Recombinant Human ICOS-L-Fc Fusion Protein

Soluble human ICOS-L (CD275) fused to an IgG1 Fc domain

Potential applications of soluble hICOS-L-Fc protein

Protein description

InvivoGen offers hICOS-L-Fc, a soluble human ICOS-L (CD275) chimera protein generated by fusing the N-terminal extracellular domain of human ICOS-L (aa 19-256) to the N-terminus of a human IgG1 Fc domain with a TEV (Tobacco Etch Virus) sequence linker.
hICOS-L-Fc has been produced in CHO cells and purified by affinity chromatography. It has an apparent molecular weight of ~70 kDa on an SDS‑PAGE gel.

ICOS-L background

Inducible Co-Stimulator Ligand (ICOS-L), also known as cluster of differentiation 275 (CD275) is a Type I transmembrane protein that is mostly expressed by antigen-presenting cells. ICOS-L is classified as an immunostimulatory immune checkpoint. It binds ICOS (CD278) which is rapidly induced in CD4⁺ and CD8⁺ T cells upon their activation [1].

 More details

Applications

• Screening of high-affinity anti-human ICOS-L monoclonal antibodies by ELISA
• Screening of anti-human ICOS monoclonal antibodies using competition assays

Quality control

• Size and purity confirmed by SDS-PAGE
• Protein validated by ELISA using an anti-hICOS-L monoclonal antibody
• Protein validated by flow cytometry using Jurkat-Lucia™ hICOS cells
• Protein potency at triggering intracellular signaling validated using Jurkat-Lucia™ hICOS cells

Reference

1. Amatore F et al. 2020. Role of ICOS in cancer immunotherapy. Expert Opin Biol Ther 20:141-150.

Figures

SDS-PAGE analysis of the hICOS-L-Fc protein. 0.5 µg of the fusion protein was loaded on a 12% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad). Detection was performed as per the manufacturer’s instructions.

Human ICOS cell surface detection using hICOS-L-Fc. ~5 x 10⁵ Jurkat-Lucia™ hICOS cells were incubated with 500 ng of hICOS-L-Fc for 30 min at 4°C. Cells were then washed and incubated with 1 µl of mouse anti-human IgG Fc antibody coupled to PE for 30 min at 4°C. Cell surface staining was analyzed by flow cytometry.

ELISA detection of hICOS-L-Fc with Anti-hICOS-L mAb. A 2-fold serial dilution of the hICOS-L-Fc fusion protein was performed and coated on ELISA plates overnight. Anti-hICOS-L-hIgG2 (red curve) or Anti-TNFa-hIgG2 control mAb (grey curve) at 5 µg/ml was added for the capture step. An HRP-labeled anti-human κ light chain antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Activation of Jurkat-Lucia™-hICOS cells using ICOS agonists. Jurkat-Lucia™-hICOS cells were incubated with increasing concentrations of recombinant human ICOS-L-Fc fusion protein, agonist Anti-hICOS armenian hamster IgG, or control Anti-βGal hIgG1 monoclonal antibodies for 6 hours. NFAT activation in the Jurkat-Lucia™-hICOS cells was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Fold responses are shown as mean + SEM.

Specifications

Protein construction: N-terminal extracellular domain of human ICOS-L (aa 19-256) with a C-terminal human IgG1 Fc tag 

Accession sequence: NP_001382847.1

Species: Human

Source: CHO cells

Tag: C-terminal human IgG1 Fc

Total protein size: 482 a.a (secreted form)

Molecular weight: ~70 kDa (SDS-PAGE)

Purification: Protein G affinity chromatography

Purity: >94% (SDS-PAGE)

Quality control:

• The protein has been validated by ELISA upon incubation with an anti-hICOS-L monoclonal antibody.
• The protein has been validated by flow cytometry using Jurkat-Lucia™ hICOS cells.
• The potency of hICOS-L-Fc at triggering intracellular signaling has been validated using Jurkat-Lucia™ hICOS cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Contents

• 50 μg of lyophilized hICOS-L-Fc protein
• 1.5 ml of endotoxin-free water

 The product is shipped at room temperature.

 Lyophilized protein should be stored at -20 ̊C.

 Resuspended protein is stable for up to 1 month when stored at 4°C, and 1 year when stored at -20°C

Avoid repeated freeze-thaw cycles.

Details

Inducible Co-Stimulator Ligand (ICOS-L), also known as cluster of differentiation 275 (CD275) is a transmembrane protein that is mostly expressed by antigen-presenting cells. ICOS-L is classified as an immunostimulatory immune checkpoint. It binds ICOS (CD278) which is rapidly induced in CD4+ and CD8+ T cells upon their activation [1]. The interaction between ICOS and ICOS-L delivers a secondary co-stimulatory signal through the activation of the transcription factor AKT, which promotes T cell proliferation and differentiation as well as the production of cytokines [1]. In tumor immunity, ICOS is involved in the amplification of the anti-tumor cytotoxic CD8+ T cell response, as well as the 'pro-tumor' function and maintenance of regulatory T cells (Tregs). Therefore, both agonistic and antagonistic monoclonal antibodies (mAbs) targeting this pathway are being investigated in combinational cancer immunotherapy [2]. Notably, ICOS agonistic mAbs have been shown to potentiate the effects of anti-CTLA4 mAbs [3]. 

​References:

1. Amatore, F. et al. 2020. Role of ICOS in cancer immunotherapy. Expert Opin Biol Ther 20, 141-150.
2. Solinas, C. et al. 2020. The rationale behind targeting the ICOS-ICOS ligand costimulatory pathway in cancer immunotherapy. ESMO Open 5.
3. Soldevilla, M.M. et al. 2019. ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity. Mol Ther 27, 1878-1891.

Citations