Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Abstract

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

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

“…These results clearly demonstrate that the identified anti-idiotypic vNARs are paratope-specific for their corresponding target antibody and that both, the heavy and the light chain variable region are engaged in binding. It is interesting to note that solely paratope-specific binders rather than variants which engage the antibody constant regions were obtained, although no selection pressure was applied during the yeast display screenings 12 .

Figure 4 Assessment of paratope-specificity of anti-idiotypic vNAR-Fc variants using Bio-Layer Interferometry.

…”

“…Analogous to the work recently published by Godar and colleagues, we wanted to investigate whether anti-idiotypic vNARs were able to identify bispecific antibodies from a heterogeneous mixture 12 . In this manner, a two-step purification procedure involving the AM-VNAR1 and AC-VNAR2 vNAR-functionalized magnetic beads should conducted.…”

“…The co-transfection of all four chains results in the generation of overall 10 different antibody constructs (Fig. 6a ) 12 . Since the identified anti-idiotypic vNARs do not interact with mispaired heavy and light chains, the first purification step should reduce the number of available antibody variants to four (Fig.…”

“…Such anti-idiotypic antibodies are of great importance in the assessment of the pharmacokinetic properties of a therapeutic antibody and can also be employed as a standard in the identification of anti-drug responses in order to analyze potential immunogenicity issues 9 . In addition to their utilization in various immunoassay formats, anti-idiotypic antibodies have been employed for the immunoaffinity enrichment of therapeutic antibodies, for mass spectrometric analyses 10 , 11 , for the purification of bispecific antibodies 12 and as cancer vaccines 13 , 14 . With regard to their employment as cancer vaccines, anti-idiotypic antibodies which represent an ‘internal image’ of the actual antigen can be used for immunizations.…”

“…In addition, the approach described in the present investigation relies on semi-synthetic instead of immune libraries, completely obviating the need for animal immunizations. This is an advantageous feature, especially when compared to the generation of camelid-derived heavy chain antibodies, which commonly involves the immunization of llamas or camelids 12 , 14 .

Figure 1 Screening of semi-synthetic, yeast-displayed vNAR libraries towards matuzumab and cetuximab.

…”