Targeting C4d to overcome resistance to antibody-based immunotherapy: an in vitro study

Tumor cells often develop defense mechanisms that render them insensitive to consecutive administrations of therapeutic monoclonal antibodies. This study aimed to produce an antibody that preferentially binds resistant tumor cells and eliminates them by the complement system attack. We considered the C4d molecule as an appropriate molecular target. C4d is the final degradation product of active complement component C4b covalently deposited on cells previously exposed to antibodies bound to their surface antigens. Because of the activity of both membrane-bound and soluble complement inhibitors, C4d persists on surviving cells and serves as a footprint of prior challenge with cell-specific antibodies. A candidate anti-C4d antibody was produced as a chimeric human-rabbit construct incorporating amino acid substitutions that enhance oligomerization. Its performance was tested on human lymphoma and breast cancer cells pretreated with standard immunotherapeutics. Application of anti-C4d antibody significantly increased the killing of lymphoma cells treated previously with anti-CD20 (rituximab, obinutuzumab) or anti-CD38 (daratumumab) compared to re-treatment using the same antibody. A similar effect was observed in lymphoma sublines resistant to rituximab, as well as in a human breast cancer cell line treated with trastuzumab, an anti-HER2 therapeutic antibody that by itself induced no measurable complement-mediated cytotoxicity. We propose antibodies specifically recognizing the C4d fragment as potent immunotherapeutic amplifiers capable of converting limited complement activation induced by a primary antibody into a robust secondary wave of complement-mediated killing of cells that survived the initial antibody treatment.