Photoswitchable CAR-T Cell Function In Vitro and In Vivo via a Cleavable Mediator

•Folate-O-FITC switch CAR-T cells to an activated state in a dose-dependent manner•CAR-T-induced cytotoxicity can be rapidly terminated by the cleavage of folate-O-FITC•CAR-T turned on/off on demand supplementation/cleavage folate-O-FITC•Such switchable regulated manner would not compromise antitumor effects Chimeric antigen receptor (CAR)-T-based therapeutics are breakthrough cancer treatment; however, they hampered constitutive activation, which leads worrisome side effects. Engineering as tightly controllable possible remains topic ongoing investigation. Here, we report photoswitchable approach that uses mediator for at-will regulation cells. This carries dual folate and fluorescein isothiocyanate moieties tethered ortho-nitrobenzyl ester photocleavable linker. were shown highly cytotoxic targeted only presence acted manner. The toxicity mediator, again resupplementation with without compromising tumor therapy. described here provides direction enhancing controllability likely applied other immunotherapies. Immunotherapy has emerged potential treatment cancer, chimeric (CAR)-T already use clinic (Grupp et al., 2013Grupp S.A. Kalos M. Barrett D. Aplenc R. Porter D.L. Rheingold S.R. Teachey D.T. Chew A. Hauck B. Wright J.F. al.Chimeric receptor-modified T acute lymphoid leukemia.New Engl. J. Med. 2013; 368: 1509-1518Crossref PubMed Scopus (2244) Google Scholar). premise is genetic modification express CAR recognition surface antigens specific individual patient. After infusion into patient, become upon contact their proceed proliferate destroy through several mechanisms, including extensive cell proliferation, increasing degree toxic living and, more importantly, causing increased secretion cellular factors, such cytokines, interleukins, growth factors (Barrett 2014Barrett D.M. Singh N. 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Zhao Yang Zhang Time-resolved activation proximal decaging systems.Nature. 569: 509-513Crossref (68) it thus becoming further enhance potentially proof concept, here, well-known folate-fluorescein (FITC) (Figure 1A) (Kim but conventional tether was replaced linker 1B) (Klan 2013Klan Solomek Bochet Blanc Givens Rubina Popik Kostikov Wirz Photoremovable protecting groups chemistry biology: reaction mechanisms efficacy.Chem. 113: 119-191Crossref (979) Scholar) render possible. system includes three key steps: (1) inert (folate-O-FITC), binding cells, respectively; (2) termination via photoirradiation breaks down folate-O-FITC; (3) reactivation supplementation needed. We find folate-O-FITC ability state, persistence forces remain state. With exposure light pulses nondetrimental wavelengths, switched off following breakdown mediator. versatile provide first synthesized modular assembly (Chang 2016Chang T.C. Adak A.K. Lin T.W. P.J. Y.J. Lai Liang C.F. 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Pritchard Osbourn J.K. Pope A.R. Earnshaw McCafferty Hodits R.A. Wilton Johnson Human sub-nanomolar affinities isolated large non-immunized phage display library.Nat. Biotechnol. 1996; 14: 309-314Crossref (841) subcloned lentiviral vector S2A) transduced previously 2016Ma Kazane Pugh H.M. Singer Sun S.B. al.Versatile controlling specificity activity cells.Proc. Natl. Acad. Sci. U S E450-E458Crossref (153) transduction efficiency ~90%, measured flow cytometry S2B). Proper folding staining anti-mouse IgG F(ab’)2-APC simultaneously. observed nearly all expressing bound (~50%), significantly higher level than nontransduced S2C), indicating expressed solvent exposed accessible ligand. Next, evaluated direct bind (FR)-positive KB assay FR-negative A549 negative found effector:target (E:T) ratio 3:1 10:1, demonstrated potency directing kill over 24 h (half maximal effective concentration = 109.5 ± 28.86 93.2 12.65 pM E:T respectively, Figure 2A). contrast, failed induce lysis No significant demonstrating selectivity Consistent results, detected during 24-h assay, levels interferon ? (IFN-?) interleukin-2 (IL-2), positively correlated case FR-positive 2B). also compared those parental folate-FITC; nonsignificant differences observed, installed little effect (Figures S3A S3B). tested monitoring markers anticipated, CD25 CD69 expression 2C). addition, selective formation aggregates, indicative crosslinking between visible cocultures any controls 2D). Clearly, switches medium explore whether could silenced photoirradiation, capacity irradiation promote detachment above, anti-CD3-APC-labeled anti-FR-PE-labeled cocultured 1:1 1 4°C. percentage PE/APC double-positive greater 3A, absence irradiated half experimental 365 nm, remaining serving control. that, 5 min, sharply decreased nonirradiated group, suggesting within effectively broken. parallel experiment detection photoirradiated Jurkat labeled corresponding fluorescent antibody performed, no obvious decrease intensity even when duration up 15 indeed ascribed S4A). Notably, analysis clusters fluorescence bright-field imaging showed mostly composed both 1:2, whereas nonspecific self-adhesion predominant 3B). Optimization time indicated 10 min 20 S4B). viability line, long 30 S4C). Upon validating feasibility mediator-induced designed course 4A) monitor vitro photoirradiation-mediated regulation. coculture 4 led marginal detectable cytotoxicity, accompanied mild IL-2 IFN-? 4B). Then, some cultured given pulse attenuation group. continuous increase release, plateaued overnight Subsequently, resting reactivated folate-O-FITC, manifested IFN-?. Impressively, exceeded after resupplementation, synchronous surpassed Concomitant progress formation, dispersion, reformation aggregates 4C), phenotypical changes indicators deactivation, 4D), respectively. observations well supplementation, UV irradiation, resupplementation. Therefore, concluded able shuttle acting regulator. Given vitro, next explored possibility photoswitching vivo. examined turn-on diffuse SKOV3 model (Song 2011Song Ye Q. Carpenito Ji Figini Coukos vivo persistence, localization, CAR-engineered enhanced costimulatory signaling CD137 (4-1BB).Cancer 71: 4617-4627Crossref (204) line weaker 5A 5B ), easily transplanted mice intravenous injection. treated doses exhibited inhibited proliferation dose 5C), successfully initiated investigated end, pharmacokinetics subcutaneous intratumoral injection tumor-bearing mice, present circulation pharmacokinetic properties, i.e., half-life (T1/2) ~3 mean retention ~4 h, comparable folate-FITC S5A S5B), remained local least 12 single S5C). Moreover, accessibility percutaneous burden ex analyses S5D). results suggest availability evaluating mouse intratumorally injected SCID/beige adequate number day 0, followed alternate-day dosing Inactivation achieved 6 each days 6, 8, 10, monitored 2 weeks 6A). 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Ahmed Jensen Mackall Current concepts diagnosis syndrome.Blood. 124: 188-195Crossref (1332) Because methods require suppression activity, necessary balance benefits suppressing inhibiting clinical applications. proteins, tocilizumab anakinra, blocking IL-6R IL-1R comprehensive suppress CRS. Aiming develop feasible technology, laser allows will, limiting comprising Folate-FITC, having UV-sensitive adopted because well-studied successful (Lu 2009Lu Klein Westrick Xu L.C. Santhapuram H.K. Bloomfield Howard S.J. Vlahov I.R. Ellis al.Strategy prevent drug-related hypersensitivity folate-targeted hapten cancer.A