An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity

•CAR-IKZF3 degron expression and CART activity can be regulated in vitro•IMiD treatment controls CART-IKZF3 anti-tumor efficacy vivo•Such strategy aims to mitigate potential toxicities associated with therapy•The IKZF3/lenalidomide pair is portable other membrane proteins The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need better control potentially fatal that arise from adverse immune reactions. Here we present ligand-controlled system, based on the IKZF3 ZF2 ?-hairpin IMiD-inducible degron, which allows for reversible levels type I proteins, including CARs. Testing this system an established mouse xenotransplantation model acute lymphoblastic leukemia, validate ability CAR19-degron target kill CD19-positive cells displaying complete control/clearance tumor. We also demonstrate vivo when dosing US Food Drug Administration-approved drug, lenalidomide. 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Gandhi A.K. induces ubiquitination CK1alpha del(5q) MDS.Nature. 523: 183-188Crossref (461) While many approaches exist, majority limitations would limit use setting. auxin-inducible (AID) utilizes plant hormone, auxin, binds TIR1, AID-tagged (Nishimura 2009Nishimura Fukagawa Takisawa Kakimoto Kanemaki auxin-based rapid nonplant cells.Nat. Methods. 917-922Crossref (799) Limiting TIR1 risk immunogenicity mammalian cells. Another could similarly limited HaloPROTACS, HaloTag (bacteria derived) interest. Degradation induced chloroalkane linked von-Hippel-Lindau (VHL) ligand, displays poor compound drug-like qualities limiting broad applicability (Buckley 2015Buckley Raina Darricarrere Hines Gustafson Smith I.E. Miah A.H. Harling Crews HaloPROTACS: PROTACs induce fusion proteins.ACS Biol. 10: 1831-1837Crossref (203) heterobifunctional degrader, dTAG, comprises IMiD ligand (AP1867) affinity FKBP12F36V point mutant, appended interest (Nabet 2018Nabet Roberts J.M. Buckley Paulk Dastjerdi Leggett Erb Lawlor Souza al.The dTAG immediate target-specific degradation.Nat. 14: 431-441Crossref (262) likely poses low PK properties than lack FDA-approved limits therapy. shown CRBN/IMiD-binding IKZF1/IKZF3 capable heterologous (Koduri 2019Koduri McBrayer S.K. Liberzon A.C. Briggs Cho Peptidic IMiD-induced proteins.Proc. 116: 2539-2544Crossref (21) Three attractive features IKZF3-degron set apart size affect activity, source concerns, two IMiDs, pomalidomide, compounds. Here, describe relies short, 60-amino-acid, peptide (IKZF3-based tag) cytosol-facing terminus CAR, combined (lenalidomide), 1A). Such should across different CARs easily translatable into clinic, aiming first sought understand if IKZF3-based was inducing lenalidomide-dependent cytosolic oriented, carboxy (C-term) proteins. This degron/IMiD pair, thus far, cytoplasmic nuclear end, utilized 6-kDa retains sensitivity, originally identified Kronke Scholar. sequence comprised amino acids 136–180, includes six residues 2, three 3, addition non-continuous 236–249, unannotated region 4. generated directly fusing 136–180 236–249 (IKZF3 136–180_236-249, hereafter referred degron) 1B). C-term each sixteen-residue, glycine-serine linker between flexibility accessibility. V5 added C-term, ease detection. chose four targets—CD3?, CD8, CD19, CD22—in order determine whether characteristics size, length tail, number lysine had impact 1C). degron-tagged were expressed Jurkat lentiviral transduction, selected antibiotic-resistant marker. transduced treated DMSO 10 ?M 24 h, western blot analysis anti-V5 antibody. Lenalidomide-dependent observed all completed assessing localized pool treatment, flow cytometry non-permeabilized Focusing naturally entire population expressing CD19-degron-V5 stained positive reduced 20% after 6 70% 16 h measured mean fluorescence intensity (MFI) 1D). These results expressed, membrane, treatment. Encouraged our showing targeted explored means regulate CAR. focused efforts CAR19, polypeptide comprising humanized anti-CD19 (scFv) CD8 domain, CD3? (illustrated Figure before, 16-residue linker. Both untagged CAR19 (CAR19-degron) stably NFAT-luciferase (JNL) antibiotic selection retain pure populations. JNL firefly luciferase under NFAT element, allowing assessment incubation CAR-expressing lines. temporal recovery time removal, performed course/washout experiment analyzed cytometry. 1 dose sampled course h. Cells then washed media, lenalidomide, placed culture, continued additional 2A, blot, near-complete degradation, reaching steady state 4 Flow revealed CAR-degron slowly decreased 2-fold steady-state loss Upon washout, noticeably assessed post-washout. Interestingly, delayed pool, compared whole-cell lysates (western blot), very similar what 2A). When treating continuously 48 there no increase S1A). lenalidomide-induced CAR19-degron, post-lenalidomide occurs short timescale reversible. Next, titration half-maximal constant (DC50) plasma-membrane CAR19-degron. CAR19-degron-expressing measure expression. dose-responsive numbers DC50 0.028 (Figures 2B S1B). contrast, effect tuned doses being reached at achievable 0.1 (Chen 2017Chen Zhou Palmisano Clinical pharmacokinetics pharmacodynamics lenalidomide.Clin. Pharmacokinet. 56: 139-152Crossref (47) pinpoint how reduction taking place, cullin-RING-ligase (CRL), proteasome, autophagy rescue experiment. dependent CRL neddylation NAE1 MLN4924 rescued 2C). addition, proteasomal MG132 bortezomib blocking bafilomycin Given known CRBN exogenously these Using did observe any change parental S1C). indeed degraded proteasome-dependent manner endogenous IKZF3. assess CAR19-degron-mediated activation, and/or presence interferes titration, same K562 (CD19 negative) NALM6 positive) 1:1 ratio 20 proceeded IC50 ?M, concentration 50% expected, neither nor CD19-negative line. Lenalidomide 2D). Taken together, findings fast manner, enabling tunable CAR-induced activation. widely prototypical line biology, significant differences downstream events, chemokine induction (Bartelt 2009Bartelt R.R. Cruz-Orcutt Houtman J.C. Comparison receptor-induced proximal functions immortalized cells.PLoS One. e5430Crossref (54) Thus, tested degron/lenalidomide human