a-nonviral-approach-to-generate-transient-chimeric-antigen-receptor-t

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

ProMab Biotechnologies

CD19 and CD37 proteins are highly expressed in B-cell lymphoma and have been successfully targeted with different monotherapies, including chimeric antigen receptor (CAR)-T cell therapy. The goal of this study was to target lymphoma with novel CD37, humanized CD37, and bi-specific humanized CD37-CD19 CAR-T cells. A novel mouse monoclonal anti-human CD37 antibody (clone 2B8D12F2D4) was generated with high binding affinity for CD37 antigen (KD = 1.6 nM). The CD37 antibody specifically recognized cell surface CD37 protein in lymphoma cells and not in multiple myeloma or other types of cancer. The mouse and humanized CD37-CAR-T cells specifically killed Raji and CHO-CD37 cells and secreted IFN-gamma. In addition, we generated bi-specific humanized hCD37-CD19 CAR-T cells that specifically killed Raji cells, CHO-CD37, and Hela-CD19 cells and did not kill control CHO or Hela cells. Moreover, the hCD37-CD19 CAR-T cells secreted IFN-gamma against CD37-positive and CD19-positive target CHO-CD37, Hela-CD19 cells, respectively, but not against CD19 and CD37-negative parental cell line. The bi-specific hCD37-CD19 significantly inhibited Raji xenograft tumor growth and prolonged mouse survival in NOD scid gamma mouse (NSG) mouse model. This study demonstrates that novel humanized CD37 and humanized CD37-CD19 CAR-T cells specifically targeted either CD37 positive or CD37 and CD19-positive cells and provides a basis for future clinical studies.

Novel CD37, Humanized CD37 and Bi-Specific Humanized CD37-CD19 CAR-T Cells Specifically Target Lymphoma.

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.

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

BCMA antigen is overexpressed in multiple myeloma cells and has been shown to be a promising target for novel cellular and antibody therapeutics. The humanized BCMA (clone 4C8A) antibody that effectively targeted multiple myeloma in a CAR (chimeric antigen receptor) format was used for designing several formats of bispecific BCMA-CD3 antibodies. Several different designs of univalent and bivalent humanized BCMA-CD3 CrossMAB and BCMA-FAB-CD3 ScFv-Fc antibodies were tested for binding with BCMA-positive cells and T cells and for killing by real time cytotoxic activity and IFN-gamma secretion with CHO-BCMA target cells and with multiple myeloma MM1S and H929 cell lines. All BCMA-CD3 antibodies demonstrated specific binding by FACS to CHO-BCMA, multiple myeloma cells, and to T cells with affinity Kd in the nM range. All antibodies with T cells specifically killed CHO-BCMA and multiple myeloma cells in a dose-dependent manner. The BCMA-CD3 antibodies with T cells secreted IFN-gamma with EC in the nM range. In addition, three BCMA bispecific antibodies had high in vivo efficacy using an MM1S xenograft NSG mouse model. The data demonstrate the high efficacy of novel hBCMA-CD3 antibodies with multiple myeloma cells and provide a basis for future pre-clinical and clinical development.

Bispecific BCMA-CD3 Antibodies Block Multiple Myeloma 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.

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

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.

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

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847  knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.

Yanwei Huang

ProMab Biotechnologies

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy