Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders

Abstract

Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast

“…In addition to indirectly monitoring enzyme production by measuring cellular green fluorescence, the GOase mediated oxidative conversion of D-galactose to D-galacto-hexodialdose can easily be followed by quantification of hydrogen peroxide formation (37,38). To investigate, whether translational readthrough occurs, which would result in the synthesis of a fusion protein rather than the synthesis of two translation products, another construct was made, where the GOase coding sequence is replaced by vNAR-6His (Supplementary Figure S6D, a shark derived antibody domain with C-terminal hexahistidine tag that can be used for protein detection by western blot analysis (39). Reporter genes were genetically implemented after 7x tet O via CasEMBLR (Figure 1A).…”

Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3

“…The efficiency of the mutagenesis was according to the expecting, considering that the technology of Kunkel mutagenesis offers efficiencies that range from 50–90% [45]. Other works report synthetic library sizes of single domains that range from 1x10 8 -3x10 9 [20, 22, 46–48]. Our libraries presented good diversity with a moderate recombinant repertoire.…”

“…Important advantages of synthetic libraries over the natural libraries are that they avoid the necessity of animal immunization, allow the relatively easy and rapid generation of recombinant antibodies with a specificity for practically any antigen [8,20,21] and also allow the incorporation of desired characteristics in the designs to allow relatively easy maturation of affinity [21, 22].…”

Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody

“…Without applying any negative selections, a few of VNAR binders that showed high specificity towards both cetuximab and matuzumab were successfully isolated using yeast display technology. Surprisingly, each anti-idiotypic VNAR binder isolated was not cross reacting to either cetuximab or matuzumab [59]. Therefore, the combination of semi-synthetic VNAR libraries with yeast surface display would have been a promising technique for rapid isolation of anti-idiotypic VNARs.…”