Abstract 6720: HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors
Authors: Kulkarni Abhishek , Paul Debasmita , Dahlberg Declan , Ligori Kostana , Rodriguez Freddys , Rozumalski Lakmal , Walcheck Bruce , Wagner Carston R. 
Published: March 2024

Abstract
Introduction/Purpose: The Wagner lab has developed a protein nanoring based platform to direct human immune cells to combat solid tumors. These nanorings are bispecific, comprised of tumor-targeting and immune cell-targeting dihydrofolate reductase (DHFR2) fusion proteins. These protein nanorings direct immune cells to target and destroy cancer cells. For treating solid tumors, we aimed to target epidermal growth factor receptor (EGFR). However, targeting EGFR could lead to potential “on-target, off-tumor” effects due to expression of EGFR in healthy tissue. Matrix metalloproteinases are overexpressed in a variety of solid tumors. Therefore, to improve tumor specificity, we hypothesized that masking the immune cell targeting DHFR2 protein through a matrix metalloproteinase-2 (MMP-2) sensitive linker will prevent engagement and activation of immune cells outside the tumor microenvironment (TME). Once these protease-activatable nanorings enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the immune cell binder, thereby crosslinking and activating immune cells, causing targeted cancer cell lysis.
Methods: We designed human histidine triad nucleotide binding protein-1 (HINT1) masked anti-immune cell targeting nanobody-DHFR2 fusion proteins. HINT1 was hypothesized to sterically block the engagement of immune cells outside the tumor. HINT1-anti-TCR-DHFR2 (TCR = T cell receptor) and HINT1-anti-CD16-DHFR2 were cloned, expressed, purified, and characterized. Their apparent binding affinities were determined using flow cytometry. The anti-tumor efficacy of these nanorings was evaluated against EGFR overexpressing A431 (human epidermoid carcinoma) cells.
Results: HINT1-anti-TCR-DHFR2 and HINT1-anti-CD16-DHFR2 rings showed reduced binding to T and NK-92-CD16 cells, respectively. In vitro MMP-2 cleavage assay showed complete removal of the HINT1 mask within 2 hours. Western blot analysis showed that both proteins were stable in human serum up to 3 days. 2D cytotoxicity assays with protein nanorings assembled using anti-EGFR-DHFR2 and anti-TCR-DHFR2 or anti-CD16-DHFR2 with T and NK-92-CD16 cells respectively, showed a potent lysis of A431 cells. The EGFR-targeted lysis was diminished in the presence of HINT1 mask, thereby providing proof-of-concept for the steric masking of the anti-immune cell targeting nanobodies.
Conclusion: Protease-activatable nanorings can selectively lyse EGFR+ tumor cells in vitro. Their antitumor efficacy and safety are currently being evaluated using a previously established in vivo mouse model. Therefore, HINT1-steric masking can serve as a simple and effective strategy to address off-tumor toxicities associated with bispecific immunotherapies.
Citation Format: Abhishek Kulkarni, Debasmita Paul, Declan Dahlberg, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Bruce Walcheck, Carston R. Wagner. HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors [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(6_Suppl):Abstract nr 6720.