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In the next 5–10 years we could see cellular-based pharmaceuticals, or cell therapy, meeting the unmet medical needs of thousands of people. How this therapy will meet these needs depends on the ability of researchers and manufacturers to successfully and cost effectively manufacture and deliver engineered cell-based therapeutic products that are safe and exhibit enhanced potency with resulting durable, meaningful clinical efficacy. The ability to engineer such enhanced potency using nonviral, cGMP-compliant, automated and closed system manufacturing processes will represent a significant advantage. To outline how such a process might work, we have summarized the application of a scalable, cGMP-compliant, electroporation platform for engineering dendritic cells (DCs), NK cells and T cells for development of cellular immunotherapies targeting hematological malignancies and solid tumors. Autologous cellular immunotherapy refers to a class of therapies that are designed to stimulate a specific immune respons...

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to “on-target off-tumor” toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of “on-target off-tumor” toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10(9) peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival. No significant off-target toxicities were observed. These data support further investigation of CARMA-hMeso as a potential treatment for ovarian cancer and other mesothelin-expressing cancers.

CD19-targeted chimeric antigen receptor (CAR)-engineered T/NK-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been less successful in solid cancers. This is partly due to specificity for shared tumor antigens also present on normal host tissues that leads to ‘on-target/off-tumor’ toxicity. We therefore developed a non-viral approach using repeated infusions of mesothelin-specific messenger RNA (mRNA) CAR-transfected T cells to permit prospective control of ‘on-target/off-tumor’ toxicity. Early trials provided preliminary evidence of the safety and anti-tumor activity of this strategy, but the ex vivo selection, activation and expansion of lymphocytes is laborious and expensive. We therefore explored the feasibility of using a rapid, automated, closed system for cGMP-compliant mRNA CAR transfection into freshly isolated peripheral blood mononuclear cells for clinical scale manufacture (CARMA). The resulting cryopreserved cellular product expressed CAR in >95% of cells, and recognized and lysed tumor cells in an antigen-specific manner. Expression of CAR was detectable for 5-7 days in vitro, with a progressive decline of CAR expression related to in vitro cell expansion. In a murine ovarian cancer model, a single intra-peritoneal (IP) injection of CARMA resulted in the dose-dependent inhibition of tumor growth and prolonged the overall survival (OS) of mice. Multiple weekly IP injections of the optimal CARMA dose enhanced disease control and further prolonged OS, both of which improved with an increasing number of injections. No significant off-tumor toxicities were observed. These data support further investigation of serial IP CARMA administration as a potential treatment for ovarian cancer and other mesothelin-expressing tumors involving the peritoneum, and provide preclinical proof of principle of CARMA for solid tumors. Citation Format: Chien-Fu Hung, Xuequn Xu, Linhong Li, Ying Ma, Qiu Jin, Angelia Viley, Cornell Allen, Pachai Natarajan, Rama Shivakumar, Madhusudan V. Peshwa, Leisha A. Emens. Development of anti-human mesothelin chimeric antigen receptor (CAR) messenger RNA (mRNA) transfected peripheral blood mononuclear cells (CARMA) for the treatment of mesothelin-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3748. doi:10.1158/1538-7445.AM2017-3748