Guest Column | August 19, 2025

Advancing CAR T Cell Therapy with Logic Gate Engineering

By Francisco Conesa Buendía

Digital logical gates-GettyImages-1409425216

CAR T cell therapy has transformed the treatment of hematologic malignancies but remains challenged by antigen heterogeneity and off-tumor toxicities, especially in solid tumors.1 Logic gating applies Boolean logic principles to CAR T cell design, requiring multiple antigen recognition events to trigger T cell activation, thus improving precision and minimizing collateral damage.2 Here, we detail the mechanistic underpinnings, clinical advances, manufacturing implications, and regulatory frameworks pertinent to logic-gated CAR T cells.

Logic Gate Types And Mechanistic Signaling


AND Gates

AND-gated CARs require simultaneous engagement of two distinct antigens to activate cytotoxic functions, thus ensuring specificity.3 Mechanistically, this is commonly achieved by co-expressing two receptors: one providing the primary activation signal (CD3ζ) and the other delivering co-stimulation (CD28 or 4-1BB).3 Activation occurs only when both receptors engage their respective antigens, triggering downstream signaling cascades synergistically.4

At the molecular level, the CD3ζ chain initiates phosphorylation of ITAM motifs, activating ZAP-70 and downstream NFAT, NF-κB, and AP-1 pathways.5 The costimulatory domain enhances cytokine production, proliferation, and survival via PI3K/AKT and MAPK pathways.5 The conjunctive activation prevents T cell activation by cells expressing a single antigen, reducing off-target effects.5


OR Gates

OR-gated CAR T cells express multiple CARs recognizing different antigens, allowing activation upon engagement with any one antigen.3,6 This design is particularly useful in heterogeneous tumors with variable antigen expression. Each CAR independently triggers canonical signaling via CD3ζ and costimulatory domains, leading to T cell activation.3,6,7


NOT Gates

NOT gates employ inhibitory CARs (iCARs) recognizing antigens expressed on normal tissues but absent on tumors, delivering suppressive signals to prevent T cell activation.3,8 These iCARs incorporate intracellular domains from immune checkpoints (e.g., PD-1, CTLA-4), which recruit SHP-1/2 phosphatases and inhibit proximal TCR signaling. This mechanism enables selective tumor targeting by overriding activation in the presence of healthy tissue antigens.9


IF-THEN Gates

IF-THEN gating enables spatiotemporal control of CAR expression, improving specificity and safety by inducing CAR activity only in the presence of a priming antigen.3,10 Typically, a SynNotch receptor detects antigen A and triggers expression of a CAR targeting antigen B, restricting CAR function to tumor sites. This approach reduces off-tumor toxicity and T-cell exhaustion while enhancing persistence.3,10 However, careful antigen pairing is crucial, as mismatched expression kinetics can compromise safety.3,10


IF-BETTER Gates

IF-BETTER gates enhance CAR T cell function by boosting activation when antigen B is present alongside a CAR-targeted antigen A.3,11 Antigen B is detected via a costimulatory receptor (CCR), which supports CAR signaling without triggering cytotoxicity alone. This design improves T cell cytokine production, persistence, and tumor control, especially when antigen A is scarce.3,11 Unlike AND-gates, IF-BETTER systems are less restrictive and avoid the toxicity risks of dual CAR approaches.3,11


Composite And Advanced Logic Gates

Recent work explores composite logic gates combining AND, OR, and NOT elements to refine activation thresholds and spatial control. “Fuzzy logic” circuits modulate T cell responses quantitatively, integrating graded antigen signals to optimize therapeutic windows.7 This system enables the cells to process multiple input signals and make context-sensitive activation decisions, rather than responding to a single antigen in a binary fashion. By doing so, the engineered T cells demonstrate improved discrimination between healthy and malignant cells, reducing off-target effects while maintaining strong anti-tumor efficacy in preclinical models. This approach offers a promising advancement toward safer and more precise cell therapies. Clinical Landscape Of Logic-Gated CAR T Cell Therapies

Clinical Trials

  • A2 Biotherapeutics: Utilizing the Tmod platform, A2 Biotherapeutics developed autologous logic-gated CAR T cell therapies incorporating a blocker receptor targeting HLA-A*02 to inhibit activity in healthy tissues. Their lead products, A2B530 (targeting CEA) and A2B395 (targeting EGFR), are under Phase 1/2 trials (EVEREST-1 NCT05736731; DENALI-1 NCT06682793), demonstrating promising safety profiles and early signals of efficacy in solid tumors.12,13
  • ImmPACT Bio: IMPT-314, a dual-antigen AND-gated CAR T cell, is in Phase 1/2 clinical evaluation for relapsed/refractory large B-cell lymphoma, following FDA IND clearance in 2023.14,15
  • Senti Bio: Their SENTI-202 CAR NK-cell product, combining logic gating with allogeneic off-the-shelf manufacturing, is in Phase 1 trials targeting AML antigens CD33 and FLT3, showing preliminary efficacy and a favorable safety profile.16
  • Autolus Therapeutics: Autolus’ AUCATZYL product, incorporating logic gating and transient antigen engagement, is being assessed in Phase 1b/2 trials for acute lymphoblastic leukemia, aiming to reduce adverse events by limiting activation duration.17,18
  • AvenCell Europe GmbH: The UniCAR platform is a switchable CAR T cell system developed to enhance safety and control in cancer therapy. Unlike conventional CAR T cells, UniCAR T cells recognize a small peptide epitope rather than a tumor surface antigen directly. Their anti-tumor activity is mediated by separate target modules (TMs) that link the UniCAR T cells to tumor cells. This design allows precise control of the therapy: when TMs are infused, the system is "ON" and active against tumors; once TMs are removed, the UniCAR T cells revert to an inactive "OFF" state. This approach helps manage side effects, including off-tumor toxicity.19,20
  • Calibr (a division of Scripps Research): Calibr’s switchable CAR T cell (sCAR T) system enables precise, reversible control of CAR T activity in vivo using an antibody-based switch. In mouse models targeting CD19+ lymphoma, sCAR T cells paired with optimized switches and costimulatory domains (especially 4‑1BB or CD28+4‑1BB) achieved complete tumor elimination and prevented relapse.21 Importantly, dosing schedules influenced CAR T expansion and memory formation, with intermittent dosing (one week on, three weeks off) promoting strong T cell expansion and durable central memory (TCM) development. These findings highlight a promising approach for safer, more flexible, and long-lasting CAR T therapies.22

Manufacturing Considerations And Scalability

Logic-gated CAR T cells necessitate the co-expression of multiple synthetic receptors, complicating vector design. Multi-cistronic viral vectors or co-transduction strategies must ensure stoichiometric expression of activating and inhibitory receptors for functional fidelity.23 Vector size constraints and transduction efficiency are critical parameters.23

Product characterization requires advanced multiparametric flow cytometry and single-cell transcriptomics to confirm receptor expression ratios and functional integrity.24,25 Moreover, logic-gated CAR T cells impose stringent quality control to verify absence of unintended receptor cross-talk or tonic signaling.23

Allogeneic logic-gated products aim to enhance scalability but require additional gene editing to prevent graft-versus-host disease, adding manufacturing complexity.

Regulatory Perspectives

The FDA and EMA have issued evolving guidance recognizing the complexity of multi-receptor CAR T cell products. Regulatory submissions must provide:

  • detailed chemistry, manufacturing, and controls (CMC) documentation describing vector constructs, expression profiles, and receptor functionality
  • robust preclinical data demonstrating gate-specific activation and absence of off-target toxicity
  • comprehensive clinical safety monitoring plans due to novel mechanisms, including long-term follow-up for insertional oncogenesis
  • risk mitigation strategies addressing potential cytokine release syndrome (CRS) and neurotoxicity.

Adaptive regulatory frameworks incorporating real-world evidence and biomarker-driven safety monitoring are anticipated to facilitate logic-gated CAR T cell approvals.24

Synergy Of Logic Gating And Armored CAR Features

Armored CAR T cells engineered to secrete cytokines (e.g., IL-12, IL-18) or express costimulatory ligands bolster T cell persistence and reprogram the immunosuppressive tumor microenvironment.28,29 Logic gating can spatially restrict these payloads, minimizing systemic toxicities and improving therapeutic indices.29

This combinatorial approach holds promise for solid tumors where hostile microenvironments and antigen heterogeneity challenge conventional CAR T cells.29

Conclusion

Logic-gated CAR T cell therapies embody a next-generation approach to refine tumor targeting, reduce toxicities, and enhance therapeutic precision. While technical and regulatory challenges remain, ongoing clinical trials and advances in synthetic biology underpin a transformative future. The integration of logic gating with armored features further enhances therapeutic potential, especially against solid tumors.

References:

  1. June, C.H., et al. (2018). CAR T cell immunotherapy for human cancer. Science, 359(6382), 1361-1365. DOI:https://doi.org/10.1126/science.aar6711
  2. Roybal, K.T., et al. (2016). Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell, 164(4), 770–779. DOI:https://doi.org/10.1016/j.cell.2016.01.011
  3. Hamieh, M., et al. (2023). Programming CAR T Cell Tumor Recognition: Tuned Antigen Sensing and Logic Gating. Cancer Discov. 3;13(4):829-843. DOI: https://doi.org/10.1158/2159-8290.cd-23-0101
  4. Kloss, C.C., et al. (2012). Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol, 31(1), 71–75. DOI: 10.1038/nbt.2459
  5. Guedan, S., et al. (2018). Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight, 3(1), e96976. DOI: 10.1172/jci.insight.96976
  6. Mardiros, A., et al. (2013). T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against acute myeloid leukemia. Blood, 122(6), 3138–3148. DOI: 10.1182/blood-2012-12-474056 
  7. Kondo, T., et al. (2025). Engineering TCR-controlled fuzzy logic into CAR T cells enhances antitumor activity while minimizing toxicity. Cell, 188(9), 2372-2389.e35. DOI: https://doi.org/10.1016/j.cell.2025.03.017 
  8. Fedorov, V.D., Themeli, M., & Sadelain, M. (2013). PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med, 5(215), 215ra172. DOI:https://doi.org/10.1126/scitranslmed.3006597
  9. Cho, J.H., Collins, J.J., & Wong, W.W. (2018). Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell, 173(6), 1426–1438.e11. DOI:https://doi.org/10.1016/j.cell.2018.03.038
  10. Morsut, L., et al. (2016). Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell, 164(4):780-91. DOI: https://doi.org/10.1016/j.cell.2016.01.012
  11. Katsarou, A., et al. (2021). Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence. Sci Transl Med. 2021 Dec 8;13(623):eabh1962. DOI: https://doi.org/10.1126/scitranslmed.abh1962
  12. A2 Biotherapeutics. (2024). Clinical pipeline and updates. https://www.a2bio.com/
  13. ClinicalTrials.gov. EVEREST-1/NCT05736731 and DENALI-1 /NCT06682793. https://clinicaltrials.gov/study/NCT05736731 and https://clinicaltrials.gov/study/NCT06682793
  14. Johnson, V. CGTlive. (2023). Logic-gated CAR T therapy IND clearance. https://www.cgtlive.com/view/logic-gated-car-t-therapy-cleared-for-lymphoma-trials
  15. Lyell Immunopharma, Inc. (2024). Lyell presents positive initial clinical data from Phase 1/2 clinical trial of IMPT-314 for the treatment of B cell lymphoma at ASH 2024 Annual Meeting. https://ir.lyell.com/news-releases/news-release-details/lyell-presents-positive-initial-clinical-data-phase-1-2-clinical/
  16. Senti Bio. (2025). SENTI-202 trial info.NCT06325748 https://clinicaltrials.gov/study/NCT06325748?intr=senti-202&rank=1
  17. Roddie, C., et al. (2024). Obecabtagene Autoleucel in Adults with B-Cell Acute Lymphoblastic Leukemia. N Engl Med, 391:2219-2230. DOI: https://doi.org/10.1056/nejmoa2406526
  18. ClinicalTrials.gov. AUTO1-AL1. https://clinicaltrials.gov/study/NCT04404660
  19. Arndt, C., et al. (2022). Development and Functional Characterization of a Versatile Radio-/Immunotheranostic Tool for Prostate Cancer Management. Cancers (Basel),14;14(8):1996. DOI: https://doi.org/10.3390/cancers14081996
  20. ClinicalTrials.gov. UniCAR02-T. https://clinicaltrials.gov/study/NCT04230265
  21. Viaud, S., et al. (2018). Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory. Proc Natl Acad Sci U S A, 115(46):E10898-E10906. DOI: https://doi.org/10.1073/pnas.1810060115
  22. ClinicalTrials.gov. CLBR001 + ABBV-461. https://www.clinicaltrials.gov/study/NCT06878248
  23. Wang, X., & Riviere, I. (2016). Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics, 3, 16015. DOI: https://doi.org/10.1038/mto.2016.15
  24. Cadinanos-Garai, A., et al. (2025). High-dimensional temporal mapping of CAR T cells reveals phenotypic and functional remodeling during manufacturing. Mol Ther, 33(5):2291-2309. DOI: https://doi.org/10.1016/j.ymthe.2025.04.006
  25. Chen, R., et al. (2025). Enhancing the potency of CAR-T cells against solid tumors through transcription factor engineering. JCI Insight, 10(14):e193048. DOI: https://doi.org/10.1172/jci.insight.193048
  26. FDA. (2023). Regulatory considerations for CAR T-cell therapies. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products
  27. EMA. (2024). Advanced Therapy Medicinal Products guidelines. https://www.ema.europa.eu/en/human-regulatory/overview/advanced-therapy-medicinal-products-overview
  28. Wang M., et al. (2025). Interleukin-enhanced CAR-engineered immune cells in tumor immunotherapy: current insights and future perspectives. Cytokine, 192:156973. DOI: https://doi.org/10.1016/j.cyto.2025.156973
  29. Chen, T., et al. (2024). Current challenges and therapeutic advances of CAR-T cell therapy for solid tumors. Cancer Cell Int, 24(1):133. DOI: https://doi.org/10.1186/s12935-024-03315-3  

About The Author:

Francisco Conesa Buendía, Ph.D., has been working in the field of advanced therapies since 2021. His research focuses on the development of ATMPs based on stem cells and chondrocytes. Currently, he is a cell manufacturing assistant working on manufacturing and process optimization of cell and gene therapies based on CAR-T and T cells at Memorial Sloan Kettering Cancer Center. Connect with him on LinkedIn.