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Animal studies were conducted with the UCSF Preclinical Therapeutics Core under a protocol approved by the UCSF Institutional Animal Care and Use Committee. NOD scid gamma (NSG) (female, 8∼12 weeks old, Jackson Laboratory #005557) mice were used for all in vivo mouse experiments.

Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board. Primary CD4+ and CD8+ T cells were isolated from anonymous donor blood after apheresis (described in METHOD DETAILS ).

Method Details

synNotch Receptor and Response Element Construct Design Grupp et al., 2013 Grupp S.A.

Kalos M.

Barrett D.

Aplenc R.

Porter D.L.

Rheingold S.R.

Teachey D.T.

Chew A.

Hauck B.

Wright J.F.

et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. Liu et al., 2015 Liu X.

Jiang S.

Fang C.

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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. Fridy et al., 2014 Fridy P.C.

Li Y.

Keegan S.

Thompson M.K.

Nudelman I.

Scheid J.F.

Oeffinger M.

Nussenzweig M.C.

Fenyö D.

Chait B.T.

Rout M.P. A robust pipeline for rapid production of versatile nanobody repertoires. Anderson et al., 1997 Anderson R.

Macdonald I.

Corbett T.

Hacking G.

Lowdell M.W.

Prentice H.G. Construction and biological characterization of an interleukin-12 fusion protein (Flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus. synNotch receptors were built by fusing the CD19 scFv (), Her2 set of scFvs (), LaG17 (lower affinity), or LaG16_2 (high affinity) GFP nanobody () to the mouse Notch1 (NM_008714) minimal regulatory region (Ile1427 to Arg1752) and Gal4VP64. All synNotch receptors contain an n-terminal CD8α signal peptide (MALPVTALLLPLALLLHAARP) for membrane targeting and a myc-tag (EQKLISEEDL) for easy determination of surface expression with α-myc A647 (cell-signaling #2233). The receptors were cloned into a modified pHR’SIN:CSW vector containing a PGK promoter for all primary T cell experiments. The pHR’SIN:CSW vector was also modified to make the response element plasmids. Five copies of the Gal4 DNA binding domain target sequence (GGAGCACTGTCCTCCGAACG) were cloned 5′ to a minimal CMV promoter. The human IL-2, IL-10, flexi IL-12 (), MIP-1α, Tbet, or TRAIL, and PD-L1_T2A_IL-10 codon optimized mRNA sequence was cloned into a MCS downstream of the Gal4 inducible promoter and 5′ of an IRES mCherry reporter. All constructs were cloned via In fusion cloning (Clontech #ST0345)).

Primary Human T Cell Isolation and Culture Primary CD4+ and CD8+ T cells were isolated from anonymous donor blood after apheresis by negative selection (STEMCELL Technologies #15062 and 15023). T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) with 20% human AB serum (Valley Biomedical, #HP1022) and 10% DMSO. After thawing, T cells were cultured in human T cell medium consisting of X-VIVO 15 (Lonza #04-418Q), 5% Human AB serum and 10 mM neutralized N-acetyl L-Cysteine (Sigma-Aldrich #A9165) supplemented with 30 units/mL IL-2 (NCI BRB Preclinical Repository) for all experiments.

Lentiviral Transduction of Human T Cells Pantropic VSV-G pseudotyped lentivirus was produced via transfection of Lenti-X 293T cells (Clontech #11131D) with a pHR’SIN:CSW transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Fugene HD (Promega #E2312). Primary T cells were thawed the same day, and after 24 hr in culture, were stimulated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies #11131D) at a 1:3 cell:bead ratio. At 48 hr, viral supernatant was harvested and the primary T cells were exposed to the virus for 24 hr. At day 4 post T cell stimulation, Dynabeads were removed and the T cells expanded until day 9 when they were rested and could be used in assays. T cells were sorted for assays with a FACs ARIA II.

Cancer Cell Lines The cancer cell lines used were K562 myelogenous leukemia cells (ATCC #CCL-243), Daudi B cell lymphoblasts (ATCC #CCL-213), MCF7 adenocarcinoma breast cancer cells (ATCC #HTB22), and HCT116 colon cancer cells (ATCC #CCL-247). K562s were lentivirally transduced to stably express human CD19 at equivalent levels as Daudi tumors. CD19 levels were determined by staining the cells with α-CD19 APC (Biolegend #302212). K562s were also transduced to stably express surface GFP (GFP fused to the PDGF transmembrane domain). All cell lines were sorted for expression of the transgenes.

In Vitro Stimulation of synNotch T Cells For all in vitro synNotch T cell stimulations, 2x105 T cells were co-cultured with sender cells at a 1:1 ratio. After mixing the T cells and sender cells in round bottom 96-well tissue culture plates, the cells were centrifuged for 1 min at 400xg to force interaction of the cells and the cultures were analyzed at 24 hr for reporter expression or expression of custom gene induction via flow cytometry with a BD LSR II. All flow cytometry analysis was performed in FlowJo software (TreeStar).

Luminex MAGPIX Cytokine Quantification Primary CD4+ T cells expressing the α-CD19 synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling either human IL-2, IL-10, IL-12, or IL-2/MIP-1α expression were stimulated as described above with K562 myelogenous leukemia cells (CD19- or CD19+). As references, CD4+ T cells expressing the α-CD19 4-1BBζ CAR were stimulated along with untransduced T cells stimulated with α-CD3/CD28 Dynabeads at a 1:3 ratio. The supernatant was collected at 24 hr and analyzed with a Luminex MAGPIX (Luminex) Human Cytokine Magentic 25-plex Panel (Invitrogen ref#LHC0009M) according to the manufacturer’s protocol. All cytokine levels were calculated based on standard curves with Her2 software (Luminex).

IL-2 Intracellular Cytokine Staining and CD69 Staining synNotch T cells controlling IL-2 production were assayed to determine if they basally produced IL-2 by intracellular cytokine stain (ICS). The synNotch T cells and untransduced T cell controls were cultured for 6 hr in the presence of GolgiPlug (BD Biosciences #555029). The T cells were then stained with α-IL-2 FITC (BD #340448) with a BD Biosciences ICS kit (#555028). The levels of IL-2 were analyzed via flow cytometry with a BD LSRII. To assess whether synNotch receptors activated the T cells, the T cells were stained after stimulation for the activation marker CD69. CD69 expression was determined by staining the cells with α-CD69 APC (Biolegend #310910).

synNotch Driven T Cell Differentiation Primary human CD4+ T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 as described above. To differentiate T cells into the T h1 subset during the activation, the cells were cultured as described above but with the addition of 2.5 ng/mL recombinant IL-12 (R&D Systems) and 12.5 μg/mL α-IL-4 clone MP4-25D2 (BD Pharmigen #554481). IL-12 and α-IL-4 were added at least twice weekly. In parallel, primary CD4 T cells were lentivirally transduced to express human Tbet T2A mCherry (TBX21, NCBI #EAW94804.1) and cultured normally in T cell medium supplemented with IL-2. CD4+ T cells expressing the α-CD19 synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling Tbet T2A GFP expression were cultured in the presence of CD19- or CD19+ K562 sender cells 24 hr after viral transduction. The synNotch T cells were cultured in the presence of K562s in T cell medium supplemented with IL-2. All T cells were cultured for 11 to 14 days and then subject to intracellular cytokine staining (ICS) to determine the percentage of T h1 T cells. For ICS, the T cells were first treated with 50 ng/mL Phorbal myristate acetate and 1 μg/mL ionomycin (both from Sigma) for 6 hr in the presence of GolgiPlug. The T cells were then stained with α-Tbet BV421 (Biolegend #644816) and α-IFNγ APC (Biolegend #502512). The levels of Tbet and IFNγ were analyzed via flow cytometry with a BD LSRII.

Sensitivity of Cancer Cell Lines to Recombinant TRAIL and synNotch Driven TRAIL Production in Primary T Cells HCT116 colon cancer cells and K562s were treated with recombinant TRAIL (from 1 to 200 ng/mL, 1:2 dilution series) for 24 hr (RND Systems #375-TEC-010). The cells were then harvested and stained with the live/dead stain, SYTOX Blue (Thermo Scientific #S34857) and the fraction of dead cells was determined by flow cytometry on a BD LSR II. The level of the death receptor 4 (DR4) expressed by K562s was assessed by staining with α-TRAIL R1 (DR4) APC (Biolegend #307208). For synNotch driven TRAIL cytotoxicity assays, primary human CD4+ T cells were transduced to express the α-GFP nanobody (LaG17) synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling the expression of LZ-TRAIL or cell surface wild-type TRAIL. The synNotch TRAIL killer cells were co-cultured with surface GFP+ or GFP- K562s for 24 hr and death was determined by staining with SYTOX Blue. Surface levels of TRAIL was determined by staining T cells with α-TRAIL (CD253) APC (Biolegend #308210). Production and secretion of LZ-TRAIL was determined by TRAIL ELISA (R&D systems #DTRL00).

Antibody Response Element Construct Design The pembrolizumab ( http://www.drugbank.ca/drugs/DB09037 ), tremelimumab ( http://www.google.com/patents/US20090074787 ), and blinatumomab ( http://www.drugbank.ca/drugs/DB09052 ) codon optimized mRNA sequences were cloned into a MCS downstream of the Gal4 inducible promoter via Infusion cloning. Both antibody constructs consist of (from n-terminus to c-terminus): an n-terminal human IgG heavy chain signal peptide (MDWTWRVFCLLAVTPGAHP) for secretion, a myc-tag for easy detection of binding to target cells with α-myc AF647, the antibody heavy chain sequence, a furin cleavage site and T2A peptide, a human IgG light chain signal peptide (MAWSPLFLTLITHCAGSWA), a HA-tag as an alternative detection means, and the antibody light chain sequence. The Blinatumomab construct contained an n-terminal human IgK signal peptide (MDMRVLAQLLGLLLLCFPGARC). The dual α-PD-1/α-CTLA-4 construct consists of (from n-terminus to c-terminus): an n-terminal human IgG heavy chain signal peptide (MDWTWRVFCLLAVTPGAHP) for secretion, the pembrolizumab heavy chain sequence, a furin cleavage site and T2A peptide, a HA-tag for detection of binding with α-HA A647 (CST #3444S), the Pembrolizumab light chain sequence, a Furin cleavage site and P2A peptide, a human IgK signal peptide, a myc-tag, and the α-CTLA-4 scFv sequence.

synNotch Driven Antibody Production Primary human CD4+ T cells were transduced to express the α-GFP nanobody (LaG17) synNotch Gal4VP64 receptor and a second vector expressing a user-defined antibody under the control of a 5x Gal4 response element. 2 × 105 transduced T cells were co-cultured 1:1 with GFP+ or GFP− K562s for 24, 48, or 72 hr to induce antibody production and secretion. Cell culture supernatant was harvested and stored for analysis at −80°C. To detect secreted antibody in cell culture supernatant, a cell-based binding assay was employed. K562 cells were lentivirally transduced to stably express human PD-1 or human CTLA-4, so that these cells could serve as ‘target cells’ for cell-based binding assays to detect antibody secreted from synNotch T cells. 5 × 104 target cells were blocked with human BD Fc Block (BD #564220) prior to incubation with cell culture supernatant from co-cultures of synNotch antibody-producing T cells and inducer K562 cells. Following incubation with cell culture supernatant, target cells were stained with α-myc AF647 to detect bound antibody (which was myc-tagged). Flow cytometry was performed to quantify the fluorescence intensity (which correlated with amount of antibody produced by synNotch T cells) of antibody-labeled target cells. To quantify the amount of secrete antibody in cell culture supernatant, a standard curve was generated for the cell-based binding assay. E6-1 Jurkat T cells (ATCC #TIB-152) were lentivirally transduced to create a stable cell line that constitutively secretes α-PD-1 pembrolizumab. Supernatant was harvested from a culture of pembrolizumab-secreting Jurkat T cells. The concentration of α-PD-1 in the supernatant was determined via human IgG4 ELISA (Life Technologies #991000). The supernatant with a known concentration of α-PD-1 was then serially diluted and incubated with PD-1+ K562 target cells to generate a standard curve. Target cell binding of supernatants from co-cultures of synNotch antibody-producing T cells and inducer K562 cells was then compared to the standard curve to determine the concentration of α-PD-1 produced by synNotch T cells co-cultured with different inducer cells.

synNotch Driven Blinatumomab Production Primary human CD4+ T cells were transduced with the α-GFP nanobody (LaG17) synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling α-CD19/CD3 BiTE, Blinatumomab expression. synNotch BiTE T cells were stimulated with either surface GFP+ or GFP- K562s for 24 hr and supernatant was harvested. The T cells were also collected and stained with α-CD69 APC (Biolegend #310910) to determine if they were activated.

synNotch Driven Flagellin Production Primary human CD4+ T cells were transduced with the α-GFP nanobody (LaG17) nanobody synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling S. Typhi FliC expression. 2 × 105 transduced T cells were co-cultured 1:1 with GFP+ or GFP− K562s for 24 hr to induce flagellin production. Cell culture supernatant was harvested 24 hr after stimulation and added to wells containing HEK-Blue hTLR5 reporter cells (Invivogen) that express secreted alkaline phosphatase (SEAP) under control of a TLR5 inducible NF-κB promoter. Recombinant FliC (Invivogen) was utilized as a positive control. Twenty-four hours later supernatant was harvested from HEK-TLR5 cells and alkaline phosphatase activity quantified in a colorimetric assay using QUANTIBLUE detection reagent (Invivogen) with a Flexstation III (Molecular Devices).

synNotch Driven PD-L1 and IL-10 Production Primary human CD4+ T cells were transduced with the α-CD19 synNotch Gal4VP64 receptor and 5x Gal4 response elements controlling human PD-L1 T2A IL-10 expression. The synNotch T cells were stimulated with either surface CD19+ or CD19- K562s for 24 hr and supernatant was harvested for IL-10 ELISA analysis (eBiosciences #BMS215/2). The T cells were also collected and stained separately for intracellular IL-10 with α-IL-10 APC (Biolegend #501410) and for surface PD-L1 with α-PD-L1 BV421 (Biolegend #329714).