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Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding.


ABSTRACT: The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.

SUBMITTER: Zwolak A 

PROVIDER: S-EPMC5680793 | biostudies-literature | 2017 Nov/Dec

REPOSITORIES: biostudies-literature

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Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding.

Zwolak Adam A   Armstrong Anthony A AA   Tam Susan H SH   Pardinas Jose R JR   Goulet Dennis R DR   Zheng Songmao S   Brosnan Kerry K   Emmell Eva E   Luo Jeffrey J   Gilliland Gary L GL   Chiu Mark L ML  

mAbs 20170912 8


The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monocl  ...[more]

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