Abstract 6871: T-Cell redirection to EGFR positive solid tumor with α-TCR nanobody chemically self-assembled nanorings

Abstract Cancer immunotherapy involves utilizing a patient’s own immune system to identify and destroy cancer cells. Recent advances in this field involve bispecific monoclonal antibodies, genetically engineered T-cells, and immune cell engagers, which have shown success in clinical settings. In this regard, the Wagner Lab has shown that prosthetic antigen receptor (PAR) T-cells, which use chemically self-assembled nanorings (CSANs) can be successfully used to redirect T-cells to target antigens upregulated on cancer cells. CSANs are composed of glycine linked dihydrofolate reductase molecules (1DD) that self-assemble into nanorings upon addition of a chemical dimerizer. This self-assembly can be reversed by the addition of the FDA approved drug, trimethoprim, thus disengaging the T-cells from tumor cells. Bispecific CSANs consisting of alphaCD3 single-chain variable fragment (scFv) recognizing CD3 on T-cells and scaffolds targeted to tumor associated antigens have shown effective eradication of solid tumors such as breast cancer and medulloblastoma both in vitro and in vivo. However, the scFvs pose challenges such as hydrophobicity, aggregation, low stability, and immunogenicity. The structural complexities make production challenging in popular bacterial expression systems. Camelid heavy-chain variable domains, also called nanobodies, have been established as a popular alternative to the scFvs due to their smaller size, increased tissue penetration, low immunogenicity, and ease of production, while retaining similar antigen binding properties. In this project, CSANs composed of 1DD units fused to an antiTCR nanobody, which binds to the T-cell receptor (TCR) on the surface of T-cells, will be used to redirect T-cells to solid tumors. Ongoing experiments show that the antiTCR nanobody has lower binding affinity as compared to antiCD3 scFv. This is beneficial as the non-specific activation of T-cells, which is a drawback of high affinity binders, is weaker in case of the antiTCR nanobody. Further, due to the stability and smaller size of nanobodies, antiTCR-1DD has been genetically modified to fuse a tumor targeting scaffold, thus generating bispecific proteins which can simultaneously engage T-cells and tumor cells. Experiments have shown that perhaps due to its geometry, the bispecific protein is highly potent even at very low doses. Bispecific proteins are clinically translatable, reduction in production load being one of the reasons. The bispecific constructs also lay the foundation of higher order immunotherapeutic tools. Citation Format: Debasmita Paul, Freddys F. Rodriguez, Declan Dahlberg, Abhishek Kulkarni, Lakmal Rozumalski. T-Cell redirection to EGFR positive solid tumor with α-TCR nanobody chemically self-assembled nanorings [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 6871.