Herceptin® was purchased from F. Hoffmann-La Roche Ltd (Pensberg, Germany) and supplied at a concentration of 21 mg/mL (i.e., clinical dose), as a neutral aqueous solution (phosphate-buffered saline, PBS).35 (link) The stock solution of Herceptin® was diluted to a concentration of 0.8 mg/mL or 250 μg/mL in PBS. The diluted solution was incubated at 37°C (i.e., native Herceptin®) and 42°C (i.e., heated Herceptin®) for 1 h in a heat block under gentle agitation (i.e., optimal mHT condition for in-vivo antibody delivery28 (link), 36 (link)).
Herceptin
Manufactured by Roche
Sourced in Switzerland, Germany, China, Canada, France, United States, United Kingdom
Herceptin is a laboratory product manufactured by Roche. It is a monoclonal antibody used for research and analytical purposes in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Mwco 40k, by Thermo Fisher Scientific (12 mentions)
Herceptin, by Thermo Fisher Scientific (9 mentions)
Erbitux, by Merck Group (8 mentions)
Perjeta, by Roche (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mabthera, by Roche (4 mentions)
Skbr3, by American Type Culture Collection (4 mentions)
Vectibix, by Amgen (3 mentions)
Kadcyla, by Roche (3 mentions)
Trastuzumab, by Roche (3 mentions)
Bt474, by American Type Culture Collection (3 mentions)
Cinqair, by Teva Pharmaceuticals (2 mentions)
Rituxan, by Roche (2 mentions)
Gamunex, by Grifols (2 mentions)
L glutamine, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Pd 10 column, by GE Healthcare (2 mentions)
Alexa fluor 680 nhs ester, by Thermo Fisher Scientific (2 mentions)
116 protocols using herceptin
1
Herceptin Thermal Stability Protocol
2
Antibody-Drug Conjugate Formation via TCEP Reduction
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Example 17
Frozen crystal of antibody Herceptin (bought from Roche) was dissolved in DIW (initial concentration: 11.36 mg/mL). 40 μL of the antibody solution was treated with 0.77 μL of TCEP (5 molar equivalent) and stirred at 37° C. for 2 hours. A desalting column was used to remove extra TCEP in the reduced Herceptin and the buffer was changed to borate buffer. 2.618 μL (17 molar equivalent) of 20 mM of Linker-drug 1 prepared in DMSO was added into the antibody solution at 25-40° C. for 2 hours (final concentration of organic solvent in the mixture solution was about 4.9%) to obtain the antibody-drug conjugate (ADC). A desalting column (ThermoFisher Scientific, MWCO: 40K) was used to purify the product ADC Herceptin-Linker-drug 1. During elution, the buffer was changed to PBS buffer (2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4-7H2O).
3
Antibody-Drug Conjugate Synthesis
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Example 27
Frozen crystal of antibody Herceptin (bought from Roche) was dissolved in DIW (initial concentration: 11.36 mg/mL). 40 μL of the antibody solution was treated with 0.77 μL of TCEP (5 molar equivalent) and stirred at 37° C. for 2 hours. A desalting column was used to remove extra TCEP in the reduced Herceptin and the buffer was changed to borate buffer. 2.002 μL (13 molar equivalent) of 20 mM of Linker-drug 14 prepared in DMSO was added into the antibody solution at 0-4° C. for 24 hours (final concentration of organic solvent in the mixture solution was about 3.8%) to obtain the antibody-drug conjugate (ADC). A desalting column (ThermoFisher Scientific, MWCO: 40K) was used to purify the product ADC Herceptin-Linker-drug 14. During elution, the buffer was changed to PBS buffer (2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4-7H2O).
4
Characterization of HER3-Targeting Antibodies
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Trastuzumab (Herceptin) was obtained from F. Hoffmann-La Roche Ltd (Basel, Switzerland). The antibodies for Western blot against EGFR, phospho (p)-EGFR (Tyr1068), HER2, p-HER2 (Tyr1248), HER3, p-HER3 (Tyr1289), AKT, p-AKT (Ser473), glyceraldehyde-3-phosphate dehydrogenase, and the corresponding secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Phycoerythrin (PE)-conjugated anti-EGFR and anti-HER3 antibodies and fluorescein isothiocyanate-conjugated goat antihuman immunoglobulin (Ig) G (IgG) were from eBioscience (San Diego, CA, USA). The PE-conjugated anti-HER2 antibody was from BD, and HER3 was from Sino Biological Inc (Beijing, China). LMAb3, a novel anti-HER3 mAb (IgG1 k) was prepared in our laboratory. The reagents for electrophoresis and hybridization nitrocellulose filter membranes were obtained from Bio-Rad (Hercules, CA, USA). The BCA protein assay and enhanced chemiluminescent (ECL) reagents were from Thermo Scientific (Waltham, MA, USA). The cell culture medium Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, Utah, USA). Human NRG1-b1/HRG1-b1 were purchased from R&D (Minneapolis, MN, USA), CCK-8 were purchased from Dojindo (Kumamoto, Japan), and agarose (cell culture grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Herceptin-Linker-Drug 9 Conjugation
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Example 23
Frozen crystal of antibody Herceptin (bought from Roche) was dissolved in DIW (initial concentration: 11.36 mg/mL). 40 μL of the antibody solution was treated with 3.07 μL of TCEP (5 molar equivalent) and stirred at 37° C. for 2 hours. A desalting column was used to remove extra TCEP in the reduced Herceptin and the buffer was changed to borate buffer. 12.5 μL of DMSO was added and mixed evenly. Then, 0.924 μL (6 molar equivalent) of 20 mM of Linker-drug 9 prepared in DMSO was added into the antibody solution at 25-40° C. for 2 hours (final concentration of organic solvent in the mixture solution was about 28.9%) to obtain the antibody-drug conjugate (ADC). A desalting column (ThermoFisher Scientific, MWCO: 40K) was used to purify the product ADC Herceptin-Linker-drug 9. During elution, the buffer was changed to PBS buffer (2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4-7H2O).
6
Trophoblast Cell Line Cytotoxicity Assays
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Trophoblast cell lines, JEG3 and HTR 8/SVneo were cultured at 37 °C in 5% CO2 humidified atmosphere in DMEM (Life Technologies, California, USA) or RPMI (Life Technologies) medium supplemented with 10% fetal bovine serum (FBS). For various experiments, cells were treated with the following drugs at the dose ranges of 0.01–100 μM vinorelbine (Sigma, Croydon, UK), vincristine (Sigma), etoposide (Sigma), gemcitabine (Sigma), 5-fluorouracil (Sigma), cisplatin (Sigma), herceptin (Roche, Basel, Switzerland), Actinomysin D (Sigma) or vehicle control. For some experiments we also treated drugs with gefitinib (Sigma) at 8 μl. All experiments were repeated at least three times in triplicate.
7
Trastuzumab Reconstitution Protocol
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Unless otherwise stated, all
chemicals and reagents were purchased from Sigma-Aldrich/Merck in
the highest available grade. LC-MS-grade solvents and additives to
mobile phases were from Merck and Honeywell. Expired leftovers of
reconstituted trastuzumab (Herceptin, Roche) were received from Multiscan
Pharma, Czech Republic.
chemicals and reagents were purchased from Sigma-Aldrich/Merck in
the highest available grade. LC-MS-grade solvents and additives to
mobile phases were from Merck and Honeywell. Expired leftovers of
reconstituted trastuzumab (Herceptin, Roche) were received from Multiscan
Pharma, Czech Republic.
8
Antibody-Mediated Protein Labeling
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All chemicals were purchased from
Merck unless stated otherwise. Therapeutic antibodies cetuximab (Erbitux,
Merck) and trastuzumab (Herceptin, Roche) were obtained via the Catherina
Hospital pharmacy in Eindhoven, The Netherlands. The NanoLuc substrate
Nano-Glo was obtained from Promega. The non-natural amino acid pBPA
was purchased from Bachem (4017646). The anti-cetuximab antibody (Clone
HCA221) was ordered at Bio-Rad. The anti-HA antibody (Clone: 5BD1D10)
was purchased from Invitrogen.
Merck unless stated otherwise. Therapeutic antibodies cetuximab (Erbitux,
Merck) and trastuzumab (Herceptin, Roche) were obtained via the Catherina
Hospital pharmacy in Eindhoven, The Netherlands. The NanoLuc substrate
Nano-Glo was obtained from Promega. The non-natural amino acid pBPA
was purchased from Bachem (4017646). The anti-cetuximab antibody (Clone
HCA221) was ordered at Bio-Rad. The anti-HA antibody (Clone: 5BD1D10)
was purchased from Invitrogen.
9
Cytotoxicity Assay of NK-92 Cells
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We determined the specific cytotoxicity of the NK-92 cell lines toward target cells using a Europium (EuTDA) cytotoxicity assay (DELFIA, PerkinElmer), following the manufacturer’s protocol. Briefly, target cells were loaded with an acetoxymethyl ester of the fluorescence-enhancing ligand (BATDA; Perkin Elmer), and then coincubated in triplicate at 10 000 cells/well with effector cells, with or without Herceptin (2 µg/mL; Roche), at the indicated E:T ratios. After a 2-hour coincubation, supernatants were collected for measurement of the fluorescent signal reflecting target cell lysis, using a VICTOR X4 fluorometer (PerkinElmer). Specific lysis was calculated using the standard formula.
10
Trastuzumab-mediated Regulation of VEGF-A
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Trastuzumab (Herceptin®) was obtained from F. Hoffmann-La Roche Ltd. (Shanghai, China). Antibodies of HIF-α, STAT3, p-STAT3, P65, p-P65, SP1, histone H3, GAPDH and corresponding secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Electrophoresis reagents and hybridization nitrocellulose filter membranes were obtained from Bio-Rad (Hercules, CA, USA). PE, DAPI, FITC and human VEGF-A Platinum ELISA kit were obtained from eBioscience (San Diego, CA, USA). Goat anti-human CD31 antibody was obtained from Abcam Biotechnology (Cambridge, MA, USA). BCA protein assay kit and enhanced chemiluminescent (ECL) reagents were purchased from Pierce (Rockford, IL, USA). Cell culture medium Dulbeccos modified Eagles medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). SP1 interference plasmids, SP1 shRNAs (1 (link)–4 (link)), were purchased from GeneChem (Shanghai, China). Female 6-week-old BALB/c nude mice were purchased from the Vital River Laboratory (Beijing, China). TransiT-2020 transfection reagent was purchased from Mirus Bio LLC (Madison, WI, USA). Transwell chamber was obtained from Merck Millipore (Darmstadt, Germany). All other chemicals were obtained from commercial sources of analytical grade.
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