Trispecific antibodies produced from mAb2 pairs by controlled Fab-arm exchange.

Veronica Natale,Gordana Wozniak-Knopp,Katharina Stadlbauer,Gerhard Stadlmayr,Filippo Benedetti,Florian Rüker

doi:10.1515/hsz-2021-0376

Veronica Natale, Gordana Wozniak-Knopp + Show 4 more

Open Access

https://doi.org/10.1515/hsz-2021-0376

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Abstract

Bispecific antibodies and antibody fragments are therapeutics of growing importance. They are clinically applied for effector cell engagement, enhanced targeting selectivity, addressing of multiple cellular pathways and active transfer of certain activities into difficult-to-reach compartments. These functionalities could profit from a third antigen specificity. In this work we have employed symmetrical bispecific parental antibodies of mAb2 format, which feature a novel antigen binding site in the CH3 domains, and engineered them with a minimal number of point mutations to guide the formation of a controlled Fab-arm exchanged trispecific antibody at a high yield after reduction and re-oxidation. Two model antibodies, one reactive with EGFR, Her2 and VEGF, and one with Fab-arms binding to Ang2 and VEGF and an Fc fragment binding to VEGF, were prepared and examined for heterodimeric status, stability, antigen binding properties and biological activity. Resulting molecules were of good biophysical characteristics and retained antigen reactivity and biological activity of the parental mAb2 constructs.

Highlights

In the last two decades, bispecific antibodies have continuously enriched the spectrum of targeted therapies by showing activity in biological situations where their unique property of containing, in one molecule, specificities for two epitopes could surpass the results of singleagent-based approaches
We first examined the consequences of introducing substitutions F405L and K409R (EU numbering) (Edelman et al 1969) into a wild-type Fc and an anti-vascular endothelial growth factor (VEGF) Fcab CT6 (Figure 1A). This Fcab is modified in the AB, CD and EF loop as well as in the Cterminus of the CH3 domain to mediate binding to VEGF with nanomolar affinity as well as inhibition of its biological activity (Wozniak-Knopp et al 2017), and can still bind to VEGF with a high affinity when expressed as a heterodimer with one wild-type CH3 domain (Lobner et al 2017b)
In immunooncology such agents can trigger a precise combination of signals that has to be delivered to molecular switches acting in various modes (Garfall and June 2019), and extend the reach of present therapeutic options to prove functional in biological situations that are currently considered difficult to address

Summary

Introduction

In the last two decades, bispecific antibodies have continuously enriched the spectrum of targeted therapies by showing activity in biological situations where their unique property of containing, in one molecule, specificities for two epitopes could surpass the results of singleagent-based approaches. Obligate connection of two specificities in a single molecule has proven invaluable in targeting two receptors on the same cell with the purpose to achieve a superior selectivity and superior safety, with such bispecifics working as either antagonists or eliciting an agonistic response (Shi et al 2018). An enzyme mimetic bispecific antibody, which replaces the missing link in an enzyme cascade by targeting simultaneously Factor IXa and Factor X, was approved for the treatment of hemophilia patients who developed antibodies against Factor VIII (Blair 2019). Even in cases where bispecific antibodies combine the activities that are achievable with the sum of their parts, they can reduce costs in comparison with multiple agents used in combination therapy

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