Miller, I. C. | bioRxiv Science

bioRxiv2026

Engineering antigen-driven co-stimulation and T helper cell activity into TCR-T cells with CD8-41BB fusion receptors enhances anti-tumor activity

1Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8{beta}, but not CD8, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8{beta}. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

synthetic biology↗

bioRxiv2020

Remote control of CAR T cell therapies by thermal targeting

The limited ability to control anti-tumor activity within tumor sites contributes to poor CAR T cell responses against solid malignancies. Systemic delivery of biologic drugs such as cytokines can augment CAR T cell activity despite off-target toxicity in healthy tissues that narrows their therapeutic window. Here we develop a platform for remote control of CAR T therapies by thermal targeting. To enable CAR T cells to respond to heat, we construct synthetic thermal gene switches that trigger expression of transgenes in response to mild elevations in local temperature (40-42 {degrees}C) but not to orthogonal cellular stresses such as hypoxia. We show that short pulses of heat (15-30 min) lead to more than 60-fold increases in gene expression without affecting key T cell functions including proliferation, migration, and cytotoxicity. We demonstrate thermal control of broad classes of immunostimulatory agents including CARs, Bispecific T cell Engagers (BiTEs), and cytokine superagonists to enhance proliferation and cell targeting. In mouse models of adoptive transfer, photothermal targeting of intratumoral CAR T cells to control the production of an IL-15 superagonist significantly enhances anti-tumor activity and overall survival. We envision that thermal targeting could improve the safety and efficacy of next-generation therapies by allowing remote control of CAR T cell activity.

bioengineering↗