Murine Anti-mCD3 mAb

Recombinant mouse mAb against murine CD3ε for in vivo use

Anti-mCD3-mIgG2a InvivoFit™ is an anti-mCD3 monoclonal antibody featuring the variable region of the previously described 145-2C11 clone [1] and a murinized IgG2a constant region.

InvivoGen’s engineered Anti-mCD3-mIgG2a InvivoFit™ antibody

CD3 is a multimeric protein complex consisting of four polypeptides (CD3ε, CD3γ, CD3δ, and CD3ζ) that assemble as three dimers (εγ, εδ, and ζζ) [2]. The CD3 complex is a marker of T cells [2]. Upon antigen recognition, the TCR/CD3 complex on T cells triggers downstream intracellular signaling and participates in T cell activation  [3]. 

More details

The anti-mCD3ε 145-2C11 mAb is commonly used for cytometry detection and in vivo depletion of the T cell population [1]. Using recombinant technology, the original 145-2C11 Armenian Hamster IgG1 constant region has been replaced with a murine IgG2a format which mediates potent cytotoxic functions [4]. This murinization also allows reduced immunogenicity of the administered mAb and risks for fatal hypersensitivity reactions [5-7].

Key features:

• Derives from the 145-2C11 clone, Armenian Hamster IgG1
• 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-mCD3-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 mCD3 has been confirmed by FACS and ELISA (see Figures).

References:

1. Leo O., et al., 1986. Identification of a monoclonal antibody specific for a murine T3 polypeptide. PNAS. 84(5):1374-1378.
2. Chetty R. & Gatter K., 1994. CD3: structure, function, and role of immunostaining in clinical practice. J. Pathol. 173(4):303-307.
3. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.
4. Nimmerjahn F. & Ravetch J.V., 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 310(5753):1510-2.
5. Mall C. et al., 2016. Repeated PD-1/PD-L1 monoclonal antibody administration induces fatal xenogenic hypersensitivity reactions in a murine model of breast cancer. Onco Immunol. 5(2):e1075114.
6. Murphy, J.T. et al., 2014. Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal antibody in mice. Blood. 123(14):2172-2180.
7. Belmar N.A. et al., 2017. Murinization and H chain isotype matching of Anti-GITR antibody DTA-1 reduces immunogenicity-mediated anaphylaxis in C57BL/6 mice. J Immunol. 198:4502-4512.

Figures

Binding of Anti-mCD3-mIgG2a InvivoFit™ mAb to coated mouse CD3ε protein.
Mouse CD3ε (10 µg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-mCD3-mIgG2a InvivoFit™ mAb (red curve) or of the Anti-βGal mIgG2a control antibody (grey curve) was performed for the capture step. An HRP-labelled anti-mIgG antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Cell surface staining of murine CD3 using Anti-mCD3-mIgG2a InvivoFit™ mAb.
~5x10⁵ Jurkat (parental) or Jurkat-mCD3 cells were incubated with 2 µg of Anti‑mCD3‑mIgG2a InvivoFit™ mAb or an 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.

Specifications

Specificity: Targets cells expressing murine CD3ε

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; ELISA

Quality control:

• Binding of Anti-mCD3-mIgG2a InvivoFit™ to mCD3ε has been confirmed using Flow cytometry
• Antibody binding has been validated by ELISA using a coated mouse CD3 protein
• 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-mCD3-mIgG2a InvivoFit™ is provided sterile, endotoxin-free, azide-free, and lyophilized.  

This product is available in two pack sizes:

• mcd3-mab10-1​: 1 mg
• mcd3-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

CD3 Background:

The cluster of differentiation 3 (CD3, formerly named T3) is a multimeric protein complex consisting of four polypeptides (CD3ε, CD3γ, CD3δ, and CD3ζ) that assemble as three dimers (εγ, εδ, and ζζ) [1]. The CD3 complex is a marker of T cells [1], which recognizes and participates in the elimination of infected cells and tumor cells through the interaction between the TCR on T cells and the MHC-peptide complex on antigen-presenting cells [2]. Because of its short cytoplasmic tail, the TCR lacks the ability to signal and requires non-covalent association with the CD3 [2]. Upon antigen recognition, the TCR/CD3 complex on T cells triggers downstream intracellular signaling and participates in T cell activation  [2]. The CD3ε polypeptide chain (~25 kDa) is targeted by OKT3, a monoclonal antibody that is clinically approved for the induction of immunosuppression in solid organ transplantation [3]. 

1. Chetty R. & Gatter K., 1994. CD3: structure, function, and role of immunostaining in clinical practice. J. Pathol. 173(4):303-307.
2. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.
3. Norman D.J., 1995. Mechanisms of action and overview of OKT3. Therapeutics Drug Monitoring. 17:615-620.