Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma

Abstract

Background: Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success of chimeric antigen receptor (CAR) T cells for the treatment of leukemia and lymphoma, significant headway into the application of CAR-T cells against solid tumors, including gliomas, is still forthcoming. The integrin complex alpha_v beta₃ (α_vβ₃) is present on multiple and diverse solid tumor types and tumor vasculature with limited expression throughout most normal tissues, qualifying it as an appealing target for CAR-T cell-mediated immunotherapy.

Methods: Patient-derived DIPG and GBM cell lines were evaluated by flow cytometry for surface expression of α_vβ₃. Second-generation CAR-T cells expressing an anti-α_vβ₃ single-chain variable fragment were generated by retroviral transduction containing either a CD28 or 4-1BB costimulatory domain and CD3zeta. CAR-T cells were evaluated by flow cytometry for CAR expression, memory phenotype distribution, and inhibitory receptor profile. DIPG and GBM cell lines were orthotopically implanted into NSG mice via stereotactic injection and monitored with bioluminescent imaging to evaluate α_vβ₃ CAR-T cell-mediated antitumor responses.

Results: We found that patient-derived DIPG cells and GBM cell lines express high levels of surface α_vβ₃ by flow cytometry, while α_vβ₃ is minimally expressed on normal tissues by RNA sequencing and protein microarray. The manufactured CAR-T cells consisted of a substantial frequency of favorable early memory cells and a low inhibitory receptor profile. α_vβ₃ CAR-T cells demonstrated efficient, antigen-specific tumor cell killing in both cytotoxicity assays and in in vivo models of orthotopically and stereotactically implanted DIPG and GBM tumors into relevant locations in the brain of NSG mice. Tumor responses were rapid and robust with systemic CAR-T cell proliferation and long-lived persistence associated with long-term survival. Following tumor clearance, TCF-1⁺α_vβ₃ CAR-T cells were detectable, underscoring their ability to persist and undergo self-renewal.

Conclusions: These results highlight the potential of α_vβ₃ CAR-T cells for immunotherapeutic treatment of aggressive brain tumors with reduced risk of on-target, off-tumor mediated toxicity due to the restricted nature of α_vβ₃ expression in normal tissues.

Keywords: T-lymphocytes; chimeric antigen; immunologic memory; immunotherapy; pediatrics; receptors.