Abstract 2837: Synthetic TGFb blockade preserves effector function and maintains stemness of GPC3 CAR-T against hepatocellular carcinoma
Authors: Chu Nina J. , Overstreet Michael G. , Gilbreth Ryan , Clarke Lori , Gesse Christina , Tu Eric , Moody Gordon , Torchia Maria Letizia Giardino 
Published: June 2022

Background: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related death worldwide. Immune checkpoint inhibitors have been recently approved in the first-line setting, however only 20-30% of patients respond to the therapy and disease progression is observed in most cases. This provides strong rationale to develop new approaches to treat this unmet medical need. We designed a chimeric antigen receptor (CAR) that targets the oncofetal antigen glypican-3 (GPC3) expressed in 70-90% of HCC and identified transforming growth factor β (TGFβ) as a major suppressive factor that could limit the extent of CAR-T expansion and function. TGFβ levels are increased in advanced HCC as a result of liver fibrosis and hypoxia, which creates an immunosuppressive milieu facilitating cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be restored by abrogating this suppressive cytokine through the co-expression of dominant-negative TGFβRII (TGFβRIIDN).

Results: GPC3 CAR-T expressing TGFβRIIDN showed minimal SMAD2/3 phosphorylation upon exposure to recombinant TGFβ and were more resistant to TGFβ-mediated suppression of IL-2 and interferon gamma (IFNγ) production in vitro, demonstrating functional attenuation of the TGFβ signaling pathway. In a xenograft model of a human HCC cell line overexpressing TGFβ, the TGFβRIIDN armored CAR-T achieved 100% tumor regression with 10/10 complete responders (CR) while the unarmored CAR-T had 4/10 CRs. Armoring GPC3 CAR-T with TGFβRIIDN doubled disease free survival compared to unarmored CAR-T, significantly delaying tumor recurrence. In three TGFβlow patient-derived xenograft (PDX) models, mice treated with unarmored and armored CAR-T achieved &gt;90% tumor growth inhibition (TGI) compared to mice treated with untransduced primary T cells. In three TGFβ+ PDX models, mice treated with unarmored CAR-T achieved no significant TGI whereas mice treated with armored CAR-T achieved 60-90% TGI. The armored CAR-T cells infiltrated TGFβ+ HCC tumors more abundantly than their unarmored counterparts, expressed lower levels of immune checkpoint markers PD1 and LAG3 and higher level of the stemness marker CD27. In line with these observations, we detected significantly more IFNγ at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, confirming in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.

Conclusions: Armoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies such as HCC. These data support the development of an agent for clinical application.

Citation Format: Nina J. Chu, Michael G. Overstreet, Ryan Gilbreth, Lori Clarke, Christina Gesse, Eric Tu, Gordon Moody, Maria Letizia Giardino Torchia. Synthetic TGFb blockade preserves effector function and maintains stemness of GPC3 CAR-T against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2837.