Chimeric antigen receptor (CAR) T-cell therapy faces persistent obstacles in acute myeloid leukemia (AML), including on-target off-tumor toxicities, antigen heterogeneity, and immune evasion. The authors previously developed an Adapter CAR (AdCAR) platform designed for transient and combinatorial targeting to improve safety and efficacy in AML. This study aimed to identify immune checkpoint receptors (ICRs) and related immunomodulatory molecules that suppress AdCAR-T-cell function and to determine whether genetic disruption of these ICRs can enhance AdCAR-T cytotoxicity.
The investigators used multiparametric flow cytometry to profile expression of immune checkpoint ligands (ICLs) across several cancer entities, including three characterized AML cell lines, primary AML bone marrow specimens, and healthy bone marrow for reference. They also examined inducibility of ICLs on AML cells after engagement with AdCAR-T cells to capture dynamic ligand regulation following immune cell interaction. The study focused on several ICRs: PD-1, CD96, LAG-3, TIM-3, and TIGIT, plus the immunomodulatory molecule CD276.
To evaluate functional consequences of removing inhibitory receptors, the team generated AdCAR-T cells with CRISPR/Cas9-mediated knockouts (KOs) targeting PD-1, CD96, CD276, LAG-3, TIM-3, or TIGIT. These modified AdCAR-T cells were used in in vitro assays to compare cytotoxic potential against leukemia and lymphoma cell lines and to assess relative impacts of genetic disruption across the selected targets.
The cytotoxic activity of each KO AdCAR-T population was assayed in vitro against target cell lines. In parallel, the study compared genetic disruption with pharmacologic immune checkpoint inhibition (ICI) by applying PD-1 or TIGIT blockade reagents and measuring resulting cytotoxicity. The assays were intended to determine whether receptor loss via gene editing produced similar or different effects compared with antibody-mediated blockade.
In the experimental setting described, PD-1 gene disruption did not significantly enhance AdCAR-T cytotoxicity against the MOLM-13 AML cell line. By contrast, TIGIT knockout significantly increased AdCAR-T-cell cytotoxicity, an effect observed across five leukemia and lymphoma cell lines evaluated in the study. When comparing approaches, pharmacologic PD-1 blockade produced greater cytotoxicity than PD-1 gene disruption in this model, indicating that systemic blockade can differ functionally from receptor gene deletion. For TIGIT, antibody-mediated blockade yielded effects comparable to TIGIT KO, supporting TIGIT inhibition as a viable strategy to augment AdCAR-T function.
The findings support targeting TIGIT, either via genetic knockout in effector cells or pharmacologic blockade, as a promising approach to enhance AdCAR-T efficacy in hematologic malignancies. The authors propose that cell-intrinsic disruption of selected ICRs could improve therapeutic potency while potentially avoiding some systemic toxicities associated with systemic immune checkpoint inhibitor therapy. The results also suggest that checkpoint-specific differences exist: PD-1 may be more effectively modulated by systemic blockade than by gene deletion in the settings tested, whereas TIGIT responded robustly to both modalities.
The source material is an abstract and reports the principal experimental approach and comparative outcomes but does not provide detailed quantitative effect sizes, assay conditions, sample numbers, or statistical measures. Specific experimental parameters, such as timing, reagent identities for blockade, or off-target assessments of CRISPR editing, were not reported in the provided text. Broader in vivo validation, safety profiling, and clinical translation steps were not described in the abstract.
This screening study identified the TIGIT–CD155 axis as a significant modulator of AdCAR-T-cell function in vitro and highlighted TIGIT knockout as a potent enhancer of cytotoxicity against leukemia and lymphoma lines. The differential outcomes between PD-1 genetic disruption and pharmacologic blockade underscore that mode of ICR inhibition can affect AdCAR-T activity. The authors conclude that immune checkpoint receptor gene disruption—particularly targeting TIGIT—represents a promising strategy to boost AdCAR-T efficacy in AML, while noting that additional detailed data and further validation steps are required beyond the abstracted report.