Anti-VISTA (Biosimilar antibody - IgG1NQ isotype)

Non-glycosylated human IgG1 monoclonal antibody (mAb) against human VISTA

VISTA in the context of immunotherapy

Anti-hVISTA-hIgG1NQ is a recombinant monoclonal antibody (mAb) featuring a variable region that recognizes human VISTA, a member of the CD28/B7 protein superfamily, and a non-glycosylated constant region of the human IgG1 (hIgG1NQ) isotype.  

V-domain Ig-containing suppressor of T-cell activation (VISTA), also known as programmed death-1 homolog (PH-1H) is a known immune checkpoint, that directly suppresses T cell activation, proliferation, and cytokine production [1]. VISTA can be considered both a ligand and a receptor, whereby it transmits inhibitory signals to T cells, either extrinsically, through expression on myeloid cells (i.e. macrophage and dendritic cells) and binding to an unknown receptor on T cells, or intrinsically, through its expression on T cells (i.e. CD4+, CD8+) [2]. In the context of cancer immunotherapy, VISTA has been found to be heightened on cells that infiltrate the tumor microenvironment (TME) and not the tumor cells themselves, therefore, VISTA blocking antibodies are of interest. These function to inhibit the engagement of VISTA with its receptor on T cells or by directly targeting VISTA expressed on naïve and effector T cells, preventing it from providing negative signaling [1, 2].  

Features of Anti-hVISTA-hIgG1NQ:

• Targets specifically human VISTA
• Features the hIgG1NQ constant region for abrogated effector function
• Functionally validated by flow cytometry

Anti-hVISTA-hIgG1NQ contains an N-glycosylation mutation in the constant region of human IgG1. Potential asparagine (N) glycosylation sites are substituted by glutamine (Q) residues, resulting in the production of a non-glycosylated antibody. Glycosylation of an antibody has no effect on antigen binding but is essential for Fc receptor-mediated activity, and therefore, the effector function of Anti‑hVISTA‑hIgG1NQ is severely compromised. Anti-hVISTA-hIgG1NQ was generated by recombinant DNA technology, produced in CHO cells, and purified by affinity chromatography with protein G.

References:

1. Nowak, E.C. et al. 2017. Immunoregulatory functions of VISTA. Immunol Rev 276, 66-79.
2. Xu, W. et al. 2018. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol 15, 438-446.

Figures

Anti-hVISTA-hIgG1NQ (1 μg) was added to Raji-null (negative control) and Raji-hVISTA cells (500 000 cells/ml) and incubated at room temperature for 30 minutes. Following this, a secondary antibody, PE, was added and incubated again at room temperature for 30 minutes. The binding affinity was then measured using flow cytometry.

Comparison of ADCC potency for native and engineered anti-human VISTA antibody isotypes: Raji-hVISTA cells were incubated with gradient concentrations of Anti-hVISTA or Anti-β-galactosidase (β-gal) mAbs for 1 hour. Jurkat-Lucia™ NFAT-CD16 effector cells were then co-incubated with target cells for 6 hours. NFAT activation, reflecting the induced ADCC response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Percentages of the maximal response normalized to the IgG1 isotype are shown.

Specifications

Specificity: Targets cells expressing human VISTA (V-domain Ig containing suppressor of T-cell activation)

Clonality: Monoclonal antibody

Isotype: Human IgG1NQ, kappa

Source: CHO cells

Formulation: 0.2 μm filtered solution in a sodium phosphate buffer with glycine, saccharose, and stabilizing agents.

Purification: Purified by affinity chromatography with protein G

Applications: Anti-hVISTA-hIgG1NQ binds and blocks ligand activation of the VISTA receptor found on the surface of cells

Quality control:

• Binding of Anti-hVISTA-hIgG1NQ to human VISTA on cells has been validated using flow cytometry.
• The complete sequence of the antibody has been verified.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Contents

Anti-hVISTA-hIgG1NQ purified monoclonal antibody is provided azide-free and lyophilized. It is available in two quantities:

• hvista-mab12: 100 µg
• hvista-mab12-03: 3 x 100 µg

Product is shipped at room temperature.

 Upon receipt, store at -20 °C.