Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA–Transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy

Abstract

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 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.

Novel, genetically induced mouse model that recapitulates the histological morphology and immunosuppressive tumor microenvironment of metastatic peritoneal carcinomatosis

“…[46][47][48][49][50] Many clinical trials and experimental therapies have targeted these two tumor markers, including vaccine, CA125/MUC16-or mesothelin-specific antibody and CAR T cell therapy. [51][52][53][54][55][56] Therefore, with these markers highly expressed in our model, this novel system physically recapitulates human HGSC development and spread, allowing for the preclinical screening of new therapies targeting CA125, mesothelin and many other markers.…”

The Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy

“…Using a model system of HIV, they generated nef-and gag-specific TCRs, and their work uncovered interesting functionality about expressing TCRs with different specificities on the same T lymphocyte. One TCR seemed to be dominant, in this case, the nef-specific TCR; in order Hodgkin's lymphoma 52 Mesothelin mesothelioma 33,34,49 mesothelioma, pancreatic cancer 35 pancreatic cancer 36 ovarian cancer 37 MCSP melanoma 40,41 Her-2/neu ovarian cancer 13,14 ErbB2 Review to have both TCRs functioning independently, the dominant TCR had to be altered with murine co-stimulatory domains. In 2018, Campillo-Davo et al 23 investigated the function of mRNA-encoded TCR after removing the endogenous TCR by RNAi.…”

“…Again, evaluation of patients' sera showed a spreading antibody response with increased antibody production against multiple proteins including several immunoregulatory molecules such as programmed cell death protein 1 (PD1), programmed death ligand 1 (PDL1), and B cell maturation antigen (BMCA). Finally, Hung et al 37 at Johns Hopkins University recently reported a new technique for production of mesothelindirected mRNA CAR T cells that uses unselected and unexpanded PBMCs that are cryopreserved after large-scale manufacturing. Using a murine model of ovarian cancer, equally good efficacy could be demonstrated when either cryopreserved or freshly made mRNA CAR T cells were injected.…”

Chimeric antigen receptor (CAR)-modified NK cells against cancer: Opportunities and challenges

“…A previous study has shown that median receptor expression 24 h after electroporation with the corresponding mRNA was 82.0% and NK cells transfected under such conditions had significant cytotoxicity in xenograft cancer model [51]. Recently, a study indicated that "on-target off-tumor" toxicity, which is a critical factor limiting the application of CAR-based immunotherapies, may be effectively avoided by transduction with mRNA [52]. Nevertheless, the anti-tumor effect of CAR-NK cells transferred by mRNA electroporation may be transient because the expression of CARs transferred in this way is no more than 3 days [53].…”