Identification of CAR-T cell therapeutic targets for solid tumors through in vivo gene screening based on artificial immune synapses
Background
Existing Chimeric Antigen Receptor T cell (CAR-T) therapies have demonstrated efficacy in hematological malignancies but have shown limitations in overcoming solid tumors. The tumor microenvironment (TME) formed by solid tumor cells inhibits immune cell infiltration and releases immunosuppressive signals, thereby neutralizing T cells. This is why research is actively underway to engineer T cells with CRISPR technology to enhance their resistance.
In vitro validation methods cannot mimic the complex immunosuppressive mechanisms of the TME. In vivo screening is emerging as an alternative because it replicates the tumor tissue environment. However, the recovery rate of tumor-infiltrating lymphocytes (TILs) that have infiltrated solid tumors has been low, posing challenges for large-scale screening. It has been difficult to identify immune cell regulatory factors at the whole-genome level in vivo.
Key Findings
The research team, led by Professor Julia Carnevale at the University of California, San Francisco (UCSF), developed an in vivo model that efficiently recovers T cells from solid tumors. They engineered a human melanoma cell line, A375, to express an anti-CD3 single-chain variable fragment (scFv) that stimulates T cells. This strategy involves forming artificial immune synapses on the surface of cancer cells to induce T cell proliferation within the tumor. This allows for the isolation and collection of tumor-infiltrating T cells within solid tumors.
Based on this model, the research team conducted whole-genome CRISPR screening. The screening was conducted in two directions: infiltration and functional activity. In the infiltration area, the purinergic receptor P2RY8 and the Gα13 signaling pathway were identified as infiltration inhibitors. In the functional activity area, the GNAS gene was found to play a leading role. Gαs, the G protein alpha subunit encoded by GNAS, acts as a hub that transmits inhibitory signals from various GPCR downstream pathways. In fact, CAR-T cells with GNAS removed maintain their ability to kill cancer cells even in an inhibitory environment rich in adenosine and prostaglandin E2 (PGE2). Furthermore, T cells with P2RY8 and GNAS simultaneously edited demonstrated superior tumor control ability compared to single editing.
Significance and Prospects
This study has been praised for discovering new immune modulation pathways that may provide a breakthrough in overcoming solid tumors. It has achieved whole-genome-level elucidation of gene function in the in vivo immunosuppressive environment, which was previously difficult to determine. In particular, the fact that blocking P2RY8 and GNAS simultaneously enhances T cell infiltration and persistence suggests that it will activate the development of combination-edited gene therapies in the future.
However, there are still points to be improved before this technology can be directly applied to patients. Additional validation is essential to determine whether the effects of artificial immune synapses observed in mouse models are reproduced in humans. Ensuring the technical stability of controlling off-target effects, which can cause unintended mutations during the gene multiplex editing process, is also a challenge to be overcome.
Nature, Published online: 12 August 2026; doi:10.1038/s41586-026-10906-9An in vivo model that is able to recover human T cells from solid tumours can identify targets to help improve CAR T cell antitumour activity.
This study provides an industrial foundation that can shorten the commercialization period of gene cell therapies for solid tumors. Existing therapeutic developers have experienced numerous failures because positive data obtained from in vitro experiments have not been reproduced in clinical trials. The introduction of a new in vivo screening platform is expected to contribute to the early identification of the efficacy of therapeutic candidates under tumor microenvironment conditions that are similar to actual clinical settings. A specific application scenario is the design of a study targeting patients with refractory pancreatic or colorectal cancer who do not respond to immune checkpoint inhibitors. This involves transplanting patient-derived cancer cells into mouse models and then administering CAR-T cells with P2RY8 and GNAS genes removed, followed by verifying infiltration and killing performance. This will lead to the creation of a therapeutic model in which an individualized gene blockade combination is selected and applied to treatment based on the tumor characteristics of each patient.