Interferon gamma responsive chimeric t cell costimulatory receptor

Abstract

The present disclosure relates to chimeric costimulatory receptors (CCR) specific to ICAM-1, e.g., the CCRs that comprise a 4-1BB (CD137) costimulatory domain and a ICAM-1 binding domain (e.g., a ICAM-1 -specific scFv). Composition comprising such CCRs and methods of using such CCRs for preventing and treating a cancer are also provided.

Description

[0001] INTERFERON GAMMA RESPONSIVE

[0002] CHIMERIC T CELL COSTIMULATORY RECEPTOR

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Patent Application serial number 63 / 655,169, filed on June 3, 2024, which is hereby incorporated by reference herein in its entirety.

[0005] STATEMENT OF RIGHTS

[0006] This invention was made with government support under grant numbers R01CA254035 and R21CA280566 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0007] FIELD OF INVENTION

[0008] The present invention relates to chimeric costimulatory receptors specific to ICAM- 1. The invention particularly relates to a chimeric costimulatory receptor targeting ICAM-1 that provides signaling for more complete T cell activation that complements natural or synthetic T cell receptors or chimeric antigen receptor modified T cells.

[0009] BACKGROUND

[0010] Immunotherapy of cancer has transformed the treatment of cancer. Naturally occurring T cells obtained from tumors, tumor infiltrating lymphocytes (TILS), the blood or lymph nodes have been shown to possess anti-tumor activity. Adoptive T cell therapy consists of administrating TILs multiplied 1000-fold ex vivo for each patient1. One of the major impediments to achieving high antitumor activity with this approach is the inherent intermediate- to low-affinity T cell receptors (TCRs) characteristic of antitumor T cells2. Low-affinity T cells can be effective if their effector functions are reinforced by signaling cascades downstream of costimulatory receptors, such as 4-1BB or CD28. However, many epithelial tumors do not generally express the cognate ligands to these costimulatory receptors such as CD80, CD86, or 4-1BBL.

[0011] Adoptive T cell therapy leverages a patient’s own T cells to help fight cancer or chronic infection (1). Current adoptive T cell therapies include T cell receptor (TCR)-T and chimeric antigen receptor (CAR)-T therapy (2,3). Typically, these therapies target a single tumor antigen. However, many solid tumors have multiple, wide-ranging inter- and intra- tumoral mutations that create clonal heterogeneity, constituting a major hurdle to successful adoptive T cell therapy (4,5). Controlling solid tumor spread is challenging when using a therapeutic designed to target a single antigen because clones not expressing the targeted antigen can expand. Even when the therapy is directed at the original clonal mutation, solid tumors can diversify and non-directed clones can expand, creating resistance. Several studies have shown that relapses can occur with this type of single antigen-redirected therapy when expression of the targeted surface antigen, neoantigen, or major histocompatibility complex (MHC) is diminished or lost (6-9). Therapeutic approaches to polyclonal targeting of tumor-associated antigens may overcome these limitations, and recent technological developments that can enhance the multi-target specificity of CAR-T cells have shown encouraging results in hematological malignancies (10). However, several challenges with this approach remain, particularly in reducing or preventing disease progression (11,12).

[0012] To date, adoptive T cell therapy is most successful when tumor-reactive T cells are isolated and harvested from tumor tissues and then expanded ex vivo by IL2 supplementation (13). When reintroduced to the patient, these tumor-reactive T cells unleash a robust immune response. The T cells recognize their specific neoantigens, which arise from somatic tumor mutations, or tumor-associated antigens. Several studies have shown that tumor-reactive T cells can be successfully isolated from the peripheral blood of cancer patients, albeit at a frequency of 1 in 103- 106cells (14-16). Indeed, an adoptive T cell therapy that uses a type of tumor-reactive T cell — tumor-infiltrating T cells (TILs) — was approved by the FDA for adult patients with advanced melanoma (1).

[0013] For solid tumors, the major challenges to achieving efficacy with T cell therapies include the technical difficulty in expanding antitumor T cells, which have inherently low- to intermediate-affinity native TCRs, while maintaining robust and persistent antitumor activity (17,18). Both tumor-reactive and native TCRs have low affinity interactions with the peptide (p) / MHC complex (KD-1-100 pM), which is aided by CD4 / 8 coreceptors, and require a secondary signal from a costimulatory receptor / ligand interaction, such as CD80 / CD28, CD86 / CD28, or CD137 / CD137L, to drive activation (17,19). However, many solid tumors have an immunosuppressive microenvironment and suppress expression of these costimulatory ligands (20,21). Without secondary signals, tumor-specific T cells may experience subthreshold activation and become ineffective cytotoxic T cells. Thus, there is a need in the art for improved adoptive immunotherapy for the treatment of cancer.

[0014] SUMMARY

[0015] The present invention relates to chimeric costimulatory receptors specific to ICAM- 1. The invention particularly relates to a chimeric costimulatory receptor targeting ICAM-1 that provides signaling for more complete T cell activation that complements natural or synthetic T cell receptors or chimeric antigen receptor modified T cells.

[0016] In some aspects, provided herein is a chimeric costimulatory receptor (CCR) comprising: a) an intercellular adhesion molecule-1 (ICAMl)-binding domain; and b) at least one costimulatory domain, wherein the CCR does not comprise a cluster of differentiation 3 zeta (CD3Q domain.

[0017] In some embodiments, the at least one costimulatory domain is selected from CD28, CD27, 4-1BB (CD137), 0X40, CD40, and ICOS. In some embodiments, the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof. In some embodiments, the at least one costimulatory domain comprises a 4- IBB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the at least one costimulatory domain comprises a CD28 costimulatory domain and a 0X40 costimulatory domain. In some embodiments, the at least one costimulatory domain comprises a 4-1BB (CD137) costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO. 10.

[0018] In some embodiments, the ICAMl-binding domain comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the ICAMl-binding domain comprises an antibody and the antibody is selected from R6.5 (BIRR-1, Enlimomab), 1 A6, MD-3, M10A12, bersanlimab, AF-647, and MSH-TP15. In some embodiments, the ICAMl-binding domain comprises an antigen-binding fragment thereof and the antigenbinding fragment of an antibody is a single-chain variable fragment (scFv). In some embodiments, the ICAMl-binding domain comprises R6.5 scFv or an I domain of the aL subunit of lymphocyte function-associated antigen-1 (LFA1). In some embodiments, the ICAMl-binding domain comprises R6.5 scFv. In some embodiments, the ICAMl-binding domain is R6.5 scFv. In some embodiments, the R6.5 scFv comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively. In some embodiments, the R6.5 scFv comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and / or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8. In some embodiments, the R6.5 scFv comprises an amino acid sequence of SEQ ID NO: 9.

[0019] In some embodiments, the CCR further comprises (c) a transmembrane domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CCR comprises R6.5 scFv and 4-1BB.

[0020] In some embodiments, the CCR comprises: a R6.5 scFv, a transmembrane domain and a 4-1BB costimulatory domain. In some embodiments, the CCR comprises: a R6.5 scFv, a CD8 transmembrane domain, and a 4-1BB costimulatory domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CCR comprises a R6.5 scFv, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CCR comprises a R6.5 scFv, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and an 0X40 costimulatory domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 16.

[0021] In some aspects, provided herein is a nucleic acid encoding the CCR described herein, optionally wherein the nucleic acid sequence comprises wherein the nucleic acid sequence comprises a nucleotide sequence selected from SEQ ID NOs: 17-19. In some aspects, provided herein is a vector comprising the nucleic acid described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is lentiviral vector. In some aspects, provided herein is a cell comprising the nucleic acid described herein.

[0022] In some aspects, provided herein is a cell expressing the CCR described herein. In some embodiments, the cell is an induced pluripotent stem cell (iPSC), a hematopoietic cell (HSC), or an immune cell. In some embodiments, the cell is a leukocyte, optionally wherein the leukocyte is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL). In some embodiments, the cell is a primary CD8+T cell. In some embodiments, the cell expresses endogenous TCRa and / or TCRp. In some aspects, provided herein is a method of generating a CAR-expressing cell comprising contacting the cell with a nucleic acid described herein, or a vector described herein.

[0023] In some aspects, provided herein is a composition comprising cells described herein.

[0024] In some aspects, provided herein is a T cell comprising the CCR described herein, optionally wherein the T cell is a CD8+ or CD4+ T cell.

[0025] In some embodiments, the T cell described herein further comprises (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen. In some embodiments, the CAR further comprises at least one costimulatory domain. In some embodiments, the at least one costimulatory region domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof. In some embodiments, the at least one costimulatory domain comprises 4- IBB and CD28 costimulatory domains. In some embodiments, the CAR further comprises a transmembrane domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the TCR or CAR binds a cancer antigen selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, Ll-CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7- H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

[0026] In some aspects, provided herein is a tumor infiltrating lymphocyte (TIL) comprising the CCR described herein.

[0027] In some embodiments, the TIL described herein further comprises (a) an engineered T cell Receptor (TCR) (e.g., CTL 16) and / or (b) a chimeric antigen receptor (CAR).

[0028] In some embodiments, the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen. In some embodiments, the CAR further comprises at least one costimulatory domain. In some embodiments, the at least one costimulatory region domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the at least one costimulatory domain is selected from 4-1BB, CD28, 0X40, and a combination of two or more thereof, optionally wherein the at least one costimulatory domain comprises 4-1BB and CD28 costimulatory domains. In some embodiments, the CAR further comprises a transmembrane domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the TCR or CAR binds a cancer antigen is selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, Ll-CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

[0029] In some aspects, provided herein is a pharmaceutical composition comprising the T cell described herein and / or the TIL described herein.

[0030] In some aspects, provided herein is a method of isolating a T cell with a tumorspecific TCR, the method comprising: a) administering to a subject having a tumor, a T cell comprising the CCR described herein; or the T cell described herein; b) harvesting the tumor from the subject; and c) isolating the T cell with the CCR from the tumor, thereby isolating the T cell with a tumor-specific TCR.

[0031] In some aspects, provided herein is a T cell isolated according to the method described herein.

[0032] In some aspects, provided herein is a method of identifying a tumor-specific TCR, the method comprising: a) isolating the T cell with a tumor-specific TCR according to the method described herein; b) sequencing the TCR to thereby identify the tumor-specific TCR.

[0033] In some aspects, provided herein is a method of preventing or treating a cancer in a subject, the method comprising administering to the subject the T cell described herein, which is optionally further expanded. In some aspects, provided herein is a method of preventing or treating a cancer in a subject, the method comprising administering to the subject the cell described herein, the composition described herein, the T cell described herein, the TIL described herein, and / or the pharmaceutical composition described herein.

[0034] In some embodiments, the T cell and / or TIL cell is autologous or allogeneic to the subject. In some embodiments, the subject is treated conjointly with at least one additional cancer therapy. In some embodiments, the subject is administered with the at least one additional cancer therapy concomitant with, prior to, or following the administration of the T cell, the TIL, and / or the pharmaceutical composition. In some embodiments, the at least one additional cancer therapy is selected from immunotherapy, checkpoint inhibitors, cancer vaccines, chemotherapy, radiation therapy, and surgery, optionally a checkpoint inhibitors.

[0035] In some embodiments, the at least one additional cancer therapy is a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a PD-L1 inhibitor (e.g., an anti-PD-Ll antibody), KD033, or any combination of two or more thereof. In some embodiments, the PD-1 inhibitor is selected from MP-514 (MED 10680), nivolimumab, cemiplimab, pembrolizumab, dostarlimab, Vopratelimab (JTX-4014), Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), INCMGA00012 (MGA012), and AMP -224. In some embodiments, the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, KN035, Cosibelimab (CK-301), AUNP12, CA-170, and BMS-986189.

[0036] In some embodiments, the cancer or tumor is selected from non-small cell lung cancer (NSCLC), gastric cancer, Melanoma, Breast cancer, Lung cancer, Esophageal cancer, Gastric cancer, Pancreatic cancer, Liver cancer, Colorectal cancer, Anal cancer, Cervical cancer, Ovarian cancer, Endometrial cancer, Prostate cancer, Bladder cancer, Kidney cancer, Testicular cancer, Penile cancer, Head and neck cancer (including oral, pharyngeal, and laryngeal cancers), Thyroid cancer, Soft tissue sarcomas, and Neuroendocrine tumors. In some embodiments, the subject is a mammal, optionally a mouse, a dog, a cat, or a human. In some embodiments, the method increases the number of tumor-infiltrating CD3+ T cells.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Fig- 1 shows a diagram of the CCR approach. Fig. 2A-Fig. 2C show CTL16 TCR and CCR-T cell activity. (Fig. 2A) A375 cellspecific killing by CTL16 and CTL16 / CCR T cells upon Aik pulsing. The percentages of target cell killing were normalized to non-transduced T cells (NT). (*, p<0.05; n=3) (Fig. 2B) The secretion level of JFNy (n=2). (Fig. 2C) The IL2 secretion level (n=2).

[0039] Fig. 3A-Fig. 3B show the functional activities of CCR and CAR transduced Jurkat T cells against target cells with varying level of ICAM1 expression. (Fig. 3 A) The NFkB activity was measured in Jurkat cells (NT) and Jurkat expressing CCR, 1’ CAR (R6.5- CD3Q, and 3’ CAR (R6.5-CD28-4-1BB-CD3Q against 293T or HeLa using luminescence assay. All cells activated NFkB when stimulated with lonomycin and PMA. (*, p<0.05; n=4).

[0040] Fig. 4A-Fig. 4B show CCR-T cell activity against 8505C ATC. (Fig. 4A) 8505C xenografts were challenged with CCR-T or NT cells. (Fig. 4B) The percentages of live 293T and 8505C cells over time after co-culture with CCR-T cells isolated from spleen, tumor, or NT cells (*, p<0.05 in NT vs. Tumor CCR-T; n=3).

[0041] Fig. 5A-Fig. 5C show allogeneic TCR against 8505C recovering using CCR. (Fig. 5A) CD69 expression in CCR-T cells recovered from the tumor of the 8505X xenograft is upregulated to the spleen CCR-T and NT cells. (Fig. 5B) Culture of the CCR-T recovered from xeno-TIL showed killing of 8505C cells and active proliferation compared to NT. (Fig. 5C) TCB and TCA sequences were determined by TCR-seq in the xeno-TIL and spleen of the 8505C xenograft, which received CCR-T cells.

[0042] Fig. 6A-Fig. 6D show that Target-directed ICCR activates NFKB but are defective in IL2 production. (Fig. 6A) Schematic maps of lentiviral constructs expressing ICCR (R6.5-137), L CAR (R6.5-3z), and 3’ CAR (R6.5-137.28.3z). (Fig. 6B) Flow cytometry plots showing expression of the transgenes in live Jurkat cells using an antibody detecting the F(ab’)2. The percentages of cells positive for transgene expression are shown. The gates are determined by fluorescence minus one (FMO) control. (Fig. 6C) Measurement of NFKB activity in Jurkat cells expressing various lentiviral constructs (or no transduction control) after coculture with 293T or HeLa cells at a 2: 1 ratio. Relative light units (RLU) were measured after 5 h of coculture with target cells. Jurkat cells without any target served as a negative control. Cells stimulated with Io (1 pM) and PMA (2.5 pg / ml) formed the positive control. Data are combined from four independent experiments (n = 5-8). (Fig. 6D) ELISA data of IL2 collected from supernatant fractions in the same conditions as in (Fig. 6C). Data are combined from three independent experiments (n = 4-5). The bar graph displays mean ± SEM. Statistical significance was determined by the one-way ANOVA followed by Tukey’s multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0043] Fig. 7A- Fig. 7J show that ICCR augments signaling cascades following TCR activation. (Fig. 7A) Schematic map of a lentiviral construct encoding CTL16. (Fig. 7B) A flow cytometry histogram plot assessing CTL16 expression in Jurkat cells using the anti- TCR Vb3 antibody that specifically binds to the Vb28*01 of the CTL16 b chain. (Fig. 7C) Schematic of the coculture system of CTL16-expressing Jurkat cells and T2 cells used to analyze TCR-driven responses after p / MHC interaction. For specific analysis, Jurkat cells were modified to express NFAT-fLuc or NFxB-fLuc. (Fig. 7D, Fig. 7E) Reporter activity of NF AT (Fig. 7D) and NF / cB (Fig. 7E) were measured in nontransduced (NT) or CTL16- transduced Jurkat cells after exposure to ALK-pulsed T2 cells. Data were normalized to the basal activity of Jurkat cells without T2 cell exposure. Controls include T2 cells pulsed with gplOO peptide, unpulsed T2 cells, and T2 cells stimulated with lo / PMA. Unpaired Student’s t-test (n = 6-9 from 3 independent experiments) was used for statistical analysis. (Fig. 7F) ELISA data of IL2 levels after ~20 h coculture of Jurkat cells with T2 or ALK-pulsed T2 cells. Unpaired t-test (n = 2-4 from two independent experiments) was used for statistical analysis. (Fig. 7G) Experimental set up with engineered Jurkat cells and T2 cells. (Fig. 7H) Expression of CTL16 and ICCR in singly- or doubly-transduced Jurkat cells were assessed by flow cytometry. (Fig. 71) NF AT activity in NT Jurkat cells and Jurkat cells transduced with engineered constructs were measured after exposure to T2 cells pulsed with ALK or gplOO. The NF AT activity for each transduced cell line was normalized to a corresponding sample without T2 cell coculture (n = 12-13 from 5 independent experiments). (Fig. 7J) Measurement of NFKB activity of engineered or NT Jurkat cells (n = 6-9 from 3 independent experiments). Except in Fig. 7D, Fig. 7E, and Fig. 7F, all statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test.

[0044] Fig. 8A- Fig. 81 show that ICCR enhances CTL16-directed killing by T cells when antigen density is low. (Fig. 8A) Experimental setup to measure engineered T cell response against A375-MA2 cells. (Fig. 8B- Fig. 8D) Percentages of viable A375-MA2 cells after coincubation with CTL16 / ICCR-T, CTL16-T, ICCR-T, NT, and 2’ CAR-T (R6.5-137.3z) cells were measured using bioluminescence assay by tracking the fLuc+A375-MA2 cells over time. T cell killing efficacy for untreated A375-MA2 cells (Fig. 8B), A375-MA2 cells pretreated with IFNy(l ng / ml) for 2 days (Fig. 8C), and A375-MA2 cells pulsed with ALK (1 pM) (Fig. 8D). For each, the ratio of E:T was 2.5: 1, and percentages of target cell viability were normalized to treatment with NT cells at each timepoint (n = 6-9 per each group). Data are combined from T cells engineered with two donors. (Fig. 8E) Percentages of CD137+CD69+- activated CD3+T cells in each cell group after exposure to A375-MA2 cells, either untreated, pretreated with IFNy, or pulsed with ALK (n = 4-6 per each group). (Fig. 8F- Fig. 8H) The amount of secreted IL2 and IFNy was measured by ELISA ~20 h after E:T setup (n = 5-8 per each group). (Fig. 81) ELISA analysis of secreted IL2 and IFNy collected from supernatant fractions after ~20 h coculture of T2 cells (ALK-pulsed, gplOO- pulsed, unpulsed) and primary T cells (engineered or NT) (n = 5-8 per each group). All statistical differences were determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

[0045] Fig. 9A- Fig. 9F show that ICCR activity requires TCR-p / MHC interaction. (Fig. 9A) Schematic of endogenous TCR KO in primary T cells. (Fig. 9B) Flow cytometry histogram plots display expression of TCRa / / 3 and CD3s in NT and TRAC knockout NT (77 C-KO) cells by CRISPR / Cas9. Gating was determined using FMO. On the right panel, ICCR expression on T cells was detected using the F(ab’)2 antibody for NT (77 C-KO) and ICCR-T (77 C-KO) cells. (Fig. 9C) The percentages of live, untreated A375-MA2 cells post 48 h incubation with engineered T cells are shown after normalization to a “No T” control (n = 7 per each group). (Fig. 9D) A diagram of HLA KO in A375-MA2 cells. (Fig. 9E) Flow cytometry histogram plots of HLA-A2, HLA-II, and b2M expression in A375-MA2 and HLA-knockout A375-MA2 (Z7A4-KO) cells. (Fig. 9F) E:T data of A375- MA2 and A375-MA2 (HLA-KO) cells as target cells, measured 48 h post-coculture. Target cells were pulsed with either ALK or gplOO peptide (1 pM) prior to E:T setup. Effector cells were added at a 2.5: 1 E:T ratio, and percentages of live cells for each experimental condition were normalized to the “No T” control (n = 3 per group). All statistical differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

[0046] Fig. 10A- Fig. 10K. ICCR increases target-directed expansion and antitumor function in a xenograft model. (Fig. 10A) Flow cytometry histogram of ICCR-transduced T and NT cells. (Fig. 10B) T cell numbers plotted over time. NT and ICCR-T cells were stimulated with 8505C cells every 2-3 days at a 5: 1 ratio of E:T. Representative of two independent experiments conducted with T cells manufactured with different donor T cells. (Fig. 10C) The percentages of live 8505C cells plotted over time after coincubation with NT, ICCR-T, and 8505C-stimulated ICCR-T* cells. The ratio of E:T was 2.5: 1, and percentages of target cell viability were normalized to target cells with “No T” cell condition (n = 4 per each group). (Fig. 10D) Representative weekly whole-body luminescence images of NSG mice xenografted with 1068505C (fLuc / GFP) cells via tail vein injection. Administration of ICCR-T, ICCR-T* cells, and NT cells (10* 106cells / mouse) started 5 days after 8505C injection into NSG mice. Weeks (w) after the xenograft are indicated on the left, symbol denotes a death due to tumor burden. T cells were produced using two different donors, and ICCR-T cells were manufactured with two lentiviral CCR constructs including either with SSTR2 or not. Animals marked with the white solid box were PET-CT scanned using the18F-NOTAOCT, and the PET-CT images are shown on the right. A white box with a broken line marks the mouse from which TILs were collected for analysis in (Fig. 10G- Fig. 10K). (Fig. 10E) Quantitation of total luminous flux, photons (P) / s, signals over time in mice with 8505C (fLuc / GFP) xenografts treated with untreated (No T; n = 12), NT cells (n = 4), ICCR-T cells (n = 5), or ICCR-T* cells (n = 8). Kruskal -Wallis test, followed by Dunn’s multiple comparisons test, was used to compare luminescence signals at Day 20-26 timepoints for each group. (Fig. 10F) Survival curve of animals with 8505C xenografts with various treatments and no treatment is shown (n = 10 for No T; n = 4 for NT; n = 5 for ICCR-T; n = 6 for ICCR-T*). Log-rank test for each treatment group to NT group. (Fig. 10G) E:T data (20 h timepoint) of TILs isolated from the 8505C xenograft in (Fig. 10D) with target 293T and 8505C cells. Unstimulated NT cells were used for comparison. (Fig. 10H) Fluorescence microscope images of CFSE-labeled TILs against 293T and 8505C after 1 day of coculture (scale = 50 pm). (Fig. 101) Flow cytometry histograms showing ICCR expression in isolated TIL and control NT cells without or with 2 days of incubation with 8505C cells. (Fig. 10 J) Distribution of TCR / 3 and TCRa clonotypes identified from ex vivo cultured TILs. (Fig. 10K) Measurement of NF / B-fLuc reporter activities of Jurkat cells expressing the TCR- 8505C with or without ICCR after 5 h challenge with the target cells (Figs. 19A and 19B). The level of activation was normalized to NT cells at a basal level. NFAT-GFP activities were measured with the same setting as in the Jurkat cells expressing the NF / B-fLuc reporter. GFP mean fluorescence intensity (MFI) of each group was normalized to the no target cell (-) control (n = 6-7 per group except for ICCR (n = 2-3)). Statistical differences in (Fig. 10D) and (Fig. 10K) were determined by one-way ANOVA and two-way ANOVA, respectively, followed by Tukey’s multiple comparisons test. Fig. 11A-11G show that OKT3-directed ICCR-T cells showed robust response in lysing target tumors. (Fig. 11 A) E:T data of NT and ICCR-T cells when 8505C or 8505C- 0KT3 cells were used as targets at a 24 h timepoint (n = 8). (Fig. 1 IB) ELISA analysis of cytokines collected from supernatant fractions after ~20 h of coculture (n = 4). (Fig. 11C) Representative whole body luminescence images and total flux analysis of 8505C-OKT3 (fLuc / GFP) xenografts (n = 7 per group). The days post-xenograft (X) and days post-cell treatment (T) are indicated to the left for each row. Mann-Whitney test was used for statistical analysis. (Fig. 11D) Tumor volume changes of 8505C-OKT3 xenografts are plotted over time. “X” denotes the point when each mouse was euthanized due to tumor burden. Kruskal -Wallis test, followed by Dunn’s multiple comparisons test, was used for analysis. (Fig. HE) Survival curves of 8505C-OKT3 xenografts with different treatment conditions. Log-rank test was used to analyze statistical differences between each treatment group. (Fig. 1 IF) ELISA analysis of blood sera collected from 8505C-OKT3 xenografts (n = 3 per treatment group, n = 1 for No T control). Student’s t-test was used to analysis statistical difference between NT and ICCR-T. (Fig. 11G) Representative flow cytometry plots of live cells isolated from 8505C-OKT3 xenograft tumors show the percentages of GFP and human CD3+T cells. Summary of flow cytometry analysis is shown as a bar graph. Statistical differences were assessed by one-way ANOVA in (Fig. 11 A) and two- way ANOVA for (Fig. 1 IB) and (Fig. 11G), followed by Tukey’s multiple comparisons test.

[0047] Fig. 12A-12H show that ICCR improved tumor-targeted T cell function in a patient- derived tumor xenograft model. (Fig. 12A) Transduction efficiency of ICCR into T cells derived from an ATC patient (RM). (Fig. 12B) Plots show the number of NT and ICCR-T cells upon stimulation with donor-matched RM-ATC cells over time. Arrows indicate the days when RM-ATC cells were added at a 5: 1 ratio of E:T. (Fig. 12C) E:T data of viable RM-ATC cells when NT, ICCR-T, and RM-stimulated ICCR-T* cells were added at a 2.5: 1 E:T ratio (n = 4). Statistical analysis was performed with one-way ANOVA followed by Tukey’s multiple comparisons test. (Fig. 12D) Survival curves of RM orthotopic xenografts treated with ICCR-T, NT cells, or no treatment (n = 4 for No T; n = 3 for NT; n = 8 for ICCR-T). Log-rank test was used to assess the difference between treatment responses with ICCR-T and NT. Experiment was terminated at when animals reached a humane endpoint (Day 60). (Fig. 12E) Representative IHC images of human CD3 (red stain) and H&E are shown for RM thyroid tumor tissues taken from xenografts treated with NT and ICCR-T cells (n = 3 per group), scale bar =150 pm. (Fig. 12F) Volcano plots of differentially expressed genes in ICCR-T vs. NT (left) and tumor-stimulated vs. unstimulated T cells (right) (Fold cutoff = log20.5 and P value < 0.05). (Fig. 12G) Gene set analysis displaying directed global significance scores. (Fig. 12H) Analysis of TCR diversity score. Statistical analysis by Mann-Whitney test.

[0048] Fig. 13 show sT2 cell phenotypic study. Flow cytometry histogram plots of HLA- A2 and ICAM1 expression in T2 cells (gray filled peaks). Gates were determined using FMO (solid black line). The percentages of cells positive for tested molecule expression are shown.

[0049] Fig. 14A-Fig. 14B show transduction rates and CD4:CD8 composition of engineered T cells. (Fig. 14A) Flow cytometry histogram plots showing lentiviral transduction levels in CTL16-T, ICCR-T, CTL16 / ICCR-T, 2’CAR-T, and NT cells. The TCR0 chain of the CTL16 construct is detected by the FITC-conjugated V03-specific antibody. Polyclonal antibody against F(ab’)2 was used to assess the expression level of R6.5, an scFv incorporated in the ICCR and 2’ CAR constructs. (Fig. 14B) Proportion of CD4+T vs. CD8+T cells in NT and engineered T cells was assessed by flow cytometry analysis. Cells were gated on live CD3+T cells.

[0050] Fig. 15A-Fig. 15B show surface expression of HLA and ICAM1 in A375-MA2 cells. (Fig. 15 A) Flow cytometry histogram plots displaying HLA-A2 (left), 02 microglobulin (02M; center), and ICAM1 (right) expression. A375-MA2 cells were untreated (UNT), prestimulated with 1 ng / ml IFNy (+IFNy) for 2 days, or pulsed with 1 pM ALK (+ALK) overnight. Positively gated cells were determined using the FMO control. Percentages of positively gated cells and fold over mean fluorescence intensity (MFI) (number within parenthesis) are shown on each plot. (Fig. 15B) Relative fold change (FC) of MAGEC2 expression in prestimulated (+IFNy) or pulsed (+ALK) vs. untreated (UNT) A375-MA2 cells was determined by qRT-PCR analysis. AACT was calculated using the Pactin as the internal control (n = 6 per group combined from 2 biological experiments). **, P < 0.01; ns, not significant.

[0051] Fig. 16 shows phenotypic analysis of T cell activation. Representative flow cytometry plots of CD137 and CD69 expression on the following T cells, left to right: NT, CTL16-T, ICCR-T, CTL16 / ICCR-T, and 2’ CAR-T. Cells were gated on live CD8+and CD4+cells separately, and FMO controls were used to gate for CD 137 and CD69 positive expression. T cells were coincubated with untreated A375 -MA2 cells or A375-MA2 that were pretreated with JFNy (1 ng / ml) for 2 days or pulsed with ALK (1 pM) overnight. CD 137 upregulation was notable in the CD8+T cell population, whereas CD69 induction was observed in both CD4+and CD8+T cells. A summary of the percentages of activated CD3+cells is presented as a graph in Fig. 8E.

[0052] Fig. 17A-Fig. 17B show generation of a Jurkat cell line expressing TCR reactive to 8505C cells. (Fig. 17A) A schematic of the lentiviral construct that incorporated the most predominant TCR0 and TCRa sequences isolated from 8505C xenograft tissue after treatment with ICCR-T cells (TCR-8505C). The gene composition of each TCR chain is indicated below each TCR chain. (Fig. 17B) Flow cytometry plots of Jurkat cells, which were transduced with reconstructed TCR-8505C, ICCR, and TCR-8505C / ICCR constructs. FITC-labeled anti-TRBV5-6 antibody was used to detect the TCR-8505C. An antibody detecting the F(ab’)2 chain was used to measure the level of ICCR transduction.

[0053] Fig. 18 shows increased NF AT activity mounted by TCR-8505C / ICCR expression. Representative flow cytometry plots display the expression levels of CD69 and NF AT promoter-driven GFP, using a reporter system described previously (7). Each Jurkat cell line (NT, TCR-8505C, ICCR, and TCR-8505C / ICCR) was coincubated with 293T and 8505C cells at a 1 : 1 ratio. After ~20 h incubation, live CD3+gated Jurkat cells were analyzed using anti-CD69-PECy7 antibody and GFP expression marking NF AT driven activation. The summary of NF AT activity data, calculated as fold increase of GFP MFI, is presented as a graph in Fig. 10K.

[0054] Fig. 19A- Fig. 19B show increased NF AT activity mounted by TCR-8505C / ICCR expression. Representative flow cytometry plots display the expression levels of CD69 and NF AT promoter-driven GFP, using a reporter system described previously (7). Each Jurkat cell line (NT, TCR-8505C, ICCR, and TCR-8505C / ICCR) was coincubated with 293T and 8505C cells at a 1 : 1 ratio. After ~20 h incubation, live CD3+gated Jurkat cells were analyzed using anti-CD69-PECy7 antibody and GFP expression marking NF AT driven activation. The summary of NF AT activity data, calculated as fold increase of GFP MFI, is presented as a graph in Fig. 10K.

[0055] Fig. 20 shows TIL analysis of 8505C-OKT3 tumors. Representative flow cytometry histogram plots show the expression level of T cell activation markers, CD69, PD1, CD137, and LAG3, on live CD3+T cells isolated from two 8505C-OKT3 xenograft tumors treated with ICCR-T cells. Analysis comparing frequencies of activated TILs were compared between NT and ICCR-T treatment groups. Statistical difference was determined using two- way ANOVA, followed by Tukey’s multiple comparisons test (n = 2 for NT; n = 4 for ICCR-T treatment group). *, P < 0.05.

[0056] Fig. 21A-Fig. 21C show phenotypes of RM-ATC and donor-matched primary T cells. (Fig. 21 A) Flow cytometry histogram plots of live 8505C and RM-ATC cells when analyzed using a PECy7-conjugated, IC AMI -specific antibody. FMO control was used to set the gate for ICAM1 expression. (Fig. 2 IB) Viability of IFNy-treated RM-ATC cells after addition of NT, unstimulated ICCR-T, and RM-stimulated ICCR-T* cells. RM-ATC cells were pretreated with IFNy (200 ng / pl) for 48 h, and the E:T ratio was 2.5: 1. (Fig. 21C) Flow cytometry plots show analysis of live CD3+T cells isolated from PBMC of an RM patient for CD4:CD8 distribution and PD1 expression. *, P < 0.05; ****, P < 0.0001.

[0057] Fig. 22 shows heatmap of differentially expressed genes. Hierarchical clustering of differentially regulated genes in comparison groups, ICCR-T and NT cells with or without target tumor stimuli (n = 2-3 per group). Allogeneic healthy donor T cells and autologous ATC patient T cells were stimulated by 8505C and RM-ATC cells, respectively, every 2-3 days for 9 days. One condition included in vivo stimulated ICCR-T cells isolated from an 8505C xenograft tumor. T cells from two donors in the same condition (+ / - target tumor stimuli, + / - ICCR-T) were combined for analysis using the Nanostring CAR-T panel.

[0058] DETAILED DESCRIPTION

[0059] In order to harness polyclonal tumor-reactive T cells present in TILs, peripheral blood or lymph nodes that might be only minimally active, we have developed an IFNy- responsive T cell stimulation strategy, via chimeric costimulatory receptor (CCR), to augment the proliferation of tumor-reactive T cells (Fig. 1). To reach maximal effector functions, cytotoxic T lymphocytes (CTLs) follow stepwise activation. T cells first release granzyme / perforin and Th 1 -type cytokines such as IFNY (a process mediated by the initial TCR / pMHC interaction), and then, stimulatory cytokines such as IL2 mediated by costimulatory signals of CD28, 4- IBB, etc.3to result in complete activation. IL2 is a crucial T cell growth factor that contributes to the persistence of T cells in vivo4. As indicated, one of the first steps in T cell activation results in IFNY production. IFNy is known to induce ICAM-1 expression in many cancers. To make use of this IFNy-induced ICAM-1 induction of tumor cells, we have designed an ICAM-1 -specific CCR to trigger NFkB activation in T cells for full activation. This method complements NF AT activation induced by TCR / pMHC contacts that may not be sufficient on their own to result in complete activation of T cells. We have designed a CCR that stimulates T cells to reach the threshold for cytokine release and proliferation only when there is also TCR activation and ZFNy secretion. While interaction between the CCR and ICAM-1 triggers IL2 release and provides a growth advantage, the cytotoxic functions of T cells are not activated by this pathway alone.

[0060] The IC AMI -specific CCR thus avoids T cell cytotoxic effects expected with traditional CARs as they lack CD3^. Traditional CARs need to be designed to be selective to tumor antigens and carry a high risk of on-target, off-tumor toxicity. In addition, our IC AMI -gated CCR approach mimics the natural interaction between ICAM1 on target cells and its ligand LFA1 on T cells. This molecular interaction contributes to the formation of the immunological synapse that sustains contact between T and target cells (e.g., tumor, APC) and increases avidity of T cells5,6. ICAM1 is one of the few surface antigens that are induced by IFNy, but is rarely immune edited in tumors7. The result of the additional NFkB signaling from this targeting modality will be to help overcome tumor resistance to T cells mediated by immune checkpoint activation. Our CCR approach is applicable to other existing genetic modification strategies as well, such as CAR and TCR, in addition to being compatible with the use of TIL for T cell immunotherapy.

[0061] Our work provides evidence supporting the use of ICCR to achieve robust activation and proliferation, thereby enabling low activity tumor-specific TCRs to surmount activation barriers present in an immunosuppressive tumor microenvironment. Intercellular adhesion molecule 1 (ICAMl)-specific chimeric costimulatory receptor augments the native tumorspecific TCRs by increasing T cell proliferation and signal activities, paving the way for improved therapies for targeting solid tumors.

[0062] Definitions

[0063] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0064] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, a cell expressing a CCR provided herein.

[0065] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Example amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0066] The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g., between a peptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and / or hydrogenbond interactions under physiological conditions.

[0067] As used herein, the term “cancer” includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bone, cartilage, blood, and vessels, including the cervix, anus, vagina, vulva, penis, tongue base, larynx, and tonsil. The term “cancer” further encompasses primary and metastatic cancers.

[0068] The term “chimeric antigen receptor” (CAR) refers to molecules that combine a binding domain against a component present on the target cell, for example an antibodybased specificity for a desired antigen (e.g., a tumor antigen) with an immune cellactivating intracellular domain to generate a chimeric protein. Generally, CARs comprise an extracellular single chain antigen-binding domain (e.g., an scFv) fused to the intracellular signaling domain.

[0069] The term “epitope” means a protein determinant capable of specific binding to an antibody or immune cell (e.g., T cell). Epitopes usually include chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a CCR, CAR or antibody is capable of binding.

[0070] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which can otherwise transcribe to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), may be transfected into cells, e.g., mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, poly adenylation sites, origins of replication, marker genes, etc.

[0071] The terms “ligand-binding domain” and “antigen-binding domain” are used interchangeably herein, and refer to that portion of a chimeric antigen receptor that binds specifically to a predetermined antigen. The term “linker” refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0072] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0073] As used herein, the phrase “pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0074] As used herein, the phrase “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0075] The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a natural or synthetic molecule, or some combination thereof, comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The polymeric form of nucleotides is not limited by length and can comprise either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides. The polynucleotide is not necessarily associated with the cell in which the nucleic acid is found in nature, and / or operably linked to a polynucleotide to which it is linked in nature.

[0076] The term “precancerous lesions” or “precancerous condition” refers to atypical cells and / or tissues that are associated with an increased risk of cancer. The term “precancerous lesions” may refer, for example, to dysplasia, benign neoplasia, or carcinoma in situ.

[0077] As used herein, a therapeutic that “prevents” a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0078] The term “specifically binds” or “specific binding”, as used herein, when referring to a polypeptide (including CCR or CAR polypeptides) refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M1(e.g., 106M 107M 108M 109M IO10M 1011M and 1012M1or more) with that second molecule.

[0079] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0080] The terms “transformation”, “transfection”, or “transduction” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell (e.g., a mammalian cell) including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0081] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0082] The term “vector” refers to the means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, to which the nucleic acid has been linked, and may or may not be able to replicate autonomously or integrate into a chromosome of a host cell. Such vectors may include any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0083] In certain embodiments, agents may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” or “administered conjointly” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0084] Chimeric Costimulatory Receptors (CCRs)

[0085] Chimeric costimulatory receptors (CCRs) are receptors comprising a targeting moiety that is associated with one or more intracellular costimulatory domains in a single fusion molecule. In some embodiments, the CCR does not comprise a cluster of differentiation 3 zeta (CD3Q domain. In certain embodiments, the binding moiety of a CCR comprises an ICAMl-binding domain, e.g., a single-chain fragment variable (scFv) comprising the light and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker. In certain embodiments, the binding moiety further comprises transmembrane (e.g., CD8 transmembrane domains) and / or hinge domains.

[0086] In some embodiments, the costimulatory domain comprises a full-length of the costimulatory domain listed in Table la and Table lb below. In some embodiments, the costimulatory domain comprises a functionally active fragment of the costimulatory region domain listed in Table la and Table lb below.

[0087] In some embodiments, the CCR disclosed herein may comprise one or more costimulatory domains listed in Table la below, and the costimulatory region domain comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% identity to an costimulatory domain amino acid sequence set forth in Table lb below.

[0088] Table la: Example costimulatory domains. | B7-H3 |

[0089] Table lb: Example costimulatory region domain amino acid sequences

[0090] In certain embodiments, the binding domain and / or extracellular domain of a CCR provided herein provides the CCR with the ability to bind to ICAM1. A binding domain (e.g., a ligand-binding domain or antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). A binding domain includes any naturally occurring, synthetic, semi -synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, and as further described herein, a binding domain may be antibody light chain and heavy chain variable regions, or the light and heavy chain variable regions can be joined together in a single chain and in either orientation (e.g., VL-VH or VH-VL). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind with a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target may be an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in tumor killing.

[0091] In one embodiment, the binding domain of the CCR is a single chain fragment variable (scFv) specific for ICAM1, and may be a murine, human or humanized scFv. Single chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target. A technique which can be used for cloning the variable region heavy chain (VH) and variable region light chain (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. Thus, in certain embodiments, a binding domain comprises an antibody-derived binding domain but can be a non-antibody derived binding domain. An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.

[0092] In some embodiments, the CCR further comprises a single chain fragment variable (scFv) domain. The scFv domain may be a scFv domain set forth in Table 2 below, or a scFv that includes CDR and / or variable region sequences set forth in Table 2. In some embodiments, the CCR comprises an antigen binding domain (e.g., a scFv domain) comprising heavy chain and light chain CDR sequences of an antibody or scFv provided in Table 2. In some embodiments, the CCR comprises an antigen binding domain (e.g., a scFv domain) comprising a heavy chain variable region sequence and a light chain variable region sequence of an antibody or scFv provided in Table 2. In some embodiments, the CCR further comprises a scFv domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, 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 100% identity to an amino acid sequence set forth in Table 2.

[0093] In some embodiments, the scFv domain is a IC AMI -specific scFv. In some embodiments, the scFv domain is a ICAM1 -specific R6.5 clone scFv. R6,5 scFv sequences are provided in Table 2. In some embodiments, the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively. In some embodiments, the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and / or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8. The heavy chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 7. The light chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the scFv domain comprises an amino acid sequence of SEQ ID NO: 9.

[0094] Table 2: scFv R6.5 sequences

[0095] In certain embodiments, the CCRs of the present disclosure may comprise a linker between the various domains, added for appropriate spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the binding domain VH or VL which may be between 1-10 amino acids long. In other embodiments, the linker between any of the domains of the CCR may be between 1-20 or more than 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers described herein are also included herein, e.g., a linker 10-30 amino acids long.

[0096] In certain embodiments, linkers suitable for use in the CCR described herein are flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0097] Example flexible linkers include glycine polymers (G)n, glycine-serine polymers, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CCRs described herein. Glycine accesses significantly more phi- psi space than even alanine, and is much less restricted than residues with longer side chains. The ordinarily skilled artisan will recognize that design of a CCR can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CCR structure.

[0098] The binding domain of the CCR may be followed by a “spacer,” or, “hinge,” which refers to the region that moves the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen-binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in a CCR is generally between the transmembrane (TM) and the binding domain. In certain embodiments, a hinge region is an immunoglobulin hinge region and may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. Other example hinge regions used in the CCRs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered.

[0099] In some embodiments, the CCR described herein further comprises a transmembrane domain. The “transmembrane” region or domain is the portion of the CCR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell, and facilitates binding of the binding domain to the target antigen. In some embodiments, the transmembrane domain may be a CD3(^ transmembrane domain. Other transmembrane domains that may be employed in some embodiments include those obtained from CD8, CD8a, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.

[0100] In some embodiments, the transmembrane domain is selected from a transmembrane domain of CD28, CD8a, ICOS, 4- IBB, CD4, Tim4, 0X40, CD27, CD2, LFA-1, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, NKG2D, NKp44, NKp46, DAP12, CD16, NKp30, FcRy, DAP10, 2B4, or DNAM-1. In some embodiments, the transmembrane domain may be selected from the transmembrane domains of Table 3 below. In certain embodiments, the transmembrane domain is a CD 8 transmembrane domain. In some embodiments, the CCR further comprises a transmembrane domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, 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 100% identity to an amino acid sequence set forth in Table 3. In specific embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 11 or 12.

[0101] Table 3: Example transmembrane domains

[0102] The CCR polypeptides encompassed by the present disclosure may comprise an amino acid sequence set forth in Table 4 below. Table 4. Example CCR polypeptides Polypeptides having substantial sequence similarities can have the same or similar catalytic and / or functional activity. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of a CCR described herein or fragment thereof may also suitable for the methods and compositions provided herein. In some embodiments, the CCR further comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, 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 100% identity to an amino acid sequence set forth in Table 4. In certain embodiments, the CCR comprises an amino acid sequence that is identical to a sequence set forth in Table 4, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and / or substitutions). In some embodiments, the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).

[0103] In some embodiments, variations or derivatives of the CCRs are provided herein. The altered CCR polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still induces antigen-specific proliferation, cytotoxicity, cytokine secretion, and / or other immune cell phenotypes, and are considered functional equivalents. As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group may typically substitute for one another without substantially affecting the function of a protein. According to certain embodiments, the derivatives, equivalents, variants, or mutants of a CCR are polypeptides that are at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% homologous to an amino acid sequence of a CCR described herein (e.g., the amino acid sequences listed in Table 4) or fragment thereof. In some embodiments, the identity is 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 more.

[0104] In some embodiments, the CCR polypeptides encompassed by the present disclosure may comprise an amino acid sequence derived from a CCR polypeptide set forth in Table 4. In some embodiments, the CCR polypeptides may include conservative or nonconservative mutations. A CCR polypeptide may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations. In some embodiments, a CCR polypeptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations.

[0105] Nucleic Acids and Vectors

[0106] In certain aspects, also disclosed are nucleic acids and polynucleotide vectors encoding the CCR polypeptides disclosed herein.

[0107] In some embodiments, the nucleic acids and / or polynucleotide vectors encoding the CCR polypeptides disclosed herein comprise an nucleic acid sequence set forth in Table 5 below.

[0108] Table 5: Example CCR-encoding nucleic acid sequences

[0109] Nucleic acid sequences encoding the disclosed CCRs, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0110] Expression of nucleic acids encoding CCRs is typically achieved by operably linking a nucleic acid encoding the CCR polypeptide to a promoter, and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0111] In certain embodiments, the polynucleotide encoding the CCR described herein is inserted into a vector. The vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and / or the cloning of the polynucleotide. Such vectors may also be referred to as “expression vectors”. The isolated polynucleotide may be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector may be digested using appropriate restriction enzymes and then may be ligated with the isolated polynucleotide having matching restriction ends. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell. In most cases, RNA molecules are then translated into a protein. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra. 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 expression vector may have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EFla promoters, ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell. Other suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and rous sarcoma virus promoter. Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In certain embodiments, inducible promoters are also contemplated as part of the vectors expressing chimeric antigen receptor. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0112] The expression vector may have additional sequence such as 6x-histidine, c-Myc, and FLAG tags which are incorporated into the expressed CCRs. Thus, the expression vector may be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest carried on the expression vector. An expression vector may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors may include a selectable marker for maintenance of the vector in the host or recipient cell.

[0113] In various embodiments, the vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional example vectors include, without limitation, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vectors (Clontrch), pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the CCRs disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells.

[0114] In certain embodiments, the nucleic acids encoding the CCR are provided in a viral vector. A viral vector can be that derived from, for example, a retrovirus (e.g., a foamy virus) or lentivirus. As used herein, the term, “viral vector,” refers 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 the coding sequence for the various chimeric proteins described herein in place of nonessential viral genes. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0115] In certain embodiments, the viral vector containing the coding sequence for a CCR described herein is a retroviral vector or a lentiviral vector. The term “retroviral vector” refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.

[0116] Retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuS V), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)). Retroviruses” also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0117] A lentiviral vector refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi; a caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). Preparation of the recombinant lentivirus can be achieved using the methods according to Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72: 8463-8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).

[0118] Retroviral vectors (i.e., both lentiviral and non-lentiviral) can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141- 6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254: 1802- 1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0119] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNAs available from commercially available sources, including the Type Culture Collection (ATCC), Rockville, Md. The sequences also can be synthesized chemically.

[0120] For expression of a CCR, the vector may be introduced into a host cell to allow expression of the polypeptide within the host cell. The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art, as described above. For example, the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EFla promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance the transcription of the polynucleotide. Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell. For cloning of the polynucleotide, the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell.

[0121] In certain embodiments, the present disclosure provides isolated host cells containing the vectors provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterob actehaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such Pseudomonas such as P. aeruginosa, and Streptomyces.

[0122] The CCRs are introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms, “transfection,” and, “transduction,” refer to the processes by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be a stable introduction. Transfection may be accomplished by a variety of means known in the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran- mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell. For example, a nucleic acid encoding a CCR carried by a retroviral vector can be transduced into a cell through infection and pro virus integration. In order to assess the expression of a CCR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.

[0123] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0124] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0125] In the case where a non-viral delivery system is utilized, an example delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; di cetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N. Y.); cholesterol (“Choi”) can be obtained from Calbiochem- Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0126] Immune effector cells

[0127] In certain aspects, also disclosed herein are cells that are engineered to express the disclosed CCR polypeptides. In some embodiments, the cells engineered to express the disclosed CCR polypeptides are immune effector cells. In some embodiments, other types of cells, such as induced pluripotent stem cells (iPSC) or hematopietic stem cells (HSC), are engineered and differentiated into immune effector cells that express the disclosed CCR polypeptides.

[0128] In some embodiments, the iPSC, HSC, or immune effector cells are obtained from the subject to be treated (i.e., are autologous). However, in certain embodiments, immune effector cell lines or donor effector cells (allogeneic) are used.

[0129] Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0130] In some embodiments, the immune effector cells is a T cell. Sometimes, the T cell is a CD8+ or CD4+ T cell. In some embodiments, the immune effector cells further comprising (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen.

[0131] In some embodiments, the TCR or CAR binds a cancer antigen selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, LI -CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

[0132] In some embodiments, the CAR further comprises at least one costimulatory domain. In some embodiments, the at least one costimulatory region domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0133] In certain embodiments, the present disclosure provides methods for making the immune effector cells which express the CCRs described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having a cancer antigen expressing tumor cell, such that the immune effector cells express one or more CCR as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CCR. In this regard, the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a CCR as described herein).

[0134] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells may be obtained from a subject. In particular, the immune effector cells for use with the CCRs as described herein comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. In one embodiment, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium, and may lack magnesium or may lack many, if not all, divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as by using a semiautomated flowthrough centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media.

[0135] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 b, CD 16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest.

[0136] PBMCs may be used directly for genetic modification with the CCRs using methods as described herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion. CD8+cells can be obtained by using standard methods. In some embodiments, CD8+cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8+cells. In embodiments, memory T cells are present in both CD62L+and CD62L' subsets of CD8+peripheral blood lymphocytes. PBMC are sorted into CD62L' CD8+and CD62L+CD8+fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, the expression of phenotypic markers of central memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

[0137] In certain embodiments, CD4+T cells are further sorted into subpopulations. For example, CD4+T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO , CD45RA+, CD62L+CD4+T cell. In some embodiments, central memory CD4+cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+cells are CD62L and CD45RO negative.

[0138] The immune effector cells, such as T cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune effector cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CCR) and then are activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874; 6,867,041; 6,797,514; W02012079000. Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2 (e.g., recombinant human IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). In other embodiments, the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177; 5,827,642; and WO2012129514.

[0139] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes, preferably cytotoxic T lymphocytes (CTLs). In some embodiments, the immune effector cell is an NK cell, a Treg, a gamma delta T cell, a macrophage, a B cell, a, NKT cell, or a plasmacytoid dendritic cell.

[0140] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including Thl, Th2, Th3, Thl7, Th9, or Tfh, which secrete different cytokines to facilitate a different type of immune response.

[0141] Cytotoxic T cells (Tc cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0142] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon reexposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0143] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described — naturally occurring Tregcells and adaptive Tregcells.

[0144] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id.

[0145] In some embodiments, the T cells comprise a mixture of CD4+cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T comprise are cytotoxic CD8+T lymphocytes.

[0146] Natural-killer (NK) cells are CD56 CD3 large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53: 1666-1676). Unlike cytotoxic CD8+T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can eradicate MHC-I-negative cells (Narni- Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects.

[0147] Therapeutic Methods

[0148] Immune effector cells expressing the CCRs disclosed herein may in some embodiments elicit a therapeutically beneficial immune response against antigen-expressing (e.g., ICAM1 -expressing) cancer cells. For example, an anti-tumor immune response elicited by the disclosed CCR-modified immune effector cells may be an active or a passive immune response. In addition, the CCR-mediated immune response may be part of an adoptive immunotherapy approach in which CCR-modified immune effector cells induce an immune response specific to a cancer antigen.

[0149] CCR-expressing immune effector cells prepared as described in some embodiments herein can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.

[0150] In some aspects, provided herein are methods of treating cancer (e.g., a solid tumor) in a subject by administering to the subject a composition comprising cells expressing a CCR polypeptide disclosed herein.

[0151] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatmenteffective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin.

[0152] A treatment-effective amount of cells in the composition is at least 2 cells (for example, at least 1 CD8+central memory T cell and at least 1 CD4+helper T cell subset), or is more typically greater than 102cells and up to 106or up to and including 108or 109cells, and can be more than 1010cells. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.

[0153] The cells may be autologous or heterologous to the patient undergoing therapy. The cells may be allogenic. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-y, IL-2, IL- 12, TNF-a, IL- 18, and TNF-P, GM-CSF, IL-4, IL- 13, Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction of the immune response.

[0154] The CCR expressing immune effector cell populations may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions disclosed herein may comprise a CCR-expressing immune effector cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions disclosed herein may be formulated for intravenous administration.

[0155] The anti -tumor immune response induced in a subject by administering CCR expressing T cells described herein using the methods described herein, or other methods known in the art, may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, by regulatory T cells, and / or by helper T cells. Humoral immune responses, mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced. A variety of techniques may be used for analyzing the type of immune responses induced by the compositions disclosed herein, which are well described in the art; e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, N.Y., N.Y.

[0156] Thus, in some aspects, provided herein are methods of treating an individual diagnosed with or suspected of having, or at risk of developing a malignancy, comprising administering to the individual a therapeutically effective amount of the CCR-expressing immune effector cells as described herein.

[0157] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratum orally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0158] In certain embodiments, the disclosed CCR-modified immune effector cells are administered to a patient in conjunction with e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to additional cancer treatments. In some embodiments, the CCR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CCR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T-cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as 0KT3 or CAMPATH. In other embodiments, the cell compositions are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells. In additional embodiments, expanded cells are administered before or following surgery to treat cancer or pre-cancerous lesions in the subject.

[0159] Indications

[0160] In certain aspects, provided herein are methods of treating cancer using a CCR- expressing cell provided herein. In certain embodiments, the cancer to be treated expresses a cancer antigen to which the CCR expressed by the immune effector cells (e.g., T cells) specifically binds. In some embodiments, the methods and compositions provided herein relate to the treatment of solid tumors.

[0161] In some embodiments, the methods and compositions provided herein relate to the treatment of a carcinoma. The term “carcinoma” refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and / or resist physiological and non-physiological cell death signals and gives rise to metastases. Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.

[0162] In some embodiments, the methods and compositions provided herein relate to the treatment of a sarcoma. The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0163] Additional exemplary neoplasias that can be treated using the methods and compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.

[0164] In some embodiments, the cancer treated is a melanoma. The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.

[0165] In some embodiments, the methods and compositions provided herein relate to the treatment of a leukemia. The term “leukemia” is meant broadly progressive, malignant diseases of the hematopoietic organs / sy stems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia.

[0166] Particular categories of tumors that can be treated using methods and compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, non-Hodgkin' s lymphoma.

[0167] ADDITIONAL EXEMPLARY EMBODIMENTS

[0168] Embodiment 1. A chimeric costimulatory receptor (CCR) comprising: a) an intercellular adhesion molecule-1 (ICAMl)-binding domain; and b) at least one costimulatory domain, wherein the CCR does not comprise a cluster of differentiation 3 zeta (CD3Q domain. Embodiment 2. The CCR of embodiment 1, wherein the at least one costimulatory domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0169] Embodiment 3. The CCR of embodiment 1 or 2, wherein the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof.

[0170] Embodiment 4. The CCR of any one of embodiments 1-3, wherein the at least one costimulatory domain comprises 4- IBB and 0X40.

[0171] Embodiment 5. The CCR of any one of embodiments 1-4, wherein the ICAM1- binding domain comprises an antibody or an antigen-binding fragment thereof.

[0172] Embodiment 6. The CCR of embodiment 5, wherein the antigen-binding fragment of an antibody is a single-chain variable fragment (scFv).

[0173] Embodiment 7. The CCR of embodiment 5 or 6, wherein the antibody is selected from R6.5 (BIRR-1, Enlimomab), 1 A6, MD-3, M10A12, bersanlimab, AF-647, and MSH- TP15.

[0174] Embodiment 8. The CCR of any one of embodiments 1-7, wherein the ICAM1- binding domain comprises R6.5 scFv or an I domain of the aL subunit of lymphocyte function-associated antigen-1 (LFA1).

[0175] Embodiment 9. The CCR of any one of embodiments 1-8, wherein the CCR comprises 4- IBB, 0X40, and R6.5 scFv.

[0176] Embodiment 10. A T cell comprising the CCR of any one of embodiments 1-9, optionally wherein the T cell is a CD8+ or CD4+ T cell. Embodiment 11. The T cell of embodiment 10, further comprising (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR).

[0177] Embodiment 12. The T cell of embodiment 11, wherein the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen.

[0178] Embodiment 13. The T cell of embodiment 12, wherein the CAR further comprises at least one costimulatory domain.

[0179] Embodiment 14. The T cell of embodiment 13, wherein the at least one costimulatory region domain is selected from CD28, 4-1BB, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0180] Embodiment 15. The T cell of embodiment 13 or 14, wherein the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof.

[0181] Embodiment 16. The T cell of any one of embodiments 11-15, wherein the TCR or CAR binds a cancer antigen selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, Ll-CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

[0182] Embodiment 17. A tumor infiltrating lymphocyte (TIL) comprising the CCR of any one of embodiments 1-9.

[0183] Embodiment 18. The TIL of embodiment 17, further comprising (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR).

[0184] Embodiment 19. The TIL of embodiment 18, wherein the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen.

[0185] Embodiment 20. The TIL of embodiment 19, wherein the CAR further comprises at least one costimulatory domain.

[0186] Embodiment 21. The TIL of embodiment 20, wherein the at least one costimulatory region domain is selected from CD28, 4-1BB, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0187] Embodiment 22. The TIL of embodiment 20 or 21, wherein the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof.

[0188] Embodiment 23. The TIL of any one of embodiments 18-22, wherein the TCR or CAR binds a cancer antigen is selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, Ll-CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, ROR1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

[0189] Embodiment 24. A pharmaceutical composition comprising the T cell of any one of embodiments 10-16 and / or the TIL of any one of embodiments 17-23.

[0190] Embodiment 25. A method of isolating a T cell with a tumor-specific TCR, the method comprising: a) administering to a subject having a tumor, a T cell comprising the CCR of any one of embodiments 1-9; or the T cell of embodiment 10; b) harvesting the tumor from the subject; and c) isolating the T cell with the CCR from the tumor, thereby isolating the T cell with a tumor-specific TCR.

[0191] Embodiment 26. The T cell isolated according to the method of embodiment 25. Embodiment 27. A method of identifying a tumor-specific TCR, the method comprising: a) isolating the T cell with a tumor-specific TCR according to the method of embodiment 25; b) sequencing the TCR to thereby identify the tumor-specific TCR.

[0192] Embodiment 28. A method of preventing or treating a cancer in a subject, the method comprising administering to the subject the T cell of embodiment 26, which is optionally further expanded.

[0193] Embodiment 29. A method of preventing or treating a cancer in a subject, the method comprising administering to the subject the T cell of any one of embodiments 10-16, the TIL of any one of embodiments 17-23, and / or the pharmaceutical composition of embodiment 24.

[0194] Embodiment 30. The method of embodiment 28 or 29, wherein the T cell and / or TIL cell is autologous or allogeneic to the subject.

[0195] Embodiment 31. The method of any one of embodiments 28-30, wherein the subject is treated conjointly with at least one additional cancer therapy.

[0196] Embodiment 32. The method of embodiment 31, wherein the subject is administered with the at least one additional cancer therapy concomitant with, prior to, or following the administration of the T cell, the TIL, and / or the pharmaceutical composition.

[0197] Embodiment 33. The method of embodiment 31 or 32, wherein the at least one additional cancer therapy is selected from immunotherapy, checkpoint inhibitors, cancer vaccines, chemotherapy, radiation therapy, and surgery, optionally a checkpoint inhibitors.

[0198] Embodiment 34. The method of any one of embodiments 31-33, wherein the at least one additional cancer therapy is a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a PD-L1 inhibitor (e.g., an anti-PD-Ll antibody), KD033, or any combination of two or more thereof. Embodiment 35. The method of embodiment 34, wherein the PD-1 inhibitor is selected from MP-514 (MED 10680), nivolimumab, cemiplimab, pembrolizumab, dostarlimab, Vopratelimab (JTX-4014), Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), INCMGA00012 (MGA012), and AMP-224.

[0199] Embodiment 36. The method of embodiment 34, wherein the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, KN035, Cosibelimab (CK-301), AUNP12, CA-170, and BMS-986189.

[0200] Embodiment 37. The method of any one of embodiments 25-36, wherein the cancer or tumor is selected from non-small cell lung cancer (NSCLC), gastric cancer, Melanoma, Breast cancer, Lung cancer, Esophageal cancer, Gastric cancer, Pancreatic cancer, Liver cancer, Colorectal cancer, Anal cancer, Cervical cancer, Ovarian cancer, Endometrial cancer, Prostate cancer, Bladder cancer, Kidney cancer, Testicular cancer, Penile cancer, Head and neck cancer (including oral, pharyngeal, and laryngeal cancers), Thyroid cancer, Soft tissue sarcomas, and Neuroendocrine tumors.

[0201] Embodiment 38. The method of any one of embodiments 25-37, wherein the subject is a mammal, optionally a mouse, a dog, a cat, or a human.

[0202] EXAMPLES

[0203] Example 1: CCR provides complementary stimulatory signals to TCR activation.

[0204] To provide additional signals to those produced by the TCR via NF AT, we developed the CCR to elicit downstream signals mediated by NFkB in response to CCR engagement with ICAM1. The induction of ICAM1 is mediated by IFNy which is released upon TCR recognition of its target, which is a signature cytotoxic synapse response. The CCR molecular design consists of an anti-ICAMl recognition domain (R6.5 mAb) fused to the costimulatory receptor 4- IBB, CD28, 0X40, or a combination of two or three of these. 0X40 signaling has been shown to contribute to inhibition of regulatory T cell development and provide anti-apoptotic signals for cell survival. In order to determine whether the CCR signals complement tumor-specific TCR to induce IL2 release and enhance target cell killing, we generated a synthetic TCR (CTL16) that reacts against A375 melanoma cells (HLA-A2+) loaded with the peptide ALKVDVEERV (ALK), which is derived from the melanoma-specific protein MAGE-C2 (336-344)8,9. CTL16-transduced primary T cells kill A375 cells when pulsed with the ALK peptide (A375 / ALK) but not A375 target cells when loaded with another melanoma-derived peptide gplOO (A375 / gpl00) (Fig. 2A). This antigen-specific killing was accompanied with release of IFNy and IL2 (Fig. 2B-C). Here, our data demonstrate that CTL16+CCR-expressing T cells secrete IL2 at a similar level to CTL16-T cells against A375 / ALK. CTL16+CCR-expressing T cells showed similar killing of A375 cells and IFNy release. Interestingly, we found that CTL16 T cells release IL2 at a substantially higher level compared to 2’ CAR (R6.5-4-1BB-CD3Q when exposed to target cells. CCR-T cells show little reactivity to target cells, but increased cytotoxicity and cytokine release is observed when CCR is added to cells expressing the CTL16 TCR, suggesting that the CCR provides an additional dimension to TCR-directed T cell function.

[0205] Example 2: CCR activates NFkB

[0206] We used Jurkat cells transduced with a lentivirus to express the CCR plus a NFkB promoter fused to a FLuc reporter for measuring NFkB activity triggered by the CCR after exposure to 293T cells (minimal or no ICAM1 expression) or HeLa cells (high ICAM1 expression). The CCR expressing Jurkat cells had the highest NFkB activity relative to NT when exposed to HeLa cells, followed by other CARs, (Fig. 3 A). None of the engineered T cells activated NFkB against ICAM1 -negative 293T cells. Importantly, only the Jurkat cells expressing CARs containing CD3^, but not CCR, secreted IL2 when exposed to HeLa cells (Fig. 3B). This finding is in an agreement with an earlier report showing that CD3^ is required for IL2 production10.

[0207] Example 3: In vivo activity of CCR T cells.

[0208] When CCR-transduced primary T cells were stimulated with MHC-mismatched HeLa (ICAM1++) or 8505C ATC cell line (ICAM1+) to mimic allogeneic TCR reactivity, the CCR-T cells targeted HeLa cells rapidly and secreted IFNY, but minimal effect was noted against 8505C cells in in vitro experiments. However, when 8505C xenografts were challenged with CCR-T cells, 8505C tumors showed a significant level of regression compared to challenge with nontransduced T cells (NT) (Fig. 4A). T cells taken from the tumor site and spleen of the 8505C xenografted mouse displayed specific killing against 8505C cells but not to 293T cells, showing that 8505C-specific T cells were selected in this setting (Fig. 4B). These data show that CCR increases the selection of tumor-targeting T cells despite the low frequency of tumor-specific T cells present in the blood.

[0209] Example 4: The utility of CCR for isolation and clonal expansion of tumor-specific TCRs.

[0210] Our data have shown that CCR-T cells administered to 8505C xenografts showed specific targeting of 8505C cells (Fig. 4). CCR-T cells taken from the tumor site (lung) of a 8505C PDX model exhibited CD69 upregulation in CD8+ T cells (Fig. 5A), killed 8505C cells specifically and showed proliferative activity (Fig. 5B). These CCR-T cells isolated from the lung and spleen were subjected to TCR-seq. TCR repertoire analysis showed that there were three dominant TCR clonotypes for both alpha and beta chains with a similar frequency in both lung and spleen (Fig. 5C). These experiments demonstrate that identification of the dominant TCR clonotypes using this platform that combines a tumor xenograft model and CCR T cells is feasible and may facilitate discovery of tumortargeting TCRs.

[0211] Example 5: The utility of the CCR to enhance CAR activity and provide further stimulatory signals to overcome immune suppression within tumors.

[0212] CCR activation in response to interaction with ICAM1, induced by IFNy release, has been shown to maximally activate the NFkB pathway. This is required for T cells with NF AT activation to generate a stimulatory immune synapse and secrete cytokines such as IL2. Further, 0X40 signaling has been shown to inhibit the effect and expansion of regulatory T cells, which exert inhibitory signals on cytotoxic T cells. CCRs co-expressed in CAR T cells to produce costimulatory signals such as those from 4-1BB and 0X40, therefore, can provide stimulatory as well as Treg suppressive signals, greatly enhancing CAR T activity against solid cancers. Given that signaling by the CCR is generated separately from those required for cell killing by the CAR (via CD28 and CD3Q, tumorspecific killing is maintained.

[0213] Example 6: Materials and Methods for Examples 7-11 a. Cell lines and generation of knockouts

[0214] The A375-MA2 (#CRL-3223; sex: female), Jurkat T (Clone E6-1, #TIB-152; sex: male), T2 (#CRL-1992; sex: female), 293T (#CRL-3216; sex: female), and HeLa (#CRM- CCL-2; sex: female) cell lines were purchased from the ATCC. The 8505C cell line (#ACC-219; sex: female) was purchased from the DSMZ. RM, ATC patient-derived cell line (sex: female) was established in our laboratory. All cells were grown were grown at 37°C under 5% CO2. All cell lines except for A375-MA2 and 293T cells were grown in complete (c) RPMI-1640 medium (Corning #10-040-CV) supplemented with 10% fetal bovine serum (FBS). A375-MA2 and 293T cells were grown in cDMEM medium (Coming #10-013-CV) containing 10% FBS. Some cell lines were transduced with an fLuc-F2A- GFP-expressing lentivirus (Biosettia #GlowCell-16-5).

[0215] A375-MA2 (HLA-KO) cells were generated using CRISPR / Cas9. Briefly, predesigned two crRNAs and tracr RNA oligos targeting b2m- and CIITA- (IDT, Inc.) were annealed in equimolar concentrations (44 pM) by heating at 95°C for 5 minutes, followed by gradual cooling to room temperature. The sequences of the guide RNAs (gRNAs) are shown in Table 1. Each gRNA duplex (final concentration of 22 pM) was then incubated with Cas9 nuclease (final concentration of 18 pM) at room temperature for 15 minutes to form the ribonucleoprotein (RNP) complex. A375-MA2 cells were mixed individually with RNP complexes and electroporated (1450 V / 10 ms / 3 pulses) using the Neon™ Transfection System (Invitrogen). Knockout was confirmed by staining with HLA-I and HLA-II antibodies. All cell lines were confirmed negative for Mycoplasma prior to the study with a mycoplasma detection kit (Lonza #LT07-l 18) according to manufacturer’s protocol. Cell lines were maintained from cryopreserved stocks made at low passage numbers, and all cells used in experiments were of a passage number <30.

[0216]

[0217] Table 1. List of gRNA sequences used in this study.

[0218] * All gRNA sequences are targeting the (-) strand of the target gene. b. Engineered T cell lentiviral constructs - generation and transduction

[0219] The CTL16 TCR was cloned into lentiviral vector using the gblock fragments of TCRP (Genbank: EU427375.1) and TCRa (GenBank: EU427374.1) (26). The ICCR construct containing the R6.5 scFv was cloned into a lentiviral backbone encoding the CD8 hinge and CD8 transmembrane domain, followed by CD137. 293T cells were transfected with transfer plasmids and LV-MAX Lentiviral Packaging Mix (ThermoFisher, #A43237) using lipofectamine 2000 transfection reagent (ThermoFisher, #11668019). At 48 h posttransfection, supernatant fractions were collected, filtered using a 0.45-pm filter (Fisher Scientific, #50-202-064), and concentrated 100-times using the Amicon Ultra Centrifugal Filter (Millipore Sigma, #UFC910008). The lentiviral titer was estimated by transducing dilutions of lentivirus into Jurkat T cells and analyzing by flow cytometry to detect surface molecules encoded by the construct.

[0220] Peripheral blood was obtained from healthy donors from commercially obtained leukopaks (AllCells) and an ATC patient under a WCM Institutional Review Board- approved protocol. Informed written consent was obtained from all donors, and research was guided by the Declaration of Helsinki. Primary T cells were isolated via MACS separation using CD4 (Miltenyi Biotec, # 130-045-101) and CD8 (Miltenyi Biotec, #130- 045-201) microbeads. T cells were cultured in TexMACS medium (Miltenyi Biotec, #170- 076-307) with 5% human AB serum 131 (Sigma, #H4522), 12.5 ng / ml IL7 (Miltenyi Biotec, #170-076-111), and 12.5 ng / ml IL15 (Miltenyi Biotec, #170-076-114). T cells were activated using Dynabeads Human T-Expander CD3 / CD28 (ThermoFisher, #11141D) and transduced twice with the same lentivirus on day 1 and day 2 after activation. T cells were maintained at a 1 million cell / ml density for 10 days before being cryopreserved in a 1 :2 mixture of T cell growth medium: CryoStor CS10 (STEMCELL Technologies, #07930). c. Chemicals and peptides

[0221] The ALK peptide (ALKDVEERV; corresponding to 336-314 a. a. of MAGEC2 gene) was synthesized from the Tufts University Core facility. The gplOO (a.a. positions 619-627 of Pmel gene) peptide (RLMKQDFSV) was purchased from Genscript (#RP20486). lonomycin (#13909) and phorbol 12-myristate 13-acetate (PMA; #P1585) were purchased from Sigma- Aldrich. IFNy was purchased from Peprotech (#300-02). For peptide pulsing, cells were incubated with the peptides overnight in culture and were washed with PBS 2-3 times the next day to remove unbound peptides. CellTrace CFSE proliferation kit (ThermoFisher, #C34554) was used to label T cells. d. NFKB and NEAT activity assays

[0222] To measure activation of NFKB and NF AT, Jurkat T cells were transduced with NFKB-fLuc lentivirus (#CLS-013L, Qiagen) or NFAT-fLuc lentivirus (#CLS-015L, Qiagen) and cultured using puromycin selection (2.0 pg / mL) for 2 weeks. Next, T2 cells were pulsed with different concentrations of ALK or gplOO peptides. After that, 50,000 transduced Jurkat T cells were cocultured with 50,000 pulsed T2, 293T, or HeLa cells in each well of a 96-well plate in a total volume of 200 pl of complete RPMI medium containing D-luciferin (150 pg / ml; Gold Biotechnology #LUCNA). Cells were cocultured for 5 to 7 h, and then luminescence was measured using a plate reader (TEC AN Infinite Ml 000 PRO). We also used NFAT-GFP expressing Jurkat cells to analyze NF AT activity by measuring GFP MFI using flow cytometry 20 h after activation (27). e. Effector to Target (E:T) assay fLuc-expressing target cells (i.e., tumor and control 293 T) were plated at a density of 5,000 cells per well in complete growth medium containing D-luciferin. NT or engineered T cells were added to the target cells at a 2.5: 1 cell ratio (1.25 x 104 cells) and 1 : 1 volume in TexMACS medium supplemented with 5% human AB serum. Target cell (i.e., tumor and control 293 T) viability was measured periodically over 0-72 h using a plate reader. The percent killing of target cells was calculated by normalizing to NT or No T control conditions. f. ELISA

[0223] Supernatant fractions from the E:T assays were collected after 18-20 h of coculture, centrifuged at 450*g for 5 min, and frozen at -80°C until needed. Human IL2 ELISA MAX™ deluxe set (Biolegend #431802) and human ZFNy ELISA MAX™ deluxe set (Biolegend #430104) were used to quantify cytokine concentrations from the supernatant fractions following manufacturer’s instructions. Mouse blood was collected in a tube containing EDTA (final concentration: 0.1 mM). Plasma was isolated by centrifugation at 300*g for 15 m, followed by second centrifugation at l,000xg for 15 m. Cleared plasma was stored at -80°C until needed. A bead-based multiplex assay (Biolegend #741186) was used to quantify the concentration of human cytokines in sera by following manufacturer’s instructions. g. Flow cytometry

[0224] Target cell lines were trypsinized and washed in PBS once, followed by wash buffer (PBS with 2% FBS) once. Tumor tissues were collected and minced with surgical scalpels in Hank’s Balanced Salt Solution (HBSS, Corning #21-023 -CM). Minced tumor bits were digested with 1 mg / mL collagenase IV and 0.1 mg / mL DNase in cRPMI medium for 1 h at 37°C. After washing with cRPMI medium, tumor-digested materials were strained through 70-pm nylon strainer. Red blood cells were lysed using ammonium-chloride-potassium (ACK) buffer. After washing, cell pellets from tumor tissues were blocked in TruStain FcX for 5-10 min before staining. Pellets from cell lines, T cells, and tumor tissues (1-3 * 105cells per tube) were stained with fluorochrome-tagged detection antibodies diluted in wash buffer for 20 min on ice in the dark. The list of the antibodies is documented in Table 2. After staining, cells were washed in excess wash buffer and analyzed using a Gallios flow cytometer (Beckman Coulter) or FACSymphony Al (BD Biosciences).

[0225] The list of the antibodies is documented in Table 2. Table 2. List of antibodies used in this study. adenotes polyclonal antibodybdenotes protein conjugate h. Mouse xenograft models

[0226] All animal work was conducted under the approvals of the Weill Cornell Medicine (WCM) and Houston Methodist Research Institute (HMRI) Institutional Animal Care and Use Committees.

[0227] NOD (NOD-scidIL2Rgnull) mice (6-8 weeks old) were purchased from the Jackson laboratory (Jackson, #00555). Both female and male were used in this study. The number of animals per group was determined using power analysis designed to detect two-fold difference in tumor burden or imaging readouts between groups with 80% power and 95% significance. Animals of the same sex, genotype, and similar body weight were procured and randomly assigned to different experimental groups. For the metastasis model, 1 x 106 8505C (fLuc / GFP) cells were injected via tail vein. After 5 days, 1 x 107 freshly thawed T cells in 100 pl of cryopreserve medium were injected via the tail vein per mouse. For the s.c. model, l x 106 8505C or 8505C-OKT3 (fLuc / GFP) cells were mixed with Matrigel (Coming #354234) at a 1 : 1 ratio and were s.c. injected into the upper right flank. Method to establish orthotopic tumor model is described herein. For the orthotopic tumor model, 1 x 105 RM-ATC cells resuspended in 10 pl of serum-free RPMI were unilaterally injected into the right thyroid gland. Six to ten million T cells were injected into each mouse via tail vein a week following the xenograft.

[0228] Analysis method of tumor volume change, tumor burden, and survival rate is described below.

[0229] (i) Orthotopic mouse xenograft model

[0230] NSG nice were anesthetized with ketamine / xylazine solution, and the neck area was shaved and cleaned with chlorhexidine solution. After making a vertical incision through the skin and s.c. tissues, another incision was made through the strap muscles to expose the right thyroid gland. U 105RM-ATC cells resuspended in 10 pl of serum-free RPMI were unilaterally injected into the right thyroid gland using a 31 -gauge insulin syringe. The incision was closed with sutures.

[0231] (ii) Tumor burden analysis using mouse xenograft models

[0232] For the metastasis model, tumor burden was quantified by weekly bioluminescence imaging (PerkinElmer IVIS-Spectrum CT). For the subcutaneous model, tumor volume was measured weekly with a digital caliper and bioluminescence imaging. Luminescence images were taken 15 minutes after intraperitoneal injection of 100 pL of 150 mg / mL D- luciferin (GoldBio, #LUCNA). Total tumor burden quantification by bioluminescence imaging was calculated with Spectrum analysis software, in which total body regions of interest (ROI) were analyzed for total luminous flux. Tumor volume was calculated by using the formula: V = (width x width x length) / 2. Mice in the survival study were euthanized once tumor burden exceeded 2.0 cm3, total body weight decreased by 20%, or if mice were suffering from difficulty breathing due to tumor burden in orthotopic tumor models. i. Gene expression and TCR analysis

[0233] Detailed methods for gene expression analysis (NanoString assay) are described herein. Total RNA was extracted from T cells and tumor tissues using RNeasy Plus Mini Kit (Qiagen #74134). Gene expression analysis (NanoString assay) was performed using the nCounter human CAR-T characterization panel (XT-CSO-HCART1-12) and CAR-T characterization standards at the New York University Genome Technology Center. Data were analyzed using nSolver 4.0 software with the “Advanced Analysis” function and standard normalization. Enhanced Volcano plots were generated using R Bioconductor using the P value cutoff = 0.05 and fold-change cutoff = log2 (0.5). The TCR diversity score, which measures the diversity of TCR beta variable regions by TCR constant gene normalization within a given sample, was generated using the ROSALIND® platform. RNA libraries from TILs were made by the WCM genomics core facility and processed for immune repertoire analysis using the Archer ImmunoVerse™ TCR panel (IDT) and NGS (Illumina). j. Statistical analysis

[0234] To compare continuous variables, the Mann-Whitney test or Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used as appropriate. For parametric variables, Student’s t-test or ANOVA test with Tukey’s multiple comparisons were used as appropriate. All error bars represent the standard error of the mean. Survival analysis was performed using the log-rank test. Statistical analyses were completed using Prism 10.0 (GraphPad). P-values < 0.05 were considered statistically significant using two-tailed statistical tests. k. Data availability

[0235] The data generated in this study are available upon request from the corresponding author. NanoString assay data are publicly available in Gene Expression Omnibus (accession number: GSE289228).

[0236] Example 7: An ICAMl-directed CCR system

[0237] Although remarkable antitumor response has been demonstrated in engineered T cell therapy, including chimeric antigen receptor (CAR)- and T cell receptor (TCR)-T cell therapies, tumor recurrence still occurs, and clinical response is suboptimal in solid tumors partly due to tumor antigen escape, immunosuppressive microenvironment, and subthreshold activation of CAR or TCRs. We hypothesized that tumor-specific T cell function could be enhanced if T cells acquire additional activation signals from intercellular adhesion molecule 1 (ICAMl)-specific chimeric costimulatory receptor (ICCR). ICAM1 is overexpressed on many solid tumor cells and is greatly induced by the T cell cytokine IFNy, which may reinforce the specificity and activity of TCRs engaged with the target tumor antigens. ICCR expression in T cell lines triggered activation of NF B and elevated NF AT activity downstream of TCR engagement. ICCR expression in primary T cells conferred significant improvement for antitumor T cell expansion and effector function, which led to better tumor targeting in two different xenografts models of anaplastic thyroid cancer when allogeneic or autologous ICCR-T cells were tested. Mechanistically, ICCR enhanced target-directed TCR selection by increasing proliferation and activating downstream signaling pathways following TCR engagement, resulting in enrichment of polyclonal tumor-specific T cells.

[0238] To provide a costimulatory signal for tumor-reactive T cells that can enhance expansion and activation, we developed an intercellular adhesion molecule 1 (ICAM1)- specific chimeric costimulatory receptor (ICCR), composed of a single-chain fragment variable (scFv) sequence fused to a costimulatory molecule CD137. We selected ICAM1 as a tumor-associated antigen based on our previous studies showing a high level of ICAM1 expression in many aggressive solid tumors and lower ICAM1 expression in normal tissues (22,23). Furthermore, expression of ICAM1 is inducible by IFNy, a major cytokine released by the T cell upon initial contact with the p / MHC complex (24,25). We hypothesized that the ICCR we designed could interact with IC AMI -expressing tumor cells and overcome poor endogenous costimulation when induced by the native TCR alone in an immunosuppressive environment. Here, we report on the impact of the ICCR on TCR- driven downstream signaling activity, cytokine secretion upon target recognition, and suppressing tumor cell growth.

[0239] Previously, we reported robust efficacy of IC AMI -specific CAR-T cells against human anaplastic thyroid cancer (ATC), for which specificity was conferred from an R6.5 scFv (22,28). Therefore, to target cancer cells that express a heterogeneous mix of tumor antigens, we designed an ICCR lentiviral construct with a scFv derived from an R6.5 mouse monoclonal antibody that binds to ICAM1 with a KD of ~10 nM (29), fused to CD137 (ICCR; Fig. 6A). We sought to compare this construct with two others: a first-generation ICAM1-CAR (F CAR) and a third-generation ICAM1-CAR with both CD137 and CD28 costimulatory receptors (3’ CAR). We transduced Jurkat cells with lentivirus expressing each construct with high efficiency (Fig. 6B).

[0240] To test whether this ICCR system can activate T cells, we measured NF KB activity, an indicator of downstream T cell signal activation, in transduced Jurkat cells. To assess NF / B activity, we transduced a Jurkat cell line with a lentiviral transcriptional reporter carrying the NF / B promoter upstream of a firefly luciferase (fLuc) gene. We cotransduced this NF KB reporter cell line with ICCR, 1’ CAR, or 3’ CAR constructs and cocultured them with one of two different target cell lines: 293T, which do not express ICAM1, and HeLa, which express high levels of ICAM1 (22). The ICCR-expressing Jurkat cells yielded the strongest NF vB response after 5 h of coincubation with HeLa cells, followed by the 1’ and 3’CAR-expressing Jurkat cells (Fig. 6C). Jurkat cells cocultured with 293T cells did not exhibit an NF / B response, indicating the activation of the ICCR and CAR systems was specific to its ICAM1 target. Pharmacological activation with lonomycin (Io) and phorbol 12-myristate 13-acetate (PMA) served as a positive control for the reporter system.

[0241] As a second indicator of T cell activation by the ICCR system, we measured the amount of IL2 secreted from each transduced Jurkat cell line cocultured with HeLa or 239T cells. We found that only the 1’- and 3’CAR-expressing cells produced IL2, while ICCR- expressing cells did not (Fig. 6D). The 1’- and 3’CAR-expressing Jurkat cells were activated at a similar level when exposed to HeLa cells. We speculate that IL2 secretion in 1’ CAR Jurkat cells may be due to costimulatory signals provided when cocultured with HeLa cells (30). These data indicate that CD3 a signaling component of the TCR complex present in 1’ CAR and 3’ CAR constructs, is a prerequisite for the IL2 production and that ICCR-directed NFKB activation alone is insufficient. Example 8: ICCR enhanced TCR downstream signaling activity

[0242] Next, we examined whether the ICCR could cooperate with the TCR to boost intracellular signaling cascades. For functional comparison, we used CTL16 (31) and transduced NFAT / NF B reporter-expressing Jurkat cells with CTL16 (Fig. 7A and B). CTL16-redirected primary T cells can mount a specific response upon activation when target cells present human leukocyte antigen (HLA)-A2*0101 restricted- ALK peptide (ALKDVEERV) derived from a melanoma-associated antigen C2 (MAGE-C2) (32). Jurkat cells were then cocultured with T2 cells, an HLA-A2 and IC AMI -expressing antigen- presenting cell line, that had been pulsed with either ALK or gplOO peptide (RLMKQDFSV) (33) (Fig. 7C; Fig. 13). ALK-pulsed T2 cells elicited robust NF AT and NFKB activation in CTL16-transduced cells, but gplOO-pulsed T2 cells did not (Fig. 7D and E). We also detected a significant amount of IL2 released from the CTL16-expressing Jurkat cells when challenged with the ALK-pulsed T2 cells compared with nontransduced T (NT) cells (Fig. 7F).

[0243] Next, we cotransduced NFAT / NFKB reporter-expressing Jurkat cells with CTL16 and ICCR lentiviruses, achieving >50% coexpression (Fig. 7G and H). We challenged the CTL16 / ICCR-expressing Jurkat cells with the ALK-pulsed T2 cells, using CTL16 only, ICCR only, or NT Jurkat cells as controls. NF AT activity was detected only in Jurkat cells expressing CTL16 and cocultured with ALK-pulsed T2 cells. NF AT activity was even higher in Jurkat cells expressing both CTL16 and ICCR (P < 0.05; Fig. 71). Jurkat cells expressing ICCR only showed significant NFkB activity irrespective of the type of peptides pulsed on T2 cells, validating that ICCR-induced NFkB activity is due to its interaction with ICAM1 expression on T2 cells but not dependent on the TCR-p / MHC interaction (P < 0.0001; Fig. 7 J). Like enhanced NF AT activation by ICCR coexpression, Jurkat cells coexpressing CTL16 and ICCR and cocultured with ALK-pulsed T2 cells yielded significantly more NF KB activation than cells expressing only CTL16 (P < 0.0001; Fig. 7 J). These results show that adding ICCR to CTL16 increases NF AT and NFKB activation, two major, important nodes of T cell signaling pathways downstream of TCR activation.

[0244] Example 9: Antigen-directed tumor lytic ability was significantly enhanced by the ICCR.

[0245] To ascertain whether the robust ICCR-assisted activation of NF AT and NFKB translates into functional enhancement, we examined the tumor-killing activity of CTL16 / ICCR-coexpressing primary T cells (Fig. 8A). Human primary T cells were activated and cotransduced with CTL16 and ICCR lentiviruses. Approximately 20% of the T cells coexpressed CTL16 and ICCR, and >50% of cells expressed CTL16 or ICCR with a single type of lentiviral transduction (Fig. 14). As a target tumor cells for CTL16-T cells, we selected A375-MA2 cells, an HLA-A2+, MAGE-C2+, and ICAM1+human melanoma cell line (Fig. 15). A375-MA2 cells were either left unstimulated (Fig. 8B), prestimulated with IFNy (Fig. 8C), or pulsed with ALK peptide for direct antigen presentation (Fig. 8D). A375-MA2 cells were challenged by each different engineered T cell types: CTL16-T, ICCR-T, and CTL16 / ICCR-T. 2’ CAR-T and NT cells were used as comparison controls. CTL16-T cells were unable to lyse unstimulated A375-MA2 cells (Fig. 8B). CTL16-T cells demonstrated robust cytotoxicity only against the ALK-pulsed A375-MA2 cells (P < 0.0001; Fig. 8D). Pretreatment of the A375-MA2 cells with IFN / had a limited effect on increasing the CTL16-T cell cytotoxicity (Fig. 8C), even though IFN / induced a significant increase of HLA-A2, / ?2-microglobulin, ICAM1, and MAGEC2 expression that may enhance their antigenicity (Fig. 15). Meanwhile, ICCR-T cells exhibited a statistically significant cytotoxicity against both untreated, IFN / -treated, and ALK-pulsed A375-MA2 cells, but with <50% of target cell lysis (P < 0.01, P <0.0001, and / J< 0.05; Fig. 8B-D). In contrast, CTL16 / ICCR-T cells demonstrated strong lytic activity against both untreated and IFN / -treated A375-MA2 cells, supported by a significant increase in lytic function compared with CTL16-T cells (P < 0.0001). Against ALK-pulsed A375-MA2 cells, the lytic activity was highest and comparable by CTL16-T and CTL16 / ICCR-T cells, indicating that CTL16 alone is sufficient for killing target cells with a high antigen density specific to the CTL16. As expected, 2’ CAR-T cells that target ICAM1 demonstrated an even level of cytotoxicity against unstimulated and IFN / - or ALK-stimulated target cells.

[0246] To determine if the CTL16 / ICCR-T cell cytotoxicity is associated with phenotypical changes in T cells, we analyzed the expression levels of T cell activation markers, CD137 and CD69, on engineered T cells and control NT cells. CTL16 / ICCR-T cells and CTL16-T cells displayed a significant increase in the CD137+CD69+T cell fraction when challenged by the ALK-pulsed A375-MA2 cells compared with unstimulated target cells (Fig. 8E; Fig. 16). The percentage of CD137+CD69+CTL16-T cells positively correlated with the target antigen density of A375-MA2 cells (untreated < IFN / -pretreated < ALK-pulsed). The activity of CTL16 / ICCR-T cells followed a similar trend as the CTL16-T cells, although the activation appeared diminished. This result may be due to the lower expression level of engineered construct in CTL16 / ICCR-T cells compared with CTL16-T cells.

[0247] Next, we measured the level of secreted IFNyand IL2 as an indicator of functional activity of engineered T cells. CTL16 / ICCR-T cells released a significantly elevated amount of IL2 when coincubated with IFNy-pretreated or ALK-pulsed A375-MA2 cells but not with untreated A375-MA2 cells (Fig. 8F-H). Against untreated and IFNy-pretreated A375-MA2 cells, 2’ CAR-T cells secreted the highest amount of IFNy(Fig. 8F and G). ALK-pulsed A375-MA2 cells induced the CTL16 / ICCR-T and CTL16-T cells to produce robust levels of IL2 and IFNy(Fig. 8H). Both types of CTL16-expressing T cells yielded much higher levels of IL2 compared with NT cells (P< 0.05 and P < 0.0001). In addition, the level of IL2 secretion was also significantly elevated in CTL16 / ICCR-T and CTL16-T cells when cocultured with ALK-pulsed T2 cells, but not with unpulsed or gplOO-pulsed T2 cells (Fig. 81). Coincubation of ICCR-T cells with ALK-pulsed T2 cells did not induce a significant amount of IL2 or IFNy(Fig. 81). Our data demonstrate that CTL16 / ICCR coexpression provides a significant advantage for targeting tumor cells with a lower antigen density, like unmodulated or IFNy-pretreated A375-MA2 cells. When exposed to target cells with a higher antigen density, such as cells directly presenting antigen after an ALK pulse, the CTL16 / ICCR-T cell killing efficacy was near the level of the 2’ CAR-T cells, but with a potentiated IL2 secretion response (Fig. 8H).

[0248] Example 10: Cytotoxic activity of ICCR depends on TCR-p / MHC interaction

[0249] Because donor T cells were not screened to ensure the HLA matched with target A375-MA2 cells, we reasoned that the noticeable level of toxicity induced by the ICCR-T cells could arise from allogeneic TCR activity. To determine if this was the case, we assessed ICCR-T cell targeting efficacy in the absence of endogenous TCR expression by disrupting the TCR. a locus (TRAC) using the CRISPR / Cas9 technology (Fig. 9A). TCRa depletion in ICCR T cells (>90%; Fig. 9B) reduced cytotoxicity significantly compared with ICCR-T cells retaining native TCR expression (P < 0.0001; Fig. 9C).

[0250] Next, we engineered A375-MA2 cells to eliminate expression of HLA, the interacting partner of the TCR, producing A375-MA2 / HLA KO (Fig. 9D). CRISPR / Cas9 disruption of the [32 m and CIITA genes reduced the expression of HLA-A2 and HLA-II by 99% and 75%, respectively (Fig. 9E). We then challenged A375-MA2 and A375- MA2 / HLA KO cells (pulsed with ALK or gplOO peptides) with our panel of engineered T cells. Both CTL16-T and CTL16 / ICCR-T cell killing activity against ALK -pulsed A375- MA2 / HLA KO cells were nearly abolished relative to A375-MA2 cells (P < 0.0001; Fig. 9F). Likewise, ICCR-T killing activity against A375-MA2 / HLA KO cells with or without ALK loading was diminished (P < 0.0001; Fig. 9F).

[0251] Altogether, these data demonstrate that ICCR-T cells exert cytotoxic function only when the TCR-p / MHC interaction occurs between the target cell and T cell, as supported by a dramatic decrease of ICCR-T cell killing activity when either TCR or MHC is disrupted. As expected, 2’CAR-T cells lysed target cells regardless of the HLA expression.

[0252] Example 11: ICCR expression in allogeneic T cells improves T cell proliferation and lytic activity

[0253] To investigate whether ICCR expression on T cells spurs selective growth of target tumor-specific TCR in a setting without an engineered TCR, we generated ICCR-T cells from healthy donors and assessed ICCR-T cell lytic activity on an ATC cell line, 8505C cells (Fig. 10A-C).

[0254] Generally, ATC have an immune-enriched microenvironment but respond poorly to immunotherapy (34). To induce antigenic interaction for tumor-specific TCRs, we challenged ICCR-T cells with 8505C cells every 2-3 days (Fig. 10B). Upon 8505C stimuli, ICCR-T cells grew faster than stimulated NT cells. Further, 8505C cell stimulation accelerated ICCR-T (ICCR-T*) cell killing against 8505C cells (P < 0.0001; Fig. 10C). Unchallenged ICCR-T cells also lysed 8505C cells better than NT cells, but to a lesser extent than stimulated ICCR-T* cells (P < 0.0001). Using this experimental design, we first examined the cytotoxicity of ICCR-T cells, with or without stimulation by 8505C cells, toward 8505C metastatic tumors grown in NOD scid gamma (NSG) mice (Fig. 10D). After 1 month of treatment, four out of five xenografts treated with unstimulated ICCR-T cells showed reduced tumor progression compared with the control group (Fig. 10D and E).

[0255] We sought to track ICCR-T cells in vivo, and thus engineered ICCR-T cells that coexpress somatostatin receptor 2 (SSTR2), which we have shown previously can be used for in vivo visualization (28). PET-CT imaging of ICCR-T cells demonstrated their colocalization at lung metastatic tumor sites. 8505C-stimulated ICCR-T* cells also targeted 8505C tumors, delaying tumor spread. Mice with 8505C xenografts that were treated with stimulated ICCR-T cells survived ~ 2 weeks longer than mice treated with NT, but mice that received unstimulated ICCR-T showed a better response and extended survival (P < 0.05 and P < 0.01; Fig. 10F). These data revealed that in vitro stimulated ICCR-T* cells did not translate to better or faster target tumor killing in vivo than unstimulated ICCR-T cells, despite superior in vitro killing efficacy.

[0256] Next, we asked whether ICCR expression can help polyclonal TCR selection, and thereby improved specificity. For this experiment, we isolated tumor tissue cells from an 8505C xenograft that showed a positive response to ICCR-T cells (Fig. 10D) and cultured them for 2 days in T cell culture medium without cytokine supplement. We tested if TILs showed specific activity for 8505C cells. TILs lysed 8505C cells selectively (Fig. 10G), coinciding with significant TIL proliferation that was not observed when TILs were cocultured with 293T cells (Fig. 10H). Over 20% of the TILs isolated from the xenograft tissue expressed ICCR, which expanded up to -70% when challenged by the 8505C cells (Fig. 101). To profile the TCR repertoire of the TILs, we analyzed TILs by TCR- sequencing. This analysis showed that over 90% of the TCR / 3 and TCRa clonal space was occupied with four predominant clonotypes (Fig. 10J). To test whether clonally expanded TCR shows reactivity toward 8505C cells, we generated a Jurkat cell line expressing the TCR / 3 and TCR. a sequences that were most prevalent (TCR-8505C) (Fig. 17) and compared the NF / cB activity level of TCR-8505C with TCR-8505C and ICCR (TCR- 8505C / ICCR) coexpressing Jurkat cells (Fig. 10K). Compared with TCR-8505C Jurkat cells, TCR-8505C / ICCR-expressing Jurkat cells showed a significant increase in NF / cB activity upon 8505C coculture (P < 0.0001). This effect was not observed when TCR- 8505C / ICCR cells were challenged by 293T cells, indicating that TCR-8505C reactivity is specific to 8505C cells. Coexpression of TCR-8505C and ICCR also enhanced

[0257] NF AT activity of the Jurkat cells upon exposure to 8505C cells (P < 0.0001), although the extent of increase was less dramatic than that observed for NF / cB (Fig. 10K; Fig. 18). These data demonstrate that ICCR drives proliferation of those T cells with weak TCR activity, consequently providing selective advantage of those ICCR-possessing T cells specific for the target antigen.

[0258] To evaluate the impact of ICCR on functional efficacy when the TCR signal is activated from the onset, we measured the cytotoxic effector function of ICCR-T cells toward 8505C cells that were engineered to express a membrane-bound OKT3, an anti- CD3e antibody (8505C-OKT3) (Fig. 19). OKT3 binding affinity to the TCR is much stronger than the native TCR-p / MHC interaction, and this interaction stimulates key downstream signaling events in T cell responses (35). ICCR-T cells lysed 8505C cells better than did NT cells (Fig. 11 A). Although this difference was not discernable when tested against 8505C-OKT3 cells due to potent T cell activation induced by 0KT3, significantly elevated amounts of IL2 and IFNy were secreted from the ICCR-T cells (P < 0.0001), substantiating the enhanced effector function of ICCR-T cells (Fig. 1 IB). When subcutaneous (s.c.) tumors established from 8505C-OKT3 (fLuc / GFP) cells were challenged, ICCR-T cells exhibited superior killing efficacy to NT cells, as shown by reduced tumor luminescence signals and tumor volume over time (P < 0.01; Fig. 11C and D).

[0259] ICCR-T cells demonstrated superior activity in controlling tumor growth and extending survival relative to NT cells (P < 0.05; Fig. 1 IE). Granzyme A and granulysin were secreted by ICCR-T cells specifically, supporting evidence of enhanced tumor lytic function (Fig. 1 IF). When xenograft tumor tissues at the endpoint were analyzed by flow cytometry, we found that the percentage of GFP+tumor cells was substantially reduced in the ICCR-T cell treatment group (P< 0.05; Fig. 11G). Two out of four analyzed tissues retained an increased fraction of CD3+human T cells, and ICCR-T cells were markedly activated (Fig. 20).

[0260] To test the ICCR efficacy in a setting closer to the clinical situation in which a patient’s own engineered T cells are administered for cancer treatment, we manufactured ICCR-T cells using peripheral T cells isolated from an ATC patient (RM) (Fig. 12A), as we have established ATC cell line from the same patient previously (36). Like ICCR-T cell responses to the 8505C, ICCR-T cells (RM) expanded upon continuous exposure to the RM-ATC cells, showing ~ 9-fold increase (Fig. 12B). Tumor-stimulated ICCR-T* cells showed an enhanced le vel of toxicity toward RM-ATC cells compared with unstimulated ICCR-T and NT cells (Fig. 12C). RM-ATC cells expressed ICAM1 at a lower level than did 8505C cells (Fig. 21 A), and preconditioning RM-ATC cells with IFNy enhanced their susceptibility to unstimulated ICCR-T cells (Fig. 21B). To investigate whether RM-derived ICCR-T cells retain augmented antitumor T cell activity, we tested these cells using RM patient-derived cell line orthotopic xenograft model by implanting RM-ATC cells into mouse thyroid tissue. Treatment with ICCR-T cells increased the survival rate of xenografted mice compared with treatment using NT cells (P < 0.05; Fig. 12D). An increased number of tumor-infiltrating CD3+T cells was found in the xenografts treated with the ICCR-T cells (Fig. 12E). Our experiments employing tumor xenograft models with allogeneic and autologous peripheral T cells support the hypothesis that ICCR expression enhances selective expansion of antitumor T cells, leading to control of solid tumor growth.

[0261] Finally, to uncover the role of ICCR on inducing changes in T cell activity and TCR selection, we analyzed the transcriptional changes of ICCR-T and NT cells stimulated by two previously used donor T cells — those used to target tumor 8505C and RM-ATC cells. Although preconditioning ICCR-T cells by target tumor stimuli triggered accelerated killing by the ICCR-T cells in vitro, but this effect was not observed in vivo: unstimulated ICCR-T cells showed better in vivo response compared to stimulated ICCR-T cells. To understand if tumor cell stimulation induced gene expression hindering the ICCR function, we analyzed gene expression changes separately for T cells upon tumor cell stimulation and ICCR activity. ICCR expression alone elicited a subtle change in overall gene expression pattern (Fig. 12F and Fig. 22). In contrast, a dramatic overall shift in the transcriptome was observed in T cells stimulated by the tumor cells. ICCR-T cells upregulated gene expression associated with cytotoxicity, TCR signaling, costimulatory molecules, and exhaustion, which are indicative of specific, TCR-mediated killing (Fig. 12G). By contrast, tumor cell stimulation induced profound changes in cellular metabolism, with increases in glycolysis and one-carbon metabolism. Further, ICCR-T cells were associated with TCR selection, resulting in lower TCR diversity score (Fig. 12H).

[0262] Collectively, we found that ICCR-T cells augment TCR selection specific to the target cells by complementing signal activation downstream of the TCR binding. This effect on TCR diversity was not observed in T cells stimulated by target tumor cells, substantiating a unique genetic program bestowed by the ICCR.

[0263] Here, we report that engineered expression of ICCR confers significant improvement for antitumor T cell expansion and effector function, resulting in better tumor targeting in two different xenografts models of ATC when allogeneic or autologous ICCR- T cells were tested.

[0264] ICCR-T cells selectively expand endogenous, tumor-selective TCRs due to its robust NFKB activation, as well as elevated NF AT signaling activity that occurs only when the TCR is engaged with its cognate target antigen. This functional improvement is more pronounced when the targetable tumor antigen density is low, as is the case in many solid tumor immune microenvironments where antigen presentation is reduced and costimulatory signaling molecules are scant. Previously, it has been shown that GD2 (diaganglioside) and PSMA (prostatespecific membrane antigen)-targeting CCR (scFv fused to CD28) provides costimulatory signaling without eliciting cytotoxicity when tested against target tumor cells (30,37). Since its conception, the CCR function was envisioned for combination with a CAR targeting a primary antigen to increase specificity and sensitivity to the target tumor (38,39) or with a TCR to provide complementary signaling for increased T cell proliferation (40). Here, we demonstrated that allogeneic and autologous primary T cells engineered to express ICCR were each effective in controlling the growth of ATC, one of the most aggressively growing solid tumors. We present the first ICAM1 -targeting CCR that was developed to capture and enhance the initial TCR interaction with a low density of p / MHC that still triggers IFNy secretion above the basal level but limited proliferation because of insufficient costimulatory ligand expression in an immunosuppressive microenvironment. Many types of solid tumors, such as ATC, gastric cancer, glioblastoma, and breast cancer, overexpress ICAM1 on the cell surface, which can be further increased with IFNy (22,23 ,41). These types of tumor cells are likely to have enhanced ICAM1 expression in response to IFNy when they are in close contact with tumor-reactive T cells. Hence, ICCR-T cell therapy may be broadly applicable to different types of solid tumors that are responsive to IFNyand have a limited number of antigens targetable by conventional adoptive T cell therapy.

[0265] Using TCR sequencing and gene expression analysis, we found that ICCR drives T cell proliferation and selectively expands T cells that possess TCRs reactive to the target antigen when stimulated. Further, the enrichment of gene expression signatures for cytotoxicity and TCR signaling, combined with decreased TCR diversity, support that ICCR is inducing intrinsic changes in T cells. Using ICCR-T -responsive tumor tissue, we were able to distinguish TCR compositions that were enriched in tumor-infiltrating T cells. Because TCR sequencing analysis was conducted with bulk tissue, we were unable to identify which TCR / 3 and TCR. a clonotypes pair to form the TCR. We rationalized that the most abundant TCR / 3 and TCRa clonotypes may represent those TCRs reactive to the 8505C tumor cells and reconstructed the TCR-8505C. TCR-8505C-transduced Jurkat cells did not elicit specific responses, but cells expressing both TCR-8505C and ICCR responded robustly. These results suggest the usefulness of ICCR in identifying tumor-specific T cells that may be insufficiently activated due to lack of costimulatory signal activation.

[0266] We engineered an ICCR with CD137 as a costimulatory molecule based on previous studies reporting the importance of CD137 in conferring improved persistence in T cells (42-44). In liquid tumors, CCR with a single costimulatory molecule (either 4-1BB or CD28) had little impact on enhancing the cytotoxicity driven by the TCR or 1’ CAR, let alone eliciting cytotoxic response by the CCR itself (39,40). In contrast, we show that redirecting the T cell population with the ICCR alone led to targeted solid tumor cell cytotoxicity, which depended on the TCR-p / MHC interaction. In particular, ICCR expression alone was sufficient to augment target tumor cell (melanoma and ATC)- directed-toxicity and T cell proliferation when the antigen density was low. ICCR-mediated cytotoxicity was robust, as demonstrated in different xenograft models of ATC. The enhanced sensitivity to the antigen density imparted by ICCR is likely to boost effector function of low activity TCR once the TCR latches onto the target antigen. One interesting question to investigate further is whether solid tumor cells are more sensitive to CCR- driven effector function than liquid tumor cells. Another consideration to explore is whether

[0267] ICAM1 as a CCR target reinforces TCR clustering and amplifies recruitment of downstream signaling molecules, boosting T cell functional activity. ICAM1 interaction with lymphocyte function-associated antigen 1 (LFA1), the natural ligand of ICAM1, is a component of an immunological synapse that stabilizes the TCR-p / MHC cluster (45). Enhanced TCR signaling and type II interferon signaling detected by gene expression analysis of ICCR-T cells compared with NT cells support the idea that ICCR confers stronger interaction for the TCR clusters.

[0268] In a study that administered ex vivo-expanded TILs from melanoma, about one- third of patients who experienced treatment failure with other modalities responded, including a complete response for -12% of patients (46). The TIL therapy was granted an FDA-approval, illustrating the power of polyclonally expanded T cells in controlling tumor growth (1). For other types of solid tumors, like advanced non-small cell lung cancers, the objective response rate and complete response rate appear to be lower than those for melanoma (47). Our data suggest three ways that ICCR could be used for adoptive T cell therapy. First, TILs manufactured to express ICCR may enhance the response rates of TIL therapy for different types of solid tumors that are less immunogenic. Second, ICCR- expressing peripheral T cells could be a novel therapeutic strategy to expand and harness the potential of antitumor T cells from a starting material with a greater diversity, given the significant enhancement we observed of target-reactive ICCR-driven T cell proliferation and TCR selection. Procuring peripheral blood for ICCR transduction is more broadly applicable to patients with unresectable solid tumors. Finally, genetically modifying T cells to coexpress TCR and ICCR could be a third strategy to boost TCR activity, particularly when the p / MHC density is low in the tumor microenvironment. We have shown that the efficacy of transgenic CTL16-T cells targeting a melanoma-specific epitope was markedly augmented by ICCR coexpression.

[0269] In this study, we demonstrated proof of concept that ICCR improves functional targeting efficacy both in vitro in melanoma cells and in vivo using anaplastic thyroid cancer xenografts. Future studies could use both human and mouse ICCR in immunocompetent models with established tumor microenvironments. These studies would reveal how the human and mouse ICCR affect tumor-associated macrophages, another IC AMI -expressing tumor component, and how this interplay shapes tumor control. A recent clinical trial of IC A I -targeting CAR-T cells (NCT04420754) utilized the engineered domain of LFA1 that binds to both human and mouse ICAM1 forms (48). Examining the tumor-targeting ability and toxicity profiles of human and mouse ICCR could provide insights into the remaining hurdles to consider when developing safe and effective tumor-targeting agents in humans. Our work provides evidence supporting the use of ICCR to achieve robust activation and proliferation in low-activity, target-specific TCRs.

[0270] These crucial metrics are essential for solid tumor control in an immunosuppressive tumor microenvironment.

Claims

WHAT IS CLAIMED IS:

1. A chimeric costimulatory receptor (CCR) comprising: a) an intercellular adhesion molecule-1 (ICAMl)-binding domain; and b) at least one costimulatory domain, wherein the CCR does not comprise a cluster of differentiation 3 zeta (CD3Q domain.

2. The CCR of claim 1, wherein the at least one costimulatory domain is selected from CD28, CD27, 4-1BB (CD137), 0X40, CD40, and ICOS.

3. The CCR of claim 1 or 2, wherein the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof.

4. The CCR of any one of claims 1-3, wherein the at least one costimulatory domain comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain.

5. The CCR of any one of claims 1-3, wherein the at least one costimulatory domain comprises a CD28 costimulatory domain and a 0X40 costimulatory domain.

6. The CCR of any one of claims 1-3, wherein the at least one costimulatory domain comprises a 4-1BB (CD137) costimulatory domain.

7. The CCR of any one of claims 1-6, wherein the 4- IBB costimulatory domain comprises the amino acid sequence of SEQ ID NO. 10.

8. The CCR of any one of claims 1-7, wherein the ICAMl-binding domain comprises an antibody or an antigen-binding fragment thereof.

9. The CCR of claim 8, wherein the ICAMl-binding domain comprises an antibody and the antibody is selected from R6.5 (BIRR-1, Enlimomab), 1A6, MD-3, M10A12, bersanlimab, AF-647, and MSH-TP15.

10. The CCR of claim 8, wherein the IC AMI -binding domain comprises an antigenbinding fragment thereof and the antigen-binding fragment of an antibody is a single-chain variable fragment (scFv).

11. The CCR of any one of claims 1-8, wherein the ICAMl-binding domain comprises R6.5 scFv or an I domain of the aL subunit of lymphocyte function-associated antigen-1 (LFA1).

12. The CCR of claim 11, wherein the ICAMl-binding domain comprises R6.5 scFv.

13. The CCR of claim 12, wherein the ICAMl-binding domain is R6.5 scFv.

14. The CCR of any one of claims 1-13, wherein the R6.5 scFv comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.

15. The CCR of any one of claims 1-14, wherein the R6.5 scFv comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and / or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.

16. The CCR of any one of claims 1-15, wherein the R6.5 scFv comprises an amino acid sequence of SEQ ID NO: 9.

17. The CCR of any one of claims 1-16, further comprising (c) a transmembrane domain.

18. The CCR of claim 17, wherein the transmembrane domain is a CD8 transmembrane domain, optionally wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11 or 12.

19. The CCR of claim 17, wherein the transmembrane domain is a CD28 transmembrane domain, optionally wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 13.

20. The CCR of claim 19, wherein the CCR comprises R6.5 scFv and 4-1BB.

21. The CCR of any one of claims 1-20, wherein the CCR comprises: a R6.5 scFv, a transmembrane domain and a 4-1BB costimulatory domain.

22. The CCR of any one of claims 1-21, wherein the CCR comprises: a R6.5 scFv, a CD8 transmembrane domain, and a 4-1BB costimulatory domain; optionally wherein the CCR comprises the amino acid sequence of SEQ ID NO: 15.

23. The CCR of any one of claims 1-21, wherein the CCR comprises a R6.5 scFv, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a 4- 1BB costimulatory domain, optionally wherein the CCR comprises the amino acid sequence of SEQ ID NO: 14.

24. The CCR of any one of claims 1-17, wherein the CCR comprises a R6.5 scFv, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and an 0X40 costimulatory domain, optionally wherein the CCR comprises the amino acid sequence of SEQ ID NO: 16.

25. A nucleic acid encoding the CCR of any one of claims 1-24, optionally wherein the nucleic acid sequence comprises a nucleotide sequence selected from SEQ ID NOs: 17-19.

26. A vector comprising the nucleic acid of claim 25.

27. The vector of claim 26, wherein the vector is an expression vector.

28. The vector of claim 26 or 27, wherein the vector is a viral vector.

29. The vector of any one of claims 26-28, wherein the viral vector is lentiviral vector.

30. A cell comprising the nucleic acid of claim 25.

31. A cell expressing the CCR polypeptide of any one of claims 1 to 24.

32. The cell of claim 30 or 31, wherein the cell is an induced pluripotent stem cell (iPSC), a hematopoietic cell (HSC), or an immune cell.

33. The cell of any one of claims 32, wherein the cell is a leukocyte, optionally wherein the leukocyte is a lymphocyte.

34. The cell of any one of claims 30 to 33, wherein the cell is a T cell.

35. The cell of any one of claims 30 to 34, wherein the cell is a cytotoxic T lymphocyte(CTL).

36. The cell of any one of claims 30 to 35, wherein the cell is a primary CD8+T cell.

37. The cell of any one of claims 30 to 36, wherein the cell expresses endogenousTCRa and / or TCRp.

38. A method of generating a CCR-expressing cell comprising contacting the cell with a nucleic acid of claim 25, or a vector of any one of claims 26-29.

39. A composition comprising cells of any one of claims 30 to 37.

40. A T cell comprising the CCR of any one of claims 1-24, optionally wherein the T cell is a CD8+ or CD4+ T cell.

41. The T cell of claim 40, further comprising (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR).

42. The T cell of claim 41, wherein the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen.

43. The T cell of claim 42, wherein the CAR further comprises at least one costimulatory domain.

44. The T cell of claim 43, wherein the at least one costimulatory region domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7- H3.

45. The T cell of claim 43 or 44, wherein the at least one costimulatory domain is selected from 4-1BB, CD28, 0X40, and a combination of two or more thereof.

46. The T cell of claim 45, wherein the at least one costimulatory domain comprises 4- 1BB and CD28 costimulatory domains.

47. The T cell of any one of claims 41-46, wherein the CAR further comprises a transmembrane domain.

48. The T cell of claim 47, wherein the transmembrane domain is a CD8 transmembrane domain.

49. The T cell of any one of claims 41-48, wherein the TCR or CAR binds a cancer antigen selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, Ll-CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

50. A tumor infiltrating lymphocyte (TIL) comprising the CCR of any one of claims 1-51. The TIL of claim 50, further comprising (a) an engineered T cell Receptor (TCR) (e.g., CTL16) and / or (b) a chimeric antigen receptor (CAR).

52. The TIL of claim 51, wherein the CAR comprises: a) a cluster of differentiation 3 zeta (CD3Q domain, and b) an antigen binding domain specific for a cancer antigen.

53. The TIL of claim 52, wherein the CAR further comprises at least one costimulatory domain.

54. The TIL of claim 53, wherein the at least one costimulatory region domain is selected from CD28, CD27, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7- H3.

55. The TIL of claim 52 or 53, wherein the at least one costimulatory domain is selected from 4- IBB, CD28, 0X40, and a combination of two or more thereof, optionally wherein the at least one costimulatory domain comprises 4-1BB and CD28 costimulatory domains.

56. The T cell of any one of claims 52-55, wherein the CAR further comprises a transmembrane domain.

57. The T cell of claim 56, wherein the transmembrane domain is a CD8 transmembrane domain.

58. The TIL of any one of claims 51-57, wherein the TCR or CAR binds a cancer antigen is selected from ICAM-1, GD2, Mesothelin, HER2, EGFR, PSMA, MUC1, LI- CAM, CD276 (B7-H3), CD44v6, IL13Ra2, EpCAM, FAP, CD133, R0R1, CD24, B7-H4, NKG2D ligands, CD47, GPC3, MAGE-C2, Claudin 18.2, and EGFRvIII.

59. A pharmaceutical composition comprising the T cell of any one of claims 40-49 and / or the TIL of any one of claims 50-58.

60. A method of isolating a T cell with a tumor-specific TCR, the method comprising: a) administering to a subject having a tumor, a T cell comprising the CCR of any one of claims 1-24; or the T cell of claim 40; b) harvesting the tumor from the subject; and c) isolating the T cell with the CCR from the tumor, thereby isolating the T cell with a tumor-specific TCR.

61. The T cell isolated according to the method of claim 60.

62. A method of identifying a tumor-specific TCR, the method comprising: a) isolating the T cell with a tumor-specific TCR according to the method of claim 60; b) sequencing the TCR to thereby identify the tumor-specific TCR.

63. A method of preventing or treating a cancer in a subject, the method comprising administering to the subject the T cell of claim 61, which is optionally further expanded.

64. A method of preventing or treating a cancer in a subject, the method comprising administering to the subject the cell of any one of claims 30-37, the composition of claim 39, the T cell of any one of claims 40-49, the TIL of any one of claims 50-58, and / or the pharmaceutical composition of claim 59.

65. The method of claim 63 or 64, wherein the T cell and / or TIL cell is autologous or allogeneic to the subject.

66. The method of any one of claims 63-65 wherein the subject is treated conjointly with at least one additional cancer therapy.

67. The method of claim 66, wherein the subject is administered with the at least one additional cancer therapy concomitant with, prior to, or following the administration of the T cell, the TIL, and / or the pharmaceutical composition.

68. The method of claim 66 or 67, wherein the at least one additional cancer therapy is selected from immunotherapy, checkpoint inhibitors, cancer vaccines, chemotherapy, radiation therapy, and surgery, optionally a checkpoint inhibitors.

69. The method of any one of claims 66-68, wherein the at least one additional cancer therapy is a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a PD-L1 inhibitor (e.g., an anti- PD-L1 antibody), KD033, or any combination of two or more thereof.

70. The method of claim 69, wherein the PD-1 inhibitor is selected from MP-514 (MEDI0680), nivolimumab, cemiplimab, pembrolizumab, dostarlimab, Vopratelimab (JTX-4014), Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBB 08), Tislelizumab (BGB-A317), Toripalimab (JS 001), INCMGA00012 (MGA012), and AMP- 224.

71. The method of claim 69, wherein the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, KN035, Cosibelimab (CK-301), AUNP12, CA-170, and BMS- 986189.

72. The method of any one of claims 60-71, wherein the cancer or tumor is selected from non-small cell lung cancer (NSCLC), gastric cancer, Melanoma, Breast cancer, Lung cancer, Esophageal cancer, Gastric cancer, Pancreatic cancer, Liver cancer, Colorectal cancer, Anal cancer, Cervical cancer, Ovarian cancer, Endometrial cancer, Prostate cancer, Bladder cancer, Kidney cancer, Testicular cancer, Penile cancer, Head and neck cancer (including oral, pharyngeal, and laryngeal cancers), Thyroid cancer, Soft tissue sarcomas, and Neuroendocrine tumors.

73. The method of any one of claims 60-72, wherein the subject is a mammal, optionally a mouse, a dog, a cat, or a human.

74. The method of 73, wherein the method increases the number of tumor-infiltrating CD3+ T cells.

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