A DUAL TARGETING CAR‐T CELL APPROACH FOR THE TREATMENT OF B CELL MALIGNANCIES

Abstract

Introduction: Chimeric Antigen Receptors (CARs) are artificial T-cell receptors which graft an antibody-like specificity onto a T cell. CAR T-cell therapies directed against CD19 have shown activity against several B-cell malignancies including chemo-refractory disease and for a proportion of patients clinical responses are sustained. More recently CAR T cells directed against CD22 have also shown activity against B-cell ALL (B-ALL). An emerging limitation of current CAR Tcell therapy for B-cell cancers is relapse due to target antigen downregulation or loss. We describe a CAR T strategy which simultaneously targets both CD19 and CD22 and thereby may reduce relapse caused by antigen escape. Methods: We constructed a bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Co-expression was achieved by a self-cleaving viral 2A peptide sequence. The antigen binding domains of both CARs were humanized to minimize immune rejection. Each CAR was optimized for maximal activity and included an OX40 co-stimulatory domain or a 41BB costimulatory domain in addition to a CD3 zeta activating domain. We enhanced the performance of the CD22 CAR by incorporating a novel pentameric spacer domain derived from the collagen oligomeric matrix protein. Standard in vitro and in vivo functional assays were performed. Results: Binding kinetics of humanized binding domains were within 0.76 nM for CD19 and 6 nM for CD22 of parental mAb. Independent expression of both CARs was demonstrated on the surface of T cells by staining with recombinant soluble CD19 and CD22. T cells expressing this dual CAR displayed cytotoxicity towards Raji cells expressing both CD19 and CD22 and the CD19-knockout variant with comparable efficacy. In addition this dual CAR could stimulate cytokine release (interferon-γ and IL2) and T cell proliferation in response to CD19 and CD22 or to CD22 alone. CAR T cells were tested against Raji cell xenograft in an NSG mouse model. Conclusion: We have designed tested and optimised a humanised dual anti-CD19/anti-CD22 CAR cassette which directs T cell responses against target cells expressing either or both CD19 and CD22. This construct will soon be tested in a phase I clinical trials for the treatment of relapsed/refractory adult and paediatric ALL and DLBCL.