New Progress in Dual-Target CAR-T Cell Therapy

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy, accounting for about 15% of childhood acute lymphoblastic leukemia (ALL) and 25% of adult ALL. Although the cure rate for pediatric patients can reach 80%, the long-term survival rate of adult patients is still less than 40%. More worryingly, more than half of patients will relapse after treatment or fail to respond to standard treatment. The median overall survival of relapsed/refractory T-ALL is only about 8 months. Current treatment methods mainly rely on high-intensity chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT), but they are highly toxic and have a high failure rate. There is an urgent need for safer and more effective targeted treatment strategies in clinical practice.

Compared with B-cell malignancies, immunotherapy for T-ALL has progressed slowly. Because malignant T cells and healthy T cells are highly similar in surface antigen expression, traditional CAR-T cell therapy faces fatal problems such as "fratricide" and T cell aplasia. In recent years, researchers have tried to knock out pan-T antigens such as CD7 through gene editing technology. Although some progress has been made, the technology is complex, the supervision is difficult, and alloHSCT is still needed for salvage treatment. Therefore, finding safe, specific, non-pan T antigen targets has become a key breakthrough in T-ALL immunotherapy.

Recently, in a research report entitled "CAR-T cells targeting CCR9 and CD1a for the treatment of T cell acute lymphoblastic leukemia" published in the international journal Journal of Hematology & Oncology, scientists from the Josep Carreras Leukemia Institute and other institutions in Spain focused on two non-pan T antigens-CCR9 and CD1a. At the same time, a dual-target CAR-T cell therapy strategy was developed and verified to overcome the problems of antigen escape, manufacturing difficulties and immunotoxicity in T-ALL treatment. Related research may have important clinical translation value.

In the article, the researchers first performed immunophenotyping on 180 T-ALL patient samples. The results showed that CCR9 was highly expressed in 73% of patients, especially in relapsed samples, with an expression rate of up to 92%. More importantly, CCR9 is almost not expressed in normal T cells, hematopoietic stem cells and other tissues, and is only weakly expressed in some thymocytes and intestinal lymphocytes, with good safety. CD1a is mainly expressed in cortical T-ALL, accounting for about 30% of all T-ALL. The combination of the two can cover 86% of patients and significantly expand the treatment population.

In the experimental design, the researchers used hybridoma technology to screen high-affinity CCR9 monoclonal antibodies and constructed mouse and humanized single-chain antibodies (scFv). It was further embedded in the second-generation CAR structure to construct three CCR9-CAR-T cells (mouse M, humanized H1 and H2). In vitro cytotoxicity experiments revealed that H2-type CAR-T cells showed the strongest activity and specificity in killing CCR9-positive T-ALL cell lines (such as MOLT4) and patient-derived xenograft models (PDX), without obvious fratricide.

Figure 1. CCR9 is a safe and specific target for T-ALL. (Tirado N, et al., 2025)

To further verify its in vivo efficacy, the researchers established a T-ALL PDX model in NSG mice, injected CCR9-positive leukemia cells and treated with different CAR-T cells. The results showed that CCR9 H2 CAR-T cells could significantly inhibit tumor progression, and 80% of mice achieved complete remission, which was better than other groups. On this basis, the researchers further developed dual-target CAR-T cells that can target both CCR9 and CD1a. Through the construction and comparison of three strategies —co-transduction, tandem CAR, and bicistronic CAR —it was ultimately found that the co-transduction strategy (i.e., transduction of CCR9 and CD1a CAR separately) performed best in terms of killing efficiency and antigen coverage. The experimental results showed that dual-target CAR-T cells can effectively eliminate leukemia cells expressing any antigen and avoid antigen escape caused by single-target treatment.

In addition, the researchers also simulated the common antigen heterogeneity in T-ALL, constructed cell lines with different CCR9 and CD1a expression levels through CRISPR/Cas9 technology and mixed them into heterogeneous populations. The results showed that dual-target CAR-T cells can still efficiently eliminate all subpopulations, while single-target CAR-T cells showed obvious antigen escape. Finally, the researchers successfully prepared CCR9/CD1a dual-target CAR-T cells from T-ALL patient samples and verified their strong killing ability against autologous leukemia cells in vitro, further proving the clinical feasibility of this strategy.

In summary, this study systematically verified the safety and effectiveness of CCR9 as a T-ALL treatment target for the first time, and innovatively proposed a CCR9/CD1a dual-target CAR-T cell treatment strategy. The three core problems faced in T-ALL immunotherapy have been successfully solved: antigen escape, manufacturing complexity and immunotoxicity. The high expression rate and good safety of CCR9 make it an ideal CAR-T target, and the combined use of CD1a further expands the treatment coverage. The dual-target strategy can not only improve the treatment efficiency of patients, but also effectively prevent the risk of recurrence caused by antigen downregulation. In addition, this strategy avoids the regulatory and safety issues brought about by gene editing and simplifies the manufacturing process, which is expected to enable widespread application of autologous CAR-T cells.

Reference

Tirado N, et al. CAR-T cells targeting CCR9 and CD1a for the treatment of T cell acute lymphoblastic leukemia. Journal of Hematology & Oncology, 2025, 18(1): 69.