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A biomimetic five-module chimeric antigen receptor(5MCAR) designed to target and eliminateantigen-specific T cellsShio Kobayashia,b,1,2, Martin A. Thelina,1
, Heather L. Parrishb, Neha R. Deshpandeb, Mark S. Leeb,Alborz Karimzadeha
, Monika A. Niewczasc,d, Thomas Serwolda,3, and Michael S. Kuhnsb,e,f,g,3
aSection of Immunobiology, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215; bDepartment of Immunobiology, TheUniversity of Arizona College of Medicine, Tucson, AZ 85724; cSection on Genetics and Epidemiology, Research Division, Joslin Diabetes Center, Boston, MA02215; dBiostatistical Consulting, Harvard Catalyst, Joslin Diabetes Center site, Boston, MA 02215; eThe BIO-5 Institute, The University of Arizona College ofMedicine, Tucson, AZ 85724; fThe Arizona Center on Aging, The University of Arizona College of Medicine, Tucson, AZ 85724; and gCancer Center, TheUniversity of Arizona, Tucson, AZ 85724
Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved October 5, 2020 (received for review June 19, 2020)
T cells express clonotypic T cell receptors (TCRs) that recognizepeptide antigens in the context of class I or II MHC molecules(pMHCI/II). These receptor modules associate with three signalingmodules (CD3γe, δe, and ζζ) and work in concert with a coreceptormodule (either CD8 or CD4) to drive T cell activation in response topMHCI/II. Here, we describe a first-generation biomimetic five-module chimeric antigen receptor (5MCAR). We show that 1) chimericreceptor modules built with the ectodomains of pMHCII assemblewith CD3 signaling modules into complexes that redirect cytotoxicT lymphocyte (CTL) specificity and function in response to the clono-typic TCRs of pMHCII-specific CD4+ T cells, and 2) surrogate corecep-tor modules enhance the function of these complexes. Furthermore,we demonstrate that adoptively transferred 5MCAR–CTLs can miti-gate type I diabetes by targeting autoimmune CD4+ T cells in NODmice. This work provides a framework for the construction of biomi-metic 5MCARs that can be used as tools to study the impact of par-ticular antigen-specific T cells in immune responses, and may holdpotential for ameliorating diseases mediated by pathogenic T cells.
CAR | TCR | pMHC | 5M-CAR | T1D
T cells scan major histocompatibility complex (MHC) moleculeson the surfaces of cells for peptide antigens (pMHC) derived
from microbes, vaccines, or tumor cells via their clonotypic T cellreceptors (TCRs). If the TCR–pMHC dwell time is sufficient,T cells becomes activated and differentiates to helper (Th), cy-totoxic (CTL), regulatory (Treg), or memory (Tm) cells that areessential for long-lived immunity (1, 2). These T cell subsets act inconcert to orchestrate complex humoral and cellular immune re-sponses; however, they can be counterproductive if activation resultsin T cell-mediated pathologies (3–6). Considerable effort has thusbeen focused on developing strategies to determine how T cells of aparticular pMHC specificity impact an immune response, enhanceT cell responses to fight infections or tumors, or mitigate T cell-mediated pathologies.Chimeric antigen receptors (CARs) have gained attention as a
technology that can redirect T cell specificity and function fornovel purposes. The archetypal design consists of a single-chainmodule (referred to here as 1MCARs) wherein ligand specificity isusually conferred via an antibody-derived Fv, while intracellularsignaling is directed through a tandem array of known signalingmotifs (Fig. 1A) (7, 8). Work to improve 1MCAR efficacy hasresulted in numerous variations on the initial design, includingfragmented domains that form 1MCARs upon final assembly (8,9). However, sensitivity and toxicity issues remain, suggesting thatthere may be practical limits to what can be achieved with variantsof this design (8–10).An alternative approach is to employ biomimetic engineering to
develop CARs that mirror the operating principles of the five-module receptors that naturally drive T cell response. In brief, the
TCR is the receptor module (module 1). It binds pMHC and relaysinformation to the immunoreceptor tyrosine-based activation motifs(ITAMs) of the associated CD3γe, δe, and ζζ signaling modules(modules 2 to 4) (11). CD4 and CD8 coreceptors represent the fifthmodule on CD4+ or CD8+ T cells; they bind MHCII or MHCI,respectively, and associate noncovalently with the Src kinase, p56Lck
(Lck) that phosphorylates CD3 ITAMs. The coreceptors sequesterLck away from TCR–CD3 complexes until either CD4 or CD8 andthe TCR bind pMHC, at which point Lck is recruited to the CD3ITAMs to initiate signaling (12–14). These five-module pMHC-receptors can signal in response to a single agonist pMHC, directCTL killing against just three pMHCs, and direct distinct T cellresponses according to the quantity and quality of the pMHC (1, 2,15, 16). Biomimetic versions of multimodule CARs could expandand enhance the applications of CAR-T cell therapy.
Significance
T cells are driven by five-module receptor complexes composed ofa T cell receptor (TCR) module, three CD3 signaling modules, and acoreceptor module. We adapted this architecture to engineer abiomimetic five-module chimeric antigen receptor (5MCAR) thatconsists of a chimeric receptor module, composed of the antigenrecognized by pathogenic T cells fused to elements of the TCR thatfacilitate assembly with the three CD3 modules, and a surrogatecoreceptor module that enhances signaling. We show that cyto-toxic T lymphocytes expressing 5MCARs can rapidly eliminatepathogenic T cells and prevent them frommediating autoimmunedisease inmice. Overall, this work describes a CAR platform and itsuse in mitigating T cell-mediated pathologies.
Author contributions: S.K., M.A.T., H.L.P., N.R.D., T.S., and M.S.K. designed research; S.K.,M.A.T., H.L.P., N.R.D., M.S.L., and A.K. performed research; S.K., M.A.T., H.L.P., N.R.D.,M.S.L., and M.S.K. contributed new reagents/analytic tools; S.K., M.A.T., H.L.P., N.R.D.,M.S.L., M.A.N., T.S., and M.S.K. analyzed data; S.K., T.S., and M.S.K. wrote the paper;and T.S. and M.S.K. directed research.
Competing interest statement: M.S.K. and T.S. have disclosed an outside interest in Mod-ule Therapeutics to the University of Arizona and the Joslin Diabetes Center. Conflicts ofinterest resulting from this interest are being managed by The University of Arizona andJoslin Diabetes Center in accordance with their policies. M.S.K. and T.S. are inventors onpatent filings covering the intellectual property tested in this study.
This article is a PNAS Direct Submission.
Published under the PNAS license.1S.K. and M.A.T. contributed equally to this work.2Present address: Department of Immunobiology, The University of Arizona College ofMedicine, Tucson, AZ 85724.
3To whom correspondence may be addressed. Email: [email protected] [email protected].
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2012495117/-/DCSupplemental.
First published November 2, 2020.
28950–28959 | PNAS | November 17, 2020 | vol. 117 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.2012495117
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Fig. 1. Structure, assembly, and function of biomimetic 5MCARs. (A) The five modules that drive pMHC-specific T cell activation (TCR, CD3γe, CD3δe, CD3ζζ, andCD4/CD8) are compared with a third-generation single-module CAR (1MCAR). (B) The TCR–CD3 complex and CD8 are compared with the CRMpMHCII
–CD3 complexand CD80–Lck ScoR of 5MCARs. (C) Flow cytometry plots showing I-Ek, CD3, and CD80 expression on parental 58α−β− cells and 5MCAR–58α−β− cells. (D) FFLISA ofTCR–CD3 and CRMpMHCII
–CD3 complexes. Anti-CD3e beads incubated without lysate (gray), or with lysates from TCR–CD3+ 58α−β− cells or 5MCAR–58α−β− cells(black), were analyzed by flow cytometry for TCRβ or CRMpMHCIIβ (GFP) and CD3ζ association. (E) IL-2 production by K5:I-Ek, MCC:I-Ek, T102S:I-Ek, T102G:I-Ek, andHb:I-Ek 5MCAR–58α−β− cells after 16-h coculture with parental M12 B cells (TCR−, CD28−), 2B4 TCR+ M12 cells, or 2B4 TCR+ CD28+ M12 cells as measured by ELISA.Results are shown as the mean ± SD of triplicates (*P < 0.05, **P < 0.01, and ****P < 0.0001, one-way ANOVA with Tukey’s posttest). (F) 5MCAR–CTL killing ofCD4+ 5c.c7 TCR Tg T cell targets. Percent killing of targets cocultured with control (Hb:I-Ek) or specific (MCC:I-Ek) 5MCAR–CTLs was measured by flow cytometry andis presented relative to number of targets cultured in the absence of 5MCAR–CTLs. Results are shown as the mean ± SD of triplicates (*P < 0.05, ***P < 0.001, and****P < 0.0001 by unpaired, two-tailed t test). All data are representative of at least two experiments. Figure is related to SI Appendix, Fig. S1.
Kobayashi et al. PNAS | November 17, 2020 | vol. 117 | no. 46 | 28951
IMMUNOLO
GYAND
INFLAMMATION
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Here, we engineered a biomimetic five-module chimeric antigenreceptor (5MCAR). The chimeric receptor module (CRM) consistsof the ectodomain of pMHCII (CRMpMHCII) and elements of theTCR that facilitate association with the three CD3 signalingmodules. This design maintained the natural receptor:ligandbinding kinetics that drive T cell activation, and enabled us to re-direct CTL specificity against clonotypic TCRs expressed onpathogenic T cells. We also engineered a surrogate coreceptor(ScoR) composed of CD80 fused to Lck as the fifth module of our5MCAR. We report that 5MCAR–CTLs can kill CD4+ T cellsin vitro and in vivo in a TCR-specific manner, and that 5MCAR–CTLs targeting autoimmune CD4+ T cells can prevent disease innonobese diabetic (NOD) mouse models. Our results demonstratethat biomimetic 5MCARs can redirect CTL specificity and function.
ResultsDesign and In Vitro Characterization of a First-Generation 5MCAR. Inkeeping with our biomimetic strategy, our first-generation 5MCARmaintained pMHCII–TCR interactions as the core recognitionevent to preserve the biophysical properties that naturally mediateantigen recognition. We engineered a pMHCII-based chimericreceptor module (CRMpMHCII, module 1) to assemble with theCD3γe, δe, and ζζ signaling modules (modules 2 to 4) into afunctional complex (Fig. 1B and SI Appendix, Fig. S1A). For allCRMpMHCII modules used herein, we fused the MHCIIα andMHCIIβ ectodomains (ECDs) to the connecting peptides (CPs),transmembrane domains (TMDs), and intracellular domains(ICDs) of the TCRα and TCRβ subunits, respectively, to mediateassembly with the CD3γe, δe, and ζζ modules. The peptide anti-gen was N-terminally tethered to the MHCIIβ region to ensureexpression of a single CRMpMHCII species, while mEGFP wastethered at the C terminus to aid in detection. We also engineereda surrogate coreceptor (ScoR, module 5) composed of the CD80ECD and TMD fused to Lck. CD80 naturally interacts with CD28on naive T cells, as well as CTLA-4 on antigen experienced T cells.The logic here is that, due to the close spatial localization ofCD28, CTLA-4, and the TCR early during immunological synapseformation, ScoR binding to CD28 or CTLA-4 would localize Lckin proximity to the CRMpMHCII
–CD3 complex (Fig. 1B and SIAppendix, Fig. S1A) (17, 18).To evaluate expression and function of our prototype 5MCAR,
we retrovirally transduced 58α−β− cells, a TCR− T cell hybridoma,to express the CRMpMHCII and the ScoR. The CRMpMHCII wasbuilt with the murine MHCII I-Ek presenting the moth cyto-chrome C peptide (MCC88–103; MCC:I-Ek). Flow cytometryanalysis revealed cell surface expression of MCC:I-Ek, CD3e, andCD80 on the transduced but not parental 58α−β− cells (Fig. 1C).Proportional expression of MCC:I-Ek and CD3e indicated that theCRMpMHCII module assembled with the CD3 modules. To con-firm that TCR–CD3 and CRMpMHCII
–CD3 complexes assembleanalogously, beads coated with anti-CD3e antibodies were incu-bated with lysates from TCR+ and 5MCAR+ 58α−β− cells and thenstained with anti-CD3ζ antibodies for analysis by flow cytometry.The TCR–CD3+ and CRMpMHCII
–CD3+ samples showed similarlevels of GFP and CD3ζ signal (Fig. 1D), demonstrating that theTCRβ–GFP and CRMpMHCIIβ
–GFP coimmunoprecipitate (IP)with CD3e and CD3ζ subunits at similar levels.To evaluate how CRMpMHCII
–TCR and ScoR–CD28 interac-tions impact responsiveness, we generated 5MCAR–58α−β− cellsexpressing CRMpMHCII built with pMHCII that bind the 2B4 TCRwith defined biophysical properties and drive defined biologicaloutputs (SI Appendix, Fig. S1 B and C): K5:I-Ek (KD, 2.9 μM; t1/2,49.5 s; strong agonist); MCC:I-Ek (KD, 8.7 μM; t1/2, 36.5 s, ago-nist); T102S:I-Ek (KD, 90.0 μM; t1/2, 3.4 s, weak agonist); T102G:I-Ek (antagonist, too weak to measure); and Hb:I-Ek (null ligand)(19). IL-2 production was measured after coculture with parentalM12 cells (TCR−CD28−) or M12 cells transduced to express the2B4 TCR and CD3 signaling modules with or without CD28. No
IL-2 was produced in response to parental M12 cells by any ofthe 5MCAR–58α−β− cells (Fig. 1E). K5:I-Ek and MCC:I-Ek
5MCAR–58α−β− cells produced IL-2 in response to TCR+CD28−
M12 cells, and the IL-2 levels were significantly increased in re-sponse to TCR+CD28+ M12 cells. T102S:I-Ek 5MCAR–58α−β−cells produced significant amounts of IL-2 only in response toTCR+CD28+ M12 cells. Finally, T102G:I-Ek and Hb:I-Ek
5MCAR–58α−β− cells produced insignificant amounts of IL-2 inresponse to any of the targets. Because M12 cells are a B celllymphoma, and do not make IL-2, the IL-2 measured in this assaywas produced by the 5MCAR–58α−β− cells. These data establishthat 5MCAR-driven responses mirror previously described rela-tionships between affinity, half-life, and potency for TCR:pMHCIIinteractions (20). Furthermore, the increased responses of5MCAR–58α−β− cells to targets expressing CD28 demonstrate thatthe ScoR enhances 5MCAR-driven responses.We next asked whether 5MCARs can redirect CTLs to kill
CD4+ T cells expressing the 5c.c7 TCR, which also recognizesMCC:I-Ek as an agonist pMHCII (19). Activated CD8+ T cellsfrom B10.A mice were transduced to generate 5MCAR–CTLsexpressing a MCC:I-Ek
–based CRMpMHCII to target 5c.c7 T cells,and 5MCAR–CTLs expressing an Hb:I-Ek
–based CRMpMHCII
were generated as negative controls (12). Flow cytometry showedmore killing of target T cells by the specific 5MCAR–CTLs whencompared with the control 5MCAR–CTLs at all effector:targetratios (Fig. 1F). No killing was observed in the control group,demonstrating that 5MCAR–CTLs kill CD4+ T cell targets in vitroin a CRMpMHCII-specific manner.
Targeting Pathogenic CD4+ T Cells with 5MCAR–CTLs In Vitro. To askwhether 5MCAR–CTLs can kill autoreactive CD4+ T cells, weused CD4+ T cells from BDC2.5 TCR transgenic (Tg) mice astargets because they recognize a self-peptide antigen derivedfrom pancreatic beta cells presented on the MHCII I-Ag7 andthey mediate beta-cell destruction and type-1 diabetes (T1D)when transferred into NOD-SCID mice (21, 22). Accordingly, wegenerated a specific CRMpMHCII built with the mimotope peptideRLGL-WE14 in I-Ag7 that binds the BDC2.5 TCR, and a negativecontrol CRMpMHCII built with a self-peptide from glucose phos-phoisomerase (GPI282–294) presented in I-Ag7 that is not associatedwith T1D (23, 24). 5MCAR–CTLs generated from NOD miceexpressed the CRMpMHCII and had increased CD80 levels com-pared with the natural levels of CD80 that are induced after acti-vation (SI Appendix, Fig. S2A). The majority of the 5MCAR–CTLswere CD44hi CD62L−, Granzyme B+, Perforin+, FasLlo, and Fas−
(SI Appendix, Fig. S2 B–D), and thus primed to kill by releasingcytotoxic granule proteins and by engaging Fas (25).To evaluate whether NOD 5MCAR–CTLs could kill their tar-
gets in a TCR-specific manner, we mixed specific (RLGL-WE14:I-Ag7) and control (GPI:I-Ag7) 5MCAR–CTLs with BDC2.5 T cellsat varying ratios and then enumerated the remaining targets afterculture. The specific, but not the control, 5MCAR–CTLs killed thetarget BDC2.5 T cells, while neither 5MCAR–CTL populationkilled polyclonal CD4+ T cells from a NOD mouse at detectablelevels (Fig. 2A). These results provide further evidence that5MCARs can redirect CTLs to specifically kill CD4+ T cells viarecognition of their clonotypic TCR.Because CTL killing requires less signaling than cytokine pro-
duction or proliferation, we investigated whether 5MCAR–CTLsmake IFNγ or proliferate in response to target CD4+ T cells (26).We found that specific 5MCAR–CTLs made IFNγ in response totarget BDC2.5 T cells, but not in response to the polyclonal NODCD4+ T cells, while neither the control 5MCAR–CTLs nor eithertarget population produced IFNγ (Fig. 2B). Additionally, specific5MCAR–CTLs proliferated in response to BDC2.5 T cells, whilethe control 5MCAR–CTLs did not (Fig. 2C). These data demon-strate that 5MCAR–CTLs can make cytokines and proliferate inresponse to CD4+ T cell targets expressing the appropriate TCR.
28952 | www.pnas.org/cgi/doi/10.1073/pnas.2012495117 Kobayashi et al.
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We also tested the contribution of the ScoR module to5MCAR–CTL function. It did not enhance killing of CD4+ T celltargets (SI Appendix, Fig. S2E), a lower-order function requiringfew cognate ligands (16, 26), but did increase the number ofspecific 5MCAR–CTLs making IFNγ after 6-h stimulation(Fig. 2D), the amount of IFNγ secreted after 24 h (Fig. 2E), andthe number of specific 5MCAR–CTLs that divided after 3 to 4 din culture (Fig. 2F). Given that the ScoR makes a significantcontribution to these important responses, we moved forwardwith the full 5MCAR system for in vivo analysis.
5MCAR–CTLs Kill Pathogenic CD4+ T Cells In Vivo. We next measured5MCAR–CTL killing of BDC2.5 T cell targets in vivo. Specific orcontrol 5MCAR–CTLs were transferred into NOD mice followed12 h later by mRaspberry (mRasp)+ BDC2.5 T cell targets mixedwith control CTV-labeled NOD CD4+ T cell targets as a refer-ence population. A separate cohort received only the mixture oftargets (no 5MCAR–CTLs). Spleens were harvested after 5.5 hand analyzed by flow cytometry (SI Appendix, Fig. S3). Killingwas quantified by measuring changes in the frequency of specifictargets relative to the reference targets (Fig. 3). The mice thatdid not receive 5MCAR–CTLs, and those that received control5MCAR–CTLs, had a mean ratio of 0.48 and 0.55 BDC2.5:NODT cell targets, respectively, while those receiving specific5MCAR–CTLs had a mean ratio of ∼0.27, indicating ∼50%killing of the target BDC2.5 T cells. 5MCAR–CTLs can thereforerapidly find and eliminate their targets in vivo.
5MCAR–CTLs Prevent Autoimmune Diabetes in NOD-SCID Mice. To askwhether 5MCAR–CTLs could prevent BDC2.5 T cell-mediatedbeta-cell destruction and diabetes, we used a model in which,after transferring BDC2.5 T cells into NOD-SCID mice, lym-phocytic infiltration of the pancreas is observed 5 d later, severeinsulitis of most islets occurs by days 6 to 8, and diabetes onsetoccurs between days 9 and 14 (SI Appendix, Fig. S4 A and B).After transferring BDC2.5 T cells into NOD-SCID mice (day 0),the mice were divided into three cohorts on day 1: BDC2.5-only(untreated), control 5MCAR-CTL–treated, and specific 5MCAR-CTL–treated (Fig. 4A). All untreated and control 5MCAR-CTL–treated mice developed diabetes within 10 d, while allmice treated with specific 5MCAR–CTLs remained diabetes-freefor the duration of the 3-wk experiment (Fig. 4B). The pan-creases of the untreated and control 5MCAR-CTL–treated dia-betic mice showed widespread lymphocytic infiltration and isletdestruction, while pancreases from mice treated with specific5MCAR–CTLs appeared free of infiltration (Fig. 4C). Also,disease amelioration corresponded with nearly complete elimi-nation of BDC2.5 T cells in the spleens of the specific 5MCAR-CTL–treated mice (Fig. 4 D and E), while the 5MCAR–CTLsremained 21 d after transfer indicating that specific 5MCAR–
CTLs have the potential for long-term engraftment (Fig. 4 Dand E).
5MCAR–CTLs Halt Ongoing Insulitis. We next asked whether5MCAR–CTLs could prevent diabetes after islets are invadedby lymphocytes and have lost structural integrity (Fig. 5A and
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Fig. 2. 5MCAR–CTLs can target autoimmune CD4+ T cells. (A) 5MCAR–CTLkilling of BDC2.5 CD4+ T cell targets (Left) or control NOD CD4+ T cells(Right) after coculture with control (GPI:I-Ag7) or specific (RLGL-WE14:I-Ag7) 5MCAR–CTLs, presented as in Fig. 1 (****P < 0.0001 by unpaired, two-tailed t test). (B) IFNγ production by 5MCAR–CTLs. 5MCAR–CTLs incubatedwith target BDC2.5 T cells or NOD T cells for 6 h were stained for intra-cellular IFNγ and gated on live cells. Representative plots are shown. Thebar graph shows the frequency of IFNγ-producing 5MCAR–CTLs gated onGFP+CD8+mRasp−CD4− cells (****P < 0.0001 by one-way ANOVA andTukey’s posttest). (C ) Proliferation of CellTrace Violet-labeled 5MCAR–CTLsafter coculture with or without target BDC2.5 T cells for 3 d. Representa-tive histograms show CellTrace Violet dilution of 5MCAR–CTLs. Bar graphsshow the number of dividing 5MCAR–CTLs (****P < 0.0001 by one-wayANOVA and Tukey’s multiple-comparison test). (D) The numbers of spe-cific 4MCAR– (without ScoR) or 5MCAR–CTLs (with ScoR) making IFNγ weremeasured by flow cytometry after coculture with BDC2.5 T cells for 6 h. (E)IFNγ was measured by ELISA for supernatants after specific 4MCAR– (withoutScoR) or 5MCAR– (with ScoR) CTLs coculture with target BDC2.5 T cells for 24 h.(F) The number of divided specific 4MCAR– (without ScoR) or 5MCAR–CTLs (withScoR) were determined by flow cytometry after coculture with target BDC2.5T cells for 3 or 4 d. Columns in A–C show mean ± SD of triplicates or qua-druplicates. Each graph is representative of three independent experiments.Paired data points (connected by lines) in D–F represent the mean ± SD oftriplicates or quadruplicates for independent experiments (*P < 0.05 by paired,two-tailed t test). Each line represents an independent experiment. Figure isrelated to SI Appendix, Fig. S2.
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Fig. 3. 5MCAR–CTLs kill target T cells in vivo. Control (GPI:I-Ag7) or specific(RLGL-WE14:I-Ag7) 5MCAR–CTLs were transferred into NOD mice followed12 h later with a mixture of mRasp+ BDC2.5 CD4+ T cell targets and CellTraceViolet-labeled NOD CD4+ T cells as a reference population. Then, 5.5 h later,the spleens were analyzed by flow cytometry to evaluate target cell killing.Plots show analysis of target and control cells in representative mice, gatedlive CD3+CD4+ cells (full gating shown in SI Appendix, Fig. S3). The graphshows the ratio of target cells/control cells as mean ± SD. Each point rep-resents the ratio from a single spleen (*P < 0.05, ***P < 0.001 by one-wayANOVA and Tukey’s posttest). Data are shown as mean ± SD of combinedfrom two independent experiments. Figure is related to SI Appendix, Fig. S3.
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Fig. 4. 5MCAR–CTLs prevent BDC2.5 CD4+ T cell-induced T1D in NOD-SCID mice. (A) BDC2.5 CD4+ T cells were adoptively transferred into NOD-SCID miceon day 0. On day 1, the mice received 5MCAR–CTLs or were left untreated. (B) Survival curve shows the percentage of diabetes-free mice that were treatedwith control (GPI:I-Ag7) 5MCAR–CTLs, specific (RLGL-WE14:I-Ag7) 5MCAR–CTLs, or left untreated (BDC2.5 only) (**P < 0.01, ***P < 0.001 by log-rank test). (C)Pancreases of representative mice from each group are shown stained with hematoxylin–eosin (magnification: 4×). Black box Inset shows clear islet (20×; scalebar, 200 μm). (D and E) Analysis of mRasp+ target CD4+ T cells and GFP+ 5MCAR–CTLs in spleens of treated and untreated mice. (D) Gating schematic andrepresentative dot plots show frequencies of targets and 5MCAR–CTLs in spleens. (E) Graphs show absolute cell counts of BDC2.5 CD4+ T cells or 5MCAR–CTLs.The days of recipient analysis are shown for each group. Each point represents an individual recipient. The horizontal lines indicate mean ± SD. **P < 0.01,****P < 0.0001 by one-way ANOVA and Tukey’s posttest (Bottom, Left) or unpaired, two-tailed t test (Bottom, Right). ns means not statistically significant.Data are representative of two similar independent experiments. Numbers of mice/group are indicated in the figure. Figure is related to SI Appendix, Fig. S4.
28954 | www.pnas.org/cgi/doi/10.1073/pnas.2012495117 Kobayashi et al.
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SI Appendix, Fig. S1 4A). BDC2.5 T cells were transferred intoNOD-SCID mice (day 0), and the recipients were divided intothree cohorts on day 7: BDC2.5-only (untreated), control5MCAR-CTL–treated, and specific 5MCAR-CTL–treated. Alluntreated and control 5MCAR-CTL–treated mice developeddiabetes by day 13, while eight of nine specific 5MCAR-CTL–treated mice remained diabetes-free (Fig. 5B). Protectedmice maintained normal weight and blood glucose throughoutthe experiment (SI Appendix, Fig. S5 A and B). Furthermore,histological sections from the pancreases of specific 5MCAR-CTL–treated nondiabetic mice showed a lack of insulitis,whereas the integrity of the islets was destroyed in the untreatedand control 5MCAR-CTL–treated mice (Fig. 5C). Importantly,for specific 5MCAR-CTL–treated animals, the BDC2.5 T cellshad been eliminated from the spleens and pancreatic lymphnodes (pLNs) (Fig. 5 D and E and SI Appendix, Fig. S5C). Evenin the one specific 5MCAR-CTL–treated animal that becamediabetic, the BDC2.5 T cells were nearly eliminated by day 11(Fig. 5E, highlighted as red symbol). 5MCAR–CTLs alsoremained in the spleens and pLNs of recipient mice for theduration of the experiment (Fig. 5 D and E and SI Appendix, Fig.S5C), confirming that they can engraft for several weeks.
5MCAR–CTLs Home to and Persist in the Pancreas.We used the sameexperimental setup to evaluate where the 5MCAR–CTLs trafficand how the numbers of targets and 5MCAR–CTLs change overtime by performing analysis on days 7, 8, 10, 15, and 36. In micetreated with control 5MCAR–CTLs, the number of BDC2.5T cells in the spleens and pLNs remained relatively constantfrom days 7 to 10, but increased from days 10 to 15 as the micedeveloped diabetes (Fig. 6A and SI Appendix, Fig. S6). In thepancreases, BDC2.5 T cells increased by day 10 and, by day 15,had expanded ∼100-fold (Fig. 6B). In mice treated with specific5MCAR–CTLs, a reduction in the frequency and number ofBDC2.5 T cells was evident by day 10 in the pLNs, in the spleensby day 15, and as early as day 8 in the pancreases. Importantly,BDC2.5 T cells were barely detectable by day 15 in any of theanalyzed tissues, and none were detected at the experimentalendpoint at day 36.Regarding the 5MCAR–CTLs, both the control and specific
populations were detected in the spleen, pLN, and inflamedpancreases within the first 24 h posttransfer (day 8) (Fig. 6 A andB and SI Appendix, Fig. S6). The numbers of control 5MCAR–
CTLs stayed relatively constant in the spleens from days 8 to 15,increased slightly over time in the pLNs, and peaked at day 10 inthe pancreases, while the number of specific 5MCAR–CTLsremained relatively constant in the spleens, pLNs, and pan-creases from days 8 to 15. The number of specific 5MCAR–CTLswas significantly lower than the control 5MCAR–CTLs at day 10in the pancreases, mirroring the loss of BDC2.5 T cells. Finally,the specific 5MCAR–CTLs were present in the spleens, pLNs,and pancreases at the termination of the experiment on day 36.Overall, these results indicate that 5MCAR–CTLs can rapidly
home to inflamed pancreases, as well as to lymphoid tissues, andeliminate their target cells from those tissues in a TCR-specificfashion. Furthermore, they can persist in these tissues for weeks.
A Panel of 5MCAR–CTLs Can Prevent Autoimmune Diabetes in NODMice. Finally, we tested the efficacy of 5MCAR–CTL treatmentin the NOD mouse model of spontaneous T1D where insulitiscan be detected by 10 wk of age, diabetes occurs starting at 12wk, and due to a sexual dimorphism of disease incidence, ∼80%of female mice become diabetic by 45 wk (27–29). In NOD miceand humans, T1D is thought to be initiated by T cells reactive toone or a small number of self-pMHC, as mice lacking a keyamino acid in an insulin peptide (INSB9–23) presented by I-Ag7
are protected from diabetes development (30, 31). Furthermore,CD4+ T cells specific for newly discovered hybrid insulin
peptides (HIPs) presented by I-Ag7, including the BDC2.5 T cellsused here, can induce diabetes in adoptive transfer models (32).We therefore generated three additional CRMpMHCII, present-ing either the insulin peptide (INSB9–23) or two hybrid insulinpeptides (HIP2.5, HIP6.9) in I-Ag7, to test whether a mixture of5MCAR–CTLs could prevent diabetes in NOD mice.To determine whether 5MCAR–CTLs that target CD4+ T cells
specific to these self-pMHCII can protect against autoimmunediabetes, we transferred a mixture of specific (INSB:I-Ag7,HIP2.5:I-Ag7, HIP6.9:I-Ag7, and RLGL-WE14:I-Ag7) or control(GPI:I-Ag7) 5MCAR–CTLs into neonatal male and female NODmice. We used neonatal mice because 1) disease-initiating au-toimmune CD4+ T cells are thought to emerge from the thymussoon after birth, 2) physiological beta-cell death at 2 wk of age isreported to trigger priming of self-reactive T cells, and 3) wewanted to eliminate the target CD4+ T cells before they couldprovide help to autoimmune CD8+ T cells (33, 34). A thirdgroup of newborn mice was left untreated to establish a baselinefor T1D incidence.To ask whether 5MCAR–CTLs persisted throughout the crit-
ical period of early autoimmune T cell development, earlyinsulitis, and beta-cell apoptosis, we assessed 5MCAR–CTL en-graftment in a cohort of male mice at 13 wk. 5MCAR–CTLs weredetected in both the control and specific groups of mice(Fig. 7A), verifying that the 5MCAR–CTLs can engraft long-termin NOD mice and were present during the period of life that isthought to be critical for spontaneous T1D in these animals.Females were followed for 315 d to assess diabetes develop-
ment. Diabetes onset and progression for those treated withcontrol 5MCAR–CTLs mirrored that of the untreated group(Fig. 7B). At day 315, 14 of 16 untreated mice and 13 of 16control 5MCAR-CTL–treated mice had developed diabetes whileonly 7 of 16 mice treated with the specific 5MCAR–CTLs de-veloped diabetes. Analysis with the Cox proportional hazardsmodel revealed that the risk of diabetes incidence in mice withthe specific 5MCAR–CTLs was approximately three times lowerwhen compared with the untreated and control mice. Hazardratios (HRs) and the respective confidence intervals (CIs) for thespecific group in comparison to the untreated group were asfollows: HR (95% CI), 0.33 (0.13, 0.82); P = 0.017. For thespecific group in comparison to the control group they were asfollows: HR (95% CI), 0.36 (0.14, 0.91); P = 0.030. There wereno differences in the diabetes incidence between the control anduntreated groups: HR (95% CI), 0.91 (0.43, 1.94); P = 0.808.These data demonstrate that a mixture of 5MCAR–CTLs tar-geting autoimmune CD4+ T cells with a limited specificity cansignificantly reduce the incidence of T1D in NOD mice.A cohort of surviving mice (four males and five females) were
killed after day 315 for analysis of pooled spleens and lymphnodes. We detected 5MCAR–CTLs in two out of nine mice (SIAppendix, Fig. S7A). To explore whether additional 5MCAR–
CTLs might be present below the limit of detection, we trans-ferred BDC2.5 T cells into the remaining diabetes-free mice topotentially expand out the BDC2.5-specific 5MCAR–CTLs. Inthese mice, two of four recipients had detectable specific5MCAR–CTLs (SI Appendix, Fig. S7B). We cannot know whetherthese 5MCAR–CTLs expanded in response to the BDC2.5T cells, or would have been detectable without them. Never-theless, the data indicate that 5MCAR–CTLs engraft up to a yearor longer in a subset of animals despite the CD8+ T cell attritionthat normally occurs with aging (35).
DiscussionHere, we evaluated the function of our first-generation biomi-metic 5MCAR. We show that 5MCARs can redirect CTL speci-ficity against clonotypic TCRs expressed by CD4+ T cells and canco-opt CTL functions, including IFNγ production, proliferation,and killing, in a TCR-specific manner. We also demonstrate that
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Fig. 5. 5MCAR–CTLs prevent diabetes after initiation of insulitis. (A) NOD-SCID mice receiving BDC2.5 CD4+ T cells on day 0 were treated with 5MCAR–CTLson day 7, or left untreated. (B) Survival curve shows the percentage of diabetes-free mice treated with either control (GPI:I-Ag7) 5MCAR–CTLs, specific(RLGL-WE14:I-Ag7) 5MCAR–CTLs, or left untreated (BDC2.5 only) (**P < 0.01, ***P < 0.001 by log-rank test). (C) Pancreases of representative mice from eachgroup, stained with hematoxylin–eosin, are shown (magnification: 4×). Black box Inset shows clear islet (20×; scale bar, 200 μm). (D and E) Analysis of mRasp+
CD4+ T cells and GFP+ 5MCAR–CTLs in spleens of treated and untreated mice. (D) Gating schematic for the analysis and representative dot plots show fre-quencies of targets and 5MCAR–CTLs in spleens. (E) Graphs show absolute cell counts of BDC2.5 T cells or 5MCAR–CTLs. The days of recipient analysis are shownfor each group. Each data point represents an individual recipient. The horizontal lines indicate mean ± SD. *P < 0.05 by one-way ANOVA and Tukey’smultiple-comparison test (Bottom, Left) or by unpaired, two-tailed t test (Bottom, Right). ns means not statistically significant. Data are combined from twoindependent experiments. Numbers of mice/group are indicated in the figure. Figure is related to SI Appendix, Fig. S5.
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5MCAR–CTLs can rapidly eliminate pathogenic CD4+ T cells andneutralize their detrimental impact in mouse models of T1D. The5MCAR design, biological implications of the data, potential ap-plications, and thoughts on the future of the technology arediscussed below.
5MCARs uniquely integrate three key biomimetic principles.First, the receptor and CD3 signaling modules assemble intocomplexes that possess a full complement of 10 ITAMs andother key motifs that mediate or regulate signaling through theTCR–CD3 complex (11, 36–41). This feature was incorporatedinto the design because the multiplicity of ITAMs that getphosphorylated during signaling via the TCR, or 1MCARs, in-fluences subsequent T cell function; furthermore, we speculatethat preserving access to the natural signaling apparatus mayserve an important safety function as improper signaling can leadto dysregulated T cells (41–44). We note that a recently reportedfour-module TruC CAR also takes advantage of the native CD3signaling modules (45).For the second key design principle, we maintained the natural
biophysical properties of receptor:ligand interactions thatevolved to mediate T cell activation. T cells are normally selected
and activated within a narrow kinetic window of TCR–pMHCinteractions (46, 47). Our 5MCAR–58α−β− cells demonstrate that5MCAR-driven responses follow defined hierarchies for TCR–
pMHCII interactions. Interestingly, we observed low, statisticallyinsignificant, levels of IL-2 made by 5MCAR–58α−β− cells inresponse to antagonist and null TCR+CD28+ targets, which mayreflect the weak intrinsic TCR–pMHCII interactions we char-acterized in a related experimental system (48). Importantly, our5MCAR–CTL data indicate that such interactions are insufficientto drive measurable responses, and we observed no evidence of5MCAR–CTL pathology in recipient mice.Finally, our biomimetic design includes a surrogate coreceptor
module. CD4 and CD8 sequester Lck away from the CD3ITAMs in the absence of cognate pMHC, meaning that theyshould keep Lck away from ITAMs associated with CARs andprevent optimal signaling (14). Our ScoR provides the oppor-tunity for Lck to be positioned proximally to the CRMpMHCII
–
CD3 ITAMs within the immunological synapse between a5MCAR–CTL and a CD28+ or CTLA-4+ target T cell. An ap-pealing aspect of using a ScoR is that alternate designs could beused to enhance targeting if needed. Altogether, the data
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Fig. 6. 5MCAR–CTLs migrate to the pancreas and eliminate BDC2.5 CD4+ T cells. (A and B) NOD-SCID mice receiving BDC2.5 T cells (day 0) were either treatedwith control (GPI:I-Ag7) 5MCAR–CTLs, specific (RLGL-WE14:I-Ag7) 5MCAR–CTLs, or killed prior to treatment on day 7. Treated groups were killed on day 8, 10, 15or 36. Flow cytometry was used to determine the frequency and number of mRasp+ CD4+ BDC2.5 T cells and GFP+ 5MCAR–CTLs in the pLNs (A) or pancreases(B). Representative dot plots show frequencies of target BDC2.5 T cells, and 5MCAR–CTLs (Top, pregated on live, CD11b− cells). Graphs show the number ofCD4+ BDC2.5 T cells (Left) or the number of 5MCAR–CTLs at each time point (Right). Data show combined results from two independent experiments asmean ± SD. Three to six mice were analyzed for each group and time point (*P < 0.05, **P < 0.01 by unpaired, two-tailed t test between the control andspecific 5MCAR–CTLs groups). † means no data. Figure is related to SI Appendix, Fig. S6.
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presented here demonstrate that the three operating principlesdiscussed above can be successfully integrated into a functional5MCAR system.While this study focused on engineering and testing our
5MCARs, we also made observations that speak to the usefulnessof 5MCAR–CTLs in research. We show that treating neonatalNOD mice with a mixture of 5MCAR–CTLs that target a limitednumber of known pMHCII-specific autoimmune CD4+ T cellscan significantly decrease diabetes incidence, consistent with theidea that one or a limited number self-pMHCII reactivities ini-tiate disease. These data indicate that 5MCAR–CTLs can be usedto eliminate specific T cell populations in mice and then study
how particular immune responses are initiated and proceed intheir absence.Our results also point to therapeutic applications for treating
diseases mediated by pathogenic T cells. We show that5MCAR–CTLs can reverse ongoing insulitis and decrease T1Ddisease incidence in NOD mice. 1MCARs targeting a singlepathogenic CD8+ T cell population can also decrease diabetesincidence in NOD mice (49); therefore, targeting a mixture ofCD4+ and CD8+ T cell clonotypes with 5MCAR–CTLs in pa-tients with preclinical disease may be effective as a preventativetherapy (50). Furthermore, another prime target for 5MCAR–
CTL therapy could be TCR+ lymphomas, which are typicallyclonal and thus would not require targeting multiple specificities(51). Significant advances have been made in identifying pMHCthat interact with a particular TCR (52, 53). Using suchmethods to identify a pMHC that binds a T lymphoma-derivedTCR would enable the rapid generation of 5MCAR–CTLs totarget TCR+ tumors. Importantly, the lack of weight loss orovert signs of distress by the mice used in our studies suggestthat the 5MCAR–CTLs themselves are not pathogenic. In ad-dition, the conservation of pMHC and TCR structures betweenmice and humans suggest that humanized 5MCARs shouldfunction similarly.Moving forward, a multipronged approach is required to ad-
vance biomimetic 5MCAR designs. Directly comparing theirperformance with other CAR designs, and their natural coun-terparts, will provide benchmarks for the iterative process ofrefinement to enhance their function according to the roadmapthat has advanced 1MCARs (7–9). In addition, a more thoroughevaluation of the impact of the CRMpMHCII affinity for its targetTCR on 5MCAR-driven responses will provide insight that couldallow for tuning of particular responses. Finally, additional basicresearch into the molecular machinery that naturally drivesT cell activation is of fundamental importance as it will provide amore complete blueprint for the development of future gener-ations of biomimetic designs.
Materials and MethodsExtended methods are described in SI Appendix. These include 5MCAR con-struction, cell lines, mice, generation of 5MCAR–CTL, retrovirus productionand cell transduction, flow cytometry, flow-based fluorophore-linked im-munosorbent assay (FFLISA), enzyme-linked immunosorbent assay (ELISA),lymphocyte preparation, in vitro killing assay, IFNγ production and prolif-eration assay, in vivo killing assay, adoptive transfer, histology, andstatistical analysis.
Data and Material Availability.All data, methods, and associated protocols arecontained in the main text and SI Appendix. Please contact correspondingauthor M.S.K. for materials.
ACKNOWLEDGMENTS. This work was supported by The University ofArizona College of Medicine (M.S.K.), the BIO5 Institute (M.S.K.), NIH/National Institute of Allergy and Infectious Diseases (NIAID) GrantR01AI101053 (M.S.K.), NIH/NIAID Grant R56AI148466 (M.S.K. and T.S.), thePew Scholars Program in the Biomedical Sciences (M.S.K.), the Cancer CenterSupport Grant CCSG-CA 023074 for flow cytometry (M.S.K.), the FleisherFamily Foundation (T.S.), the Alexander and Margaret Stewart Trust (T.S.),the Iacocca Family Foundation (S.K. and M.A.T.), and the Swedish Society ofMedicine and the Swedish Society of Medical Research (M.A.T.). Statisticalanalysis (M.A.N.) was conducted with support from Harvard Catalyst–TheHarvard Clinical and Translational Science Center (NIH Award UL1TR002541). The Joslin Diabetes Center Flow Cytometry Core is supportedby NIH grants (P30-DK-036836 and S10OD021740). We thank Amy Wagersfor additional support and critical feedback. We thank David Duron, KarenHernandez, and Lacey Orsini for technical assistance with generation of con-structs. We also thank Deepta Bhattacharya, Alfred Bothwell, Michael Wor-obey, and Joonsoo Kang for critically reading the manuscript.
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Fig. 7. Treatment with an oligoclonal set of specific 5MCAR–CTLs decreasesdiabetes incidence in NOD mice. Newborn NOD mice received control (GPI:I-Ag7) 5MCAR–CTLs, a mixture of specific 5MCAR–CTLs targeting four pop-ulations of T1D-related autoimmune T cell (INSB:I-Ag7, HIP2.5:I-Ag7, HIP6.9:I-Ag7, and RLGL-WE14:I-Ag7), or were left untreated. (A) A subset of male micewere killed at 13 wk of age, and their spleens were analyzed by flowcytometry. 5MCAR–CTLs were identified by CD8 and GFP expression (pre-gated on live, CD3+ cells). Plots from representative engrafted mice areshown (five of five control 5MCAR–CTL recipients engrafted; five of six spe-cific 5MCAR–CTL recipients engrafted). (B) Female mice were screened weeklyfor glycosuria from day 32 to day 315 and cumulative incidence of diabetes(survival probability plots based on the Kaplan–Meier method) is shown withexact P values determined by log-rank test. Data show the combined resultsfrom three independent cohorts. Numbers of mice/group are indicated inthe figure. Figure is related to SI Appendix, Fig. S7.
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