Abstract LB125: Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells
Authors: Ma Wei , Wang Nuan , Choi Hye Yeon , Ziying Lin , Fajardo Nicolette , Ilaga Royce , Machida Risa , Lin Zhou , Ramrakhiani Ambika , Chen Yvonne Y. , Sher Linda , Tahara Stanley M. , Machida Keigo 
Published: April 2024

Abstract
Chemotherapy does not sufficiently eliminate tumor-initiating cells (TICs) of hepatocellular carcinoma (HCC). To remove TICs and achieve personalized cancer therapy, dendritic cells (DC) were vaccinated against tumor germline mutations, but metastatic cancers recurred in many cases. Emerging data suggest that nanoparticle-based siRNA delivered targeting of stemness transcription factor NANOG (pluripotency transcription factor) potentiates anti-tumor immunity. We hypothesized that pre-treatment of selective TIC inhibitors targeting stemness genes sensitized the efficacy of immunotherapy targeting of liver TICs. Novel TIC-targeting immunoglobulin, variable chain fragments were identified by phage-display library screening. The top single chain camelid antibody (scFv) candidates were identified and individually cloned into mammalian expression vectors to test binding to TICs. A Tri-specific T cell engager (TriTE) was constructed to connect TIC-targeting scFv with anti-dendritic cell (DC) scFv (anti-CD14) and CD3. Such scFv vectors were tested for target cell lysis. Candidate ligands of anti-TIC ScFvs present on the tumor cell surface were identified through a series of bioinformatic analysis., The latter provided the preconditions for one of our aims to identify the target, tumor surface ligand(s) of anti-TIC ScFv to better understand the mechanism of TriTE action upon a tumor cell. IP-Mass spectrometry analyses demonstrated that two target surface antigens, TBCE and CEP170, along with their respective mRNAs were highly expressed in CD133+ TIC Huh7 cells when compared to CD133- non-TIC Huh7 cells. This accounted for specific TriTE binding to TIC cells, but not to CD133(-) Huh7 cells or primary hepatocytes. These data demonstrated that these unique tumor antigens, primarily arising during neoplastic transformation, elicited T and/or monocyte immunity capable of protecting the host from cancer progression. These conjunctive with PD1 antibody therapeutic modality leads to the personalized immune/chemotherapy of HCC patients. Human monocyte-derived DCs, TIC and T cells were incubated with TriTE to measure IFNγ production during CTL killing effects against TICs. TriTE treatment significantly reduced tumor growth of PDX tissues in NSG-SG3 mice with humanized immunity. Other novel surface antigens on TIC were identified by mass spectroscopy analyses. These data demonstrated that the presence of unique tumor antigens, primarily arising during neoplastic transformation, which elicited T and/or myeloid cell immunity capable of protecting the host from cancer progression. Self-adapting antigen-targeting machinery with Trite therapy is adaptable and fine-tuning next-generation immunotherapy.
Citation Format: Wei Ma, Nuan Wang, Hye Yeon Choi, Lin Ziying, Nicolette Fajardo, Royce Ilaga, Risa Machida, Zhou Lin, Ambika Ramrakhiani, Yvonne Y. Chen, Linda Sher, Stanley M. Tahara, Keigo Machida. Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB125.