Recombinant Human CTLA-4-Fc Fusion Protein

Soluble human CTLA-4 (CD152) fused to an IgG1 Fc domain

Potential applications of soluble hCTLA4-Fc protein

Protein description

InvivoGen offers hCTLA4-Fc, a soluble human CTLA-4 (CD152) chimera protein generated by fusing the N-terminal extracellular domain of human CTLA-4 (aa 36-160) to the N-terminus of a human IgG1 Fc domain with a TEV (Tobacco Etch Virus) sequence linker.
hCTLA-4-Fc has been produced in CHO cells and purified by affinity chromatography. It has an apparent molecular weight of ~47 kDa on an SDS‑PAGE gel.

CTLA-4 background

The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as cluster of differentiation 152 (CD152) is a Type I transmembrane protein expressed by activated and regulatory T cells. CTLA-4 is an inhibitory receptor and immune checkpoint. It exerts competitive binding to the co-stimulatory receptor CD28 ligands (i.e. CD80 and CD86) expressed by antigen-presenting cells. Thereby CTLA-4 upregulation by T cells prevents overstimulation [1-3].

 More details

Applications

• Screening of high-affinity anti-human CTLA-4 monoclonal antibodies by ELISA
• Screening of anti-human CD80 or CD86 monoclonal antibodies using competition assays

Quality control

• Size and purity confirmed by SDS-PAGE
• Protein validated by ELISA using an anti-hCTLA-4 monoclonal antibody
• Protein validated by flow cytometry using Raji-APC-hPD-L1 cells
• Protein potency at blocking the CD28/CD80- and CD28/CD86-mediated intracellular signaling validated using Jurkat-Lucia™ TCR-hPD-1 cells

References

1. Wilson, R.A.M. et al. 2018. Immune checkpoint inhibitors: new strategies to checkmate cancer. Clin. Exp. Immunol. 191(2):133-148.
2. Wei, S.C. et al. 2018. Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov. 8(9):1069.
3. Marin-Acevedo J.A. et al. 2018. Next generation of immune checkpoint therapy in cancer: new developments and challenges. J. Hematol. Oncol. 11(1):39.

Figures

SDS-PAGE analysis of the hCTLA4-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 CD80/86 cell surface detection using hCTLA4-Fc. ~5 x 10⁵ Raji-APC hPD-L1 cells were incubated with 2 µg of hCTLA4-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 hCTLA4-Fc with Anti-hCTLA4 mAb. A 2-fold serial dilution of the hCTLA4-Fc fusion protein was performed and coated on ELISA plates overnight. Anti-hCTLA4-hIgG1 (red curve) or Anti-βGal-hIgG1 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™ TCR-hPD-1 cells. ~2.5 x 10⁵ Raji-APC hPD-L1 cells were incubated with hCTLA4-Fc (50 µg/ml) or hICOS-Fc control for 1 h at 37°C. 10⁵ Jurkat-Lucia™ TCR-hPD-1 cells were then added either alone or with Anti-hCTLA4-hIgG1 (10 µg/ml). After 6 hours of incubation at 37°C, the NFAT activation in the Jurkat-Lucia™ TCR-hPD-1 cells was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™ 4 Lucia/Gaussia. Responses are shown as relative light units (RLUs; mean + SEM.)

Specifications

Protein construction: N-terminal extracellular domain of human CTLA-4 (aa 36-160) with a C-terminal human IgG1 Fc tag 

Accession sequence: NP_005250.2

Species: Human

Source: CHO cells

Tag: C-terminal human IgG1 Fc

Total protein size: 370 a.a (secreted form)

Molecular weight: ~47 kDa (SDS-PAGE)

Purification: Protein G affinity chromatography

Purity: >97% (SDS-PAGE)

Quality control:

• The protein has been validated by ELISA upon incubation with an Anti-hCTLA4-hIgG1 monoclonal antibody.
• The protein has been validated by flow cytometry using Raji-APC-hPD-L1 cells.
• The potency of hCTLA4-Fc at blocking the CD28/CD80- and CD28/CD86-mediated intracellular signaling has been validated using Jurkat-Lucia™ TCR-hPD-1 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 hCTLA4-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

The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) is an inhibitory receptor and immune checkpoint expressed by activated and regulatory T cells [1, 2].

The current paradigm is that full activation of T cells requires at least 2 signals upon contact with antigen-presenting cells (APCs) [3, 4]. Signal 1 is delivered upon the interaction of the T cell receptor (TCR) with antigenic peptides bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). Signal 2 is delivered upon the interaction of the co-stimulatory receptor CD28 with the B7 family ligands, B7-1 (CD80) and B7-2 (CD86), on APCs.

Signal 1: TCR and [HLA::peptide]

The 'classical' and most represented TCR is an 80 to 90 kDa heterodimer composed of one α chain and one β chain. The αβTCR is a transmembrane protein expressed by developing and mature T cells. It features an extracellular ligand-binding pocket and a short cytoplasmic tail. Each αβTCR is restricted to a specific complex made of an antigenic peptide and a class I or class II MHC molecule. Human MHC molecules are also known as HLA (human leukocyte antigen). Because of its short cytoplasmic tail, the TCR, once engaged, cannot signal and requires non-covalent association with the CD3 to trigger downstream intracellular signaling and T cell activation [3, 4]. Importantly, signal 1 without co-stimulation results in T cell unresponsiveness or 'anergy', a tolerance mechanism that guards against premature activation.

Signal 2: CD28 and CD80/86

CD28 is a homodimeric and transmembrane protein expressed by T cells. Nearly all human CD4+ T cells and 50% of human CD8+ T cells express CD28. The CD28 interaction with CD80 (aka B7-1) or CD86 (aka B7-2) on APCs, in conjunction with TCR engagement, triggers a co-stimulation signal (signal 2). It results in T-cell proliferation, cytokine production, cell survival, and cellular metabolism [3, 4].

IC signal: CTLA-4 and CD80/86

CTLA-4 exerts competitive binding to the co-stimulatory receptor CD28 ligands (i.e. CD80 and CD86) expressed by antigen-presenting cells. Thereby CTLA-4 upregulation by T cells prevents overstimulation.
Anti-CTLA-4 monoclonal antibodies (mAbs), as well as other immune checkpoints targeting mAbs, are extensively investigated to treat various cancers [2, 5, 6].

​References:

1. Ribas A. and Wolchock J.D. 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350.
2. Wei, S.C. et al. 2018. Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov. 8(9):1069.
3. Budd R.C. & Fortner K.A., 2017. Chapter 12 - T Lymphocytes. Kelley and Firestein's Textbook of Rheumatology (Tenth Edition). pages 189-206.
4. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.
5. Wilson, R.A.M. et al. 2018. Immune checkpoint inhibitors: new strategies to checkmate cancer. Clin. Exp. Immunol. 191(2):133-148.
6. Marin-Acevedo J.A. et al. 2018. Next generation of immune checkpoint therapy in cancer: new developments and challenges. J. Hematol. Oncol. 11(1):39.