The following reagents were purchased: T-DM1 (Roche), LY294002 (Selleck, S1105), 3-MA (Selleck, S2767), and Cyto-ID (Enzo Life Sciences, ENZ-51031-K200). The following major antibodies were obtained for immunoblot analysis: anti-HER (Code: 2165), anti-SQSTM1 (Code: 8025), anti-Beclin-1 (Code: 3495), anti-caspase-9 (Code: 9502), anti-LC3 (Code: 3868), anti-p-Akt (Ser473; Code: 4060), anti-PARP (Code: 9532), anti-GAPDH (Code: 5174), anti-β-actin (Code: 3700), anti-caspase-3 (Code: 9665), anti-phospho-4E-BP1/2/3 (Thr46; Code: 4923), anti-phosphop70S6 kinase (Ser371; Code: 9208), anti-Ki67 (Code: 9449), and anti-p-mTOR (Ser2448; Code: 2971) from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from MR Biotech. The major antibodies for immunofluorescence and immunohistochemistry were purchased from Servicebio (Wuhan, China).
T dm1
Manufactured by Roche
Sourced in Switzerland
T-DM1 is a monoclonal antibody-drug conjugate (ADC) developed by Roche. It is designed to target and deliver a chemotherapeutic agent specifically to HER2-positive cancer cells.
Automatically generated - may contain errors
Lab products found in correlation
Trastuzumab, by Roche (7 mentions)
Anti sqstm1, by Cell Signaling Technology (3 mentions)
Anti lc3, by Cell Signaling Technology (3 mentions)
Anti beclin 1, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Cyto id, by Enzo Life Sciences (3 mentions)
Anti p mtor, by Cell Signaling Technology (3 mentions)
Anti phospho p70 s6 kinase, by Cell Signaling Technology (3 mentions)
Anti her, by Cell Signaling Technology (3 mentions)
Anti phospho 4e bp1 2 3, by Cell Signaling Technology (3 mentions)
Anti ki67, by Cell Signaling Technology (3 mentions)
Ly294002, by Selleck Chemicals (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti caspase 9, by Cell Signaling Technology (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti parp, by Cell Signaling Technology (3 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (3 mentions)
Dylight 488 nhs ester, by Thermo Fisher Scientific (3 mentions)
Sulforhodamine b, by Merck Group (3 mentions)
17 protocols using t dm1
1
Molecular Mechanisms of T-DM1 Efficacy
2
Molecular Mechanisms of T-DM1 Efficacy
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The following reagents were purchased: T-DM1 (Roche), LY294002 (Selleck, S1105), 3-MA (Selleck, S2767), and Cyto-ID (Enzo Life Sciences, ENZ-51031-K200). The following major antibodies were obtained for immunoblot analysis: anti-HER (Code: 2165), anti-SQSTM1 (Code: 8025), anti-Beclin-1 (Code: 3495), anti-caspase-9 (Code: 9502), anti-LC3 (Code: 3868), anti-p-Akt (Ser473; Code: 4060), anti-PARP (Code: 9532), anti-GAPDH (Code: 5174), anti-β-actin (Code: 3700), anti-caspase-3 (Code: 9665), anti-phospho-4E-BP1/2/3 (Thr46; Code: 4923), anti-phosphop70S6 kinase (Ser371; Code: 9208), anti-Ki67 (Code: 9449), and anti-p-mTOR (Ser2448; Code: 2971) from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from MR Biotech. The major antibodies for immunofluorescence and immunohistochemistry were purchased from Servicebio (Wuhan, China).
3
Molecular Mechanisms of T-DM1 Efficacy
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The following reagents were purchased: T-DM1 (Roche), LY294002 (Selleck, S1105), 3-MA (Selleck, S2767), and Cyto-ID (Enzo Life Sciences, ENZ-51031-K200). The following major antibodies were obtained for immunoblot analysis: anti-HER (Code: 2165), anti-SQSTM1 (Code: 8025), anti-Beclin-1 (Code: 3495), anti-caspase-9 (Code: 9502), anti-LC3 (Code: 3868), anti-p-Akt (Ser473; Code: 4060), anti-PARP (Code: 9532), anti-GAPDH (Code: 5174), anti-β-actin (Code: 3700), anti-caspase-3 (Code: 9665), anti-phospho-4E-BP1/2/3 (Thr46; Code: 4923), anti-phosphop70S6 kinase (Ser371; Code: 9208), anti-Ki67 (Code: 9449), and anti-p-mTOR (Ser2448; Code: 2971) from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from MR Biotech. The major antibodies for immunofluorescence and immunohistochemistry were purchased from Servicebio (Wuhan, China).
4
Investigating Antibody-Drug Conjugate Mechanisms
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Both T‐DM1 and trastuzumab were purchased from F. Hoffmann‐La Roche (Basel, Switzerland). DM1 was provided by Jiangsu Hengrui Pharmaceutical Co. (Lianyungang, China). Bafilomycin A1 was obtained from Selleck Chemicals (Houston, TX, USA). DyLight 488 NHS Ester and LysoTracker Deep Red were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Propidium iodide, sulforhodamine B, and DAPI were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Acridine orange was purchased from China National Pharmaceutical Industry Corp (Beijing, China). Hertuzumab‐vc‐MMAE was obtained from Rongchang Pharmaceuticals, Ltd (Yantai, China).
Antibodies against HER2, GAPDH, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody specific for β‐tubulin was purchased from Sigma‐Aldrich. The antibody against β‐actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488‐conjugated goat anti‐mouse IgG was purchased from Invitrogen (Carlsbad, CA, USA).
Antibodies against HER2, GAPDH, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody specific for β‐tubulin was purchased from Sigma‐Aldrich. The antibody against β‐actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488‐conjugated goat anti‐mouse IgG was purchased from Invitrogen (Carlsbad, CA, USA).
5
Cell Cycle Analysis of T-DM1 and LCB-ADCs
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JIMT‐1 and N87 cells were plated in a 60 mm tissue culture dish at a density of 5 × 105 cells plate−1 and incubated overnight. Cells were treated with T‐DM1 (Kadcyla, 10172048, Roche, Basel, Switzerland) or LCB‐ADCs (provided by LegoChem Bioscience) at a dose of 0.25 µg mL−1 based on the amount of antibody for 24–72 h. The cells were collected and suspended in 100% cold‐ethanol for fixation, stained with propidium iodide (PI), and then the cell cycle distribution was analyzed by flow cytometry (BD FACSCancto II).
6
Evaluating Antibody Cytotoxicity in Cell Lines
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Viability assays were performed to determine the antiproliferative effects of trastuzumab, pertuzumab and T-DM1 (all from Roche, Basel, Switzerland), using the Cell Proliferation Reagent WST-1 (Abcam, Cambridge, United Kingdom) and following the manufacturer’s instructions. Briefly, cell lines were seeded in 96-well plates to obtain a confluency of 90%, after 24 h (5 × 103 cells/well for CAT-MT and FMCp, 15 × 103 cells/well for FMCm and 10 × 103 cells/wells for SKBR-3), and then exposed to increasing concentrations of each antibody (Table 1 ), with the control wells left unexposed. Phosphate buffered saline (PBS; Corning) was used as a vehicle for mAbs and ADC. After 72 h of exposure, the WST-1 reagent (Abcam) was added, followed by an incubation period of 4 h, at 37 °C, and absorbance was measured at 440 nm using a plate reader (FLUOStar Optima, BMG LabTech, GmbH, Ortenberg, Germany). Triplicate wells were used to determine each data point and three independent experiments were performed.
For the combined assays: trastuzumab plus pertuzumab, trastuzumab plus lapatinib (Sigma-Aldrich, Darmstadt, Germany) and pertuzumab plus lapatinib, a similar methodology was used, testing concentrations that covers different cytotoxic responses (Table 2 ).
For the combined assays: trastuzumab plus pertuzumab, trastuzumab plus lapatinib (Sigma-Aldrich, Darmstadt, Germany) and pertuzumab plus lapatinib, a similar methodology was used, testing concentrations that covers different cytotoxic responses (
7
Linker Synthesis and Purification
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Starting materials for the linker
synthesis were purchased from
commercial suppliers in the highest purity grade available. TFA.H2N-Val-Cit-PABC-MMAE was obtained from Levena Biopharma. Acros
Organics and Carlo Erba were respectively providers of dry and classical
solvents. The Hospital Pharmacy of the Tours Teaching Hospital supplied
Trastuzumab (Herceptin, Genentech) and T-DM1 (Kadcyla, Roche). The 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded
with a Bruker UltraShield 300 spectrometer. Chemical shifts are reported
in parts per million (ppm, δ), and are referenced to solvent
signal. Coupling constants are reported in hertz (Hz). Semipreparative
HPLC was carried out on a Gilson PLC 2050 system ARMEN V2 (pump) equipped
with ECOM TOYDAD600 (UV) for UV detection at 254 nm at 25 °C;
a Waters XBridge C-18; 5 μm (250 mm × 19.00 mm) column
was used; compounds were eluted with 0.1% TFA in water (solvent A),
and acetonitrile (solvent B); as a gradient from 20 to 100% B over
32 min then 100% B for 6 min at 17.1 mL/min. Flash purifications were
carried out by chromatography on silica gel columns on an ISCO purification
unit, Combi Flash RF 75 PSI, with Redisep flash silica gel columns
(60 Å, 230–400 mesh, grade 9385).
synthesis were purchased from
commercial suppliers in the highest purity grade available. TFA.H2N-Val-Cit-PABC-MMAE was obtained from Levena Biopharma. Acros
Organics and Carlo Erba were respectively providers of dry and classical
solvents. The Hospital Pharmacy of the Tours Teaching Hospital supplied
Trastuzumab (Herceptin, Genentech) and T-DM1 (Kadcyla, Roche). The 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded
with a Bruker UltraShield 300 spectrometer. Chemical shifts are reported
in parts per million (ppm, δ), and are referenced to solvent
signal. Coupling constants are reported in hertz (Hz). Semipreparative
HPLC was carried out on a Gilson PLC 2050 system ARMEN V2 (pump) equipped
with ECOM TOYDAD600 (UV) for UV detection at 254 nm at 25 °C;
a Waters XBridge C-18; 5 μm (250 mm × 19.00 mm) column
was used; compounds were eluted with 0.1% TFA in water (solvent A),
and acetonitrile (solvent B); as a gradient from 20 to 100% B over
32 min then 100% B for 6 min at 17.1 mL/min. Flash purifications were
carried out by chromatography on silica gel columns on an ISCO purification
unit, Combi Flash RF 75 PSI, with Redisep flash silica gel columns
(60 Å, 230–400 mesh, grade 9385).
8
Combination Therapy Evaluation in Cancer
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Doxorubicin (Aurovitas), trastuzumab (Roche), T-DM1 (Roche), and T-DXd (Daichii Sankyo) were obtained from a local pharmacy. SYD985 was obtained from Byondis B.V. Cdk4/6i palbociclib (LC Laboratories) and Ca-074Me (SIGMA) were purchased.
9
Site-Specific ADC Conjugation of Trastuzumab
10
Antibody-drug Conjugate T-DM1 Internalization
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The T‐DM1 and trastuzumab were purchased from F. Hoffmann‐La Roche (Basel, Switzerland). Napabucasin and DM1 were purchased from Meilunbio Inc. (Dalian, China). LysoTracker Deep Red and DyLight 488 NHS ester were purchased from Thermo‐Fisher Scientific (Waltham, MA, USA). Propidium iodide, sulforhodamine B, and the antibody against β‐tubulin were purchased from Sigma‐Aldrich (St Louis, MO, USA). Antibodies against HER2, P‐glycoprotein (P‐gp), phospho‐STAT1 (Tyr701), STAT1, phospho‐STAT3 (Tyr705), STAT3, phospho‐STAT5 (Tyr694), STAT5, phospho‐EGFR (Tyr845), phospho‐HER3 (Tyr1289), phospho‐c‐MET (Tyr1234/1235), phospho‐FGFR1 (Tyr653/654), phospho‐histone H3 (Ser10), PARP and c‐Myc were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against leukemia inhibitory factor receptor (LIFR), interleukin‐6 receptor (IL‐6R), granulocyte‐macrophage colony‐stimulating factor receptor (GM‐CSFR), β‐actin, and caspase‐3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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