Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses

•A split, universal, and programmable (SUPRA) CAR system for T cell therapy•SUPRA CAR can fine-tune T cell activation strength to mitigate toxicity•SUPRA CAR can sense and logically respond to multiple antigens to combat relapse•SUPRA CAR can inducibly control cell-type-specific signaling T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical “upgrades,” such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system. T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical “upgrades,” such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system. The transfer of chimeric antigen receptor (CAR)-expressing T cells to patients is a promising approach for cancer immunotherapy (Brentjens et al., 2011Brentjens R.J. Rivière I. Park J.H. Davila M.L. Wang X. Stefanski J. Taylor C. Yeh R. Bartido S. 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Lacey S.F. et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.N. Engl. J. Med. 2014; 371: 1507-1517Crossref PubMed Scopus (3554) Google Scholar). Despite these encouraging results, safety and efficacy continue to be major hurdles that hinder CAR T cell therapy development (Brentjens et al., 2013Brentjens R.J. Davila M.L. Riviere I. Park J. Wang X. Cowell L.G. Bartido S. Stefanski J. Taylor C. Olszewska M. et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci. Transl. Med. 2013; 5: 177ra38Crossref PubMed Scopus (1502) Google Scholar, Kochenderfer et al., 2012Kochenderfer J.N. Dudley M.E. Feldman S.A. Wilson W.H. Spaner D.E. Maric I. Stetler-Stevenson M. Phan G.Q. Hughes M.S. 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To improve overall effectiveness and safety of CAR T cell therapy, there is an urgent need for a better system that can finely tune T cell activation, enhance tumor specificity, and independently control different signaling pathways and cell types. The CAR T cells used in clinical trials typically have a rigid design that is difficult to alter without re-engineering the T cells. Current CAR designs are composed of a fixed antigen-specific single-chain variable fragment (scFv) and intracellular signaling domains (CD3ζ and costimulatory domains). When the constant antigen-specific CAR binds to the target antigen, these invariable signaling domains are activated simultaneously at a predetermined level. Due to the fixed design that limited the controllability of CAR T cell activation level, managing CAR T cell-related toxicities have proven to be challenging (Brentjens et al., 2013Brentjens R.J. Davila M.L. Riviere I. Park J. Wang X. Cowell L.G. Bartido S. Stefanski J. Taylor C. Olszewska M. et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci. Transl. Med. 2013; 5: 177ra38Crossref PubMed Scopus (1502) Google Scholar, Brudno and Kochenderfer, 2016Brudno J.N. Kochenderfer J.N. Toxicities of chimeric antigen receptor T cells: recognition and management.Blood. 2016; 127: 3321-3330Crossref PubMed Scopus (767) Google Scholar, Davila et al., 2014Davila M.L. Riviere I. Wang X. Bartido S. Park J. Curran K. Chung S.S. Stefanski J. Borquez-Ojeda O. Olszewska M. et al.Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci. Transl. Med. 2014; 6: 224ra25Crossref PubMed Scopus (1760) Google Scholar). In addition to constraining the controllability of CAR T cell activity, this fixed CAR design also restricts the antigen specificity and affinity. High-affinity scFvs are often used in the CAR design to ensure high antigen specificity. However, CARs made with high-affinity scFvs have limited capacity in discriminating antigen density, which has led to dangerous reactivity against healthy organs expressing a low level of antigens (Bonifant et al., 2016Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 3: 16011Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, Morgan et al., 2010Morgan R.A. Yang J.C. Kitano M. Dudley M.E. Laurencot C.M. Rosenberg S.A. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol. Ther. 2010; 18: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar). Using a scFv with lower antigen affinity allowed better antigen density discrimination (Caruso et al., 2015Caruso H.G. Hurton L.V. Najjar A. Rushworth D. Ang S. Olivares S. Mi T. Switzer K. Singh H. Huls H. et al.Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity.Cancer Res. 2015; 75: 3505-3518Crossref PubMed Scopus (259) Google Scholar, Liu et al., 2015Liu X. Jiang S. Fang C. Yang S. Olalere D. Pequignot E.C. Cogdill A.P. Li N. Ramones M. Granda B. et al.Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice.Cancer Res. 2015; 75: 3596-3607Crossref PubMed Scopus (318) Google Scholar), but antigen specificity may be compromised. Thus, modulation of CAR components other than scFv affinity may be needed for improving CAR T cell specificity. Recently, several studies have demonstrated the importance of regulating CD3ζ and the different costimulatory pathways independently to achieve optimal T cell response (Kloss et al., 2013Kloss C.C. Condomines M. Cartellieri M. Bachmann M. Sadelain M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat. Biotechnol. 2013; 31: 71-75Crossref PubMed Scopus (624) Google Scholar, Lanitis et al., 2013Lanitis E. Poussin M. Klattenhoff A.W. Song D. Sandaltzopoulos R. June C.H. Powell Jr., D.J. Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo.Cancer Immunol. Res. 2013; 1: 43-53Crossref PubMed Scopus (246) Google Scholar, Zhao et al., 2015Zhao Z. Condomines M. van der Stegen S.J.C. Perna F. Kloss C.C. Gunset G. Plotkin J. Sadelain M. Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells.Cancer Cell. 2015; 28: 415-428Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). 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CD137 stimulation delivers an antigen-independent growth signal for T lymphocytes with memory phenotype.Blood. 2007; 109: 4882-4889Crossref PubMed Scopus (68) Google Scholar), demonstrating the value of CAR design that allows independent control of different signaling domains. The composition of the T cell subsets, such as the ratio of CD4+ and CD8+ T cells, has also been shown to be an important parameter for enhancing the antitumor response of CAR T cells (Turtle et al., 2016Turtle C.J. Hanafi L.A. Berger C. Gooley T.A. Cherian S. Hudecek M. Sommermeyer D. Melville K. Pender B. Budiarto T.M. et al.CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.J. Clin. Invest. 2016; 126: 2123-2138Crossref PubMed Scopus (1243) Google Scholar). Given the fact that our immune system is composed of many different T cell subtypes with distinct effector functions (Golubovskaya and Wu, 2016Golubovskaya V. Wu L. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy.Cancers (Basel). 2016; 8Crossref PubMed Scopus (280) Google Scholar, Vignali et al., 2008Vignali D.A. Collison L.W. Workman C.J. How regulatory T cells work. Nat. Rev.Immunol. 2008; 8: 523-532PubMed Google Scholar, Vivier et al., 2008Vivier E. Tomasello E. Baratin M. Walzer T. Ugolini S. Functions of natural killer cells.Nat. Immunol. 2008; 9: 503-510Crossref PubMed Scopus (2499) Google Scholar), regulating the activity of T cell subtypes independently may be an attractive strategy for optimizing the efficacy of CAR T cell therapy (Sadelain et al., 2017Sadelain M. Rivière I. Riddell S. Therapeutic T cell engineering.Nature. 2017; 545: 423-431Crossref PubMed Scopus (465) Google Scholar). However, current fixed CAR design limits independent and inducible activation of different signaling domains or different T cell subsets to achieve user-defined diverse T cell response. New receptor designs have been developed to address some of the deficiencies (e.g., controllability, flexibility, specificity) in current CAR T cell therapies. For instance, drug-inducible ON and kill switches have been developed to regulate CAR activity (Di Stasi et al., 2011Di Stasi A. Tey S.-K. Dotti G. Fujita Y. Kennedy-Nasser A. Martinez C. Straathof K. Liu E. Durett A.G. Grilley B. et al.Inducible apoptosis as a safety switch for adoptive cell therapy.N. Engl. J. Med. 2011; 365: 1673-1683Crossref PubMed Scopus (1041) Google Scholar, Wu et al., 2015Wu C.Y. Roybal K.T. Puchner E.M. Onuffer J. Lim W.A. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.Science. 2015; 350: aab4077Crossref PubMed Scopus (454) Google Scholar). Also, to afford greater flexibility in antigen recognition, CARs have been split such that the antigen recognition motif is dissociated from the signaling motif of the CAR. This split CAR configuration uses a universal receptor as the common basis for all interactions, allowing a large panel of antigens to be targeted without re-engineering the immune cells (Cartellieri et al., 2016Cartellieri M. Feldmann A. Koristka S. Arndt C. Loff S. Ehninger A. von Bonin M. Bejestani E.P. Ehninger G. Bachmann M.P. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts.Blood Cancer J. 2016; 6: e458Crossref PubMed Scopus (146) Google Scholar, Rodgers et al., 2016Rodgers D.T. Mazagova M. Hampton E.N. Cao Y. Ramadoss N.S. Hardy I.R. Schulman A. Du J. Wang F. Singer O. et al.Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.Proc. Natl. Acad. Sci. USA. 2016; 113: E459-E468Crossref PubMed Scopus (261) Google Scholar, Tamada et al., 2012Tamada K. Geng D. Sakoda Y. Bansal N. Srivastava R. Li Z. Davila E. Redirecting gene-modified T cells toward various cancer types using tagged antibodies.Clin. Cancer Res. 2012; 18: 6436-6445Crossref PubMed Scopus (168) Google Scholar, Urbanska et al., 2012Urbanska K. Lanitis E. Poussin M. Lynn R.C. Gavin B.P. Kelderman S. Yu J. Scholler N. Powell Jr., D.J. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor.Cancer Res. 2012; 72: 1844-1852Crossref PubMed Scopus (211) Google Scholar). In addition, to increase tumor specificity, CARs were developed that allow combinatorial antigen sensing (Kloss et al., 2013Kloss C.C. Condomines M. Cartellieri M. Bachmann M. Sadelain M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat. Biotechnol. 2013; 31: 71-75Crossref PubMed Scopus (624) Google Scholar, Lanitis et al., 2013Lanitis E. Poussin M. Klattenhoff A.W. Song D. Sandaltzopoulos R. June C.H. Powell Jr., D.J. Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo.Cancer Immunol. Res. 2013; 1: 43-53Crossref PubMed Scopus (246) Google Scholar, Roybal et al., 2016Roybal K.T. Rupp L.J. Morsut L. Walker W.J. McNally K.A. Park J.S. Lim W.A. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.Cell. 2016; 164: 770-779Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar) or target two tumor-specific antigens that can reduce tumor antigen escape rate (Grada et al., 2013Grada Z. Hegde M. Byrd T. Shaffer D.R. Ghazi A. Brawley V.S. Corder A. Schönfeld K. Koch J. Dotti G. et al.TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy.Mol. Ther. Nucleic Acids. 2013; 2: e105Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, Zah et al., 2016Zah E. Lin M.-Y. Silva-Benedict A. Jensen M.C. Chen Y.Y. T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells.Cancer Immunol. Res. 2016; 4: 498-508Crossref PubMed Scopus (355) Google Scholar). All of these features are arguably vital to ensure a safe and effective CAR T therapy. However, none of these advanced CARs has incorporated all of these features into one system. Additionally, the signaling pathways and cell types that can be activated are also fixed, thus limiting the diverse immune responses that can be achieved. To enhance the specificity, safety, and programmability of CARs, we develop a split, universal, and programmable (SUPRA) CAR system composed of a universal receptor expressed on T cells and a tumor-targeting scFv adaptor molecule (Figure 1A). The activity of SUPRA CARs can be finely regulated via multiple mechanisms to limit overactivation. SUPRA CARs can also logically respond to multiple antigens for improving tumor specificity. We show that the SUPRA CAR system is effective against two different tumor models, demonstrating the broad clinical potential of this system. In addition, we show that SUPRA components can be humanized to reduce potential immunogenicity. Furthermore, we use orthogonal SUPRA CARs to inducibly regulate multiple signaling pathways or different human T cell subtypes to increase the range of the immune responses that can be achieved. Together, the SUPRA CAR system is a feature-rich system with inducible and logical control capabilities that can improve the safety and efficacy of current cellular cancer immunotherapy. The SUPRA CAR is a two-component receptor system composed of a universal receptor (zipCAR) expressed on T cells and a tumor-targeting scFv adaptor (zipFv) (Figure 1A). The zipCAR universal receptor is generated from the fusion of intracellular signaling domains and a leucine zipper as the extracellular domain. The zipFv adaptor molecule is generated from the fusion of a cognate leucine zipper and a scFv. The scFv of the zipFv binds to the tumor antigen, and the leucine zipper binds and activates the zipCAR on the T cells (Figures S1A and S2). Unlike the conventional fixed CAR design, the SUPRA CAR modular design allows targeting of multiple antigens without further genetic manipulations of a patient’s immune cells (Figure 1B, left). To test the ability of the SUPRA CAR system targeting multiple antigens with the same batch of T cells expressing the zipCAR, we first engineered human primary CD8+ T cells to express an RR zipCAR (RR leucine zipper with CD28, 4-1BB co-stimulatory and a CD3ζ signaling domain; Figure S1A). Next, we designed three different zipFvs to target three common tumor antigens (α-Her2, α-Axl, and α-Mesothelin; Figure S1A) by fusing the corresponding scFvs to an EE leucine zipper, which binds to the RR zipCAR on T cells. The engineered CD8+ T cells were co-cultured in vitro with K562 myelogenous leukemia cells that express Her2, Axl, or Mesothelin tumor antigens. The CD8+ zipCAR T cells killed the corresponding tumor cells when the matching zipFvs were added (Figure 1B, right).Figure S2Comparison of SUPRA CAR with Conventional α-Her2 CAR and Characterization of zipFvs, Related to Figures 1Show full caption(A) (Left) Schematic of SUPRA CAR (EE-RR pair) and a-Her2 CAR. (Right) Forward- and side-scatter FACS plots of the cell mixture after 24 hours co-culture of T cells (blue) with Her2+ K562 tumor cells (orange) (representative of three biological replicates).(B and C) The CD69 expression and IFN-γ measurement after 24hr of co-culturing with RR zipCAR / α- Her2 CAR and Her2+ K562 target cells (n = 3, data are represented as mean ± SD).(D and E) Denaturing SDS-PAGE and western blot images of the different zipFvs used in the paper. (Gel images have been cropped for visualization purposes.)(F) Table of expected protein mass (Da) of different zipFvs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) (Left) Schematic of SUPRA CAR (EE-RR pair) and a-Her2 CAR. (Right) Forward- and side-scatter FACS plots of the cell mixture after 24 hours co-culture of T cells (blue) with Her2+ K562 tumor cells (orange) (representative of three biological replicates). (B and C) The CD69 expression and IFN-γ measurement after 24hr of co-culturing with RR zipCAR / α- Her2 CAR and Her2+ K562 target cells (n = 3, data are represented as mean ± SD). (D and E) Denaturing SDS-PAGE and western blot images of the different zipFvs used in the paper. (Gel images have been cropped for visualization purposes.) (F) Table of expected protein mass (Da) of different zipFvs. A unique feature of the split CAR design is that it has multiple tunable variables, such as (1) the affinity between leucine zipper pairs, (2) the affinity between tumor antigen and scFv, (3) the concentration of zipFv, and (4) the expression level of zipCAR, that can be used to modulate the T cell response (Figure 1C). We first characterized the effect of zipFv concentration and zipper affinity on T cell activation. We generated three zipFvs with the same α-Her2 scFv but fused to leucine zippers (SYN5, SYN 3, and EE) that have different affinity to the RR zipCAR (Reinke et al., 2010Reinke A.W. Grant R.A. Keating A.E. A synthetic coiled-coil interactome provides heterospecific modules for molecular engineering.J. Am. Chem. Soc. 2010; 132: 6025-6031Crossref PubMed Scopus (125) Google Scholar, Thompson et al., 2012Thompson K.E. Bashor C.J. Lim W.A. Keating A.E. SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains.ACS Synth. Biol. 2012; 1: 118-129Crossref PubMed Scopus (119) Google Scholar). The amount of zipFv required to activate T cells to half-maximal interferon (IFN)-γ secretion and cytotoxicity inversely correlated with the affinity of leucine zipper pairs where α-Her2-EE zipFv showed the lowest EC50 and α-Her2-SYN5 zipFv showed the highest EC50 value (Figures 1D and S1C). Also, the maximum level of IFN-γ secretion or killing efficiency correlated with the affinity of leucine zipper pairs. We next investigated the effect of scFv-tumor antigen affinity, leucine zipper affinity, and zipCAR expression levels on the IFN-γ secretion and cancer-killing efficiency by the SUPRA CAR T cells (Figures 1E and S1D). We created 12 different zipFvs (three different leucine zippers with different affinity and four scFvs against Her2 [G98, C65, ML39, and H3B1] with Kd ranging from 3.2 × 10−7 to 1.2 × 10−10 M) (Chmielewski et al., 2004Chmielewski M. Hombach A. Heuser C. Adams G.P. Abken H. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity.J. Immunol. 2004; 173: 7647-7653Crossref PubMed Scopus (185) Google Scholar). We also generated two batches of T cells with high or low RR zipCAR expression level using fluorescence-activated cell sorting (FACS) (Figure S1B). Cells expressing higher levels of zipCAR exhibited greater cytokine secretion when activated (Figure 1E). The affinity of scFv to Her2 correlated weakly with cytokine secretion or target cell lysis (Chmielewski et al., 2004Chmielewski M. Hombach A. Heuser C. Adams G.P. Abken H. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity.J. Immunol. 2004; 173: 7647-7653Crossref PubMed Scopus (185) Google Scholar). The affinity between leucine zippers, however, correlated well with cellular activation regarding cancer cell killing efficiency and cytokine secretion. As high-affinity scFv CARs often over-activate and show severe toxicities in clinical trials (Bonifant et al., 2016Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 3: 16011Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, Brudno and Kochenderfer, 2016Brudno J.N. Kochenderfer J.N. Toxicities of chimeric antigen receptor T cells: recognition and management.Blood. 2016; 127: 3321-3330Crossref PubMed Scopus (767) Google Scholar), the SUPRA platform can mitigate these toxicities by controlling other factors (e.g., zipFv concentration, affinity between leucine zipper pairs) to regulate T cell activation level. Together, these results demonstrate the tunable and modular nature of the SUPRA CAR design. As many patients treated with CAR T cell therapy face cytokine release syndrome, which can be life threatening (Maude et al., 2014bMaude S.L. Barrett D. Teachey D.T. Grupp S.A. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. 2014; 20: 119-122Crossref PubMed Scopus (517) Google Scholar, Morgan et al., 2010Morgan R.A. Yang J.C. Kitano M. Dudley M.E. Laurencot C.M. Rosenberg S.A. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol. Ther. 2010; 18: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar), it is important to prevent CAR T cell activity when necessary. Thus, we explored the possibility of inhibiting the SUPRA CAR T cell activation through the addition of a competitive zipFv that can bind to the other zipFv, thus preventing zipCAR from being activated (Figure 2A, left). To test this approach, we screened several competitive zipFvs with different affinities for the EE zipFv (strong, medium, and weak) (Figures S3B and S3C). Human CD8+ T cells were transduced with RR zipCAR and co-cultured with Her2+ K562 cells. Then, EE zipFv (22.5 nM, red) was subsequently added to activate the T cells. Without the competitive zipFv, the EE zipFv alone could activate T cells to destroy Her2+ cancer cells (Figure 2A, right). However, when the competitive zipFv (SYN4, SYN 47, or SYN 13) was also introduced (90 nM, green), it bound to the EE zipFv and prevented the EE zipFv from activating the zipCAR T cells. By utilizing this competitive approach, we were able to inhibit primary CD8+ T cell activation in vitro with the strong competitive zipFv (SYN4). Furthermore, we were able to tune the activation levels with weaker binding zippers (SYN 47 and SYN 13). To understand inhibition dynamics, we varied the amount of competitive zipFv and timing of its addition. Increasing the amount of competitive zipFv or delaying competitive zipFv addition did not affect inhibition strength greatly (Figure S3D).Figure S3Competitive zipFv Screen to Tune SUPRA CAR Activity and Using SUPRA as a Cell Selector, Related to Figure 2Show full caption(A) Leucine zippers with different affinities to EE leucine zipper.(B) EE zipCAR expressing Jurkat T cells were co-cultured with Her2 expressing K562. Then, different zipFvs (α-Her2-SYN2, α-Her2-SYN4, α-Her2-SYN47, or α-Her2-SYN13) were added. GFP expression was measured after 24 hours to quantify the NFAT promoter activity.(C) Normalized NFAT promoter activity measured by GFP expression of different zipFvs (n = 2, data are represented as mean ± SD).(D) RR zipCAR expressing CD8+ T cells were co-cultured with Her2 expressing K562. α-Her2-EE zipFv (22.5nM) was added to activate T cells. Then, different amount of competitive zipFv (90nM, 45nM, and 22.5nM) was added at a different time after EE zipFv was added (n = 3, data are represented as mean).(E) Cell selector with zipFv (α-Axl-SYN13) that does not bind strongly to α-Her2-EE zipFv (n = 3, data are represented as mean ± SD).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Leucine zippers with different affinities to EE leucine zipper. (B) EE zipCAR expressing Jurkat T cells were co-cultured with Her2 expressing K562. Then, different zipFvs (α-Her2-SYN2, α-Her2-SYN4, α-Her2-SYN47, or α-Her2-SYN13) were added. GFP expression was measured after 24 hours to quantify the NFAT promoter activity. (C) Normalized NFAT promoter activity measured by GFP expression of different zipFvs (n = 2, data are represented as mean ± SD). (D) RR zipCAR expressing CD8+ T cells were co-cultured with Her2 expressing K562. α-Her2-EE zipFv (22.5nM) was added to activate T cells. Then, different amount of competitive zipFv (90nM, 45nM, and 22.5nM) was added at a different time after EE zipFv was added (n = 3, data are represented as mean). (E) Cell selector with zipFv (α-Axl-SYN13) that does not bind strongly to α-Her2-EE zipFv (n = 3, data are represented as mean ± SD). Antigen escape is a major challenge for targeted cancer therapies (Scott et al., 2012Scott A.M. Wolchok J.D. Old L.J. Antibody therapy of cancer.Nat. Rev. Cancer. 2012; 12: 278-287Crossref PubMed Scopus (1573) Google Scholar), including adoptive T cell therapies (Hegde et al., 2013Hegde M. Corder A. Chow K.K. Mukherjee M. Ashoori A. Kew Y.