Binding domain on CD22 molecules contributing to the biological activity of T cell-engaging bispecific antibodies

CD22, as the B-cell malignancies antigen, has been targeted for immunotherapies through CAR-T cells, antibody-drug conjugates (ADCs) and immunotoxins via interaction of antibodies with binding domains on the receptor. We hypothesized that avidity and binding domain of antibody to target cells may have significant impact on the biological function in tumor immunotherapy, and T cell-engaging bispecific antibody (TCB) targeting CD22 could be used in the therapy of hematologic malignancies. So, to address the question, we utilized the information of six previously reported CD22 mAbs to generate CD22-TCBs with different avidity to different domains on CD22 protein. We found that the avidity of CD22-TCBs to protein was not consistent with the avidity to target cells, indicating that TCBs had different binding mode to the protein and cells. In vitro results indicated that CD22-TCBs mediated cytotoxicity depended on the avidity of antibodies to target cells rather than to protein. Moreover, distal binding domain of the antigen contributed to the avidity and biological activity of IgG-[L]-scfv-like CD22-TCBs. The T cells' proliferation, activation, cytotoxicity as well as cytokine release were compared, and G5/44 BsAb was selected for further in vivo assessment in anti-tumor activity. In vivo results demonstrated that CD22-TCB (G5/44 BsAb) significantly inhibited the tumors growth in mice. All these data suggested that CD22-TCBs could be developed as a promising candidate for B-cell malignancies therapy through optimizing the design with avidity and binding domain to CD22 target in consideration.