes were hybridized with RNA by PCR for 18 hours at 65°C according to NanoString protocol. nCounter gene expression assays (NanoString Technologies)

[0372] #14645213vlwere performed using NanoString XT CAR-T Panel Standard + Customized PLUS panel (CAR-T) or nCounter PanCancer IO 360 panel (tumor). Hybridized RNA was loaded into nCounter MAX cartridges, run on the nCounter MAX and quantified using nSolver or nSolver Advanced software. For advanced analysis, donors as covariates were considered. Data is displayed as volcano plots, normalized gene counts (heatmaps / scatter plots) and cell type profiling.

[0373] NSG xenograft mouse models: hematologic malignancies. NSG mice were purchased from Jackson Laboratory and bred under pathogen-free conditions at the MGH Center for Comparative Medicine. 6-10-week-old NSG mice were intravenously (IV) injected with le6JeKo-1 or Nalm6 CBG-GFP+cells. Seven days later, mice were left untreated (tumor only; TO) or IV injected with le6CAR T cells and tumor burden was measured by bioluminescence using an Ami spectral imaging apparatus and analyzed with IDL software following intraperitoneal (IP) injection of D-Luciferin substate solution (30mg / ml). For most experiments, mice were bled on days 2 and 14 post-CAR-T injection to monitor IFNg expression / cytokine profiles and CAR-T persistence, respectively. In all experiments, bioluminescence was measured weekly.

[0374] NSG xenograft mouse models: pancreatic cancer. NSG mice were purchased from Jackson Laboratory and bred under pathogen-free conditions at the MGH Center for Comparative Medicine. 6-10-week-old NSG mice were subcutaneously injected with 1.5e6AsPC-1 CBG-GFP+cells (resuspended in 1:1 ratio of sterile PBS and Matrigel (Coming)) 14 days prior to IV administration of 3e6mesothelin- or EFGR-targeting CAR-T. Caliper measurements were conducted weekly to assess tumor burden and CAR-T persistence in the blood was assessed at days 7, 14, 21, 28 and 35 post-CAR-T injection. Mice treated with EGFR-specific CAR-T that demonstrated curative responses (3 / 5 KO CAR and 3 / 5 IFNgRKO CAR) were subcutaneously re-challenged on day 70 with 1.5e6AsPC-1 tumor cells (1:1 sterile PBS and Matrigel (Coming)) in the opposing flank to primary tumors and were monitored for tumor growth using weekly caliper measurements. All experiments were performed according to protocols approved by the MGH Institutional Animal Care and Use Committee.

[0375] Syngeneic C57BL / 6 mouse model: triple-negative breast cancer. C57BL / 6 mice, including CD45.2+(Stock No. 000664), CD45.2+Ifngr KO (Stock No. 003288) and CD45.U (Stock No. 002014), were purchased from Jackson Laboratory. E0771 mB7H3 OE cells (3e5) were injected into the 4thmammary fat pad in a total volume of 100ml of 1:1 PBS / Matrigel solution. Tumor-bearing mice were conditioned with an I.V. injection of lOOmg / g cyclophosphamides on day 2, followed by I.V. administration of 5e6CD8+CAR T cells four days post-tumor implantation. 25 days post-CAR T cell treatment, tumors were collected from mice and dissected by mincing tumor tissue prior to digestion with Collagenase D (2.5mg / ml)

[0376] #14645213vlfor 20 minutes at 37°C. Tumors were then disassociated to single cell suspension by passing tissue through a 70mM filter prior to centrifugation and analysis by flow cytometry. All experiments were performed according to protocols approved by the MGH Institutional Animal Care and Use Committee.

[0377] Statistical Analysis. All analyses were performed with GraphPad Prism vlO software. Data were presented as means ± s.e.m. with statistically significant differences determined by tests as indicated in the Brief Description of the Drawings. For experiments with multiple groups, correction for multiple comparisons was applied. All n values given are biologic replicates unless otherwise stated.

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[0427] #14645213vl

Claims

CLAIMSWhat is claimed is:

1. An immune cell comprising a first polynucleotide encoding a chimeric antigen receptor and / or an engineered immune cell receptor, wherein the immune cell is deficient in interferon y receptor (IFNyR) expression and / or signaling.

2. The immune cell of claim 1, comprising a mutation in one or more IFNyR gene loci of the immune cell.

3. The immune cell of claim 1 or clam 2, comprising an insertion and / or deletion in one or more IFNyR genes of the immune cell.

4. The immune cell of claim 3, wherein the insertion and / or deletion results in a frameshift of the one or more IFNyR genes.

5. The immune cell of any one of claims 1-4, wherein the immune cell comprises a loss of function mutation in one or more IFNyR genes of the immune cell.

6. The immune cell of any one of claims 1-5, wherein the immune cell comprises a knockout of one or more IFNyR genes of the immune cell.

7. The immune cell of any one of claims 1-6, wherein the immune cell comprises an insertion and / or deletion, a frameshift, or a knockout of each IFNyR gene of the immune cell.

8. The immune cell of any one of claims 1-7, wherein the immune cell comprises a loss of function mutation in each IFNyR gene of the immune cell.

9. The immune cell of any one of claims 1-8, further comprising:(i) a functional RNA comprising a portion that is complementary to an IFNyR gene and / or an mRNA transcribed from an IFNyR gene; and / or(ii) a second polynucleotide encoding the functional RNA.#14645213vl10. The immune cell of claim 9, wherein the functional RNA is an siRNA, a micro-RNA, a shRNA, an antisense oligonucleotide, a ribozyme, or a guide RNA.

11. The immune cell of claim 10, wherein the functional RNA is a guide RNA comprising a homology region that is complementary to a strand of an IFNyR gene of the immune cell.

12. The immune cell of claim 11, wherein the guide RNA comprises a sequence of any one of SEQ ID NOs: 32-47.

13. The immune cell of any one of claims 1-12, wherein the immune cell is deficient in TRAC expression.

14. The immune cell of any one of claims 1-13, further comprising:(i) a TRAC-targeted guide RNA comprising a homology region that is complementary to a TRAC gene of the immune cell; and / or(ii) a third polynucleotide encoding the TRAC-targeted guide RNA.

15. The immune cell of any one of claims 9-14, further comprising ribonucleoprotein (RNP) comprising:(i) the guide RNA of claim 9 or claim 10 or the TRAC-targeted guide RNA of claim 11.2; and(ii) a Cas protein.

16. The immune cell of claim 15, wherein the Cas protein is a Cas9 protein.

17. The immune cell of any one of claims 1-16, further comprising a transcription activatorlike effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a meganuclease that targets one or more IFNyR genes of the immune cell.

18. The immune cell of any one of claims 1-17, wherein two or more of the first polynucleotide, the second polynucleotide, and the third polynucleotide are part of the same contiguous polynucleotide.#14645213vl19. The immune cell of any one of claims 1-18, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to a promoter.

20. The immune cell of claim 19, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to different promoters.

21. The immune cell of claim 19, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are each operably linked to the same promoter.

22. The immune cell of any one of claims 1-21, wherein the CAR comprises:(i) an extracellular antigen-binding domain,(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

23. The immune cell of claim 22, wherein the extracellular antigen-binding domain comprises a single-chain antibody fragment (scFv) that binds a cell surface protein.

24. The immune cell of claim 22 or claim 23, wherein the extracellular antigen-binding domain binds CD19, B7H3, BCMA, TACI, CD79b, CD22, CD30, CS1, Claudin 18.2, GPC3, GD2, GPCR, PSMA, mesothelin, MUC1, MUC16, EGFR, IL-13Ralpha2, EGFRvIII, CD20, CD79a, or combinations thereof.

25. The immune cell of claim 24, wherein the extracellular antigen-binding domain binds B7H3.

26. The immune cell of claim 24, wherein the extracellular antigen-binding domain is a CD 19 binding extracellular domain.

27. The immune cell of claim 26, wherein the CD19 extracellular antigen-binding domain comprises:(a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 24; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 25; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 26; and#14645213vl(b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 27; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 28; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 29.

28. The immune cell of claim 26 or 27, wherein the CD19 extracellular antigen-binding domain comprises a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 50.

29. The immune cell of claim 24, wherein the extracellular antigen-binding domain is a mesothelin binding extracellular domain.

30. The immune cell of claim 29, wherein the mesothelin extracellular antigen-binding domain comprises:(a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 4; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 5; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 6; and(b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 7; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 8; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 9.

31. The immune cell of claim 29, wherein the mesothelin extracellular antigen-binding domain comprises:(a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 13; the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 14; and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 15; and(b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises an amino acid sequence of SEQ ID NO: 16; the CDR-L2 comprises an amino acid sequence of SEQ ID NO: 17; and the CDR-L3 comprises an amino acid sequence of SEQ ID NO: 18.#14645213vl32. The immune cell of any one of claims 22-31, wherein the transmembrane domain is a transmembrane domain of an alpha chain of a immune cell receptor, beta chain of a immune cell receptor, zeta chain of a immune cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

33. The immune cell of any one of claims 22-32, wherein the transmembrane domain is a CD8 transmembrane domain.

34. The immune cell of any one of claims 22-33, wherein in the intracellular signaling domain comprises an intracellular signaling domain from 4-1BB, CD27, CD28, 0X4, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

35. The immune cell of any one of claims 22-34, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

36. The immune cell of any one of claims 22-35, wherein the intracellular signaling domain further comprises a TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

37. The immune cell of any one of claim 36, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain and a CD3-zeta intracellular signaling domain.#14645213vl38. The immune cell of any one of claims 22-37, wherein the extracellular antigen-binding domain further comprises a leader.

39. The immune cell of any one of claims 22-38, wherein the CAR comprises any amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98.

40. The immune cell of claim 39, wherein the CAR comprises any amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98.

41. The immune cell of claim 39, wherein the CAR comprises any amino acid sequence of any one of SEQ ID NOs: 1, 10-12, 22-23, and 55-98.

42. The immune cell of any one of claims 1-41, wherein the immune cell is a T cell.

43. The immune cell of any one of claims 1-41, wherein the immune cell is a natural killer (NK) cell.

44. A polynucleotide encoding:(i) a chimeric antigen receptor (CAR) of any one of claims 22-43; and(ii) a guide RNA comprising a homology region that is complementary to IFNgR.

45. The polynucleotide of claim 44, further comprising nucleic acid encoding a guide RNA that comprises a homology region that is complementary to TRAC.

46. A method of treating cancer in a subject, the method comprising administering the immune cell of any one of claims 1-43, or an immune cell comprising the polynucleotide of claim 34 or claim 35.

47. The method of claim 46, wherein the CAR comprises a CD28 intracellular signaling domain.

48. The method of claim 46 or claim 47, wherein the cancer is a CD 19 expressing cancer, the CAR comprises an extracellular antigen binding domain that bind to CD 19.#14645213vl49. The method of any one of claims 46-48, wherein the cancer is a blood cancer.

50. The method of claim 49, wherein the blood cancer is leukemia or lymphoma.

51. The method of claim 46 or claim 47, wherein the cancer is a mesothelin expressing cancer, the CAR comprises an extracellular antigen binding domain that bind to mesothelin.

52. The method of any one of claims 46, 47 or 51, wherein the cancer is a solid tumor.

53. The method of claim 52, wherein the cancer is pancreatic cancer.

54. The method of any one of claims 46-47, wherein the cancer is breast cancer.

55. The method of claim 54, wherein the breast cancer is triple negative breast cancer (TNBC).

56. The method of claim 54 or 55, wherein the triple negative breast cancer is B7H3 positive and the CAR-T cell comprises a B7H3 binding CAR.

57. The method of any one of claims 54-56, wherein the CAR comprises an extracellular antigen binding domain that binds to B7H3.

58. A method of increasing the persistence of a CAR-immune cell in a subject, the method comprising decreasing the expression of interferon y receptor (IFNyR) in the immune cell.

59. The method of claim 58, further comprising knocking out one or more interferon y receptor (IFNyR) genes in the immune cell.

60. The method of claim 59, wherein knocking out one or more IFNyR genes comprises introducing a loss of function mutation into the one or more IFNyR genes.

61. The method of claim 60, wherein knocking out one or more IFNyR genes comprises introducing an insertion, a deletion, and / or a frameshift into the one or more IFNyR genes.#14645213vl62. The method of any one of claims 58-61, wherein decreasing the expression of IFNyR in the immune cell comprises decreasing the expression using CRISPR.

63. The method of claim 62, wherein decreasing the expression using CRISPR, comprising introducing a loss of function mutation into one or more IFNyR genes of the immune cell using CRISPR.

64. The method of any one of claims 58-63, wherein decreasing the expression of IFNyR in the immune cell comprises decreasing the expression using RNA interference.

65. The method of any one of claims 58-63, wherein decreasing the expression of IFNyR in the immune cell comprises decreasing using an antisense oligonucleotide (ASO).

66. The method of any one of claims 59-65, wherein the CAR comprises a CD28 intracellular signaling domain.

67. A method of increasing the persistence of a CAR-immune cell in a subject, the method comprising contacting the CAR-immune cell with an anti-IFNgR antibody.

68. A method of increasing the persistence of a CAR-immune cell in a subject, the method comprising administering the CAR-immune cell and an anti-IFNgR antibody.#14645213vl

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