Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models

Aran F Labrijn,Anthony A Armstrong,Sandeep Somani,Janine Schuurman,Işil Altintaş,Matthew Bunce,Sandra Verploegen,Joyce I Meesters,Paul W H I Parren,Tom C Nesspor,Mark L Chiu

doi:10.1038/s41598-017-02823-9

Abstract

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.

Highlights

May hamper correct engagement of the mouse effector functions, compromising proper translation of therapeutic concepts
The reaction condition and matched point-mutations described for controlled Fab-arm exchange were optimized for the generation of human IgG1-based bispecific antibodies[21, 22], the approach can be used to generate bsAbs based on human IgG2, IgG3 and IgG4 subclasses[21]
To investigate whether controlled Fab-arm exchange (cFAE) could be applied to generate mouse bsAbs, the matched F405L and K409R mutations were introduced into chimeric antibodies containing the variable regions of human mAbs 2F824 and 7D825, respectively, and mouse (Mus musculus, mm) IgG1, IgG2a, IgG2b or IgG3 constant regions

Summary

Introduction

May hamper correct engagement of the mouse effector functions, compromising proper translation of therapeutic concepts. Tandem single chain variable fragments (scFvs)[13,14,15] or chemically-crosslinked Fab fragments[16,17,18,19] were used as surrogates. Their lack of Fc region, causes these fragment-based formats to have poor pharmacokinetic properties and disqualifies them for therapeutic concepts that require interaction with effector proteins. The recombination is driven by two matching point-mutations, F405L and K409R (EU-numbering)[23], one in each parental IgG, that weaken the non-covalent CH3-CH3 interaction in the parental Abs enough to allow for the dissociation of half-molecules, but at the same time, strongly favor the heterodimerization that promotes bsAb yield and post-exchange stability upon re-oxidation. To facilitate the investigation of dual-targeting strategies in immunocompetent mouse models, we applied and optimized cFAE for the efficient generation of murine bsAbs

Methods

Results

Conclusion