InvivoGen’s engineeredAnti-mPD-1-mIgG1e3 InvivoFit™ antibody

Recombinant mouse mAb against murine PD-1 for in vivo use

Anti-mPD-1-mIgG1e3 InvivoFit™ is a recombinant 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 apply 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 ~85% 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 cytolytic effector function [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™:

• Sequence is ~85% mouse origin (constant region) and ~15% rat origin (variable region)
• Features the effectorless IgG1e3 (IgG1 with a D265A point mutation)
• Blocks the murine PD-1 receptor without causing T cell depletion
• Guaranteed sterile, endotoxin level < 1 EU/mg
• Specifically designed for in vivo studies in mice
• Low aggregation < 5%
• Produced in both 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™ and anti-mPD-1 (clone RMP1-14) was used for 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 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 

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)
• Guaranteed sterile and <1 EU/mg (determined by the LAL assay)

Contents

Anti-mPD-1-mIgG1e3 InvivoFit™ is provided sterile, endotoxin-free, azide-free and lyophilized.  

This product is available in two pack sizes:

• mpd1-mab15-1​: 1 mg
• mpd1-mab15-10​: 10 mg (2 x 5 mg)

 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

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.