CAR T Cells Generated UsingSleeping BeautyTransposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor ActivityIn VitroandIn Vivo

Abstract

These authors contributed equally to this work.Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBf CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBf CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

Three-Compartment Model of CAR T-cell Immunotherapy

“…The model was able to represent tumor elimination after immunotherapy with CAR T 123 cells even in case of a new tumor challenge due to memory T cells longterm protection for HDML-2 target. The change of CAR T cells from effector to memory cells and their long-term persistency as CAR T memory cells were also demonstrated by [44] and our previous work with RS4;11 B-ALL model using 19BBz CAR T [10]. For the CAR T 19 therapy and RAJI target scenario, the model represented well the tumor dynamics with or without IDO inhibitor.…”

Development of CAR-T Cell Therapy for B-ALL Using a Point-of-Care Approach

“…On this occasion we purified T cells from PBMCs to ensure no antitumor effect by natural killer (NK) cells and ensure cell composition homogeneity in both groups by injecting only CD3+ cells. As previously described by our group, NK cells may have accounted for improved survival of the mock group compared to PBS treated animals when the infused product contains this lymphocyte population (15). T cells were electroporated and infused into the animals after 24 hours in the POC group.…”

“…Children's Research Hospital, Memphis, TN). The sequence was codon optimized and synthesized by Genscript (Piscataway, NJ) and a myc-tag was added between CD8ɑ signal peptide and scFv to allow flow cytometry-based detection as described elsewhere (15). The transgene was cloned using AgeI/NotI sites in the transposon vector pT3 provided by Dr. Richard Morgan (NIH).…”

“…Nalm-6 and RS4;11 were modified by lentivirus to express a fluorescent GFP protein as previously described (15) and were selected by fluorescence-activated cell sorting (FACS). These cells were cultivated RPMI (Gibco,CA) complete medium supplemented with 10% fetal calf serum, 2 mM L-Glu and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, MO) at 37°C, 5% CO2.…”

“…We and others have shown that the integrative, non-viral Sleeping Beauty (SB) transposon system is a suitable alternative to viral vectors in the process of CAR-T cell production (15)(16)(17)(18). CAR-T cells generated by electroporation of mononuclear cells with SB plasmids (one encoding the CAR transgene and the other encoding the SB100x transposase) have antitumor activity in vitro and in vivo, while presenting long-term CAR expression.…”