Chicken‐derived CD20 antibodies with potent B‐cell depletion activity

Abstract

Summary We report four novel anti‐human CD20 (hCD20) monoclonal antibodies (mAbs) discovered from a phylogenetically distant species—chickens. The chicken–human chimaeric 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 chimaeric anti‐hCD20 antibody rituximab (RTX). Thus, to our knowledge these mAbs are the first to significantly outperform RTX 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 RTX and retain efficacy in vivo. One of the mAbs, AC1, can bind mouse CD20, indicating specificity for a novel hCD20 epitope inaccessible to current (mouse‐derived) anti‐hCD20 mAbs. A humanized version of one antibody, hAC11‐10, was created by complementarity‐determining region (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. These 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 chimaeric antigen receptor‐engineered T (CAR‐T) cells.

Methods for Engineering Binders to Multi-Pass Membrane Proteins

“…Screening the hybridoma supernatant against rat cells overexpressing CCR6 yielded the antibody 1C6, which is able to bind the N-terminal domain of CCR6 and block its signaling (IC 50 10.23 nM). In another example, to engineer antibodies targeting CD20, we immunized chickens with chicken HD11 cells overexpressing CD20 (day 1 and day 27) and constructed a chicken Fab phage library [67,68]. After four rounds of sequential positive and negative selection against CD20 + and naïve CHO or HEK cells, respectively, we identified four chicken antibodies with high affinity to CD20 and 20-100-fold superior whole-blood B cell depletion ability relative to the clinically used anti-CD20 antibody rituximab.…”