Chicken-derived anti-CD20 antibodies with potent B cell depletion activity
Introduction
Monoclonal antibodies (mAbs) targeting the human B cell marker CD20 have transformed the treatment of lymphoid malignancies. Existing anti-CD20 mAbs remain only partially effective, often requiring extended treatment and/or combination with chemotherapy or other immunotherapies. Additionally, most patients develop recurrent disease that is refractory to retreatment and as many as 50% of patients fail to show any response. Existing anti-CD20 Abs in the clinic derive from immunization of a mouse host. The high (67%) sequence homology between the CD20 ectodomains from mice and humans makes it challenging to discover anti-CD20 mAbs with new specificities, and ostensibly new therapeutic potential. Not surprisingly, the majority of existing anti-CD20 Abs found via mouse immunization bind a highly similar overlapping epitope on CD20. We sought to identify novel anti-CD20 mAbs that (i) can bind CD20 epitopes not limited to the 33% of sequence space divergent between humans and mice, and (ii) exhibit enhanced B- cell depletion activity, by utilizing a phylogenetically distant species— chickens— as a host for immunization.
Methods
An antibody Fab library was created from the immune repertoire of human CD20-immunized chickens. The Fab phage display library was panned on whole cells displaying human CD20 to identify monoclonal anti-CD20 Fab candidates which were subsequently re-formatted to full-length chicken-human chimeric antibodies containing a human IgG1 Fc domain. One of the antibodies was humanized by grafting of chicken complementarity-determining regions (CDRs) onto human variable region frameworks. The anti-tumor potential of anti-CD20 mAbs candidates was characterized in vitro in terms of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), whole-blood B cell depletion activities, and in vivo in a mouse peritoneal lymphoma model. Cross-reactivity with mouse CD20 was evaluated in mouse CD20-transfected HEK293 cells. Rituximab (Rituxan) and the clinically used second-generation anti-CD20 mAbs ofatumumab (Arzerra) and obinutuzumab (Gazyva) were used as benchmarks in these studies.
Results
Four novel anti-CD20 mAbs were discovered from the immune repertoire of chickens. The chicken–human chimeric antibodies exhibit at least 10-fold enhanced antibody-dependent cellular cytotoxicity (ADCC) and 4-8-fold stronger complement-dependent cytotoxicity (CDC) relative to the clinically used mouse-human chimeric anti-hCD20 antibody rituximab. Thus, to our knowledge these mAbs are the first to significantly outperform rituximab in both Fc-mediated mechanisms of action. The antibodies show 20-100-fold superior depletion of B cells in whole blood from healthy humans relative to rituximab and retain efficacy in vivo. One of the mAbs, AC1, can bind mouse CD20, indicating specificity for a novel human CD20 epitope inaccessible to current (mouse-derived) anti-CD20 mAbs. A humanized version of one antibody, hAC11- 0, was created by CDR grafting into a human variable region framework and this molecule retained the ADCC, in vitro human whole-blood B cell depletion, and in vivo lymphoma cell depletion activities of the parent.
Conclusion
Our chicken-derived anti-CD20 mAbs represent promising monotherapy candidates for improving upon current less-than-ideal clinical outcomes in lymphoid malignancies and provide an arsenal of biologically relevant molecules for the development of next-generation CD20-mediated immunotherapies including bispecific T-cell engagers (BiTE), antibody-drug conjugates (ADC) and chimeric antigen receptor-engineered T (CAR-T) cells.