Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness
Wannakorn Khopanlert, Pongsakorn Choochuen, Kajornkiat Maneechai, Nawaphat Jangphattananont, Socheatraksmey Ung, Shingo Okuno, Peter Steinberger, Judith Leitner, Surasak Sangkhathat, Pongtep Viboonjuntra, Seitaro Terakura, Jakrawadee Julamanee
Abstract
CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2 , was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37 + tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.