TAC-T CELLS PERSIST AND REMAIN FUNCTIONAL DURING AND AFTER REPEATED TUMOR EXPOSURE IN VITRO AND IN VIVO

Heather L. MacGregor, Suzanna L. Prosser, Stacey X. Xu, Kyle MacDonald, Aksha Kadri, Ritu R. Randhawa, Swati Shey, Sailaja Pirati, Deepika

ABSTRACT TAC SCIENCE

Background Antigen Binding Domain T cell antigen coupler (TAC) is a chimeric receptor that redirects T cells (TAC-T) towards surface-expressed tumor antigens to create safe and durable anti-cancer immune responses. The TAC receptor activates T cells by co-opting the endogenous T cell receptor machinery via a CD3e- CD3 e specific binding motif and a cytoplasmic co-receptor tail. TAC01-HER2, a first-in-class TAC-T product targeting HER2 (ERBB2), has entered a Binding phase I/II clinical trial. Here, we show that TAC-T cells retain their cytotoxicity capacity during and after repeated tumor challenges in vitro and in vivo.

Materials and Methods

The membrane-bound TAC ...binds directly to the targeted The robustness of anti-tumor T cell responses were assessed in vitro in a recursive killing assay by repeatedly exposing HER2-specific TAC-T receptor interacts directly tumor antigen. Clustering of cells to HER2-expressing tumor cells for 11 successive rounds (39 days). T cells were characterized by flow cytometry to correlate T cell Lck Interaction with the TCR-CD3 epsilon TAC-TCR complexes leads to phenotypes with anti-tumor activity. In vivo, ongoing tumor control established by a single infusion of TAC-T cells was assessed in a tumor Site domain and recruitment of kinases (Lck) + rechallenge experiment. MHC I/II-deficient NSG mice were engrafted subcutaneously with HER2 tumor cells and rechallenged with the same via the cytoplasmic co-tumor cell line 28 days later. TAC-T cells were isolated from mice at various time points for phenotypic and functional characterization.

Results Key features of TAC technology: TAC-T products controlled tumor cell growth through 11 rounds of tumor cell challenge in vitro. Signs of reduced functionality were observed at round 11, which coincided with the emergence of a dysfunctional phenotype. During in vivo tumor rechallenge experiments, a single infusion of TAC-T cells led to complete clearance of the solid tumor xenograft and protected mice from a second tumor challenge 28 days after adoptive MHC T cell transfer. TAC-T cells isolated from tumor sites at various time points exhibited phenotypic markers of activation, whereas TAC-T cells isolated from blood and spleen appeared to be antigen-experienced cells but lacked markers indicative of chronic activation and exhaustion.

• TAC activates T cells via the endogenous TCR.
• TAC-T cells isolated from spleens before and after the rechallenge were able to proliferate and kill tumor cells ex vivo.

Conclusions Here we report evidence that TAC-T cells controlled tumor cell growth through 11 rounds of repeated tumor rechallenge in vitro, protected mice against tumor rechallenge, and demonstrated long-term ex vivo proliferative and cytotoxic capabilities. These data indicate long-lasting T cell persistence and functionality against solid tumors.

Figures and Data

Figure 1: HER2-TAC T cells

Figure 3: HER2-TAC T cells in peripheral reservoir maintained over 56-day in vivo rechallenge study without rechallenge

NSG mice were injected subcutaneously with NCI-N87 tumor cells before receiving a single ACT dose of HER2-TAC T cells. Twenty-eight days after ACT, mice were rechallenged in the opposite flank with the same tumor cell line. Tissues were harvested at various timepoints to track the phenotype of HER2-TAC T cells throughout tumor rejection.

A: DOE outlining dosing and tissue harvest schedule for rechallenge assay. B: Tumor volumes of primary (magenta) and rechallenge (orange) tumors. C: Proportions of CD4 and CD8 T cells isolated from spleen, tumor, and rechallenge tumor. D: Proportion of T cells expressing CD69, PD-1, or an exhausted phenotype (CD39 PD-1 TIM3) isolated from mouse tissues.