Adoptive lymphocyte transfer increases intra-tumoral T cell counts during lymphocytic sequestration and augments immunostimulatory therapy in the CNS of mice with established glioma

Introduction: We have previously described development of a novel brain bi-specific T cell engager (BRiTE) targeting EGFRvIII in Glioma. Despite BRiTEs impressive potency, efficacy in solid tumors is highly dependent on tumor-infiltrating lymphocytes (TILs). We evaluated if peripheral adoptive lymphocyte transfer (ALT) increases TILs in immunocompetent mice, even during glioma-induced sequestration.

Methods: C57/Bl6 mice were implanted with 3 x 10^4 cells syngeneic glioma (CT-2AvIII) and treated with 1 x 10^7 IV CD45.1+ ALT on Day 15. The brain was sub-divided into tumor and non-tumor bearing hemispheres and analyzed each day for exogenous (CD45.1+) CD3/4/8 counts. Survival experiments used combination ALT and anti-4-1BB therapy in the same model. Mice were treated with IV ALT on Day 5, along with 200µg of IP anti-4-1BB . IP mAb Injections were given every 3 days for 4 total cycles (D5, D8, D11 & D14).

Results: Peripherally administered lymphocytes (CD45.1+) were detected and accumulated in tumor bearing hemispheres at significantly higher levels compared to non-tumor bearing hemispheres at both 3- and 48-hours following administration (p=0.0022, p=0.0286 respectively, Mann-Whitney U). Exogenous T cell entry into tumor-bearing CNS overwhelmingly consisted of CD8+ T cells, with minimal presence of exogenous CD4+ lymphocytes detected 48 hours following administration (p=0.0286). Evaluation at these time points was selected as this correlated with peak sequestration of immune cells as evidenced by splenic involution. For survival studies, only combination ALT and anti-4-1BB produced significantly increased survival benefit compared to ALT alone (median survival 45.5 days for combination versus 25 days in ALT only group, p=0.0134, chi-square test).

Conclusion : These findings demonstrate the feasibility and benefit of combination ALT/antibody-based therapy in pre-clinical glioma, as well as outlining a novel approach for simultaneously delivering both immune cells and antibody-based immunotherapy. Combination therapy with BRiTE and ALT will be explored as part of an upcoming clinical trial (NCT04903795).