Abstract LB192: Design and validation of PD-L1 transferrin receptor targeting chimera (TransTAC) protein degraders

Abstract Background: Immune checkpoint blockade (ICB), the blocking of immune checkpoint molecules using therapeutic antibodies, has transformed cancer care for over a decade. But while highly efficacious for several malignancies, challenges remain for the widespread adoption of ICB therapy including primary and acquired resistance and immune-related adverse events. Targeted protein degradation represents an alternative therapeutic modality in which immune checkpoint molecules can be degraded from cell surfaces in a targeted manner, thereby preventing their activation and downstream regulatory effects on T-cells. Recently, the Zhou lab has developed transferrin receptor targeting chimeras (TransTACs); bispecific antibodies which take advantage of the overexpression and exceptionally fast endocytosis of transferrin receptors (TfRs) in cancer cells to simultaneously degrade surface proteins. Aim: The aim of this study was to investigate the molecular determinants of TransTAC design and evaluating their efficacy in PDL1 internalization, degradation, and overall TransTAC function. Methods: Using standard protein engineering techniques, several TransTACs targeting the immune checkpoint PDL1 were designed, and their internalization and degradation efficacies were evaluated using flow cytometry and immunoblotting respectively. Bispecific T-cell engager (BiTE) assays were also conducted to determine the functional effect of TransTAC-mediated PDL1 degradation on T-cell activation. Results & Discussion: Internalization and degradation assays of several TransTAC variants demonstrated that increased affinity to PDL1 is positively correlated with degradation efficiency and that a cleavable linker may not be required for PDL1 degradation. Furthermore, the BiTE assay recapitulated the expected increase in T-cell activation following PDL1 internalization from the cell surface of Raji cancer cells. Finally, a degradation screening identified one variant containing a mutated TfR1 binder with greater degradation efficiency than comparable constructs. Conclusion: Our findings demonstrate three key molecular determinants of TransTAC design- affinity to the protein of interest, the presence or absence of a cleavable linker, and TfR1 binding - which may potentially affect the therapeutic efficacy of TransTAC technology. Citation Format: George Morcos, Lawrence Shue, Dingjingyu Zhou, Johely Duque-Jimenez, Xin Zhou. Design and validation of PD-L1 transferrin receptor targeting chimera (TransTAC) protein degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB192.