Optimization of an Automated Phage Display Workflow and Isolation of Fc Knob-into-Hole specific binders

Antibodies are one of the most important entities on the therapeutics market, where modified formats such as Fabs (fragment antigen binding), the bispecifics (bsAb), or VHHs (Nanobodies) gain more and more relevance in comparison to their parental IgG formats. This is due to their various advantageous traits. In particular, bsAbs provide an opportunity to target two different antigens with a single therapeutic thanks to two heterodimeric variable domains: this does not only enable the simultaneous blocking of two different pathways but it can also redirect specific immune cells to the tumour cells, enhancing their elimination. High purity is one of the challenges in the development of bsAbs therapeutics. Therefore, several protein engineering approaches have been developed to increase the correct assembly of the desired product, favouring the right heavy chain – light chain pairing. However, a high percentage of heavy chain – light chain mispairing and other impurities may be still produced together with the desired bsAb. This problem is reduced by modified Fc domains of the heavy chains, recreating a compatible lock-key structure (Knob-into-Hole – KiH technology). Despite the KiH construction, knob-knob (KK) and hole-hole (HH) homodimeric chain pairs can still contaminate the desired product. Finding a binder that specifically recognizes KiH formation and excludes the undesired homodimers could be useful for cell line development and side product characterization in the bsAbs production. In this study, we combined the quest for this binder with the implementation of the automated phage display protocol: given the importance of antibodies, there is a need to speed up the Ab discovery platforms. In this sense, in the Biologics Discovery and Engineering team of the Large Molecule Research department of Roche, a versatile liquid handling robot was programmed to perform panning automatically. Recently, two new devices were purchased aiming to eliminate the cross-contamination issues experienced in the past. In the first part of the project, the robustness of the existing panning protocol was investigated, and some operating parameters optimized. Next, we tested the optimized protocol i) to check whether the cross-contamination issues were solved and ii) to isolate specific KiH binders. This first phage campaign yielded two potential candidate VHHs that specifically bind to KiH-Fc. Binders were characterized by ELISA, SPR, DSF, HIC, and SEC. In terms of cross-contaminations, we observed that the problem was not solved. However, a second phage campaign where the phage library choice was optimized, proved that no cross-contamination occurred.