CD5 is a characteristic surface marker found on malignant T cells, present in approximately 80% of cases of T-ALL and T-cell lymphomas (32). The Maksim’s group engineered a CD5-targeting CAR-T construct that includes an extracellular anti-CD5 single-chain variable fragment (scFv) derived from clone H65, an IgG Fc transmembrane domain, and intracellular segments from CD28 and TCRζ chains. Notably, CAR expression led to the down-regulation of CD5 on CAR-T cells, effectively reducing fratricide and enhancing in vivo expansion. Co-culture assays with CD5-positive T-ALL cell lines such as Jurkat, CCRF, and MOLT4 showed that CD5 CAR-T cells eliminated over 95% of target cells within 48 hours, with no detectable target cells after 7 days, indicating a high specificity and cytotoxic capability towards malignant T cells (33).

Further development by Dai’s group introduced fully human CD5-targeting heavy-chain variable (FHVH) domains to create a biepitopic CAR, termed FHVH3/VH1, which includes FHVH1 and FHVH3 domains that bind to different epitopes of the CD5 antigen. Additionally, they employed CRISPR-Cas9 technology to produce CD5 knockout (CD5^KO) CAR-T cells, which reduces fratricide among the CD5 CAR-T population. This strategy not only enhanced the CD5 binding affinity of CAR-T cells but also significantly improved their therapeutic efficacy against CD5-expressing T-cell malignancies (34).

Moreover, integrating insights from the most recent advances, such as the development of a CH2CH3 hinge region that enhances the cytotoxicity of CD5 CAR-T cells (35) to optimize the efficacy and durability of CD5 CAR-T therapies. These enhancements not only bolster the CAR constructs’ direct anticancer activities but also refine their persistence in the hostile tumor microenvironment.

Overall, the evolving landscape of CD5 CAR-T cell research points to a promising future for the treatment of T-cell malignancies, with ongoing innovations aimed at improving efficacy, safety, and treatment accessibility (36).

CD7 is highly expressed on the surface of T-ALL cells, making it a potent target for T-ALL therapy. Although CD7 is also found on NK cells and normal T cells, research in CD7 knockout mice has shown normal immune development, indicating that targeting CD7 does not impairing the essential functions of T and NK cells (37–41).

Diogo’s group used CRISPR-Cas9 to knock out CD7 expression in CAR-T cells, developed second-generation CAR-T cells incorporating CD28 and CD3ζ as intracellular signaling domains. These CD7 CAR-T cells demonstrated high efficacy in eliminating various T-ALL cell lines (Jurkat, CCRF-CEM, MOLT-4, Hut 78, SupT1, and Raji), as well as primary T-ALL cells, and exhibited therapeutic effects in mouse models of T-ALL (42). Jiang’s group employed a novel technique using EF1α to specifically disrupt the CD7 site on CAR-T cells. This approach aims to prevent fratricide, enhancing both the specificity and safety of CAR-T cell therapies (43). Conversely, Peihua Lu’s group adopted a different approach by employing a natural selection method to mask or sequence CD7 molecules on the cell surface using a specialized scFv, developing a novel CD7-targeting CAR (NS7CAR) -T cell. This method, though less invasive than gene editing, might not completely eliminate the risk of fratricide as the masking could be incomplete under certain physiological conditions. In a phase I trial (NCT04572308), NS7CAR was administered to 20 patients with refractory/relapsed T-ALL (r/r T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). Nineteen patients achieved MRD negativity at Day28, attained complete remission, and became eligible for allo-HSCT (44). Additionally, Zhang’s group engineered CD7-targeted CAR-T cells that blocked CD7 expression by coupling CD7 nanoantibody VHH6 to an ER/Golgi-retention motif peptide. In a phase I clinical trial (NCT04004637), eight patients with r/r T-ALL received this treatment, achieving a CR rate of 87.5% at 3 months post-infusion, with detectable CAR-T cells persisting for up to 270 days (45).

Notably, Wong’s group introduced the (protein expression blocker) PEBL technology, which provides a groundbreaking solution to the problem of fratricide in CD7-targeted CAR-T therapy. By engineering a PEBL that retains CD7 within the Golgi apparatus, PEBL effectively prevents CD7 from being presented on the cell surface, thereby safeguarding CAR-T cells from fratricide. This approach not only maintains the efficacy of the CAR-T cells but also improves their survival and persistence in the patient’s body. The PEBL technology offers significant advantages over previous methods, including its non-invasive nature and the avoidance of potential gene editing complications. It represents a safer alternative that could potentially be applied to other CAR-T therapies targeting antigens expressed on the T cells themselves. A phase I is ongoing with these CAR T-cells in children and adults with relapse/refractory T-ALL in Singapore (CARTALL, NCT05043571). However, the scalability of this technology and its adaptability to other antigens require further investigation (46).

CCR9, a G protein-coupled receptor with a 7-transmembrane structure, is expressed on approximately 5% of normal circulating T and B cells (47). Recent studies have demonstrated a significant upregulation of CCR9 in r/r T-ALL cases, compared to its minimal expression on normal T cells. This differential expression profile marks CCR9 as a viable target for CAR-T cell therapy for T-ALL. Paul’s team knocked out CCR9 of T cells and developed CCR9-targeted CAR-T cells. These CAR-T cells demonstrated strong cytotoxicity against both T-ALL cell lines and primary T-ALL blasts in vitro, confirming their potent anti-leukemic properties. Further exploration into the functionality of these CCR9-targeted CAR-T cells revealed significant therapeutic efficacy in preclinical studies, including standardized mouse models with T-ALL cell lines and patient-derived xenograft (PDX) models representing high tumor burden in r/r T-ALL (48). These studies confirmed that targeting CCR9 can lead to substantial reduction of tumor burden without causing significant toxicity to the normal T cell population.

CD38, a cell surface glycoprotein part of the ribocylase family, is crucial in various cellular functions including cell migration, receptor-mediated adhesion, extracellular metabolism, and intracellular Ca2+ signaling. Its expression is significantly elevated in multiple myeloma (MM), NHL,T-ALL, and NK/T cell tumors, yet remains low in normal lymphocytes, bone marrow cells, and some non-hematopoietic tissues (49, 50). Importantly, CD38 is not present in multipotent hematopoietic stem cells, which are essential for bone marrow recovery, making it an attractive target for the treatment of various hematologic malignancies (51).

Wang’s group engineered T cells from PBMCs to express an anti-CD38 scFv fragment, controlled by an EF1α promoter and enhanced with co-stimulatory domains from 4-1BB and CD3ζ, developing a CD38 CAR-T. It demonstrated significant targeted killing capabilities against MM, B cell lymphoma, T-ALL, and NK/T cell lymphoma both in vitro and in vivo (52). Glisovic-Aplenc’s group further confirmed these findings, demonstrating the effectiveness of CD38 CAR-T in treating primary hematological malignant cells (53).

In 2023, Liao’s group introduced an innovative approach by integrating CD38-specific CARs into the CD38 gene locus itself, driven by different promoters. This novel approach led to the creation of both CAR-T and CAR-NK cells that exhibited robust anti-tumor activities. The use of native CD38 promoters for expression control potentially enhances the physiological relevance and tumor specificity of these CAR-modified cells, minimizing off-target effects and improving safety profiles (54).

CD1a is predominantly expressed on cortical T-ALL (coT-ALL) cells, but is absent on CD34-positive progenitor cells and mature T cells. This distinction provides a unique therapeutic advantage by minimizing off-target cytotoxic effects, significantly reducing the risk of damaging non-malignant hematopoietic cells (55–57).

Sanchez-Martinez’s group developed CD1a-targeting CAR-T cells, demonstrating strong and specific cytotoxicity when co-cultured with coT-ALL cell lines and primary lymphoid cells derived from coT-ALL patients. In NSG mouse xenograft models generated using Jurkat cells or T-ALL patient-derived tumor cells, it demonstrated significant anti-leukemic efficacy, with a marked reduction in tumor burden and prolonged survival in treated mice. These promising outcomes underscore the therapeutic potential of CD1a CAR-T cells and advocate for their further clinical development for this challenging leukemia subtype (58).

Expression of T-cell receptor beta chain constant domain1 (TRBC1) and T cell receptor beta chain constant domain 2 (TRBC2) are mutually exclusive, with TRBC1 T cells comprising 25% to 47% of the T-cell population in healthy individuals. Maciocia’s group has made significant advancements in immunotherapy by developing a CAR-T therapy targeting the TRBC1. They engineered CAR-T cells that selectively recognize and eliminate TRBC1-expressing cells, both normal and malignant, while preserving TRBC2 positive cells, maintaining essential immune functions. This selective targeting strategy is advantageous over nonselective methods that deplete the entire T-cell population, thereby avoiding severe immunosuppression (59). However, a challenge identified by Zhang’s group highlights the complexity of CAR-T cell production: the anti-TRBC1 CAR gene might unintentionally transfer to malignant T cells during manufacturing, leading to a self-binding scenario where the CAR interacts with TRBC1 on malignant cells, thus shielding them from cytotoxic action (60). This underscores the need for meticulous control measures to prevent cross-contamination and ensure the purity of therapeutic cells.

In the evolving landscape of CAR-T cell therapy, it is essential to fully explore these technologies in larger, more diverse patient populations. Addressing challenges related to manufacturing, regulatory approval, and cost-effectiveness is equally important. Investigating PEBL and similar technologies in clinical trials will provide valuable insights into their long-term safety and efficacy, potentially revolutionizing treatment for T-ALL and other T-cell malignancies. Continuous innovations, like the integration of CAR constructs into specific gene loci demonstrated by Liao’s team, offer more regulated expression and could reduce the risk of cytokine release syndrome and other side effects. This advancement points to a future where CAR therapies are finely tuned for maximum efficacy and minimal toxicity. Targeting strategies focused on CD3, CCR7, CD4, and CD8 molecules have shown initial efficacy, broadening the scope of therapeutic targets. However, despite these advancements, challenges such as non-targeted cell damage and fratricide highlight the need for ongoing optimization. These developments underscore the dynamic and promising future of CAR cell therapy, aiming for greater precision and effectiveness in treating T-ALL and other T-cell malignancies (61).

As previously mentioned, Liao’s group integrated CD38-specific CARs into the CD38 gene locus, creating CAR-NK cells with native CD38 promoters that demonstrated robust anti-tumor activities and enhanced specificity while minimizing off-target effects. To address CD38 expression on normal NK cells and prevent fratricide, they employed a “two-in-one” CRISPR/Cas9 strategy, giving CAR-NK cells anti-fratricide capabilities. This allowed efficient elimination of primary T-ALL cells, showing the significant therapeutic effects (54). Furthermore, Clara’s group utilized CRISPR/Cas9 technology to disrupt CD38 expression in ex vivo expanded NK cells and integrated high-affinity CD16 into the edited CD38 locus. The integration of CD16 is based on its role as a high-affinity Fc receptor, which allows NK cells to bind to the Fc region of antibodies attached to target cells. This binding facilitates antibody-dependent cellular cytotoxicity (ADCC), enhancing the NK cells’ ability to identify and destroy malignant cells. This innovative approach not only circumvented NK cell autologous attack but also significantly enhanced antitumor efficacy by improving targeting and elimination of leukemic cells (63).

Looking forward, the potential of CD38 CAR-NK cells, with their targeted integration and controlled expression facilitated by CRISPR/Cas9, offers a promising alternative to conventional CAR therapies. However, the real-world efficacy of these cells requires comprehensive clinical trials to assess their stability, persistence, and effectiveness across various tumor environments.

CD5 is recognized as a critical characteristic marker of T-ALL cells and is expressed on nearly all normal T cells, yet is absent on NK cells. This distinct expression profile enables the utilization of CD5 CAR-NK cell immunotherapy to bypass the fratricide effects often seen with CAR-T therapies. Xu’s group harnessed NK-92 cells as a platform for CD5-CAR modification, engineering anti-CD5 CAR constructs that incorporated different costimulatory domains: one with the T-cell-associated 4-1BB (BB.z) activating receptor and another with the NK cell-associated 2B4 (2B4.z) activating receptor. In vitro studies showed that both BB.z-NK and 2B4.z-NK cells exhibited targeted cytotoxicity against CD5-positive malignant cell lines, primary malignant cells, and normal T cells. In a T-ALL mouse xenograft model, both types of CAR-NK cells demonstrated significant anti-tumor effects, with 2B4.z-NK cells displaying enhanced anti-CD5 activity compared to BB.z-NK cells (64).

Further analysis by Voynova’s group compared the efficacy of CD3/CD5-directed CAR-NK and CAR-T therapies in T-ALL. Their findings highlighted that NK cells, when used as effector cells, effectively reduced the fratricide effect and addressed complications related to T cell aplasia and long-term immunodeficiency resulting from targeting molecules expressed on healthy T cells. CD3/CD5-directed CAR-NK cells do not cause long-term immunodeficiency due to their distinct biological characteristics and shorter lifespan compared to T cells. Unlike T cells, NK cells have a reduced risk of prolonged immune suppression and do not undergo extensive clonal expansion or persistence, minimizing their impact on long-term immune function. In contrast, CAR-T cells, being part of the adaptive immune system, can persist and expand over time, potentially leading to sustained depletion of target cells and subsequent immunodeficiency. Voynova’s group observed that although CD3 CAR-NK cells demonstrated robust efficacy against lymphoma cells in vitro, their performance in xenograft tumor models was compromised due to significant down-regulation of target antigens on malignant cells after the infusion of CD3 CAR-NK cells (65). In contrast, therapies directed towards CD5 present a potentially safer and more effective alternative. The addition of DAP10 co-stimulation imparts memory-like features to CAR-NK cells targeting CD5, enhancing their efficacy and persistence. This design significantly bolsters the therapeutic potential of CD5 CAR-NK cells as a treatment for T-ALL (66, 67).

Zhu’s group developed CD7 CAR-NK-92MI cells, which exhibited significant cytotoxicity against CD7-positive hematological malignancies, including T-ALL, AML, and T-cell lymphoma, compared to their non-modified counterparts. These modified NK cells exhibited increased secretion of cytokines (e.g., IL-2, IFN-γ, granzyme B) play a critical role in mediating immune responses and enhancing cytotoxic activities (68). Jiang’s group developed a CD7-targeting CAR-NK by integrating the CAR directly into the CD7 gene locus of NK cells, using EF1α as the intracellular activation segment. This approach prevents the elimination of NK cells caused by their own CD7 expression, effectively addressing issues related to cell fratricide. These CD7-specific CAR-NK cells exhibit promising anti-tumor activity with reduced risk of off-target effects and enhanced tumor specificity (43).