Abstract 3184: Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action

Abstract Background: Tumor immunotherapy, especially the combination of PD-1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody-drug conjugates (ADCs) are exceptional as a target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, we designed the ADC based on a bispecific anti-PD-L1/anti-AXL antibody with toxic MMAE (Monomethyl auristatin E) for precise targeting of cancer cells and reduction of the side effects. Materials and Methods: The CPL976-MMAE conjugate was prepared with site-specific conjugation technology of Fc-glycan remodeling and click-chemistry. The binding to the extracellular domain of AXL and PD-L1 was confirmed with use of the Surface Plasmon Resonance (SPR). After the conjugation with MMAE, cytotoxicity and selectivity of the ADC were evaluated on the human cancer cell lines with different expression levels of AXL and PD-L1. The ADC's ability to force internalization of targets was shown on a panel of cell lines. The effectiveness of ADC was evaluated based on changes in the cell cycle and of treated cells. The high efficacy of the conjugates was confirmed in a mouse model of MDA-MB-231 xenografts in bi-weekly intravenous injection of 6 doses. Results: CPL976-MMAE showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the cancer cell lines with different expression of the targets AXL/PD-L1. We confirmed ADCs efficacy using cells expressing high levels of AXL and PDL-1 shown as arrest in G2-M phase of the cell cycle and dose-depended apoptosis. The in vivo experiment on a mouse xenograft model confirmed complete tumor reduction (8/8) after ADC treatment, despite the application of an 11 times lower dose in comparison to the free-MMAE control group and was well-tolerated by mice. Importantly, even after the withdrawal of 976-MMAE for 3 weeks, no tumor regrowth was observed. Conclusions: We presented a new, ADC based on bispecific anti-PD-L1/anti-AXL antibody conjugated with MMAE, that combines the effects of cancer growth inhibition by interaction with AXL and immunotherapy by disruption of the PD-1/PD-L1 axis, with selective delivery of MMAE to cancer cells. The simultaneous action of delivering the toxin and acting on both therapeutic targets provides a potent synergistic anti-tumor effect, that can lead to complete tumor eradication and potentially overcome the primary and secondary resistance to PD-1/PD-L1 targeted therapies. Citation Format: Krysztof Lacek, Anna Jablonska, Olga Abramczyk, Martyna Janowska, Filip Mitula, Aleksandra Sowinska, Agnieszka Bojko-Matuszek, Tomasz Kornatowski, Damian Kolakowski, Magdalena Bojko, Monika Skupinska, Wiktor Pyra, Michal Gorka, Joanna Hucz-Kalitowska, Emilia Mroz, Malgorzata Choros, Jerzy Pieczykoland, Maciej Wieczorek, Delfina Popiel. Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3184.