Enhancing Rituximab's Anti-Tumor Efficacy: The Development and Validation of KPMW135, a CD3 Bispecific Agent with T Cell-Mediated Cytotoxicity

KPMW135 is a newly developed CD20 x CD3 bispecific molecule that integrates a CD3-specific ScFv (SP34) with rituximab (RTX), a monoclonal antibody targeting CD20 for the treatment of B-cell lymphomas and leukemias. This molecule is created through a novel MATE platform that facilitates the chemical conjugation of existing antibodies without genetic engineering, offering a more precise and efficient alternative to previous methods.

The SP34 ScFv was expressed in FreeStyle 293 cells, purified, and characterized for its binding to CD20 using Bio-Layer Interferometry. The binding affinity was found to be 0.75nM, indicating that the conjugation did not compromise the CD20 binding ability. Additional ELISA tests confirmed that KPMW135 can bind to CD3εδ, FcRn, and CD16a, demonstrating that the conjugation process did not hinder its interaction with these proteins.

In vitro assays using PBMCs revealed that KPMW135 can effectively mediate T cell cytotoxicity against CD20+ B lymphoblast cells, with an EC50 of 0.03-0.07nM for prestimulated PBMCs and 0.09-0.21nM for freshly thawed PBMCs. This indicates that KPMW135 has a potent ability to induce target cell death through T cell engagement.

In vivo studies in cynomolgus monkeys demonstrated that KPMW135 can activate T cells, as evidenced by increased expression of CD69 and CD44, and induce a rapid and pronounced depletion of B cells. This was in contrast to RTX, which showed a transient depletion followed by a quicker recovery.

In conclusion, KPMW135, developed using the MATE technology, retains the native properties of RTX, including its binding to FcRs and CD20, while adding the capability to mediate T cell cytotoxicity. The in vitro and in vivo data suggest that KPMW135 is more effective than RTX in targeting B lymphoid malignancies and can be a promising candidate for the development of "off-the-shelf" biosuperior antibodies.