Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

Abstract

We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR) specific for vascular endothelial growth factor receptor 2 (VEGFR2), demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP) and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6–12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

mRNA as novel technology for passive immunotherapy

“…Later, contact to target cells strongly down-regulated receptor on the cell surface while lentiviral expression remained constant [ 261 ]. In comparison to a single transfer of cells with retroviral CAR expression, an mRNA-encoded receptor required three consecutive lymphocyte infusions to obtain a comparable antitumor effect [ 262 ]. These observations and considerations may be put into perspective at least in part by the finding that transferred T cells can become tolerized rather rapidly, thereby losing their ability to function in the tumor microenvironment [ 263 ].…”

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

“…is considered local delivery, again emphasizing one of the major considerations when mRNA CAR T cell is used to eradicate solid tumor. Additional novel target explorations for mRNA CAR T cell therapy have included melanoma-associated chondroitin sulfate proteoglycan (MCSP), CEA, and vascular endothelial growth factor receptor 2 (VEGFR2) for melanoma; [40][41][42] epithelial cell adhesion molecule (EpCAM) for gastric and ovarian cancer; 43 epidermal growth factor receptor (EGFR) for glioblastoma; 44 and Natural Killer group 2D ligand (NKGD2L) for Ewing's sarcoma. 45 Tchou et al 46 recently targeted c-Met, a cell-surface protein present in a variety of solid tumors.…”

Predicting the Efficacy and Safety of TACTICs (Tumor Angiogenesis-Specific CAR-T Cells Impacting Cancers) Therapy for Soft Tissue Sarcoma Patients

“…On the other hand, as in epithelial carcinomas, STSs induce angiogenesis from nearby parent vessels as the tumor grows, and vascular endothelial cells comprising these tumor vessels have been shown to strongly express vascular endothelial growth factor receptor 2 (VEGFR2) [ 13 , 14 ]. Previously, we have developed adoptive immunotherapy using T cells expressing a chimeric antigen receptor (CAR) that targets VEGFR2 and reported that anti-VEGFR2 CAR-T cells could inhibit tumor growth based on tumor vascular injury in a variety of tumor-bearing mouse models [ 15 , 16 ]. Our TACTICs (Tumor Angiogenesis-specific CAR-T cells Impacting Cancers) therapeutic strategy has the potential to be an all-around effective treatment against diverse STSs and to prevent hematogenous metastases via vascular injury.…”

“…Therefore, the safety management of subjects should be carried out continuously and consistently, as the complete elimination of transferred CAR-T cells from the body can be difficult when signs of serious adverse effects are observed [ 18 ]. Therefore, we selected electroporation (EP) transfer of mRNA encoding the CAR gene as a method for generating CAR-T cells for clinical research [ 16 ]. The mRNA-EP method can reduce the risk of side effects by allowing the reversion of CAR-T cells to their original phenotype over time; however, in this context, the efficacy of CAR-T cells is obviously transient.…”

“…A previous study has confirmed that the mRNA-EP method was an efficient way to express CAR on T cells without affecting their viability and functions. Further, the study revealed that multiple doses of anti-VEGFR2 CAR-mRNA-transfected T cells exhibited strong anti-tumor activity, comparable to that provided by a single dose of CAR-T cells generated via viral vector-mediated gene transfer [ 16 ].…”