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

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 beta (αβ) 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 αβ. Second-generation CAR-T cells expressing an anti-αβ 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 αβ CAR-T cell-mediated antitumor responses.

RESULTS

We found that patient-derived DIPG cells and GBM cell lines express high levels of surface αβ by flow cytometry, while αβ 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. αβ 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-1αβ CAR-T cells were detectable, underscoring their ability to persist and undergo self-renewal.

CONCLUSIONS

These results highlight the potential of αβ 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 αβ expression in normal tissues.