Recombinant anti-mouse PD-1 antibody - Clone RMP1-14 (D265A)

InvivoGen’s engineered
Anti-mPD-1-mIgG1e3 InvivoFit™ antibody

Recombinant murinized PD-1 antibody for in vivo use

Anti-mPD-1-mIgG1e3 InvivoFit™ is a recombinant murinized anti-mouse monoclonal antibody (mAb) featuring the variable region of the previously described anti-mPD-1 RMP1-14 mAb [1, 2]. The original RMP1-14 hybridoma was obtained by immunizing rats with cells expressing murine programmed cell death 1 (mPD-1; also known as CD279). The use of xenogeneic sequences (i.e. rat origin) for mAbs renders them immunogenic upon injection in mice [3]. Moreover, special attention should be paid to mAbs targeting the PD-1/PD-L1 axis, as repeated injections of xenogeneic anti-PD-1 or anti-PD-L1 in tumor-bearing mice were shown to induce fatal hypersensitivity reactions [4].  To overcome this issue, Anti-mPD-1-mIgG1e3 InvivoFit™ was generated by recombinant DNA technology so that it is ~65% murine. It contains the constant region of mouse IgG1 with a D265A point mutation (replacement of aspartic acid by alanine at position 265) resulting in the complete loss of unwanted Fc-associated effector functions [2, 5].
Anti-mPD-1-mIgG1e3 is provided in an InvivoFit™ grade, a high-quality standard specifically adapted to in vivo studies.

Key features of Anti-mPD-1-mIgG1e3 InvivoFit™:

• Derives from the RMP1-14 clone, rat IgG2a,κ
• Features mouse IgG1e3 isotype (constant region)
• mIgG1e3 (IgG1 with a D265A point mutation) is effectorless
• Blocks the murine PD-1 receptor without causing T cell depletion
• Filter-sterilized (0.2 µm), endotoxin level < 1 EU/mg
• Specifically designed for in vivo studies in mice
• Low aggregation < 5%
• Produced in animal-free facilities and defined media

Anti-mPD -1-mIgG1e3 InvivoFit™ is produced in Chinese hamster ovary (CHO) cells, purified by affinity chromatography with protein A, and its binding is validated by flow cytometry and ELISA.

References:

1. Ribas A. & Wolchock J.D., 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350-55. 
2. Yamazaki T. et al.,2005. Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production. J Immunol. 175(3):1586-92.
3. Brüggemann M. et al., 1989. The immunogenicity of chimeric antibodies. J. Exp. Med. 170:2153-2157.
4. Mall C. et al., 2016. Repeated PD-1/PD-L1 monoclonal antibody administration induces fatal xenogeneic hypersensitivity reactions in a murine model of breast cancer. Oncoimmunology. 5(2):e1075114.
5. Baudino L. et al., 2008. Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions. J. Immunol. 181(9):6664-9.
 

Figures

ELISA binding of Anti-mPD-1-mIgG1e3 InvivoFit™ to recombinant murine PD-1. 

A dilution series of Anti-mPD-1-mIgG1e3 InvivoFit™ (red curve) and anti-mPD-1 (clone RMP1-14; blue c) was used for the detection of coated mPD-1 antigen at 2 µg/ml. Detection of bound antibody was performed using an HRP-conjugated anti-murine IgG secondary antibody for Anti-mPD-1-mIgG1e3 InvivoFit™ and an HRP-conjugated anti-rat IgG2a secondary antibody for anti-mPD-1 (clone RMP1-14). HRP activity was measured by a colorimetric assay using a Tetramethylbenzidine (TMB) substrate. Optical density was measured at 490 nm. Data are shown as percentage absorbance.

Specifications

Specificity: Targets cells expressing murine PD-1

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 IgG1e3 (D265A mutation; no effector function), kappa 

Control: mIgG1e3 InvivoFit™ isotype control

Source: CHO cells

Purity: Purified by affinity chromatography with protein A

Tested applications: Flow cytometry and ELISA

Quality control:

• Binding confirmed by flow cytometry
• The complete sequence of this antibody has been verified
• < 5% aggregates (confirmed by size exclusion chromatography)
• Endotoxin level <1 EU/mg (determined by the LAL assay)

Contents

Anti-mPD-1-mIgG1e3 InvivoFit™ is filter-sterilized (0.2 µm), endotoxin-free, azide-free, and lyophilized.  

This product is available in three pack sizes:

• mpd1-mab15-1: 1 mg
• mpd1-mab15-10​: 10 mg
• mpd1-mab15-50​: 50 mg (5 x 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

Programmed cell death 1 (PD-1; also known as CD279) is a type I transmembrane protein expressed at the cell surface of activated and exhausted conventional T cells. PD-1 is an inhibitory immune checkpoint that prevents T-cell overstimulation and host damage.
PD-1 interaction with its ligands PD-L1 (programmed cell death ligand 1) or PD-L2 induces inhibition of T-cell receptor signaling. Blockade of PD-1 with mAbs has allowed unprecedented remissions in patients with metastatic melanoma or non-small cell lung cancer. 

Citations