title: Development of Innovative Bioconjugation Strategies for the Generation of Bi-Specific Antibodies Enabling Targeted Protein Degradation
Creators: name: Lehmann, Tanja Monika
Publisher: name: UNSPECIFIED
Year: 2024

description: More than forty years after description of the first technology for generating monoclonal antibodies, they have become a well-established drug class for treating various diseases. Protein engineering efforts and combining forces with classical medicinal chemistry have paved the way to a plethora of antibody-derived molecules that are now being applied, particularly in cancer therapy. Two main classes are antibody-drug conjugates (ADC), meant to deliver cytotoxic payloads to target cells, and bispecific antibodies (bsAb), that are being used for various approaches, e.g. for enhanced tissue specificity or to recruit immune cells to malignant tissue. This work aimed to expand the current toolbox of antibody modalities and develop modular strategies their generation. In the first part of the work, antibodies were used as vehicles to deliver so-called “proteolysis-targeting chimeras (PROTACs)” into tumor cells. PROTACs are bivalent small-molecules that recruit E3 ligases to intracellular protein of interest (POI) and thereby label them for elimination through the endogenous cellular proteasomal degradation mechanism. Despite their promise, PROTACs face challenges such as limited selectivity, cell permeability or poor pharmacokinetics. A possible solution is to equip antibodies with PROTAC-binding domains, e.g. "camelid single-domain antibody" (VHH), and thereby create bispecific antibodies that can deliver PROTACs in a targeted, ADC-like manner. Such bispecifics can be generated by genetic fusion, which however requires individual production of every antibody-VHH combination. Hence, a modular strategy to promptly produce bispecifics capable of delivering PROTACs was developed. This approach involves enzymatically coupling PROTAC-binding VHHs with commercially available antibodies. The resulting conjugates maintain their target binding and internalization properties. Upon incubation with PROTACs, defined antibody-PROTAC complexes were formed. These complexes selectively induce POI degradation, thereby inducing cytotoxicity selectively in cells expressing the target of the antibody. The second part of the work aims on generation of bispecific antibodies for the recruitment of transmembrane E3 ligases to cell surface receptors. Simultaneous binding can induce a spatial proximity between both proteins which ultimately results in degradation of the receptor via the endosome-lysosome pathway. Bispecific antibodies for this approach are typically produced by antibody engineering and recombinant production. Herein, a chemo-enzymatic approach to generate such molecules from already available antibodies has been developed. Therefore, singlechain variable domains (scFv) and antibodies were first equipped with reactive handles using enzymatic conjugation and afterwards connected using SPAAC click reaction. The scFv-antibody conjugates were analyzed for their antigen binding properties and regarding the degradation of different targets. The chemo-enzymatic conjugation used in this work for generation of bispecific antibodies offers flexible and effective solution using commercially available antibodies. While the resulting molecules have been primarily employed for targeted protein degradation, these conjugation strategies can also be harnessed to extend antibodies with additional binder moieties for various other applications.