Developing exhaustion-resistant CAR-T cells for the treatment of solid tumors

Paula Barbao Carrasco

Dra. Sònia Guedan Carrió and Dr.Aleix Prat Aparicio

Cellular immunotherapies for cancer and Translational genomics and targeted therapies in solid tumors

CAR-T therapy has demonstrated unprecedented success in the treatment of hematological diseases, leading to the approval in Europe of seven CAR-T therapies for the treatment of patients with certain types of lymphoma, leukemia and multiple myeloma. The next key step is to translate CAR-T therapy into the context of solid tumors, which are responsible for the majority of cancer deaths worldwide. The results of clinical studies conducted so far indicate that CAR-T therapy against solid tumors can be safe, but efficacy in most cases has been limited. One of the main difficulties is the functional exhaustion of CAR-T cells in the tumor environment, which reduces their ability to eliminate cancer cells. Overcoming the exhaustion of CAR-T cells is key to improving their persistence and efficacy in the treatment of solid tumors. Currently, the mechanisms that lead to CAR-T cell exhaustion are not fully characterized and only two clinical trials are incorporating strategies specifically aimed at avoiding cell exhaustion in CAR-T therapy. In this thesis, we have established a mouse model to study the mechanisms that drive CAR-T cells to a state of dysfunction, using human CAR-T cells against HER2. We have characterized the transcriptional profile of dysfunctional CAR-T cells and have performed an in vivo functional screening using a CRISPR-KO library to identify genes directly involved in exhaustion. Using this strategy, we have identified 14 new molecular targets, five of which have been selected by individual validation, and all of them have shown a significant increase in antitumor activity and persistence of CAR-T cells. The ZC3H12C gene stands out as the main candidate, demonstrating a significant increase in efficacy in several cancer models and with different CAR designs. This strategy defines a new CAR-T therapy that is resistant to exhaustion and more effective against solid tumors. The project is in an advanced stage of preclinical development, with the characterization of the main target and the validation of four additional targets that improve the efficacy of CAR-T therapy against solid tumors. To ensure clinical translation and commercial exploitation, the project has a European patent filed in April 2025, aimed at protecting the identified targets. In parallel, work is being done to design a strategy for exploiting this technology in the form of a technological platform and generating a product portfolio in the context of the growing market for CAR-T therapies. In conclusion, the technology developed has strong potential to offer a new therapeutic option against cancer, which could be capable of generating a relevant social impact by reducing healthcare costs and improving the quality of life of patients with solid tumors.

The thesis by Paula Barbao Carrasco focuses on overcoming a major limitation of CAR-T cell therapy for solid tumors: T-cell exhaustion, which reduces persistence and antitumor activity in complex tumor microenvironments. Key points: Clinical Context: CAR-T therapy has revolutionized hematologic cancer treatment, but no approved CAR-T exists for solid tumors, which cause 90% of cancer deaths globally. Problem: Sustained antigen exposure in solid tumors leads to CAR-T exhaustion, impairing proliferation and cytotoxicity. Research Achievements: Developed a mouse model with HER2+ ovarian cancer to study CAR-T dysfunction. Identified >2000 differentially expressed genes between effective and exhausted CAR-T cells. Created a CRISPR-KO library targeting 300 overexpressed genes; in vivo screening revealed 14 candidate genes whose deletion improves CAR-T persistence and efficacy. Validated top candidates, notably ZC3H12C, which significantly enhances antitumor activity across multiple tumor models (ovary, pancreas, lung) and CAR designs (CD28, 4-1BB). Technology: A novel CAR-T therapy incorporating genetic modifications to eliminate exhaustion-related genes, improving persistence and therapeutic efficacy. Commercial Potential: Solid tumors represent the largest growth opportunity in the CAR-T market (projected $16B by 2030). Strategy includes academic clinical trials, spin-off creation, and licensing to CAR-T companies. Development Stage: Patent filed (April 2025) covering 14 genes and combinations. Preclinical validation ongoing; PCT patent submission planned for April 2026. Supported by PERIS funding and integrated into Hospital Clínic de Barcelona’s CAR-T program. Impact: Offers a transformative approach for solid tumor treatment, with potential applications in hematologic CAR-T, TCR-engineered T cells, and tumor-infiltrating lymphocytes.

CAR-T therapy, Solid tumors, Tumor microenvironment, T-cell exhaustion, HER2, Ovarian cancer, Pancreatic cancer, Lung cancer, Genetic modification, CRISPR-KO, Gene editing, ZC3H12C, Candidate genes, Immunotherapy, Adoptive cell therapy, Chimeric antigen receptor, CD28, 4-1BB, Persistence, Antitumor activity, Tumor-infiltrating lymphocytes, TIL, TCR-engineered T cells, Functional improvement, Transcriptomic analysis, RNA sequencing, Differential gene expression, In vivo screening, Preclinical validation, Patent protection, PCT, Hospital Clínic de Barcelona, ARI-HER2, Clinical trial, Phase I, PERIS funding, Spin-off, Tech transfer, Licensing strategy, Market growth, Advanced therapies, Personalized medicine, Cancer immunotherapy, Translational research, Therapeutic innovation, High-impact oncology, Regulatory pathway, AEMPS, Manufacturing protocols, Safety studies, ADME-Tox, Global health impact.