Robust antitumor activity and low cytokine production by novel humanized Anti-CD19 CAR T cells

Abstract

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (h1CAR19-8BBz). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/ refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBz T cells produced lower levels of cytokines (IFNg, TNFa) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBz T cells. There was a comparable proliferation of h1CAR19-8BBz T cells and mCAR19-8BBz T cells upon repeated antigen encounter. Finally, h1CAR19-8BBz T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.