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Ofatumumab

Manufactured by GlaxoSmithKline
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Ofatumumab is a monoclonal antibody targeting the CD20 antigen on the surface of B cells. It is designed for use in laboratory research and preclinical studies.

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Lab products found in correlation

Obinutuzumab, by Roche (1 mentions) Rituximab, by InvivoGen (1 mentions) Rituximab, by Roche (1 mentions) Normal human serum, by Innovative Research (1 mentions) Aim 5 media, by Thermo Fisher Scientific (1 mentions) Synergy h1, by Agilent Technologies (1 mentions) Ca2 mg2, by Biowest (1 mentions) 96 well microplate, by Thermo Fisher Scientific (1 mentions) Np 40, by Merck Group (1 mentions) Calcein am, by Merck Group (1 mentions)

5 protocols using ofatumumab

1

Ofatumumab Administration in Humanized Mice

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Ofatumumab (GlaxoSmithKline) from the NIH Pharmacy (Bethesda, MD) and pharmaceutical grade 0.9% sodium chloride (MedVet International, Mettawa, IL) were used in these studies. Ofatumumab was diluted in saline to concentrations of 0.2–0.75 mg/mL prior to intravenous administration to reduce the likelihood of immediate hypersensitivity reactions occurring, as has been reported on the drug label. Humanized mice were administered Ofatumumab (n = 48) intravenously in the tail vein by slow manual injection. Control humanized mice (n = 8) were administered saline in volumes consistent with Ofatumumab injection.
Semple K.M., Gonzaléz C.M., Zarr M., Austin J.R., Patel V, & Howard K.E. (2019). Evaluation of the Ability of Immune Humanized Mice to Demonstrate CD20‐Specific Cytotoxicity Induced by Ofatumumab. Clinical and Translational Science, 12(3), 283-290.
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2

Anti-CD20 Antibody Comparative Study

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The cell lines used in this study were Raji, Jurkat (both human) and the mouse cell line BW5147.
The following anti-CD20 antibodies were used: rituximab and obinutuzumab (Roche), ofatumumab (GSK) and rituximab isotypes (anti-hCD20 isotype collection, InvivoGen). Details of the antibodies used are included in the supplemental methods.
Elias S., Kahlon S., Kotzur R., Kaynan N, & Mandelboim O. (2018). Obinutuzumab activates FcγRI more potently than other anti-CD20 antibodies in chronic lymphocytic leukemia (CLL). Oncoimmunology, 7(6), e1428158.
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3

Complement-Dependent Cytotoxicity Assay Protocol

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For analysis of complement-dependent cytotoxicity (CDC), cells were incubated with or without 10μg/ml ofatumumab (commercial supply - GlaxoSmithKline) for two hours in the presence of 33% normal human serum (Innovative Research, Novi, MI) as a source of complement. The proportion of lysed CD19+ cells was assessed by TO-PRO3 (Life Technologies, Frederick, MD) staining. Complement deposition was measured using the FITC labeled 1H8 mAb (Cedarlane, Burlington, NC) that binds all forms of C3 (27 (link)). Cell surface expression levels were measured on CD19+ cells using PE labeled mAbs against CD55, CD46 and CD59 (BD Biosciences). Samples were analyzed using FACS Canto II and Fortessa LSR flow cytometers (BD Biosciences) and FlowJo 10 software (TreeStar). For all ex vivo experiments, CLL cells were cultured in AIM V media (Life Technologies).
Skarzynski M., Niemann C.U., Lee Y.S., Martyr S., Maric I., Salem D., Stetler-Stevenson M., Marti G.E., Calvo K.R., Yuan C., Valdez J., Soto S., Farooqui M.Z., Herman S.E, & Wiestner A. (2015). Interactions between ibrutinib and anti-CD20 antibodies; competing effects on the outcome of combination therapy. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(1), 86-95.
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4

Immunomodulatory Compound Evaluation

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The following compounds were used: rituximab (RTX; Roche), ofatumumab (OFA; GSK), histamine dihydrochloride (HDC; 100μM), ranitidine (100μM), catalase (200U/ml), interleukin-2 (IL-2; 500U/ml; Chiron) and diphenylene iodonium chloride (DPI; 3μM; Sigma).
Werlenius O., Aurelius J., Hallner A., Akhiani A.A., Simpanen M., Martner A., Andersson P.O., Hellstrand K, & Thorén F.B. (2016). Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells. Oncotarget, 7(22), 32046-32053.
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5

Ofatumumab-Mediated CDC Assay

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CD20-positive cells were harvested, suspended in complete medium to yield 106 cells/ml and calcein-AM (Sigma) was added to the final concentration of 1 µg/ml. After 30 min incubation at standard culture conditions, cells were washed with PBS buffer with Ca2+/Mg2+ (Biowest), loaded into the V-shape wells of 96-well microplate (Nunc) at 105 cells (or more, as indicated separately in the text) per well and pelleted. Pellets were overlaid with PBS w. Ca2+/Mg2+ containing desired concentration of ofatumumab (GlaxoSmithKline) and NHS, in a total volume of 50 µl. Microplates were incubated for 30 min. at 37 °C and shaken at 650 rpm, then overlaid with another 50 µl of PBS buffer and centrifuged. Eighty microliter of the supernatant was transferred into flat-bottom plate and fluorescence 485/515 nm was measured in Synergy H1 (Biotek) reader. Fluorescence readout obtained for cells loaded with calcein-AM and lysed with 2% NP40 (Sigma) was considered as full lysis.
Felberg A., Urban A., Borowska A., Stasiłojć G., Taszner M., Hellmann A., Blom A.M, & Okrój M. (2019). Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics. Cancer Immunology, Immunotherapy, 68(4), 587-598.
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