Murine Anti-mCD20 mAb

Recombinant mouse mAb against murine CD20 for in vivo use

InvivoGen’s engineered Anti-mCD20-mIgG2a InvivoFit™ antibody

Anti-mCD20-mIgG2a InvivoFit™ is a mouse anti-mouse monoclonal antibody (mAb) featuring the variable region of the previously described anti-mCD20 18B12 clone [1]. Using recombinant technology, the original 18B12 murine IgG1 constant region has been replaced with a murine IgG2a format which mediates potent cytotoxic functions [2].

CD20 is a cell surface protein mainly expressed by resting and activated B lymphocytes, but not plasma cells [3]. CD20 is also expressed by most malignant B cells, which makes it an ideal target for antibody-based immunotherapies against lymphoid malignancies [3-6]. 

 More details

The anti-mCD20 18B12 mAb is commonly used for in vivo depletion of the CD20+ B cell population to study the role of B cells in various immune responses, including auto-immune or tumoral contexts [1, 7].

Key features of Anti-mCD20-mIgG2a InvivoFit™:

• Derives from the 18B12 clone, mouse IgG1, κ
• Features the mIgG2a isotype (constant region)
• Guaranteed sterile, endotoxin level < 1 EU/mg
• Suitable for parental delivery in mice (azide-free)
• Low aggregation < 5%
• Produced in animal-free facilities and defined media

Anti-mCD20-mIgG2a InvivoFit™ is produced in Chinese hamster ovary (CHO) cells, purified by affinity chromatography with protein A, and provided in an InvivoFit™ grade, a high-quality standard specifically adapted to in vivo studies. The specific binding of this mAb to mCD20 has been confirmed by FACS (see Figure).

References:

1. Ahuja A. et al., 2007. Depletion of B cells in murine lupus: efficacy and resistance. J. Immunol. 179(5):3351-3361.
2. Nimmerjahn F. & Ravetch J.V., 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 310(5753):1510-2.
3. Uchida J. et al., 2004. Mouse CD20 expression and function  Int Immunol. 16(1):119-29.
4 Cvetković R.S. & Perry C.M., 2006. Rituximab. Drugs. 66:791-820.
5. Freeman C. L. & Sehn L. H., 2018. A tale of two antibodies: obinutuzumab versus rituximab. Br. J. Haematol. 182(1):29-45.
6. Soe Z. N. & Allsup D., 2017. The use of ofatumumab in the treatment of B-cell malignancies. Future Oncol. 13(29):2611-2628. 
7. Maglioco A. et al., 2017. B cells inhibit the antitumor immunity against an established murine fibrosarcoma. Oncology letters. 13(5):3225-3232.

Figures

Cell surface staining of murine CD20 using Anti-mCD20-mIgG2a InvivoFit™ mAb.
~5x10⁵ HEK293 (parental) or HEK-mCD20 cells were incubated with 2 µg of Anti‑mCD20‑mIgG2a InvivoFit™ mAb or isotype control for 1h at 4°C. Cells were then washed and incubated with 0.25 µg of goat anti-mIgG2a coupled to PE for 1h at 4°C. Cell surface staining was analyzed by flow cytometry.

Analysis of CD19+ regulatory T cell population in mice upon injection of Anti-mCD20-mIgG2a InvivoFit™ mAb.
SWR/Jmice were injected intraperitoneally (IP) at day 0, +2, +4, and +7 with 200 μg of the depleting Anti-mCD20-mIgG2a InvivoFit™ mAb or Mouse IgG2a control. At day +7 and +14, peripheral blood monocytes (PBMCs) and splenocytes were stained with the following conjugated antibodies: Anti-CD20-PE (clone SA271G2), Anti-CD19-PeCy7 (clone 6D5), according to standard procedures. Cell surface staining was analyzed by flow cytometry.
(A) Frequency of CD20+CD19+ cells among PBMCs and splenocytes at day+7 and day+14.
(B) Representative FACS staining of PBMCs at day +7.

Specifications

Specificity: Targets cells expressing murine CD20

Formulation: Lyophilized from 0.2 μm filtered solution in 150 mM sodium chloride, 20 mM sodium phosphate buffer with 5% saccharose

Clonality: Monoclonal antibody

Isotype: Murine IgG2a, kappa

Source: CHO cells

Purity: Purified by affinity chromatography with protein A

Applications: Flow cytometry; in vivo depletion

Quality control:

• Binding of Anti-mCD20-mIgG2a InvivoFit™ to mCD20 has been confirmed using Flow cytometry
• The complete sequence of the antibody construct has been verified
• < 5% aggregates (confirmed by size exclusion chromatography)
• Guaranteed sterile and <1 EU/mg (determined by the LAL assay)

Contents

Anti-mCD20-mIgG2a InvivoFit™ is provided sterile, endotoxin-free, azide-free, and lyophilized.  

This product is available in two pack sizes:

• mcd20-mab10-1​: 1 mg
• mcd20-mab10-10​: 10 mg

 The product is shipped at room temperature.

 Store lyophilized antibody at -20 °C.

 Lyophilized product is stable for at least 1 year

 Avoid repeated freeze-thaw cycles.

Details

CD20 Background:

The cluster of differentiation 20 (CD20, aka Ly-44, or B-lymphocyte antigen) is a ~35 KDa cell surface protein mainly expressed by resting and activated B lymphocytes, but not plasma cells [1]. CD20 is also expressed by most malignant B cells, which makes it an ideal target for antibody-based immunotherapies against lymphoid malignancies [1-4]. For example, Rituximab, Obinutuzumab, and Ofatumumab are FDA-approved monoclonal anti-human CD20 antibodies for the treatment of B-cell non-Hodgkin’s lymphoma and B-cell chronic lymphocytic leukemia [1-4]. These antibodies mediate the target cell destruction through different mechanisms including direct signaling of apoptosis, complement activation (CDC), and cell-mediated cytotoxicity (ADCC).

References:

1. Uchida J. et al., 2004. Mouse CD20 expression and function  Int Immunol. 16(1):119-29.
2. Cvetković R.S. & Perry C.M., 2006. Rituximab. Drugs. 66:791-820.
3. Freeman C. L. & Sehn L. H., 2018. A tale of two antibodies: obinutuzumab versus rituximab. Br. J. Haematol. 182(1):29-45.
4. Soe Z. N. & Allsup D., 2017. The use of ofatumumab in the treatment of B-cell malignancies. Future Oncol. 13(29):2611-2628.