Tvingsholm, S. | bioRxiv Science

bioRxiv2025

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

The engineering of autologous T cells to express 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 in clinical trials for solid tumours. However, clinical manufacturing of engineered T cells is resource-intensive and often yields highly differentiated, exhausted effector T cells. 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. 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.

immunology↗

bioRxiv2025

Antigen-scaffolds loaded with hyper-stable Neoleukin-2/15 expand antigen-specific T cells with a favorable phenotype for adoptive cell therapy

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 overcome this, we loaded the engineered cytokine Neoleukin-2/15 (Neo2/15) on our recently established artificial antigen-presenting scaffolds (Ag-scaffolds) to expand antigen-specific T cells. Neo2/15 selectively binds to IL-2R{beta}/{gamma} receptors, enhancing CD8+ T cell proliferation while limiting regulatory T cell expansion. Our study assessed the efficacy of Neo2/15-loaded Ag-scaffolds (Ag-Neo2/15 scaffolds) in expanding antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors. We optimized Ag-scaffold configurations by varying the number of Neo2/15 molecules loaded on Ag-scaffolds and evaluated their impact on T-cell expansion and functionality. We showed that Ag-Neo2/15 scaffolds promoted significant T-cell expansion, with a comparable frequency of antigen-specific CD8+ T cells compared to IL-2/IL-21-loaded Ag-scaffolds (Ag-IL2/21 scaffolds). The CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited potent TNF and IFN{gamma} production and expressed high levels of 4{beta}7 integrin, a homing molecule which is important for directing T cells to specific tissues, 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. 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.

immunology↗