Adoptive transfer of tumor antigen–specific T cells using engineered T cell receptors (TCR-T cells) can access a broad set of cancer antigens, including neoantigens, for both hematologic malignancies and solid tumors. However, most tumor microenvironments provide little or no co-stimulatory signaling, which attenuates T cell responses. By contrast, clinically validated second-generation CAR-T cells incorporate intracellular co-stimulatory domains such as 41BB or CD28, which support persistence and function. The authors sought to build antigen-driven co-stimulation and T helper functionality into TCR-T cells to overcome these limitations.
Using CRISPR-Cas9, the team performed targeted integration of chimeric CD8 constructs that include intracellular co-stimulatory domains. The approach aimed to couple HLA-Class I recognition by TCRs with engineered co-stimulatory signals delivered through modified CD8 co-receptors, thereby activating costimulatory pathways only in antigen-engaged T cells.
The investigators evaluated expression of wild-type CD8 isoforms in HLA-Class I–restricted TCR-T cells. They found that expression of wild-type CD8αβ, but not CD8αα, could promote CD4+ T cell activities when the CD4+ cells were engineered to express HLA-Class I–restricted TCRs. Despite this gain of helper function, CD8αβ expression alone did not yield durable anti-tumor responses in stringent tumor mouse models when used with a high-affinity WT1-TCR.
To amplify co-stimulatory signaling beyond that provided by wild-type CD8αβ, several chimeric CD8 constructs were designed incorporating intracellular domains from CD28 or 41BB. These CD8 co-stimulatory fusion constructs were screened for the capacity to enhance TCR-T cell function. The screen identified two CD8-41BB chimeras that substantially increased TCR-T cell activity relative to wild-type CD8αβ.
WT1-directed TCR-T cells co-expressing the CD8-41BB fusion receptors showed multiple functional improvements. CD4+ T cell activity was enhanced, with strong and polarized Th1-type cytokine secretion. CD8+ cytotoxic T lymphocytes (CTLs) co-expressing the fusions exhibited increased proliferation, greater cytokine release, and enhanced cytotoxicity compared with counterparts expressing wild-type CD8αβ. These enhancements indicate that the engineered co-receptors potentiate both helper and effector arms of the engineered T cell response.
To further overcome suppressive tumor signals, the team combined CD8-41BB expression with disruption of the TGFBR2 gene. In mouse models using an established cell line–derived ovarian tumor, WT1-TCR-T cells co-expressing CD8-41BB and harboring TGFBR2 disruption achieved complete tumor regression. These cells demonstrated robust in vivo expansion and persistence and conferred long-term protection against tumor rechallenge. These in vivo results indicate that antigen-driven co-stimulation via CD8-41BB can be synergistic with ablation of TGF-β signaling to produce durable anti-tumor effects in this preclinical setting.
Importantly, expression of CD8-41BB did not alter the antigen specificity of the WT1-directed TCR. The WT1-TCR’s HLA-A*02:01 restriction and its WT1 peptide recognition motif were preserved after CD8-41BB engineering, indicating that the enhanced function did not come at the cost of target selectivity.
To simplify clinical translation and cell manufacturing, the investigators configured a homology-directed repair (HDR) cassette that enables insertion of both the TCR and CD8-41BB transgenes into the TRAC locus in a single CRISPR-Cas9 editing step. The authors report an insertion efficiency greater than 80% for this simultaneous knock-in approach, which could streamline production of engineered TCR-T cell products.
The platform’s applicability beyond WT1 was tested using a second clinically relevant TCR targeting PRAME. Enhanced activity conferred by CD8-41BB expression was validated with this PRAME-directed TCR, supporting the potential generalizability of CD8-41BB co-receptors to multiple TCR specificities.
This study demonstrates that engineering antigen-driven co-stimulation and T helper function into TCR-T cells via CD8-41BB fusion receptors can substantially enhance both CD4+ and CD8+ T cell activities, improve in vivo efficacy in a stringent tumor model when combined with TGFBR2 disruption, and be implemented via a single-step TRAC-directed HDR cassette with reported >80% efficiency. The authors note that many contributors are current or former employees of Intellia Therapeutics and that patent applications related to CD8 co-receptors have been filed by some authors. Additional disclosed relationships and funding are reported in the source.