Abstract 5036: Induction of highly efficacious anti-tumor activity and modulation of tumor microenvironment: Cell-free off the shelf therapeutic modality
Authors: Thakur Archana , Yarlagadda SriVidya , Ji Kyungmin , Schalk Dana L. , Ung Johnson , Bliemeister Edwin T. , Aboukameel Amro , Casarez Eli , Sloane Bonnie F. , Lum Lawrence G. 
Published: August 2019

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) show promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occur upon engagement with tumor cells via the bispecific antibody bridge which stimulates BATs to release not only the lytic and cytotoxic molecules (perforin/granzyme) but also cytokines, chemokines and other signaling molecules extracellularly. We hypothesized that the release of extracellular soluble factors by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune activating conditioned media (CM). In a 3D tumorsphere model, tumor+BATs-CM (n=10) showed potent cytotoxicity (p&lt;0.001) against multiple breast (MDA-MB-231, BT-20, SK-BR-3 and MCF-7) and other cancer cell lines (p&lt;0.001) compared to control tumor-CM or BATs-CM. Tumor+BATs-CM (n=6) was able to reduce the proportion of CD44hi/CD24lo cancer stem like cells to 0.7% compared to 4.9% in control CM. The addition of tumor+BATs-CM decreased the proportions of T regulatory cells (5% to 1.1%; p&lt;0.02) and myeloid derived suppressor cells (3.8% to 1.2%; p&lt;0.03), but increased activation and proliferation of effector T cells in 3D cultures compared to control CM (n=3). Size based CM factionation showed that most activity is retained in the &lt;50kDa but &gt;10kDa fraction. Multiplex analysis showed high levels of IL-2, IL-15, IFN-γ, TNF-α, GM-CSF, granszyme B (GZB), IL-13, MIP-1β, IP-10, MIG and RANTES. These factors are likely responsible for the cytolytic and immune activating effects. Phospho-specific signaling protein arrays showed enhanced JAK1/STAT-1/STAT-5A, Rac/cdc42/STIM-1) pathways in tumor+BATs-CM (n=3). Exosomal microRNA (miR) in tumor+BAT-CM showed higher expression of several miRs that are associated with T cell function and activation compared to control CM (n=2). Simulations using cocktails of multiple cytokines were done to test anti-tumor activity, IFN-γ/TNF-α/GZB showed potent cytotoxicity directed at breast (58-78%) and pancreatic cancer (50-72%) cell lines compared to 45-65%, 20-27%, 18-25% with IFN-γ, TNF-α and GZB individually, respectively. In a xenograft breast cancer model, IV and intra-tumoral injections of 10x concentrated tumor+BAT-CM (3x/week for 4 weeks;150μl CM/injection) was able to inhibit tumor growth significantly (p&lt;0.01) compared to the control CM treated mice (n=10 mice/group). Therapeutic advantages of CM include: 1) a ready off-the-shelf product; 2) a decrease in regulatory and manufacturing costs. In summary, BATs-Tumor complex derived CM provides a clinically controllable cell-free platform to target various tumor types with diverse anti-cancer immune activating mediators regardless of the heterogeneous nature of the tumor cells and mutational burden as a novel and potent off-the-shelf therapeutic modality.

Citation Format: Archana Thakur, SriVidya Yarlagadda, Kyungmin Ji, Dana L. Schalk, Johnson Ung, Edwin T. Bliemeister, Amro Aboukameel, Eli Casarez, Bonnie F. Sloane, Lawrence G. Lum. Induction of highly efficacious anti-tumor activity and modulation of tumor microenvironment: Cell-free off the shelf therapeutic modality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5036.