Robust anti-tumor 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 (CRS) 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 scFv derived from a murine FMC63 mAb and combining it with CD8a transmembrane domain, 4-1BB costimulatory domain and CD3ζ signalling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics (MD) simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared to murine scFv (mCAR19). Ex vivo studies with CAR-T cells generated from healthy donors and patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable anti-tumor activity and proliferation. More importantly, h1CAR19-8BBζ-T cells produced lower levels of cytokines (IFN-g, TNF-a upon antigen encounter and reduced the induction of IL-6 cytokine from monocytes than mCAR19-8BBζ-T cells. There was a comparable proliferation of h1CAR19-8BBζ-T cells and mCAR19-8BBζ-T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ-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.