Insights into next-generation immunotherapy designs and tools: molecular mechanisms and therapeutic prospects

Abstract

Immunotherapy has revolutionized the oncology treatment paradigm, and CAR-T cell therapy in particular represents a significant milestone in treating hematological malignancies. Nevertheless, tumor resistance due to target heterogeneity or mutation remains a Gordian knot for immunotherapy. This review elucidates molecular mechanisms and therapeutic potential of next-generation immunotherapeutic tools spanning genetically engineered immune cells, multi-specific antibodies, and cell engagers, emphasizing multi-targeting strategies to enhance personalized immunotherapy efficacy. Development of logic gate modulation-based circuits, adapter-mediated CARs, multi-specific antibodies, and cell engagers could minimize adverse effects while recognizing tumor signals. Ultimately, we highlight gene delivery, gene editing, and other technologies facilitating tailored immunotherapy, and discuss the promising prospects of artificial intelligence in gene-edited immune cells.

Keywords: Adapter CAR; Chimeric antigen receptor; Gene delivery; Gene editing; Logic gate; Multi-targets.

Fig. 1

CAR production. A. Autologous CAR-immune cells. The process includes isolating immune cell populations from the patient, followed by transduction of these cells with viral or non-viral vectors to express the CAR. After ex vivo expansion, the engineered immune cells are infused back into the patient. B. CAR generation in vivo. Injecting nanoparticles containing mRNA that encodes the CAR could achieve CAR expression in vivo. C. Allogeneic CAR-immune cells. Immune cells are isolated from healthy donors, undergo genetic editing to reduce immunogenicity and minimize immune rejection, and are then produced at a large scale with rigorous quality control before being applied in clinical settings

Fig. 2

Timeline of key events. [1] Eshhar, Z., Waks, T., Gross, G. & Schindler, D. G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A 90, 720–724 (1993). [2] NCT00924326 [3] Hegde, M. et al. Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma. Mol Ther 21, 2087–2101 (2013). [4] Fedorov, V. D., Themeli, M. & Sadelain, M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med 5, 215ra172 (2013). [5] https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-blinatumomab-consolidation-cd19-positive-philadelphia-chromosome-negative-b-cell [6] Hegde, M. et al. Tandem CAR T cells targeting HER2 and IL13Ralpha2 mitigate tumor antigen escape. [7] Ruella, M. et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J Clin Invest. 126, 3814–3826 (2016). [8] Roybal, K. T. et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419–432 e416 (2016). [9] Bielamowicz, K. et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. [10] Sukumaran, S. et al. Enhancing the Potency and Specificity of Engineered T Cells for Cancer Treatment. Cancer Discov 8, 972–987 (2018). [11] Pan, J. et al. Sequential CD19-22 CAR T therapy induces sustained remission in children with r/r B-ALL. Blood 135, 387–391 (2020). [12] Liu, E. et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med 382, 545–553 (2020). [13] Au, K. M., Park, S. I. & Wang, A. Z. Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy. Sci Adv 6, eaba8564 (2020). [14] NCT04660929 [15] NCT04606433 [16] A first-in-human study of CD123 NK cell engager SAR443579 in relapsed or refractory acute myeloid leukemia, B-cell acute lymphoblastic leukemia, or high-risk myelodysplasia.—ASCO [17] Wang, X. et al. Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis. Cell 187, 4890–4904 e4899 (2024). [18] Wang, X. et al. Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis. Cell 187, 4890–4904 e9 (2024). [19] Zhou, J.-e. et al. Lipid nanoparticles produce chimeric antigen receptor macrophages (CAR-M) in situ for the treatment of solid tumors. Nano Today 61, 102,610 (2025)

Fig. 3

Overview of CAR. A. Structure of CAR The structure of a CAR includes the extracellular domain, transmembrane domain, and intracellular domain. The extracellular domain comprises the single-chain variable fragment (scFv), which is composed of VH, VL, and a flexible linker, along with the hinge region. The intracellular domain includes co-stimulatory domains and signaling domains. B. The CAR evolution The evolution of CAR structures primarily involves modifications to the intracellular domain. First-generation CARs contain a simple CD3ε domain. The second-generation and third-generation CARs introduce one and two co-stimulatory domains, respectively. Fourth-generation CARs incorporate additional functional domains, such as those promoting cytokine release. The fifth-generation CARs integrate JAK/STAT signaling to enhance immune cell persistence and sensitivity to immunosuppressive microenvironments

Fig. 4

FDA-approved CAR-T products

Fig. 5

Overview of logic gates. A. OR gate Dual CAR (with complete co-stimulatory and activation domains per receptor), Tandem CAR and Trivalent CAR belong to the OR-gate design, where activation of CAR-immune cells can be triggered by either one of the targeted antigens. B. AND gate Dual CAR (with separated co-stimulatory and activation domains), On-Switch and SynNotch CAR belong to the AND gate design, requiring the simultaneous presence of two targeted antigens to activate CAR-immune cells. ON switch: The structure of CAR is divided into two parts, one carrying the intracellular structural domain of FKBP12 and the other carrying the structural domain of FRB. As an inducer, rapamycin bridges FKBP12 and FRB to assemble a functional CAR that is inactivated (OFF) in the absence of rapamycin and activated (ON) upon addition to achieve spatiotemporal regulation of toxic effects. C. IF-BETTER gate The presence of Tumor-Associated Antigen (TAA) 2 facilitates the activation of CAR-immune cells in the absence of TAA1. However, it is not a necessary condition for activation when TAA1 is present in sufficient amounts. D. NOT gate Binding of TAA2 to the CAR induces the activation of immune cells, whereas this activation does not occur in the presence of TAA1

Fig. 6

SUPRA CAR, multi-specific antibody, and multi-specific cell engager. A. SUPRA CAR a. Structure of SUPRA The structure of SUPRA involves a zipCAR and a zipFv, with the scFv targeting TAAs. b. SUPRA CAR based on OR gate Any zipFv with scFvs targeting different TAAs can bind to the zipCAR, mediating the activation of gene-edited immune cells. c. SUPRA CAR based on AND gate The zipFv with scFvs targeting different TAAs must simultaneously bind to the corresponding zipCAR to promote the activation of gene-edited immune cells. d. SUPRA CAR based on NOT gate Introducing zipFvs targeting two different TAAs, where TAA2 is absent on tumor cells (expressed only on normal cells), allows gene-edited immune cells to target and kill tumor cells. In normal tissues, the presence of TAA2 results in dimerization of the zipFv targeting TAA1 with the zipFv targeting TAA2, preventing the activation of gene-edited immune cells and reducing toxicity to normal tissues. e. OFF switch Introducing inhibitory zipFvs that competitively bind to the zipCAR with the zipFv targeting TAA can inhibit the activation of gene-edited immune cells. B. Multi-specific antibody a. BsAb The Fab segments of a BsAb target CD3 and a TAA, respectively, linking immune cells and tumor cells to promote tumor killing. b. TsAb TsAb introduces an additional anti-CD28 fragment, which, while linking immune cells, also binds to co-stimulatory domains, enhancing the oncolytic effect. c. MicAbody Based on natural killer group 2 member D (NKG2D), MicAbody is a specific type of bispecific antibody. NKG2D triggers the activation and proliferation of immune cells by recognizing and binding to activating ligands expressed on immune cells, thereby promoting the killing of target cells. C. Multi-specific cell engager a. Bispecific cell engager One arm of the bispecific antibody targets the CD3ε/CD16 chain to activate T/NK cells, while the other arm targets a TAA, promoting the formation of a cytolytic synapse to mediate tumor cell killing. b. TriKE TriKE contains scFvs that specifically bind to the activating receptor CD16 on NK cells and TAA, linked to the cytokine IL-15. IL-15 induces the activation and expansion of NK cells while avoiding off-target toxicity

Fig. 7

Emerging novel strategies. A. Lipid nanoparticle (LNP)-mediated expression of CAR. LNPs enter cells via endocytosis and then degrade, releasing mRNA encoding the CAR, which is subsequently translated to promote CAR expression. Immune cells expressing CARs exert CAR-mediated anti-tumor effects, but this function gradually diminishes as the mRNA degrades. B. Application of gene editing technology in improving the function of immune cells C. Strategies for gene delivery a. Viral vectors. Lentivirus: Single-stranded RNA genome, enveloped, conical core capsid, capable of host genome integration. Adenovirus: Double-stranded linear DNA genome, non-enveloped, icosahedral capsid, episomal persistence without integration. Retrovirus: Single-stranded RNA genome, enveloped, spherical capsid, capable of host genome integration. b. Electroporation-mediated CAR mRNA delivery Electric field-induced membrane permeabilization enables intracellular uptake of CAR-encoding mRNA, followed by ribosomal translation to produce CAR proteins. c. Transposon-based genomic integration CAR-containing donor DNA and transposase are co-delivered into immune cells. The transposase recognizes specific sequences on the donor DNA, excises CAR sequences, and then mediates their site-specific integration into the host genome via sequence recognition. d. Nanoparticle-mediated plasmid delivery Nanoparticles serve as carriers to encapsulate CAR-encoding plasmid DNA

Fig. 8

Application of Artificial Intelligence (AI) in Immunotherapy. A. AI plays a pivotal role in the production, storage, and logistics of CAR-immune cells by leveraging IoT sensors. B. AI enhances the precise identification of CAR targets through integration with CRISPR/Cas9 technology. C. AI continuously monitors immunotherapy patients via the detection of biochemical indicators. D. AI is capable of analyzing baseline information, as well as genomic and proteomic data from patients. E. AI facilitates the discovery of novel targets and the optimization of CAR construct structures. F. AI can screen suitable candidates for various gene-edited cell therapies, develop personalized treatment plans, and predict treatment outcomes