Fully Mouse PD-1 Antibodies - For in vivo use

Mouse anti-mouse PD-1 antibodies in InvivoFit™ grade

Fully mouse anti-mouse PD-1 InvivoFit™ antibodies

InvivoGen offers fully mouse antibodies targeting the mouse (m)PD-1 (programmed cell death ligand 1) in two formats:

• Anti-mPD-1-mIgG1e3 (4C11) featuring a mutated mIgG1 constant region
  for no effector functions.
   
• Anti-mPD-1-mIgG1 (4C11) featuring a native mIgG1 constant region.

Both monoclonal antibodies (mAbs) are 100% murine, as the original clone 4C11 was raised in PD-1 knockout mice using a proprietary method, designed to overcome the limitation of using xenogeneic mAbs in vivo. 

In contrast, the original RMP1-14 clone, widely used in PD-1 pathway studies, was developed in rats by immunizing with mPD-1-expressing cells [1]. Repeated administrations of such rat-derived mAbs often trigger host anti-rat immune responses, which can compromise antibody performance or lead to immunogenic events [2-3]. Indeed, in a syngeneic tumor mouse model, treatment with the 100% murine Anti-mPD1-mIgG1e3 (4C11) clone resulted in improved survival and tumor regression compared to the RMP1-14 clone (see figures). The 4C11 clone achieved a higher rate of tumor-free mice (4/9 vs. 1/9) and delayed tumor progression more effectively. 

 In the Fc region of Anti-mPD-1-mIgG1e3 (4C11) InvivoFit™, a point mutation was introduced changing the aspartic (D) acid at position 265  to an alanine (A) (D265A). This alteration abolished any unwanted Fc-mediated effector functions, ensuring a more controlled and specific PD-1 pathway blockage [4]. Together, these design characteristics make the 4C11-derived mAbs ideally suited for in vivo studies, enhancing both safety and experimental reproducibility [3]. 

Key features

• Each lot is functionally tested and validated
• The complete sequence of the antibody construct was verified.
• The absence of endotoxins was determined using the EndotoxDetect™ assay.
• InvivoFit™ grade: specifically designed for in vivo studies in mice

Applications

• In vivo experiments
• Fc-mediated effector function studies
• Comparable studies using different anti-mPD-1 mAbs
   

Programmed cell death ligand 1 (PD-L1; also called B7-H1 or CD274) binds to programmed cell death protein 1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, the engagement of PD-1 on T cells and PD-L1 on malignant cells is associated with the immune escape of tumors. Clinical trials have highlighted the anti-tumor efficacy of blockades targeting the PD-1/PD-L1 pathway [5].  

 More details

References:

1. 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.
2. Brüggemann M. et al., 1989. The immunogenicity of chimeric antibodies. J. Exp. Med. 170:2153-2157.
3. 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.
4. 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.
5. McDermott D. & Atkins M. 2013. PD-1 as a potential target in cancer therapy. Cancer Med. 2(5): 662–673.

Figures

Tumor growth after treatment with different Anti-mPD-1 mAbs. 40 BALB/c female mice were subcutaneously implanted with CT-26 WT cells (2.0 x 10⁵ per mouse) into the right flank. When the mean tumor volume reached approximately 60 mm³, animals were randomized into treatment groups (n = 9/group) and dosing was initiated on Day 13 post-cell inoculation. Survival rate, tumor size, and body weight (data not shown) were measured three times weekly. During the observation period, animals bearing oversized tumors exceeding 1500 mm³ were sacrificed. Negative control antibodies Anti-β-Gal-mIgG1e3, Anti-mPD-1-mIgG1e3 (clone 4C11), and Anti-mPD-1-mIgG1e3 (clone RMP1-14) (10 mg/kg) were administered intraperitoneally on day 13, 16, 20, 23, 27, 30, 34, 37, 41 and 44. 

Percent survival of mice depicted by the Kaplan–Meier plot.  Mice were treated as described before. Tumor growth was monitored for45 days. 

Cell surface staining of mouse PD-1. EL4-mPD-1 cells were incubated with 250 ng of Anti-mPD-1-mIgG1e3 (4C11), Anti-mPD-1-mIgG1 (4C11), or isotype control for 1h at 4°C. Subsequently, a secondary PE-labeled antibody was added and incubated at 4°C for 30 minutes. Cell surface staining was analyzed by flow cytometry.

Specifications

Application: In vivo studies, flow cytometry, ELISA

Isotype:  Mouse IgG1e3 (D265A mutation; no effector function), kappa or mouse IgG1, kappa 

Recommended isotype control: Mouse IgG1e3 or mouse IgG1

Target: Mouse PD-1

Species reactivity: Mouse

Clone: 4C11

Sterility: 0.2 µm filtration

Source: CHO cells 

Production: Animal-free

Purification: Protein A

Physical form: Lyophilized

Formulation buffer: Sodium chloride buffer, sodium phosphate buffer, saccharose

Preservative: Azide-free

Reconstitution buffer: Sterile water (not provided)

Purity: ≥ 95 %

Quality control: Each lot is functionally tested and validated

Contents

Please note: each mAb is sold separately. 
All products are filter-sterilized (0.2 µm), endotoxin-free, azide-free, and lyophilized. 

Anti-mPD-1-mIgG1e3 (4C11) InvivoFit™

• mpd1c2-mab15-1: 1 mg
• mpd1c2-mab15-10: 10 mg
• mpd1c2-mab15-50: 50 mg (5 x 10 mg)

Anti-mPD-1-mIgG1 (4C11) InvivoFit™

• mpd1c2-mab9-1: 1 mg
• mpd1c2-mab9-10: 10 mg
• mpd1c2-mab9-50: 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

Immune checkpoint signal: PD-1 and PD-L1

— PD-1 (programmed cell death 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 and PD-L2 induces inhibition of TCR signaling [1].

— PD-L1 (programmed cell death ligand 1; also known as CD274 or B7-H1) is a transmembrane protein expressed at the cell surface of hematopoietic and non-hematopoietic cells and is induced by pro-inflammatory cytokines, such as in the tumor microenvironment  [1].  PD-L1 is one ligand for PD-1, an inhibitory immune checkpoint receptor that is expressed by activated and exhausted T cells. PD-1:PD-L1 interaction induces inhibition of TCR signaling, thereby preventing T-cell overstimulation and host damage [1].

PD-L1 expression is an immune evasion mechanism exploited by various malignancies and is generally associated with poorer prognosis [2]. Specifically, over-expressed PD-L1 on tumor cells and tumor-infiltrating immune cells, such as macrophages, can bind to PD-1 on cytotoxic T cells, and ultimately inhibit the anti-tumor T cell response [3, 4]. Thus, due to PD-L1’s instrumental role in immune evasion by cancer cells, there are numerous inhibitors in development as promising immuno-oncology therapies. Notably, Atezolizumab (also known as MPDL3280A), a fully humanized IgG1 (N298A) mAb that blocks the interaction of PD-L1 with PD-1 and induces anti-tumor immune reactivation, has been approved by the FDA for combinational use in the treatment of lung and breast cancer [3, 5].

References

1. Ribas A. and Wolchock J.D., 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350-55.
2. Sun, C. et al. 2018. Regulation and Function of the PD-L1 Checkpoint. Immunity 48, 434-452. 
3. Kythreotou, A. et al. 2018.  PD-L1. J Clin Pathol 71, 189-194.
4. Lau, J. et al. 2017. Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nat Commun 8, 14572.
5. Heimes, A.S. & Schmidt, M. 2019. Atezolizumab for the treatment of triple-negative breast cancer. Expert Opin Investig Drugs 28, 1-5.