CAR-NK Cells Generated with mRNA-LNPs Kill Tumor Target Cells In Vitro and In Vivo.

Natural killer (NK) cells are cytotoxic lymphocytes that are critical for the innate immune system. Engineering NK cells with chimeric antigen receptors (CARs) allows CAR-NK cells to target tumor antigens more effectively. In this report, we present novel CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect NK cells expanded from primary PBMCs and to generate functional CAR-NK cells. CD19-CAR mRNA and BCMA-CAR mRNA were embedded into LNPs that resulted in 78% and 95% CAR expression in NK cells, respectively. BCMA-CAR-NK cells after transfection with CAR mRNA-LNPs killed multiple myeloma RPMI8226 and MM1S cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner in vitro. In addition, CD19-CAR-NK cells generated with CAR mRNA-LNPs killed Daudi and Nalm-6 cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner. Both BCMA-CAR-NK and CD19-CAR-NK cells showed significantly higher cytotoxicity, IFN-gamma, and Granzyme B secretion compared with normal NK cells. Moreover, CD19-CAR-NK cells significantly blocked Nalm-6 tumor growth in vivo. Thus, non-viral delivery of CAR mRNA-LNPs can be used to generate functional CAR-NK cells with high anti-tumor activity.

mRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth.

The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.

Novel CS1 CAR-T Cells and Bispecific CS1-BCMA CAR-T Cells Effectively Target Multiple Myeloma.

Multiple myeloma (MM) is a hematological cancer caused by abnormal proliferation of plasma cells in the bone marrow, and novel types of treatment are needed for this deadly disease. In this study, we aimed to develop novel CS1 CAR-T cells and bispecific CS1-BCMA CAR-T cells to specifically target multiple myeloma. We generated a new CS1 (CD319, SLAM-7) antibody, clone (7A8D5), which specifically recognized the CS1 antigen, and we applied it for the generation of CS1-CAR. CS1-CAR-T cells caused specific killing of CHO-CS1 target cells with secretion of IFN-gamma and targeted multiple myeloma cells. In addition, bispecific CS1-BCMA-41BB-CD3 CAR-T cells effectively killed CHO-CS1 and CHO-BCMA target cells, killed CS1/BCMA-positive multiple myeloma cells, and secreted IFN-gamma. Moreover, CS1-CAR-T cells and bispecific CS1-BCMA CAR-T cells effectively blocked MM1S multiple myeloma tumor growth in vivo. These data for the first time demonstrate that novel CS1 and bispecific CS1-BCMA-CAR-T cells are effective in targeting MM cells and provide a basis for future clinical trials.

Transferrin epitope-CD19-CAR-T cells effectively kill lymphoma cells in vitro and in vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated clinical success in treatment of B-cell hematologic cancers. In this study, we compared human Transferrin epitope tagged CAR-T cells with non-tagged CAR-T cells for cytotoxicity, IFN-gamma secretion and tumor clearance in NSG mice. CD19-TF-CAR-T cells had similar cytotoxicity in vitro to CD19-CAR-T cells against cells expressing CD19 antigen: exogenously CD19+ Hela cells and endogenously CD19+ Raji cells. In addition, CD22-TF CAR-T cells were similarly cytotoxic against CD22+ CHO cells and CD22+  Raji cells. Both CD19-TF or CD22-TF-CAR-T cells secreted less IFN-gamma as compared to non-tagged CAR-T cells. In a Raji xenograft NSG mouse model, CD19-TF-CAR-T cells were as effective as CD19-CAR-T cells in reducing tumor growth and extending mouse survival. The results show that CD19-TF-CAR-T cells can be monitored using TF antibody in vitro and ex vivo, and that these cells effectively killed Raji cells in vitro and in vivo with reduced secretion of IFN-gamma. Thus, these TF-tagged CAR-T cells might have improved safety and provide a basis for future clinical studies.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

Generation of CAR-T Cells for Cancer Immunotherapy.

T cells engineered with chimeric antigen receptors (CARs) are emerging as powerful cancer immunotherapies. Remarkable efficacies have been demonstrated in treating B-cell malignancies with CAR-T cells, leading to the FDA's first approval of gene therapy. Currently, numerous clinical trials for hematological malignancies and solid tumors are underway worldwide. Production of CAR-T cells with proper qualities is essential for CAR-T success in vivo. Here we detail optimized protocols for the generation of CAR-T cells for preclinical studies using lentiviral gene transfer, expansion of CAR-T cells in culture, detection of CAR expression, and evaluation of CAR-T cellular cytotoxicity in vitro.

CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth.

The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-γ in vitro. BCMA-dependent cytotoxicity and IFN-γ secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

GITR domain inside CAR co-stimulates activity of CAR-T cells against cancer.

T cells expressing Chimeric antigen receptors or CAR-T cells are used as a novel treatment against hematological and solid cancers. In this report, we designed CAR with glucocorticoid-induced TNFR-related protein (GITR) co-stimulatory domain to study its ability to co-activate CAR-T cells. EGFR-GITR-CD3 CAR-T cells were cytotoxic against EGFR-positive: pancreatic and ovarian cancer cells but not against EGFR-negative cancer cells. The cytotoxic activity of EGFR-GITR-CD3 CAR-T cells was comparable or better than EGFR-28-CD3 or EGFR-41BB-CD3 CAR-T cells. We designed also EGFR-CD3-GITR-CAR and EGFR-ΔGITR-CD3 with deleted 184-192 amino-acids of co-stimulatory GITR domain, and showed that EGFR-GITR-CD3 had significantly higher cytotoxic activity against EGFR-positive cells. The EGFR-GITR-CD3 cells secreted significantly higher levels of IFN-gamma than EGFR-CD3-GITR and EGFR-ΔGITR-CD3 cells. In addition, Mesothelin-GITR-CD3 CAR-T cells also killed mesothelin-positive ovarian cancer cell lines, and pancreatic cancer cells. Moreover, CD19-GITR-CD3 CAR-T cells had significant cytotoxic activity against CD19-positive cancer cells in vitro and in Raji xenograft tumors in vivo. Thus, our results clearly show that GITR co-stimulatory domain can be used as a novel co-stimulatory domain in CAR-T cells.

CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth.

CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells in vitro and in vivo.

Autologous T cells expressing chimeric antigen receptors (CARs) specific for CD19 have demonstrated remarkable efficacy as therapeutics for B cell malignancies. In the present study, we generated FLAG-tagged CD19-specific CAR-T cells (CD19-FLAG) and compared them to their non-tagged counterparts for their effects on solid and hematological cancer cells in vitro and in vivo. For solid tumors, we used HeLa cervical carcinoma cells engineered to overexpress CD19 (HeLa-CD19), and for hematological cancer we used Raji Burkitt's lymphoma cells, which endogenously express CD19. Like non-tagged CD19 CAR-T cells, CD19-FLAG CAR-T cells expanded in culture >100-fold and exhibited potent cytolytic activity against both HeLa-CD19 and Raji cells in vitro. CD19-FLAG CAR-T cells also secreted significantly more IFN-gamma and IL-2 than the control T cells. In vivo, CD19-FLAG CAR-T cells significantly blocked the growth of HeLa-CD19 solid tumors, increased tumor cleaved caspase-3 levels, and expanded systemically. CD19-FLAG CAR-T cells also significantly reduced Raji tumor burden and extended mouse survival. These results demonstrate the strong efficacy of FLAG-tagged CD19 CAR-T cells in solid and hematological cancer models.

Major Highlights of the CAR-TCR Summit, Boston, 2016.

Cellular immunotherapies such as CAR-T cell therapy and TCR-T cell therapy are relatively new, highly promising approaches for the treatment of cancer. In CAR-T cell therapy, a patient's T cells are engineered to express chimeric antigen receptors targeting tumor-associated cell surface antigens. In TCR-T cell therapy, the patient's T cells are engineered to express receptors targeting intracellular antigens. This report will summarize presentations from the recent CAR-TCR summit in Boston on September 13-16, 2016. These presentations were given by leaders in the field and many were divided into three streams: Discovery and Genetic T Cell Engineering; Translation and Clinical Development; and Manufacturing, Supply Chain and Commercialization. The report summarizes major pharmaceutical companies developing these novel therapies and provides challenges and perspectives for future therapeutic developments.