Open research questions in CAR-T cell therapy research

What the literature leaves open

• Driven by continuous breakthroughs, gene editing technology, exemplified by CRIPSR/Cas, is rapidly developing toward higher precision, lower off-target effects, larger-fragment targeted integra- tion, and safer in vivo delivery. Progressing from basic tool optimization to clinical translation and cross-disciplinary integra- tion, it has already been widely applied in diverse fields, such as clinical therapeutics, agricultural breeding, and ecological gover- nance, and offers substantial potential. Meanwhile, as an innovative cellular immunotherapy, CAR-T cell therapy has achieved remark- able breakthroughs in the treatment of hematological malignancies, providing renewed hope for patients with relapsed or refractory cancer. While CAR-T cell therapy faces multiple challenges in solid tumors, it also holds great promise beyond cancer, including for autoimmune, cardiac, and senescence-associated diseases (176). Optimizing this therapy through gene editing to address the limitations in solid tumors and expand the scope of application thus represents a future direction with tremendous potential. Several promising trends are noteworthy: (1) Comprehensive anal- ysis of complex regulatory systems: Systematic analysis of tran- scriptional regulation, epigenetic modification, metabolic fitness, and protein-protein interactions will uncover new targets, thereby offering fundamental guidance for designing and optimizing CAR- T cell therapy. (2) Development of diverse gene editing technolo- gies: Gene editing technology is evolving from single-site correction to multiple types of gene modification continuously, enabling versatile rewiring of CAR-T cells. Combined with multiplex gene editing and synergistic therapeutic strategies, CAR-T cell therapy is developing toward greater efficacy and safety. (3) Development of predictive models: The establishment of individualized predictive models is emerging as an important direction. By integrating patients’ clinical characteristics and cellular parameters, personal- ized treatment strategies can be precisely formulated, significantly improving therapeutic efficacy and safety. In the future, gene editing technology will continue to progress toward higher efficiency and greater precision, thereby overcoming current bottlenecks and further expanding the applications of CAR-T cell therapy, provid- ing revolutionary breakthroughs in treating a broader spectrum of diseases.

  Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy · 2026 · DOI

• While strictly tumor-specific antigens are ideal, they are scarce, and clinically relevant targets like B7-H3 often exhibit basal expression in normal organs.

  Structure-Guided Computational Engineering of pH-Selective B7-H3 CAR-T Cells to Mitigate On-Target/Off-Tumor Toxicity 2328499 · 2026 · DOI

• Abstract Introduction Chimeric antigen receptor (CAR) T cell therapy in solid tumors is limited by the immunosuppressive tumor microenvironment (TME).

  T-bet CAR T cells: A new avenue for NSCLC therapy analyzed in lung cancer-derived organoids 2258880 · 2026 · DOI

• T cell-directed immunotherapy has transformed hematological oncology, but the next gains will depend on measuring and preserving the immune substrate as carefully as the tumor target. Relapse, limited persistence, immune-reconstitution failure and infection reflect both tumor biology and the state of the recruited or engineered T cells (165–167). A central message of this Review is that T cell dysfunction in hematological cancer should not be reduced to exhaustion alone. Exhaustion, senescence and terminal differentiation are biologically related but mechanistically distinct (16). Exhaustion is largely driven by chronic antigen stimulation and stabilized by transcriptional and epigenetic programmes involving factors such as TOX and NR4A (168). Senescence reflects ageing, replicative history, telomere attrition, DNA damage responses and loss of proliferative capacity.

  Beyond exhaustion: T cell fitness for next generation of immunotherapy for hematological cancer · 2026 · DOI

• This article outlines the biological mechanisms underlying CD8+ T-cell-mediated tumor killing and explores the major limitations faced by T cells in the treatment of solid tumors, including a limited number of tumor-specific antigens, poor tumor infiltration capacity, an immunosuppressive microenvironment, metabolic stress, and T-cell exhaustion.

  T-Cell-Based Immunotherapy for Solid Tumors: Challenges, Mechanisms, and Combination Strategies · 2026 · DOI

• Although gamma-secretase inhibitors (GSI) significantly increase cell surface BCMA density and decrease soluble BCMA (sBCMA), their effects on BCMA trogocytosis and the resulting impact on CAR T-cell function remain incompletely understood.

  Enhanced BCMA Antigen Density Increases Trogocytosis and Attenuates CAR T cell Function · 2026 · DOI

• Although numerous relevant studies have been published, no standardized multi-database bibliometric analysis has illustrated the overall development trajectory, cooperation patterns and research theme evolution of this field from 2006 to 2025.

  T cell-mediated immune function in gastric cancer: a bibliometric overview of the last 20 years (2006-2025) · 2026 · DOI

• As highlighted in the previous section, significant gaps in the current literature continue to limit our understanding of CAR T cell–related CV toxicity and its optimal management. First, available data on risk factors are limited and primarily derived from small, retrospective cohorts treated with heterogeneous CAR T cell products and across various malignancies. These factors hamper the clinical applicability of findings and precludes the identification of therapy- specific cardiotoxicity risk profiles. Future research should prioritize large, prospective cohorts of patients treated with a single CAR T cell product to enable robust identification and external validation of risk factors. These efforts would form the basis for the development of a structured, CAR T cell–specific risk stratification tool. Second, the optimal approach to cardiovascular monitoring in patients undergoing CAR T cell therapy—especially over the long term—remains poorly defined. Given the distinct inflammatory pathophysiology—most notably characterized by CRS—the immediate post-infusion period appears to be the most critical window for surveillance. In this context, the potential role of serum biomarkers in detecting early cardiotoxicity remains to be clarified. Randomized controlled trials comparing different monitoring strategies are urgently needed to determine whether deviations from 16 ARTICLE IN PRESS ARTICLE IN PRESS standard cardio-oncology surveillance principles are justified in this unique clinical setting. If proven effective, such deviations could lay the foundation for dedicated CAR T cell–specific surveillance protocols, tailored to the timing and mechanisms of toxicity observed with these therapies. Third, current therapeutic approaches to CAR T cell–associated cardiotoxicity are largely extrapolated from conventional management strategies for HF and arrhythmias, despite the distinct clinical features of this population. The use of guideline-directed medical therapy for HF may be limited by hemodynamic instability, particularly hypotension. Similarly, AF in this setting is often inflammation-driven and self-limiting, raising questions about the long-term utility of antiarrhythmic agents and oral anticoagulation. Randomized trials are needed to define evidence- based treatment algorithms that reflect the specific needs and risks of this population. Finally, the risk of late-onset cardiotoxicity and the long-term prognostic significance of early cardiac events remain poorly characterized. Longitudinal follow-up studies are essential to address these knowledge gaps and to inform comprehensive, evidence-based strategies for long-term CV surveillance and management in CAR T cell recipients.

  Chimeric antigen receptor T cell therapy cardiotoxicity: a narrative review · 2026 · DOI

• DNA methylation is a major driver of T cell exhaustion and inhibition of de novo methylation can block exhaustion and restore T cell function in chronic viral infections and other cancers but is understudied in AML.

  Azacytidine restores T cell function in AML by modulating DNA methylation · 2026 · DOI

• 9510 Background: Although acute toxicities associated with adoptive cellular therapy using lifileucel are well described and largely occur within the first 2 weeks of treatment, adverse events beyond this early window remain incompletely characterized.

  Real-world safety of adoptive cellular therapy with lifileucel in patients with advanced melanoma. · 2026 · DOI

• A synthetic immune niche (SIN) composed of immobilized CCL21 and ICAM1 was shown to improve T cell expansion while preserving cytotoxicity; however, it remains unclear which specific step of the T cell killing process is enhanced by the SIN stimulation.

  Decoupling T Cell Cytotoxicity: A CCL21+ICAM1-Based Synthetic Immune Niche Enhances Tumor Elimination by Accelerating Lytic Hit Delivery · 2026 · DOI

• However, its access to a broader population has been limited by the unavailability of an off-the-shelf product derived from an allogeneic donor that can evade immune rejection, which is mediated by polymorphic class I and class II human leukocyte antigens (HLAs).

  HLA-G engineering reprograms CAR-T cells with an immune privilege · 2026 · DOI

• CRISPR screens have mapped the core circuitry by which T cells achieve anti-tumor activity, repeatedly converging on JAK/STAT cytokine signaling, proximal and distal TCR/NF-kB pathways including RNA stability programs, and transcriptional/epigenetic checkpoints that shape differentiation and persistence. Looking forward, a stage-aligned, context-aware-design strategy as presented herein provides a practical approach for the selection of targets and pathways to optimally enhance T cell therapies. As dual editing of identified target combinations can integrate complementary bene- fits into T cell therapies, combinatorial editing will be an important tool in this approach. These insights provide a rational foundation for engineering adoptive cell therapies.

  CRISPR-screen informed engineered T cell therapies · 2026 · DOI

• CAR-T cell therapy has demonstrated excellent efficacy in treating hematologic malignancies, and CAR-T-based personalized immunotherapy has led to notable remissions. However, this mode of immunotherapy is still in its early stages, and specific challenges persist. For example, the CAR-T-associated CRS and neurotoxicity are significant side effects. Additionally, noteworthy factors include the limited persistence of CAR-T cells, their failure to home to tumor sites, inhibitory factors within the TME, exhaustion induced by metabolic stress, and inhibitory signals. Addressing these barriers will move the field one step closer to translating CAR-T cell therapy into a durable and widely applicable cancer treatment. Recent advances reviewed here highlight a shift toward rational, multi-layered engineering strategies. Self-regulating CAR designs and switch-on/switch-off CAR systems provide critical safety controls to mitigate CRS and neurotoxicity.

  Advanced strategies to enhance the safety, persistence, and efficacy of CAR-T cells in solid tumors · 2026 · DOI

• Cell-based immunotherapies have emerged as a central component of contemporary oncology by enabling therapeutic mechanisms that are difficult to achieve with conventional modalities, functional adaptability within including in vivo expansion, dynamic tumor ecosystems, and the potential for durable immune surveillance. The clinical success of CAR T-cell therapy in hematologic malignancies has validated this paradigm and catalyzed the development of a broader spectrum of cellular tumorplatforms, infiltrating lymphocytes, NK cell–based therapies, dendritic cell vaccines, and macrophage-directed strategies [121–123]. Collectively, these approaches expand the scope of precision immunooncology by engaging complementary immune functions, including antigen-specific cytotoxicity, innate effector activity, antigen presentation and repertoire shaping, and active modulation of the tumor microenvironment. encompassing TCR–engineered T Despite this progress, the principal limitations of the field are now well defined. Antigen escape and heterogeneous target expression continue to compromise response durability, particularly for single-antigen strategies. In solid tumors, immune exclusion, suppressive myeloid programs, metabolic constraints, and physical barriers collectively limit cellular trafficking, persistence, and effector function, even in the presence of tumor- In parallel, manufacturing remains reactive immune cells. both a biological and logistical bottleneck; variability in starting material, prolonged vein-to-vein timelines, and high production costs restrict scalability and broad clinical access [125, 126]. These challenges indicate that future advances will depend less on incremental refinement of individual platforms and more on integrated strategies that simultaneously address target recognition, cellular fitness, tumor accessibility, and safety. Several development priorities are likely to shape the next phase of cellular immunotherapy. Multiplex targeting and logic-gated designs provide promising solutions to antigen escape while improving on-target specificity. Cell-state engineering—supported by optimized manufacturing conditions, epigenetic and metabolic conditioning, and precise genome editing—offers a pathway to enhanced persistence and resistance to exhaustion [128, 129]. Off-the-shelf approaches, including allogeneic donor–derived products and iPSC–based platforms, have the potential to reduce production timelines and improve scalability, provided that immune rejection and product consistency can be effectively controlled [53, 130, 131]. rational combination strategies are increasingly In addition, essential; likely require coordinated interventions integrate immune improved trafficking, and tumor microenvironment priming, modulation while maintaining an acceptable therapeutic index.

  Cell-based cancer immunotherapy: milestones, mechanistic insights, and emerging therapeutic directions · 2026 · DOI

• DC vaccines remain exceptionally safe and highly customizable; however, their clinical efficacy is constrained by several well- defined limitations, including variable cross-presentation effi- ciency, limited functional persistence within immunosuppressive environments, suboptimal migration and lymphoid homing, and persistent immune exclusion in solid tumors. Accordingly, the most impactful advances are likely to arise from integrative, mechanism-driven strategies rather than incremental refinement of conventional maturation protocols. Mechanistically grounded approaches—such as ER–directed antigen routing to enhance cross-presentation and endothelial signaling programs that promote vascular normalization—suggest that DC vaccination should be conceptualized as a coordinated system with explicit control over both antigen processing and In parallel, the development of biomarker immune-cell access. frameworks capable of distinguishing “priming-limited” from “access-limited” tumors represents a critical priority for guiding the rational selection of adjuvants, delivery routes, and treatment scheduling. If these principles can be operationalized alongside scalable manufacturing platforms and standardized immune-monitoring strategies, DC vaccines may evolve from safe but modest monotherapies into durable priming backbones that reliably establish tumor-specific T-cell repertoires and facilitate their effective delivery into otherwise immune-excluded tumors. More- over, integration of DC vaccination with T cell–based therapies or immune checkpoint inhibitors is expected to further enhance the tumor antigen–specific T-cell magnitude and durability of responses, thereby maximizing overall antitumor efficacy. Acta Pharmacologica Sinica (2026) 0:1 – 18 NK CELL THERAPIES IN CANCER IMMUNOTHERAPY NK cells have emerged as a complementary pillar of cellular immunotherapy, distinguished by rapid cytotoxic activity, innate tumor recognition, and a clinical deployment paradigm that is often more “drug-like” than that of autologous T cell–based therapies. In contrast to T cells, NK cells can be administered in an allogeneic setting with minimal risk of graft-versus-host disease (GVHD). Moreover, clinical experience across multiple platforms consistently demonstrates a lower incidence of severe CRS and ICANS compared with CAR T-cell therapies [93]. These attributes position NK cells as attractive candidates for off-the-shelf manufacturing, repeat dosing, and rational combination regimens.

  Cell-based cancer immunotherapy: milestones, mechanistic insights, and emerging therapeutic directions · 2026 · DOI

• This was a prospective study with serial collection of PROs using psychometrically robust measures. The inclusion of equal propor- the first tions of comparison of PROs between FDA-approved CAR-T treatments.

  Patient-reported outcomes after idecabtagene vicleucel vs. ciltacabtagene autoleucel CAR-T for multiple myeloma · 2026 · DOI

• The manufacturing workflow for iPSC-derived Tregs is still in its early stages and lacks detailed information on achieving stable and efficient differentiation into CD4+ or CD8+ Tregs.

  Regulatory T cell therapies: biological foundations, engineering strategies, and clinical translation · 2026 · DOI

• The EIGHT Treg study lacks a control group for conventional induction immunosuppression, making it difficult to assess the efficacy of CD8+ Tregs in preventing renal graft rejection.

  Regulatory T cell therapies: biological foundations, engineering strategies, and clinical translation · 2026 · DOI

• The study measured serum ALT and AST activities to quantify hepatotoxicity, but did not report comprehensive histopathological scoring, immune cell infiltration patterns, or mechanistic assessment of hepatocyte damage pathways in response to αCD137 treatment with or without LTβR-Fc blockade. Detailed liver tissue analysis would clarify whether LTβR blockade prevents hepatocyte injury, reduces pathogenic T cell infiltration, or modulates both processes.

  Selective elimination of circulating effector CD8 T cells via LTβR blockade separates anti-CD137 efficacy from toxicity · 2026 · DOI

• The tumor models used MC38 murine colorectal carcinoma cells and MC38-OVA cells, but validation of the LTβR blockade strategy across additional syngeneic tumor types (e.g., melanoma, lymphoma) or human-derived xenografts remains absent. Testing whether selective elimination of circulating effector CD8 T cells via LTβR-Fc generalizes beyond colorectal carcinoma models is necessary for broader therapeutic applicability.

  Selective elimination of circulating effector CD8 T cells via LTβR blockade separates anti-CD137 efficacy from toxicity · 2026 · DOI

• Adoptive cell transfer experiments transferred either total splenocytes or purified CD8 T cells, but the paper does not report whether effector CD8 T cell subsets (e.g., CD62L−CCR7− circulating effector versus tissue-resident memory cells) were differentially transferred to assess their specific contributions to anti-CD137 toxicity versus tumor control.

  Selective elimination of circulating effector CD8 T cells via LTβR blockade separates anti-CD137 efficacy from toxicity · 2026 · DOI

• The bone marrow chimera experiments used only LTβR−/− or LTβRf/f donor cells to examine the contribution of LTβR signaling on hematopoietic versus stromal cells in anti-CD137-induced hepatotoxicity. However, the paper does not specify whether LTβR expression was systematically ablated in specific immune subsets (e.g., B cells, dendritic cells, T cells) to identify which hematopoietic populations drive the observed toxicity reduction.

  Selective elimination of circulating effector CD8 T cells via LTβR blockade separates anti-CD137 efficacy from toxicity · 2026 · DOI

• The bulk RNA sequencing analysis characterized intrahepatic CD11c+TSLE and CD11c−TSLE cells in response to αCD137 antibody treatment, but the study only sorted these two cell populations from liver tissue. A more comprehensive single-cell RNA sequencing approach across multiple immune cell subsets and tissue compartments would be needed to fully map the transcriptomic changes underlying LTβR blockade-mediated separation of anti-CD137 efficacy from hepatotoxicity.

  Selective elimination of circulating effector CD8 T cells via LTβR blockade separates anti-CD137 efficacy from toxicity · 2026 · DOI

• Despite transformative advances in adult oncology and pedi- atric hematologic malignancies, immunotherapy for pedi- atric solid tumors faces distinct biological challenges that demand tailored solutions. Two fundamental barriers define this therapeutic landscape: exceptionally low TMB with sparse neoantigen generation, and profoundly immunosup- pressive TME dominated by myeloid suppressors, regulatory T cells, and stromal barriers that exclude effector cells and blunt therapeutic responses. The field has nonetheless achieved meaningful progress across multiple platforms. Anti-GD2 monoclonal antibodies have established a validated paradigm in high-risk neuro- blastoma, demonstrating that antibody-mediated cytotoxicity can improve survival when integrated into multimodal regi- mens. Next-generation antibody platforms, including B7-H3 and GD2-targeted ADCs, and bispecific T cell engagers, are 146 Page 16 of 20 Cancer Immunology, Immunotherapy (2026) 75:146 translating preclinical promise into early clinical trials, with engineered payloads and dual-targeting strategies addressing limitations of conventional antibodies. CAR T cell therapy has shown clinical activity against GD2, HER2, and B7-H3 targets, with locoregional delivery and advanced engineering (third-generation CARs, cytokine armoring, bispecific con- structs) enabling responses in select patients, though tumor infiltration, persistence, and antigen escape remain rate- limiting. ICIs, while largely ineffective as monotherapy in unselected populations, induce durable responses in molecu- larly defined subsets, hypermutated tumors, mismatch repair deficiency, and high TMB, establishing biomarker-driven selection as essential for rational ICI deployment. The future of pediatric solid tumor immunotherapy lies in rational combinations that simultaneously address multi- ple resistance mechanisms. Emerging data support integrat- ing TME modulators, oncolytic viruses, NK cell engagers, myeloid-reprogramming agents, and TGF-β inhibitors with ICI or cellular therapies to convert cold tumors into immu- nologically active lesions. Personalized neoantigen vac- cines combined with ICIs represent a compelling strategy to expand tumor-reactive T cell repertoires in otherwise non- immunogenic cancers. Critically, comprehensive immune profiling using spatial transcriptomics, multiplex imaging, and longitudinal immunomonitoring will be essential for stratifying patients, predicting responses, and guiding adap- tive treatment strategies. Realizing the full potential of immunotherapy in pedi- atric solid tumors requires continued investment in pediat- ric-specific trials, novel target discovery through system- atic antigen screening, optimization of delivery routes and dosing schedules, and mechanistic studies elucidating age- dependent immune ontogeny and tumor–host interactions. As platforms mature and combinations are refined through iterative clinical–translational cycles, immunotherapy stands poised to fundamentally alter outcomes for children with these historically refractory malignancies.

  Immunotherapy for pediatric solid tumors: overcoming biological barriers through rational multimodal combinations · 2026 · DOI

Most-cited papers in CAR-T cell therapy research

• Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia · New England Journal of Medicine · 2013 · 3,059 citations
• Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors · New England Journal of Medicine · 2020 · 1,988 citations
• Intraventricular CARv3-TEAM-E T Cells in Recurrent Glioblastoma · New England Journal of Medicine · 2024 · 399 citations
• Unveiling the mechanisms and challenges of cancer drug resistance · Cell Communication and Signaling · 2024 · 356 citations
• Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results · Nature Medicine · 2024 · 341 citations
• Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial · Nature Medicine · 2024 · 299 citations
• Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis · Cell · 2024 · 274 citations
• IL-10-expressing CAR T cells resist dysfunction and mediate durable clearance of solid tumors and metastases · Nature Biotechnology · 2024 · 264 citations
• Secondary Cancers after Chimeric Antigen Receptor T-Cell Therapy · New England Journal of Medicine · 2024 · 255 citations
• T cell lymphoma and secondary primary malignancy risk after commercial CAR T cell therapy · Nature Medicine · 2024 · 250 citations

Most recent work

• Regulatory T cell therapies: biological foundations, engineering strategies, and clinical translation · Frontiers in Immunology · 2026
• Memory programming and new manufacturing paradigms in CAR-T cell therapy · Cancer Gene Therapy · 2026
• PRECISE-seq reveals disease-relevant TCR repertoires with phenotypic plasticity · Journal of Experimental Medicine · 2026
• Circadian engineering of in vivo CAR T cell therapy for precision oncology · npj Precision Oncology · 2026
• Single-cell multiomics reveals regulatory mechanisms of CAR T-cell persistence and dysfunction in multiple myeloma · Blood Neoplasia · 2026
• LVV SMRTcap reveals extensive proviral variation in lentiviral vector-transduced CAR T cells · bioRxiv · 2026
• Kidney Transplantation after Clearing Anti-HLA Antibodies with CD19 CAR T Cells · New England Journal of Medicine · 2026
• Kidney Transplantation in Two Highly Sensitized Candidates after CAR T-Cell Therapy · New England Journal of Medicine · 2026
• Extracorporeal blood purification in the management of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome following chimeric antigen receptor T cell therapy: a case report · Acta Haematologica Polonica · 2026
• Oncostatin-M ligand-based CAR-T therapy displays robust anti-tumor activity against osteosarcoma · BMC Medicine · 2026

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