Armored car constructs

Abstract

Disclosed and claimed are CAR constructs and therapeutic applications thereof. The CAR constructs include antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) or folate receptor (FOLR2) and a payload comprising at least one cytokine or cytokine variant.

Description

Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCARMORED CAR CONSTRUCTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. provisional patent application 63 / 788,954, filed on April 15, 2025, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant no. R01AT011326 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.BACKGROUND

[0003] The concept of using T cells in cell therapy has gained significant attention in recent years, particularly through the development of chimeric antigen receptor (CAR) T-cell therapy. CAR T-cell therapy involves genetically modifying a patient’s T cells to express a CAR, allowing these cells to recognize and eliminate cells, such as tumor cells, more effectively than normal T cells. These CAR constructs allow T cells to recognize tumor-associated antigens directly, bypassing the traditional major histocompatibility complex (MHC)-dependent antigen recognition system. As a result, CAR T-cell therapies are capable of targeting and eliminating the targeted cells with high specificity and efficacy.

[0004] Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a protein that is primarily expressed on the surface of myeloid cells, including macrophages and dendritic cells, as well as certain other immune cells. Due to its involvement in various diseases, TREM2 is being studied as a potential therapeutic target for treating conditions such as solid tumors, Alzheimer’s disease, autoimmune diseases, and other inflammation-related disorders. Notably, TREM2 expression coincides with immunosuppressive program of tumor associated macrophage in multiple cancers including non-small cell lung carcinoma, breast cancer and colorectal carcinoma.

[0005] Folate receptor 2 (FOLR2) is a protein that binds folate and its derivatives, playing a key role in folate uptake and cellular metabolism. FOLR2 is involved in receptor-mediated endocytosis, where it facilitates the transport of folate into cells, which is essential for DNA synthesis and cellular function. Due to its overexpression in certain cancers, FOLR2 hasMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCbecome a target for developing targeted therapies and drug delivery systems. Additionally, its expression in the immune system suggests potential roles in immune modulation and inflammatory responses. In particular, FOLR2 expression coincides with immunosuppressive program of tumor associated macrophage in multiple cancers including ovarian cancer, pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma.BRIEF SUMMARY

[0006] One aspect of the disclosure includes a Chimeric Antigen Receptor (CAR) construct comprising: a first antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) comprising a light chain variable region, a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, a co-stimulatory domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0007] One aspect of the disclosure includes a CAR comprising a first antigen-binding domain capable of binding to folate receptor 2 (FOLR2) comprising a heavy chain variable region, a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, a co-stimulatory domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0008] One aspect of the disclosure includes a CAR construct comprising: a first antigenbinding domain capable of binding to TREM2 comprising a light chain variable region, a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, a co-stimulatory domain, and a payload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0009] One aspect of the disclosure includes a CAR construct comprising a first antigenbinding domain capable of binding to FOLR2 comprising a heavy chain variable region, a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, a co-stimulatory domain, and aMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCpayload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0010] In an aspect, the polynucleotide comprises internal ribosome entry sites (IRES)

[0011] In an aspect, the flexible linker is a (G4S)nlinker, wherein n is selected from the group consisting of 1, 2, 3, 4, or 5.

[0012] In an aspect, the self-cleavable linker is a peptide linker. In an aspect, the self-cleavable linker is selected from the group consisting of P2A, T2A, E2A and F2A.

[0013] In an aspect, the at least one cytokine is selected from the group consisting of interleukins (ILs), interferons (IFNs), chemokines, and tumor necrosis factor (TNF).

[0014] In an aspect, the cytokine variant comprises muteins derived from at least one cytokine. In an aspect, the at least one cytokine is an IL selected from the group consisting of IL-12, IL-1, IL-2, IL-6, IL-10, IL-17, IL-18, IL-15, IL-9, IL-11, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-13, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, and IL-38.

[0015] In an aspect, the payload promotes immunomodulation at disease sites.

[0016] In an aspect, the CAR construct further comprises a protease cleavable linker, wherein the protease cleavable linker is downstream of the payload. In an aspect, the protease cleavable linker is engineered to be recognized and cleaved by a protease. In an aspect, the protease is selected from the group consisting of cysteine proteases, serine proteases, or metalloproteinases. In an aspect, the protease is selected from the group consisting of matriptase, legumain, matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-7 (MMP7), matrix metalloproteinase-9 (MMP9), urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase 10 (ADAMI 0), a disintegrin and metalloproteinase 17 (ADAMI 7), and membrane-type 1 matrix metalloproteinase (MT1-MMP).

[0017] In an aspect, the CAR construct further comprises an additional transmembrane region, wherein the additional transmembrane region is downstream of the protease cleavable linker. In an aspect, the additional transmembrane region comprises CD8, B7-1T (CD80 transmembrane domain), or CD28.

[0018] In an aspect, the CAR construct further comprises a small molecule-induced degron domain and / or a mutated FK506-binding protein sequence, wherein the small molecule-induced degron domain and / or the mutated FK506-binding protein sequence is downstream of the additional transmembrane region.

[0019] In an aspect, the CAR construct further comprises a peptide tag.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0020] In an aspect, the CAR construct further comprises a destabilizing domain, wherein the destabilizing domain modulates the payload activity.

[0021] In an aspect, the antigen-binding domain is a single-chain variable fragment (scFv).

[0022] In an aspect, the antigen-binding domain is derived from a monoclonal antibody or a nanobody.

[0023] In an aspect, the transmembrane domain comprises CD28 or CD8.

[0024] In an aspect, the activation domain comprises a co-stimulatory domain CD28 and a signaling domain CD3C or a variant thereof.

[0025] In an aspect, the co-stimulatory domain comprises CD28, 4 IBB, 0X40, ICOS, CD40, and combinations thereof.

[0026] One aspect of the disclosure includes a pharmaceutical composition comprising a CAR construct according to an aspect of the disclosure and a pharmaceutically acceptable carrier.

[0027] One aspect of the disclosure includes a nucleic acid encoding a CAR construct according to an aspect of the disclosure.

[0028] One aspect of the disclosure includes a plasmid, vector, or cell comprising a nucleic acid according to an aspect of the disclosure.

[0029] In an aspect, the CAR construct is expressed in a cell. In an aspect, the cell is an immune cell. In an aspect, the immune cell is selected from the group consisting of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell. In an aspect, the T cells are regulatory T cells (Tregs).

[0030] One aspect of the disclosure includes a method of producing CAR-expressing immune cells, the method comprising: engineering the immune cells, wherein the engineered immune cells comprise a CAR construct according to an aspect of the disclosure, and expanding the engineered immune cells to produce a population of CAR-expressing immune cells.

[0031] In an aspect, the immune cells are selected from the group consisting of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell. In an aspect, the T cells are regulatory T cells (Tregs).

[0032] In an aspect, engineering the immune cells comprises transfecting or transducing the CAR construct into the immune cells.

[0033] One aspect of the disclosure includes a method of treating an autoinflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any aspect of the disclosure.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0034] In an aspect, the autoinflammatory disease is characterized by the accumulation of macrophages.

[0035] In an aspect, the macrophages comprise TREM2+ macrophages or FOLR2+ macrophages.

[0036] One aspect of the disclosure includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any aspect of the disclosure.

[0037] In an aspect, the cancer is selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

[0038] One aspect of the disclosure includes a method for treating solid tumors in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any aspect of the disclosure.

[0039] In an aspect, the solid tumor is selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

[0040] One aspect of the disclosure includes a method of enhancing anti-tumor activity of engineered immune cells, the method comprising administering to the subject an effective amount of a population of cells according to any aspect of the disclosure, wherein the payload enhances the immune response to tumor cells and / or modulates the CAR construct activity.

[0041] In an aspect, any one of the methods disclosed is cell therapy.

[0042] One aspect of the disclosure includes the use of the CAR construct according to any aspect of the disclosure in the manufacturer of a medicament for the treatment of cancer or an autoinflammatory disease.

[0043] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0045] FIGs. 1A- 1E are exemplary CAR constructs according to various aspects of the disclosure. FIG. 1A illustrates a IL- 12 TREM2.CAR construct according to an aspect of this disclosure. FIG. IB illustrates a destabilization domain (DD) IL-12 TREM2.CAR constructMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCaccording to an aspect of this disclosure. FIG. 1C illustrates a protease-cleavable membranebound IL- 12 TREM2.CAR construct according to an aspect of this disclosure. FIG. ID illustrates a cleavable membrane -bound IL-12 TREM2.CAR construct with a FKPBmutF36v fused domain according to an aspect of this disclosure. FIG. IE illustrates a dTAG system for degrading proteins that are fused to an FKPBmut domain of FIG. ID.

[0046] FIGs. 2A- 2C illustrates how a cleavable membrane -bound IL-12 according to an aspect of the disclosure reduces IL- 12 armored aTREM2.CAR toxicity at higher doses and mediates similar tumor growth delay. FIG. 2A is a schematic showing experimental setup for FIGs. 2B and 2C. FIG. 2B are graphs showing body weight changes of KPI NSCLC tumorbearing mice treated with 3xlOA5 IL-12.aTREM2.CAR-T, CleavMbIL-12.aTREM2.CAR-T, or control T cells (n=6 mice / group). FIG. 2C are survival curves of mice treated in FIG. 2B.

[0047] FIGs. 3A-3K illustrate how FOLR2.CAR-T cells localize in tumor lesions, kill FOLR2+ macrophages, and have an anti-tumoral effect. FIG. 3A is a UMAP plot of myeloid cell subsets of ID8 tumors. FIG. 3B illustrates a Control and aFOLR2.CAR retroviral vector structure. FIG. 3C is a representative flow cytometry plots: aFOLR2.CAR and GFP expression on transduced T cells. FIG. 3D shows in vitro cytotoxicity assay of control and aFOLR2.CAR-T cells (effector cells) with FOLR2+ RAW macrophages as target cells. 5 technical replicates, representative of 2 independent experiments. FIG. 3E shows flow analysis of FOLR2 expression on RAW macrophage and ID8-BVluc cells. FIG. 3F shows gene expression of Wtl and Folr2 in cells from ID8 tumor based on scRNA-seq analysis. FIG. 3G shows imaging (right) and quantification (left) of total macrophages (F4 / 80+ ) and FOLR2+ macrophages in ID8 tumors at 3 days post-treatment (n = 5 mice per group).Representative of 2 independent experiments. FIG. 3H shows FOLR2+ F4 / 80+ cells in peritoneal lavage samples collected 3 days post-treatment and measured (mean ± s.d.) by flow cytometry (n = 5 mice per group). Representative of 2 independent experiments. FIG.31 shows BALB / c RAG1. / . mice (n = 5 per group) bearing subcutaneous ID8 tumors intravenously injected on day 8 with luciferase-expressing aFOLR2.CAR or control T cells. Dorsal bioluminescence images acquired at the indicated time points. FIG. 3J shows survival curve of ID8 ovarian tumor-bearing mice treated with aFOLR2.CAR or control T cells (n = 5 mice / group), 2Gy radiation prior to T cell injection. FIG. 3K shows KPC pancreatic tumor burden treated with aFOLR2.CAR (n = 6) or control T cells (n = 4) with 2Gy radiation prior to injection. Pancreas collected at day 15. (Left). Mean ± s.d. percent of FOLR2+ macrophages in the pancreas, (Right) Primary tumor weights. *p < 0.05, **p < 0.01, ***p <Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC0.001, ****p < 0.0001, ns > 0.05 in unpaired two-tailed t test parametric, except otherwise stated. All survival significance determined by log-rank test.

[0048] FIGs. 4A-4L show CAR and payload functionality, related to FIGs. 3A-3K. FIG. 4A: (left) Measure of specific killing of FOLR2+ cells in co-cultures as described in method, (right) Measurement IFNy+ T cells as percentages of GFP+ cells. Graph shows mean±s.d. (n=3 / group, 2 independent experiments). FIG. 4B: (left) Measurement of specific killing of FOLR2+ macrophages isolated from peritoneal lavage collected 2 days after thioglycolate i.p. injection, (right) Frequency of IFNy+ T cells in the co-culture cells. Graph shows mean±s.d. (n=3 / group, 2 experiments). FIG. 4C is a representative flow cytometry plots showing expression of FOLR2 on macrophage in ascites collected from ID8 ovarian tumor-bearing mice peritoneal co-culture ex vivo with control T cells or aFOLR2.CAR-T cells. FIG. 4D: Graphs shows the relatively percent of GFP+ T cells in the blood (as fraction of CD8+ cells) 3 days after injection of T cells (2Gy lymphodepletion) in ID8 tumor bearing mice (n=5-6 mice / group). FIG. 4E: Representative image of ID8 ovarian tumor (no luciferase) stained for FOLR2. ID8 cells implanted subcutaneously in BALB / c RAG1- / - mice. FIG. 4F:Bioluminescence from the ventral view was taken at the time points indicated from subcutaneously ID8-inoculated mice treated 8 days later with luciferase-expressing control or aFOLR2.CAR-T cells. FIG. 4G: Representative flow cytometry plots showing expression of aFOLR2.CAR and GFP in control or armored CAR-T cells transduced CD8 T cells. Shown are the percentages of transduced cells. FIG. 4H: CAR expression of Sushi.IL-15.aFOLR2.CAR measured by flow with anti-rat antibody. FIG. 41: Flow cytometric measurement from intracellular staining of TNFa, and Granzyme B expressed in control or CAR-T cells in killing assays of the indicated T cells co-cultured with FOLR2+ target cells and non-target cells. Data shown as mean±s.d. of the percent of GFP+ (transduced) T cells (Three technical replicates, representative of 3 independent experiments). FIG. 4J: Flow cytometric measurement from intracellular staining of TNFa expressed in control or IFNa.aFOLR2. CAR-T cells in killing assays, where T cells were co-cultured with targeted FOLR2+ cells and non-target cells. IFNa.aFOLR2. CAR-T cells were transduced from IFNAR KO mice. Data shown as mean±s.d. of the percent of GFP+ (transduced) T cells. (Three technical replicates, representative of 2 independent experiments). FIG. 4K:Representative plots measured by flow cytometry of GFP, a marker of transduction, and intracellular IFNy indicative of T cell activation. Comparison of control IL- 12. aFOLR2. CAR-T and IFNy.aFOLR2. CAR-T cells, when cocultured with targeted FOLR2+Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcells, vs. when cultured alone. FIG. 4L: (Left) schematic explaining the collection of supernatant from CAR-T cells 24h culture to be applied to ID8 tumor cells. (Right) Percentage of PD-Llhigh ID8 cells out of total cells when supernatant from recombinant IFNy or supernatant from control, aFOLR2.CAR-T cells, IFNcr.CAR-Tor IFNy.aFOLR2 T cells is applied, measured by flow cytometry. Three technical replicates and a representative experiment of two were performed. *p<0.05, **p<0.01, ***p<0.001, ****p pO.OOOl, ns>0.05 in unpaired two-tailed t-test parametric,except otherwise stated.

[0049] FIGs. 5A -5G illustrate how an armored aFOLR2.CAR T cells kill FOLR2+cells and secrete functional payload. FIG. 5A is a schematic of the armored aFOLR2.CAR retroviral vector with cytokine payload. FIG. 5B: (Left) Experiment schematic. (Center) Specific killing of targeted FOLR2+cells for each of the indicated CAR-T cells was measured by flow. (Right) IFNy-expressing control or CAR-T cells. Three technical replicates, representative of 3 independent experiments. FIG. 5C: (Left) Same experiment setting as (FIG. B) for IFNa.aFOLR2. CAR-T cells transduced into T cells from IFNAR knockout mice. (Right) IFNy-expressing control or CAR-T cells. Three technical replicates, representative of 2 independent experiments. FIG. 5D: (Left) Schematic of the experiment. (Right) Mean± s.d of MHC class I expression on ID8 cells cultured with recombinant IFNy or the indicated supernatant (n = 3 / group, representative of 2 independent experiments). FIG. 5E: ( (Left) Schematic of the experiment. (Center) Mean ± s.d of IFNy-expressing naive T cells after exposure to indicated cells culture supernatant (n = 3 / group). (Right) Flow cytometric measurement (mean ± s.d) Ki67+and CD62L+ / CD44+naive T cells after exposure to recombinant IL- 15 or indicated supernatant. Three technical replicates, 2 independent experiments. FIG. 5F: ( (Left) Experiment setting. (Right) Survival curves of ID8 ovarian tumor-bearing mice treated with 1.5 x 106of indicated CAR-T cells following 2Gy irradiation (n = 5-7 mice / group). FIG. 5G: (Survival curves of ID8 ovarian tumor-bearing mice treated with 1.5 x 105Sushi.IL-15.aFOLR2 T or control T cells with no lymphodepletion (n = 5 mice / group). (Center) Ascites development curve. (Right) Survival curve. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns > 0.05 in unpaired two-tailed t test parametric, except otherwise stated. All survival significance determined by log-rank test.

[0050] FIGs. 6A- 6D show IL- 12 and Sushi-IL-15 armored aFOLR2. CAR-T cells' efficiency and toxicity at different doses with and without lymphodepletion. FIG. 6A:Experimental schematic for 1D8 ovarian tumor-bearing mice treated with 1.5xl06Sushi.lL-Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC15.aFOLR2.CAR-T (Sushi.IL-15.aFOLR2) or control T cells following total body irradiation 2Gy (TBI) lymphodepletion (n=8,9 mice / group). (Left) Weight measurement over time. FIG.6B: Frequency of GFP+ T cells (marker of transduction) in the blood of ID8 tumor-bearing mice at 6 days after injection 1.5xl06of Sushi.IL-15.aFOLR2 or control T cells. FIG. 6C: (Left) Experimental schematic: ID8 ovarian tumor-bearing mice treated with IxlO6Sushi.IL-15.aFOLR2 or control with no lymphodepletion (n=4, 3 mice / group). (Right) Survival curve. Statistical p-value determined by log-rank. FIG. 6D: (Left) Experimental schematic for ID8 ovarian tumor-bearing mice treated with 1.5xl06IL-12.aFOLR2.CAR-T (IL-12.aFOLR2) or control T cells with lymphodepletion. (Center) GFP+ T cells in blood (% CD8 T cells).(Right) Weight change over time and survival curve (n=2 mice / group). *p<0.05, **p<0.01, ***p<0.001, ****p p<0.0001, ns>0.05 in unpaired two-tailed t-test parametric, except otherwise stated.

[0051] FIGs. 7A- 7M show low-dose IL-12-armored anti-TAM CAR-T achieves long-lasting survival in mice with ovarian cancer with no severe toxicity. FIG. 7A: Destabilization Domain (DD) fused to IL-12 in armored aFOLR2.CAR (DD.IL-12.aFOLR2) construct. FIG.7B: Experimental setting for C-D. ID8 ovarian tumor-bearing mice treated with 106IL-12. aFOLR2, DD.IL-12.aFOLR2, or control T cells (n = 4-5 mice / group). FIG. 7C: Body weight percentage changes overtime. FIG. 7D: Survival curves per group. FIG. 7E:Experimental setting for (F-H). ID8 ovarian tumor-bearing mice treated with 105IL-12. aFOLR2 or control T cells, (n = 5 mice / group, representative of 3 independent experiments). FIG. 7F: Body weight percentage changes overtime. FIG. 7G: (Left) Quantification of bioluminescence signal overtime. (Right) Representative IVIS images, IVIS on day (-1) is a composite image of mice prior to randomization. FIG. 7H: Survival curves of mice. FIG. 71: Spleen weights (mean of ±s.d.) of mice treated as in FIG. 7E were measured 10 days post-treatment. FIG. 7J: Spleen size area (mean of ±s.d.) of mice treated as in FIG. 7E measured in control mice (n = 6) at 4 days post-treatment or IL-12. aFOLR2-treated mice (n = 3) at 163 days post-treatment. Representative of 2 independent experiments.FIG. 7K: FOLR2+ macrophages in the liver of CAR-T cell treated as in (E). (Left) Representative images of liver sections stained for FOLR2 on day 13 post-treatment. (Right) Quantification (mean ± s.d.) of FOLR2+ macrophage density in the liver (n = 5 mice / group). Representative of 3 independent experiments. FIG. 7L: Serum alanine transaminase (ALT) level measured on day 10 post T cell treatment as in FIG. 7E (n = 5-6 mice / group). Group mean shown. Representative of 2 independent experiments. FIG. 7M: Survival curves of KPC pancreatic tumor-bearing mice treated with the indicated T cells (n = 4 mice / group).Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCSchematic on the left shows the treatment design. No lymphodepletion used *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns > 0.05 in unpaired two-tailed t test parametric, except otherwise stated. All survival significance determined by log-rank test.

[0052] FIGs. 8A-8K show In vitro functionality and in vivo tissue distribution of DD.IL 12.CAR-T cells. FIG. 8A: Schematics explaining Shield-1 small molecule action on FKPB-mut destabilization domain. FIG. 8B: Representative plots measuring by flow cytometry GFP expression, a transduction marker, and intracellular IFNy expression indicative of T cell activation. Comparison of aFOLR2.CAR-T cells, IL-IL-12.aFOLR2 T cells and DD.IL-12.aFOLR2 T cells when cocultured with targeted FOLR2+ cells vs. when cultured alone. Graph shows IFNy+ cells % in each group mean±s.d (n=3 / group). FIG. 8C: IL-12 secretion in supernatant of DD-IL-12.aFOLR2 T cells vs. control cells, measured by IL- 12 ELISA kit.FIG. 8D: Intracellular IFNy expression levels measured by flow cytometry after 24-hour culture of CAR-T cells or control T cells. IL-12. aFOLR2 and DD.IL-12.aFOLR2 cells were treated with incremental doses of the Shield- 1 molecule. Graph shows mean±s.d. (3 technical replicates). FIG. 8E: Frequency of IFNy+ T cells measured by flow cytometry in co-cultures of FOLR2+target cells with CAR or control T cells. Cells were treated with incremental doses of the Shield- 1 molecule Graph shows mean±s.d. (3 technical replicates). FIGs. 8F-8J:Experimental setup: ID8 ovarian tumor-bearing mice treated on day 8 post-inoculation with IxlO6IL-12. aFOLR2 T, DD.IL-12.aFOLR2-T, or control T cells. Tissues were collected at day 4 and at day 11 post-treatment, sections of FFPE-processed tissue were stained via MICSSS, imaged, and quantified, except for FIG. 8J where spleens were processed and analyzed by flow cytometry. Mean±s.d. of CAR-T cells per 105 of total cells were measured for FIG. 8F: omentum. FIG. 8G: liver. FIG. 8H: lung. FIG. 81: brain, FIG. 8 J: spleen. FIG. 8K: Serum alanine transaminase (ALT) in the mice from FIG. 8J measured on day 6 post-treatment (n=2-4 mice / group). Group mean shown. *p<0.05, **p<0.01, ***p<0.001, ****p pO.OOOl, ns>0.05 in unpaired two-tailed t-test parametric, except otherwise stated.

[0053] FIGs. 9A-9I show IL-12.NoCAR, IL-12. aFOLR2 compared efficacy, and IL-12. CARaFOLR2 CAR-T treatment effect on spleen and omentum. FIG. 9A: Schematic of the retroviral vectors encoding CMV driven IL- 12 with no CAR construct (IL-12.NoCAR).FIG. 9B: IL-12 secretion in supernatant from 5xl05CAR-T cells 24h culture of IL- 12. aFOLR2 compared to IL-12.No-CAR, measured by IL-12 ELISA kit. FIG. 9C: Control or CAR-T cells cultured alone and stained for CD8 and IFNy by flow cytometry, representative plot charts, and summary bar charts (3 technical replicates). FIG. 9D: Control or CAR-T cells cocultured with non-target A20 B cells and FOLR2+ cells, macrophages were stainedMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCfor FOLR2 by flow, representative plot charts, and summary bar charts (3 technical replicates). FIG. 9E: Measurement of ID8-BVluc ovarian tumor growth in mice treated with IL-12. aFOLR2, IL-12.NoCAR-T or control T cells (n=5 mice / group). Shown is quantification of bio luminescence signal from the abdominal region at the indicated time points. FIG. 9F: Survival curves of mice from (e). Significance determined by log-rank test.FIG. 9G: Experimental setup : ID8 ovarian tumor-bearing mice treated on day 8 postinoculation with 1.5xl05IL-12. aFOLR2 T or control T cells (n=5 mice / group). Spleens were collected at 11 days post-treatment, weighted and processed for flow analysis. Spleen weight at day 11 post-treatment. Spleen immune cell composition analyzed by flow cytometry, mean±s.d. measurement shown. FIG. 9H-9I: Experimental setup: ID8 ovarian tumor-bearing mice treated on day 8 post-inoculation with 1.5xl05IL-12.aFOLR2 T or control T cells (n=5 mice / group). Livers were collected at day 4 and 11 post-treatment, and sections of FFPE-processed tissue were stained via MICSSS, imaged, and quantified. FIG. 9H: (Left) Representative MICSSS image of liver at day 4 post-treatment showing CD8 (magenta), F4 / 80 (yellow), FOLR2 (red), and nuclei (blue) staining. (Right) Graph shows the mcan±s.d. of CD8 T cells and FOLR2+ macrophages in % of total cells. FIG. 91: (Left) Representative MICSSS image of liver at day 11 post-treatment showing CD8 (magenta), F4 / 80 (yellow), FOLR2 (red), and nuclei (blue) staining. (Right) Graph shows the mean±s.d. of CD8 T cells and FOLR2+ macrophages in % of total cells. *p<0.05, **p<0.01, ***p<0.001, ****p p<0.0001, ns>0.05 in unpaired two-tailed t-test parametric, except otherwise stated.

[0054] FIGs. 10A- 10I show IL-12-armored anti-FOLR2 CAR-T promotes a reset and reprogram of tumor macrophages to an immunostimulatory state ID 8 ovarian tumor-bearing mice treated with 105 IL-12.aFOLR2 or control T cells. Omentum collected 10 days posttreatment, tissue sections analyzed by Xenium in situ spatial transcriptomics (ST) profiling using a custom panel of 480 gene probes (n = 3 mice per group, mice 2L, 2LL and 2RR in the control group and mice 3L, 3N, 3R in the IL-12. aFOLR2 treated group). FIG. 10A: (Left) UMAP representation of full cohort (n = 492,584 confident cells) from control and treated omentum sections, with annotated cell subtypes indicated. (Center) Absolute abundance of cells per cell-type for each sample / mouse. (Right) Spatial scatterplot of representative Xenium-profiled omentum resections for control and treated mice. FIG. 10B: Dot plot for ST data: marker genes of annotated myeloid subtypes, with cells grouped by subtype and expression information provided. (I) Folr2 high and Folr2 low, (II) Folr2- Argl+, (III) Illb macrophages, (IV) 1112 signature, (V) immuno-regulatory markers, and (VI) pro-inflammatory and antigen-presenting genes. FIG. 10C: (Left) UMAP representations of theMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCmacrophage compartment (n = 145,329 confident cells) in ST data: treated vs. control and annotated macrophage subtypes. (Center) Absolute abundance of cells per macrophage subtype for each sample / mouse. (Left) Relative abundance of cells per macrophage subtype.FIG. 10D: UMAP projections of macrophage compartment, color-coded according to their respective scaled expression of Folr2 and Cxcl9 in each cell. FIG. 10E: Folr2 expression in representative ST-profiled samples for control and treated mice. FIG. 10F:Immunosuppressive macrophage score (left) and response to IL- 12 signature (right) representative samples. FIG. 10G: Spatial scatter of macrophage subtypes in representative samples for control and treated mice. FIG. 10H: Argl and Cxcl9 expression in representative samples for one treated mouse. FIG. 101: Spatial arrangement of Folr2- Argl+ macrophages, Cxcl9+ macrophages, and B cells in a region of a representative treated mouse.

[0055] FIGs. 11A-11K show wildtype C57BL / 6 mice were inoculated with 5x106 ID8-BVluc cells and treated 8 days later with 1.5xl05with either IL-12.aFOLR2.CAR or control T cells intraperitoneal injection with no prior lymphodepletion. Omentum was collected at 10 days post-treatment, FFPE processed, and tissue sectioned for Xcnium in situ spatial transcriptomics (ST) profiling (3 mice analyzed per group). FIG. 11 A: (Left) Relative abundance of cells per cell type based on UMAP across each group of samples: control vs. treated (Right) Spatial scatter plot of all ST-profiled samples for control and treated samples, color-coded according to their cell type assignment. FIG. 11B: Relative abundance of cells per cell type based on UMAP for all cell subtypes across each group of samples: control vs. treated. FIG. 11C: (Left) Folr2 expression, immunosuppressive macrophage score, and (Right) IL- 12 signature score in all Xenium-profiled samples for control and treated mice. The immunosuppressive macrophage score was calculated using the combined expression of Argl, Cd274, Folr2, Hmoxl, 1110, Igfl, Mertk,Mrcl, Tgfbl, Trem2, and 114. The IL-12 response score was calculated using the available genes in the ST panel (CxcllO, Cxcl9, CC15, Irf7, Statl) from the ‘macrophage response to IL-12’ signature in the Immune Dictionary. FIG. 11D: UMAP projections of macrophage compartment, color-coded according to their respective scaled expression of Argl and II lb in each cell. FIG. HE: UMAP representations of the macrophage compartment in ST data: color-coded according to their respective score of response to IL- 12 (IL- 12 signature). The program score is transferred from the RNA annotation. The dotted line delineates the cells from IL- 12. aFOLR2. CAR-treated samples. FIG. HF: Spatial representation of Cxcl9+, Folr2- Argl+ macrophages and B cells in a representative Xenium-profiled region for one treated mouse. Generated by lOx Genomics Xenium Explorer 4.0.0. FIG. 11G: Dot plot for Xenium ST data: MacrophageMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCmarker genes representative of myeloid signaling, with cells grouped by subtype and expression information provided. FIG. 11H: Dot plot for Xenium ST data: marker genes of broadly annotated B cells, plasma cells, myeloid cells, T and NK cells, cancer cells, and stromal cells, with cells grouped by subtype and expression information provided. The subtypes are I: B cells, II: Plasma cells, III: Myeloid cells, IV: T cells, V: Tregs, VI:Exhausted T cells, VII: NK cells, VIII: Tumor epithelial, IX: Fibroblast, X: Endothelial. FIG. Ill: ST-profiled samples for control and treated mice, color-coded according to Gzmb expression in each cell. FIG. 11 J: Dot plot for Xenium ST data: Effector memory CD8 T cell marker genes, with cells grouped by control and treated, and by subtype, expression information provided. FIG. 11K: UMAP projections of T andNK cells compartment, color-coded according to their respective expression of effector memory CD8 T cell makers in treated vs. control. Chart shows quantification of number of effector memory CD8 T cells subset and activated CD8 T cells in treated and control subset.

[0056] FIGs. 12A-12G show MICSS analysis on omentum and flow analysis on peritoneal lavage and diaphragm for low-dose IL-12. aFOLR2 treatment. Experimental setup: Wild-type C57BL / 6 mice were inoculated with 5x106 ID8-BVluc cells and treated 8 days later with 1.5x105 with either IL-12. aFOLR2.CAR (IL-12. aFOLR2) or control T cells intraperitoneal injection with no prior lymphodepletion. FIG. 12A: (Top) Representative image of ID8 tumor lesions in the omentum, collected day 10 post treatment; stained by MICSSS for F4 / 80, FOLR2 and Tumor cells. (Bottom) Associated cells measurement by MICSSS. Graphs show the mean±s.d. (n=5 mice / group). Representative of two independent experiments. FIG. 12B:Graphs showing quantification macrophages in the peritoneal lavage of ID8 tumor-bearing mice at 10 days post-treatment. Cell frequency determined by flow cytometry. Graphs show the mean±s.d. (n=4-6 mice / group). Representative of two independent experiments. FIG. 12C: Graphs showing quantification macrophages in the diaphragm of ID8 tumor-bearing mice at 10 days post-treatment. Cell frequency determined by flow cytometry. Graphs show the mean±s.d. (n=6 mice / group). Representative of two independent experiments. FIG. 12D:(Top) Representative image of ID8 tumor lesions in the omentum, collected day 10 post treatment; stained by MICSSS for CD8 (yellow), Granzyme B (pink) and nuclei (blue) staining. (Bottom) Graph shows the mean±s.d. of CD8+ and CD8+ GrB+ cells in the tumors. Graphs show the mean±s.d. (n=6 mice / group). Representative of two independent experiments. FIG. 12E: (Top) Representative image of ID8 tumor lesions in the omentum, collected day 10 post treatment; stained by MICSSS for FOXP3 and CD4. (Bottom) Associated double positive FOXP3+ and CD4+ Treg cells measurement. Graphs show theMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCmean±s.d. (n=6 mice / group). FIG. 12F: Graphs showing quantification of T cells in the peritoneal lavage of ID8 tumor-bearing mice at 10 days post-treatment with the indicated vector. Cell frequency determined by flow cytometry. Graphs show the mean±s.d. (n=6 mice / group). Representative of two independent experiments. FIG. 12G: Graphs showing quantification of T cells in the diaphragm of ID8 tumor-bearing mice at 10 days posttreatment with the indicated vector. Cell frequency determined by flow cytometry. Graphs show the mean±s.d. (n=6 mice / group). Representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p pO.OOOl, ns>0.05 in unpaired two-tailed t test parametric, except otherwise stated.

[0057] FIGs. 13A - 13D show IL-12-armored anti-FOLR2 CAR-T promotes the recruitment and activation of CD8 T cells. FIG. 13A: Dot plot ST data: marker genes of annotated T and NK cell subtypes, with cells grouped by subtype and expression information provided. FIG.13B: (Left) UMAP representations of the T and NK cells (n = 63,397 confident cells) in ST data: control vs. treated and annotated T and NK cell subtypes. (Right) Absolute and relative abundance for T and NK cell subtypes for each samplc / mousc. FIG. 13C: UMAP projections of T and NK cell compartments, color-coded according to their respective scaled expression. Chart Gzmb+ Cd8+ cells in treated and control subsets. FIG. 13D: Gzmb expression in representative samples for control and treated mice. Mean ± s.d quantification of Gzmb+ CD8 T cells in control and IL-12.aFOLR2 treated.

[0058] FIGs. 14A - 14E show the effect of IL-12aFOLR2 and contribution of NK cell and FAS in anti-tumoral efficacy. FIG. 14A: (Top) schematic showing experimental setup: wild type ID8 ovarian tumor-bearing mice treated with 1.5x105 IL-12.aFOLR2.CAR-T or control (n=6 mice / group). (Left) Omentum tissues were collected 10 days post-treatment, sections of FFPE-processed tissue were stained via MICSSS, imaged, and quantified. (Right) Peritoneal lavages were collected 10 days post-treatment and processed for flow analysis. Mean±s.d. measurement shown. FIG. 14B: (Top) schematic showing experimental setup: ID8 ovarian tumor-bearing mice treated with IxlO5IL-12.aFOLR2.CAR-T or control T cells with anti-NK1.1 antibody injection one day prior CAR-T treatment and every other day after (n=5 mice / group). (Bottom) Survival curve. Significance determined by log-rank test. FIG. 14C:Assessment of PD-L1 expression on ovarian cancer cells from ID8 tumors from mice injected 10 days prior with the indicated treatment. Single cell suspensions were made from tumorbearing diaphragm, stained for the indicated markers and analyzed by flow cytometry.Percent shown as a fraction of the ID8 cancer cells. Graphs show the mean±s.d. (n=6 mice / group). FIG. 14D: Wildtype C57BL / 6 mice were injected i.p. with FAS- / - ID8-BVlucMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcells and after 8 days injected with Control or IL12.aFOLR2.CAR-T cells (n=6 mice / group, representative of 2 independent experiments). Tumor burden was assessed over time by luciferase measurement using non-invasive imaging. FIG. 14E: Comparison of TREM2 expression in TREM2ecto KPI cells vs. wild-type KPI cells stained with TREM2 antibody except for FMO by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, ****p pO.OOOl, ns>0.05 in unpaired two-tailed t test parametric, except otherwise stated.

[0059] FIGs. 15A- 15F show IL-12.aFOLR2 therapeutic benefit is driven by tumor-antigen reactive endogenous CD 8 T cells and by FAS-mediated tumor cell death. FIG. 15A:Xenium-profiled treated sample, color-coded according to Krtl8, Gzmb, and Cd8a transcript expression in each cell overlapped with cell segmentation color-coded according to juxtaposed cancer-T cells score, image generated using Xenium Explorer 4 (lOx Genomics). Mean ± s.d. quantification of the number of Gzmb+ juxtaposed cells in treated and control subsets. FIG. 15B: Tumor-reactive T cell frequency from ID8 ovarian tumor-bearing mice treated with 1.5 x 105IL-12. aFOLR2 or control T cells as indicated in the schematic (left) for the two independent following experiments. FIG. 15C: IFNy Elispot performed by applying CD8+T cells isolated from spleens to irradiated ID8 cells (n = 3 mice / group). Positive control: activated CD8 T cell. Graphs show the mean ± s.d. FIG. 15D: Isolated CD8+T cells from ID8 tumor-bearing mice spleens collected 11 days post-treatment with IL-12.aFOLR2 or control (n = 5 mice per group) cocultured for 24h with irradiated ID8 cancer cells. (Right) Flow analysis mean ± s.d. (fraction and of total cells). FIG. 15E: FAS and MHC-I expression on ID8 ovarian cancer cells 10 days after treatment. Single-cell suspensions from tumorbearing diaphragms were stained and analyzed by flow cytometry. Graphs show mean ± s.d. (n = 6 mice / group). (n = 6 mice per group). FIG. 15F: Wildtype C57BL / 6 mice were injected i.p. with FAS- -ID8-BVluc cells, and after 8 days, injected with control or IL12.aFOLR2.CAR-T cells (n = 6 mice / group). The animals were monitored for survival. Representative of 2 independent experiments. *p < 0.05, **p < 0.01, *** / ? < 0.001, **** / ? < 0.0001, ns > 0.05 in unpaired two-tailed t test parametric, except otherwise stated. All survival significance determined by log-rank test.

[0060] FIGs. 16A- 161 show IL- 12 armored anti-TREM2 CAR-T cells improve survival in a model of metastatic lung cancer. FIG. 16A: aTREM2.CAR and IL-12.aTREM2.CAR retroviral vector constructs. FIG. 16B: Flow cytometry plots: aTREM2.CAR and GFP expression on indicated T cells. FIG. 16C: Flow cytometry analysis of TREM2 expression on the indicated cells. FIG. 16D: (Left) Specific killing of targeted TREM2+cells for indicated T cells. (Right) Percent of IFNy+ GFP+ T cells by flow (mean ± s.d). ThreeMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCtechnical replicates (representative of 2 independent experiments). FIG. 16E: (Top) Experimental Setting: KPI tumor-bearing mice treated with aTREM2.CAR or control T cells (n = 5 mice / group) with 2 Gy irradiation prior to T cell injection. (Bottom) Survival curve.FIG. 16F: (Top) Experimental Setting. Lungs were collected 18 days after tumor inoculation (11 days post-treatment). (Left) Representative H&E-stained sections are shown. (Right) Mean ± s.d lung tumor lesion area. FIG. 16G: (Top) Experimental Setting. Survival curves of KPI lung tumor-bearing mice treated with IL-12.aTREM2.CAR or GFP+ control T cells (n = 8-10 mice / group combination of two independent experiments). No prior lymphodepletion. Representative of 3 independent experiments. FIG. 16H: Experimental Setting. FIG. 161: Tumors were collected at 18 days post-tumor inoculation from each lung, stained, and analyzed by flow cytometry. Graphs show the mean ± s.d. (n = 5-6 mice / group). *p < 0.05, **p < 0.01,mp < 0.001,m*p < 0.0001, ns > 0.05 in unpaired two-tailed t test, except otherwise stated. All survival significance is determined by the log-rank test.DETAILED DESCRIPTIONI. Introduction

[0061] Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant clinical efficacy in hematological malignancies, where lineage-restricted and stably expressed antigens enable effective and selective tumor cell targeting. By contrast, application of CAR-T therapies to solid tumors has yielded limited and inconsistent clinical success. A principal challenge is the lack of tumor-associated antigens (TAAs) that are both specific to malignant cells and durably expressed. Targeting such antigens can result either in immune escape through antigen mutation or downregulation, or in on-target, off-tumor toxicity due to shared antigen expression between tumor and healthy tissues, as exemplified by severe toxicities observed when targeting antigens such as HER2.

[0062] Even in cases where cancer-cell-restricted targets are identified, CAR-T therapies face an additional and substantial barrier in the form of the highly immunosuppressive tumor microenvironment (TME) characteristic of solid tumors. The TME actively limits T-cell infiltration, activation, and persistence, thereby impairing anti-tumor efficacy. A key contributor to this immunosuppressive milieu is the presence of tumor-associated macrophages (TAMs), which are recruited and polarized by tumors to support immune evasion and disease progression.

[0063] Transcriptomic analyses, including single-cell RNA sequencing, have revealed that immunosuppressive TAM subsets share conserved molecular features across multiple solidMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCtumor types and are distinct from macrophages found in healthy tissues. In particular, pro-tumoral TAMs are characterized by elevated expression of markers such as triggering receptor expressed on myeloid cells 2 (TREM2) and folate receptor beta (FOLR2). These markers are highly enriched on TAMs across diverse cancers, including ovarian, pancreatic, lung, breast, and colorectal malignancies, and are minimally expressed by most other cell types, making them attractive therapeutic targets.

[0064] Given the central role of TAMs in establishing and maintaining the immunosuppressive TME, substantial efforts have focused on strategies to deplete or modulate these cells. Approaches such as inhibition of macrophage survival pathways have shown limited clinical benefit, likely due to insufficient selectivity and disruption of beneficial macrophage populations. More recently, CAR-T cells targetingmacrophage-associated antigens have been developed to selectively eliminate TAMs. While these approaches have demonstrated anti-tumor activity in preclinical models, tumor control has generally been incomplete or transient, with tumors frequently rebounding.

[0065] Accordingly, a need exists for improved therapeutic strategics that not only target tumor-associated macrophages with high specificity but also actively reprogram the tumor microenvironment to support durable anti-tumor immunity. The present invention addresses this need by providing cytokine-armored CAR immune cells that selectively target TAM-associated antigens, such as TREM2 or FOLR2, and locally deliver immunostimulatory cytokines within the tumor microenvironment. As described herein, such engineered cells are capable of depleting or reprogramming immunosuppressive macrophages, inducing sustained remodeling of the tumor microenvironment, and promoting endogenous immune-mediated tumor clearance. Notably, the disclosed approach enables durable anti-tumor effects without direct targeting of tumor cells and without reliance on prolonged CAR-cell persistence, thereby overcoming key limitations associated with prior CAR-T therapies for solid tumors.II. Definitions

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0067] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0068] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0069] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0070] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0071] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0072] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0073] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwiseMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCindicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0074] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure.Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0075] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims arc to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0076] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0077] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0078] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0079] The terms "Chimeric Antigen Receptor" or "CAR" refer to a molecule engineered to comprise a binding motif and a means of activating immune cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof), NK cells, and other cell types upon antigen binding. CARs arc also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell.

[0080] The antigen-binding domain of a CAR typically consists of an extracellular region that targets a surface antigen on tumor cells, and an intracellular domain to derive T cell activation. The antigen-binding domain of a CAR commonly comprises a targeting moiety, such as an antibody single chain variable fragment (scFv) or a nanobody, which is directed against the target antigen. The T cell-activating domain of a CAR is intracellular and activates the T cell in response to the antigen-binding domain interacting with its target antigen. A T cell activating domain can contain one or more co-stimulatory domains, and a signaling domain which are the intracellular domains of known activating T cell receptors. The selection and positioning of costimulatory domains within a CAR construct influence CAR T cell function and survival, as costimulatory domains have impacts on CAR-T cell, cytotoxic function, and safety profile. The extracellular antigen-binding and intracellular T cell- activating domains of CARs are linked by a transmembrane domain, hinge, and optionally, a spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on the tumor cell. It may beMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCused alone or in conjunction with a spacer domain that locates the scFv away from the T cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface

[0081] "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to reside outside of the cell membrane, in the extracellular space.

[0082] The binding domain of the CAR may be followed by a "spacer," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. The spacer may further comprise a hinge region or domain. The hinge region is typically membrane proximal, and is between the transmembrane (TM) and the binding domain. In certain embodiments, a hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as IgG (c.g., IgGl, IgG2, IgG3, and IgG4), CD8a, CD4, CD28, 4- IBB, and CD7, which may be wild- type hinge regions from these molecules or may be altered.

[0083] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell. The transmembrane domain may be for example those obtained from CD8a, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, CD3zeta, and CD154.

[0084] In one embodiment, the transmembrane domain is the transmembrane domain of CD28. In certain embodiments, the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.

[0085] The “intracellular domain” comprises one or more costimulatory domain, and one or more intracellular signaling domains. The intracellular costimulatory domain can be from, e.g., 4- IBB and / or CD28.

[0086] The "intracellular signaling domain" or "signaling domain" refers to the part of the chimeric antigen receptor protein 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 with antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of the cell. Effector function of the T cell, forMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCexample, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the terms "intracellular signaling domain" or "signaling domain," are used interchangeably herein and refer to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. The intracellular signaling domain is also known as the, "signal transduction domain," and is typically derived from portions of the human CD3 or FcR chains. The intracellular signaling domain can be, e.g., derived from CD3z.

[0087] It is known that signals generated through the T cell receptor alone are generally insufficient for full activation of the T cell and that a secondary, or costimulatory signal, is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen dependent primary activation through the T cell receptor (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or ITAMs.

[0088] Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the disclosure include those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0089] As used herein, the term, "costimulatory signaling domain," or "costimulatory domain", refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule. “Costimulatory molecules” are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The inclusion of one or more costimulatory signaling domains may enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain. Suitable costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134,Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCCD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1- la, CDl-lb, CDl-lc, CD1-Id, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CDlla / CD18), MEW class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), 0X40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0090] As will be appreciated, although scFv-based CARs engineered to contain a signaling domain from CD3 or FcRgamma have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of a concomitant costimulatory signal. Thus, CARs containing a binding domain, a hinge, a transmembrane and the signaling domain derived from CD3zeta or FcRgamma together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD278) may more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR expressing T cells in vitro, and in animal models and cancer patients. See Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420 and Carpenito et al., PNAS, 2009; 106:3360-3365).

[0091] As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0092] As used herein, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percentMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCidentity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0093] Suitable software programs are available from various sources and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI).

[0094] Sequence alignments can be conducted using methods such as, but not limited to, MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), or MUSCLE.

[0095] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity.

[0096] As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0097] As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0098] As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances (e.g., nucleotide sequence or protein sequence) can have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some aspects, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. The term “substantially isolated” means that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof.

[0099] A polynucleotide, vector, polypeptide, cell, or any composition disclosed herein which is “isolated” is a polynucleotide, vector, polypeptide, cell, or composition which is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the degree that they are no longer in a form in which they are found in nature. In some aspects, a polynucleotide, vector, polypeptide, or composition that is isolated is substantially pure.

[0100] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0101] As used herein, a “native” or “naturally occurring” polynucleotide sequence means a polynucleotide sequence existing in nature without artificial aid.

[0102] The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a continuous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0103] The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprises a polymer of nucleotides. These polymers are often referred to as polynucleotides. Example nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs) or deoxyribonucleic acids (DNAs).

[0104] The phrase “nucleotide sequence encoding” refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence that encodes a polypeptide. As used herein, the terms “coding region” and “coding sequence”, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression, yields a polypeptide or protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.

[0105] The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA.

[0106] As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0107] In addition to messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and / or translation, and immunostimulation. Within the present disclosure the term "RNA" further encompasses any type of single stranded (ssRNA) or double stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulating / immunostimulatoryMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0108] The T bases in the codon maps disclosed herein are present in DNA, whereas the T bases may be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in vitro translation (IVT) template, may have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding RNA sequences (comprising U) are considered codon-optimized nucleotide sequences of the present disclosure. Equivalent codon-maps can be generated by replacing one or more bases with non-natural bases. Thus, e.g., a TTC codon (DNA map) may correspond to a UUC codon (RNA map).

[0109] Standard A-T and G-C base pairs form under conditions that allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3, and C4-NH2, of cytidine and the C2-NH2, N' — H and C6-oxy, respectively, of guanosine. Thus, for example, guanosine (2-amino-6-oxy-9-P-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-P-D-ribofuranosyl-purine). Such modification results in a nucleoside base, which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (l-P-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-P-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine.

[0110] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine).

[0111] The term “polypeptide,” as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer, or tetramer. They can alsoMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcomprise single chain or multichain polypeptides. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. In some aspects, a “peptide” can be less than or equal to about 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0112] The terms "immunotherapy" or “cell therapy” refer to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapies or NK cell therapies. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, and allogeneic T cell transplantation.

[0113] One of skill in the art will recognize techniques to enhance the effectiveness of cell therapy using, e.g., preconditioning techniques such as those found in U.S. Patent Nos.9,855,298 and 10,322,146, the contents of which arc hereby incorporated by reference in their entirety.

[0114] The T cells of the immunotherapy can come from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation and / or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No.2013 / 0287748, which is herein incorporated by references in its entirety.

[0115] As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions.

[0116] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCBiological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0117] The term “synthetic” or “engineered” means produced, prepared, and / or manufactured by the hand of man. Synthesis of CAR constructs, CAR T cells, or other molecules of the present disclosure can be chemical or enzymatic.

[0118] As used herein, “transfection” refers to the introduction of a polynucleotide into a cell wherein a polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates a cellular function (e.g., siRNA, miRNA). As used herein, “expression” of a nucleic acid sequence refers to the translation of a polynucleotide (e.g., an mRNA) into a polypeptide or protein and / or post-translational modification of a polypeptide or protein.

[0119] As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified can, but does not always, refer to the wild type or native form of a biomolecule. Molecules can undergo a series of modifications whereby each modified molecule can serve as the “unmodified” starting molecule for a subsequent modification.

[0120] As used herein “in silico” refers to the use of modeling software or simulation software to generate a result. For example, in silico includes optimizing a polynucleotide sequence in an iterative manner using the software to optimize several values and parameters simultaneously that are entered by a user.

[0121] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0122] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of theMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCdisclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0123] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and / or decrease the number, frequency, or severity of clinical symptoms and / or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0124] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0125] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCAlternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0126] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Armored CAR Constructs

[0127] Prior approaches employing chimeric antigen receptor (CAR) T cells targeting macrophages have primarily focused on depleting tumor-associated macrophages (TAMs), which are known to promote tumor progression through multiple mechanisms, including suppression of effector T-cell activity. However, as reported previously and confirmed in the studies described herein, macrophage depletion alone yields only modest therapeutic benefit in preclinical cancer models. Similarly, clinical trials evaluating macrophage-depleting agents, such as antibodies targeting colony-stimulating factor 1 receptor (CSF1R), have not demonstrated a significant impact on cancer progression, likely due to insufficient selectivity and disruption of beneficial macrophage populations.

[0128] In light of these limitations, the inventors sought to evaluate anti-TAM CAR-T cells not merely as cytotoxic agents, but as vehicles for actively remodeling the tumor microenvironment (TME) through localized delivery of immunostimulatory cytokines. This objective is particularly relevant for improving immunotherapy outcomes in immunologically “cold” tumors. A longstanding challenge associated with pro-inflammatory cytokines is the difficulty of achieving tumor-restricted delivery, as systemic exposure is associated with significant toxicity. Indeed, administration of recombinant interleukin- 12 (IL- 12) has been associated with severe adverse events, and prior efforts to engineer tumor-infiltrating lymphocytes to express IL- 12 resulted in unacceptable toxicity. Strategies involving transient cytokine expression, such as RNA-based IL- 12 delivery, demonstrated feasibility in preclinical studies but did not provide durable therapeutic effects.

[0129] Early efforts to armor CAR-T cells with IL- 12 showed enhanced anti-tumor activity and, in some cases, improved CAR-T persistence through autocrine mechanisms. However, clinical studies targeting tumor-associated antigens (TAAs) with IL-12-armored CAR-T cells have thus far demonstrated limited efficacy. Moreover, CAR design has been shown to significantly influence cytokine expression profiles and functional outcomes. For example, IL- 12 expression driven by internal ribosome entry site (IRES) elements has been reported toMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCrequire target engagement to induce interferon-y (IFNy) production, whereas alternative expression architectures may result in higher basal cytokine secretion.

[0130] In parallel, identification of suitable tumor-specific surface antigens for CAR targeting in epithelial-derived solid tumors has proven challenging. Broadly expressed antigens raise the risk of on-target, off-tumor toxicity, while cancer cells can evade targeted therapies through antigen downregulation or loss. The inventors reasoned that TAMs provide an advantageous therapeutic target for CAR-T-mediated TME reprogramming, as TAMs play a pivotal role in establishing immunosuppression and express surface markers, such as folate receptor beta (FOLR2) and triggering receptor expressed on myeloid cells 2 (TREM2), that are selectively enriched on TAMs and not expressed by most healthy cells. Moreover, unlike tumor cells, TAMs are not clonally evolving but are continually replenished from circulating monocytes, making them substantially less capable of developing resistance through antigen loss or mutation.

[0131] Using this rationale, the inventors established a means of remodeling the TME and promoting tumor clearance by targeting TAM-spccific surface markers with IL-12-sccrcting CAR-T cells. This approach resulted in improved survival in orthotopic models of metastatic ovarian and lung cancer. Importantly, therapeutic efficacy was achieved withoutpre-conditioning and with relatively low CAR-T cell doses. The IL-12-armored anti-TAM CAR-T cells trafficked to tumor sites and induced profound changes within the TME, including depletion of immunosuppressive macrophage populations, polarization of myeloid cells toward an immunostimulatory phenotype, and increased infiltration and activation of endogenous T cells.

[0132] Mechanistic analyses further demonstrated that treatment induced significant upregulation of FAS on tumor cells and that FAS signaling was essential for therapeutic benefit. Tumor clearance was therefore mediated not by direct CAR-T cytotoxicity against cancer cells, but by reprogramming of the TME to support endogenous immune -mediated tumor killing. Collectively, these findings demonstrate that cytokine-armored anti-TAM CAR-T cells provide a robust and durable strategy for overcoming immunosuppression in solid tumors by resetting the tumor microenvironment to an immunoreactive state.

[0133] One aspect of the disclosure includes a chimeric antigen receptor (CAR) construct comprising a first antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) comprising a light chain variable region, a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain areMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCconnected via a flexible linker, a transmembrane domain, an activation domain comprised of a signaling domain and a co-stimulatory domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0134] One aspect of the disclosure includes a CAR construct comprising a first antigenbinding domain capable of binding to folate receptor 2 (FOLR2) comprising a heavy chain variable region, a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigenbinding domain are connected via a flexible linker, a transmembrane domain, an activation domain comprised of a signaling domain and a co-stimulatory domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0135] One aspect of the disclosure includes a chimeric antigen receptor (CAR) construct comprising a first antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) comprising a light chain variable region, a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain comprised of a signaling domain and a co-stimulatory domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0136] In an aspect, the payload promotes immunomodulation at disease sites. In certain embodiments, the immunomodulation may include a strategic regulation of immune response in a subject after administration of the CAR construct to said subject. In certain embodiments, the immunomodulation may include the control of inflammation, enhancement of immune defense, or suppression of harmful immune activity in the subject.

[0137] Another aspect of the disclosure includes a CAR construct comprising: a first antigenbinding domain capable of binding to TREM2 comprising a light chain variable region, a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain comprised of a signaling domain and a co-stimulatory domain, and a payload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0138] One aspect of the disclosure includes a CAR construct comprising a first antigenbinding domain capable of binding to folate receptor 2 (FOLR2) comprising a heavy chain variable region, a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigenbinding domain are connected via a flexible linker, a transmembrane domain, an activation domain comprised of a signaling domain and a co-stimulatory domain, and a payload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload connected to the signaling domain via a self-cleaving linker.

[0139] In an embodiment, the polynucleotide comprises internal ribosome entry sites (IRES). As used herein, IRES refers to sequences of RNAthat allow for cap-independent translation initiation in eukaryotic cells. Unlike the standard translation initiation mechanism, which begins at the 5' cap of mRNA and involves scanning for the AUG start codon, IRES sequences enable translation to begin at specific locations within the mRNA, bypassing the need for the 5' cap.

[0140] FIGs. 1A- 1D arc exemplary CAR constructs according to various aspects of the disclosure.

[0141] One embodiment of exemplary CAR construct (FIG. ID) is a cleavable membranebound IL- 12 which incorporates an FKPBmut sequence the intracellular domain. This allows for the degradation of the entire protein using a small-molecule degradation TAG (dTAG). dTAG is an established system for degrading proteins that are fused to an FKPBmut domain. A small-molecule dTAG binds to both the FKBPmut domain and an E3 ligase, bringing the protein of interest in close proximity to the E3 ligase allowing it to be ubiquitinated for degradation (FIG. IE). This application of the dTAG system could provide the cleavable membrane -bound IL- 12 with a small molecule-controlled expression, further reducing the exposure of healthy tissue to IL- 12 before CAR T cells localize in the tumor microenvironment (TME) and allowing for controlling the spatial and temporal release of IL-12 in the TME.

[0142] In certain aspects, the antigen-binding domain is a single-chain variable fragment (scFv). As used herein, the terms “single-chain Fv,” “single-chain antibody,” and “scFv” refer to a single-polypeptide-chain antibody fragment that comprise the variable regions from both the heavy and light chains but lack the constant regions. Generally, a single-chain antibody further comprises a peptide linker connecting the variable heavy and variable light domains which enables it to form the desired structure to bind to antigen. In specific embodiments, scFv can also be bispecific, multispecific, human, humanized and / or synthetic.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0143] In certain aspects, the antigen-binding domain is derived from a monoclonal antibody or a nanobody. As used herein “antibody” refers to a glycoprotein which exhibits binding specificity to a specific antigen. An antibody can include naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, single-domain antibodies, bifimctional, nanobody, and humanized antibodies. An antibody often comprises a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen. Within each variable domain are three complementarity determining regions (CDR) which form loops in the heavy chain variable domain (VH) and light chain variable domain (VL) that contact the surface of the antigen. Antibodies herein also include “antigen binding portion” or fragments of the antibody that are capable of binding to the antigen. As used herein “nanobody” refers to a single-domain antibody derived from the antibodies found in camelids (such as camels, llamas, and alpacas).

[0144] In a certain aspect, the transmembrane domain comprises CD28 or CD8. In certain aspect, the activation domain comprises a co-stimulatory domain and a signaling domain CD3C or a variant thereof. In some embodiments, the co-stimulatory domain comprises CD28, 41BB, 0X40, ICOS, CD40, and combinations thereof.

[0145] In some aspects, the flexible linker includes a (G4S)n(i.e., Gly-Gly-Gly-Gly-Ser) linker, wherein n is selected from the group consisting of 1, 2, 3, 4, or 5. In an embodiment, the flexible linker is (G4S)I. In an embodiment, the flexible linker is (648)2. In an embodiment, the flexible linker is (648)3. In an embodiment, the flexible linker is (648)4. In an embodiment, the flexible linker is (648)5. However, it would be appreciated by one of ordinary skill that any suitable length of flexible linker may be used in the CAR construct.

[0146] In an aspect, the self-cleavable linker includes a specific sequence that is recognized by a cleavage mechanism. For example, the self-cleavable linker may be cleaved via proteolytic cleavage. In another example, the self-cleavable linker may be cleaved via chemical reaction. In an embodiment, the self-cleavable linker is a peptide linker. In certain embodiments, the self-cleavable linker is a Porcine Tescho virus- 1 2A derived peptide (P2A), a Thosea asigna Virus 2A derived peptide (T2A), an Equine Arteritis Virus 2A derived peptide (E2A), or a Foot-and-Mouth Disease Virus 2A derived peptide (F2A).

[0147] In an aspect, the at least one cytokine is selected from the group consisting of interleukins (ILs), interferons (IFNs), chemokines, and tumor necrosis factor (TNF). In some embodiments, the cytokine variant is a mutein. A mutein may be derived from or engineeredMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCfrom a cytokine. In some embodiments, the mutein is a modified version of a cytokine that has been engineered to alter its properties, such as activity, receptor binding, half-life, or solubility. Non- limiting examples include sushi-IL-15 muteins and IL-2 muteins.

[0148] Cytokines are a diverse group of small proteins or peptides that regulate the immune system and cell signaling. Cytokines have demonstrated therapeutic activity in, for example patients suffering from cancer, autoimmune diseases, or inflammatory diseases. However, some subjects treated with cytokine immunotherapy may develop a hyperactive immune response, known as a cytokine storm, which may result in severe symptoms, including fever, organ dysfunction and failure, hypotension, and acute respiratory distress syndrome (ARDS). As described herein, a cytokine armored macrophage-targeted CAR-T may limit or eliminate this hyperactive immune response.

[0149] Interleukins (ILs) are a subgroup of cytokines primarily produced by leukocytes (white blood cells) and play crucial roles in intercellular communication within the immune system. They are involved in various immune responses, including cell activation, differentiation, proliferation, and regulation of immune cells like T cells, B cells, and macrophages. Some interleukins promote inflammatory responses, while others are involved in immunosuppression or the resolution of inflammation.

[0150] In an aspect, the at least one cytokine is IL- 12. In an aspect, the at least one cytokine is IL-1. In an aspect, the at least one cytokine is IL-2. In an aspect, the at least one cytokine is IL-6. In an aspect, the at least one cytokine is IL- 10. In an aspect, the at least one cytokine is IL- 17. In an aspect, the at least one cytokine is IL- 18. In an aspect, the at least one cytokine is IL-15. In an aspect, the at least one cytokine is IL-9. In an aspect, the at least one cytokine is IL-11. In an aspect, the at least one cytokine is IL-3. In an aspect, the at least one cytokine is IL-4. In an aspect, the at least one cytokine is IL-5. In an aspect, the at least one cytokine is IL-7. In an aspect, the at least one cytokine is IL-8. In an aspect, the at least one cytokine is IL-9. In an aspect, the at least one cytokine is IL-13. In an aspect, the at least one cytokine is IL- 19. In an aspect, the at least one cytokine is IL-20. In an aspect, the at least one cytokine is IL-22. In an aspect, the at least one cytokine is IL-23. In an aspect, the at least one cytokine is IL-24. In an aspect, the at least one cytokine is IL-26. In an aspect, the at least one cytokine is IL-27. In an aspect, the at least one cytokine is IL-28. In an aspect, the at least one cytokine is IL-29. In an aspect, the at least one cytokine is IL-30. In an aspect, the at least one cytokine is IL-31. In an aspect, the at least one cytokine is IL-32. In an aspect, the at least one cytokine is IL-33. In an aspect, the at least one cytokine is IL-34. In an aspect, the at least one cytokine isMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCIL-35. In an aspect, the at least one cytokine is IL-36. In an aspect, the at least one cytokine is IL-37. In an aspect, the at least one cytokine is IL-38.

[0151] In an aspect, the cytokine variant comprises muteins derived from IL- 12, in particular in this embodiment the IL-12p40(beta) linked to IL-12p35(alpha) by a flexible linker GGGGGGS. In an aspect, the cytokine variant comprises muteins derived from IL-1. In an aspect, the cytokine variant comprises muteins derived from IL-2. In an aspect, the cytokine variant comprises muteins derived from IL-6. In an aspect, the cytokine variant comprises muteins derived from IL- 10. In an aspect, the cytokine variant comprises muteins derived from IL- 17. In an aspect, the cytokine variant comprises muteins derived from IL- 18. In an aspect, the cytokine variant comprises muteins derived from IL- 15. In an aspect, the cytokine variant comprises muteins derived from IL-9. In an aspect, the cytokine variant comprises muteins derived from IL-11. In an aspect, the cytokine variant comprises muteins derived from IL-3. In an aspect, the cytokine variant comprises muteins derived from IL-4. In an aspect, the cytokine variant comprises muteins derived from IL-5. In an aspect, the cytokine variant comprises mutcins derived from IL-7. In an aspect, the cytokine variant comprises muteins derived from IL- 8. In an aspect, the cytokine variant comprises muteins derived from IL-9. In an aspect, the cytokine variant comprises muteins derived from IL-13. In an aspect, the cytokine variant comprises muteins derived from IL- 19. In an aspect, the cytokine variant comprises muteins derived from IL-20. In an aspect, the cytokine variant comprises muteins derived from IL-22. In an aspect, the cytokine variant comprises muteins derived from IL-23. In an aspect, the cytokine variant comprises muteins derived from IL-24. In an aspect, the cytokine variant comprises muteins derived from IL-26. In an aspect, the cytokine variant comprises muteins derived from IL-27. In an aspect, the cytokine variant comprises muteins derived from IL-28. In an aspect, the cytokine variant comprises muteins derived from IL-29. In an aspect, the cytokine variant comprises muteins derived from IL-30. In an aspect, the cytokine variant comprises muteins derived from IL-31. In an aspect, the cytokine variant comprises muteins derived from IL-32. In an aspect, the cytokine variant comprises muteins derived from IL-33. In an aspect, the cytokine variant comprises muteins derived from IL-34. In an aspect, the cytokine variant comprises muteins derived from IL-35. In an aspect, the cytokine variant comprises muteins derived from IL-36. In an aspect, the cytokine variant comprises muteins derived from IL-37. In an aspect, the cytokine variant comprises muteins derived from IL-38.

[0152] Interferons (IFNs) are cytokines primarily produced in response to viral infections but also play a role in immune regulation and modulation of cell activity. They can be classifiedMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCinto three main types: Type I interferons (IFN-a and IFN-|3), Type II interferons (IFN-y), and Type III interferons (IFN-X). Interferons help control the replication of viruses and activate immune cells like macrophages and natural killer (NK) cells. They also influence antiviral defense, immune cell signaling, and inflammation.

[0153] Chemokines are a specialized subset of cytokines primarily involved in directing the migration of immune cells to specific sites within the body, a process called chemotaxis. They play a critical role in the recruitment of leukocytes to areas of infection, inflammation, and tissue injury. Chemokines are classified into four families based on the arrangement of conserved cysteine residues in their structure: CC, CXC, CX3C, and XC. They bind to specific receptors on immune cells, influencing the movement of cells to tissues that require immune surveillance or response.

[0154] Tumor Necrosis Factor (TNF) is a group of cytokines involved in systemic inflammation and primarily known for their role in inducing cell death (apoptosis), inflammation, and the regulation of immune responses. TNF-a, is produced mainly by macrophages and T cells and acts to promote inflammatory pathways. TNF is also important in the pathogenesis of various diseases, including autoimmune disorders, chronic inflammatory diseases, and cancer.

[0155] In an embodiment, the CAR construct further includes a protease cleavable linker, wherein the protease cleavable linker is downstream of the payload. In an embodiment, the protease cleavable linker is engineered to be recognized and cleaved by a protease. Nonlimiting examples of proteases include cystine proteases, serine proteases, or metalloproteinases. In some embodiments, the proteases may be selected from matriptase, legumain, matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-7 (MMP7), matrix metalloproteinase-9 (MMP9), urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase 10 (ADAMI 0), a disintegrin and metalloproteinase 17 (ADAMI 7), and membrane -type 1 matrix metalloproteinase (MT1-MMP). However, depending on the proteolytic process required to disengage the protease cleavable linker from the payload and / or CAR construct, any suitable linker may be used.

[0156] In an aspect, the CAR construct further includes an additional transmembrane region, wherein the additional transmembrane region is downstream of the protease cleavable linker. In an embodiment, the additional transmembrane region includes CD8, B7-1T (CD80 transmembrane domain), and / or CD28.

[0157] In an aspect, the CAR construct further includes a small molecule-induced degron domain, wherein the small molecule-induced degron domain is downstream of the additionalMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCtransmembrane region. In certain embodiments, the small molecule-induced degron domain includes an engineered protein or peptide sequence bound to a small molecule degron sequence. These small molecule-induced degron domains are able to selectively target a protein of interest for degradation. In an aspect, the CAR construct further comprises a peptide tag.

[0158] In certain embodiments, the present disclosure provides for a polynucleotide (including an isolated polynucleotide) which may encode a CAR construct according to an aspect of this disclosure.

[0159] The polynucleotide may be configured for administration directly, configured for administration in a composition, be encoded in one or more polynucleotides for expression in a cell, and / or may be encoded in DNA, RNA, or mRNA for administration. The polynucleotide can comprise a circular or linear structure.

[0160] In some aspects, the polynucleotide may be an expression construction. “Expression construct” refers to an engineered DNA or RNA molecule designed to drive the production of a specific protein or RNA in a host cell. It typically contains regulatory elements such as a promoter to initiate transcription, an enhancer to increase expression levels, a coding sequence for the gene of interest, and a terminator to end transcription. Additional features like selectable markers, tags for protein purification, or sequences for proper localization can also be included. In an embodiment, the expression construct is a RNA. In an alternative embodiment, the expression construct is an mRNA. In an alternative embodiment, the expression construct is an cDNA.

[0161] In some embodiments, the expression construct may have the following formula:

[0162] 5’UTR — coding region — 3’ UTR — Poly(A).

[0163] In some embodiments, the expression construct may have the following formula:

[0164] 5’UTR — signal — coding region — 3’ UTR — Poly(A).

[0165] In some embodiments, the expression construct may have the following formula:

[0166] 5’CAP-5’UTR — signal — coding region — 3’UTR — Poly(A).

[0167] In some embodiments, the expression construct may have the following formula:

[0168] 5’CAP-5’UTR — signal — coding region - localization — 3’ UTR — Poly(A).

[0169] where “UTRs” are the untranslated regions located at the 5’ and 3’ ends of an mRNA construct, and “Poly(A)” refers to the polyadenylation site of the mRNA.

[0170] As used herein “5'-CAP” (or a 5'-CAP-Structure) is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain implementations, the 5'-CAP is added using a 5'-5'-triphosphate linkage (alsoMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCnamed m7GpppN). In the context of the present disclosure, a 5' CAP structure may also be formed in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cap analogues, or a cap structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits).

[0171] Signal” refers to a suitable nucleic acid sequence which encodes a signal sequence, leader sequence, sorting sequence, in frame with and upstream of the antigenic or coding region. The signal sequence is a short peptide chain, typically found at the N-terminus of a newly synthesized protein, that directs the protein to specific cellular compartments, such as the endoplasmic reticulum (ER), mitochondria, or chloroplasts. Once the protein reaches the target organelle or membrane, the signal sequence is often cleaved off by specific proteases. In an example, the signal encodes a secretion signal and / or a helper epitope.

[0172] ‘ ‘Localization” reference to a suitable nucleic acid sequence which encodes a short peptide segment within a protein that directs the protein to its specific location within the cell, such as the nucleus, mitochondria, endoplasmic reticulum, or other organelles. For example, a nuclear localization signal (NLS) directs proteins to the nucleus, while a mitochondrial targeting sequence guides proteins to mitochondria.IV. Therapeutic Applications

[0173] One aspect of the disclosure includes an engineered cell wherein the engineered cell expressed the CAR construct. In some aspects, the cell is an immune cell. Non-limiting examples of suitable immune cells include T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cells. In some embodiments, the T cells are regulatory T cells (Tregs).

[0174] One aspect of the disclosure includes the use of the CAR construct to direct (or redirect) the specificity of an immune cell to a cancer cell, solid tumor, and / or cells characterized by the accumulation of macrophages, such as TREM2+ macrophages.

[0175] One aspect of the disclosure includes a type of cellular therapy where immune cells are engineered to express a CAR and the CAR-immune cells are administered to a subject in need. Unlike antibody therapies, CAR-immune cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control and / or inflammatory control.

[0176] One aspect of the disclosure includes a method of treating cancer in a subject in need thereof, the method including administering to the subject an effective amount of a population of CAR-immune cells.

[0177] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may include non-solidMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCtumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.

[0178] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas and can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0179] Types of cancers to be treated with the CARs of the disclosure include, but are not limited to, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0180] In certain embodiments, the disclosure provides for methods of enhancing anti-tumor activity of engineered immune cells, the method comprising administering to the subject an effective amount of a population of CAR-immune cells wherein the payload enhances the immune response to tumor cells and / or modulates the CAR construct activity

[0181] One aspect of the disclosure includes a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a subject by administering to the subject a T cell that expresses a CAR. By targeting TREM2 or FOLR2, the CAR T cells enriched in tumors and minimally in healthy tissue. With anti-TAM CAR T cells, IL- 12 armoring mediated therapeutic benefit from paracrine activity of IL- 12 on cells within the tumor.

[0182] Notably, tumor regression was maintained long after the IL-12-secreting anti-TAM CAR-T cells had contracted, indicating that continuous TAM depletion or prolonged CAR-TMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCpersistence was not required for sustained therapeutic benefit. Examination of tumors after CAR-T retraction revealed that transient, localized release of IL- 12 had durably reprogrammed the tumor microenvironment (TME). Treated tumors exhibited a persistent reduction in immunosuppressive tumor-associated macrophages (TAMs) and regulatory T cells, together with increased infiltration of cytotoxic lymphocytes and immunostimulatory macrophage populations. These findings indicate that IL- 12, in combination with selective targeting of TREM2+or FOLR2+macrophages, promotes infiltration and expansion of endogenous anti-tumor immune cells and establishes a self-sustaining immunostimulatory TME.

[0183] Consistent with this observation, an interferon-y (IFNy) / IL- 12 response signature persisted within the TME following CAR-T contraction, reflecting stable resetting of immune cross-talk networks. In particular, the treatment promoted reciprocal interactions between CXCL9+and IL1B+macrophages and activated CD8+T cells, wherein macrophages provided chemoattractants and activation signals, including CXCL9, CD40, CD80, and IL- 18, while CD8+T cells supplied IFNy to reinforce the immunostimulatory macrophage phenotype. The increased abundance of macrophages in CAR-treated tumors further suggests recruitment from circulating monocytes rather than polarization of tissue-resident macrophages.

[0184] For example, in an in-vivo experiment with cleavable membrane -bound IL- 12 anti-TREM2 CAR-T cells (FIGs. 2A-2C), lower toxicity was observed despite the higher dose treatment, as indicated by no decrease in weight compared to the IL-12.aTREM2.CAR. Notably, the cleavable IL- 12 configuration maintained anti-tumoral efficacy comparable to constitutively secreting IL- 12 anti-TREM2 CAR-T cells, thereby improving the therapeutic window.

[0185] Mechanistic analyses demonstrated that a substantial component of the anti-tumor effect was dependent on FAS signaling. Deletion of FAS from tumor cells markedly reduced therapeutic efficacy, despite the presence of cytotoxic T cells. While antigen-dependent T-cell killing typically relies on perforin- and granzyme-mediated pathways, T cells can also mediate killing through FAS-FASL interactions, including bystander killing. These findings suggest that IL-12-driven upregulation of FAS on tumor cells, together with recruitment and activation of endogenous T cells, enables FAS-mediated tumor clearance. This mechanism provides a plausible explanation for effective control of tumors with low antigenicity or low mutational burden and supports the applicability of the approach to cancers that are otherwise poorly responsive to antigen-directed immunotherapies.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0186] In addition to T-cell-mediated effects, immunostimulatory macrophages induced by IL-12-armored anti-TAM CAR-T cells may directly contribute to tumor clearance. These macrophages expressed high levels of CYBB (GP91-PHOX), associated with reactive oxygen species production, and may mediate cytotoxicity through cytokine release, phagocytic mechanisms, or other innate effector functions. Further investigation is warranted to delineate the relative contributions of T-cell-dependent and myeloid-mediated tumor killing.

[0187] Safety considerations remain paramount for CAR-T therapies, particularly those incorporating potent cytokines such as IL- 12. At the doses evaluated, no clear evidence of systemic toxicity was observed. To further enhance safety, the inventors incorporated a drug-dependent destabilization domain into IL- 12 as a controllable safety switch. This strategy reduced baseline IL- 12 activity, enabled higher CAR-T dosing without concomitant toxicity, and resulted in complete tumor clearance with long-term survival in preclinical models. While the destabilization domain approach provides an effective safety mechanism, alternative or complementary strategics — such as tumor-microcnvironmcnt-triggcrcd cytokine release, membrane-bound cytokines, NFAT-regulated expression systems, or inducible suicide genes — may also be employed to further optimize safety and clinical translatability.

[0188] FOLR2. CAR-T localize in tumor lesions, kill FOLR2+macrophages, and have anti-tumoral effect

[0189] The inventors sought to utilize FOLR2-directed CAR-T cells as a delivery vehicle for immunostimulatory factors within the tumor microenvironment (TME), where FOLR2-positive macrophages are located. To determine whether FOLR2. CAR-T cells selectively home to tumors and deplete FOLR2 -positive tumor-associated macrophages (TAMs), an advanced-stage ovarian cancer model was employed. Specifically, an aggressive variant of the ID8 ovarian cancer cell line (ID8-BV), containing a breal deletion, was used. Upon intraperitoneal (i.p.) administration, ID8-BV cells formed disseminated tumors throughout the peritoneal cavity, and the associated immunosuppressive TAM population expressed FOLR2, as shown in FIG. 3 A.

[0190] An anti-FOLR2 CAR construct (aFOLR2.CAR) was generated, along with a control vector encoding green fluorescent protein (GFP), as illustrated in FIG. 3B. Surface expression and assembly of the aFOLR2.CAR on transduced CD8+T cells were confirmed (FIG. 3C). The aFOLR2. CAR-T cells were functionally activated upon co-culture with FOLR2 -positive target cells, as evidenced by interferon-y (IFNy) production, and mediatedMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcytotoxicity against FOLR2-expressing macrophages. This included efficient killing of FOLR2 -positive macrophages isolated from ascites fluid of ID8 ovarian tumor-bearing mice (FIG. 3D and FIGs. 4A-4C).

[0191] When aFOLR2.CAR-T cells were administered to mice bearing ID8 ovarian tumors, in which FOLR2 expression was restricted to TAMs and absent from cancer cells (FIG. 3E and FIG. 4F), the transferred CAR-T cells underwent in vivo expansion following mild total body irradiation (TBI) (FIG. 4D). Consistent with antigen specificity, FOLR2 -positive macrophages were selectively depleted within the tumor and peritoneal lavage, whereas FOLR2 -negative macrophages were preserved (FIGs. 3G and 3H).

[0192] To assess tumor homing capability, luciferase-expressing CD8+T cells were isolated and transduced with either the aFOLR2.CAR or the control vector. RAGl_ / _ mice were subcutaneously implanted with ID8-BV tumor cells lacking luciferase expression, with subcutaneous implantation selected to enhance luminescence detection. Tumor analysis confirmed the presence of abundant FOLR2 -positive macrophages (FIG. 4E). Within three days of adoptive transfer, aFOLR2. CAR-T cells demonstrated significant accumulation within tumor lesions, with minimal to no detection in healthy tissues (FIG. 31 and FIG. 4F).

[0193] The anti-tumoral efficacy of aFOLR2. CAR-T cells was further evaluated in an immunocompetent ovarian cancer model. Mice received an intraperitoneal injection of 5 x 106ID8-BV cells expressing luciferase (ID8-BVluc). Eight days later, mice underwent lymphodepletion via total body irradiation and were administered 1.5 x 106aFOLR2. CAR-T cells or control T cells. Treatment with aFOLR2.CAR-T cells resulted in a modest delay of survival compared to controls (FIG. 3 J), consistent with prior reports of anti-FOLR2 CAR-T therapy.

[0194] The ability of aFOLR2.CAR-T cells to deplete FOLR2 -positive macrophages was also evaluated in an orthotopic pancreatic ductal adenocarcinoma (PDAC) model. Mice were implanted with 5 x 104KPC tumor cells (KrasG12D; Trp53R172H) into the pancreas (FIG.3K). Following total body irradiation, 1.5 x 106aFOLR2.CAR-T cells or control T cells were administered. Ten days post-treatment, overall tumor burden did not significantly differ between groups; however, a reduction of approximately 75% in FOLR2 -positive macrophages was observed within pancreatic tumors. These findings indicate that aFOLR2. CAR-T cells trafficked efficiently to tumors and selectively eliminated FOLR2-expressing macrophages even within poorly vascularized tumor tissue (FIG. 3K).Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0195] Collectively, these results demonstrate that aFOLR2.CAR-T cells selectively home to tumor sites and effectively deplete FOLR2 -positive macrophages, while exhibiting modest direct anti-tumoral activity.

[0196] Armored Anti-FOLR2 CAR-T Cells Secrete Functional Cytokine Payloads but Exhibit Dose-Limiting Toxicity or Reduced Efficacy at Higher Doses.

[0197] The inventors reasoned that the selective homing of anti-FOLR2 chimeric antigen receptor (CAR) T cells to tumor-associated macrophages (TAMs) could be exploited as a means for targeted delivery of pro-inflammatory cytokines into the tumor microenvironment (TME). To evaluate this approach, a panel of “armored” CAR vectors was generated, each encoding an anti-FOLR2 CAR (aFOLR2.CAR) in combination with a cytokine payload, including interferon-a (IFNa), interferon-y (IFNy), interleukin- 12 (IL- 12, expressed as an IL-12p40 / IL-12p35 fusion), or sushi-interleukin- 15 (sushi-IL-15) (FIG. 5A). These cytokines were selected based on their known anti-tumor activity, as well as their documented systemic toxicity when administered exogenously, making them suitable candidates for localized delivery via anti-TAM CAR-T cells.

[0198] CD8+T cells were transduced with each armored CAR construct (FIGs. 4G-4H). In co-culture assays, IFNa-, IFNy-, IL-12-, and sushi-IL-15-expressing aFOLR2. CAR-T cells lysed greater than 90% of FOLR2+macrophages (FIGs. 5B and 5C). All armored CAR-T cell populations exhibited activation upon target engagement, as evidenced by increased frequencies of T cells producing IFNy, tumor necrosis factor-a (TNF ), and granzyme B relative to control T cells (FIGS. 5B-5C and S1I-S1J). Among the tested constructs, the IL-12-expressing CAR induced particularly robust IFNy production by the CAR-T cells (FIG. 5B). Functional assays confirmed that each armored CAR-T cell type secreted its respective cytokine payload with the expected biological activity (FIGs. 5D-5E and 4K-4L).

[0199] The therapeutic efficacy and tolerability of the armored aFOLR2. CAR-T cells were next evaluated in an ovarian cancer model. Immunocompetent mice were intraperitoneally injected with 5x106ID8-BVluc tumor cells. Tumor engraftment was confirmed by bioluminescence imaging eight days post- inoculation, after which animals were randomized into treatment groups. Mice received 2 Gy total body irradiation followed by intraperitoneal administration of 1.5x106CAR-T cells or control T cells. Treatment withIFNa. aFOLR2. CAR-T or IFNy. aFOLR2. CAR-T cells did not result in improved survival relative to controls, indicating a lack of therapeutic benefit under these conditions (FIG. 5F).Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0200] In contrast, mice treated with sushi-IL-15.aFOLR2.CAR-T or IL-12. aFOLR2.CAR-T cells exhibited accelerated mortality compared to control-treated animals, consistent with severe treatment-related toxicity (FIG. 5F). In both groups, mice experienced significant weight loss, and expansion of CAR-T cells in peripheral blood exceeded 50% of CD8+T cells, compared to approximately 20% observed with non-armored aFOLR2. CAR-T cells (FIGs. 6A-6B and 6D).

[0201] For sushi-IL-15.aFOLR2.CAR-T cells, omission of lymphodepletion did not mitigate toxicity (FIG. 6C). Reducing the administered dose to 1.5 xlO5cells decreased toxicity; however, this lower dose failed to produce measurable anti-tumor activity (FIG. 5G). These results indicate that, in this configuration, sushi-IL-15-expressing aFOLR2. CAR-T cells exhibit a narrow therapeutic index, limiting their utility as an effective and tolerable treatment.

[0202] Low-Dose IL-12 Anti-TAM CAR-T Cells Achieve Long-Lasting Survival in Ovarian Cancer Models

[0203] The inventors next evaluated whether eliminating lymphodcplcting preconditioning and reducing the administered dose of IL-12. aFOLR2. CAR-T cells would reduce treatment-associated toxicity while preserving therapeutic efficacy. In parallel, an alternative strategy was explored to reduce IL- 12 output from the CAR vector. To this end, a chimeric IL- 12 construct incorporating a destabilization domain (DD) was generated (FIG. 7A). The destabilization domain permits translation of the fused protein while promoting its proteasomal degradation, thereby reducing steady-state protein levels. This enhanced degradation can be reversibly inhibited by administration of a small-molecule ligand, Shield- 1, enabling pharmacologic modulation of cytokine output without affecting CAR expression.

[0204] The DD.IL-12.aFOLR2.CAR construct retained the ability to produce IL-12 and to induce interferon-y (IFNy) production in T cells; however, IL- 12 protein levels were approximately 75% lower than those produced by IL-12.aFOLR2.CAR (FIGs. 8C and 9B).Consistent with reduced basal cytokine secretion, a 90% reduction in IFNy+T cells was observed when DD.IL-12.aFOLR2.CAR-T cells were cultured in the absence of target cells (FIGs. 8B and 8D). Upon co-culture with FOLR2+cells, IFNy+T-cell frequencies were reduced by approximately 50-65% (FIGs. 8B and 8E), indicating that CAR-mediated activation enhanced IL- 12 output even in the presence of the destabilization domain.Administration of Shield- 1 resulted in a dose-dependent increase in both IL- 12 protein levelsMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCand IFNy+T-cell frequencies (FIGs. 8D and 8E), confirming functional regulation of cytokine expression by the DD / Shield-1 system.

[0205] Wild-type mice were intraperitoneally injected with ID8-BVluc ovarian tumor cells and, eight days later, were treated with IxlO6IL-12.aFOLR2.CAR-T cells,DD.IL-12.aFOLR2.CAR-T cells, or control T cells without lymphodepleting preconditioning. Mice treated with DD.IL-12.aFOLR2.CAR-T cells exhibited complete tumor remission, with all but one animal surviving beyond 100 days post-treatment and no measurable loss of body weight, indicating favorable tolerability (FIGs. 7C and 7D). Mice treated withIL-12.aFOLR2.CAR-T cells at the same dose and without lymphodepletion demonstrated a similar survival benefit; however, several animals experienced body-weight loss and one animal died ten days after treatment, indicating residual toxicity.

[0206] Tissue distribution of DD. IL-12. aFOLR2.CAR-T cells and IL-12.aFOLR2.CAR-T cells was evaluated at four and eleven days post-treatment. Minimal infiltration was detected in the lungs or brain, and modest accumulation was observed in the liver at day four, which resolved by day eleven (FIGs. 8F-8I). Scrum alanine transaminase (ALT) levels, a marker of hepatotoxicity, were not elevated between treatment groups (FIG. 8K). A small expansion of CAR-T cells was detected in the spleen of mice treated with IL-12.aFOLR2.CAR-T cells, but not in those treated with DD.IL-12.aFOLR2.CAR-T cells (FIG. 8 J). Transient infiltration of CAR-T cells was observed in the omentum, with minimal residual CAR-T cells detectable by day eleven post-treatment.

[0207] Because IL-12.aFOLR2.CAR-T cells demonstrated comparable efficacy but evidence of toxicity at the 1x106-cell dose, the inventors further evaluated whether reducing the dose could improve tolerability. Ovarian tumor-bearing mice were treated with 0.1 MO6IL-12.aFOLR2.CAR-T cells without preconditioning. No loss of body weight was observed, indicating reduced toxicity (FIG. 7F). By 27 days post-treatment, tumor burden was reduced by approximately 5- to 9-fold, as measured by bioluminescence imaging (FIG. 7G). Tumor regression was durable, with treated mice exhibiting a median survival of 102 days, compared to 43 days for control-treated mice (FIG. 7H). This marked survival benefit was observed despite the aggressive nature of the tumor model, the low CAR-T cell dose administered, and the lack of responsiveness of this model to immune checkpoint blockade. Collectively, these results demonstrate that IL-12-armored anti-FOLR2 CAR-T cells can mediate sustained tumor clearance at low cell doses without overt toxicity.

[0208] As an additional comparison, a vector encoding IL- 12 driven by a cytomegalovirus (CMV) promoter and lacking a CAR domain (IL-12.noCAR) was generated (FIG. 9A). CD8+Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCT cells transduced with this construct produced IL- 12 at levels lower thanIL-12.aFOLR2.CAR-T cells but comparable to DD. IL-12. aFOLR2.CAR-T cells. The frequency of IFNy+T cells induced by IL- 12.noCAR was similar to that observed with IL-12.aFOLR2.CAR-T cells, indicating comparable biological activity (FIG. 9C).IL-12.noCAR-transduced T cells did not deplete FOLR2+cells (FIG. 9D).

[0209] Immunocompetent mice bearing ID8-BVluc tumors were treated with transduced CD8+T cells following confirmation of equivalent tumor burden by bioluminescence imaging. IL-12.aFOLR2.CAR-T cells induced tumor regression (FIG. 9E) and extended survival for several months (FIG. 9F). In contrast, administration of IL-12-expressing T cells lacking a CAR had no measurable effect on tumor growth or survival, consistent with prior observations that IL- 12 expression alone is insufficient to control ID8 tumor growth in the absence of CAR-mediated targeting of cells within the tumor microenvironment.

[0210] Low-Dose IL-12.aFOLR2.CAR-T Cells Do Not Result in Hepatotoxicity

[0211] Although folate receptor beta (FOLR2)-positive macrophages are highly enriched within tumors, such cells can also be present in non-tumor tissues. To assess the impact of anti-FOLR2 CAR-T cells outside the tumor microenvironment, mice were administered IxlO5IL-12.aFOLR2. CAR-T cells or control T cells. This construct was selected for further characterization because it produced a strong therapeutic response at low dose without acute lethality, rendering it particularly suitable for translational applications.

[0212] At ten days post-treatment, an increase in spleen size and weight was observed (FIG.71; FIG. 9G). This splenomegaly was primarily attributable to expansion of endogenous T cells, as evidenced by an increase in CD45.2+T cells (FIG. 9G). Notably, only minimal numbers of CAR-T cells were detected in the spleen (FIG. 9G). At later time points, spleen size returned to baseline, indicating resolution of the transient splenomegaly (FIG. 7J).

[0213] In the liver, no reduction in Kupffer cells, which express FOLR2, was observed at either four or ten days post-treatment. Similarly, no significant increase in endogenous CD8+T cells was detected at these time points (FIG. 7K; FIGs. 9H and 91). Importantly, serum alanine transaminase (ALT) levels, a marker of hepatotoxicity, were not significantly elevated in treated animals relative to controls (FIG. 7L). These findings indicate that administration of low-dose IL-12.aFOLR2. CAR-T cells does not induce measurable hepatotoxicity.

[0214] IL-12.aFOLR2.CAR-T Cells Delay Pancreatic Tumor Growth

[0215] To evaluate therapeutic activity in an additional solid tumor context, 5x104KPC pancreatic cancer cells were orthotopically implanted into the pancreas of immunocompetent mice. Seven days later, animals were treated with 0.1x106control T cells orMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCIL-12. aFOLR2.CAR-T cells administered either intraperitoneally or intravenously. Both routes of administration resulted in significant anti-tumor activity, with survival extended by more than one week in this highly aggressive pancreatic cancer model (FIG. 7M).

[0216] These results demonstrate that IL-12.aFOLR2.CAR-T cells are capable of mediating therapeutic benefit within the pancreatic tumor microenvironment, including following systemic administration. The observed efficacy further supports the ability of anti-TAM CAR-T cells to localize to tumors and exert anti- tumoral effects beyond the site of injection, although additional optimization may further enhance therapeutic outcomes.

[0217] IL-12.aFOLR2.CAR-T Cells Reset and Reprogram Tumor-Associated Macrophages to an Immunostimulatory State.

[0218] IL-12.aFOLR2.CAR-T cells extended survival without directly targeting cancer cells. To elucidate the mechanisms underlying this effect, the tumor microenvironment (TME) of ovarian tumors treated with IxlO5control T cells or IL-12. aFOLR2.CAR-T cells was examined. Ten days post-treatment, tumor-bearing omentum was harvested and analyzed using Xcnium spatial transcriptomics (ST) with a customized 480-gcnc panel designed for TME characterization. Following data processing, 492,584 cells were clustered and annotated based on canonical cell-type markers.

[0219] A marked reduction in cancer cells was observed in tumors treated withIL-12.aFOLR2.CAR-T cells. Control-treated tumor sections contained 29,414 cancer cells, representing approximately 15% of the tumor mass, whereas IL-12.aFOLR2.CAR-T-treated tumor sections contained only 1,021 cancer cells, accounting for less than 0.3% of total cells (FIG. 10A and FIG. 11A).

[0220] Substantial differences in TME composition were evident between control-treated and CAR-T-treated tumors. Most notably, IL-12.aFOLR2.CAR-T treatment resulted in a significant increase in T cells and myeloid cells, accompanied by a reduction in stromal components, including fibroblasts and endothelial cells, as well as plasma cells (FIG. 10A and FIGs. 11A and 11B). Neutrophil abundance remained largely unchanged and constituted a minor fraction of the TME.

[0221] Given that tumor-associated macrophages (TAMs) were the intended CAR target, the macrophage compartment was examined in detail. Five macrophage clusters were identified and annotated based on established markers (FIG. 10B). These included Folr2Ahigh macrophages, characterized by expression of immunosuppressive genes such as 1110 and Tgfb, and Cxcl9+macrophages, which expressed immunostimulatory genes associated with antigen presentation (Tapi, Psmb8), T-cell recruitment (Cxcl9, Ccl5), and T-cell activationMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC(1118). Folr2Ahigh macrophages were nearly completely eliminated followingIL-12.aFOLR2.CAR-T treatment, decreasing from 4,427 cells in control-treated tumors to 58 cells in CAR-T-treated tumor sections (FIGs. 10C-10E and FIG. 11C). In contrast, Folr2Alow macrophages were less efficiently depleted, suggesting a threshold level of antigen expression is required for IL-12.aFOLR2.CAR-T-mediated clearance (FIGs. 10C-10E).

[0222] Concomitant with depletion of Folr2Ahigh macrophages, there was a pronounced expansion of Cxcl9+macrophages, which constituted more than 70% of macrophages within the CAR-T-treated TME (FIGs. 10C and 10D). These macrophages exhibited a robust IL-12 / IFNy transcriptional signature, including genes involved in antigen presentation and T-cell recruitment (FIGs. 10E and 10F and FIGs. 11C-11G). Cxcl9+macrophages also expressed elevated levels of Cybb, encoding gp91-phox, a key component of reactive oxygen species production (FIG. 10B). A closely related population of Illb+macrophages emerged in CAR-T-treated tumors and was largely absent from control-treated tumors (FIG. 10C and FIG. 11D). These Illb+macrophages shared transcriptional features with Cxcl9+macrophages but exhibited increased expression of Tnf and Cd40. Both macrophage populations demonstrated upregulated Cd274 (PD-L1), consistent with elevated IFNy levels in the TME (FIG. 11G).

[0223] At the periphery of Cxcl9+macrophage clusters, a small population of Folr2_ macrophages expressing Argl was detected (FIGs. 10H and 101 and FIG. 11F). Analysis of the relative abundance of immunostimulatory versus immunosuppressive macrophages revealed a marked shift following IL-12. aFOLR2.CAR-T treatment, with the ratio increasing from approximately 0.5 in control-treated tumors to 44 in CAR-T-treated tumors (FIGs. 10C and lOF and FIG. 11B). This observation is notable, as an increased ratio of CXCL9+immunostimulatory macrophages to SPP1+immunosuppressive macrophages has been associated with improved prognosis in multiple human cancers.

[0224] Reprogramming of the macrophage compartment was further confirmed by protein-level analyses, including immunostaining, multiplex imaging of omental tissue sections, and flow cytometric analysis of peritoneal lavage and tumor-bearing diaphragms (FIGs. 12A-12G). Consistent with spatial transcriptomics findings, IL-12.aFOLR2.CAR-T treatment resulted in depletion of FOLR2+macrophages. Residual FOLR2 staining detected by immunohistochemistry likely corresponded to Folr2Alow macrophages, as immunohistochemical methods do not provide quantitative resolution. Depletion of FOLR2+macrophages was accompanied by an overall increase in macrophage abundance, including an approximately three-fold increase in MHCIIAhigh CD80+macrophages (FIGs. 12A-12C),Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcorresponding to the Cxcl9+and II 1 b+macrophage populations identified by spatial transcriptomics. Similar macrophage reprogramming was observed in both the omentum and diaphragm, two major sites of ovarian tumor growth.

[0225] Collectively, these results demonstrate that IL-12.aFOLR2.CAR-T cells induce a profound and sustained reprogramming of the myeloid compartment within the tumor microenvironment, shifting it from an immunosuppressive state to an immunostimulatory state conducive to durable anti-tumor immunity.

[0226] Recruitment and Activation of CD8 T Cells in the Tumor Microenvironment by IL-12.aFOLR2. CAR-T Cells

[0227] IL-12-armored anti-FOLR2 CAR-T cells (IL-12.aFOLR2.CAR-T) promote robust recruitment, expansion, and activation of endogenous CD8+T cells within the tumor microenvironment (TME). Spatial transcriptomics (ST) analysis demonstrated that the remodeling of the myeloid compartment induced by IL-12.aFOLR2.CAR-T treatment was accompanied by pronounced alterations in the lymphoid compartment of treated tumors (FIG. 13A, FIG. 13B).

[0228] Specifically, CAR-treated tumors exhibited a marked increase in activated CD8+T cells, with a greater than seven- fold elevation compared to control-treated tumors. Activated CD8+T cells comprised approximately 80% of total T cells within the TME following CAR-T treatment. This expansion was accompanied by a concomitant reduction of regulatory T cells (Tregs), which were decreased by approximately 76% relative to controls (FIG. 13B).The activated CD8+T cell population displayed increased expression of interferon gamma (Ifng) and granzyme B (Gzmb) (FIG. 13C, FIG. 13D, FIG. Ill), molecular signatures consistent with enhanced cytotoxic effector function. These findings align with the known biological activity of IL- 12 signaling, which promotes T cell activation and IFN-y production.

[0229] In addition to increased effector activity, ST analysis revealed a substantial expansion of CD8+T cells with memory phenotypes. Effector memory T cells increased approximately 17-fold and comprised approximately 8% of the activated effector CD8+population (FIGs.11J-11K). Furthermore, stem-like memory CD8+T cells increased by approximately 50% in CAR-treated tumors (FIG. 13A, FIG. 13B). In contrast, natural killer (NK) cell frequencies were reduced in CAR-treated mice according to ST analysis (FIG. 13B).

[0230] These results demonstrate that IL-12.aFOLR.CAR-T promotes a reprogramming of the ovarian TME, including significant infiltration / expansion of endogenous activated cytotoxic T cells along with Cxcl9+macrophages. As the TME shift persisted after CAR-TMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcontraction, these results suggest that elimination of FOLR2+ macrophages and delivery of IL- 12 can induce a self-sustaining IL-12 / IFNy program in the tumor.

[0231] Orthogonal Validation ofT Cell Remodeling by Imaging and Flow Cytometry

[0232] Multiplex immunofluorescence imaging of omental tumors, combined with flow cytometric analysis of diaphragm tissue and peritoneal lavage, confirmed the CAR-induced remodeling of the T cell compartment. These analyses demonstrated an approximately eight- fold increase in endogenous CD8+T cells following IL-12.aFOLR2.CAR-T treatment, with a substantial fraction expressing Granzyme B, indicative of cytotoxic effector differentiation (FIG. 12D). A corresponding decrease in FOXP3+regulatory T cells was also observed (FIG. 12E).

[0233] Notably, the expanded CD8+T cell population consisted almost entirely of endogenous T cells rather than CAR-T cells, as indicated by the absence of GFP expression, which is encoded by the CAR vector. By day 10 post-treatment, CAR-T cells had contracted to less than 1% of CD8+T cells within the peritoneal cavity (FIG. 12D). These findings indicate that IL-12. aFOLR2.CAR-T cells act as transient immune modulators that promote sustained endogenous anti-tumor immunity rather than persisting as the dominant effector population.

[0234] Consistent with a pro-inflammatory TME shift, CAR-treated mice displayed a reduced CD4:CD8 T cell ratio and increased expression of activation markers CD69 and KLRG1 on endogenous CD8+T cells. These observations correlated with the presence of a memory-like T cell cluster identified by ST analysis in the omentum (FIG. 13B, FIG. 11J-K, FIG. 12F, FIG. 12G).

[0235] NK cell reduction was independently confirmed by flow cytometry in peritoneal lavage samples (FIG. 14A). Furthermore, depletion of NK cells in ovarian tumor-bearing mice did not abrogate the therapeutic benefit of IL-12. aFOLR2. CAR-T treatment, as treated animals still achieved long-term survival (FIG. 14B), demonstrating that NK cells are not required for CAR-mediated tumor control.

[0236] Collectively, these data establish that IL-12.aFOLR2.CAR-T cells induce durable reprogramming of the ovarian TME, characterized by substantial infiltration and expansion of endogenous cytotoxic CD8+T cells in concert with Cxcl9+immunostimulatory macrophages. Persistence of this immunologic shift following CAR-T contraction suggests that elimination of FOLR2+macrophages combined with IL- 12 delivery initiates a self-sustainingIL-12 / IFN-y signaling axis within the tumor.

[0237] IL-12.aFOLR2.CAR-T Cells Promote Expansion of Cancer-Reactive CD8+T CellsMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0238] The expansion of endogenous CD8+T cells, including populations with memory phenotypes, together with prolonged animal survival after CAR-T contraction, suggested induction of tumor-specific adaptive immunity. This conclusion was supported by ST data showing enrichment of cancer cells directly juxtaposed with Gzmb+CD8+T cells inIL-12. aFOLR2. CAR-treated tumors (FIG. 15A), a spatial arrangement indicative of antigen-dependent cytotoxic engagement.

[0239] To directly assess tumor reactivity, ovarian tumor-bearing mice were treated with 1.5xl05IL-12.aFOLR2. CAR-T or control T cells. Eleven days post-treatment, splenic CD8+T cells were isolated and co-cultured with irradiated ID8 tumor cells. IFN-y ELISPOT analysis demonstrated only background reactivity in control samples, whereas CD8+T cells from CAR-treated mice produced robust IFN-y responses (FIG. 15C). Flow cytometric confirmation revealed a greater than three-fold increase in CD44+IFN-y+CD8+tumor-reactive T cells in IL- 12. aFOLR2. CAR-treated animals (FIG. 15D). These findings demonstrate that IL-12-armored anti-TAM CAR-T cells promote functional education and expansion of endogenous tumor-specific T cells.

[0240] Partial Dependence of Tumor Control on Cancer Cell FAS Expression

[0241] Although endogenous CD8+T cells in treated tumors expressed Granzyme B, additional mechanisms of tumor cell vulnerability were identified. Flow cytometry and ST analysis revealed upregulation of FAS, MHC-I, and PD-L1 on tumor cells followingIL-12.aFOLR2.CAR-T treatment (FIG. 15E, FIG. 14C). Given prior evidence that antigen-specific T cells mediate tumor killing via FAS-dependent mechanisms, including bystander killing, the functional role of FAS was evaluated.

[0242] ID 8 tumor cells lacking FAS expression (ID8AFas- / --BVluc) were generated and implanted intraperitoneally into immunocompetent mice. Following treatment withIL-12.aFOLR2.CAR-T cells, therapeutic efficacy was markedly reduced relative to control ID8 tumors (FIG. 15F, FIG. 14D). These results indicate that IL-12 anti-TAM CAR-T therapy promotes FAS upregulation on cancer cells and that tumor control is, at least in part, FAS-dependent.

[0243] IL-12-Armored Anti-TREM2 CAR-T Cells in Metastatic Lung Cancer

[0244] TREM2 is another macrophage-enriched target expressed by immunosuppressive tumor-associated macrophages (TAMs) across multiple cancers. In the KP lung cancer model, TREM2+macrophages are abundant and suppressive. Accordingly, CAR constructs targeting TREM2, with or without IL-12 armoring (aTREM2.CAR and IL-12.aTREM2.CAR), were developed (FIG. 16A). CAR expression was confirmed in transduced mouse CD8+T cellsMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC(FIG. 16B), and cytotoxic activity against TREM2+macrophages was verified in vitro (FIG.16C, FIG. 16D).

[0245] Therapeutic efficacy was evaluated using the KPI lung cancer model. KPI tumor cells were confirmed to lack TREM2 expression prior to treatment (FIG. 16C, FIG. 14E).High-dose aTREM2.CAR-T cells with lymphodepletion provided only modest survival benefit (FIG. 16E). In contrast, low-dose IL-12.aTREM2.CAR-T cells administered without pre-conditioning significantly reduced tumor burden, with treated mice exhibiting less than 5% lung involvement compared to greater than 30% in controls (FIG. 16F). This reduction translated into a survival extension of approximately two weeks (FIG. 16G).

[0246] Analysis of lungs at day 10 post-treatment revealed a decreased CD4 / CD8 ratio and increased CD69 expression on endogenous CD8+T cells, indicative of activation (FIG. 161).Macrophages exhibited increased MHC-II and CD80 expression, consistent with an immunostimulatory phenotype. Tumor cells displayed elevated FAS expression, reinforcing the mechanistic link between IL- 12 anti-TAM CAR-T treatment and enhanced susceptibility to T cell-mediated killing (FIG. 161).

[0247] These findings demonstrate that IL-12-armored anti-TAM CAR-T cells targeting TREM2 effectively remodel the lung tumor microenvironment and provide therapeutic benefit in disseminated solid tumor models.

[0248] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLES

[0249] Example 1: Cloning of CAR constructs

[0250] CAR T constructs were cloned into the vector from a previous study originally based on an MSGV CD28-CD3z all ITAM intact (Addgene #107226) including a GFP sequence separated by a T2A self-cleaving sequence (MSGV vector). The rat anti-mouse FOLR2 sequence (CLIO) of the heavy and light chain portion of the antibody variable domain was synthesized by GenArt (Gene Synthesis). The aFOLR2.CAR vector was constructed byMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCstepwise Gibson Assembly (New England Biolabs #E2611S) using the cDNA of the heavy and light chain were separated by a 3xGGGGS linker.

[0251] The heavy and light chain of a mouse TREM2 antibody sequences were provided by the Colonna Lab as part of a collaboration agreement with Mount Sinai and each variable chain domain were synthetized by Twist Bioscience, CA, USA. The heavy and light chain were cloned into aFOLR2.CAR vector using Nsil-HF (NEB #R3127S) and Xhol (NEB #R0146S) replacing aFOLR2 scFV by the TREM2 scFV light and heavy chains separated by a 4x GGGGS linker. A Myc-tag sequence (EQKLISEEDL) was added on the C terminal of the anti-TREM2 scFV sequence. GFP and CAR expression was verified by flow cytometry, staining the anti-FOLR2 CAR using an anti-rat and the anti-TREM2 CAR through identification of Myc-tag positive cells.

[0252] IFNa, IFNy, was sequenced using Uniprot in mus musculus species respectively P01580, P48346 with their endogenous signal peptide. IL-12p70 (IL- 12) sequence is derived from the fusion of IL-12p40 unit (sub-unit Beta) with its endogenous signal peptide, the GGGGGGS flexible linker and the IL-12p35 unit (Sub-unit Alpha) as described in Lieschke G et al. Sushi-IL-15 sequence was created using the sushi domain (or IL-15Ralpha binding domain) sequence from UniProt (P01658) with a linker used in Mortier E et al. cloned upstream to the IL- 15 sequence downstream of its pro-peptide domain. A mus musculus IgGk signal peptide was added to the sequence. All payload protein for armored CAR with the addition of a P2A self-cleaving sequence upstream of the N terminal were then synthetized by Twist Bioscience (CA, USA) and cloned between the CD3z domain and the T2A-GFP sequence using using Ecorl-HF (NEB # R3101S) and Hindlll-HF (NEB R3104S) fortheir respective restriction sites.

[0253] Truncated anti-FOLR2 CAR sequence was generated by truncating the heavy chain and light chain of the anti-FOLR2 sequence and was cloned into aFOLR2.CAR and IL-12. aFOLR2. CAR vectors using Nsil-HF and Xhol restriction enzyme replacing anti-FOLR2 sequence with the truncated anti-FOLR2 sequence.

[0254] For the CMV driven IL-12 vector, the inventors used pMSCVsin-PGK-GFP plasmid, a gift from Patrick Salmon (Addgene plasmid # 35615),) as backbone and inserted CMV promoter sequence from lenti retroviral vector pCCL.sin.PPT.GFP previously described with downstream of the CMV promoter, the IL- 12 sequence described above preceded by a Kozak sequence. The inventors cloned both inserts using In-Fusion Snap Assembly Master Mix (Takara #638948) as per manufacturer protocol.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0255] Using the destabilization domain described in Banaszynski L. et al, the inventors sequence the FK506 - rapamycin-binding protein FKBP with a point mutant (L106P) and use Twist Bioscience to synthetize a fused destabilization domain with IL-12p70 (DesD-IL-12 insert) flanked by restriction site Ecorl-HF and Hindlll-HF. The inventors cloned as previously described the insert replacing IL-12 sequence in the IL-12.aFOLR2.CAR vector with the fused protein DesD-IL-12 insert.

[0256] For control purposes, the inventors used a retroviral HSC1 vector coding for GFP under the EFla promoter.

[0257] Example 2: Retroviral CAR vector production

[0258] Phoenix-Eco cells were seeded into 10 or 15 cm tissue culture plates 24h prior to achieve an approximate cell density of 70% at the moment of transfection. Transfection was carried out using the calcium-phosphate method. Briefly, CAR or control plasmid constructs were suspended in O.lx TE and 0.25M CaC12; one volume of 2x HBS was added on a dropwise fashion while continually vortexing and the resulting solution was immediately added onto Phoenix-Eco cells and allowed to sit overnight. IMDM medium was replaced the next morning and supernatants collected and 0.2 mm-filtered 24-30h after that. Supernatant aliquots were stored at -80°C until use.

[0259] Example 3: T-cell transduction

[0260] CD8 T cells were isolated from mouse spleens using the EasySep™ Mouse CD8+ T Cell Isolation Kit (StemCell #19853). Activation was carried out at a cell density of IM / ml in 100 U / ml Recombinant Murine IL-2 (Peprotech #212-12). Dynabeads™ Mouse T- Activator CD3 / CD28 (ThermoFisher #11453D) were used to activate cells at a 1 :4 bead-to-cell ratio for 24h before transduction. Non-treated culture plates (NUNCTM, ThermoFisher) were coated overnight at 4°C with 20 mg / ml Retronectin (Takara #T100B). Viral supernatant was spun for 90 minutes at 2,000g and 30°C onto the plated retronectin and a third of the supernatant volume removed carefully after spinning. T cells were resuspended in fresh RPMI medium with IL2, added onto the supernatant-containing wells (final IL-2 concentration 50U / ml), and allowed to sit for 24h. After this, Dynabeads were magnetically removed, and T cells were resuspended in fresh RPMI medium at 50 U / ml IL-2. New RPMI medium containing 50 U / ml IL-2 was added daily to keep T cells at a concentration between 0.8-1.25*106 cells / ml until use, typically within 5 days of isolation. Detection of GFP by flow cytometry was employed to quantify the efficiency of transduction.

[0261] Example 4: Cloning of cell linesMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0262] Murine FOLR2 ectodomain was obtained from Uniprot (Pl 4276) and reverse codon optimized sequence. The FOLR2 ectodomain was fused with CD28 transmembrane sequence and the inventors added downstream a P2A self-cleaving sequence followed by mCherry fluorophore sequence. The resulting sequence was synthetized by Twist Bioscience and cloned into a lenti retroviral vector coding for GFP under the hPGK promoter described above (pCCL.sin.PPT.GFP).

[0263] Murine TREM2 was obtained from Uniprot (Q99NH8) and reverse codon optimized sequence was created using Twist Bioscience. The inventors introduce an S158T mutation to impair the cleavable SH peptide site in the TREM2 ectodomain and truncated the cytosolic domain deleting amino acids from 197 to 222 to impair the protein signaling domain. A P2A self-cleaving sequence followed by mCherry fluorophore sequence was added as a transduction marker. The resulting sequence was cloned into the lenti retroviral vector described above.

[0264] For the generation of ID8-BVluc, the inventors used pRC49-U6-DR-crRNA-BsmbI(x2)-6T; EFS-Puro-2A-Fluc-2A-EGFP_NLS-WPRE plasmid, a gift from Sidi Chen (Addgene plasmid #123363) (79). The plasmid has puro-2a-luciferase-2a-GFP-NLS, the inventors replaced GFP-NLS with mCherry-NLS using Kpnl (NEB # R3142S) and Mlul (NEB # R3198S) restriction enzymes and their respective restriction sites resulting in a mCherry-Luc plasmid.

[0265] All new cell line plasmid were transfected using calcium chloride transfection as described below using 293T packaging cells for retroviral particle production. Briefly the inventors seed in a 6-well plate 5x105 cells one day before transection and incubate at 37°. The day of transfection the inventors change the media, after warming it at 37°, 2 hours before transfection. The inventors add 6ug of lenti retroviral vector, and the following lenti retroviral packaging plasmid pVSV (Img), pMDLg / pRRE (2mg), pRSV-REV (Img) in 0.1 TE medium for a total of 500ul. The inventors add 2.5 M CaC12. In a volume of 50ul, vortexing, and incubate at room temp for 10 minutes. The inventors then add 500ul of 2x HBS solution (281 nM NaCl, lOOmM HEPES, 1.5 mM Na2HPO4, pH 7.05) in a dropwise manner while vortexing. The resulting mixture is added to the cells in a dropwise manner. The inventors incubate at 37° overnight. The next morning, the inventors replace with warm media and incubate at 37° for 30 hours. The inventors collect the supernatant after filtering through a 0.2mm disc filter and store at -80°.

[0266] ID8-BV and RAW264.7 were transduced with FOLR2 truncated-mCherry lenti retrovirus and the mCherry-luciferase lenti retrovirus derived from the plasmid describedMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCabove. KPI and RAW264.7 were transduced with non-cleavable and non-signaling TREM2 Lenti retrovirus.

[0267] All new cells were transduced with Cells were seeded 24 hours prior to transduction at a density of 50,000 cells per well in 6-well plates. The following day the cells were transduced with lenti retroviral vectors in the presence of 5 mg / ml polybrene (Millipore) at low multiplicity of infection (MOI), with each well transduced by the corresponding lenti retrovirus. Each cell population was select based on their mCherry+ percentage to reach 20% of total populations. Cells were sorted based on mCherry positivity to reach >99% purity using BD bioscience CSM5 sorter machine.

[0268] FAS Knock out ID8-BVluc (FAS -I- ID8-BVluc) was generated through CRISPR ribonucleoprotein (RNP) nucleofection. Using CAS9 nuclease (Synthego) and Fas sgRNA 5’-GGCGUCCCAAAGCUUACCAG-3' synthetized by Synthego mixed at room temperature with a ratio of 1 : 1.5 for 10 min and diluted in IX TE buffer to constitute Cas9: sgRNA RNP. Subsequently the resulting RNP was mixed with 400,000 ID8-BVluc cells in nucleofector solution (5:1 SF Cell Line solution: Supplement 1, Lonza cat no. V4XC-2032) in a 20pLtubc adapted for Lonza 4D-Nucleofector® X machine. Cells were electroporated using two different programs (EH-115 or CM- 138). Cells incubate at 37° for 3 days. KO efficiency was evaluated by flow cytometry using FAS antibody (see Flow antibody table) and then sorted using FAS antibody staining selecting FAS negative cells. Sorted cells were incubated for 3 days at 37°. FAS knock out was confirmed via flow cytometry using FAS antibody and by rtPCR. Cells were frozen in nitrogen for long terms storage.

[0269] Example 5: Cell culture

[0270] All cell culture media were supplemented with 10% FBS (Gibco 26140-079), 2 mM Glutamine (Gibco 25030-081) and 1% Pen / Strep (Gibco 15140-122).

[0271] Phoenix-Eco cells (ATCC CRL-3214TM) and 293T cells (ATCC CRL-3216) were cultured in supplemented IMDM medium (Gibco 12440-053)).

[0272] RAW264.7 (ATCC TIB-71), A20 and FAS- / - A20 (kindly provided by the Dr. Joshua Brody lab, Icahn School of Medicine at Mount Sinai), HKP1 (80), TREM2+ KPI (see below), FC 1245 PDAC cells (KPC cells, in this manuscript) generated from a primary tumor in a KrasLSL-G12D / +; Trp53LSL-R172H / +; Pdxl-cre mouse and kindly provided by D. Tuveson (Cold Spring Harbor Laboratory) (81) and primary mouse CD8 T cells were cultured in supplemented RPMI 1640 medium (Gibco 22400-089) plus 500 mm b-mercaptoethanol (Gibco 21985-23).Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0273] IDSBrcal -deficient overexpressing VGEF and Defensin (ID8-BV) cell line was kindly provided by Jean J. Zhao (Harvard University).

[0274] All ID8 cells (82) were cultured in supplemented RPMI plus 2500 nM Sodium Pyruvate (Coming 25-000-CI).

[0275] For killing assays, effector:target:non-target cocultures (at an 1:1:1 ratio) were cultured for 24h in 96-well culture plates in supplemented RPMI with 1 / 1000 IL-2. The inventors used alternatively as target cells FOLR2 expressing RAW264.7 cells (see above) or FOLR2 expressing ID8 cells (see above), TREM2 expressing RAW264.7 cells (see above) or TREM2 expressing KPI cells (see above), adding an equal number of A20 B cells as nontarget controls. In separate experiments, C57BI 6 CAR-T cells were cocultured with thioglycate-elicited peritoneal macrophages (see below). The inventors also cocultured C57B1 / 6 CAR-T with collected ascites from mice inoculated with ID8-BVluc. To detect cytokine secretions from our CAR T cells via flow cytometry, 4 hours before the end of the culture, the inventors added 3 mg / ml Brefeldin A (Invitrogen 00-4506-51) to the cultures. Specific lysis was calculated as “([T / NT]control_avcragc-[T / NT]samplc) / ([T / NT]control_average*100)”, with T and NT representing the number of target and nontarget cells in each sample.

[0276] In experiment using Shield- 1 (Takara 632189), the molecule was added to co-culture for 24h at ImM, 3mM, and lOrnM diluted in supplemented RPMI to reach intended concentration.

[0277] For in-vitro experiment using CAR T supernatant, the inventors cultured 5x105 CAR T cells in supplemented RPMI for 24h, spin cells at 350g for 5 minutes and collect supernatant. The inventors applied 200ul of supernatant to cells reporting functionality of secreted cytokines from CAR T cells for 24h and analysis via flow cytometry phenotype of reporting cells. As positive control the inventors apply recombinant mouse IFNy (Peprotech 315-05) at 20ng / ml concentration and mouse recombinant IL-15 (Peprotech 210-15) at 1 Ong / ml concentration.

[0278] Generation of thio-elicited macrophages was carried out as previously described (83). Briefly, a 3% solution of Thioglycolate Broth (ThermoFisher CM0173B) was prepared in distilled water, autoclaved and stored at 4°C until use. 1.5 ml of thioglycolate solution was injected intraperitoneally (i.p.) into C57B1 / 6 mice. 72h later, mice were euthanized, and peritoneal lavage was performed for co-culture with CAR or control transduced T cells or for flow cytometry (see below).

[0279] Example 6: Mouse experimentationMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0280] C57B1 / 6, CD45.1+ C57B1 / 6, Balb / c, , FVB-Tg(CAG-luc,-GFP)LG85Chco / J (Mice expressing luc-eGFP L2G85 transgene), C.129S7(B6)-RagltmlMom / J (Balb / c RAG1) female mice were obtained from Jackson laboratories (Jackson strain: 000664, 002014, 000651, 008450 and 003145 respectively). Luc-eGFP expressing mice were backcrossed with Balb / c mice and the inventors use at the 4th or later generation for our experiments.

[0281] All mice were kept in the Mount Sinai vivarium during use. All mouse experiments were carried out under institutional IACUC approval. All mice were randomized before treatment administration.

[0282] For ID8-BVluc intraperitoneal tumor inoculation, ID8 cells were trypsinized, washed twice with PBS by centrifuging cells at 350g for 5 minutes and resuspend with ice-cold sterile PBS at a 25x106cells / ml concentration. 5x106ID8 tumor cells were injected intraperitoneally into female mice in a volume of 200ul using a 27G needle.

[0283] T cells were injected intraperitoneally in a 200pl volume with a 27G needle. Number of CAR T cell varied depending on experiments as described in results and total number of T cells injected were adjusted depended on transduction level. Also depending on experiment, mice were irradiated with a 2Gy prior to T cells injections.

[0284] Mice were weighted and taken to IVIS for bioluminescence measurement twice weekly from tumor inoculation. The inventors also monitored mice visually for the development of peritoneal ascites.

[0285] In time points experiment, mice were euthanized at the indicated time points using CO2 inhalation. Omentum, diaphragm and spleens were extracted, weighed, and either processed for flow cytometry or processed for histology or MICSSS (see below).

[0286] For tumor inoculation, HKP1 cells were trypsinized, washed twice with PBS by centrifuging cells at 350g for 5 minutes and resuspend with ice-cold sterile PBS and resuspended in ice-cold sterile PBS at a concentration of 7.5xl05cells / ml. Mice were injected with 1.5x105cells into the tail vein in a volume of 200 ml using a 27G needle after being heated for 3-5 min using a heating lamp.

[0287] T cells were injected intravenously via tail vein injection in a volume of 200 pl PBS using 27G needles. The number of T cells to inject in each experimental group was adjusted for transduction level to reach the number of CAR T cells indicated in each of the experiment. Also depending on experiment, mice were irradiated with a 2Gy prior to T cells injections.

[0288] Mice were weighted and taken to IVIS for bioluminescence measurement twice weekly from tumor inoculation. In time points experiment, mice were euthanized at theMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCindicated time points using CO2 inhalation. Lungs and spleens were extracted, weighed, and processed for immunofluorescence or flow cytometry or processed for histology or MICSSS (see below).

[0289] For KPC tumor inoculation, KPC cells were trypsinized, washed twice with PBS by centrifuging cells at 350g for 5 minutes and resuspend with ice-cold sterile PBS at a concentration of 2x106cells / ml. To generate pancreatic orthotopic tumors, KPC cancer cells (50’000, 1:1 in a PBS:matrigel solution) were orthotopically grafted into the pancreas, following established protocol (84). T cells were injected intraperitoneally or intravenously via tail vein in a volume of 200 pl PBS using 27G needles. The number of T cells to inject in each experimental group was adjusted for transduction level to reach the number of CAR T cells indicated in each of the experiment. Depending on experiment, mice were irradiated with a 2Gy prior to T cells injections. In time points experiment, mice were euthanized at the indicated time points using CO2 inhalation. Primary tumors were extracted, weighed, and processed for immunofluorescence or flow cytometry or processed for histology or MICSSS (sec below).

[0290] For ID8 subcutaneous tumor inoculation, the inventors process ID8 cells similarly to ID8-BVluc and injected 2.5x106subcutaneously ID8 cells in lOOpl PBS volume on the flank of RAG1- / - mice. T cells were injected 8 days after following the same procedure than in intraperitoneal tumor inoculation.

[0291] All blood samples were drawn where indicated using maxillary vein puncture or from tail when necessary and stained for flow cytometry.

[0292] Serum was collected by first collecting blood via intracardiac puncture after euthanizing the mice. Collected blood was left at room temp for 30 minutes and spin at 2,000g for 10 minutes. Seram was transferred by collecting clear supernatant and kept at -20c until analysis. Serum analysis was performed by Center for Comparative Medicine and Surgery (CCMS) Comparative Pathology Laboratory, Mouse Blood analyses.

[0293] For all experiments, tumor cells were inoculated in mice aged between 8 and 12 weeks.

[0294] Example 7: ELISPOT

[0295] The inventors seed 2x106ID8-BVluc cells in a 15cm plate in ID8 medium supplemented with 100IU of IFNy (Peprotech 315-05)) and incubate overnight at 37°. Next day, the inventors irradiate the cells with 120Gy, the inventors collect the cells, wash twice with PBS and seed 4x104irradiated ID8-BVluc in a 96-well ELISPOT well (Abeam #ab64029). The inventors harvest the spleen of each mouse, and the inventors isolate CD8 TMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCcells as previously described. The inventors seed 8x104CD8 T cell per well mixing them to the ID8-BVluc cells. The cells were cultured for 24h at 37° and the plates were developed according to Abcam's protocol. The plate was scan and analyzed with ImmunoSpot Series 3 Analyzer (Cellular Technology Ltd).

[0296] Example 8: ELISA

[0297] For measurement of IL- 12 secretion, an enzyme-linked immunosorbent assay (ELISA) was performed on supernatants collected from CAR-T cells cultured alone or co-cultured with their respective target cells or non-target cells. CAR-T cells were seeded based on the number of positively transduced cells at a concentration of 1 xlO6cells / mL in supplemented RPMI medium as described above.

[0298] The inventors utilized a Legend Max™ Mouse IL- 12 (p70) ELISA Kit (BioLegend, Cat. No. 433607) and followed the manufacturer’s instructions. Briefly, the provided IL-12 (p70) standard was serially diluted from 200 pg / mL to 3.2 pg / mL. Collected cell culture supernatants were diluted as needed in Assay Buffer A. Samples and standards were incubated in the ELISA plate for 2 hours at room temperature with shaking at 200 rpm.

[0299] Following incubation, plates were washed with wash buffer, and 100 pL of Mouse IL-12 (p70) Detection Antibody solution was added to each well. Plates were incubated for 1 hour at room temperature with shaking at 200 rpm. After washing, 100 pL of Avidin-HRP A solution was added and incubated for 30 minutes at room temperature with shaking at 200 rpm. Plates were washed again, followed by addition of 100 pL of Substrate Solution E and incubation for 15 minutes at room temperature in the dark. The reaction was stopped by adding 100 pL of Stop Solution.

[0300] Absorbance was measured at 450 nm and 570 nm using a Polarstar Omega microplate reader with Omega software (BMG Labtech), with the 570 nm reading subtracted from the 450 nm reading. A standard curve was generated, and regression analysis was used to determine the IL- 12 concentration in each well. For diluted samples, the calculated concentration was multiplied by the corresponding dilution factor.

[0301] For IL-12 ELISA assays performed on serum samples, serum was collected as described above and analyzed using the same protocol as for cell culture supernatants, with the exception that samples were diluted in the provided Matrix A solution, in accordance with the manufacturer’s protocol for serum samples.

[0302] Example 9: IVIS

[0303] Mice were subjected to anesthesia using inhaled isoflurane and received 10 pl / g with a concentration of 15 mg / ml luciferase through intraperitoneal injection. Luciferase-based inMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCvivo bioluminescence was acquired in an IVIS Imaging System (Perkin Elmer) at the BioMedical Engineering and Imaging Institute (BMEII) at Mount Sinai. Images were taken 1 min after injection and then every 2 minutes until reaching peak bio luminescence. Images were processed using Living Image software (Perkin Elmer).

[0304] Example 10: Flow cytometry

[0305] Collected organs were minced and digested for 30 min in Collagenase IV (C5318, Sigma Aldrich) (with exception of spleen for which no digestions performed) before being mashed through a 70 mm cell strainer. Cell suspensions were spun at 350g for 5 min at 4°C. Pellets were resuspended in RBC Lysis Buffer (eBioscience 00-4300-54) for 3 minutes at room temperature to lyse red blood cells. 9 volume of FACS buffer (PBS, 5% BSA and 2 mM EDTA) was added to the cells suspension, spun at 350g for 5 min at 4°C and resuspended in flow buffer.

[0306] Single cell suspension was obtained from peritoneal cavity by injecting intraperitoneally 5 ml PBS 2mM EDTA to collect the peritoneal lavage. The lavage was RBC-lyscd and resuspended in RPMI medium containing 10 ng / ml murine M-CSF (Peprotech 315-02).

[0307] Sample were surface stain, using the mouse antibodies listed in supplemental table 1 at 4°C for 20 minutes, washed with flow buffer. Extracellular stainings were performed for 20 minutes at 4°C with mouse anti-body at a 1 / 100 to 1 / 300 dilution in flow buffer. For intracellular cytokine staining, the inventors added in the cells in culture at 37° 3 pg / ml Brefeldin A 3-5h before staining. Cells were sequentially stained for extracellular antigens, fixed and permeabilized using BD Cytofix / Cytoperm™ Fixation / Permeabilization Solution Kit before being resuspended in Flow Buffer for acquisition. Where applicable, the inventors use AccuCheck counting beads (Life Technologies) for cell counting. The inventors use FACS Diva in a BD Fortessa flow cytometer for acquistion and FlowJo (BD) for analysis.

[0308] Example 11: Histology

[0309] Collected organs were washed in PBS and then fixed overnight at 4°C in 4% PFA and then sent in 70% EtOH to the Biorepository and Pathology Core at Mount Sinai to generate paraffin tissue embedded blocks and generate 5mm H&E sections and 5mm FFPE tissue sections. H&E sections were scanned in a Leica Aperio AT2 whole-slide scanner. The inventors quantify lung tumor burden on H&E slides using QuPath software, defining tumor nodules by morphological features. The inventors calculate tumor burden based on the sum of the areas of all tumor nodules divided by the total lung area.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0310] Example 12: Multiplexed immunohistochemical consecutive staining on a single slide (MICSSS)

[0311] MICSSS was executed following a previously provided detailed protocol. Briefly, FFPE tissue sections, 5 pm thick, were first treated to remove paraffin using xylene and then rehydrated in decreasing concentrations of ethanol (100%, 90%, 70%, and 50%). To retrieve antigens, the slides were immersed in an Antigen Retrieval Solution (pH 9) (Dako #S236784-2) and heated to 95°C for 15-30 minutes, then cooled at room temperature (RT) for 30 minutes and rinsed with Tris Buffer Saline (TBS) (Fisher Scientific #BP24711). Next, the slides were immersed in 3% hydrogen peroxide for 15 minutes at RT, followed by a 30-minute incubation at RT with Serum-Free Protein Block (Dako #X090930-2). Then, the inventors applied primary antibody, diluted in Antibody Diluent, Background Reducing (Dako #S302283-2), for 1 hour at RT. After washing the slides with TBS 0.04% Tween 20, the inventors incubated them with HRP-conjugated secondary antibodies at RT for 30 minutes based on primary antibody host (i.e., EnVision+ System- HRP Labelled Polymer Anti-mouse (Dako #K400111-2), EnVision+ System- HRP Labelled Polymer Anti-rabbit (Dako #K400311-2), VisUCyte HRP Polymer Goat IgG Antibody (R&D Systems #VC004-25), ImmPRESS HRP Goat Anti-Rat IgG, Mouse Absorbed (Vector Laboratories #MP-74444-15). Antigen detection was performed using AEC Peroxidase Substrate kit (Vector Laboratories #SK-4200) and counterstained briefly with Hematoxylin Solution, Harris Modified (Sigma- Aldrich #HHS16). The slides were mounted with coverslips using Glycergel Mounting Media (Agilent #C056330-2) and then captured using an Aperio AT2 slide scanner (Leica) at a magnification of 20X or 40X. In subsequent staining, coverslips were removed by incubating in 60°C water. AEC and Hematoxylin slides were removed with a series of ethanol solutions (i.e., 50%, 70%-l% HCI 12N, and 100%). Subsequently, staining procedure was repeated slides for consecutive target antigen, with the addition of an extra blocking step corresponding to the antibody host used. This involved incubating the slides with one of the following reagents at room temperature for 30 minutes before primary antibody staining, selected based on the host species of the primary antibody used that day: AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch #715-007-003), AffiniPure Fab Fragment Donkey Anti- Rabbit IgG (H+L) (Jackson ImmunoResearch #711-007-003), AffiniPure Fab Fragment Donkey Anti-Goat IgG (H+L) (Jackson ImmunoResearch #705-007-003), AffiniPure Fab Fragment Donkey Anti-Rat IgG (H+L) (Jackson ImmunoResearch #112-007-003).

[0312] Example 13: MICSSS procession and visualizationMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0313] The inventors registered and segmented the MICSSS images using ImageJ and QuPath softwares, following a detailed protocol previously outlined(85). Briefly, the inventors registered all the consecutively stained images using ImageJ Linear Stack Alignment with SIFT tool. Then, the inventors deconvoluted the images on ImageJ using the Hematoxylin / AEC color vector. One hematoxylin image, plus all AEC images were pseudocolored and stacked to create image composites. Cell nuclear and cell segmentation was performed on QuPath with optimized parameters. The inventors apply consistent segmentation parameters to all images within the same experiment.

[0314] Example 14: Mouse scRNA-seq analysis

[0315] scRNA-seq analysis was performed on the dataset GSE263461 as previously described ( Mollaoglu et al.). Briefly, scRNA-seq data from control ID8 tumors were analyzed using the Scanpy package (vl.9.4). Initial quality control steps included filtering out cells with fewer than 1500 total counts or more than 40,000 total counts. Cells with mitochondrial gene content exceeding 20% of total counts were excluded to account for potential stress or degradation. Cells expressing fewer than 200 genes were removed.Doublets were identified using Scrublet (vO.2.3) with default parameters and excluded based on a threshold doublet score of 0.04. After filtering, 17,767 cells were retained for downstream analysis. For gene filtering, genes expressed in fewer than 20 counts across all cells were removed, resulting in 16,066 genes available for analysis. Data normalization was performed using a log-transformation with a pseudocount of 1. Highly variable genes (n = 4,000) were identified using Scanpy’s highly variable genes function. The processed dataset underwent principal component analysis (PCA) for dimensionality reduction, followed by Leiden clustering and UMAP visualization for embedding. Annotation of cell types was performed based on the expression of defined markers.

[0316] The dot plot, used to visualize marker genes (Wtl and Folr2) and showing the mean value and the percentage of cells expressing the gene across multiple clusters, was generated with the scanpy.pl. dotplot function of Scanpy.

[0317] Example 14: Spatial Transcriptomics Analysis Using Xenium In Situ Gene Expression

[0318] Sectioning and Slide Preparation

[0319] Formalin- fixed paraffin-embedded (FFPE) tissue sections (5 pm thickness) were cut using a rotary microtome, floated on a 40-42 °C water bath, and mounted ontoXenium-compatible, positively charged glass slides (lOx Genomics). Slides were baked atMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC60 °C for 1 hour, cooled to room temperature, and stored in a desiccated environment at 4 °C for up to three days prior to use.

[0320] Xenium Probe Panel Design (Custom Add-On)

[0321] Spatial in situ gene expression was performed using the Xenium In Situ Gene Expression vl chemistry (lOx Genomics) with a custom probe panel. Panel design, including input review and quality control, was conducted by the 1 Ox Genomics panel design team. The custom panel comprised 480 target genes (Table SI) selected to enable identification of all major cell types present in lung, pancreas, and ovarian cancer tissues, based on three reference single-cell RNA sequencing datasets.

[0322] Probes were designed by the manufacturer in accordance with Xenium specifications, screened for off-target binding and isoform cross-reactivity, and supplied ready for use. Each experimental run included manufacturer-provided positive controls (housekeeping genes) and negative controls (non-mammalian sequences).

[0323] Deparaffinization and Pre-Treatment

[0324] Slides were equilibrated to room temperature and deparaffinized by two sequential 10-minute incubations in fresh xylene, followed by rehydration through graded ethanol solutions (100%, 95%, and 70%; 2-3 minutes each) and rinsing in distilled water.Manufacturer-recommended pre-treatment and decrosslinking steps for FFPE tissue were performed on-slide according to the Xenium vl FFPE workflow, including heat-mediated decrosslinking in proprietary buffer and enzymatic permeabilization. Permeabilization time was determined in advance using a Xenium Tissue Optimization experiment performed on matched tissue and was applied uniformly across all samples in the study.

[0325] In Situ Hybridization, Ligation, and Amplification

[0326] Following pre-treatment, slides were loaded into the Xenium instrument (lOx Genomics) and processed using Xenium In Situ Gene Expression vl chemistry.Target-specific padlock-style probe pairs were hybridized to RNA molecules in situ, followed by proximity-dependent ligation to generate circularized probe barcodes. Rolling circle amplification was then performed to generate localized amplicons suitable for optical detection. All incubations, washes, and reagent exchanges were executed automatically by the instrument according to the manufacturer’s protocol.

[0327] Cycled Reporter Hybridization and Imaging

[0328] Cycled fluorescent reporter hybridizations were carried out on-instrument to decode barcoded transcripts across multiple imaging cycles. Imaging was performed using the Xenium optical system at 20 x magnification, capturing multi-channel images for each cycleMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCacross the full tissue area defined during experimental setup. Autofluorescence suppression and background subtraction were performed using manufacturer-default settings.

[0329] Cell Segmentation and Transcript Assignment

[0330] Nuclei were segmented using the onboard nuclear channel and Xenium Onboard Analysis (XOA). Where specified, membrane signal enhancement was performed using the Xenium Cell Segmentation Add-On Kit according to the manufacturer’s instructions.Transcripts were assigned to individual cells based on segmentation boundaries and default transcript-assignment rules.

[0331] Raw Spatial Transcriptomic Data Pre-Processing

[0332] Xenium spatial transcriptomics data were segmented using the Xenium In Situ Multimodal Cell Segmentation Workflow, incorporating nuclear staining (DAPI), boundary protein staining (ATP1A1, E-Cadherin, CD45), interior RNA staining (18S rRNA), and interior protein staining (aSMA, Vimentin). Xenium Explorer 4 was used to identify and exclude non-omental tissue regions, such as mouse pancreas, which were removed from downstream analyses but retained for spatial arrangement visualizations.

[0333] Data were pre-processed using SpatialData and Scanpy to ensure inclusion of high-quality cells only, following recommended best-practice guidelines (Marcos-Salas et al., 2025, Nature Methods). Cells were filtered based on quality control metrics, excluding potential empty droplets defined by low transcript counts (<40) or low gene counts (<15). Remaining counts were normalized to a target sum of 100 and log-transformed without scaling. Highly variable genes were selected using a minimum mean expression of 0.3, maximum mean of 7, and minimum dispersion of -0.5. Cells were subsequently clustered using the Louvain algorithm.

[0334] Cell-Type and Subtype Annotation

[0335] Clusters were manually annotated based on expression of canonical marker genes. Broad cell classes included cancer cells (Krt8, Krtl8, Krtl9, Krt7), stromal cells (Cdh5, Pecaml, Plvap, Fltl, Vegfa, Kdr, Collal, Col3al, Acta2), T and NK cells (Cd3d, Cd3e, Cd4, Cd8a, Trac, Tcf7, Nkg7, Klrkl, Prfl, Gzmb), myeloid cells (Cd68, Cdl4, Clqc, Csflr, Clec9a), B cells (Cd79b, Ms4al), and plasma cells (Igkc, Iglc3, Xbpl). Cells lacking marker expression from other broad lineages were classified as confident. Cells exhibiting marker co-expression consistent with doublets were labeled ambiguous or adjacent and excluded from downstream analyses, except where adjacency analysis between cancer and T cells was required.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0336] For subtype identification, each broad compartment was re-clustered using the Louvain algorithm within the same scVI latent space used for initial clustering. Myeloid cells were further subdivided into macrophages (Clqa, Clqc, Mertk, Mrcl, Trem2, Folr2, Marco), monocytes (Ly6c2, Ccr2, Cdl4), dendritic cells (Xcrl, Clec9a), and neutrophils (Csf3r, Cxcr2, Pglyrpl). Macrophages were further re-clustered, resulting in identification of five macrophage subtypes: Folr2-High, Folr2-Low, Folr2 Argl , Cxcl9+, and Ill b+macrophages.

[0337] T and NK cells were sub-clustered into CD4 T cells (Cd4), regulatory T cells (Foxp3, I12ra), activated CD8 T cells (Cd8a, Prfl, Gzmb, Ccl5), memory CD8 T cells (Ccr7, Tcf7, Sell, I17r, Cd44), and NK cells (Nkg7, Klrkl, Klrblc). Stromal cells were subdivided into fibroblasts (Collal, Colla2, Col3al, Pdgfra), myofibroblastic CAFs (Acta2, Myhll), endothelial cells (Pecaml, Fltl, Kdr, Plvap), and adipocytes (Pparg).

[0338] Macrophage Response Signatures

[0339] Module scores were calculated using Scanpy’s sc.tl.score_genes function with custom gene dictionaries. Immunosuppressive macrophage scores were calculated based on expression of Argl, Cd274, Folr2, Trcm2, Hmoxl, 1110, Igfl, Mertk, Mrcl, Tgfbl, and 114. The macrophage response to IL- 12 signature was derived using genes present in the custom Xenium panel (CxcllO, Cxcl9, Ccl2, Irf7, Statl) selected from the Immune Dictionary’s macrophage IL- 12 response gene set.

[0340] Example 15: Statistical analyses

[0341] Statistical analyses were carried out using GraphPad Prism 10 software. Log-rank analysis was used to compare survival curves. T-student comparison was used for column analyses, except where indicated: Mann- Whitney rank-based analyses were used to include placeholder data points representing mice that succumbed to tumor development before sacrifice (indicated in figures with an “x” datapoint symbol).

[0342] Example 16: Data visualization

[0343] QuPath open-source software, Fiji opensource software were used for MICSSS images. FlowJo 10 was used to analyze and visualized flow cytometry data. Bar charts and survival plots were created in GraphPad Prism 10.

[0344] Exemplary Embodiments

[0345] Several exemplary embodiments are listed in the following examples.

[0346] Example Clause A: A Chimeric Antigen Receptor (CAR) construct may include: a first antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) may include a light chain variable region, a second antigen-binding domain capable of binding to TREM2 may include a heavy chain variable region, where theMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCfirst antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain may include of a co-stimulatory domain and a signaling domain, and a payload may include at least one cytokine or cytokine variant, where the payload is connected to the signaling domain via a self-cleaving linker.

[0347] Example Clause B: The CAR construct of any one of Example Clause A to 5, where the flexible linker is a (G4S)n linker, where n is selected from the group having of 1, 2, 3, 4, or 5.

[0348] Example Clause C: The CAR construct of any one of Example Clause A or Example Clause B to 6, where the self-cleavable linker is a peptide linker.

[0349] Example Clause D: The CAR construct of any one of any one of Example Clauses A-C to 7, where the self-cleavable linker is selected from the group having of P2A, T2A, E2A and F2A.

[0350] Example Clause E: The CAR construct of any one of any one of Example Clauses A-D to 8, where the at least one cytokine is selected from the group having of interleukins (ILs), interferons (IFNs), chcmokincs, and tumor necrosis factor (TNF).

[0351] Example Clause F: The CAR construct of any one of Example Clauses A-E or of any one of Example Clauses A-E, where the at least one cytokine is an IL selected from the group having of IL-12, IL-1, IL-2, IL-6, IL-10, IL-17, IL-18, IL-15, IL-9, IL-11, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-13, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, and IL-38.

[0352] Example Clause G: The CAR construct of any one of any one of Example Clauses A-F to 11, where the payload promotes immunomodulation at disease sites.

[0353] Example Clause H: The CAR construct of any one of any one of Example Clauses A-G to 12, where the CAR construct further may include a protease cleavable linker, where the protease cleavable linker is downstream of the pay load.

[0354] Example Clause I: The CAR construct of any one of Example Clauses A-H, where the protease cleavable linker is engineered to be recognized and cleaved by a protease.

[0355] Example Clause J: The CAR construct of any one of Example Clauses A-I, where the protease is selected from the group having of cysteine proteases, serine proteases, or metalloproteinases.

[0356] Example Clause K: The CAR construct of any one of Example Clauses A- J, where the protease is selected from the group having of matriptase, legumain, matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-7 (MMP7), matrix metalloproteinase-9 (MMP9), urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase 10Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC(ADAMI 0), a disintegrin and metalloproteinase 17 (ADAMI 7), and membrane -type 1 matrix metalloproteinase (MT1-MMP).

[0357] Example Clause L: The CAR construct of any one of any one of Example Clauses A-K to 16, where the CAR construct further may include an additional transmembrane region, where the additional transmembrane region is downstream of the protease cleavable linker.

[0358] Example Clause M: The CAR construct of any one of Example Clauses A-L, where the additional transmembrane region may include CD 8, B7-1T (CD 80 transmembrane domain), or CD28.

[0359] Example Clause N: The CAR construct of any one of any one of Example Clauses A-M to 18, where the co-stimulatory domain may include CD28, 41BB, 0X40, ICOS, CD40, and combinations thereof.

[0360] Example Clause O: The CAR construct of any one of any one of Example Clauses A-N to 19, where the signaling domain may include CD3z or variants thereof.

[0361] Example Clause P: The CAR construct of any one of any one of Example Clauses A-O to 20, where the CAR construct further may include a small molecule-induced degron domain and / or a mutated FK506-binding protein sequence, where the small molecule-induced degron domain and / or the mutated FK506-binding protein sequence is downstream of the additional transmembrane region.

[0362] Example Clause Q: The CAR construct of any one of any one of Example Clauses A-P to 21, where the CAR construct further may include a peptide tag.

[0363] Example Clause R: The CAR construct of any one of any one of Example Clauses A-Q to 22, where the CAR construct further may include a destabilizing domain, where the destabilizing domain modulates the payload activity.

[0364] Example Clause S: The CAR construct of any one of any one of Example Clauses A-R to 23, where the antigen-binding domain is a single-chain variable fragment (scFv).

[0365] Example Clause T: The CAR construct of any one of any one of Example Clauses A-S to 24, where the antigen-binding domain is derived from a monoclonal antibody or a nanobody.

[0366] Example Clause U: The CAR construct of any one of any one of Example Clauses A-T to 25, where the transmembrane domain may include CD28 or CD8.

[0367] Example Clause V: The CAR construct of any one of any one of Example Clauses A-U to 26, where the co-stimulatory domain may include CD3C or a variant thereof.

[0368] Example Clause W: A pharmaceutical composition may include the CAR construct of any one of any one of Example Clauses A-V to 27 and a pharmaceutically acceptable carrier.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0369] Example Clause X: A nucleic acid encoding a CAR construct according to any one of any one of Example Clauses A-W to 27.

[0370] Example Clause Y: A plasmid, vector, or cell may include the nucleic acid of any one of Example Clauses A-X.

[0371] Example Clause Z: The CAR construct of any one of any one of Example Clauses A-Y to 27, where the CAR construct is expressed in a cell.

[0372] Example Clause AA: The CAR construct of any one of Example Clauses A-Z, where the cell is an immune cell.

[0373] Example Clause AB: The CAR construct of any one of Example Clauses A-AA, where the immune cell is selected from the group having of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell.

[0374] Example Clause AC: The CAR construct of any one of Example Clauses A-AB, where the T cells are regulatory T cells (Tregs).

[0375] Example Clause AD: A method of treating an autoinflammatory disease in a subject in need thereof, the method may include administering to the subject an effective amount of a population of cells according to any one of any one of Example Clauses A- AC to 38.

[0376] Example Clause AE: The method of any one of Example Clauses A- AD, where the autoinflammatory disease is characterized by the accumulation of macrophages.

[0377] Example Clause AF: The method of any one of Example Clauses A- AE, where the macrophages may include TREM2+ macrophages or FOLR2+ macrophages.

[0378] Example Clause AG: The method of any one of any one of Example Clauses A- AF to 48, where the method is cell therapy.

[0379] Example Clause AH: A method of treating cancer in a subject in need thereof, the method may include administering to the subject an effective amount of a population of cells according to any one of any one of Example Clauses A- AG to 38.

[0380] Example Clause AE The method of any one of Example Clauses A- AH, where the cancer is characterized by the accumulation of macrophages.

[0381] Example Clause AJ: The method of any one of Example Clauses A- Al, where the macrophages may include TREM2+ macrophages or FOLR2+ macrophages.

[0382] Example Clause AK: The method of any one of any one of Example Clauses A- AJ to 44, where the cancer is selected from the group having of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC

[0383] Example Clause AL: A method for treating solid tumors in a subject in need thereof, the method may include administering to the subject an effective amount of a population of cells according to any one of any one of Example Clauses A-AK to 38.

[0384] Example Clause AM: The method of any one of Example Clauses A- AL, where the solid tumor is selected from the group having of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

[0385] Example Clause AN: A method of enhancing anti-tumor activity of engineered immune cells, the method may include administering to the subject an effective amount of a population of cells according to any one of any one of Example Clauses A- AM to 38, where the payload enhances the immune response to tumor cells and / or modulates the CAR construct activity.

[0386] Example Clause AO: A method of producing CAR-expressing immune cells, the method may include: engineering the immune cells, where the engineered immune cells may include the CAR construct of any one of any one of Example Clauses A- AN to 27, and expanding the engineered immune cells to produce a population of CAR-expressing immune cells.

[0387] Example Clause AP: The method of any one of Example Clauses A- AO, where the immune cells are selected from the group having of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell.

[0388] Example Clause AQ: The method of any one of Example Clauses A- AP, where the T cells are regulatory T cells (Tregs).

[0389] Example Clause AR: The method of any one of any one of Example Clauses A-AQ to 36, where engineering the immune cells may include transfecting the CAR construct into the immune cells.

[0390] Example Clause AS: The use of the CAR construct of any one of any one of Example Clauses A- AR to 27 in the manufacturer of a medicament for the treatment of cancer or an autoinflammatory disease.

[0391] Example Clause AT: The CAR construct of any one of any one of Example Clauses A-AS to 8, where the cytokine variant may include muteins derived from at least one cytokine.

[0392] Example Clause AU: A CAR construct may include: a first antigen-binding domain capable of binding to folate receptor 2 (FOLR2) may include a heavy chain variable region, a second antigen-binding domain capable of binding to FOLR2 may include a light chain variable region, where the first antigen-binding domain and second antigen-binding domainMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCare connected via a flexible linker, a transmembrane domain, an activation domain may include of a co-stimulatory domain and a signaling domain, and a payload may include at least one cytokine or cytokine variant, where the payload is connected to the signaling domain via a self-cleaving linker.

[0393] Example Clause AV: A CAR construct may include: a first antigen-binding domain capable of binding to TREM2 may include a light chain variable region, a second antigenbinding domain capable of binding to TREM2 may include a heavy chain variable region, where the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain may include of a costimulatory domain and a signaling domain, and a payload may include at least one polynucleotide encoding a cytokine or cytokine variant, where the payload is connected to the signaling domain via a self-cleaving linker.

[0394] Example Clause AW: The CAR construct of Example Clause AV or of Example Clause AV, where the polynucleotide may include internal ribosome entry sites (IRES)

[0395] Example Clause AX: A CAR construct may include: a first antigen-binding domain capable of binding to FOLR2 may include a heavy chain variable region, a second antigenbinding domain capable of binding to FOLR2 may include a light chain variable region, where the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain may include of a costimulatory domain and a signaling domain, and a payload may include at least one polynucleotide encoding a cytokine or cytokine variant, where the payload is connected to the signaling domain via a self-cleaving linker.

[0396] Example Clause AY: A Chimeric Antigen Receptor (CAR) construct may include: a first antigen-binding domain capable of binding to an antigen expressed on the surface of tumor macrophages may include a light chain variable region, a second antigen-binding domain capable of binding to the antigen may include a heavy chain variable region, where the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker, a transmembrane domain, an activation domain may include of a costimulatory domain and a signaling domain, and a payload may include at least one cytokine or cytokine variant, where the payload is connected to the signaling domain via a selfcleaving linker.

[0397] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will nowMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCoccur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCCLAIMS1. A Chimeric Antigen Receptor (CAR) construct comprising:a first antigen-binding domain capable of binding to Triggering Receptor Expressed on Myeloid cells 2 (TREM2) comprising a light chain variable region,a second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker,a transmembrane domain,an activation domain comprised of a co-stimulatory domain and a signaling domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload is connected to the signaling domain via a self-cleaving linker.

2. A CAR construct comprising:a first antigen-binding domain capable of binding to folate receptor 2 (FOLR2) comprising a heavy chain variable region,a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker,a transmembrane domain,an activation domain comprised of a co-stimulatory domain and a signaling domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload is connected to the signaling domain via a self-cleaving linker.

3. A CAR construct comprising:a first antigen-binding domain capable of binding to TREM2 comprising a light chain variable region,Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCa second antigen-binding domain capable of binding to TREM2 comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker,a transmembrane domain,an activation domain comprised of a co-stimulatory domain and a signaling domain, and a payload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload is connected to the signaling domain via a self-cleaving linker.

4. A CAR construct comprising:a first antigen-binding domain capable of binding to FOLR2 comprising a heavy chain variable region,a second antigen-binding domain capable of binding to FOLR2 comprising a light chain variable region, wherein the first antigen-binding domain and second antigen-binding domain are connected via a flexible linker,a transmembrane domain,an activation domain comprised of a co-stimulatory domain and a signaling domain, and a payload comprising at least one polynucleotide encoding a cytokine or cytokine variant, wherein the payload is connected to the signaling domain via a self-cleaving linker.

5. The CAR construct of claim 3 or claim 4, wherein the polynucleotide comprises internal ribosome entry sites (IRES)6. The CAR construct of any one of claims 1 to 5, wherein the flexible linker is a (G4S)nlinker, wherein n is selected from the group consisting of 1, 2, 3, 4, or 5.

7. The CAR construct of any one of claims 1 to 6, wherein the self-cleavable linker is a peptide linker.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC8. The CAR construct of any one of claims 1 to 7, wherein the self-cleavable linker is selected from the group consisting of P2A, T2A, E2A and F2A.

9. The CAR construct of any one of claims 1 to 8, wherein the at least one cytokine is selected from the group consisting of interleukins (ILs), interferons (IFNs), chemokines, and tumor necrosis factor (TNF).

10. The CAR construct of any one of claims 1 to 8, wherein the cytokine variant comprises muteins derived from at least one cytokine.

11. The CAR construct of claim 9 or claim 10, wherein the at least one cytokine is an IL selected from the group consisting of IL-12, IL-1, IL-2, IL-6, IL-10, IL-17, IL-18, IL-15, IL-9, IL-11, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-13, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, and IL-38.

12. The CAR construct of any one of claims 1 to 11, wherein the payload promotes immunomodulation at disease sites.

13. The CAR construct of any one of claims 1 to 12, wherein the CAR construct further comprises a protease cleavable linker, wherein the protease cleavable linker is downstream of the payload.

14. The CAR construct of claim 13, wherein the protease cleavable linker is engineered to be recognized and cleaved by a protease.

15. The CAR construct of claim 14, wherein the protease is selected from the group consisting of cysteine proteases, serine proteases, or metalloproteinases.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC16. The CAR construct of claim 15, wherein the protease is selected from the group consisting of matriptase, legumain, matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-7 (MMP7), matrix metalloproteinase-9 (MMP9), urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase 10 (ADAMI 0), a disintegrin and metalloproteinase 17 (ADAM17), and membrane -type 1 matrix metalloproteinase (MT1-MMP).

17. The CAR construct of any one of claims 13 to 16, wherein the CAR construct further comprises an additional transmembrane region, wherein the additional transmembrane region is downstream of the protease cleavable linker.

18. The CAR construct of claim 17, wherein the additional transmembrane region comprises CD8, B7-1T (CD80 transmembrane domain), or CD28.

19. The CAR construct of any one of claims 1 to 18, wherein the co-stimulatory domain comprises CD28, 4 IBB, 0X40, ICOS, CD40, and combinations thereof.

20. The CAR construct of any one of claims 1 to 19, wherein the signaling domain comprises CD3z or variants thereof.

21. The CAR construct of any one of claims 17 to 20, wherein the CAR construct further comprises a small molecule-induced degron domain and / or a mutated FK506-binding protein sequence, wherein the small molecule-induced degron domain and / or the mutated FK506-binding protein sequence is downstream of the additional transmembrane region.

22. The CAR construct of any one of claims 13 to 21, wherein the CAR construct further comprises a peptide tag.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC23. The CAR construct of any one of claims 1 to 22, wherein the CAR construct further comprises a destabilizing domain, wherein the destabilizing domain modulates the payload activity.

24. The CAR construct of any one of claims 1 to 23, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

25. The CAR construct of any one of claims 1 to 24, wherein the antigen-binding domain is derived from a monoclonal antibody or a nanobody.

26. The CAR construct of any one of claims 1 to 25, wherein the transmembrane domain comprises CD28 or CD8.

27. The CAR construct of any one of claims 1 to 26, wherein the co-stimulatory domain comprises CD3C or a variant thereof.

28. A pharmaceutical composition comprising the CAR construct of any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A nucleic acid encoding a CAR construct according to any one of claims 1 to 27.

30. A plasmid, vector, or cell comprising the nucleic acid of claim 29.

31. The CAR construct of any one of claims 1 to 27, wherein the CAR construct is expressed in a cell.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC32. The CAR construct of claim 31 , wherein the cell is an immune cell.

33. The CAR construct of claim 32, wherein the immune cell is selected from the group consisting of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell.

34. The CAR construct of claim 33, wherein the T cells are regulatory T cells (Tregs).

35. A method of producing CAR-expressing immune cells, the method comprising: engineering the immune cells, wherein the engineered immune cells comprise the CAR construct of any one of claims 1 to 27, andexpanding the engineered immune cells to produce a population of CAR-expressing immune cells.

36. The method of claim 35, wherein the immune cells are selected from the group consisting of T cells, innate lymphoid cells (ILCs), myeloid progenitors, and dendritic cell.

37. The method of claim 36, wherein the T cells are regulatory T cells (Tregs).

38. The method of any one of claims 35 to 36, wherein engineering the immune cells comprises transfecting the CAR construct into the immune cells.

39. A method of treating an autoinflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any one of claims 31 to 38.

40. The method of claim 39, wherein the autoinflammatory disease is characterized by the accumulation of macrophages.Mount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PC41. The method of claim 40, wherein the macrophages comprise TREM2+ macrophages or FOLR2+ macrophages.

42. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any one of claims 31 to 38.

43. The method of claim 42, wherein the cancer is characterized by the accumulation of macrophages.

44. The method of claim 43, wherein the macrophages comprise TREM2+ macrophages or FOLR2+ macrophages.

45. The method of any one of claims 42 to 44, wherein the cancer is selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

46. A method for treating solid tumors in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of cells according to any one of claims 31 to 38.

47. The method of claim 46, wherein the solid tumor is selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

48. A method of enhancing anti-tumor activity of engineered immune cells, the method comprising administering to the subject an effective amount of a population of cellsMount Sinai Ref. No. 250207G Lathrop Ref. No. 776011: MTST-976PCaccording to any one of claims 31 to 38, wherein the payload enhances the immune response to tumor cells and / or modulates the CAR construct activity.

49. The method of any one of claims 39 to 48, wherein the method is cell therapy.

50. The use of the CAR construct of any one of claims 1 to 27 in the manufacturer of a medicament for the treatment of cancer or an autoinflammatory disease.

51. A Chimeric Antigen Receptor (CAR) construct comprising:a first antigen-binding domain capable of binding to an antigen expressed on the surface of tumor macrophages comprising a light chain variable region,a second antigen-binding domain capable of binding to the antigen comprising a heavy chain variable region, wherein the first antigen-binding domain and second antigenbinding domain are connected via a flexible linker,a transmembrane domain,an activation domain comprised of a co-stimulatory domain and a signaling domain, and a payload comprising at least one cytokine or cytokine variant, wherein the payload is connected to the signaling domain via a self-cleaving linker.

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