In recent years, chimeric antigen receptor–modified T cell (CAR T cell) therapy has proven to be a promising approach against cancer. Nonetheless, this approach still faces multiple challenges in eliminating solid tumors, one of which being the immunosuppressive tumor microenvironment (TME). Here, we demonstrated that knocking out the endogenous TGF-β receptor II ( TGFBR2 ) in CAR T cells with CRISPR/Cas9 technology could reduce the induced Treg conversion and prevent the exhaustion of CAR T ce lls. Meanwhile, TGFBR2 -edited CAR T cells had better in vivo tumor elimination efficacy, both in cell line–derived xenograft and patient-derived xenograft solid tumor models, whether administered locally or systemically. In addition, the TGFBR2 -edited CAR T cells could eliminate contralaterally reinoculated xenografts in mice effectively, with an increased proportion of memory subsets within circulating CAR T cells of central memory and effector memory subsets. In conclusion, we greatly improved the in vitro and in vivo function of CAR T cells in TGF-β–rich tumor environments by knocking out endogenous TGFBR2 and propose a potentially new method to improve the efficacy of CAR T cell therapy for treating solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy targeting antigens shared with normal T cells requires genetic modifications to prevent fratricide. This phase 1 trial evaluates autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5⁺ hematologic malignancies. The overall response rate is 85.7%, including four complete responses. All patients experience cytokine release syndrome (six grade 1-2, one grade 3), and two patients develop immune effector cell-associated neurotoxicity syndrome. The most common grade ≥3 adverse events are cytopenia and infection, with unique observations of rash and autoimmune-related events. Post-infusion immunophenotyping shows persistent depletion of CD5⁺ T cells and CD19⁺ B cells, with reduced CD4/CD8 ratios. The human CD5 knockin murine model reveals skin lesions without significant vital organ involvement. These findings demonstrate CT125A's therapeutic potential in CD5⁺ malignancies while highlighting the need for safety optimization. The trial has been registered at ClinicalTrials.gov (NCT04767308).

Background: While chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment landscape for certain hematologic malignancies, therapeutic resistance and disease relapse highlight the critical need to improve the durability of clinical responses. The limited in vivo persistence and antitumor efficacy of CAR-T cells remain major barriers to achieving sustained therapeutic outcomes. Although CD5 has been extensively studied as a therapeutic target in cancers, particularly T-cell malignancies, its role as an immunomodulatory molecule in T cell-based immunotherapy remains poorly understood. Here, we developed a CD5-deficient T cell-based immunotherapy using the CRISPR-Cas9 system to address these limitations and enhance antitumor potency. Methods: Employing green fluorescent protein knock-in murine models alongside clinical specimens, we examined the in vivo persistence of CD5-deficient T cells and their influence on T-cell receptor (TCR) clonality diversity. The antitumor efficacy of CD5-deficient engineered T cells was assessed in tumor cell line-derived xenograft murine models. To elucidate underlying mechanisms, we performed a comprehensive evaluation of the activation, expansion and infiltration of CD5-deficient engineered T cells in response to antigen stimulation, as well as their exhaustion dynamics under conditions of repeated antigen exposure. Results: Our study identifies CD5 as a bona fide inhibitory immunomodulatory molecule. CD5 ablation significantly enhances T cell functionality by enhancing the activation level, mitigating exhaustion, promoting CD8 ⁺ T cell expansion, and improving in vivo persistence. Single-cell transcriptomic profiling of patient-derived CD5-deficient T cells revealed distinct effector subsets with elevated cytotoxicity markers and cell cycle regulators, such as STMN1, which correlate with enhanced expansion while preserving clonal diversity, as evidenced by TCR repertoire analysis. Conclusions: These collective findings establish CD5 ablation as a viable strategy to circumvent the intrinsic limitations of current T cell-based therapies, providing a mechanistic rationale for clinical translation.

Poly(ADP-ribose) polymerase inhibitors (PARPis) are a class of agents targeting DNA damage repair that have become standard therapy for epithelial ovarian cancer (EOC) and multiple other solid tumors. In addition to targeting DNA damage repair, PARPis actively modulate antitumor immune responses, with efficacy being partially dependent on T cell activity. Here, we found that patient T cells sustain DNA damage during PARPi treatment, which reduces treatment efficacy. Leveraging paired pre- and posttreatment tumor samples from a clinical trial of patients with EOC treated with neoadjuvant niraparib as monotherapy, we showed that the PARPi caused DNA damage, slowed proliferation, and increased apoptosis in T cells, which we validated both in vitro and in mouse models. A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) knockout screen in primary human T cells identified PARP1 as the principal mediator of PARPi-induced T cell death. T cell–specific deletion of PARP1 or mutating Parp1 at its binding sites in transgenic mice led to reduced T cell DNA damage during PARPi treatment, resulting in improved efficacy of PARPis, alone or in combination with immune checkpoint inhibition. We then engineered PARPi-tolerant CAR T cells using cytosine base editing, which decreased PARPi-induced PARP1 trapping and led to reduced PARPi-induced DNA damage, resulting in superior antitumor efficacy in xenograft models compared with parental CAR T cells. This study highlights the relevance of PARPi-induced DNA damage to T cells and suggests opportunities to improve the efficacy of PARPis as monotherapy or in combination with immunotherapy.

Background: The therapeutic application of chimeric antigen receptor (CAR) T cells in T-cell malignancies faces substantial limitations owing to fratricide and potential T cell aplasia, primarily attributed to the shared expression of target antigens, such as CD5, between normal and malignant T cells. Although natural killer (NK) cell-based immunotherapy is a promising alternative approach, its efficacy in treating hematologic malignancies remains to be fully elucidated. Methods: CD5-targeted CAR-modified primary NK cells, T cells and NK92 cell lines were generated and comprehensively evaluated for their anti-tumor efficacy through in vitro cytotoxicity assays and xenograft mouse models. Furthermore, preliminary investigation of the herpes simplex virus-1 thymidine kinase (HSV-TK) suicide switch system in CAR-NK cells were conducted using ganciclovir (GCV) as the activating agent. Results: CAR-NK cells exhibited significantly increased cytotoxic activity against CD5-positive cell lines and primary tumor cells, compared to NK, CAR-NK92, and CAR-T cells. Moreover, CAR-NK cells effectively decreased the leukemic burden and extended survival in murine model. Additionally, an off-switch utilizing the HSV-TK switch system successfully eradicated CAR-NK cells for safety considerations. Conclusions: This study developed a controllable CD5 CAR-NK cells that exhibit high efficacy against T-cell malignancies, although further validation is necessary to assess their clinical potential.