Recombinant anti-mouse CD20 antibody

Recombinant murinized CD20 antibody 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]. 

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].
Anti-mCD20-mIgG2a is provided in an InvivoFit™ grade, a high-quality standard specifically adapted to in vivo studies.
 

Key features of Anti-mCD20-mIgG2a InvivoFit™:

• Derives from the 18B12 clone, mouse IgG1, κ
• Features the mIgG2a isotype (constant region)
• Filter-sterilized (0.2 µm), 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 and purified by affinity chromatography with protein A. The specific binding of this mAb to cell surface mCD20 and its in vivo depleting function have been confirmed (see Figures).

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

Control: mIgG2a  InvivoFit™ isotype control

Source: CHO cells

Purity: Purified by affinity chromatography with protein A

Applications: Flow cytometry; in vivo depletion

Quality control:

• The 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)
• Endotoxin level <1 EU/mg (determined by the LAL assay)

Contents

Anti-mCD20-mIgG2a InvivoFit™ is filter-sterilized (0.2 µm), 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.

InvivoFit™

InvivoFit™ is a high-quality standard specifically adapted for in vivo studies. InvivoFit™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and a TLR2 and TLR4 reporter assay.

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.