Functional genomics–guided design of CAR-T and CAR-NK therapies in hematological malignancies: aligning cellular engineering with immune escape and microenvironmental resistance
Aug 2026· Frontiers in Genome Editing· Vol 8· 0 citations· 127 references
Medicine
TL;DR
Future CAR-T and CAR-NK development should link each modification to a measurable resistance mechanism, a feasible biomarker, and a clinically testable benefit, before resistance-matched cellular immunotherapy can be broadly integrated into clinical practice.
Abstract
Background Chimeric antigen receptor T-cell therapy has changed the treatment landscape of relapsed or refractory hematological malignancies, but primary non-response and post-infusion relapse remain frequent clinical problems. In aggressive B-cell lymphomas, acute leukemias, and multiple myeloma, treatment failure is often driven by overlapping mechanisms rather than a single resistance pathway. These include antigen loss or reduced antigen density, impaired immune recognition, defective inflammatory signaling, checkpoint-mediated suppression, metabolic stress, and limited effector-cell persistence within suppressive disease niches. Main Body Genome engineering has become an important tool for both identifying and addressing these resistance mechanisms. CRISPR-based functional screening, single-cell perturbation approaches, and multi-omics profiling allow immune escape and tumor microenvironment-mediated resistance to be defined more functionally, rather than inferred only from correlative datasets. These insights can inform the design of CAR-T and CAR-NK therapies through multi-target or logic-gated receptors, checkpoint or exhaustion-pathway editing, cytokine-supported and armored constructs, metabolic fitness enhancement, and selected multiplex-editing strategies. In parallel, CAR-NK cells, universal allogeneic CAR-T products, and stem-cell-derived platforms may provide additional options in relapse-prone or heavily pretreated patients, particularly when autologous T-cell fitness, manufacturing feasibility, or repeat dosing is a concern. Conclusion A resistance-guided approach may help align engineered cellular therapy design with the dominant mechanisms of treatment failure in high-risk hematological malignancies. Rather than simply increasing engineering complexity, future CAR-T and CAR-NK development should link each modification to a measurable resistance mechanism, a feasible biomarker, and a clinically testable benefit. Prospective validation, genomic safety assessment, manufacturing consistency, and long-term monitoring will be essential before resistance-matched cellular immunotherapy can be broadly integrated into clinical practice.
Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment landscape for relapsed or refractory hematologic malignancies, producing high remission rates in otherwise treatment-resistant patients. However, primary resistance and disease relapse remain common, particularly in solid tumors, limiting long-term benefit and broader clinical applicability. As the population of patients failing therapy grows, there is an urgent need for an integrated understanding of resistance mechanisms and a structured approach to salvage therapy. This review proposes a conceptual “Why-How-What if” framework to navigate the complexities of treatment failure. We first address “Why” therapy fails, identifying multifactorial drivers including tumor-intrinsic factors like antigen loss and immune evasion, T cell-intrinsic dysfunction such as exhaustion and limited persistence, and extrinsic constraints imposed by an immunosuppressive tumor microenvironment. We then explore “How” to enhance efficacy through mechanism-based strategies. These include rational combination approaches with immune checkpoint inhibitors or small molecule inhibitors, and next-generation engineering such as dual-target, armored, and in vivo generated CAR-T cells aimed at overcoming metabolic and physical barriers. Finally, we address the “What if” of treatment failure by summarizing individualized salvage options, for which current clinical evidence is derived predominantly from hematologic malignancies. These strategies range from target-switching and bispecific antibodies to emerging cellular platforms like CAR-natural killer cells and consolidation via allogeneic hematopoietic stem cell transplantation. By integrating mechanisms of failure with evolving optimization and salvage strategies, this framework provides a practical roadmap for clinical and translational progress. Future success will depend on biomarker-guided combinations and the continued diversification of adoptive cell therapy platforms.
Bi-Jing Wu, Jia-Hui Wang, Qihua Zou et al.· Biomarker Research· 0 citations
Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of hematological malignancies, yet its broader application to solid tumors, chronic viral infections, and autoimmune diseases remains constrained by antigen heterogeneity, immunosuppressive tissue microenvironments, T-cell exhaustion, limited persistence, and treatment-associated toxicities. These challenges have shifted the field from optimizing individual receptor constructs toward engineering CAR-T cells as programmable immune systems capable of adapting to diverse disease contexts. This review synthesizes recent advances in molecular engineering strategies that enhance CAR-T cell function beyond conventional receptor design. We discuss how receptor engineering, genome editing, transcriptional and epigenetic regulation, metabolic reprogramming, synthetic gene circuits, and safety-control platforms collectively reshape CAR-T cell fate, persistence, and therapeutic efficacy. Rather than functioning independently, these engineering strategies are increasingly integrated to generate context-specific cellular therapies capable of adapting to diverse disease environments, including cancer, autoimmune diseases, and chronic viral infections. We also highlight the potential for translation into clinical practice or clinical translation and discuss the major challenges associated with clinical implementation. Next-generation CAR-T therapies will increasingly integrate molecular engineering strategies or will rely on molecular engineering strategies to integrate antigen recognition, cellular fitness, immune regulation, and longevity rather than simply maximizing cytotoxic activity. Recent advances in programmable cellular engineering coupled with rigorous clinical evaluation as well as scalable manufacturing technologies or scalable manufacturing platforms in the treatment of other diseases beyond oncology will facilitate the development of safer, more durable, and broadly applicable cellular therapies. Not applicable.
Hany E. Marei, G. Pozzoli, S. Caratelli et al.· Stem cell research & therape...· 0 citations
Glioblastoma (GBM) remains the most aggressive primary malignant brain tumor in adults and is characterized by poor prognosis, profound intratumoral heterogeneity, and a highly immunosuppressive tumor microenvironment (TME). Although chimeric antigen receptor (CAR)-T cell therapy has shown remarkable efficacy in hematologic malignancies, clinical responses in GBM remain inconsistent and rarely durable. This review critically evaluates how receptor architecture, antigen selection, multi-antigen targeting, TME- and exhaustion-resistant engineering, locoregional delivery, alternative CAR-cell platforms and computational design address the major biological and anatomical barriers of GBM. What should be given a particular attention is not only the translational gap between preclinical activity and limited clinical efficacy, but also differences in target specificity, antigen escape, safety, delivery route and CAR-T persistence. However, the available evidence indicates that no single engineering strategy is sufficient to overcome the interconnected barriers. The strongest approach is likely to combine several strategies. These should improve antigen coverage, maintain CAR-T cell function, increase safety and improve delivery within the CNS. Future progress in GBM CAR therapy will therefore depend on combining complementary solutions rather than improving only one CAR feature.
Aleksandra Królikowska, Katarzyna Białkowska· International Immunopharmaco...· 0 citations
Chimeric antigen receptor-engineered natural killer (CAR-NK) cells have emerged as a promising off-the-shelf platform for cancer immunotherapy, with a favorable safety profile in early clinical trials and potent antitumor activity demonstrated in relapsed/refractory (R/R) hematologic malignancies. However, their clinical efficacy in solid tumors remains severely limited by interconnected resistance mechanisms. In this review, we systematically dissect the pathological basis of CAR-NK therapy failure in solid tumors and propose an integrative cascade resistance framework that delineates three core bottlenecks: tumor microenvironment (TME)-mediated functional exhaustion, structural defects of non-natural killer (NK)-cell-adapted chimeric antigen receptors (CARs), and trogocytosis-driven immune escape. We further characterize the context-dependent regulatory roles of the natural killer group 2 member A–human leukocyte antigen E (NKG2A-HLA-E) immune checkpoint axis and the intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 (ICAM-1/LFA-1) adhesion pathway within this cascade model, with clear stratification of evidence strength across all mechanistic conclusions. Centered on the activating-inhibitory dual-module CAR (aCAR-iCAR) system, we summarize its design principles, preclinical validation status, and potential to mitigate cascade resistance, with explicit distinction between killer cell immunoglobulin-like receptor (KIR)-based (Level 1 evidence) and NKG2A-based (Level 2–3 evidence) inhibitory CAR backbones. We then outline an end-to-end artificial intelligence (AI)-driven rational design framework covering target screening, structural optimization, and signaling balance calibration, and introduce the AI-nanosymbiont concept as an exogenous synergistic strategy to address TME delivery barriers. Building on the molecular heterogeneity of solid tumors, we propose a four-subtype precision stratification framework to match tumor features with tailored therapeutic regimens, and summarize core translational challenges including manufacturing constraints, regulatory gaps, and safety considerations. Overall, this review provides a balanced, evidence-graded theoretical framework for next-generation CAR-NK development against solid tumors, and generates testable hypotheses for future mechanistic and clinical investigations.
Chenru Ma, Yafei Zhuang, Song-Chen Han et al.· Frontiers in Immunology· 0 citations
Endometrial cancer (EC) is characterized by significant molecular and immunological heterogeneity, which influences both disease progression and response to therapy. Although immune checkpoint blockade has improved the clinical management of selected EC subtypes, durable responses remain limited in many patients, particularly in tumors with poorly inflamed or mismatch repair-proficient/microsatellite-stable profiles. In this context, natural killer (NK) cells represent an important but still insufficiently explored component of anti-tumor immunity. NK cells can recognize transformed or stressed cells independently of antigen-specific priming, which may be relevant in EC tumors characterized by altered antigen presentation, immune exclusion, or limited T-cell responsiveness. Within the EC tumor microenvironment, NK-cell dysfunction is likely shaped by a complex interplay between defective recruitment, altered receptor–ligand interactions, suppressive cytokine networks, and tumor-driven immune remodeling. These mechanisms may reduce NK-cell cytotoxicity and favor immunoregulatory or tolerance-like phenotypes, some of which resemble programs involved in maternal–fetal immune tolerance. Here, we examine how EC may reshape NK-cell recruitment, phenotype, and function, and discuss how these alterations intersect with molecular tumor heterogeneity, immune escape, and emerging NK-directed therapeutic strategies. Particular attention is given to how NK-directed and NK-complementary approaches, including cytokine-based activation, NK-cell engagers, adoptive NK-cell transfer, chimeric antigen receptor natural killer cell (CAR-NK) platforms, and their integration with immune checkpoint blockade, may help address resistance in EC.
Alessandro Poggi, V. Bruno, Anna Di Spirito et al.· Frontiers in Immunology· 0 citations
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