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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE329498 | GEO | 2026/05/01

REPOSITORIES: GEO

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Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.

Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion. 24 samples were analyzed in total. 6 biological replicates represented by cells from different donors. 4 different samples per donor: freshly isolated NK cells (d0) and NK cells expanded for 14 days under 3 different conditions: in co-culture with EBV-LCL feeder cells and 500 U/mL IL2 either cultivated manually in T75 flasks (T) or automatically using the CliniMACS Prodigy system (P); NK cells cultured with 500 U/ml IL2 without feeder cells (I)

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

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