Publications

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18 nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc. Further, three different species variants of this antibody were generated to allow the use of either anti-human, anti-mouse or anti-rabbit Fc secondary antibodies for detection. We characterized the specificity of both antibodies in depth: individual amino acid exchange mapping demonstrated that the recognized epitope was not changed by the in vitro evolution process. A laser printed array of 29,127 different epitopes representing all human linear B-cell epitopes of the Immune Epitope Database allowing to chart unwanted reactivities with mimotopes showed these to be very low for both antibodies and not increased for Hyper-Myc despite its improved affinity. The very low background reactivity of Hyper-Myc was confirmed by staining of myc-tag transgenic zebrafish whole mounts. Hyper-Myc retains the very high specificity of Myc1-9E10 while allowing myc-tag detection at lower concentrations and with either anti-mouse, anti-rabbit or anti human secondary antibodies.

Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.