WO2025072372A1

WO2025072372A1 - Car-t cells expressing vegf binding proteins

Info

Publication number: WO2025072372A1
Application number: PCT/US2024/048466
Authority: WO
Inventors: Marcela V. Maus; Mark LEICK
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 2023-09-26
Filing date: 2024-09-25
Publication date: 2025-04-03
Anticipated expiration: 2026-03-26
Also published as: WO2025072372A9

Abstract

This application is directed to, in part, T cells and CAR-T cells expressing VEGF binding proteins or comprising VEGF gene knockouts, and methods of use thereof in treating cancer.

Description

CAR-T CELLS EXPRESSING VEGF BINDING PROTEINS RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63/585,484, filed September 26, 2023, entitled “CAR-T Cells Expressing VEGF Binding Proteins”, the entire contents of which are incorporated herein by reference. FEDERALLY SPONSORED RESEARCH This invention was made with Government support under R01CA238268-04, awarded by the National Institute of Health. The Government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (M105370050WO00-SEQ-ARM.xml; Size: 97,345 bytes; and Date of Creation: September 24, 2024) is herein incorporated by reference in its entirety. BACKGROUND While CAR-T cell therapy is effective at treating some types of hematological cancer, far less success has been achieved using CAR-T cells to treat solid tumors. Treating solid tumors is challenging at least because the solid tumor microenvironment can limit CAR-T cell expansion and decrease CAR-T cell persistence. SUMMARY The inventors recognized that CAR-T cell efficacy in solid tumors could be enhanced by engineering the CAR-T cells to express VEGF binding proteins. Without being bound to theory, these VEGF binding proteins enhance CAR-T cell function in solid tumor in at least two ways. First, CAR-T cells expressing VEGF binding proteins are more resistant to exhaustion. Second, results show that CAR-T cells expressing VEGF binding proteins also increase CAR-T cell cytotoxicity, presumably by decreasing tumor angiogenesis which is critical to the growth of many solid tumors. Additionally, the inventors discovered that knocking out VEGF in CAR-T cells (e.g., using CRISPR) can also increase CAR-T cell function similar to using a VEGF binding protein. Overall, these results indicate that T cell therapy persistence and cytotoxicity may be enhanced by engineering T cell to secret VEGF binding proteins and/or by knocking out VEGF in T cells. In aspects, this disclosure describes a T cell comprising a heterologous polynucleotide encoding a Vascular Endothelial Growth Factor (VEGF) binding protein. In some embodiments, the VEGF binding protein is secreted by the cell. In some embodiments, the VEGF binding protein comprises a secretory signal peptide. In some embodiments, the secretory signal peptide comprises an Igk signal peptide. In some embodiments, the VEGF binding protein comprises an antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an antigen-binding fragment (Fab), a Fab’, or a F(ab’)2, a fragment variable (Fv), or a single chain variable fragment (scFv). In some embodiments, the antibody fragment is an scFv. In some embodiments, the antibody comprises: (i) a VH domain comprising three complementary determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 70, CDR-H2 comprises SEQ ID NO: 71, and CDR-H3 comprises SEQ ID NO: 72; and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 73, CDR-L2 comprises SEQ ID NO: 74, and CDR-L3 comprises SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence of SEQ ID NO: 76. In some embodiments, the VEGF binding protein comprises a VEGF A binding protein. In some embodiments, the VEGF binding protein is operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is a EF1-alpha promoter. In some embodiments, the antibody comprises the VH domain n-terminal to the VL domain. In some embodiments, the antibody comprises the VL domain n-terminal to the VH domain. In some embodiments, this disclosure describes a T cell comprising a VEGF gene knockout. In some embodiments, the T cell comprises a VEGF gene knockout. In some embodiments, the VEGF gene knockout comprises a VEGF-A gene knockout. In some embodiments, the VEGF gene knockout is a VEGF gene deletion. In some embodiments, the VEGF gene knockout comprises a VEGF loss of function mutation. In some embodiments, a T cell comprises a polynucleotide encoding a guide RNA polynucleotide comprising a homology region that is complementary to a polynucleotide encoding VEGF. In some embodiments, a T cell further comprises a polynucleotide encoding a guide RNA comprising a homology region that is complementary to a polynucleotide encoding VEGF. In some embodiments, the polynucleotide encoding VEGF encodes VEGF-A. In some embodiments, the polynucleotide encoding the guide RNA homology region comprises a polynucleic acid of SEQ ID NO: 79 or SEQ ID NO: 80. In some embodiments, the T cell is a chimeric antigen receptor (CAR)-T cell. In some embodiments, the T cell comprises a polynucleotide encoding a chimeric antigen receptor. In some embodiments, the CAR comprises: (i) an antigen binding domain; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain. In some embodiments, the antigen binding domain binds to any one of CD19, CD37, CD79b, Claudin 18.2, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19/CD79b, BCMA/TACI, or is a TriPRIL antigen binding domain. In some embodiments, the antigen binding domain comprises an antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an antigen-binding fragment (Fab), a Fab', or a F(ab')2, a fragment variable (Fv), or a single chain variable fragment (scFv). In some embodiments, the antigen binding domain binds to CD70. In some embodiments, the antigen binding domain comprises CD27. In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the antigen binding domain binds to mesothelin. In some embodiments, the antibody comprises an amino acid sequence of any one of SEQ ID NOs: 3-4. In some embodiments, the transmembrane domain is selected from the group consisting of alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C transmembrane domains. In some embodiments, the transmembrane domain comprises a CD8 or a CD27 transmembrane domain. In some embodiments, the co-stimulatory domain comprises a 4-1BB, CD27, CD28, OX40, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, or ZAP70 costimulatory domain. In some embodiments, the co-stimulatory domain comprises a 4-1BB. In some embodiments, the intracellular signaling domain comprises a CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, or CD66d intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain. In some embodiments, the CAR further comprises a leader sequence. In some embodiments, the leader sequence comprises a CD8 leader sequence. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-47. In some embodiments, the CAR comprises: (i) a CD70 binding antigen binding domain; (ii) a CD27 transmembrane domain; (iii) a 4-1BB costimulatory domain; and (iv) a CD3-zeta intracellular signaling domain. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 30-35. In some embodiments, the CAR comprises: (i) a mesothelin binding antigen binding domain; (ii) a CD8 transmembrane domain; (iii) a 4-1BB costimulatory domain; and (iv) a CD3- zeta intracellular signaling domain. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. In some embodiments, the polynucleotide encoding the CAR and the heterologous polynucleotide encoding the VEGF binding protein are encoded on the same polynucleic acid. In some embodiments, a self-cleaving peptide or an internal ribosomal entry site is encoded in the polynucleic acid between the CAR and the VEGF binding protein. In some embodiments, the self-cleaving peptide is a 2A peptide. In some embodiments, the polynucleic acid encodes an amino acid sequence of any one of SEQ ID NOs: 77-78. In some aspects, this disclosure describes a polypeptide encoding a CAR and the VEGF binding protein. In some aspects, this disclosure describes a polypeptide comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some aspects, this disclosure describes a polynucleic acid encoding a polypeptide encoding a CAR and the VEGF binding protein. In some aspects, this disclosure describes a polynucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some aspects, this disclosure describes a vector comprising the polynucleic acid. In some aspects, this disclosure describes a cell comprising the polynucleic acid or the vector. In some aspects, this disclosure describes a method of treating cancer in a subject, the method comprising administering a T cell described herein (e.g., a CAR-T cell) the subject. In some embodiments, the method comprises administering a T cell described herein to the subject. In some embodiments, the CAR comprises a CD70 antigen binding domain and the cancer expresses CD70. In some embodiments, the cancer is renal cell carcinoma, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, acute myeloid leukemia, or adenoid cystic carcinoma (ACC). In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, or papillary renal cell carcinoma (pRCC). In some embodiments, the CAR comprises a mesothelin binding antigen binding domain and the cancer expresses mesothelin. In some embodiments, the cancer is a pancreatic cancer, a lung cancer, an ovarian cancer, an endometrial cancer, a biliary cancer, a gastric cancer, or mesothelioma. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the VEGF binding protein inhibits binding of VEGF to VEGFR1 and/or VEGFR2. In some embodiments, the VEGF binding protein inhibits binding of VEGF-A to VEGFR1 and/or VEGFR2. In some aspects, this disclosure describes a method of decreasing CAR-T cell exhaustion, the method comprising transfecting the CAR-T cell with a polynucleotide encoding a VEGF binding protein. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG.1 shows VEGF-pathway member expression in CAR-T cells. FDA approved CD19 CAR-T cell products VEGFA, FLT1 (VEGFR1), and NRP1 expression by scRNAseq (left), and VEGFR1 expression of 70^VEGF and 70¹⁹ by flow (right); Tconv-Tconventional, Treg- Tregulatory, axi-cel-axicabtagene ciloleucel, tisa-cel-tisagenlecleucel; UTD-untransduced T cell CAR-T cells were co-cultured with CD70 expressing K562 cells (Activated) at ratio of 1:1 or alone for 72 hours followed by assessment by flow cytometry. Symbols represent unique T cell donors. P-values represent 1-way ANOVA with multiple hypotheses correction. Bar represents the median. FIG.2 shows secreted ^^VEGF abrogates VEGFR signaling. VEGFR reporter activity ± S.E.M., p-value via 2-way ANOVA. Technical triplicates. Supernatant from 70^VEGF or 70¹⁹ was collected and added to HEK293 VEGFR luciferase reporter cells with recombinant VEGFA. FIG.3 shows higher short proliferation kinetics of CD70 CAR-T cells expressing an anti-VEGF scFv (70^VEGF) CAR-T cells compared to CD70 CAR-T cells expressing an anti- CD19 scFv (70¹⁹) controls. Expansion with plate-bound recombinant CD70 ± S.E.M., p-value via 2-way ANOVA.3- donors’ T cells in technical triplicates. Coating with recombinant CD70 protein and real time incucyte monitoring were performed. FIG.4 shows 70^VEGF CAR-T demonstrate superior killing of RCC in vitro. ± S.E.M., p- value via 2-way ANOVA.3- donors T cells in technical triplicate. Luciferase killing assay with 786O RCC cells after 18hrs. FIG.5 is a schematic of a CD70 anti-VEGF-scFv CAR-T cell (70^VEGF) identifying putative sites of VEGF action within the TME (1) autocrine or paracrine secretion by tumor cells (2) paracrine secretion by CAR-T cells or tumor cells thereby enhancing angiogenesis and/or cytokine release syndrome (3) autocrine/ paracrine /intracrine CAR-T cell signaling. FIG.6 is a schematic of constructs for hU6, H1, EF1a are promoters; TRACg- CRISPR guide to constant chain of the TCRĮ gene; trCD27-truncated CD27. FIG.7 shows VEGF secretion by CAR-T cells ± S.E.M., p-value via 1-way ANOVA. Two donors T cells; technical triplicate. Supernatant was collected at the end of expansion for VEGF ELISA FIG.8 shows live imaging using nLuc. Liver specific transgene expression via delivery of AAV8 nLuc-nanoluciferase; AAV8-adeno associated virus serotype 8; FFz-flurofurimazine Twenty-one days after AAV8 with 7.5e11 GCs containing nLuc. IP injection of 50uL of FFz at 0.44^moles FIG.9 is a schematic of the experimental approach for CAR-T monitoring of various mice groups. FIG.10 is a schematic of the experimental approach for serial tumor monitoring of various mice groups. FIG.11 shows ex-vivo CAR-T generation from humanized mice. Humanized NOD- Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl (NCG) mice used to generate human CAR-T cells ex vivo with successful transduction, expansion, and cytotoxicity. Twenty-four-week-old humanized NCG mice were pre-humanized with CD34+ selected cord blood. One cohort was euthanized, and spleens were harvested and underwent standard CAR-T production. FIG.12 is a schematic of the experimental approach for mice engraftment studies. FIG.13 is a schematic of the experimental approach for anti-VEGF scFv injections. FIG.14 shows greater short term proliferation of 70^VEGF CAR-T cells compared to 70¹⁹ controls. Expansion with plate-bound recombinant CD70 ± S.E.M., p-value via 2-way ANOVA. 3- donors’ T cells in technical triplicates. Coating with recombinant CD70 protein and real time incucyte monitoring. FIG.15 shows greater longer term proliferation of 70^VEGF CAR-T cells compared to 7019 controls. Expansion with CD70 expressing K562 cells ± S.E.M., p-value via 2-way ANOVA.1- donor’s T cells in technical triplicates. CAR-T cells were recursively stimulated with a 1:1 ratio weekly of CD70 expressing K562s. FIGs.16A-16B show greater longer term proliferation of mesothelin CAR-T cells expressing an anti-VEGF scFv (Meso^VEGF) CAR-T cells compared to mesothelin CAR-T cells expressing an anti-CD19 scFv Meso¹⁹ controls. FIG.16A shows a schematic of the Meso^VEGF and Meso¹⁹ CARs. FIG 16B shows Meso^VEGF CAR T cells have greater long term proliferation than Meso¹⁹ CAR T cells. Expansion with Mesothelin expressing K562 cells ± S.E.M., p-value via 2-way ANOVA.1- donor’s T cells in technical triplicates. CAR-T cells were recursively stimulated with a 1:1 ratio weekly of mesothelin expressing K562s. FIG.17 show MesoVEGF^VEGF CAR-T completely suppress VEGF and produce higher levels of inflammatory cytokines compared to Meso¹⁹ controls. Supernatant from killing assay with SKOV3 ovarian tumor cells. Data reflects 3:1 E:T ratio and two normal donors in technical duplicate. Values are mean ± S.E.M., p-value via t-test. FIG.18 shows Meso^VEGF CAR-T have enhanced expansion relative to Meso¹⁹ controls in co-culture with non-small cell lung cancer A549 targets. Coculture of CAR-T cells with A549 lung cancer targets. Data reflects 2 healthy donors at an E:T ratio of 3:1. Plot represents mean+/- SEM. P-value represents 2-way ANOVA. FIG.19 show Meso^VEGF CAR-T have enhanced in vivo tumor control relative to Meso¹⁹ controls in a metastatic model of ovarian cancer. On day -7, 1e6 A549 lung cancer cells were injected by tail vein.7 days later, on day 0, 3e6 CAR-T cells or an equivalent number of untransduced T cells (UTD) were injected. Mice were followed by bioluminescent imaging of the tumor. FIGs.20A-20C relate to expression of VEGF signaling family members by CAR-T cells. FIG.20A shows expression of VEGFR1, VEGFR2, NRP1, and NRP2 at baseline or in the infusion product (IP) or monocytes from responding and non-responding patients that received Tisa-cel or Axi-cel. P-values represent a wilcoxon ranksum test, the q-values are FDR-corrected by Benjamini-Hochberg. FIG.20B shows mean fluorescence intensity (MFI) of VEGFR1 expression in Meso-targeting CD8+CAR+ T cells (or untransduced T cells, UTD) with (+) and without (-) stimulation with CD70+K562s for 96 hours and representative histograms (Symbols represent technical triplicates from 2 normal donors, bars represent mean ± S.D., p values by unpaired T tests). FIG.20C shows VEGF concentration in culture supernatant following 2 week mesothelin CAR-T cell production measured by ELISA. (Symbols represent technical triplicates from two normal donors, bars represent mean ± S.E.M, p value by one-way ANOVA). FIGs.21A-21F relate to blockade of VEGF signaling and angiogenesis in vitro by CARĮ^VEGF cells. FIG.21A is an exemplary schematic of CARĮ^CD19 and CARĮ^VEGF. FIG.21B is an exemplary schematic of an HEK293T VEGF reporter assay used to detect VEGF signaling. FIG.21C shows luminescence of supernatant from confluent VEGF-producing 786-0 tumor cells (points represent technical triplicates, lines represent the median and p-value by one-way ANOVA). FIG.21D is an exemplary schematic of in vitro angiogenesis assay to visualize disruption of blood vessel formation upon blocking VEGF. GFP+ human umbilical vein endothelial cells (HUVECs) were cocultured with normal human dermal fibroblasts (NHDFs), rVEGF, and culture supernatant from CARĮ^CD19 or CARĮ^VEGF constructs and primitive blood vessel formation was imaged over time on the incucyte. FIG.21E shows HUVEC blood vessel network length (length of blood vessel network (mm) per mm2 in image) detected with the Incucyte Angiogenesis software package. (data representative of 2 technical replicates, mean with SD, 2way ANOVA). FIG.21F shows representative images of GFP+ HUVECs with vessels labeled by the unbiased automated incucyte angiogenesis software package. FIGs.22A-22J relate to the enhancement of CAR-T cell proliferation and cell effector function across target antigens and tumor types in vitro by CARĮ^VEGF. FIG.21A shows MFI of CD69 surface expression of Meso-targeting CAR-T cell constructs cultured with SKOV3 for 18 hours (Points represent separate donors, error bars represent mean ± S.E.M., p-value by ratio paired T test. FIG.22B shows CD69 surface expression of CD70-targeting CAR T cell constructs cultured with plate bound CD70 antigen for x hours (data displays 2 donors and 2 technical replicates, ratio paired T test). FIGs.22C and 22D, Cytotoxicity and proliferation of Meso-targeting CAR-T cells cultured with SKOV3 at 3:1 E:T ratio (2 normal donors) (FIG. 22C) and OVCAR3 at 3:1 E:T (2 normal donors) (FIG.22D). Plots show mean ± S.E.M. with p- values by 2 way ANOVA. FIG.22E shows cytotoxicity and proliferation of Meso-targeting CAR-T cells cultured with Nomo-1 at 3:1 E:T (1 normal donor, representative of 2 normal donors). FIG.22F shows fold expansion of MESOĮ^CD19 and MESOĮ^VEGF CAR-T cells from restimulation assays with Meso+K562 (data displays 2 normal donors, mean ± S.E.M., p-values by 2 way ANOVA). FIGs.22G-22J show cytotoxicity assays of mCherry+ CD70Į^CD19 and CD70Į^VEGF CAR-T cells cultured with GFP+ 786o at 2:1 E:T (data displays 3 normal donors) (FIG.22G), SKOV3 at 3:1 E:T (data displays 2 normal donors) (FIG.22H), and Nomo-1 at 3:1 E:T (data displays 1 normal donor) (FIG.22I). Plots show mean ± S.E.M. with p-values by 2 way ANOVA. FIG.22J shows concentration of VEGF from supernatant of cytotoxicity assay in FIG.22H (data displays 2 donors with 2+ technical replicates, mean with SEM, one way ANOVA). Cytotoxicity assays were performed using incucyte imaging system and wells were imaged every 1-2 hours. Data represents ^m/image of red or green fluorescence area. FIGs.23A-23C relate to enhancement of anti-tumor activity against metastatic orthotopic models of lung and ovarian cancer by VEGF scFv-secreting CAR-T cells. FIG.23A shows bioluminescent (BLI) quantification inclusive of 2 separate experiments, n=15 mice/group. P- values represent 2-way ANOVA. FIG.23B shows results from NSG mice injected intraperitoneally with 1e6 SKOV3 tumor cells. After 7 days of engraftment, mice were treated with 5e6 mesothelin-targeted CAR T cells. FIG.23C shows BLI quantification inclusive of mice pooled from 3 distinct experiments with two separate donors, n=20 mice per group. P-values represent 2way ANOVA. FIGs.24A-B show that expression of VEGFR1 was unchanged following CAR-T cell stimulation with phorbol 12-myristate 13-acetate (PMA) on anti-Meso CAR-T cells (FIG.24A) and anti-CD70 CAR-T cells (FIG.24B). Symbols represent technical triplicates of normal donors. Bars represent mean ± S.D. FIGs.25A-25C show that supernatant containing a VEGF-blocking scFv is not sufficient to impair tumor growth in vitro. FIG.25A is a growth curve of OVCAR3 cells. FIG.25B is a growth curve of Nomo-1 cells. FIG.25C is a growth curve of 786o cells. Cell lines were co- cultured with supernatant from CAR-T cells producing ĮCD19 scFv of ĮVEGF scFv. Data represent technical triplicates, mean ± S.E.M., p-values determined by 2-way ANOVA. FIGs.26A-26C demonstrate that anti-Meso and ant-CD70 CAR-T cells secreting ĮCD19 scFv and/or ĮVEGF scFv are phenotypically similar and show no difference in expression of exhaustion markers after CAR-T cell production. FIG.26A shows the phenotypic profile of CAR-T cells following 14 days of scFv production. Fig.26B shows the expression of exhaustion-associated markers on CAR-T cells following 14 days of scFv production. FIG.26C shows exemplary gating schema for FIGs.26A and 26B. Data show mean ± S.E.M. DETAILED DESCRIPTION In some aspects, this disclosure describes a T cell (e.g., a CAR-T cell) comprising a heterologous polynucleotide encoding a Vascular Endothelial Growth Factor (VEGF) binding protein. A “T cell” refers to a T lymphocyte. In some embodiments, the T cell is a cytotoxic T- cell. In some embodiments, the T cell is CD8+. In some embodiments, the T cell is a helper T cell. In some embodiments, the T cell is CD4+. In some embodiments, the T cell is CD8+ and CD4+ positive. In some embodiments, the T cell is regulatory T cell. In some embodiments, the T cell is a memory T cell. In some embodiments, the T cell is an innate-like T cell or unconventional T cell. In some embodiments, the T cell is a natural killer T cell. In some embodiments, the T cell is a mucosal associated invariant T cell. In some embodiments, the T cell is a gamma delta T cell. In some embodiments, the T cell is a T cell therapy and is an autologous T cell therapy. In some embodiments, the T cell is a T cell therapy and is an allogeneic T cell therapy. In some embodiments, the T cell is modified to comprise a chimeric antigen receptor (e.g., the T cell is a CAR-T cell). CAR-T cells of this disclosure are described throughout this application including in the section, “Chimeric Antigen Receptors (CARs)”. A "polynucleotide" is used herein interchangeably with "nucleic acid molecule" or “polynucleic acid” to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double- stranded forms (and complements of each single-stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein the term, “heterologous nucleic acid molecule” refers to a nucleic acid molecule that does not naturally exist within a given cell. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated. A “heterologous polynucleotide” refers to a polynucleotide that does not naturally exist within a given cell. The term “encoding” when used in the context of a polynucleotide encoding a protein (e.g., a heterologous polynucleotide encoding a VEGF binding protein), means the heterologous polynucleotide comprises a polynucleic acid encoding the protein. For example, the heterologous polynucleotide encoding the VEGF binding protein may also comprise an additional polynucleotide encoding additional proteins or polynucleic acids, including, but not promoters, terminators, signal peptides, linkers, and chimeric antigen receptors (CARs). A "vector," refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. In some embodiments, a vector may comprise a polynucleotide described herein (e.g., a polynucleotide comprising a polynucleic acid sequence encoding a VEGF binding protein and/or a CAR). As used herein, the term "expression vector" may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. As used herein, the term "viral vector" may refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. By "recombinant vector" may be a vector that includes a heterologous nucleic acid sequence or "transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration. VEGF binding proteins “Vascular Endothelial Growth Factor (VEGF)” refers to a protein or family of proteins that are growth factors for vascular endothelial cells and an angiogenic factor. VEGF is also known to play a role in numerous physiological functions including bone formation, hematopoiesis, wound healing, and development. VEGF is also upregulated in many cancers and promotes vascularization of cancer. VEGF can exert physiological effects by binding to VEGF receptor 1 (VEGFR1) and/or VEGF receptor 2 (VEGFR2). In some embodiments, VEGF refers to a family of VEGF proteins (e.g., VEGF-A, VEGF-B, VEGF-C, and VEGF-D) and isoforms thereof. A “Vascular Endothelial Growth Factor (VEGF) binding protein” refers to protein that is capable of binding to a VEGF protein family member. In some embodiments, the VEGF binding protein binds to VEGF-A, VEGF-B, VEGF-C, and VEGF-D. In some embodiments, the VEGF binding protein binds to VEGF-A. In some embodiments, the VEGF binding protein binds selectively to VEGF-A over VEGF protein family members. In some embodiments, the VEGF binding protein inhibits the function of VEGF (e.g., VEGF-A). For example, a VEGF binding protein, upon binding VEGF, may inhibit the activity of VEGF on a cell by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) compared to a cell where VEGF binding protein is not present. In some embodiments, the VEGF binding protein inhibits the activity of VEGF to below detectable levels. In some embodiments, the VEGF binding protein inhibits activity of VEGF in a cell to a level that is similar to or equivalent to the level achieved by knocking out VEGF. VEGF activity may be determined measuring VEGFR1 and/or VEGFR2 signaling effects (e.g., changes in cytokine expression). In some embodiments, the VEGF binding protein inhibits binding of VEGF (e.g., VEGF- A) to VEGFR1 or VEGFR2. For example, a VEGF binding protein, upon binding VEGF, may inhibit the binding of VEGF to VEGFR1 or VEGFR2 by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) compared binding of VEGF in the absence of a VEGF-binding protein. In some embodiments, the VEGF binding protein comprises an antibody. VEGF antibodies are known in the art, e.g., as described in US9421256, US11111291, US10072075, US9777059, US2015024612, and US8092797, the antibody amino acid sequences (including CDR sequences, VH sequences, VL sequences, and an antibody fragment sequences) are incorporated by reference in their entirety. In some embodiments, the VEGF binding protein (e.g., a VEGF binding antibody or antibody fragment) comprises an amino acid sequence of a VEGF binding antibody (e.g., a CDR sequence (i.e., CDR1-H1, CDR2-H2, CDR3-H3, CDR1- L1, CDR2-L2, and/or CDR3-L3), a VH sequence, or a VL sequence) of any one of US9421256, US11111291, US10072075, US9777059, US2015024612, and US8092797. In some embodiments, the VEGF binding protein comprises an antibody fragment (e.g., an antigen-binding fragment (Fab), a Fab’, or a F(ab’)2, a fragment variable (Fv), or a single chain variable fragment (scFv)). The antibody fragment may comprise portions of the antibody sequences (e.g., CDR sequences, VH sequences, VL sequences, and an antibody fragment sequences) of US9421256, US11111291, US10072075, US9777059, US2015024612, and US8092797. In some embodiments, the VEGF binding protein comprises an scFv. In some embodiments, the VEGF binding protein is an scFv. In some embodiments, the VEGF binding protein (e.g., a VEGF binding scFv) comprises (i) a VH domain comprising three complementary determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 70, CDR-H2 comprises SEQ ID NO: 71, and CDR-H3 comprises SEQ ID NO: 72, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 73, CDR-L2 comprises SEQ ID NO: 74, and CDR-L3 comprises SEQ ID NO: 75. In some embodiments, the VH and/or the VL comprise 1 or more mutations in framework regions of the VH and VL. In some embodiments, the VEGF binding protein (e.g., a VEGF binding scFv) comprises a peptide linker between the VH and the VL. In some embodiments, the VEGF binding protein comprises an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity with SEQ ID NO: 76. In some embodiments, the VEGF binding protein comprises an scFv having an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity with SEQ ID NO: 76. In some embodiments, the VEGF binding protein (e.g., VEGF scFv) comprises from N- terminal to C-terminal the VH domain followed by the VL domain. In some embodiments, the VEGF binding protein comprises from N-terminal to C-terminal the VL domain followed by the VH domain. In some embodiments, the heterologous polynucleotide encoding the VEGF binding protein is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is a tissue- specific promoter (e.g., an immune cell-specific promoter). In some embodiments, the promoter is an inducible promoter (e.g., a tet or lac promoter). In some embodiments, the promoter is selected from the group consisting of a CMV promoter, an EF1-alpha promoter, a CAG promoter, a PGK promoter, H1 promoter, or a U6 promoter. In some embodiments, the U6 promoter is from a non-human species. In some embodiments, the U6 promoter is from a human U6 promoter. In some embodiments, the U6 promoter is from cow, mice, rat, pig, yeast, dog, cat, drosophila, or C. elegans. In some embodiments, the promoter is a EF1-alpha promoter. In some embodiments, the T cell comprising the heterologous polynucleotide encoding a VEGF binding protein secretes the VEGF binding protein. In some embodiments, the heterologous polynucleotide encoding the VEGF binding protein comprises a polynuceic acid encoding a VEGF binding protein and a polynucleic acid encoding a secretory signal peptide. In some embodiments, the secretory signal peptide is Human OSM signal peptide, VSV-G signal peptide, Mouse Ig Kap signal peptide, Mouse Ig Heavy signal peptide, BM40 signal peptide, Secrecon signal peptide, Human IgKVIII signal peptide, CD33 signal peptide, tPA signal peptide, Human Chymotrypsinogen signal peptide, Human trypsinogen-2 signal peptide, Human IL-2 signal peptide, Gaussia luc signal peptide, Albumin(HSA) signal peptide, Influenza Haemagglutinin signal peptide, Human insulin signal peptide, or Silkworm Fibroin signal peptide. In some embodiments, the secretory signal peptide comprises a IgK secretory signal peptide. In some embodiments, the secretory signal peptide comprises an amino acid sequence of SEQ ID NO: 56. VEGF knockout T cells In some aspects, this disclosure describes a T cell comprising a VEGF knockout. A “VEGF knockout” refers to a VEGF gene deletion or a VEGF gene comprising a loss of function mutation. A VEGF knockout may be specific to a particular VEGF family member (e.g., VEGF-A). A VEGF knockout may be general knocking out most or all VEGF family members. In some embodiments, the loss of function mutation inhibits VEGF activity (e.g., VEGF binding to VEGFR). For example, the loss of function mutation may inhibit the activity of VEGF on a cell by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) compared to a cell where VEGF binding protein is not present. In some embodiments, the loss of function mutation is a point mutation. In some embodiments, the loss of function mutation is an early stop codon. In some embodiments, the loss of function mutation is an insertion or a deletion. In some embodiments, the loss of function mutation is an insertion or deletion that results in a frameshift. In some embodiments, a VEGF gene deletion refers to removal of the entire gene (i.e., a specific VEGF family member like VEGF-A, or deletion of all VEGF family members) from the chromosome. In some embodiments, a VEGF knockout comprises a knockout of at least one VEGF gene (e.g., knockout of VEGF-A. In some embodiments, a VEGF knockout comprise a knockout of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D. In some embodiments, this disclosure describes a CAR-T cell (e.g., as described herein) comprising a VEGF knockout. In some embodiments, the CAR-T cell comprises a CAR that binds to CD70 and a VEGF knockout. In some embodiments, the CAR-T cell comprises a CAR that binds to mesothelin and a VEGF knockout. In some embodiments, the T cell VEGF knockout is produced using a CRISPR (e.g., a Cas protein (e.g., as described herein) and a corresponding guide RNA that comprises a homology region complementary to VEGF. VEGF targeting CRISPR guide RNAs In some embodiments, this application discloses a T cell comprises a polynucleotide encoding a guide RNA that is complementary to VEGF. In some embodiments, the T cell is a CAR-T cell. In some embodiments, the CAR-T cell is a CD70 CAR-T cell or a Mesothelin CAR-T cell. In some embodiments, the CAR-T cell comprises a polynucleotide encoding the CAR and the guide RNA. As used herein the term “clustered regularly interspaced short palindromic repeats” or “CRISPR” may refer to a gene editing system that comprises a guide RNA component and a CRISPR associated (Cas) protein component. The guide RNA polynucleotide may comprise a homology region that is complementary to a target gene and a stem loop region that is capable of binding to a Cas protein. The Cas protein may comprise a guide RNA binding site and nuclease activity. The Cas protein and the guide RNA may form a complex that is capable of binding to the target gene (based on the homology region), and cleaving the DNA (using the nuclease activity of the Cas protein). In some embodiments, the Cas protein guide RNA complex binds to sequence that is adjacent to and downstream of a protospacer adjacent motif (PAM). Cleavage results in a DNA strand break and repair of that strand break may introduce a mutation (e.g., single nucleotide polymorphism, insertion, or deletion). In some embodiments, the Cas protein is any suitable Cas protein for mutating and/or altering the expression of a target gene (a Cas protein may also be referred to as a CRISPR protein herein). In some embodiments, the Cas protein is selected from the group consisting of a Cas9 protein, a Cas12 protein, or a Cas13 protein. The skilled person will understand that Cas proteins (e.g., Cas9) may have many different orthologs (e.g., SpyoCas9, spCas9, spyCas9, and geoCas9). In some embodiments, the Cas protein is SpyoCas9. Cas proteins and orthologs thereof are well known in the art as discussed in Gasiunas, Giedrius, et al, Nature communications 11.1 (2020): 1-10; and Fancheng Y et al., Cell Biology and Toxicology 35.6 (2019): 489-492, each of which is incorporated by reference in its entirety. Methods for designing guide RNAs (e.g., selecting homology region sequences for targeting a specific gene) are also well known in the art as described in Liu, Guanqing L. et al., Computational and Structural Biotechnology Journal 18 (2020): 35-44, which is incorporated by reference in its entirety. In some embodiments, gRNAs (encoded by gRNA polynucleotides) are designed using CRISPick (portals.broadinstitute.org /gppx/crispick/public), which performs as described in Doench et al., Nature Biotechnology, 34(2), 184-191 (2016) and Sanson et al., Nature Communications, 9(1), 5416 (2018), both of which are incorporated by reference in their entirety. As used herein, the term “guide RNA (gRNA) polynucleotide” refers to a DNA or an RNA polynucleotide that encodes a guide RNA (gRNA). A guide RNA polynucleotide comprises a sequence that binds to a clustered regularly interspaced short palindromic repeats (CRISPR) protein or CRISPR-related protein and a sequence and comprises an additional sequence that is complementary to a target polynucleotide (i.e., a homology region). For example, a guide RNA polynucleotide may be a Cas9 protein guide RNA polynucleotide or a Cas12 protein guide RNA polynucleotide. Cas9 protein guide RNAs are compatible with Cas9 CRISPR proteins and are well known in the art e.g., as described in Adli et. al., Nature communications 9.1 (2018): 1-13, which is incorporated by reference in its entirety. Cas12 protein guide RNA polynucleotides are compatible with Cas12 CRISPR proteins and are well known in the art e.g., as described in Zetsche et al., Cell 163.3 (2015): 759-771, which is incorporated by reference in its entirety. In some embodiments, a guide RNA polynucleotide is a base editor guide RNA polynucleotide. In some embodiments, the gRNA is a prime editing guide RNA polynucleotide. In some embodiments, a guide RNA polynucleotide encodes a homology region (e.g., spacer) and a region that binds to a CRISPR protein (e.g., a direct repeat). In some embodiments, the guide RNA polynucleotide is a single guide RNA polynucleotide comprising a homology region and a region that binds to a CRISPR protein. In some embodiments, the homology region comprises a sequential series of about 10-30 or about 15-25 nucleotides. In some embodiments, the homology region comprises about 20 nucleotides. In some embodiments, the homology region is complementary to a target gene (e.g., a gene associated with immune cell function). In some embodiments, the gRNAs are designed using an algorithm (e.g., CRISPick). CRISPick is described in Kim et al., Nat Biotechnology 36, 239–241 (2018); Doench et al. Nature Biotechnology, 34(2), 184-191 (2016); and Sanson et al., Nature Communications, 9(1), 5416 (2018), each of which are incorporated by reference in their entirety. In some embodiments, the gRNA polynucleotides are not cross-reactive or minimally cross-reactive. Cross-reactive gRNA polynucleotides refer to guide RNA polynucleotides comprising homology regions having sufficient complementarity with more than one target polynucleotide (e.g., gene sequence) such that the gRNA may induce a CRISPR mutation in more than one target polynucleotide. Design algorithms may be used in guide RNA polynucleotide design to decrease the chances of cross-reactivity (e.g., gRNAs may be scored for on-target activity using Rule Set 3(RS3) with sequence and target information and the Chen2013 tracr (Chen, Baohui, et al. Cell 155.7 (2013): 1479-1491). gRNAs may also be scored for off-target activity using Tier-agnostic 1 mismatch aggregated Cutting Frequency Determination (CFD) scores). The skilled person will understand the gRNA polynucleotides designed with such an algorithm may still have some degree of cross-reactivity, however, the risk of cross reactivity is expected to be decreased or may be specified at a selected threshold in the algorithm. In some embodiments, cross reactivity is a function of complementarity. In some embodiments, gRNA polynucleotides that are not cross-reactive do not have greater than 80% complementarity to more than 1 gene. In some embodiments, gRNA polynucleotides that are not cross-reactive do not have greater than 85% complementarity to more than 1 gene. In some embodiments, gRNA polynucleotides that are not cross-reactive do not have greater than 90% complementarity to more than 1 gene. In some embodiments, gRNA polynucleotides that are not cross-reactive do not have greater than 95% complementarity to more than 1 gene. The term “complementary” as described herein refers to the degree of Watson-Crick base pairing between two polynucleotides. For example, two polynucleotides may be 90% complementary if 9/10 nucleotides of each of the polynucleotides form a Watson Crick base pair. In some embodiments, complementary may refer to at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of nucleotides in a first polynucleotide Watson- Crick base pairing with a second polynucleotide. In some embodiments, a homology region of a gRNA is complementary to a gene sequence when the homology region is capable of hybridizing to the gene sequence and at least a threshold percentage (e.g., at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of nucleotides are Watson Crick base pairs to the gene sequence. In some embodiments, a homology region is complementary to a gene sequence when the homology region is capable of hybridizing to the gene sequence and initiating cleavage of the gene sequence by a CRISPR protein and at least a threshold percentage (e.g., at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of nucleotides are Watson Crick base pairs to the gene sequence. In some embodiments, a homology region is complementary to a target gene sequence when the nucleotides of the homology region are 100% complementary to a sequential portion of the target gene sequence (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 sequential nucleotides of the target gene sequence, e.g., 20 sequential nucleotides). In some embodiments, the homology region is complementary to the sense strand of the target gene sequence. In some embodiments, the homology region is complementary to the anti-sense strand of the target gene sequence. In some embodiments, a homology region that is complementary to a gene encoded by a sequence may refer to a homology region that is complementary to either the sense strand of the gene sequence or the antisense strand of the gene sequence. In some embodiments, the homology region is complementary to a region of the target gene sequence this is adjacent to a protospacer adjacent motif (PAM). In some embodiments, the homology region is complementary to a region of the target gene sequence that is downstream of and adjacent to a protospacer adjacent motif (PAM). In some embodiments, the guide RNA comprises a homology region that is complementary to polynucleotide encoding a VEGF family protein e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D. In some embodiments, the guide RNA comprises a homology region that is complementary to VEGF-A (e.g., VEGF-A (Ensembl:ENSG00000112715 MIM:192240; AllianceGenome:HGNC:12680)). Chimeric Antigen Receptors (CARs) In some embodiments, the T cell comprising a heterologous polynucleotide encoding a Vascular Endothelial Growth Factor (VEGF) binding protein further comprises a chimeric antigen receptor (CAR). In some embodiments, the heterologous polynucleotide comprises a polynucleic acid encoding the VEGF binding protein and a polynucleic acid encoding the CAR. In some embodiments, the heterologous polynucleotide comprises, from 5’ to 3’, a polynucleic acid encoding the VEGF binding protein and a polynucleic acid encoding the CAR. In some embodiments, the heterologous polynucleotide comprises, from 5’ to 3’ a polynucleic acid encoding the CAR and a polynucleic acid encoding the VEGF binding protein. The terms "chimeric antigen receptor" or "CAR" or "CARs", as used herein, refer to engineered T cell receptors, which graft a ligand or antigen specificity onto T cells. In some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells or combinations thereof. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. A CAR places an antigen binding domain that specifically binds a target, e.g., a polypeptide, expressed on the surface of a cell to be targeted for a T cell response, onto a construct including a transmembrane domain and intracellular domain(s) of a T cell receptor molecule. In some embodiments, the antigen binding domain includes the antigen domain(s) of an antibody that specifically binds an antigen expressed on a cell to be targeted for a T cell response. In some embodiments, the antigen binding domain includes a ligand that specifically binds an antigen expressed on a cell to be targeted for a T cell response. As used herein, a "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, CARs have the ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Any cell-surface moiety can be targeted by a CAR. Often, the target will be a cell- surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. In some embodiments, the antigen binding domain binds to any one of CD19, CD37, CD70, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19/CD79b, BCMA/TACI, or is a TriPRIL antigen binding domain, e.g., as described in PCT/US2020/065733, PCT/US2020/036108, PCT/US2018/013215, PCT/US2018/013213, PCT/US2018/027783, PCT/US2018/013221, PCT/US2018/022974, PCT/US2019/042268, PCT/US2019/038518, PCT/US2019/066357, PCT/US2019/013103, PCT/US2019/017727, PCT/US2020/051018, and/or PCT/US2018/013095. Antigen binding domain As used herein, the term "antigen binding domain" refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding to a target. In some embodiments, the CARs described herein comprise an antigen binding domain. The antigen binding domain will specifically bind to its binding partner, i.e., the target. As non-limiting examples, the antigen binding domain can include an antigen domain of an antibody, or a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule that binds specifically to a portion of a protein and/or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand:cognate partner binding can result in the alteration of the ligand-bearing receptor, or activate a physiological response, for example, the activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a conserved function across at least two species. Any cell-surface moiety can be targeted by a CAR (e.g., the antigen binding domain of the CAR). In some embodiments, the target will be a cell-surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. To target Tregs, antibodies can be targeted against, e.g., Glycoprotein A Repetitions Predominant (GARP), latency-associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB, and LAG-3. In some embodiments, the CAR vector comprises a CAR polynucleotide encoding an antigen binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19/CD79b, BCMA/TACI, or is a TriPRIL antigen binding domain. In some embodiments, the CAR vector comprises a CAR comprising an antigen binding domain that binds mesothelin. In some embodiments, the mesothelin CAR comprises a polynucleotide encoding an antigen binding domain comprising a mesothelin antibody (e.g., scFv). In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 2, or a variant thereof. In some embodiments, the mesothelin scFv comprises SEQ ID NO: 3 or SEQ ID NO: 4, or a variant thereof. In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 5 and a VL domain of SEQ ID NO: 6, or a variant thereof. In some embodiments, the CAR vector comprises a CAR comprising an antigen binding domain that binds CD70. In some embodiments, the CD70 binding domain comprises CD27 or a CD70-binding fragment of CD27 (e.g., comprises SEQ ID NO: 66 or an CD70-binding variant thereof). In some embodiments, the CD70 binding domain comprises the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 65. In some embodiments, the CD70 binding domain comprises the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 66. Hinge and Transmembrane Domains In some embodiments, the CAR polypeptide further comprises a transmembrane domain, or a hinge/transmembrane domain, which joins the antigen binding domain to the intracellular signaling domain. The binding domain of the CAR is, in some embodiments, followed by one or more "hinge domains," which plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding (by the antigen binding domain) and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8alpha), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, the CAR comprises polynucleotide encoding CD8alpha hinge/transmembrane domain. In some embodiments, the CAR comprises a polynucleotide encoding a 41BB intracellular domain. In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin like protein (e.g., lgA, lgD, lgE, lgG, or lgM), CD28, or CD8. In some embodiments, the hinge domain includes a CD8a hinge region. As used herein, "transmembrane domain" (TM domain) refers to the portion of the CAR that fuses the extracellular binding portion, in some embodiments via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and intracellular signaling domain) and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR, which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used herein, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR. As used in relation to a transmembrane domain of a protein or polypeptide, "fragment thereof" refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface. In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T cell receptor, CD2, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4- 1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. As used herein, a "hinge/transmembrane domain" refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge/transmembrane domain can be derived from the hinge/transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge/transmembrane domain is a CD27 hinge/transmembrane domain (e.g., SEQ ID NOs: 68 and 69). In some embodiments, the hinge/transmembrane domain of a CAR or fragment thereof is derived from or includes the hinge/transmembrane domain of CD8 (e.g., SEQ ID NO: 49, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha or CD8a) and beta (CD813 or CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and/or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD8 sequence. In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO: 49; or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 49. Co-stimulatory Domains Each CAR described herein optionally includes the intracellular domain of one or more co-stimulatory molecule or co-stimulatory domain. As used herein, the term "co-stimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fe receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4-1BB, CD27, CD28, or OX40. In one example, a 4-1BB intracellular domain (ICD) can be used (see, e.g., below and SEQ ID NO: 53, or variants thereof). Additional illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB.4-1BB (CD137; TNFRS9) is an activation induced costimulatory molecule and is an important regulator of immune responses. 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily.4-1BB is expressed on activated T lymphocytes.4-1BB sequences are known for a number of species, e.g., human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5).4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BB can refer to the 4-1BB of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and/or orthologs of human 4-1BB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4-1BB sequence. Intracellular Signaling Domains In some embodiments, the CAR comprises a polynucleotide encoding a CD3zeta intracellular signaling domain. The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target/antigen binding domains(s) render the receptor sensitive to signaling activation when the chimeric target/antigen binding domain binds the target/antigen on the surface of a targeted cell. With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3-zeta signals upon antigen binding by the antigen binding domain. So-called "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3-zeta;) domains, and so-called "third-generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3-zeta). In various embodiments, the CAR is selected to have high affinity or avidity for the target/antigen - for example, antibody-derived target or antigen binding domains will generally have higher affinity and/or avidity for the target antigen than would a naturally occurring T cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific T cell targeting by CAR-T cells. CARs as described herein include an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is from CD3-zeta; (see, e.g., below). Additional non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)- containing intracellular signaling domains that are of particular use in the technology include those derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d. CD3 is a T cell co-receptor that facilitates T lymphocyte activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co-stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammalian CD3 consists of a CD3-gamma chain, a CD3delta chain, and two CD3-epsilon chains. These chains associate with a molecule known as the T cell receptor (TCR) and the CD3- zeta to generate an activation signal in T lymphocytes. A complete TCR complex includes a TCR, CD3-zeta, and the complete CD3 complex. In some embodiments of any aspect, a CAR polypeptide described herein includes an intracellular signaling domain that includes an Immunoreceptor Tyrosine-based Activation Motif or ITAM from CD3-zeta, including variants of CD3-zeta such as ITAM-mutated CD3- zeta, CD3-eta, or CD3-theta. In some embodiments of any aspect, the ITAM includes three motifs of ITAM of CD3-zeta (ITAM3). In some embodiments of any aspect, the three motifs of ITAM of CD3-zeta are not mutated and, therefore, include native or wild-type sequences. In some embodiments, the CD3-zeta sequence includes the sequence of a CD3-zeta as set forth in the sequences provided herein, e.g., a CD3-zeta sequence of SEQ ID NO: 54, or variants thereof. For example, a CAR polypeptide described herein includes the intracellular signaling domain of CD3-zeta. In some embodiments, the CD3-zeta intracellular signaling domain corresponds to an amino acid sequence of SEQ ID NO: 54 or includes a sequence of SEQ ID NO: 54; or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence of SEQ ID NO: 54. In some embodiments, the intracellular domain is the intracellular domain of a 4-1BB. In some embodiments, the 4-1BB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO: 53; or includes a sequence selected from SEQ ID NO: 53; or includes at least 75%, at least 80%, at least 85%, 35 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 53. Individual CAR and other construct components as described herein can be used with one another and swapped in and out of various constructs described herein, as can be determined by those of skill in the art. Each of these components can include or consist of any of the corresponding sequences set forth herein, or variants thereof. A more detailed description of CARs and CAR-T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol.32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res.18:6436-6445, 2012; each of which is incorporated by reference herein in its entirety. Signal Peptide In some embodiments, a CAR polypeptide as described herein includes a signal peptide. Signal peptides can be derived from any protein that has an extracellular domain or is secreted. A CAR polypeptide as described herein may include any signal peptides known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, e.g., a CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO: 55 or including an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 55. In further embodiments, a CAR polypeptide described herein may optionally exclude one of the signal peptides described herein, e.g., a CD8 signal peptide of SEQ ID NO: 55 or an IgK signal peptide of SEQ ID NO: 56. Linker Domain In some embodiments, the CAR further includes a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains/regions of the CAR as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Linker sequences may be from 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids in length, and include any suitable linkers known in the art. For instance, linker sequences may include, but are not limited to, glycine/serine linkers, e.g., SEQ ID NOs: 57-60 as described by Whitlow et al., Protein Eng.6(8):989-95, 1993, the contents of which are incorporated herein by reference in its entirety; the linker sequence of SEQ ID NO: 61 as described by Andris-Widhopf et al., Cold Spring Harb. Protoc.2011 (9), 2011, the contents of which are incorporated herein by reference in its entirety; as well as linker sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing Cre-Lox recombination site as described by Sblattero et al., Nat. Biotechnol.18(1):75-80, 2000, the contents of which are incorporated herein by reference in its entirety. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Furthermore, linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 62) and T2A (SEQ ID NO: 63), 2A-like linkers or functional equivalents thereof and combinations thereof. In various examples, linkers having sequences as set forth herein, or variants thereof, are used. It is to be understood that the indication of a particular linker in a construct in a particular location does not mean that only that linker can be used there. Rather, different linker sequences (e.g., P2A and T2A) can be swapped with one another (e.g., in the context of the constructs of this disclosure), as can be determined by those of skill in the art. In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. Linkers such as these can be used in the context of polyproteins, such as those described below. For example, they can be used to separate a CAR component of a polyprotein from a therapeutic agent (e.g., an antibody, such as a scFv, single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) component of a polyprotein (see below). In some embodiments, a P2A linker sequence comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, a T2A linker sequence comprises the amino acid sequence of SEQ ID NO: 63. Exemplary CARs In some embodiments, the CAR is selected from a group consisting of (1) a CAR that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, (2) a CAR that binds to any pair of CD19/CD79b, BCMA/TACI, or (3) is a TriPRIL antigen binding domain. In some embodiments, the CAR polypeptide comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity of a sequence selected from any one of SEQ ID NOs: 7-47. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-47. In some embodiments, the CAR polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 7-47. In some embodiments, the CAR polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 7-47. In some embodiments, the CAR comprises a polynucleotide encoding a Mesothelin scFv, a CD8alpha hinge/transmembrane, a 41BB intracellular domain, and a CD3zeta signaling domains. In some embodiments, the CAR polypeptide comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to a sequence selected from any one of SEQ ID NOs: 7-10. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. In some embodiments, the CAR polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. In some embodiments, the CAR comprises a polynucleotide encoding a CD70 binding CD27 fragment, a CD27 transmembrane domain, a 4-1BB costimulatory domain, and a CD3- zeta intracellular signaling domain. In some embodiments, the CAR polypeptide comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to SEQ ID NOs: 30-35. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 30-35. In some embodiments, the heterologous polynucleotide encodes an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, In some embodiments, the heterologous polynucleotide encodes an amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, the heterologous polynucleotide comprises a polynucleic acid encoding an amino acid sequence of a CAR (e.g., as described herein) and a polynucleic acid encoding a guide RNA that comprises a homology region complementary to VEGF (e.g., VEGF-A). In some embodiments, the guide RNA comprises a homology region that is complementary to human VEGF-A (Ensembl:ENSG00000112715 MIM:192240; AllianceGenome:HGNC:12680). In some embodiments, the polynucleotide comprises from 5’ to 3’, the polynucleic acid encoding an amino acid sequence of a CAR and then a polynucleic acid encoding a guide RNA that comprises a homology region complementary to VEGF. In some embodiments, the polynucleotide comprises from 5’ to 3’, then a polynucleic acid encoding a guide RNA that comprises a homology region complementary to VEGF and the polynucleic acid encoding an amino acid sequence of a CAR. In some embodiments, the polynucleic acid encoding the CAR and the polynucleic acid encoding the VEGF-binding protein are operably linked to different promoters. In some embodiments, the polynucleic acid encoding the CAR and the polynucleic acid encoding the VEGF-binding protein are operably linked to the same promoter. In some embodiments, the polynucleotide encodes a cleavable linker between the CAR and the VEGF-binding protein (e.g., a 2A peptide as described herein). Table 1: Sequences SEQ^ID^ Description^ Sequence^ NO:^ ^ ^ Y L V S S Y L V D S S S L G I S S R S T G C M S T P G F L G S SK LK

GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSH NEDYTIVEQYERAEGRHSTGGMDELYK^ ^ ^ T K N P H A R A R V K R N G L A S T G C M S T P G A L G S SK LK H T K N P H A R A Y

APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM G V Y SS G I RL TL G T G T L D G R P L P P K L I V G R T E G R T RL TL G T

TYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGG SYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLT L D G R P L P P K L I V G R T E G R T L I V R K S K S F V C S P

APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC E G V VI A S R S V VI S E L V VI S E A S FT Y K T G E I S R V V

hinge^+^TM/4^ TITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGS 1BB/CD3^ɺ]^ GSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRTTT W G E I S T G K S F T S R D P G F L G S R V V T G K P L C D S T Q P K

RTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE G A S R T G S T D R E Q K A S V H A S C G Q K S I V LL N R A R V K

^ EGFRvIII^CAR^ EIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLE (3C10)^ WIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAV M R H G K G Y E T Q V P R R P GL C N E M E R R D T S E G A C N F Q G Y

^ CD70^CAR^4^ ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQC DPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRN S L C E G C N C G C N P N L S R P V Q R G P LY D A E A G Q T R K S K G Y

FCQQSKEAPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSEV QLQQSGPELVKPGASVKISCKTSGYTFTESTIHWVKQSHGKSLEWI Y SL C F G Q G V C F G S R P V Q R G P LY D A S R P V Q R A Q A S T T M L SE G

Leader^–^anti^ SGTDFRLNIHPLEEDDTGMYFCQQSKEAPPTFGGGTKLEIKGGGG TACI^L^H^–^linker^ SGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKTSGYTF ^ ^ ^ ^ K G S R P V Q R G P LY D A S R P V Q R Q G T K K Q R Q Q G V C F G S R P V Q R A

GTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV K S R P V Q G E S E G A P S Y SE G G TF K G S R P V Q R A R V K S R P V Q G Y D G

GSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKTSGYTFTESTI HWVKQSHGKSLEWIGGISPNNGGSPFNQKFKGKATLTVDKSSSTV P L C D G V T S T R R Q Q IY IY S^ Q Q E D

^ CD27^ MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQM CEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHC S H S F L A E G Y C P G E R R Q TL V G G A

TWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSG WGLDIWGQGTLVTVSSHHHHHH^ ^ ^ ^ T K N P H A R A R V K F S A L D

Methods of Treatment and Administration In some aspects, this disclosure describes a method of treating cancer in a subject, the method comprising administering a T cell described herein (e.g., a CAR-T cell comprising a polynucleotide encoding a CAR and a VEGF binding protein). “Treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease, a symptom of the target disease, or a predisposition toward the target disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease. In some embodiments, the method comprises treating a subject having a solid tumor. The term “solid tumor” as used herein refers to a tumor characterized by a solid mass of cancer cells. Solid tumors include, but are not limited to, lung cancer, brain cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, testicular cancer, skin cancer, throat cancer, liver cancer, breast cancer, and colon cancer. Solid tumors do not include liquid tumors. In some embodiments, the method comprises treating a subject having a liquid tumor (e.g., a leukemia, lymphoma or myeloma). In some embodiments, the method comprises administering a CAR-T cell to the subject that comprises a CAR antigen binding domain, which binds to an antigen expressed by tumor cells of the subject. For example, administering a CD70 binding CAR-T cell to a subject having a CD70 expressing cancer. Cancers that express CD70 are known in the art and include, but are not limited to, bladder cancer, breast cancer (e.g., breast invasive carcinoma), cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC). In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is a B-cell Non-Hodgkin Lymphoma (NHL), mantle cell lymphoma, Burkitt’s lymphoma, B-cell lymphoblastic lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma, or T-cell lymphoma. In some embodiments, the cancer is a leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), or T-cell leukemia. In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the cancer is renal cell carcinoma. For example, administering a mesothelin binding CAR-T cell to a subject having a mesothelin expressing cancer. Mesothelin expressing cancer are known in the art and include, but are not limited to, pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary cancer, gastric cancer, and mesothelioma. In some embodiments, the method comprises administering the T cell to a subject having a cancer associated with increased VEGF expression (e.g., increase relatively to healthy cells of the same type as the cancer cells). Cancer associated with increased VEGF expression are known in the art, and include, but are not limited to colorectal carcinoma, gastric carcinoma, pancreatic carcinoma, breast cancer, prostate cancer, lung cancer, melanoma, and renal cell carcinoma (RCC). In some embodiments, the method comprises administering to a subject having a CD70 expressing cancer, a T cell comprising a heterologous polynucleotide, the heterologous polynucleotide comprising a polynucleic acid encoding a CD70 CAR and a VEGF binding protein (e.g., a VEGF-A binding scFv). In some embodiments, the method comprises administering to a subject having a CD70 expressing renal cell carcinoma, a T cell comprising a heterologous polynucleotide, the heterologous polynucleotide comprising a polynucleic acid encoding a CD70 CAR and a VEGF binding protein (e.g., a VEGF-A binding scFv). In some embodiments, the method comprises administering to a subject having a mesothelin expressing cancer, a T cell comprising a heterologous polynucleotide, the heterologous polynucleotide comprising a polynucleic acid encoding a mesothelin CAR and a VEGF binding protein (e.g., a VEGF-A binding scFv). In some embodiments, the method comprises administering to a subject non-small cell lung cancer, a T cell comprising a heterologous polynucleotide, the heterologous polynucleotide comprising a polynucleic acid encoding a mesothelin CAR and a VEGF binding protein (e.g., a VEGF-A binding scFv). Subject A "subject" refers to a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a CD70 expressing cancer or a mesothelin expressing cancer described herein) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. Dosage "Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example, a unit dosage form can be an amount of therapeutic disposed in a delivery device, e.g., a syringe or intravenous drip bag. In some embodiments, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously. In some embodiments, the activated CAR T cells described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject. A pharmaceutical composition including the T cells described herein can generally be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. If necessary, T cell compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med.30319:1676, 1988). In certain aspects, it may be desired to administer activated CAR-T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom as described herein, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from 3510 cc to 400 cc. In certain aspects, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Administration In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a mammalian cell including any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein, or a nucleic acid encoding any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein. The CAR-T cells described herein include mammalian cells including any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein, or a nucleic acid encoding any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein. Subjects having a condition can be identified by a physician using current methods of diagnosing the condition. Symptoms and/or complications of the condition, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fatigue, persistent infections, and persistent bleeding. Tests that may aid in a diagnosis of, e.g., the condition, but are not limited to, blood screening and bone marrow testing, and are known in the art for a given condition. A family history for a condition, or exposure to risk factors for a condition can also aid in determining if a subject is likely to have the condition or in making a diagnosis of the condition. The compositions described herein may be administered to a subject having or diagnosed as having a disease described herein (e.g., cancer). In some embodiments, the methods described herein include administering an effective amount of activated CAR-T cells described herein to a subject in order to alleviate a symptom of the disease. As used herein, "alleviating a symptom of the disease" is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of a tumor. The term "effective amount" as used herein refers to the amount of activated CAR-T cells needed to alleviate at least one or more symptom of the disease or disorder and relates to a sufficient amount of the cell preparation or composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of activated CAR-T cells that is sufficient to provide a particular anti-condition effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a condition), or reverse a symptom of the condition. Thus, it is not generally practicable to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of activated CAR-T cells, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for bone marrow testing, among others. Modes of Administration Modes of administration can include, for example intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient transarterially, intratumorally, intranodally, intraperitoneally or intramedullary. In some embodiments, the compositions of T cells may be injected directly into a tumor, or lymph node. In some embodiments, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid). In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates can be expanded by contact with an artificial APC, e.g., an aAPC expressing anti-CD28 and anti-CD3 CD Rs, and treated such that one or more CAR constructs of the technology may be introduced, thereby creating a CAR-T cell. Subjects in need thereof can subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. Following or concurrent with the transplant, subjects can receive an infusion of the expanded CAR-T cells. In some embodiment, expanded cells are administered before or following surgery. In some embodiments, lymphodepletion is performed on a subject prior to administering one or more CAR-T cell as described herein. In such embodiments, the lymphodepletion can include administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. In some embodiments, a single treatment regimen is required. In others, administration of one or more subsequent doses or treatment regimens can be performed. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. In some embodiments, no additional treatments are administered following the initial treatment. The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. Methods of decreasing T cell exhaustion In some aspects, the inventors discovered engineering T cells (e.g., CAR-T cells) to express VEGF binding proteins (e.g., a VEGF binding scFv) decreases CAR-T cell exhaustion compared to a control CAR-T cell. In some embodiments, a method of decreasing T cell exhaustion comprises transfecting the CAR-T cell with a polynucleotide encoding the VEGF binding protein (e.g., as described herein). Transfecting refers to the process of artificially introducing polynucleotides into a cell (e.g., a T cell). Methods of transfecting cell (e.g., T cells) are well known in the art, e.g., as described in Kumar et al. Advanced Materials 33.21 (2021): 2007421. In some embodiments, the method of deceasing CAR-T cell exhaustion comprises knocking out VEGF in a CAR-T cell (e.g., by introducing a guide RNA polynucleotide complementary to VEGF and a Cas protein into the cell). In some embodiments, the method of decreasing T cell exhaustion comprises engineering a T cell to express a VEGF binding protein. In some embodiments, the method of decreasing T cell exhaustion comprises knocking out the T cell VEGF gene (e.g., using a CRISPR) as described herein. EXAMPLES Example 1 Renal cell carcinoma (RCC) can be an aggressive primary tumor with over 82,000 cases and 15,000 deaths yearly in the United States. Chimeric antigen receptor (CAR)-T cells have demonstrated substantial potency in B cell malignancies but have had limited success in solid tumors. CD70 has emerged as a tumor marker that is overexpressed in almost all cases of clear- cell RCC. While safe, CAR-T cells targeting CD70 alone are insufficient to drive high rates of response in early phase I RCC clinical trials. Separately, decades of foundational research have identified that vascular endothelial growth factor (VEGF)-targeting agents improve outcomes in RCC. Increasing recognition is being paid to the combination of VEGF-targeting agents and modern immunotherapy approaches with significant success in RCC, though half of patients still relapse. Furthermore, renal and cardiovascular toxicities limit doses of systemically administered VEGF-targeted agents. Without the thoughtful unification of CAR-T and VEGF- blockade (^VEGF), synergy between these two potent forms of therapy will not be realized in RCC and other VEGF-dependent malignancies. Determine the effects of secreted VEGF blockade on CD70-CAR-T cell biology Single-cell RNA sequencing (scRNAseq) of the infusion product CAR-T cells from two leading FDA approved CAR-T cells for aggressive lymphomas was recently performed (Harvadhala*, Leick*, et al., Nature Medicine, 2022). Reanalysis of VEGF-pathway members for this application (FIG.1) revealed elevated FLT1 (VEGFR1) and VEGFA among the activated CAR-T product cells among 41BB signaling but not CD28-costimulated CAR-T products. Given the more apparent VEGF pathway flux in 41BB co-stimulated CAR-T cells from the data in FIG.1, a ligand-based CD70 targeted 41BB CAR with a modified hinge to mitigate proteolytic cleavage (Leick et al.2022) was utilized and further modified to secrete a single chain variable fragment (scFv) targeting VEGFA (70^VEGF) or CD19 (70¹⁹) as a control. This was confirmed using the 70^VEGF CAR-T cells, finding elevated VEGFR1 levels on the surface of CAR-T cells activated through the CAR (FIG.1). FIG.1 shows activated CAR-T cells do make VEGF-A and express a cognate receptor (FLT1/VEGFR1) at the RNA level. This was recapitulated in a different context with the 70^VEGF CAR-T cells at the protein level via flow cytometry upon CAR-T stimulation. These suggest signaling through the VEGF-pathway within CAR-T cells for the first time. Using this system and a VEGFR reporter assay, it was shown that the 70^VEGF CAR (but not 70¹⁹) blocked VEGF at a wide range of concentrations (FIG.2). The data in FIG.2, show that the 70^VEGF CAR-T cells secreted a functional scFv sufficient to abrogate VEGFR pathway signaling across nearly 2-logs of biologically relevant concentrations. These data help suggest that the 70^VEGF CAR-T cells generate a functional scFv that blocks VEGF signaling. The effect of ^VEGF scFv on 70^VEGF short term proliferation and long term proliferation was assessed. Plate-bound CD70 stimulation resulted in increased expansion of the 70^VEGF over the 70¹⁹ control CAR-T cells in both the short term (FIGs.3 and 14) and the long term (FIG. 15). From the data in FIG.3, the ^VEGF scFv confers greater short-term expansion capabilities to the 70^VEGF CAR-T cells. These data suggest a tangible fitness advantage conferred by ^VEGF scFv’s when generated by CD70 CAR-T cells. Determining the capacity for CD70-CAR-T cells with secreted VEGF blockade to modulate RCC tumor proliferation, angiogenesis, and endothelial activation in vivo. The anti-RCC potency of 70^VEGF CAR-T cells was demonstrated in an in vitro luciferase based killing assay (FIG.4). Compared to untransduced T cells (UTD) and 70¹⁹ control CAR-T cells, the 70^VEGF CAR-T cells had superior RCC tumor killing across effector to target ratios. FIG.4 shows that the ^VEGF scFv confers superior in vitro cytotoxicity against 786-O RCC targets. These data suggest increased tumor susceptibility to secreted ^VEGF scFv targeting. Example 2 Determining the effect of secreted VEGF blockade on CD70-CAR-T cell biology Studies are performed to ascertain the effect of a secreted ^VEGF scFv on CD70 CAR-T biology. To attain this objective, it is assessed whether ^VEGF prevents VEGF-mediated CAR- T exhaustion through intracrine, paracrine, or autocrine pathways and leads to enhanced expansion and cytotoxicity. The approach is to block VEGF at multiple levels through (1) the secreted scFv, (2) CRISPR-Cas9 genetic knockouts and (3) exogenous administration of ^VEGF scFv. Validated assays of CAR-T performance including long-term proliferation, phenotyping, and cytokine production are performed and assessment of how these different levels of ^VEGF affect in vivo CAR-T trafficking are made. Completion of this study will contribute additional knowledge regarding the effects of VEGF signaling on CAR-T cell function which may help further elucidate a CAR-T armoring strategy. Various levels are identified paracrine, autocrine, and/or intracrine) upon which VEGF acts on CD70 CAR-T cells, as well as the effects of secreted ^VEGF on proliferation, phenotype, exhaustion, and cytokine production. Such findings can help inform the development of CAR-T cells agnostic of tumor type and contribute to alternate ways to manipulate VEGF-associated signaling pathways in immune effector cells. While the ^VEGF scFv may be acting on CAR-T cells in an autocrine or paracrine manner, there is also evidence that the VEGF-VEGFR signaling occurs in tumor cells in an intracrine manner prior to exocytosis of VEGFA (Bhattacharya et al.2016). To better ascertain the effect of a secreted anti-VEGF scFv on CD70 CAR-T biology, 4 different CAR-T cell constructs were generated (FIG.6).70^{VEGF,scramble} and 70^{CD19,scramble} represent the same constructs used to generate the other data but have the added CRISPR-Cas9 scramble non-targeting guide control and for guides for constant chain of the TCRĮ gene (TRAC) to allow selection of edited cells through standard CD3 magnetic bead kits.70^{CD19,VEGF KO1/KO2} contain two separate guides targeting proximal exons in VEGFA. These modifications were ligated onto the optimized trCD27-41BB ligand-based CD70-targeting backbone with a modified hinge region to prevent protease mediated decapitation (Leick et al.2022). This approach for generating CRISPR-Cas9 CAR-T cells using the TRAC knockout selection approach was previously described (Larson et al.2022). VEGFA knockouts were confirmed by both sequencing and VEGF ELISA (FIG.7). Threewell-established long-term assays of CAR-T cell fitness ± exogenous VEGF scFv administration were used: (1) Long-term proliferative capacity by recursive weekly stimulation with human K562 antigen presenting cells expressing CD70, (2) Cytokine secretion related to CAR-T function (e.g. IFNȖ and TNFĮ), (3) Flow cytometric profiling of differentiation state (CCR7 and CD45RA to define naïve, central memory, effector memory, and effector phenotypes) and exhaustion state (PD1, LAG3, TIM3) over time. To visually identify locations of binding to VEGF, ^VEGF scFv is fluorescently labeled using an included 6x histidine tag and confocal microscopy is performed to assess intracellular VEGF-VEGFR binding. Based on the data collected, the 70^{VEGF,scramble} CAR-T cells to are expected to have superior long term proliferative capacity, secretion of cytokines, and delayed progression to a terminally differentiated and exhausted phenotype relative to the 70^19,scramble CAR-T cells. Furthermore, the 70^{CD19,VEGF KO1/KO2} CAR-T cells are expected to phenocopy the 70^{VEGF,scramble} constructs, while exogenous addition of the VEGF scFv is expected to be insufficient to recapitulate this phenotype due to intracrine binding effects. Higher levels of 70^{VEGF,scramble} CAR-T trafficking to the tumor than 70^19,scramble and 70^{CD19,VEGF KO1/KO2} are expected due to the added effects of local modification of the vasculature from a secreted ^VEGF (rather than just genetic ablation). Defining the capacity for CD70-CAR-T cells with secreted VEGF blockade to modulate RCC tumor proliferation, angiogenesis, and endothelial activation in vivo The objective of these experiments is to ascertain the effect of secreted VEGF scFv blockade on tumor biology, angiogenesis, and endothelial activation. It is expected that CAR-T cells secreting ^VEGF achieve higher levels of ^VEGF blockade within the tumor compared to systemic ^VEGF administration, and that a locally secreted ^VEGF strategy prevents tumor growth, angiogenesis, and limits organ toxicity and endothelial activation compared to a systemically administered ^VEGF strategy. Studies compare the 70^VEGF CAR to the control 70¹⁹ in three in vivo models: (1) An orthotopic RCC model to assess for anti-tumor and anti- angiogenesis effects, (2) a cytokine release syndrome (CRS) model to evaluate endothelial activation, and (3) a cardiovascular toxicity model to evaluate nephrotoxicity endpoints. Anti-tumor, and angiogenesis assessment in an RCC patient derived xenograft (PDX): High- quality PDX’s were generated using the public repository of xenografts (PROXE) to model acute myeloid leukemia and RCC PDX’s in this database suitable for orthotopic modeling in NSG mice were identified. Orthotopic modeling, while more technically challenging, better recapitulates the trafficking and tissue niche features of tumors including RCC. CT scanners available at the center helped track the disease state including metastasis (Linxweiler et al. 2017). PDX RCC tumor seeds were orthotopically injected within the renal capsule followed by CAR-T cell injection by tail vein 90 days later after mouse randomization (FIG.10). An additional 3 mice per group were engrafted with a planned early collection of kidneys at day +21 to assess tumor CAR-T cell infiltration, angiogenesis endpoints using CD31 as an endothelial marker and NGD2 or PDGFRȕ as pericyte markers (D. K. Kim et al.2022). VEGF Blockade of endothelial activation in a CRS model: To determine the effect of ^VEGF on endothelial activation during cytokine release syndrome (CRS), a modified humanized CRS model is used. Prior experience with ex-vivo humanized CAR-T generation is used as a foundational step for this experiment (FIG.11). To adapt the model to the CAR-T system and avoid anti-tumor effects of the CD19-scFv secreting control CAR, CD19^KO Nalm6 acute lymphoblastic leukemia (ALL) cells transduced with CD70 are injected to ensure adequate CAR-T homing and activation. After irradiation and co-infusion of all with cord blood stem cells, CAR-T cells are injected 7 weeks later (FIG.12). CRS mortality is defined as death preceded by:ௗ>ௗ15% body weight loss, ǻTௗ>ௗ2ௗ°C and serum IL- 6ௗ>ௗ1,500ௗpg/ml. Lethal neurotoxicity is death in the absence of CRS criteria and preceded by either paralysis or seizures. The blood and sera are serially assessed for factors that induced endothelial activation (IL6, IFNȖ, and TNFĮ), as well as markers of endothelial activation in CAR-T patients (ANG2, and vWF) or are associated with CRS such as serum amyloid A (SAA, mouse homolog of CRP) (Gust et al.2017; Norelli et al.2018). In vivo systemic anti-VEGF-scFv toxicity model. The 70^VEGF versus 70¹⁹ CAR-T cells is tested with systemically administered ^VEGF scFv treatment. First, the 786O RCC cells are orthotopically implanted into the renal capsule in 5 mice per group into 8-week-old NSG mice. After 3 weeks of engraftment, mice are randomized and then treated with either CAR-T constructs or UTD cells along with systemic ^VEGF scFv or vehicle control for 4 weeks by tail vein (FIG.13), followed by humane sacrificing and collection of both kidneys for evaluation by immunohistochemistry and scanning electron microscopy (SEM) for pathogenic glomular changes. Blood is evaluated for changes in renal function (creatinine and albumin), and urine is evaluated for proteinuria (albumin:creatinine ratio [uACR]). Supporting the feasibility of this experiment, systemically administered VEGF-antibody blockade resulted in renal-toxic endpoints as ascertained through development of ascites, kidney, and glomerular changes as seen by immunohistochemistry and SEM (Gerber et al.2007). These experiments are repeated with T cells from three healthy individuals and mice of both female and male sexes. To capture mouse urine, ‘metabolic mouse cages’ which separate urine from feces for serial collection of urine are used. Example 3 Additional experiments were performed to determine the effects VEGF binding protein CAR-T expression in Mesothelin CAR-T cells (Meso^VEGF) (FIG.16) and targeting a mesothelin expressing lung cancer. Like the 70^VEGF CAR-T cells, the Meso^VEGF CAR also increased T cell expansion (FIGs.16 and 18) compared to a Meso¹⁹ CAR-T cell control. Additionally, Meso^VEGF CAR-T cells completely suppressed VEGF and produce higher levels of inflammatory cytokines compared to Meso¹⁹ CAR-T cell controls. In vivo mouse experiments showed that Meso^VEGF CAR-T cells are more effective at treating lung cancer than corresponding Meso¹⁹ CAR-T cell controls. Overall, these results and the CD70^VEGF CAR-T cell results indicate that modifying CAR-T cells to express and secrete anti-VEGF antibodies is a general strategy from improving CAR-T cell function. And further provides evidence that expressing VEGF binding proteins in T cells can increase T cell persistence and efficacy when treating solid tumors. Example 4. Secretion of a VEGF blocking scFv to enhance CAR-T cell potency. CAR-T cell therapy has become an effective treatment strategy for many B-cell malignancies. However, its efficacy in solid tumors remains limited. VEGF-targeted drugs have been used as adjunct anti-tumor agents for decades to target abnormal tumor vasculature. Unfortunately, cardiovascular toxicities associated with systemic VEGF blockade limit the maximal therapeutic benefit of these drugs. VEGF may play a role in the immunosuppressive tumor microenvironment (TME), possibly through direct induction of T-cell effector dysfunction. Herein, CAR-T cells from patients treated with FDA-approved CAR-T products were shown to express members of the VEGF signaling pathway and, moreover, expression of these members was found to correlate with patient non-response. To overcome putative VEGF- induced CAR-T dysfunction and deliver local VEGF blockade, CAR-T cells that secrete a VEGF-targeting scFv to block both T-cell and tumor-derived VEGF within the TME were developed and characterized. Secreted VEGF-blockade endowed these CAR-T cells with enhanced activation, cytotoxicity, proliferation, and effector function across different antigen and solid tumor contexts. Finally, VEGF scFv-secreting CAR-T cells had improved tumor control in immunocompromised murine orthotopic metastatic models of lung and ovarian cancer. These findings suggest that secreted VEGF blockade is a viable strategy to augment CAR-T cell performance, leverage the benefit of VEGF inhibition, and avoid systemic toxicity. Introduction Chimeric antigen receptor (CAR)-T cells drive dramatic and durable remissions in patients with B-cell malignancies. However, obtaining responses in solid tumors has been more challenging, and is constrained by T-cell exhaustion, limited suitable tumor-specific antigens, and suppression by the tumor microenvironment (TME). Blockade of vascular endothelial growth factor (VEGF) signaling to limit tumor angiogenesis and tumor autocrine/paracrine signaling is a long-standing anti-tumor strategy, with FDA-approved drugs employing this approach in a variety of solid tumor types. The success of these agents is predicated on an imbalance of pro-angiogenic signals (like overproduction of VEGF) in the tumor microenvironment leading to the formation of abnormal tumor vasculature. This deranged milieu promotes tumor growth and induces immunosuppression. Currently, there are more than 10 FDA-approved products targeting the VEGF signaling axis with many more in clinical trials. Patients treated with VEGF blocking agents have seen benefits in progression-free and overall survival across a range of cancers including non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), ovarian cancer, and glioblastoma. However, doses of systemically administered VEGF-targeted agents are limited by cardiovascular and renal toxicities, constraining their maximal theoretical anti-tumor benefit. Beyond an angiogenic-specific effect, VEGF signaling may limit the anti-tumor potency of T cells. VEGF has also been shown to impair T cell proliferation in vitro, which can be ameliorated with VEGF blockade. Dose-dependent increases in exhaustion-associated T-cell markers (PD-1, Tim3, Lag3) after co-culture with VEGF have been observed to coincide with loss of hallmarks of effector function. CAR-T cells represent a class of highly efficient living delivery vehicles capable of deploying biologics to the tumor microenvironment when they would otherwise be toxic with systemic administration. Leveraging this concept, CAR T cells were engineered to secrete a VEGF-blocking scFv (CARĮ^VEGF). To achieve this, an anti-Mesothelin 4-1BB CAR an anti- CD70 targeting 4-1BB CAR with a modified hinge were employed in a CAR-T cell that secretes the VEGF-blocking scFv . Using this approach, it was observed that activation-dependent VEGFR upregulation in CAR-T cells is recapitulated in the CAR-T cells of patients treated with FDA-approved CAR-T cell therapies. Additionally, it was found that secreted VEGF-blockade results in VEGF signaling abrogation across a range of physiologically relevant concentrations and interruption of in vitro angiogenesis. CAR^VEGF displayed enhanced activation, cytotoxicity, proliferation, and production of effector cytokines across both targets and a range of histologies, including both solid and liquid tumors. Finally, CAR^VEGF drove enhanced anti-tumor responses against orthotopic metastatic models of lung and ovarian cancer. Results To understand VEGF pathway member expression in CAR-T cell patients, a reanalysis of a single-cell RNA sequencing dataset was conducted on datasets previously generated by the inventors from patients treated with two FDA-approved CAR-T cell products: axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel). Samples were collected at baseline prior to CAR-T cell production, from the infusion product itself, and at day 7 post-infusion. It was found that the CAR-T cells in the infusion products had expression of VEGF signaling pathway members, namely Flt1 (VEGFR1), VEGFA, and NRP2 (FIG.1). Interestingly, this expression was almost exclusively restricted to Tisa-cel (CD19 CAR with 41BB costimulatory domain) (FIG.1). Furthermore, infusion product CAR-T cells from non-responding tisa-cel patients had significantly higher expression of VEGFR1 than responding patients (FIG.20A). To corroborate these findings and determine if they could be extended to other antigen contexts, an in vitro validation using antigen-expressing K562s to stimulate CAR-T cells was performed. Upon stimulation through the CAR with CD70 or Mesothelin (Meso) expressing K562s , Meso- (FIG.20B) and CD70- (FIG.20C) targeting 41BB CAR-T cells increased expression of VEGFR1. However, VEGFR1 expression in activated Meso-targeting CD28z CAR-T cells remained unchanged on activation, paralleling the results in the tisa-cel vs. axi-cel patient CAR-T scRNAseq data. CAR-independent stimulation with Phorbol 12-myristate 13- acetate (PMA) did not affect the expression of VEGFR1 in Meso nor CD70-targeting in CAR-T cells with either costimulatory domain (FIGs.24A-24B). In addition to the expression of the receptor, it was also found that activated T cells and CAR-T cells themselves secrete VEGF, further indicating that the VEGF signaling pathway may be relevant to CAR-T cell behavior (FIG.7). Given the previously established effector dysfunction induced by VEGF on T cells and the finding that VEGF receptor was upregulated in non-responding tisa-cel (41BB- but not CD28- costimulated) CAR-T patients, Mesothelin and CD70-targeted 41BB-costimulated CAR- T cells that secrete a VEGF-blocking single chain variable fragment (scFv, 70Į^VEGF and MesoĮ^VEGF) (or CD19 scFv control, 70Į^CD19 and MesoĮ^CD19) were generated (FIGs.6, 16A, and 21A). To prove that the construct resulted in the secretion of a biologically active inhibitor of VEGF signaling Į^VEGF-containing supernatant from 70Į^VEGF was utilized it in a HEK293T VEGFR reporter assay, demonstrating blockade of VEGF signaling across several log-fold of physiologically relevant concentrations (FIGs.2 and 21B). Į^VEGF also completely blocked signaling from VEGF containing-supernatant taken from the VHL deficient 786O RCC cell line which has unrestrained VEGFA production (FIG.21C). Given the known role of VEGF in promoting angiogenesis, the inventors next sought to determine if the secreted Į^VEGF scFv was sufficient to inhibit angiogenesis in vitro. To model in vitro angiogenesis, human umbilical vein endothelial cells (HUVECs) on a normal human dermal fibroblasts (NHDFs) matrix were used (FIG.21D). HUVECs and NHDFs were cultured in the presence or absence of recombinant VEGF and the VEGF signaling inhibitor, suramin. Į^VEGF-containing supernatant significantly reduced (p=0.009) blood vessel network length (primitive vessels per square millimeter (mm/mm2)) relative to the ĮCD19 containing supernatant control during 70 hours of co-culture (FIGs.14 and 21E-21F). After demonstrating that secreted Į^VEGF effectively abrogates VEGF signaling in reporter and angiogenesis assays at physiologically relevant concentrations, experiments performed endeavored to characterize how secreted VEGF blockade affects CAR-T cell activation. In a short-term co-culture looking at the activation marker CD69, higher expression in both MesoĮ^VEGF and 70Į^VEGF was observed compared to their control counterparts (FIGs.22A-22B). To understand the cytolytic and proliferative capacity of Į^VEGF secreting CAR-T cells, co-cultures with constructs and various solid and liquid tumor lines were performed. Upon co- culture with the natively Meso-expressing SKOV3 and OVCAR3 ovarian cancer cell lines, MesoĮ^VEGF CAR-T cells demonstrated enhanced anti-tumor activity compared to controls. This was coupled with a significant increase in CAR-T cell expansion of the Į^VEGF-secreting CAR-T cells (FIGs.16B and 22C-22D). Interrogation of the supernatant following these cocultures revealed complete blockade of detectable VEGF (p<.0001) by the Į^VEGF CAR-Ts and an increase in effector cytokines such as IFNȖ, TNFĮ, and Granzyme B (FIG.17). Similar results were seen with other endogenously expressing mesothelin cell lines Nomo-1 (AML, FIG.22E), and A549 (lung cancer, FIG.18). Culture of tumor cell lines with Į^VEGF-containing supernatant alone had no effect on tumor growth, suggesting that the anti-tumor effect is CAR-T cell- dependent (FIGs.25A-25C). Longer term co-culture of meso CAR-T cells with recursive stimulation with irradiated mesothelin expressing K562 cells for 12 days revealed significantly higher proliferation of MesoĮ^VEGF (p=0.0017)(FIG.22F). Subtle differences in exhaustion- associated marker expression were observed at days 3 and 6, however at day 9 MesoĮ^VEGF had significantly higher TIM3 expression than MesoĮ^CD19. To validate the enhanced anti-tumor and proliferative benefits of CAR-T cell-secreted VEGF blockade in the context of another antigen target, CD70Į^VEGF CAR-T cells were cultured with natively CD70-expressing tumor cells. Culture with 786O RCC, SKOV3 ovarian cancer, and Nomo-1 AML cell lines resulted in greater anti-tumor activity, enhanced proliferation, and substantial decrease in detectable VEGF by the Į^VEGF-secreting CAR-T cells (FIGs.15 and 22G- 22J), demonstrating VEGF-blockade induced proliferative and cytotoxic benefits are applicable in multiple CAR-antigen contexts. Analysis of phenotypic and exhaustion-associated markers in CAR-T cells revealed no differences between Į^CD19 and Į^VEGF in either Meso or CD70 CAR-T cells (FIGs.26A-26C). The anti-tumor activity of the MesoĮ^VEGF in vivo in NSG models of lung and ovarian cancer was tested. As a model of NSCLC, mice injected IV with the A549 cell line, which engrafts directly in the lungs of the mice, were utilized (FIG.19, top). After a 7 day engraftment period, the mice were treated with the meso CAR-T cells. In this model, mice treated with MesoĮ^VEGF CAR-T cells demonstrated significantly improved tumor control compared to Į^CD19- secreting Meso CAR-T cell controls (FIG.19, middle and bottom, p=0.005 by day +20). The effects of the Į^VEGF CAR-T cell in a metastatic model of ovarian cancer were also tested. In this model, NSG mice were injected intraperitoneally with SKOV3 ovarian cancer cell line and given 7 days for the tumor to engraft, followed by intravenous CAR-T cell treatment (FIG. 23B). MesoVEGF-treated mice had enhanced tumor control (FIG.23C). Discussion In this study, the first characterization of the expression of VEGF pathway members in the context of CAR-T cells was conducted. It was found that elevation of VEGFR1 in non- responding patients after CD19-targeted CAR-T cell therapy and, along with data suggesting that VEGF induces effector dysfunction, a strategy of CAR-T secreted VEGF blockade was pursued. CARĮ^VEGF secreted a highly potent, biologically active inhibitor of VEGF signaling and angiogenesis in vitro and displayed superior cytotoxicity, proliferation, and effector cytokine production across antigen and tumor contexts. Additionally, targeted delivery of VEGF blockade improved anti-tumor activity in vivo against orthotopic metastatic models of lung and ovarian cancer in immunocompromised mice. A key finding of this work was the previously unknown impact of VEGF on CAR-T cells. VEGF has now been demonstrated to disrupt T cell effector function, however, this study presents the first characterization of VEGF effects specifically on CAR-T cells. Intriguingly, while VEGFR2 has been identified as the dominant receptor responsible for VEGF-mediated signaling in T cells, only elevated VEGFR1 expression in CAR-T cell products from patients was found. Furthermore, elevation of VEGFR1 was exclusively seen in 41BB costimulated (but not CD28 costimulated) CAR-T cells and was higher among non-responding than responding patients. The finding that VEGFR1 is elevated on activated 41BB costimulated CAR-T cells in vitro was recapitulated in this study. VEGFR1 is typically thought to act as a decoy receptor and modulator of VEGF-signaling flux, having higher binding affinity than VEGFR2, but weaker RTK signaling activity. With localized delivery only at the site of the tumor, the CARĮ^VEGF technology described herein represents a strategy that avoids systemic toxicities but imparts the same benefits of combined CAR-T cell and systemic VEGF blockade. Compared to standard VEGF-blocking antibodies used in clinically like bevacizumab, the scFv is much smaller (at 26 KDa compared to bevacizumab at 149 KDa) and has a stronger binding affinity to VEGF. ScFvs are ideal secreted inhibitors since they have substantially greater tissue penetration and rapid systemic clearance compared to antibodies and are more easily packaged into small lentiviral vector payloads. Methods Cell lines Cell lines were obtained from the American Type Culture Collection (ATCC) (786o, SKOV3, A549, U87, U251). All cells except A549 were cultured in R10 medium, RPMI1640 medium (Thermo Fischer Scientific, 61870036) supplemented with 10% FBS and 1% Pen Strep. A549 cell lines were cultured in EMEM with L-glutamine (ATCC, 30-2003) supplemented with 10% FBS and 1% Pen Strep. Cells were genetically engineered via lentiviral transduction to express click beetle green and green fluorescent protein (CBG-GFP) and sorted to purity. Cell lines were validated using short tandem repeat (STR) testing. K562 cell lines were obtained from xxx and cultured in r10 medium. They were transduced with Meso and CD70 using lentiviral transduction. CAR Construction Mice were immunized with human mesothelin and a novel mesothelin-targeting monoclonal antibody, termed A2A11, was generated by LifeTein. Sequencing of the antibody variable domain of the A2A11 hybridoma was performed by GenScript and the resulting sequence was incorporated into the second-generation CAR construct. Lentiviral Production HEK 293 T cells were used to produce Lentivirus harboring CAR plasmids. HEKs at 80% confluence were transfected with CAR plasmid using Lipofectamine 3000 transfection reagent (Invitrogen). Lentivirus was harvested at 24 and 48 hours and concentrated via 2 hour ultracentrifugation. CAR-T cell Production Primary T cells were obtained from healthy donor leukapheresis products purchased from the Massachusetts General Hospital blood bank and isolated (Stem cell tech). Isolated Primary T cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% Pen strep. T cells were activated using CD3/28 beads (supplier) and 20 IUs/mL of human recombinant IL2 (Peprotech). Cells were transduced with lentivirus 24 hours after activation at an MOI of 5. CD3/28 beads were removed via magnetic debeading on day 6. Media was doubled every 2-3 days until day 14 where transduction efficiency was measured and cells were frozen. Stimulation Assays CAR T cells were stimulated with irradiated CD70 or Mesothelin expressing K562s at a 1:1 ratio for 96 hours or cell stimulation cocktail (eBioscience) for 4 hours. After incubation, cells were stained for CD8, VEGFR1, and VEGFR2 on flow cytometry. Reporter Assays Reporter assays were performed using VEGF BioAssay Kit from Promega according to manufacturer's instructions. Jurkats transduced with CD70a19 and CD70aVEGF plasmids were seeded at equivalent concentrations and expanded for 1 week. Supernatant was collected and filtered to obtain cell-free Į19 or ĮVEGF-containing medium. VEGF from 786o cell line was harvested from a confluent plate after 1 week of culture. Angiogenesis Assay Normal Human Dermal Fibroblasts (Lonza) were seeded at 15,000 cells/well in a flat bottom 96 well plate in EGM-2 medium (without VEGF added, Lonza) for 3 days until confluent. After 3 days, Human Umbilical Vein Endothelial Cells (HUVECs, Angio Proteomie) were seeded onto the confluent layer of NHDFs at 5,000 cells/well and treatment conditions were added (VEGF at 4 ng/ml, suramin at 0.25 mg/ml (Millipore Sigma), culture supernatant from confluent 70Į^VEGF, 70Į¹⁹, or untransduced Jurkat T cells diluted 1:4). Media was changed every two days. The assay was imaged and analyzed using the Sartorius Incucyte Angiogenesis Software package every 6 hours for 14 days. Long Term Proliferation Assays CAR-T cells were stimulated with irradiated (100 grays) Mesothelin expressing K562s at a 1:1 ratio. Every 3 days, CAR T cells were quantified and restimulated at 1:1 ratio and phenotyped for expression of PD1, TIM3, and LAG3 surface markers via flow cytometry. Cytotoxicity Assays All cytotoxicity assays were performed using the Incucyte Live Cell Analysis System (Incucyte SX5). CBG-GFP expressing tumor cells were plated and given 4-6 hours to adhere. For suspension tumor cell lines, plates were pre-coated with CD71 unconjugated antigen to ensure suspension tumor adhesion. CAR T cells were thawed and cultured in IL2 overnight before being plated onto adhered tumor cells. Plates were imaged every 1-2 hours using the green and red fluorescence channels. Analysis was performed using the Incucyte masking and analysis software. Fluorescence values normalized to time 0 are shown where specified. Cytokine Analysis Cytokines were measured using Ella Automated Immunoassay System with Multianalyte assay chips (Biotechne). Supernatant was collected from cytotoxicity assays at 72 hours, or as specified, and frozen until day of quantification. Assay was performed following manufacturers protocol. ELISA VEGF ELISA (Invitrogen) was performed following manufacturers instructions. Supernatant was collected from CAR T cells on day 13 of production. Supernatant was centrifuged and filtered to eliminate cells. Flow Cytometry The following antibodies were used: VEGFR1 (R&D systems, FAB321V-100ug), VEGFR2 (Biolegend, 359910), Mouse IgG1k Isotype (biolegend-400136, R&D systems- IC002V), CD69 (Biolegend, 310918), CD8 (BD-560179, BD-647458), CD4 (BD-651850), CD3 (641406), CD71 (Biolegend-334104), PD1(BD Horizon-563789), TIM3 (BD Horizon-565566), LAG3 (BD Pharmingen-565716), CCR7 (BD Pharmingen-561271), CD45RA (Biolegend- 304126), CD33 (Biolegend-366620), CD45 (Bioledgend-368562). Cells were stained for 10 minutes at room temperature in the dark followed by 2x washes with FACS buffer (2% FBS in PBS). Cells were resuspended in DAPI-containing FACS buffer to determine live/dead cell separation. Cells were flowed on a Fortessa Cytometer and analysis was performed using Flow Jo software. In vivo models NOD-scid gamma (NSG) mice were obtained from Jackson Laboratories and bred under pathogen-free conditions at the MGH Center for Comparative Medicine. For A549 model, 1e6 CBG-GFP A549 tumor cells were injected via tail vein on day -7. On day -1, animals were randomized and injected IV with the indicated quantity of CAR T cells. Once per week, mice were injected IP with luciferin to image tumor bioluminescence (BLI) using Spectral Ami HT imaging system. For SKOV3 model, 1e6 CBG-GFP SKOV3 tumor cells were injected IP on day -7. On day -1, animals were randomized and injected IV with indicated quantity of MESO CAR T cells. Animals were imaged once per week to record BLI. All mouse procedures were performed by a technician who was blinded to experimental groups. Statistical Methods All analysis was performed using GraphPad prism (version 10.2.3). Data indicates mean values and error represents standard error of mean. P values are reported on figures and significance was attributed to p value less than 0.05.

Claims

CLAIMS What is claimed is: ^ 1. A T cell comprising a heterologous polynucleotide encoding a Vascular Endothelial Growth Factor (VEGF) binding protein.

2. The T cell of claim 1, wherein the VEGF binding protein is secreted by the cell.

3. The T cell of claim 1 or 2, wherein the VEGF binding protein comprises a secretory signal peptide.

4. The T cell of claim 3, wherein the secretory signal peptide comprises an Igk signal peptide.

5. The T cell of any one of claims 1-4, wherein the VEGF binding protein comprises an antibody.

6. The T cell of claim 5, wherein the antibody is an antibody fragment.

7. The T cell of claim 5, wherein the antibody fragment is an antigen-binding fragment (Fab), a Fab’, or a F(ab’)2, a fragment variable (Fv), or a single chain variable fragment (scFv).

8. The T cell of claim 7, wherein the antibody fragment is an scFv.

9. The T cell of any one of claims 3-8, wherein the antibody comprises: (i) a VH domain comprising three complementary determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 70, CDR-H2 comprises SEQ ID NO: 71, and CDR-H3 comprises SEQ ID NO: 72, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 73, CDR-L2 comprises SEQ ID NO: 74, and CDR-L3 comprises SEQ ID NO: 75.

10. The T cell of claim 8, wherein the scFv comprises an amino acid sequence of SEQ ID NO: 76.

11. The T cell of claim any one of claims 1-10, wherein the VEGF binding protein comprises a VEGF A binding protein.

12. The T cell of claim 1-11, wherein the VEGF binding protein is operably linked to a promoter.

13. The T cell of claim 12, wherein the promoter is a constitutively active promoter.

14. The T cell of claim 13, wherein the promoter is a EF1-alpha promoter.

15. The T cell of any one of claims 9-14, wherein the antibody comprises the VH domain n- terminal to the VL domain.

16. The T cell of any one of claims 9-14, wherein the antibody comprises the VL domain n- terminal to the VH domain.

17. A T cell comprising a VEGF gene knockout.

18. The T cell of any one of claims 1-16, comprising a VEGF gene knockout.

19. The T cell of claim 17 or claim 18, wherein the VEGF gene knockout comprises a VEGF-A gene knockout.

20. The T cell of any one of claims 17-19, wherein the VEGF gene knockout is a VEGF gene deletion.

21. The T cell of any one of claims 17-20, wherein the VEGF gene knockout comprises a VEGF loss of function mutation.

22. A T cell comprising a polynucleotide encoding a guide RNA polynucleotide comprising a homology region that is complementary to a polynucleotide encoding VEGF.

23. The T cell of any one of claims 1-21, further comprising a polynucleotide encoding a guide RNA comprising a homology region that is complementary to a polynucleotide encoding VEGF.

24. The T cell of claim 22 or 23, wherein the polynucleotide encoding VEGF encodes VEGF-A.

25. The T cell of any one of claims 22-24, wherein the polynucleotide encoding the polynucleotide encoding the guide RNA homology region comprises a polynucleic acid of SEQ ID NO: 79 or SEQ ID NO: 80.

26. The T cell of any one of claims 1-25, wherein the T cell is a chimeric antigen receptor (CAR)-T cell.

27. The T cell of any one of claims 1-26, wherein the T cell comprises a polynucleotide encoding a chimeric antigen receptor.

28. The T cell of claim 27, wherein the CAR comprises: (i) an antigen binding domain; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain.

29. The T cell of claim 28, wherein the antigen binding domain binds to any one of CD19, CD37, CD79b, Claudin 18.2, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, binds to any pair of CD19/CD79b, BCMA/TACI, or is a TriPRIL antigen binding domain.

30. The T cell of claim 28 or claim 29, wherein the antigen binding domain comprises an antibody.

31. The T cell of claim 30, wherein the antibody is an antibody fragment.

32. The T cell of claim 31, wherein the antibody fragment is an antigen-binding fragment (Fab), a Fab', or a F(ab')2, a fragment variable (Fv), or a single chain variable fragment (scFv).

33. The T cell of any one of claims 28-32, wherein the antigen binding domain binds to CD70.

34. The T cell of any one of claims 28-32, wherein the antigen binding domain comprises CD27.

35. The T cell of claim 34, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 66.

36. The T cell of any one of claims 28-32, wherein the antigen binding domain binds to mesothelin.

37. The T cell of claim 36, wherein the antibody comprises an amino acid sequence of any one of SEQ ID NOs: 3-4.

38. The T cell of claim any one of claims 27-37, wherein the transmembrane domain is selected from the group consisting of alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C transmembrane domains.

39. The T cell of claim 38, wherein the transmembrane domain comprises a CD8 or a CD27 transmembrane domain.

40. The T cell of any one of claims 27-39, wherein the co-stimulatory domain comprises a 4- 1BB, CD27, CD28, OX40, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, or ZAP70 costimulatory domain.

41. The T cell of any one of claims 27-40, wherein the co-stimulatory domain comprises a 4- 1BB.

42. The T cell of any one of claims 27-41, wherein the intracellular signaling domain comprises a CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, or CD66d intracellular signaling domain.

43. The T cell of any one of claims 27-42, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain.

44. The T cell of any one of claims 27-43, wherein the CAR further comprises a leader sequence.

45. The T cell of claims 44, wherein the leader sequence comprises a CD8 leader sequence.

46. The T cell of claim 28, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-47.

47. The T cell of claim 28, wherein the CAR comprises: (i) a CD70 binding antigen binding domain; (ii) a CD27 transmembrane domain; (iii) a 4-1BB costimulatory domain; and (iv) a CD3-zeta intracellular signaling domain.

48. The T cell of claim 47, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 30-35.

49. The T cell of claim 28, wherein the CAR comprises: (i) a mesothelin binding antigen binding domain; (ii) a CD8 transmembrane domain; (iii) a 4-1BB costimulatory domain; and (iv) a CD3-zeta intracellular signaling domain.

50. The T cell of claim 49, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 7-10.

51. The T cell of anyone of claims 28-50, wherein the polynucleotide encoding the CAR and the heterologous polynucleotide encoding the VEGF binding protein are encoded on the same polynucleic acid.

52. The T cell of claim 51, wherein a self-cleaving peptide or an internal ribosomal entry site is encoded in the polynucleic acid between the CAR and the VEGF binding protein.

53. The T cell of claim 51, wherein the self-cleaving peptide is a 2A peptide.

54. The T cell of any one of claims 51-53, wherein the polynucleic acid encodes an amino acid sequence of any one of SEQ ID NOs: 77-78.

55. A polypeptide encoding the CAR of any one of claims 27-50 and the VEGF binding protein of any one of claims 1-14.

56. A polypeptide comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78.

57. A polynucleic acid of any one of claims 51-54.

58. A vector comprising the polynucleic acid of claim 57.

59. A cell comprising the polynucleic acid of claim 57 or the vector of clam 58.

60. A method of treating cancer in a subject, the method comprising administering the T cell of any one of claims 1-54 to the subject.

61. The method of claim 60, comprising administering the T cell of any one of claims 27-54 to the subject.

62. The method of claim 61, wherein the CAR comprises a CD70 antigen binding domain and the cancer expresses CD70.

63. The method of claim 62, wherein the cancer is renal cell carcinoma, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, acute myeloid leukemia, or adenoid cystic carcinoma (ACC).

64. The method of claim 63, wherein the cancer is renal cell carcinoma.

65. The method of claim 64, wherein the renal cell carcinoma is clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, or papillary renal cell carcinoma (pRCC).

66. The method of claim 61, wherein the CAR comprises a mesothelin binding antigen binding domain and the cancer expresses mesothelin.

67. The method of claim 60 or claim 61, wherein the cancer is a pancreatic cancer, a lung cancer, an ovarian cancer, an endometrial cancer, a biliary cancer, a gastric cancer, or mesothelioma.

68. The method of any one of claims 60-67, wherein the cancer comprises a solid tumor.

69. The method of any one of claims 60-68, wherein the VEGF binding protein inhibits binding of VEGF to VEGFR1 and/or VEGFR2.

70. The method of any one of claims 60-68, wherein the VEGF binding protein inhibits binding of VEGF-A to VEGFR1 and/or VEGFR2.

71. A method of decreasing CAR-T cell exhaustion, the method comprising transfecting the CAR-T cell with a polynucleotide encoding the VEGF binding protein of any one of claims 1- 16.