EDITORIAL: Achilles's Heel of CAR T-Cell Therapy

Fabio Grizzi^1*Maurizio Chiriva-Internati²

• ¹Humanitas Research Hospital, Rozzano, Milan, Italy
• ²The University of Texas MD Anderson Cancer Center, Houston, United States

metasta3c renal cell carcinomas using autologous T cells engineered to express MOv-γ and scFv(G250) chimeric receptors, respec3vely. Although these treatments were well tolerated, they did not reduce tumor burden, likely due to the limited in vivo persistence of the infused CAR-T cells.T-cell ac3va3on requires two signals: an3gen recogni3on through the TCR-pep3de-MHC (pMHC) interac3on and co-s3mula3on via receptors such as CD28. To address this limita3on, Dr. Michel Sadelain's group at MSKCC developed a chimeric receptor incorpora3ng both CD3ζ and CD28 signaling domains, thereby providing dual ac3va3on signals that enhanced an3gen-dependent prolifera3on, IL-2 secre3on, and cytotoxic ac3vity in vitro (Mitra et al., 2023). Second-genera3on CD19 CAR-T cell therapy has since revolu3onized the treatment of B-cell malignancies. However, in chronic lymphocy3c leukemia (CLL), response rates and durability remain inferior to those observed in other indolent B-cell lymphomas. In 2024, Derigs et al. reported early clinical results from the HD-CAR-1 trial, evalua3ng academically manufactured third-genera3on CAR-Ts in pa3ents with relapsed/refractory CLL (Derigs et al., 2024). Third-genera3on an3-GD2 CAR-T cells (GD2-CART01) showed promising efficacy in children with high-risk metasta3c, relapsed, or refractory neuroblastomas in a phase 1/2 trial. The Locatelli et al. (Locatelli et al., 2025) final report results from 54 children, confirming that GD2-CART01 can induce durable remissions in this popula3on. This Research Topic aimed to tackle the key challenges in developing sustainable and cost-effective CAR-T cell therapies. Its main objectives were to identify promising strategies for reducing manufacturing costs, automate production processes to minimize human error, and enhance the therapeutic efficacy of CAR-T cells. The research also sought to explore how to lower CAR immunogenicity, improve infiltration into the tumor microenvironment, and prevent antigen escape. By addressing these critical areas, the work aimed to advance the field toward more effective, accessible, and safer cancer treatments.In their manuscript, Harer et al. (Harrer et al., 2023) discussed upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse. Specifically, CAR-T cells are engineered "living drugs" designed to recognize specific tumor an3gens and eliminate malignant cells through targeted immune ac3va3on. Despite their success in trea3ng B cell malignancies, many pa3ents experience relapse due to an3gen loss or T cell exhaus3on, which limits long-term efficacy. To overcome these challenges, current research focuses on developing next-genera3on CARs with enhanced an3gen sensi3vity, enabling the detec3on and elimina3on of cancer cells expressing low levels of target an3gens. Cancer cells can evade CAR-T cell therapy by reducing the expression of target an3gens, rendering them invisible to immune aqack and leading to tumor relapse. Although enhancing CAR-T cell sensi3vity to low an3gen levels could address this issue, it also raises significant safety concerns, as many tumor-associated an3gens are shared with healthy 3ssues, increasing the risk of on-target off-tumor toxicity. Consequently, ongoing research focuses on balancing efficacy and safety through strategies such as logic-gated CAR designs, coopera3ve targe3ng approaches, and the careful selec3on of tumor-selec3ve an3gens to minimize adverse effects while maximizing therapeu3c benefits. CAR-T cell therapy has made remarkable strides in trea3ng hematological malignancies. However, the widespread adop3on of CAR-T cell therapy is hindered by several challenges. Li et al. (Li et al., 2024) comprehensively examined the clinical challenges of CAR-T cell therapy and outlined strategies to overcome them, aiming to chart pathways beyond its current Achilles' heels. CAR T cell therapies have achieved remarkable success in trea3ng hematologic cancers, yet their broader use remains limited by high costs, long manufacturing 3mes, safety issues, and variable efficacy. Advancements in gene edi3ng technologies and delivery systems are essen3al to overcome these barriers and redefine the development of next-genera3on CAR-T cells. As these tools con3nue to evolve, they hold the promise of crea3ng safer, more potent, and more accessible CAR-T cell therapies, transforming them into rou3ne and affordable treatment op3ons for a wider range of pa3ents. Although CAR-T cell therapy remains one of the most innova3ve immunotherapeu3c approaches with remarkable clinical success, its broader applica3on is limited by lengthy manufacturing 3mes, high costs, and pa3ent-to-pa3ent variability (Chen et al., 2024). Despite notable advances in the development of universal CAR T (U-CAR T) cells, a stable and standardized cell bank has yet to be achieved. Mohammad et al. (Mohammad et al., 2024) systema3cally reviewed and evaluated the efficacy of modular (universal) CAR T-cell plasorms in xenograt mouse models. Across 33 studies encompassing 15 dis3nct plasorm designs, modular CAR-T cells were shown to significantly reduce tumor burden and improve survival compared to nega3ve controls, achieving outcomes comparable to conven3onal CAR-T cell therapies. Overall, these findings suggest that modular CAR T-cell plasorms are effec3ve and represent a promising, flexible, and controllable approach for next-genera3on cancer immunotherapy. Select pa3ents with relapsed/refractory aggressive B cell lymphoma may benefit from bridging radia3on (bRT) prior to an3-CD19-directed CAR-T. Manzar et al. (Manzar et al., 2025) evaluated 51 adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who received bridging radia3on therapy (bRT) prior to an3-CD19 CAR-T cell therapy. Just over half (51%) received comprehensive bRT to all disease sites, and 29% also received systemic therapy. The overall response rate at 30 days post-CAR-T was 82.4%.Median overall survival (OS) was 22.1 months, and median progression-free survival (PFS) was 7.4 months. One-year OS and PFS rates were 80% and 78%, respec3vely, while two-year rates were 59% and 54%. Comprehensive bRT was associated with improved OS and PFS (p ≤ 0.04). Poor outcomes were linked to ECOG performance status ≥2, advanced stage (III/IV), high IPI score (≥3), non-GCB histology, and radia3on doses ≤30 Gy. Relapse occurred in 51% of pa3ents, with 46% of relapses within the radia3on field, especially among those with bulky disease or poor early CAR-T response.Overall, the study concludes that bRT before CAR-T is an effec3ve and feasible strategy for selected pa3ents with aggressive B-cell lymphomas, and that comprehensive radia3on to all disease sites improves survival outcomes. CAR-T cell therapy has transformed cancer treatment, but key challenges remain, including an3gen loss and op3mizing CAR design as discussed by Gomez-Melero et al. (Gómez-Melero et al., 2025). To address these issues, mul3-targeted approaches, such as tandem CAR-T (TanCAR-T) cells, have been developed. These engineered cells can recognize mul3ple tumor an3gens simultaneously, reducing relapse risk and improving treatment efficacy. Preclinical and clinical studies in both hematologic cancers and solid tumors have shown that TanCAR-T therapies are effec3ve, safe, and associated with rela3vely low relapse rates. Despite these promising results, several challenges persist, such as determining the op3mal CAR construct, selec3ng the best an3gen targets, and improving transduc3on efficiency. Overall, tandem CAR-T cells represent a promising advancement in immunotherapy, with ongoing research needed to refine their design and maximize their clinical benefits.Li et al. (Li et al., 2025b) outlined the limita3ons of CD28-based CAR T-cell therapies, evaluated current strategies designed to op3mize CD28-based CAR constructs, and discussed future direc3ons and clinical prospects for enhancing their therapeu3c poten3al. CAR-T cell therapy, which engineers T cells to specifically target cancer cells, has achieved major advances in recent years. Current approved CAR-T products are second-genera3on designs that include co-s3mulatory domains, essen3al for T-cell ac3va3on and func3on. Among these, CD28-based co-s3mulatory molecules provide strong cytotoxic effects but are limited by high relapse rates, short-lived efficacy, and severe side effects. Recent research has focused on improving CD28 func3on by muta3ng its signaling mo3fs, combining co-s3mulatory domains, and op3mizing other CAR components to enhance an3tumor ac3vity and minimize toxicity. Yin et al. (Yin et al., 2025) have explored the use of sodium citrate to reduce exhaus3on and enhance the func3on of CAR-T cells. While CAR-T therapy is effec3ve against blood cancers, its success in solid tumors is limited by T cell exhaus3on, oten driven by tonic signaling and calcium ac3vity during cell expansion. They generated an3-CD70 and an3-mesothelin (MSLN) CAR-T cells and cultured them with sodium citrate. Results showed that citratetreated CAR-T cells had reduced exhaus3on, higher memory T cell levels, and improved an3-tumor efficacy, both in vitro and in vivo. Treated CAR-T cells also demonstrated beqer persistence and lower tumor recurrence. This study highlights the poten3al of sodium citrate to overcome a major limita3on of CAR-T cell therapy in solid tumors. Sodium citrate-pretreated CAR-T cells (CITR CAR-T) showed stronger persistence, greater an3-tumor efficacy, and preven3on of tumor recurrence in vivo compared to untreated cells. Mechanis3c studies revealed that sodium citrate suppresses CamkII phosphoryla3on, thereby inhibi3ng mTORC1 signaling and glycolysis, pathways linked to T cell exhaus3on. These effects collec3vely promote the forma3on of memory T cells and sustain CAR-T ac3vity. Although the experiments were performed in a cell-derived xenograt (CDX) model, which may not fully mimic human tumors, these findings suggest that sodium citrate could be a simple, safe, and cost-effec3ve strategy to improve CAR-T therapy for solid tumors. The study concludes that sodium citrate enhances CAR-T persistence and func3on by modula3ng calcium, mTOR, and metabolic signaling, offering a promising avenue for future clinical applica3ons. This study highlights an urgent need to standardize CAR-T cell func3onal potency assays and to iden3fy predic3ve biomarkers for treatment response, relapse, and toxicity. It also found inconsistent CAR-T cell monitoring during pa3ent follow-up. This first pan-European survey provides a snapshot of current CAR-T cell analy3cal prac3ces and emphasizes the importance of harmoniza3on across centers to improve the safety, efficacy, and accessibility of CAR-T cell therapies in Europe. The survey revealed widespread heterogeneity and lack of standardiza3on across all phases of CAR-T cell therapy in Europe, from apheresis collec3on to pa3ent monitoring. T2Evolve highlights the need to harmonize analy3cal methods, quality control assays, lymphodeple3on protocols, and immune-monitoring standards to ensure consistent product quality, pa3ent safety, and equitable access to CAR-T cell therapies across Europe. Currently, no standardized protocols exist for leukapheresis collec3on, quality assessment, or cryopreserva3on, and only a few centers rou3nely characterize leukapheresis products. About 66% of respondents emphasized standardiza3on of apheresis and cryopreserva3on, while iden3fying biomarkers predic3ng manufacturing success as a research priority. All centers use viral transduc3on, but over half (52%) called for standardized assays, including vector copy number, sterility, and flow cytometry tests.Rapid quality tes3ng was also seen as essen3al to shorten manufacturing 3me. Most centers use fludarabine and cyclophosphamide (Flu/Cy), though alterna3ves like Bendamus3ne are being explored. Lymphodeple3on regimens and monitoring prac3ces vary widely, with fewer than half performing addi3onal tests during toxici3es, underscoring the lack of consensus on predic3ve biomarkers such as IL-6, IFN-γ, and IL-1.Bolsée et al. (Bolsée et al., 2025) have inves3gated a dual-targe3ng CAR-T cell strategy to address an3gen escape, a major cause of relapse in B-cell malignancies such as acute lymphoblas3c leukemia (B-ALL). The authors designed tandem CAR-T cells that simultaneously recognize CD19 and NKG2D ligands (NKG2DL), stress-induced molecules commonly expressed on cancer cells but not on healthy 3ssues. Three tandem CAR constructs were developed, and two demonstrated strong an3-tumor ac3vity against both CD19 + and CD19 -cancer cells. Compared to conven3onal CD19 CAR-T cells, these tandem CARs maintained similar cytokine produc3on, cytotoxicity, and prolifera3on when engaging CD19 + targets, while retaining effec3veness against CD19 -cells. In experiments with primary B-ALL samples and xenograt models mimicking CD19 -relapse, the selected CD19/NKG2DL tandem CAR-T cells successfully controlled tumor growth and prevented relapse. This study provides proof-of-concept that NKG2D-based tandem CAR T-cells can overcome CD19 an3gen loss and enhance long-term therapeu3c efficacy in B-cell malignancies. The Authors conclude that CD19/NKG2DL tandem CAR-T cells provide a promising strategy to prevent an3gen escape, extend therapeu3c reach, and maintain an3-tumor efficacy in B-cell malignancies. Furthermore, the broad expression of NKG2DL suggests poten3al applicability in solid tumors and tumor-associated microenvironments, offering a versa3le plasorm for mul3specific CAR T-cell therapies. Muthuvel et al. (Muthuvel et al., 2025) describes a safer and efficient method for genera3ng an3-CD19 CAR-T cells using self-inac3va3ng (SIN) len3viral vectors for adop3ve immunotherapy. The CAR construct included a CD8α hinge, CD28 transmembrane and co-s3mulatory domains, and CD3ζ signaling, and T-cells were pre-ac3vated via CD3/CD28 beads before transduc3on. The resul3ng CAR-T cells achieved a transduc3on efficiency of approximately 28% at an MOI of 10 using high-3ter len3viral vectors (~9.85×10⁷ TU/ml). The T-cells expanded about 148-fold over 12 days in serum-free media, maintaining high viability (>87%) and exhibi3ng a predominantly CD8+ effector memory phenotype by days 7-12. Func3onally, the CAR-T cells demonstrated specific an3tumor ac3vity, lysing CD19 + NALM6 cells (~28% at a 1:1 ra3o) and producing robust an3gen-specific responses, including IFN-γ secre3on and CD107a degranula3on. Safety modifica3ons included removal of WPRE, GFP, and P2A sequences from the CAR construct. Overall, this study establishes a reproducible workflow for genera3ng func3onal, safe, and scalable an3-CD19 CAR-T cells, suitable for applica3ons in cancer and autoimmune diseases involving CD19 + B-lineage cells.CAR-based cell therapies have revolu3onized cancer treatment by enabling precise, an3gen-specific immune ac3va3on against malignant cells. Since the first FDA approvals of CD19-directed CAR-T cell therapies in 2017, adop3ve cell therapy has progressed from a conceptual innova3on to a transforma3ve clinical modality. CAR-T cell therapy u3lizes pa3ent-derived T cells gene3cally engineered to express synthe3c receptors that redirect specificity toward tumor-associated an3gens (Maude et al., 2014, June et al., 2018). This approach has shown cura3ve poten3al in hematologic malignancies; however, its efficacy in solid tumors remains limited by an3gen heterogeneity, immunosuppressive tumor microenvironments, and manufacturing complexity. Globally, over 1,000 clinical trials are inves3ga3ng strategies to broaden CAR therapy's reach, including the development of "off-the-shelf" universal plasorms. Recent advances in CAR-natural killer (CAR-NK) cells and induced pluripotent stem cell (iPSC)-derived CAR immune cells offer promising avenues to overcome current challenges in scalability, safety, and cost (Qian and Liu, 2025, Zhang et al., 2025, Schindler-Wnek et al., 2025, Kumar et al., 2025, Daher and Rezvani, 2025). CAR-NK cells enable allogeneic use with lower toxicity risk, while iPSC-derived immune cells facilitate the produc3on of homogeneous, programmable effector popula3ons at an industrial scale. Together, these innova3ons signal a paradigm shit toward universal, programmable, and ethically scalable CAR-based immunotherapies. Supported by CRISPR-mediated gene edi3ng and refined an3gen-targe3ng strategies, next-genera3on CAR plasorms are poised to expand the therapeu3c fron3er to both hematologic and solid malignancies. MCI: Wri3ng -review & edi3ng, Wri3ng -original drat, Supervision, Conceptualiza3on. FG: Wri3ng -review & edi3ng, Wri3ng -original drat, Supervision, Conceptualiza3on.