Abstract B121: CPL976, an innovative bispecific antibody targeting AXL and PD-L1 axis as a potential new anticancer therapeutic
Authors: Bojko-Matuszek Agnieszka , Kołakowski Damian , Bojko Magdalena , Sowińska Aleksandra , Mituła Filip , Lacek Krzysztof , Popiel Delfina , Kwiatkowski Sebastian , Kornatowski Tomasz , Kliszcz Beata , Jabłońska Anna , Flis Krzysztof , Wiernicki Bartosz , Wieczorek Maciej , Pieczykolan Jerzy , Abramczyk Olga 
Published: December 2023

Abstract
Background: Bispecific antibodies rapidly emerge as a powerful therapeutic tool in cancer treatment. Usage of the bispecific antibodies in oncology therapies targeting more than one antigen on cancer cells enhances the specificity of the therapy, increasing its effectiveness and decreasing the resistance rate. This approach may be used to modulate the mechanism of the defense and development of cancer cells, such as high-level expression on the cell surface the ligands of the immune checkpoints like PD-L1, and growth factor receptors like EGFR, VEGFR, or AXL. The latest reports reveal the correlation between the expression level of PD-L1 and AXL/EGFR1 in cancer. Notably, the disruption of the signaling pathway of AXL leads to a reduction of the PD-L1 on the cell surface. Adoption of this mechanism into oncology therapies may enhance the immune response against cancer cells. Moreover, the use of the bispecific antibodies simultaneously targeting PD-L1 and AXL may provoke internalization which maximizes the biological effect of the inhibition of the receptor/ligand complexes (AXL/GAS6, PD-1/PD-L1) formation.
Materials and Methods: Bispecific antibody CPL976 was developed as a VHH-Fc-VHH format, with two VHHs raised against PD-L1 and AXL, selected from immune libraries generated after llamas’ immunization. Antibodies' biological activity and selectivity were studied by the Western blot technique on breast cancer cell line (MDA-MB-231) and functional assays constructed on Jurkat E6.1 and Raji cells with stable expression of the PD-1 and PD-L1, respectively. The immunomodulatory functions were also verified in cancer cell lines and PBMC co-cultures. Bispecific binding potential to AXL and PD-L1 and its effect on receptor internalization were evaluated with the use of surface plasmon resonance, Western blot technique, flow cytometry, and pH-dependent dye assays.
Results: CPL976 (PD-L1 × AXL biAb), presents excellent binding kinetics with subnanomolar KDs and an innovative mechanism of action based on simultaneous internalization and degradation of both therapeutic targets at 150 nM. The compound also modulates the cell viability and migration in the breast cancer line stronger than the single agents. This data is supported by the observed decrease of the EMT (Epithelial-Mesenchymal Transition) markers. The immunomodulatory effect was confirmed in the co-culture, where the T-cell activity is blocked. The efficacy of the antibody in the tumor was checked in a murine cancer model.
Conclusions: We present CPL976, an innovative bispecific antibody, with excellent binding parameters, strong biological effect on receptor presentation on the cell surface, and promising in vitro and in vivo results. These combined properties may establish a new generation of anticancer antibodies, that effectively block cancer development and break the PD-1/PD-L1 axis in patients with primary and secondary resistance to PD-1/PD-L1 targeted therapies. Such unique properties might also be later used as an effective carrier of toxic payloads in antibody-drug conjugate format.
Citation Format: Agnieszka Bojko-Matuszek, Damian Kołakowski, Magdalena Bojko, Aleksandra Sowińska, Filip Mituła, Krzysztof Lacek, Delfina Popiel, Sebastian Kwiatkowski, Tomasz Kornatowski, Beata Kliszcz, Anna Jabłońska, Krzysztof Flis, Bartosz Wiernicki, Maciej Wieczorek, Jerzy Pieczykolan, Olga Abramczyk. CPL976, an innovative bispecific antibody targeting AXL and PD-L1 axis as a potential new anticancer therapeutic [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B121.