Abstract
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.
Highlights
Screening of large numbers of BsAbs is required to find relevant lead candidates, but the selection process can be challenging due to the need to develop custom purification steps to purify the BsAb from the undesired antibody molecules that can have similar biophysical characteristics as the desired BsAb
It was shown that mutation of human IgG1 CH3 domain residues H435 and Y436 to the IgG3 residues R435 and F436 could abolish binding to protein A and this effect has been exploited for differential protein A-based purification of human IgG1 bsAbs[30]
Since mouse IgG2 binds to Z-domain weaker than human IgG1 while both bind to FcRn17, we identified sites in the Z-domain binding interface of human IgG1 Fc which are not conserved in mouse IgG2 (Fig. 1A)
Summary
Screening of large numbers of BsAbs is required to find relevant lead candidates, but the selection process can be challenging due to the need to develop custom purification steps to purify the BsAb from the undesired antibody molecules that can have similar biophysical characteristics as the desired BsAb. One method for generating BsAbs, termed controlled Fab-arm exchange (cFAE), involves introduction of a pair of complementary mutations (either F405L or K409R) into the CH3 region of two parental mAbs[14] These sites make important stabilizing intermolecular interactions in human IgG1 and the mutations impart a destabilizing effect on heavy chain homodimers. We describe a) the design and preparation of mutations in the CH2 domain of the Fc designed to disrupt Z-domain interaction while retaining FcRn binding, b) in vitro analysis of the binding between the human IgG mutants and both Z-domain and FcRn, c) development of methods to prepare the BsAbs by differential protein A purification either from controlled Fab arm exchange of purified parental mAbs, culture supernatants; or co-transfection of parental mAbs, and d) pharmacokinetic analysis of the serum lifetime of the mutant BsAbs
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