Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of 'Almost Natural' Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection.

Daniel Klewinghaus,Lukas Pekar,Bernhard Valldorf,Harald Kolmar,Stefan Zielonka,Paul Arras,Simon Krah

doi:10.3389/fimmu.2021.801368

Daniel Klewinghaus, Lukas Pekar + Show 5 more

Open Access

https://doi.org/10.3389/fimmu.2021.801368

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Abstract

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

Highlights

The human body is continuously exposed to potentially lifethreatening opponents such as bacteria, viruses or cancerous cells
As already described earlier [31] we amplified ultralong CDR-H3 regions from cDNA obtained from the peripheral blood mononuclear cell (PBMC) repertoire of cattle that were immunized with recombinant human NKp30 ECD
Afterwards, the resulting diploid yeast cell Fab library was screened by fluorescence activated cell sorting (FACS) to isolate ultralong CDR-H3 common light chain paratopes specific to

Summary

Introduction

The human body is continuously exposed to potentially lifethreatening opponents such as bacteria, viruses or cancerous cells. The high specificity for a given antigen in conjunction with humoral and cellular effector functions mediated by the Fcpart of IgG isotypes renders this class of Abs as very promising molecules for therapy [1, 2]. This is exemplified by the fact that as of 2021 around 100 therapeutic antibody derivatives have been granted marketing approval by the FDA [3]. An unprecedented multitude of different bsAb formats has been engineered [15], including bi- and multispecifics derived from camelids [16,17,18] or sharks [19,20,21]

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