Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity

Abstract

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.

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

“…We have previously described the generation of a platform process for the isolation of ultralong CDR-H3 antibodies by combining cattle immunization and yeast surface display ( 31 ). The same strategy involving the same library was applied in this study for the isolation of NKp30-specific antibodies.…”

“…The same strategy involving the same library was applied in this study for the isolation of NKp30-specific antibodies. In brief, as already described earlier ( 31 ) we specifically 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. Subsequently, a heavy chain library was constructed by grafting the amplified CDR-H3 diversity onto a fixed bovine IGHV1-7 scaffold fused to human domain CH1 and AGA2P by gap repair cloning into S. cerevisiae strain EBY100.…”

“…As previously described, three cattle ( Bos taurus ) with approximately one year of age were immunized using recombinant human NKp30 extracellular domain comprising a C -terminal hexahistidine tag (ECD; produced in-house) in a cocktail approach with recombinant C- terminal his-tagged EGFR ECD (produced in-house) at preclinics GmbH, Germany ( 31 ). Animal care and invasive procedures were in accordance with local animal welfare protection laws and regulation (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES), Dezernat 33 – Tierschutzdienst.…”

“…The library construction process involving strains, reagents as well as plasmids has been described in detail elsewhere ( 31 ). In brief, S. cerevisiae strain EBY100 MATa (URA3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL (pIU211:URA3 )) was utilized for the generation of the heavy chain diversity, whereas BJ5464 cells ( MATα URA3-52 trp1 leu2Δ1his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL ) harboring the light chain plasmid (pLC) encoding for a specific VLλ30 ( Supplementary Figure S1 ) was exploited.…”

“…EGFR is a receptor tyrosine kinase overexpressed in an array of different tumors (28)(29)(30). We have recently described the generation of a platform process for isolating ultralong CDR-H3 antibodies targeting EGFR by combining cattle immunization with yeast surface display (31). To this end, bovine ultralong CDR-H3 regions were PCRamplified and grafted onto a fixed IGHV1-7 scaffold.…”