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Antigen-scaffolds drive preferential expansion of functional genetically engineered CAR and TCR T cells

ABSTRACT: The engineering of autologous T cells for the expression of chimeric antigen receptors (CARs) can induce profound clinical responses in haematological malignancies, while T cell receptor-engineered T (TCR T) cells have led to durable responses to solid tumours in clinical trials. However, clinical manufacturing of engineered T cells is resource-intensive and frequently yields highly differentiated, exhausted effector T cell products. To circumvent this, we have developed an antigen-scaffold (Ag-scaffold) technology to preferentially expand genetically engineered T cells. Such Ag-scaffolds present cognate antigen together with stimulatory factors such as cytokines. By providing a specific and receptor-engaging stimulation to CAR/TCR T cells, the expanded product is highly enriched for engineered T cells with a favourable proliferative and efficacious phenotype. Here, we expand CRISPR/Cas9- and lentiviral-engineered TCR T and CAR T cells. We expanded TCR T cells with Ag-scaffolds presenting peptide MHC (pMHC), and anti-CD19 CAR T cells with Ag-scaffolds presenting CD19 antigen. By applying cognate pMHC Ag-scaffolds, we achieved >80% antigen-specific T cells (83.62%±9.2%) after 14 days of culture with a distinct cytotoxic, proliferative phenotypical profile. Ag-scaffold expansion enhanced initial TCR and CAR cytotoxicity; sustained control was observed after repeated rechallenges of CAR T cells. In vivo, Ag-scaffold-expanded CRISPR/Cas9-engineered anti-CD19 CAR T also showed complete tumour eradication in a B-cell lymphoma xenograft model with a low dose of CAR T cells, which was not achieved using IL2/7/15 expansion.

ORGANISM(S): Homo sapiens

PROVIDER: GSE314938 | GEO | 2026/09/25

REPOSITORIES: GEO

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Project description:Adoptive cell therapy (ACT) has shown promising results in cancer treatment, however, achieving effective ex vivo expansion of potent, functionally active, and cytotoxic T cells remains challenging. To address this challenge, we recently developed artificial antigen-presenting scaffolds (Ag-scaffolds) capable of expanding antigen-specific T cells with phenotypes favorable for ACT. In this study, we compared the established technology using IL2/IL21-loaded Ag-scaffolds (Ag-IL2/21) with Neoleukin-2/15-loaded Ag-scaffolds (Ag-Neo2/15). Neoleukin-2/15 (Neo2/15) is an engineered cytokine that selectively binds to IL-2Rβ/γ receptors, enhancing CD8+ T cell proliferation while limiting regulatory T cell expansion. Based on its favorable signaling properties, we hypothesized that incorporating Neo2/15 as part of the Ag-scaffolds would improve the quality and functionality of expanded T cells. We showed that Ag-Neo2/15 scaffolds promoted significant T-cell expansion, with frequencies of antigen-specific CD8+ T cells comparable to those achieved with Ag-IL2/21 scaffolds. The CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited potent TNFα and IFNγ, potentially enhancing their therapeutic potential. T cells expanded with Ag-Neo2/15 scaffolds had superior and durable cytotoxicity against tumor target cells compared to T cells expanded with Ag-IL2/21 scaffolds. These findings were further supported by our single-cell analysis revealing that T cells expanded with Ag-Neo2/15 scaffolds had higher cytotoxic scores and lower dysfunctionality scores compared to T cells expanded with Ag-IL2/21 scaffolds. The single-cell analysis also indicated increased expression of genes linked to cell division and enhanced proliferative capacity in Ag-Neo2/15 expanded T cells. Furthermore, TCR clonality analysis demonstrated that Ag-Neo2/15 scaffolds promoted the expansion of functionally superior T-cell clones, as the top clones of CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited a favorable phenotype, essential for effective antigen recognition and sustained T-cell mediated cytotoxicity. Our findings suggest that Ag-Neo2/15 scaffolds represent an advancement in ACT by producing high-quality, functional antigen-specific T cells. This method has the potential to improve clinical outcomes in cancer therapy by generating large numbers of highly functional T cells, thereby optimizing the balance between cytotoxicity and proliferation capacity with less exhausted T-cells in expansion protocols.

Project description:Engineered T cell therapies are limited by progressive dysfunction under chronic tumor antigen exposure, and genetic perturbation of negative regulators of T cell function has emerged as a promising strategy to sustain long-term effector activity. We reanalyzed a genome-wide CRISPR knockout screen in primary human T cells under repetitive tumor stimulation and conducted protein-protein interaction mapping that revealed seven members of the Cullin-RING Ligase 5 (CRL5) complex as a significantly enriched hit cluster. Knockout of any of these CRL5 complex members (CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3) enhanced tumor serial killing, and CUL5, the complex scaffold, emerged as the strongest target. CUL5 knockout (KO) TCR-T cells exhibited sustained cytotoxicity, elevated effector cytokine production, and a reduced percentage of exhaustion marker positive cells while retaining antigen specificity and cytokine dependence. We used quantitative ubiquitylation proteomics in resting and chronically stimulated T cells to map CRL5-dependent substrates and identified candidates beyond the canonical JAK/STAT axis including nutrient transporters, receptor trafficking proteins, and TCR signaling components. The functional benefit of CUL5 knockout extended across CARs targeting CD19, BCMA, and HER2 and containing different costimulatory domains, and to 1XX-modified CAR and HLA-independent TCR (HIT) architectures. Mechanistically, combining CUL5 ablation with a tumor-induced IL-2 synNotch circuit produced tumor control across nine rounds of repetitive stimulation, whereas either genetic modification alone eventually failed. In vivo, CUL5 KO BCMA CAR T cells were enriched in the bone marrow following repeated tumor rechallenge and exhibited enhanced ex vivo cytotoxicity after nearly 90 days in tumor-bearing mice. These findings position CUL5 perturbation as a strategy to improve T cell persistence and the efficacy of adoptive cell therapies, potentially in combination with engineered cytokine support.

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Antigen-scaffolds drive preferential expansion of functional genetically engineered CAR and TCR T cells

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