Beyond CD19 and BCMA: Diversifying CAR targets in cell and gene therapy

Fourth-generation CAR constructs are now moving past classic B-cell antigens. 2025 ASCO data on the CD70-directed, allogeneic product ALLO-316 reported a 31% confirmed objective response rate (ORR) after a single infusion in heavily pre-treated clear-cell renal-cell carcinoma (ccRCC), with responses persisting beyond 12 months¹. Other solid-tumor targets in active trials include GD2 (neuroblastoma, glioblastoma), CLDN18.2 (gastric cancer), and MUC1 (triple-negative breast cancer), each selected for high tumor-restricted expression and limited normal-tissue cross-reactivity^2,3. Parallel efforts with CAR-NK cells are exploiting the innate cytotoxicity and minimal graft-versus-host potential of NK cells while using the same modular antigen-binding logic.

Key R&D themes

• Epitope mapping to avoid fratricide (eg, CD70 knock-out in ALLO-316).
• Dual-CAR and tandem-CAR designs to thwart antigen escape.
• Armored CARs secreting IL-12 or PD-1-blocking scFvs to remodel suppressive stroma.

Cracking solid tumors: Trafficking and micro-environment engineering

Solid tumors account for >90% of global cancer incidence but remain refractory to cell therapy because of antigen heterogeneity, fibrosis, hypoxia, and checkpoint-rich milieus. These hostile environments can induce cell exhaustion, which reduces the effectiveness and persistence of CAR T cells. In the first randomized trial of CAR-T for gastric/GEJ cancer, median overall survival improved by 40% versus standard chemotherapy; separate Phase I work in recurrent glioblastoma documented 62% radiographic regressions, an unprecedented signal in that disease^4,5,6.

Allogeneic, “Off-the-Shelf” CAR T cell therapies shift the economics

Autologous CAR-T remains constrained by 3-5-week vein-to-vein times, personalized QC, and >$400k COGs. Allogeneic approaches edit healthy-donor T or NK cells to create allogeneic CAR T cells that are genetically modified using advanced gene editing technologies to reduce immune rejection and improve safety:

1. Eliminate endogenous TCRα/β (TALENs, CRISPR) to prevent graft-versus-host disease.
2. Insert antigen-specific CARs and, frequently, CD52-KO to permit anti-CD52 lymphodepletion.
3. Inactivate HLA-I/II or over-express HLA-E/B2M fusion proteins to evade host NK clearance.

Gene editing technologies such as CRISPR/Cas9, TALENs, and ZFNs are employed to knock out T cell receptor and HLA genes, and viral vectors are often used to introduce chimeric antigen receptor constructs.

Healthy donor T cells can be sourced from peripheral blood, umbilical cord blood, or bone marrow, and induced pluripotent stem cells and pluripotent stem cells are emerging as alternative sources for generating allogeneic CAR T cell products. Compared to traditional therapies using autologous cells, allogeneic CAR T cells derived from healthy donors offer increased standardization, immediate availability, and the potential for improved manufacturing outcomes.

ALLO-316 exemplifies clinical traction, showing rapid manufacturing (<5 days) and on-demand dosing with no grade ≥3 cytokine-release syndrome (CRS) in its ccRCC cohort⁷. Universal iPSC-derived CAR-NK products are entering the clinic and may deliver even lower COGs because of single master cell banks; genome editing is used to create standardized, universal CAR T cell products with reduced risk of immune rejection.

Engineered TCRs and next-gen TILs: Precision for intracellular and neo-antigens

TCR-transduced T cells

The NCI’s personalized neoantigen program (NCT03412877) reported durable peripheral engraftment of retrovirally encoded TCRs (>20% CD3⁺ cells at 2 years) with objective responses in metastatic colorectal cancer⁸. Persistence and expansion of specific cell populations, such as memory and naive T cell subsets, are critical for therapeutic efficacy. Unlike CARs, TCRs recognize intracellular peptides presented by HLA, vastly expanding the target universe (KRAS^G12D, p53^R175H, etc.).

Refined TIL therapy

FDA accelerated approval of lifileucel (Amtagvi™) in February 2024 validated large-scale, centralized TIL manufacturing for melanoma⁹. Second-generation TIL platforms now incorporate:

• Selective expansion of neoantigen-reactive clonotypes via tetramer sorting or barcode-guided tracking, involving the isolation and enrichment of specific cell subsets with superior antitumor activity.
• IL-2-independent ex vivo cultures to mitigate capillary-leak toxicity¹⁰.
• Gene-edited “armored” TILs co-expressing IL-12 or down-regulating PD-1 to enhance intra-tumor persistence.

Safety and control: Logic gates, SynNotch circuits, and inducible switches

The field is converging on programmable safety devices to reduce on-target/off-tumor toxicities and lethal CRS/ICANS:

• AND-gate circuits (SynNotch → CAR): CAR activation occurs only when two antigens are co-expressed, sharply improving selectivity for heterogeneous tumours¹¹.
• Small-molecule “suicide” or dormancy switches: Rimiducid-activated iCaspase-9 purges >90% of CAR-T cells within 30 minutes in human studies of graft-versus-host disease¹², by triggering programmed cell death to eliminate CAR-T cells in case of severe adverse events rapidly.
• Drug-turnable ON/OFF systems: Reversible Src kinase blockade with dasatinib transiently arrests CAR signaling and abrogates CRS in murine models, a paradigm now in Phase I evaluation¹³.
• Logic-integrated “universal” CARs: Dockable adaptor platforms (e.g., SpyTag/SpyCatcher) permit precise dose titration of tumor-targeting adapters, effectively adding a pharmacokinetic dial¹⁴.

Synthetic-biology toolkits such as genome-integrated Boolean circuits and post-translational degron tags are rapidly maturing, supported by falling DNA-synthesis costs and high-throughput pooled screening.

In vivo gene editing: From CRISPR-Cas9 to base and prime editors

The first ex vivo CRISPR therapy (CASGEVY™) is already approved for haemoglobinopathies, but direct in vivo editing is the next frontier. These advances hold promise to treat genetic diseases at their source.

Base editors avoid double-strand breaks, dramatically lowering p53 activation and chromothripsis risk, while prime editors offer scarless installation of any 1-to-100 bp edit. Remaining bottlenecks are tissue-selective LNPs (lipid chemistry, charge shielding) and immune tolerance to editor proteins. Parallel work on stealth Cas variants and transient self-replicating RNA circuits aims to expand editing to the heart, CNS, and lung.

Biomarkers: Guiding target selection and response prediction

Biomarkers are transforming the landscape of CAR T cell therapies by enabling a more precise and personalized approach to cancer treatment. In the development of CAR T cell therapy, biomarkers serve as critical tools for identifying which tumor-associated antigens are most suitable for targeting, ensuring that engineered T cells selectively attack cancer cells while sparing healthy tissue. This molecular profiling allows researchers to design CAR constructs that are tailored to the unique antigenic landscape of each patient’s tumor, increasing the likelihood of effective cell therapy.

Beyond target selection, biomarkers are increasingly used to predict which patients are most likely to benefit from CAR T cell therapy. For example, the expression levels of certain immune checkpoints or the presence of specific genetic mutations within cancer cells can inform clinicians about the potential responsiveness to cell therapies. Monitoring biomarkers throughout the course of treatment also provides real-time insights into disease progression and therapeutic efficacy, enabling timely adjustments to the treatment plan.

By integrating biomarker analysis into the clinical workflow, researchers and clinicians can optimize CAR T cell therapies, minimize adverse effects, and improve overall patient outcomes. This biomarker-driven strategy is paving the way for more effective, individualized cell therapy regimens in both hematologic malignancies and solid tumors.

Combination therapies: Synergizing CARs with checkpoint inhibitors, cytokines, and beyond

The next frontier in CAR T cell therapies lies in combination strategies that harness the power of multiple modalities to achieve superior clinical outcomes. By pairing CAR T cells with checkpoint inhibitors, researchers can disrupt immune-suppressive pathways, such as PD-1/PD-L1, that often limit the effectiveness of T cell therapy. This synergy can reinvigorate exhausted T cells and enhance their ability to eliminate cancer cells, particularly in solid tumors where immune evasion is a major challenge.

Cytokine support, using agents like IL-2 or IL-15, is another promising avenue. These cytokines can boost the proliferation and persistence of CAR T cells within the patient’s body, sustaining a robust immune response against tumor cells. Innovative gene therapies, including prime editing, are being explored to further engineer CAR T cells for improved specificity, resistance to exhaustion, and enhanced function, expanding the reach of cell therapies to a broader range of human diseases, including genetic disorders.

Additionally, combining CAR T cell therapy with other cell therapies, such as NK cells, offers a multi-pronged attack on the tumor microenvironment. NK cells bring innate cytotoxicity and can target cancer cells that may escape T cell recognition, providing complementary mechanisms of action. These combination approaches are being evaluated in preclinical models, with the goal of overcoming resistance, reducing relapse rates, and delivering more durable remissions.

By integrating CAR T cell therapies with checkpoint inhibitors, cytokines, gene therapies, and NK cells, the field is moving toward comprehensive, multi-modal treatment regimens that address the complexity of cancer and other human diseases at multiple levels.

Outlook: Convergence toward curative, accessible medicines

Modular gene circuits, universal donor platforms, and non-viral delivery vehicles are coalescing into an industrialized “cell-and-gene foundry” model. For clinicians, this heralds:

• 24-hour on-demand dosing of cryopreserved allogeneic cells.
• Algorithm-driven target selection leveraging single-cell multi-omics and spatial transcriptomics.
• Adaptive control loops that sense cytokine flux and auto-regulate effector output.

For regulators, the shift from bespoke autologous lots to master-cell-bank economies will require new CMC and potency-assay frameworks but promises significantly lower cost of goods and broader global access. Ongoing clinical trial data will be critical in demonstrating the ability of these therapies to treat cancer and other serious diseases.

The bottom line is that next-generation cell and gene therapies are accelerating from proof-of-concept to platform, bringing the prospect of durable remissions, or outright cures, for malignancies and Mendelian diseases that were untreatable a decade ago. These therapies are designed to target and eliminate malignant cells and diseased cells at their source, with the immune system playing a central role in mediating their effects.

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