Recombinant anti-mouse GITR antibody

Recombinant murinized GITR antibody for in vivo use

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

Anti-mGITR-mIgG2a InvivoFit™ is a recombinant murinized anti-mouse monoclonal antibody (mAb) featuring the variable region of the previously described anti-mGITR clone DTA-1 [1]. The rat-derived constant region was replaced by a murine constant region, making the mAb ~65% of murine origin. This feature allows for reduced immunogenicity and risks of fatal hypersensitivity reactions upon repeated mAb injections into mice [2-4]. Anti-mGITR-mIgG2a is provided in an InvivoFit™ grade, a high-quality standard specifically adapted to in vivo studies. 

This agonist mAb has been reported to increase T cell and NK cell activity, cytotoxicity, and degranulation in vivo [4]. It shows strong antitumor efficacy by modulating both effector (Teffs) and regulatory (Tregs) T cells [4-5]. The Anti-mGITR mAb efficacy is moderate on its own, thus it is more often used in combination with other immune checkpoint mAbs (e.g. CTLA4, PD-1) [4]. Notably, the anti-GITR (DTA-1) antibody seems to trigger anaphylaxis in C57BL/6 mice in a target/isotype-dependent manner [4]. Thus, the correct selection of a strain-matched heavy chain isotype prior to in vivo experiments is crucial (IgG2a for Balb/c, swiss mice & IgG2c for C57BL/6 mice). 

Key features of Anti-mGITR-mIgG2a InvivoFit™:

• Specific binding to mouse GITR
• Derived from the DTA-1 clone
• Features the IgG2a isotype (associated with high antitumor activity) 
• Filter-sterilized (0.2 µm), endotoxin level < 1 EU/mg
• Specifically designed for in vivo studies in mice
• Free from non-relevant mAbs found in hybridoma-based productions
• Produced in animal-free facilities and defined media
• Low aggregation < 5%

GITR (Glucocorticoid-Induced TNFR-Related protein) is a co-stimulatory immune checkpoint protein found on a variety of immune cells including T cells, natural killer (NK) cells and some myeloid cells. Interaction of GITR with its ligand GITRL can modulate the pathogenesis of tumors, inflammation, and autoimmune diseases [5].

 More details

References:

1. Cohen AD, et al., 2010. Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation. PLoS One.;5(5):e10436.
2. 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.
3. Murphy, J.T. et al., 2014. Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal antibody in mice. Blood. 123(14):2172-2180.
4. 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.
5. Tian J, et al., 2020. The Role of GITR/GITRL Interaction in Autoimmune Diseases. Front Immunol.;11:588682.

Figures

Binding of Anti-mGITR-mIgG2a InvivoFit™ mAb to coated murine GITR protein. Murine GITR (5 µg/ml) was coated on ELISA plates overnight. A 2-fold serial dilution of the Anti-mGITR-mIgG2a InvivoFit™ (red curve) or of the Anti-β-Gal-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. Data are shown as optical density (OD) at 490 nm (mean ± SEM).

Specifications

Target: Cells expressing murine GITR

Specificity: No cross-reactivity with human GITR

Clone: DTA-1

Source: CHO cells

Isotype: Murine IgG2a, lambda

Control: Murine IgG2a Isotype Control

Formulation: Lyophilized from 0.2 μm filtered solution in a sodium phosphate buffer with 5% saccharose

Purity: Purified by affinity chromatography with protein A

Tested application: ELISA

Quality control:

• This product has been validated using ELISA binding assay.
• The complete sequence of this antibody has been verified.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK‑Blue™ TLR4 cells.
• < 5% aggregates (confirmed by size exclusion chromatography)
• Endotoxin level <1 EU/mg (determined by the LAL assay)

Contents

Anti-mGITR-mIgG2a InvivoFit™ is filter-sterilized (0.2 µm), endotoxin-free, azide-free, and lyophilized.  

This product is available in two pack sizes:

• mgitr-mab10-1​: 1 mg
• mgitr-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

GITR

The glucocorticoid-induced TNF receptor–related protein GITR (also known as CD357 or TNFRSF18) is an immune receptor that belongs to Tumor Necrosis Factor Receptor Superfamily (TNFRSF). The engagement with its ligand GITRL (TNFSF18, AITRL, CD357L) has been shown to modulate both the acquired and innate immune system [1]. GITR is mainly expressed on activated CD4+, CD8+, and FoxP3+ T cells. To a lesser extent, it is also found on natural killer (NK) cells, B cells, mast cells, and keratinocytes. The ligand GITRL is primarily found in various APCs, including dendritic cells (DCs) subsets, B cells, monocytes, endothelial cells, and interestingly in human tumor cells [2].  
Given the divers expression patterns, the GITR/GITRL axis may play a role not only in regulating immune responses but also in mediating leukocyte adhesion and migration [3]. Indeed, GITR activation and function is cell type and context dependent, and therefore can have divers effects [3].  

GITR signaling

GITR signaling is mediated through the TRAF (TNF receptor-associated factors )-dependent activation of NF-κB as well as members of the MAPK pathway, including ERK, p38 and JNK [1,3]. On activated CD8+ effector T cells (Teffs), GITR has a co-stimulatory function by enhancing cell activation, cytokine secretion, proliferation, and survival [3]. In contrast, GITR activation on murine regulatory T cells (Tregs) abrogates their suppressive function on Teffs leading to increased Teffs survival and therefore, enhanding antitumor responses [3]. In vivo data suggests, that GITR signaling is involved in the development of autoimmune diseases, graft-versus-host disease, and in the immune response against infectious pathogens. Moreover, in murine tumor models, GITR modulation has been reported to influence animal survival and even lead to the eradication of tumors [4]. However, several studies show severe differences of GITR/GITRL between human and mice regarding expression profile, signaling and structure [4,5]. Therefore, additional studies are required to elucidate the possible differences between GITR/GITRL-modulating reagents in human and mice [5]. 

References:

1. Tian J, et al., 2020. The Role of GITR/GITRL Interaction in Autoimmune Diseases. Front Immunol.;11:588682.
2. Nocentini G, Riccardi C., 2009. GITR: a modulator of immune response and inflammation. Adv Exp Med Biol. 2009;647:156-73.
3. Knee DA, et al,. 2016. Rationale for anti-GITR cancer immunotherapy. Eur J Cancer.;67:1-10. 
4. Baltz KM, et al., 2008. Neutralization of tumor-derived soluble glucocorticoid-induced TNFR-related protein ligand increases NK cell anti-tumor reactivity. Blood. 2008 Nov 1;112(9):3735-43.
5. Baltz KM, et al., 2007. Cancer immunoediting by GITR (glucocorticoid-induced TNF-related protein) ligand in humans: NK cell/tumor cell interactions. FASEB J.;21(10):2442-54. 
6. Wang, F. et al., 2021. Structures of mouse and human GITR–GITRL complexes reveal unique TNF superfamily interactions. Nat Commun 12, 1378.