Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor

Abstract

Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα+ cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα+ cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.

Targeting folate receptor alpha for cancer treatment

“…Completely human Fab fragments against FRα were produced by using phage display and among them one, named C4, exhibited good specificity [117]. The human C4 in scFv format, after adequate optimization, resulted in CAR redirection [118]. …”

“…In particular, first generation CARs failed because of poor T cell expansion and adverse effects; new second and third generation CARs have solved at least in part these problems and good pre-clinical in vivo results, i.e. tumor regression, longer persistence in circulation and better localization towards the tumor, have been obtained with MOv19-based CAR constructs containing the co-stimulatory motif of CD137 or CD27 [118, 135]. …”

Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform

“…[12] In parallel, non-viral gene transfer methods have recently been developed with the goal of overcoming high manufacturing costs, regulatory hurdles and scale-up complexities, which have limited so far the range of application of CAR-based immunotherapy with respect to other easier approaches such as monoclonal antibodies (mAbs). [13] However, commonly available non-viral methods are based on transient transfection by mRNA electroporation [14, 15] or stable, integrative methods that have limited transfection efficiency. In this context, the Sleeping Beauty (SB) transposon plasmid system [16] is quite inexpensive and easy to produce and purify.…”

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

“…injected mRNA GD2 CAR T cells did not fully penetrate disseminated tumors, suggesting the transient nature of the RNA will require local delivery of the mRNA-transduced cells for full efficacy. Schutsky et al39 reported similar findings when using mRNA CAR directed against folate receptor for ovarian cancer treatment. In their murine model, localized flank tumors treated with intratumoral CAR T cell injections significantly halted tumor growth.…”