Hoppstädter, Jessica

Kiemer, Alexandra K.

Signal Transduction and Targeted Therapy

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Universaar

Control of CAR-T cell activity in space and time: the next level of anti-tumor action

Scientific publications of the Saarland University

RESEARCH HIGHLIGHTḌ OPENControl of CAR-T cell activity in space and time: the next levelof anti-tumor actionJessica Hoppstädter1 and Alexandra K. Kiemer 1✉Signal Transduction and Targeted Therapy           (2023) 8:150 ; https://doi.org/10.1038/s41392-023-01424-5In two recent papers published in Science, advanced syntheticbiology techniques were used to overcome the limitations ofchimeric antigen receptor (CAR)-T cell therapy: In one case, CAR-Tcells were successfully introduced into and maintained in solidtumors,1 and in another approach, CAR-T cell proliferation andactivation could be turned on and off in a time-dependentmanner by drug administration.2 These approaches mightimprove therapy for a large number of solid malignancies.Cell-based therapies using CAR T cells have introduced syntheticbiology into the clinic to treat various types of blood cancers. Thepatient’s T cells are engineered with a synthetic receptor, i.e., the CAR,that helps recognize and attack cancer cells. The genetically modifiedT cells are then reinfused into the patient’s body, and a significantproportion of patients have been shown to be cured of difficult-to-treat cancers. Nevertheless, there are several limitations: On the onehand, the therapy can cause significant collateral damage by inducinga cytokine storm and attacking normal, non-cancerous cells. On theother hand, the efficiency of CAR-T cells may decrease over time, aphenomenon linked to a process denoted as T cell exhaustion.3While CAR-T cells have shown impressive success in hematolo-gical cancers, solid tumors represent the majority of malignanciesin clinical practice. They often harbor an immunosuppressivetumor microenvironment (TME), which prohibits the success ofCAR-T cell-based therapies. In contrast to hematological malig-nancies, solid tumors also raise the problem of finding the idealtarget antigen: Tumor-associated antigens overexpressed in solidtumors are expressed in many human body tissues, albeit to alesser extent. Without tumor antigen specificity, the risk ofsignificant on-target off-tumor toxicity increases substantially.4To overcome the immunosuppressant environment in malig-nant diseases, recombinant cytokines, such as interleukin (IL)-2,which is essential for T cell proliferation and activation, can beadministered. However, cytokine administration can induce severesystemic side effects resembling the clinical manifestation ofseptic shock and including symptoms such as hypotension,supraventricular tachycardia, or acute respiratory distress syn-drome. Accordingly, local production of IL-2 to promote T cellproliferation and activity would be desirable (Fig. 1).In order to make sure that T cells are activated within solidtumors and only there, Allen et al.1 employed modular syntheticNotch receptors (synNotch). Modular synNotch receptors werefirst described in 2016 as general systems that allow customizedsensing/response behaviors and, in particular, are able to drive Tcell cytokine profiles.5By coupling TCR/CAR activation and synNotch-driven IL-2production in an autocrine synNotch→IL-2 circuit, Allen et al.1have now been able to selectively propagate the therapeutic T cellpopulation within solid tumors, as shown in the syngeneic KPCpancreatic cancer model in immunocompetent C57BL/6mice. Unlike in immunodeficient mice, a paracrine approach inwhich IL-2 and CAR were expressed in separate cell populationswas ineffective in these animals. The authors speculated thatautocrine cells have preferential access to self-produced IL-2,which is particularly important in environments with competingIL-2-consuming cells (e.g., Tregs, naive T cells, and other cellswithin the TME), also referred to as IL-2 sinks, that are present onlyin the immunocompetent model. The study also showed thatconstitutive or TCR/CAR-activation-induced IL-2 expression in CAR-T cells was less efficient, most likely due to reduced T cell viabilityand the persisting sensitivity to the suppressive TME, respectively.Of note, expressing the autocrine synNotch→IL-2 circuit in CART cells improved anti-tumor activity not only in the KPC but also ina B16-F10 melanoma model. Despite its potency, the treatmentshowed no evidence of systemic cytokine toxicity or exacerbationof CAR T-cell toxicity, as the required recognition of dual antigeninputs, i.e., CAR and synNotch antigens further increased thespecificity of tumor targeting. In conclusion, combining a tumor-responsive TCR/CAR with an autocrine synNotch→IL-2 circuitresults in a potent and localized anti-tumor response, which mayprove extremely useful for the clinical use of CAR T cells.In another approach, FDA-approved small molecules were usedin order to regulate programmable orthogonal gene circuits.2 Theadvantage of this system is that the desired activities can beinduced and titrated by different drugs at specific stages oftherapy. This is realized by the use of zinc fingers (ZFs), i.e., smallDNA binding domains that can be programmed to recognize newmotifs. Activation of respective synthetic zinc finger transcriptionregulators (synZiFTR) can be regulated by safe compounds, suchas the antiviral protease-inhibiting drug grazoprevir, the breastcancer drug 4-hydroxytamoxifen/tamoxifen, or the plant hormoneabscisic acid. synZiFTRs can be employed to induce CARexpression but also to enhance CAR-T cell functions by supplyingimmunoregulatory cytokines, such as IL-2 and IL-12. With IL-2representing an autocrine T cell mitogen, activating drugs mightallow to switch T cell proliferation on and off. In addition,cytokines, such as IL-12, which are typically produced by innateimmune cells, can promote T cell activity.The authors could prove orthogonal patterns of geneactivation in a time-dependent manner: the proliferation ofprimary human T cells could be switched on beforeCAR expression was induced. The resulting dual-switchcells showed an enhanced cytotoxic activity against humanReceived: 26 February 2023 Revised: 22 March 2023 Accepted: 24 March 20231Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbrücken, GermanyCorrespondence: Alexandra K. Kiemer (pharm.bio.kiemer@mx.uni-saarland.de)www.nature.com/sigtransSignal Transduction and Targeted Therapy© The Author(s) 20231234567890();,:HER2-positive MCF10A breast cancer cells in 3D spheroids. Invivo mouse experiments using a NALM6 leukemia model inNSG, i.e., immunodeficient mice showed that dual-switch cellsare more effective than constitutive CAR-expressing cells.However, sequential production of IL-2 and CAR could leadto the loss of an advantage that activated CAR T cells have overIL-2 sinks: Allen et al.1 showed that after activation, CAR T cellsupregulate CD25, a subunit of the IL2 receptor. Thus, thisattractive novel switchable approach still has to be verified inan immunocompetent in vivo model, i.e., in the presence ofpotentially competing IL-2-consuming cells, and its suitabilityfor the therapy of solid malignancies needs to be evaluatedin vivo.Possible future developments could combine these new technol-ogies that allow controlled spatial and temporal activation of CART cells: on the one hand, proliferation and activation can be spatiallyrestricted to tumors, and on the other hand, the expression of CAR,stimulatory cytokines, or additional innate immune system adju-vants, can be controlled in a titrated and time-dependent manner.Although further studies are needed to evaluate the feasibility of thepresented systems in humans, the studies by Allen et al. and Li et al.represent significant steps for the development of advanced andindividualized CAR-T cell therapies.AUTHOR CONTRIBUTIONSJ.H. and A.K.K. wrote the article and prepared the figure. All authors have read andapproved the article.FUNDINGOpen Access funding enabled and organized by Projekt DEAL.ADDITIONAL INFORMATIONCompeting interests: The authors declare no competing interests.REFERENCES1. Allen, G. M. et al. Synthetic cytokine circuits that drive T cells into immune-excluded tumors. Science 378, eaba1624 (2022).2. Li, H.-S. et al. Multidimensional control of therapeutic human cell function withsynthetic gene circuits. Science 378, 1227–1234 (2022).3. Good, C. R. et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell 184,6081–6100.e6026 (2021).4. Marofi, F. et al. CAR T cells in solid tumors: challenges and opportunities. Stem CellRes Ther. 12, 81 (2021).5. Roybal, K. T. et al. Engineering T cells with customized therapeutic responseprograms using synthetic notch receptors. Cell 167, 419–432.e416 (2016).Fig. 1 Two novel synthetic biology approaches aim to overcome present limitations of CAR-T cell therapy. Upper panel (1): To overcometumor microenvironment (TME) suppression of CAR-T cells in solid malignancies, Allen et al.1 developed circuits in which tumor-specificsynthetic Notch (synNotch) receptors locally induced the production of IL-2 trigged by tumor antigens independent from TCR/CAR activation,thereby restricting IL-2 secretion to the tumor site and bypassing the suppressive effects of the TME. Subsequently, autocrine IL-2 signallingpromoted CAR-T cell proliferation. This approach increased CAR-T cell infiltration and tumor clearance in immunocompetent models ofpancreatic cancer and melanoma. Lower panel (2): Li et al.2 developed compact synthetic zinc finger transcriptional regulators (synZiFTRs) tocontrol the transcription of therapeutically relevant genes in CAR-T cells using FDA-approved small molecule inducers. In this proof-of-principle study, it was shown that it is possible to instruct T cells by the use of such drugs to sequentially activate multiple cellular programssuch as proliferation and antitumor activity. Studies performed in vitro in breast cancer cells and in vivo in immunodeficient mice showed thata sequential induction of superIL-2 (sIL-2, an enhanced version of IL-2 with stronger IL-2 receptor affinity) and CAR can be beneficial for theanti-tumor effect. Created with BioRender.comControl of CAR-T cell activity in space and time: the next level of. . .Hoppstädter and Kiemer2Signal Transduction and Targeted Therapy           (2023) 8:150 Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2023Control of CAR-T cell activity in space and time: the next level of. . .Hoppstädter and Kiemer3Signal Transduction and Targeted Therapy           (2023) 8:150 

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Control of CAR-T cell activity in space and time: the next level of anti-tumor action

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