Daratumumab

Manufactured by Johnson & Johnson

Sourced in United States

Daratumumab is a monoclonal antibody used as a laboratory research tool. It binds to the CD38 protein, which is expressed on the surface of various cell types. Daratumumab is utilized in research applications to study the role of CD38 in cellular processes and disease pathways.

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Lab products found in correlation

Nivolumab, by Bristol-Myers Squibb (2 mentions) Venetoclax, by Selleck Chemicals (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) 7 aad, by Thermo Fisher Scientific (1 mentions) Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions) Cytochalasin d, by Merck Group (1 mentions) Lenalidomide, by MedChemExpress (1 mentions) Dexamethasone (dex), by Kyoritsu Seiyaku (1 mentions) Migg2a clone c1.18.4, by BioXCell (1 mentions) Elotuzumab, by Bristol-Myers Squibb (1 mentions) Pierce bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions) 7 aad viability staining solution, by Thermo Fisher Scientific (1 mentions) Erbitux, by Merck Group (1 mentions) Mopc 21, by BioLegend (1 mentions) Cetuximab, by Merck Group (1 mentions) Darzalex, by Johnson & Johnson (1 mentions) Red blood cell lysis buffer, by BioLegend (1 mentions) Mm cell lines, by American Type Culture Collection (1 mentions)

14 protocols using daratumumab

Daratumumab Cytotoxicity Against NK Cells

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Peripheral blood NK cells and iNK cells were cultured for 3 hours in the presence of daratumumab (Janssen) at concentrations ranging from 0-30 μg/ml. Cells were then stained with a fluorescently conjugated anti-CD56 antibody, fixable viability dye, and 7-AAD (Thermo Fisher) for flow cytometry analysis. NK cells were gated based on CD56 expression, and viable cell percentages were determined based on exclusion of the dead cell dye and 7-AAD staining. Specific cytotoxicity was calculated as (% specific death - % spontaneous death) / (1- % spontaneous death) x 100.

Woan K.V., Kim H., Bjordahl R., Davis Z.B., Gaidarova S., Goulding J., Hancock B., Mahmood S., Abujarour R., Wang H., Tuininga K., Zhang B., Wu C.Y., Kodal B., Khaw M., Bendzick L., Rogers P., Ge M.Q., Bonello G., Meza M., Felices M., Huffman J., Dailey T., Lee T.T., Walcheck B., Malmberg K.J., Blazar B.R., Bryceson Y.T., Valamehr B., Miller J.S, & Cichocki F. (2021). Harnessing Features of Adaptive NK Cells to Generate iPSC-Derived NK Cells for Enhanced Immunotherapy. Cell stem cell.

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Daratumumab-Mediated ADCP of MM Cells

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MM cell lines were fluorescently labeled using the CellTrace™ CFSE Cell Proliferation Kit (Thermofisher Scientific). Cells were stained as outlined in the manufacturer’s instructions. Stained cells were co-cultured with control or CTX-TCS conditioned macrophages at an effector to target (E:T) ratio of 2:1 for 18 hours. MM cells were pre-incubated in the presence of 1 μg/ml daratumumab (Janssen, PA, USA), an IgG1 anti-CD38 antibody, or the relevant isotype control and then added to cultures. After 18 h incubation, cells were imaged by fluorescent microscopy and then detached by pipetting to single cell suspensions. MM cell clearance by ADCP was assessed by ﬂow cytometry. Percentage daratumumab-specific cell clearance was calculated as follows; [100–100*(% CFSE labeled MM cells (daratumumab treated)/% CFSE labeled MM cells (isotype control))]. To confirm that clearance was due to macrophage-mediated phagocytosis, experimental wells containing macrophages were pre-incubated with 1 μg/ml Cytochalasin D (Sigma), which inhibits actin polymerization and thus inhibits phagocytosis. When investigating the role played by FcγRI/CD64 in daratumumab-specific killing, macrophages were pre-incubated with anti-CD64 blocking antibody (eBiosciences, CA, USA).

Naicker S.D., Feerick C.L., Lynch K., Swan D., McEllistrim C., Henderson R., Leonard N.A., Treacy O., Natoni A., Rigalou A., Cabral J., Chiu C., Sasser K., Ritter T., O’Dwyer M, & Ryan A.E. (2021). Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis. Oncoimmunology, 10(1), 1859263.

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Combination Therapy for Myeloma

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TAS4464 was synthesized at Taiho Pharmaceutical. Bortezomib was purchased from LKT Laboratories. Lenalidomide was purchased from MedChemExpress. Dexamethasone was purchased from Kyoritsu Seiyaku. Daratumumab was purchased from Janssen‐Cilag. Elotuzumab was purchased from Bristol‐Myers Squibb GmbH & Co. KGaA.

Muraoka H., Yoshimura C., Kawabata R., Tsuji S., Hashimoto A., Ochiiwa H., Nakagawa F., Fujioka Y., Matsuo K, & Ohkubo S. (2019). Activity of TAS4464, a novel NEDD8 activating enzyme E1 inhibitor, against multiple myeloma via inactivation of nuclear factor κB pathways. Cancer Science, 110(12), 3802-3810.

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Investigating Monoclonal Antibody Therapies

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Daratumumab was provided by Janssen Pharmaceuticals (Beerse, Belgium) and nivolumab by Bristol-Myers Squibb (New York City, NY, USA). The nonspecific human IgG1 antibody CNTO 3930 (Janssen Pharmaceuticals) was used as the isotype control.
The following monoclonal antibodies (mAbs) were used in mouse (m) tumor models: anti-mCD38-FcγR-engaging mAb (mIgG2a isotype; this antibody mediates ADCC/ADCP of mCD38⁺ tumor cells); anti-mCD38 FcγR-inert mAb (mIgG1-D265A isotype; this antibody has no FcγR-mediated effector function); and anti-mPD-1 FcγR-inert mAb (mIgG1-D265A isotype; blocks mPD-1:mPD-L1/mPD-L2 interactions [42 (link),43 (link)]). Control antibodies included mIgG2a (clone C1.18.4, BioXCell, Lebanon, NH, USA) and mIgG1-D265A (Bristol-Myers Squibb).

Verkleij C.P., Jhatakia A., Broekmans M.E., Frerichs K.A., Zweegman S., Mutis T., Bezman N.A, & van de Donk N.W. (2020). Preclinical Rationale for Targeting the PD-1/PD-L1 Axis in Combination with a CD38 Antibody in Multiple Myeloma and Other CD38-Positive Malignancies. Cancers, 12(12), 3713.

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Monoclonal Antibody Binding Assay

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Daratumumab (Janssen Pharmaceuticals, Inc. NJ, USA) and elotuzumab (Bristol-Myers Squibb, New York City, NY, USA) were purchased from the research pharmacy at UPMC. Daratumumab is in 20 mg/mL solution. elotuzumab is reconstituted with water to obtain a concentration of 25 mg/mL per manufacturer instructions. Both drugs were stored at 2–8 °C and used before their expiration date. Stability was confirmed by continued appearance of the monoclonal band at the appropriate location by electrophoresis.
Magnetic beads were from Invitrogen (Dynabeads, Catalog No 10104D, Carlsbad, CA). His-tag human CD38 protein (Catalog No 10818-H08H) and his-tag human SLAMF7 (11691-H08H) were from Sino Biological Inc. (Wayne, PA). Both recombinate proteins were purchased in lyophilized aliquots and were reconstituted within 1 week of use. All reagents were used before their expiration date. Bead binding of antigen was verified by performance of a Pierce bicinchoninic acid assay (Thermo Fisher Scientific, Waltham MA, USA) to determine protein concentration before and after antigen binding to the beads. tmAb spiked patient sera was utilized as a control to ensure beads coated with cognate antigen continued to remove the anticipated concentration of tmAb.

Liu L., Shurin M.R, & Wheeler S.E. (2019). A Novel Approach to Remove Interference of Therapeutic Monoclonal Antibody with Serum Protein Electrophoresis. Clinical biochemistry, 75, 40-47.

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Daratumumab and Isotype Control Protocol

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Daratumumab was provided by Janssen Pharmaceuticals. Human IgG1-b12 (Genmab), a human mAb against an innocuous antigen (HIV-1 gp120), was used as an isotype control as described previously(13 (link)).

Krejcik J., Frerichs K.A., Nijhof I.S., van Kessel B., van Velzen J.F., Bloem A.C., Broekmans M.E., Zweegman S., van Meerloo J., Musters R.J., Poddighe P.J., Groen R.W., Chiu C., Plesner T., Lokhorst H.M., Sasser A.K., Mutis T, & van de Donk N.W. (2017). Monocytes and granulocytes reduce CD38 expression levels on myeloma cells in patients treated with daratumumab. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(24), 7498-7511.

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NK-92 Cytotoxicity Assay against Daratumumab-Treated Cells

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NK-92—dependent tumor cell cytotoxicity was quantified by flow cytometry. Target cells were labeled using CellTrace™ Violet (Thermo Fisher Scientific, Waltham, MA, USA) and were incubated for 20 min at 37 °C in humidified atmosphere with 5% CO₂ at an effector-to-target ratio of 10:1. After washing, target cells were re-suspended in supplemented media and mixed in co-culture with effector cells in the presence of 10 µg/mL of Daratumumab [24 (link)] (Janssen Pharmaceuticals, Spring House, PA, USA) in 96-well plates for 6 h at 37 °C in a humidified atmosphere with 5% CO₂. After 6 h, dead violet dye-labeled tumor cells were measured with 7-AAD Viability Staining Solution (Thermo Fisher Scientific, Waltham, MA, USA) by flow cytometry 5 min after its addition. Specific cytotoxicity was determined by the equation: % dead tumor cells − % spontaneous tumor cell death. The leukemia cell line K562 was used as a negative control for NK-92—dependent cytotoxicity. The gating strategy is described in Figure S1.

Lejeune M., Duray E., Peipp M., Clémenceau B., Baron F., Beguin Y, & Caers J. (2021). Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma. Cancers, 13(12), 3072.

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Antibody Variants for CD38 and CD47 Studies

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The approved CD38 antibody daratumumab (human IgG1, DARA-IgG1, clone 005, DARZALEX^®) was from Janssen Biotech (Horsham, PA, USA). An IgA2 variant of daratumumab (DARA-IgA2) was generated de novo (see below). Isotype control antibodies were the EGFR antibody cetuximab (human IgG1, clone 225; Erbitux^®), which was obtained from Merck (Kenilworth, NJ, USA), and its IgA2 variant generated as described (22 (link)). The Fc silent CD47 antibody variant 5F9-IgG2σ with V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions and the soluble SIRPα-IgG2σ protein (referred to as SIRPα-Fc) were produced as described (17 (link), 23 (link)). Murine antibodies against human CD38 (clone HB-7), CD47 (clone CC2C6) as well as the isotype control antibody (clone MOPC-21) were from BioLegend (San Diego, CA, USA). Murine antibody against CD47 (clone B6H12) was from Thermo Fisher Scientific (Waltham, MA, USA). All-trans retinoic acid (ATRA) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Baumann N., Arndt C., Petersen J., Lustig M., Rösner T., Klausz K., Kellner C., Bultmann M., Bastian L., Vogiatzi F., Leusen J.H., Burger R., Schewe D.M., Peipp M, & Valerius T. (2022). Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab. Frontiers in Immunology, 13, 949140.

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Multiparametric Analysis of Myeloma Cells

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Lipophilic cell tracer (DiO) was purchased from Invitrogen (Eugene, OR). All antibodies used for flow cytometry were purchased from BD Biosciences (San Jose, CA) unless otherwise noted. Red Blood Cell (RBC) lysis buffer was purchased from Biolegend (San Diego, CA). All chemicals were purchased from Millipore Sigma (Burlington, MA). All drugs were purchased from Selleck Chemicals (Houston, TX), except Daratumumab (Janssen, Beerse, Belgium), which was generously provided to us by the pharmacy at Washington University. MM cell lines were obtained from American Type Culture Collection (Manassas, Virginia).

Alhallak K., Jeske A., de la Puente P., Sun J., Fiala M., Azab F., Muz B., Sahin I., Vij R., DiPersio J.F, & Azab A.K. (2021). A pilot study of 3D tissue-engineered bone marrow culture as a tool to predict patient response to therapy in multiple myeloma. Scientific Reports, 11, 19343.

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Anti-CD47 mAb Production Protocol

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Daratumumab was purchased from Janssen Biotech Inc. Anti-CD47 monoclonal antibody (hu5F9) was produced in house by transient expression into suspension-adapted HEK293-EBNA cells (catalog no. ATCC-CRL-10852, LGC Standards, Teddington, UK) using polyethyleneimine. Briefly, equal quantities of each engineered chains vectors were co-transfected. The culture was fed with the same volume of Ex-cell 293 media and incubated for 5 days at 37 °C, 150 rpm. The supernatant was then harvested by centrifugation at 3220 × g for 15 min and sterile filtered (0.22 μm) prior purification by Protein A and cation exchange chromatography, as described below.

Grandclément C., Estoppey C., Dheilly E., Panagopoulou M., Monney T., Dreyfus C., Loyau J., Labanca V., Drake A., De Angelis S., Rubod A., Frei J., Caro L.N., Blein S., Martini E., Chimen M., Matthes T., Kaya Z., Edwards C.M., Edwards J.R., Menoret E., Kervoelen C., Pellat-Deceunynck C., Moreau P., Mbow M.L., Srivastava A., Dyson M.R., Zhukovsky E.A., Perro M, & Sammicheli S. (2024). Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma. Nature Communications, 15, 2054.

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