title: Anti-idiotype antibodies for biomedical applications
Creators: name: Habermann, Jan
Publisher: name: UNSPECIFIED
Year: 2024

description: Therapeutic antibodies have become an indispensable part of today's medicine. In addition to the implementation of antibodies in the treatment of a variety of different diseases, the most important area of application is cancer treatment. Approved antibodies all share the disadvantage of addressing tumor related antigens, which are overexpressed on tumor cells, but can also be found on the surface of healthy cells. Due to this fact, severe side effects can occur during antibody treatment. In order to decrease the risks of side effects, several approaches have been pursued including bispecific, immunomodulatory, pH-dependent and protease activatable antibodies. They are later activated by tumor-associated proteases that are weakly expressed in healthy tissue. Additionally, a masking domain is required. These masking domains can either consist of bulky domains interfering with target by steric hinderance or tailor-made affinity-based masking domains.
In this work different approaches were pursued to obtain masking domains based on shark-derived single domain antibodies (vNARs) and chicken-derived single-chain fragment variables (scFvs) directed against different antibodies of therapeutic relevance. In case of the vNARs one approach was based on an existing vNAR directed against matuzumab, which displayed an inhibitory effect on the matuzumab-EGFR interaction. Histidine-doping of the CDR3 of the vNAR resulted in a pH-dependent binding behavior with strongly decreased affinity. The loss of affinity could be overcome neither using a SEED-based nor a heavy or light chain fusion approach.
Another part of this project was aimed at the de novo isolation of pH-dependent vNARs directed against rituximab (anti-CD20 antibody) and 5F9 (anti-CD47 antibody). Isolation of pH-dependent vNARs was successful for both antibodies. Two vNARs directed against 5F9 were isolated, while four vNARs directed against rituximab were obtained; one of which has been further characterized. Both vNARs directed against 5F9 and the one analyzed vNAR against rituximab exhibited masking properties in co-incubation experiments of the respective antibody with the respective target cell line. While the masking properties of the vNAR directed against 5F9 could not be observed in any SEED or light chain construct, the vNAR directed against rituximab displayed masking effects in SEED and light chain constructs. The vNAR was fused to the light chain of rituximab via several MMP-9 cleavable linkers. In case of the SEED constructs pH-dependent restoration of binding was observed, while binding of light chain fusions was restored upon MMP-9 cleavage. In case of one further analyzed light chain fusion, the masking effect in the light chain construct could be attributed to the linker and not the vNAR itself.
The third project in this work was aimed at the isolation of anti-idiotype like vNARs directed against a T cell receptor (TCR). During the screening process, four vNARs were obtained, but only one revealed specific TCR binding, while the other variants displayed binding towards constant domains.
The last project in this work was aimed at the isolation of anti-idiotype chicken scFvs from an immune library based on the immunization of a chicken with the scFv of the therapeutic antibody 6G11 (anti-CD32b antibody). By implementing CD32b into the screening process, scFvs interfering and non-interfering with the 6G11-CD32b interaction could be isolated. Fusion of the scFv obtained from the interfering screening approach resulted in 2700-fold reduction of binding compared to the unmasked 6G11. Upon MMP-9 cleavage, binding was restored, but partly removal of the scFv was required for further restoration of binding.