hs578ts i 8 lysate  (Danaher Inc)

Journal: Nature chemical biology

Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

doi: 10.1038/s41589-021-00932-1

Figure Lengend Snippet: (a) SEAKER CAR constructs encoding secreted prodrug-activating enzymes: CPG2–19BBz (CPG2/α-CD19/4–1BB/CD3ζ) and β-Lac-19BBz (β-Lac/α-CD19/4–1BB/CD3ζ). LTR = long terminal repeat, Ψ = psi packaging element, CD8 ss = CD8 signal peptide (gray), HA tag = hemagglutinin epitope tag (yellow), P2A = 2A self cleaving peptide, α-CD19 scFv = CD19-specific single chain variable fragment, 4–1BB = 4–1BB costimulatory domain (blue), CD3ζ = CD3 zeta chain (red). (b) Cytolytic activity of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells against Raji (CD19+) target cells expressing firefly luciferase (18 h, bioluminescence assay; mean ± s.d. of n = 3 biological replicates/sample; representative data from 3 independent donors). (c) Antitumor efficacy of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells, without prodrugs, against Raji xenografts in NSG mice (day 16 post-tumor engraftment, bioluminescent imaging (left) and Kaplan–Meier curve (right). (Log-rank (Mantel–Cox) test: *p<0.05; untreated vs. β-Lac-19BBz: p = 0.003; untreated vs. CPG2–19BBz: p = 0.023; Untreated vs. 19BBz: p = 0.03. Experiment was performed once.) (d) SEAKER enzyme expression in cocultures of anti-CD19 SEAKERS with Raji (CD19+) or SET2 (CD19−) cells (CPG2: ELISA assay; β-Lac: nitrocefin cleavage UV assay; mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001; representative of 3 experiments). (e) Trans-cytotoxicity of supernatant fluids from standard 19BBz CAR-T cells and SEAKER cells, with or without the corresponding prodrug, against SET2 target cells (48 h, CellTiter-Glo) (mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001).

Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

Techniques: Construct, Activity Assay, Expressing, Luciferase, ATP Bioluminescent Assay, Imaging, Enzyme-linked Immunosorbent Assay, UV Assay, Two Tailed Test

Journal: Nature chemical biology

Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

doi: 10.1038/s41589-021-00932-1

Figure Lengend Snippet: (a) CPG2 enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted NSG mice treated with CPG2–19BBz SEAKER cells or β-Lac-19BBz SEAKER cells (negative control), based on methotrexate cleavage assay (fold-change; mean ± s.d. of n = 5 mice per group; Student’s two-tailed t-test: *p<0.05; representative data from 2 independent experiments). (b) β-Lac enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted mice treated with CPG2–19BBz SEAKER cells (negative control) or β-Lac-19BBz SEAKER cells, based on nitrocefin cleavage assay (fold-change; mean ± s.d. of n = 4 mice per group; Student’s two-tailed t-test: **p<0.01; representative data from 2 independent experiments). (c) Experimental scheme to assess efficacy of SEAKER–prodrug combinations in intraperitoneal tumor model (AMS-Glu (2): 50 mg/kg, ip, bid, 12 doses total; Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (d,e) Tumor bioluminescence was monitored over time (representative images shown; experiment in panel e was repeated with similar results) (see also Extended Data Fig. 3). (f) Experimental scheme to assess efficacy in a heterogeneous tumor model (2 × 106 total cells, 1:1 Nalm6-mCherry/gLuc (CD19+) and Nalm6-eGFP/fLuc (CD19−). (Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (g,h) Antitumor efficacy against CD19− Nalm6 cancer cells (fLuc) engrafted within the heterogeneous tumor with CD19+ Nalm6 cancer cells (see also Extended Data Fig. 4), only in mice receiving β-Lac-19BBz SEAKER cells plus Ceph-AMS prodrug (3) (representative images shown on day 17 from 20-day study; one mouse in prodrug-treated group died between day 14 and day 17 and is omitted) (mean ± s.d. of n = 5 mice in SEAKER-treated group and n = 4 mice in SEAKER+prodrug-treated group; Student’s two-tailed t-test: *p<0.05; experiment was performed once). (i) Retention of β-Lac enzyme activity in β-Lac-19BBz SEAKER cells expressing T-cell exhaustion markers (TIM3, LAG3, PD1) after 26 days in Raji-engrafted mice (flow cytometry analysis: FMO = fluorescence minus one control; β-Lac substrate = CCF2-AM) (representative data shown for one of n = 4 mice; representative data from 2 independent experiments).

Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

Techniques: Activity Assay, Negative Control, Cleavage Assay, Two Tailed Test, Expressing, Flow Cytometry, Fluorescence, Control

Journal: Nature chemical biology

Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

doi: 10.1038/s41589-021-00932-1

Figure Lengend Snippet: (a) Nitrocefin cleavage-based quantitation of tumor β-Lac concentration in a subcutaneous Raji tumor from a mouse treated with 3 × 106 β-Lac-19BBz SEAKER cells (IV) (representative data from two of n = 5 mice per group in 3 independent experiments). (b) anti-β-Lac immunohistochemistry imaging of subcutaneous Raji tumors extracted on day 15 from mice that were untreated, or received 3 × 106 β-Lac-19BBz SEAKER cells (IV) (top panels – left: isotype control; center: untreated mouse stained with anti-β-Lac antibody; right: β-Lac-19BBz-treated mouse stained with anti-β-Lac antibody). Increased magnification highlights diffuse β-Lac staining throughout the tumor environment (bottom panels) (representative data shown from one of the 3 remaining mice from the n = 5 group used in panel a). (c) Experimental scheme to assess therapeutic efficacy of SEAKER–prodrug combinations in a subcutaneous solid tumor model. Raji tumor cells were engrafted sq on day 0 followed by SEAKER cells iv on day 7. The corresponding prodrug was administered beginning on day 15 (AMS-Glu (2): 50 mg/kg, ip, bid, days 15–20, 12 doses total, or Ceph-AMS (3): 4 mg/kg, ip, bid every other day, days 15, 17, 19, 6 doses total) and mice were monitored for survival. (d) Survival analysis of mice engrafted with subcutaneous Raji tumors receiving subtherapeutic doses of CPG2–19BBz SEAKER cells plus AMS-Glu (2) (left panel), or β-Lac-19BBz SEAKER cells plus Ceph-AMS (3) (right panel). (Arrows denote beginning and end of the prodrug administration period; n = 5 mice/group; log-rank (Mantel–Cox) test: *p<0.05; CPG2–19BBz vs. CPG2–19BBz + AMS-Glu: p = 0.023; β-Lac-19BBz vs. β-Lac-19BBz + Ceph-AMS: p = 0.048; experiment was repeated with similar results).

Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

Techniques: Quantitation Assay, Concentration Assay, Immunohistochemistry, Imaging, Control, Staining, Drug discovery

Journal: Nature chemical biology

Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

doi: 10.1038/s41589-021-00932-1

Figure Lengend Snippet: Assessment of SEAKER cell immunogenicity in an immunocompetent mouse model. (a) Experimental scheme to assess immunogenicity of β-Lac-MUC28z SEAKER cells in a syngeneic intraperitoneal ID8 tumor model. Sera were collected on Days 21, 24, 28, and 31 and tested for anti-β-Lac antibodies in panel b. In a separate experiment, ascites were recovered on Day 28 by peritoneal lavage and tested for the presence of SEAKER cells in panels c,d and β-Lac enzyme activity in panel e. (b) Detection anti-β-Lac antibodies in sera over 10 days following SEAKER cell engraftment (Days 21–31) (median with lines representing each individual mouse of n = 12; experiment was performed once). (c,d) Flow cytometry analysis of SEAKER cell (myc+) persistence among T cells (CD3+) and (e) nitrocefin cleavage-based quantitation of β-Lac enzyme activity in ascites recovered 7 days after SEAKER cell engraftment (Day 28) (representative data shown from n = 2 mice per group; on average, 25% of T cells were SEAKER-positive; experiment was performed once). SSC = side scatter. (f) In a third experiment, mice were treated as in panel a, but without cyclophosphamide pretreatment to maximize the antibody response, then sera were recovered 5 days after SEAKER cell engraftment (Day 26) and analyzed for anti-β-Lac antibodies (n = 1 mouse in untreated group; n = 5 mice in treated group; mean ± s.d. of n = 3 technical replicates from each mouse; experiment was performed once). Sera from the 4 mice showing anti-β-Lac antibodies were used for the ex vivo enzyme activity experiment in panel g. (g) Nitrocefin cleavage-based quantitation of enzyme activity of recombinant β-Lac treated with sera from untreated or SEAKER-treated mice from panel f (n = 2 mice in untreated group; mean ± s.d. of n = 4 mice in treated group; experiment was performed once).

Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

Techniques: Immunopeptidomics, Activity Assay, Flow Cytometry, Quantitation Assay, Ex Vivo, Recombinant