Abstract 1810: Anti-human TNFR2 antagonist induces T cell proliferation and restores T cell balance to promote tumor killing in TNFR2 HuGEMM™ engrafted with MC38/OVA tumor cells

Introduction: Tumor necrosis factor receptor 2 (TNFR2) is a member of the TNF-receptor superfamily, which is abundantly expressed in regulatory T cells (Treg) and effector T cells (Teff) in the tumor microenvironment (TME) upon T cell activation. TNFR2 is also highly expressed on the surface of many human tumors. For hot tumors, the TME is infiltrated by immunosuppressive Treg cells that suppress Teff cells to allow tumor growth. However, antagonistic anti-TNFR2 inhibitors are believed to break this imbalance by promoting T cell activation and eliminating Tregs to improve cytotoxic T cell function. Taken together, TNFR2 is becoming a promising potential immunotherapy target in the TNF-receptor superfamily along with GITR, OX40, 4-1BB, etc. whether by stimulating T cells to restore Teff/Treg balance in the TME or by directly inducing tumor cell killing though NF-kappaB signaling. We have developed a humanized TNFR2 mouse model (TNFR2 HuGEMM) to evaluate antagonistic anti-TNFR2 inhibitors to induce tumor killing. Methods: We established TNFR2 HuGEMM in both C57BL/6 background and BALB/c background by inserting chimeric h/m TNFR2 CDS in the mouse Tnfrsf1b locus via CRISPR/Cas9-based gene engineering, in which the mouse TNFR2 extracellular domain was replaced by human counterparts, leading to expression of chimeric TNFR2 receptors with human extracellular domain and mouse trans-membrane and intracellular domains. Results: We profiled and characterized the surface expression of human/mouse chimeric TNFR2 on FoxP3+ Treg cells as well as on granulocytes (CD11b+Gr1hi/F4/80- and CD11b+Gr1-/F4/80+) and monocytes (CD11blowGr1-/F4/80low) by FACS analysis of peripheral blood and spleen in the heterozygous and homozygous TNFR2 HuGEMM mice. The expression of chimeric m/hTNFR2 on Tregs in HuGEMM mice can proportionally recapitulate the expression of mouse TNFR2 in wild-type mice as well as human TNFR2 expression in human PBMC. We then inoculated MC38-OVA syngeneic tumors into the female homozygous HuGEMM mice and treated the mice with anti-human TNFR2 antagonists (Ab1 and Ab2). Anti-hTNFR2 Ab1 led to ~80% tumor growth inhibition (TGI) and 14% of the mice (1/7) were eventually cured. By contrast, anti-hTNFR2 Ab2 only led to ~50% TGI and 28% of the mice (2/7) were cured, but 3 out of 7 mice were found dead upon treatment with Ab2. At the efficacy endpoint, FACS analysis of tumor infiltrating lymphocytes indicated that both anti-hTNFR2 antibodies promoted T cell proliferatio