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 lifethreatening 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.

Safety and efficacy of autologous and allogeneic humanized CD19-targeted CAR-T cell therapy for patients with relapsed/refractory B-ALL

“…Research has shown that a murine single-chain variable fragment (scFv) in the CAR structure causes an HLA-restricted T-cell-mediated immune response, 7 resulting in the disappearance or loss of CAR-T cells. A preclinical study by Dwivedi et al 8 revealed that a humanized scFv generated by altering the CAR framework or non-complementarity-determining region reduced the immunogenicity of CAR, thereby reducing cytokine release and enhancing antitumor efficacy. However, the immunity to CAR is associated with treatment failure in some, not all clinical trials.…”

15‐color highly sensitive flow cytometry assay for post anti‐CD19 targeted therapy (anti‐CD19‐CAR‐T and blinatumomab) measurable residual disease assessment in B‐lymphoblastic leukemia/lymphoma: Real‐world applicability and challenges

“…Of 43 B‐ALL patients, 21 were relapsed B‐ALL, 17 were refractory B‐ALL with persistent MRD positivity, and five patients were referral cases with no available clinical details. Among them, 25 received blinatumomab, eight received indigenously developed anti‐CD19‐CAR‐T, 41 and in 10 patients, BM assessment was performed pre‐anti‐CD19 immunotherapy. Among the 25 patients on blinatumomab, MRD analysis was performed on samples after 33–35 days (37 samples), 75–77 days (46 samples), and 204 days (2 samples) of starting therapy.…”

CAR‐T cell therapy in hematological malignancies: Where are we now and where are we heading for?

“…Surprisingly, none of the individuals required tocilizumab therapy or admission to ICUs 58 . In addition, some studies have also emphasized the application of humanized or fully human scFvs to improve the safety and efficacy of CAR‐T cell therapy 59,60 . Affinity‐tuned CAR designs, therefore can potentially aid CAR‐T cells operate within their therapeutic window while minimizing on‐target/off‐target toxicity.…”