Abstract 3105: Unveiling the potential of humanized bispecific antibody: a novel approach to tumor suppression in oncology

Abstract Background: AXL is a critical regulator in tumor progression, contributing to proliferation, invasiveness, epithelial-mesenchymal transition (EMT), angiogenesis, and immune modulation. High AXL levels are associated with poor prognosis in aggressive cancers. Concurrently, PD-L1 can suppress the antitumor immunity by binding to PD-1 on immune cells, a pathway already targeted by several immunotherapies. However, recent studies indicated that PD-L1's role extends beyond PD-1 binding. It seems to be involved in regulation of tumor growth, stemness, immune modulation, and DNA damage responses, many independently of PD-1. Our humanized bispecific antibody (BsAb), CPL976H, offers a unique dual-target approach by inducing internalization and lysosomal degradation of both PD-L1 and AXL, effectively diminishing their cell surface expression. Methods: The BsAb CPL976H was engineered in a VHH-Fc-VHH format, incorporating dual VHH domains against PD-L1 and AXL. The antibody’s activity, specificity, internalization, and target degradation were assessed using Western blot and flow cytometry across multiple cancer cell lines with varying target expression profiles (e.g., MDA-MB231, MCF7, A549, H1299), and after target induction with INFγ or its silencing with siRNA. RT-PCR and Western blot analyses were performed to assess impact on EMT and immune-modulating pathways. Results: CPL976H demonstrated specific, high-affinity binding to PD-L1 and AXL on diverse cancer cell lines, inducing rapid internalization and lysosomal degradation of these targets. What is important, we were able to achieved efficient targets degradation in sub-nanomolar concertation’s. This degradation led to downstream modulation of critical pathways, particularly those governing EMT and immune evasion. Notably, CPL976H impacted PD-L1 and AXL signaling disrupting cellular processes essential for tumor survival, regardless of PD-1 interaction, indicating its potential efficacy in PD-L1/PD-1 blockade-resistant cancers. Conclusions: CPL976H represents a groundbreaking advancement in oncology by targeting PD-L1 and AXL through a novel mechanism that extends beyond traditional PD-L1/PD-1 inhibition. This bispecific antibody not only mitigates immune escape but also disrupts crucial survival pathways within the tumor, offering a potent therapeutic option for patients with resistance to conventional PD-L1/PD-1 therapies. Its pronounced internalization capabilities make CPL976H a promising candidate for antibody-drug conjugate (ADC) development, which could further amplify its antitumor effects. Notably, in vivo studies confirmed the high efficacy of CPL976H, both as a standalone antibody and as an ADC, demonstrating significant tumor-suppressive effects. This in vivo validation underscores CPL976H’s potential to reshape the therapeutic landscape for challenging oncologic cases. Citation Format: Anna Jablonska, Damian Kolakowski, Tomasz Kornatowski, Aleksandra Sowinska, Sylwia Wieczorek, Krzysztof Lacek, Filip Mitula, Bartosz Wiernicki, Olga Abramczyk, Maciej Wieczorek, Delfina Popiel. Unveiling the potential of humanized bispecific antibody: a novel approach to tumor suppression in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3105.