VSIG4: a Prominent B7 Family Costimulatory Molecule or Complement Receptor in Cancers and Inflammatory Diseases!

1. What are Costimulatory Molecules in the B7 Family?

The B7 family was the first co-signaling molecule shown to play a key role in T-cell activation. The B7 family now includes at least 10 members, namely B7-1/CD80, B7-2/CD86, and B7-DC, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7/HHLA2. In 1970, Bretscher and Cohn proposed the idea that T cell activation requires two signals: the first signal (antigen-stimulated signal) is generated by the TCR recognizing the pMHC presented by the APC, resulting in initial T-cell activation; the second signal (co-stimulated signal) is generated by the interaction of the APC with co-stimulatory molecules on the T-cell surface, resulting in full T-cell activation ^[1-3].

The known co-signaling molecules are divided into three families: the B7 family (immunoglobulin family), the TNF family (tumor necrosis factor family) and integrin family. The B7 family is one of the most important costimulatory molecules, widely expressed in tumor cells. B7 family members positively or negatively regulate T cell function and is essential for maintaining immune regulation and self-tolerance. No matter it was used alone or with other therapies, the B7 family is seen as "the most promising target for immunotherapy" ^[2] (Figure 1).

Figure 1. B7 family proteins and cancer ^[2]

2. What is VSIG4?

2.1 VSIG4 Structure

VSIG4 (V-set and immunoglobulin domain containing 4) is a recently identified B7 family co-stimulatory molecule, also known as Z39Ig or complement receptor of the immunoglobulin family (CRIg). Both human and murine VSIG4 genes are localized to Xq12 of chromosome X and are type I transmembrane proteins. The human VSIG4 extracellular region contains two structural domains, IgV and IgC2. A transmembrane region consists of 25 amino acids and a cytoplasmic tail exists (Figure 2) ^{[1, 4]}.

Figure 2. The structure of human or murine VSIG4 ^[1]

2.2 VSIG4 Expression

VSIG4, a novel B7 family-related protein, is currently found to be expressed mainly in tissue resting macrophages. VSIG4 mRNA is highly expressed in various tissues, especially the liver, lung, placenta, and possibly the central nervous system. But VSIG4 protein form is restricted to the surface of macrophages, where it is partly responsible for maintaining immune tolerance, anti-inflammation, host defense, and other immune regulation. Therefore, VSIG4 is a key component of the innate immune system.

2.3 VSIG4 Function

Studies have found an association between VSIG4 and a variety of inflammatory diseases such as neurological disorders, type I diabetes, and kidney disease ^[5-7]. In addition, numerous studies have demonstrated that VSIG4 is overexpressed in hepatocellular carcinoma, lung cancer, ovarian cancer, breast cancer, glioma, and multiple myeloma. To this end, VSIG4 may act as an immune checkpoint molecule, or a tumor-independent prognostic factor ^[8-9]. Currently, there are several clinical agents targeting VSIG4 in development, mainly for solid tumor treatment. Thus, VSIG4 is becoming a novel therapeutic target for inflammatory diseases and multiple tumors!

3. VSIG4-Related Mechanisms of Action

3.1 VSIG4 as a Suppressor of T Cell Activation

VSIG4 functions as an immune checkpoint regulator, suppressing T‐lymphocyte function. Much as the exact mechanism by which VSIG4 inhibits T cells is not fully known, the available studies suggest that i) VSIG4 may bind to yet unknown receptors on the surface of T cells, leading to inhibition of T cell activation; ii) VSIG4 and CR1 or CR3 on the surface of T cells bind to their co-ligands C3b and iC3b, initiating T cell inhibitory signaling; iii) VSIG4 inhibits T cell activation by downregulating G1-phase cycle protein-dependent kinases, like if CDK2, CDK4, or CDK6, induces G0/G1 phase block, and that leads to the inhibition of T cell proliferation (Figure 3) ^[10-12].

For example, in VSIG4 knockout mice group (VSIG4^-/-), it was found that the degree of interstitial inflammatory cell infiltration and heavy tubular injury in mice was higher than in the VSIG4 normal expression group (VSIG4^+/+). Meanwhile, in the VSIG4 group^-/-, the expression levels of TGF-β1, IL-2, IFN-γ, IL-10, and TNF-α were significantly increased ^[13]. In the experimental autoimmune uveitis (EAU) model, VSIG4-Ig fusion, which leads to a decrease in inflammatory cytokines secreted by T cells, such as IFN-γ, TNF-α, IL-6, and IL-17 ^[13]. Recent studies suggest that the inhibitory effect of VSIG4 does not only downregulate the activated T cell response but is able to prevent T cell activation in normal tissues ^[12].

Figure 3. VSIG4 inhibits the T cell activation ^[12]

3.2 VSIG4 as a Receptor for Complement C3

The complement system serves as an important effector system and effector amplification system in vivo. The complement system promotes phagocytosis of pathogens by working on the surface of pathogens, like disrupting the cell membranes. C3 is central to the action of complement regulators. It can be degraded to produce C3a and C3b fragments by the different proteases. These complement fragments bind to complement receptors on the surface of pathogens and induce phagocytosis. It has been identified that VSIG4 could bind to degraded fragments of complement C3, such as C3b and iC3b. To this end, VSIG4 is the fifth complement C3 receptor after CR1/CD35, CR2/CD21, CR3/CD18, CR4 (Figure 4) ^[12-15].

Studies have confirmed that VSIG4 is exclusively expressed on macrophages, effectively clears C3-regulated pathogens, thereby reducing host infection and mortality. Another study found that VSIG4 signaling not only promotes the capture of Listeria monocytogenes (LM), but also activates Chloride Intracellular Channel 3 (CLIC3), which activates phagosomes and promotes intracellular Listeria clearance ^[16]. Thus, VSIG4 acts as a novel receptor of complement C3 with potential as a therapeutic agent.

Figure 4. VSIG4 acts as a receptor for complement C3 ^[12]

3.3 VSIG4 as an Inhibitor of the Alternative Complement Pathway

There are three pathways of complement activation: the classical pathway, the MB-lectin pathway, and the alternative pathway. The alternative pathway (AP) is the main pathway of complement system activation. The AP is activated when C3 undergoes spontaneous hydrolysis and forms the initial AP C3 convertase, C3(H2O)Bb, in the presence of Factors B and D, leading to additional C3 cleavage and eventual formation of the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b). Activation of complement releases large amounts of complement fragments, such as C3a and C5a, which are chemokines and activators of neutrophils and inflammatory macrophages ^{[12, 14]}.

Even though complement activation facilitates the clearance of pathogens, excessive activation of the complement system may also be involved in the pathogenesis of autoimmune diseases. Studies suggest that VSIG4 impedes the activity of C3 convertase and C5 convertase in the alternative pathway ^{[12, 14]}, but not in the classical pathway. Namely, VSIG4 selectively inhibits the activation of the alternative pathway without affecting the classical and MBL pathways. It offers the possibility of VSIG4-targeted therapy against the AP activation.

4. The Roles of VSIG4 in Tumors and Other Diseases

4.1 VSIG4 and Lung Cancer

VSIG4-deficient in a mouse tumor model induced by constructing Lewis lung cancer cell lines had smaller tumor growth than wild type (VSIG4^+/+). For the lung cancer tissue samples, VSIG4 expression was detected by immunohistochemistry, suggesting that VSIG4⁺ cells were heavily infiltrated. Double fluorescent staining implied that VSIG4 was present in CD68+macrophages but not in CD3+T cells, CD31+endothelial cells, and CK-18+epithelial cells ^[17].

In addition, when VSIG4 transfected human renal epithelial cells, VSIG4 binds to unknown receptors on T cells. It further inhibits the proliferation of CD4+and CD8+T cells, as well as the production of cytokines, such as IL-2 and IFN-γ ^[17]. Existing studies have identified that VSIG4+tumor-associated macrophages induce the development of lung cancer. Therefore, specific targeting of VSIG4 may be a novel and effective approach to treat lung cancer.

4.2 VSIG4 and Ovarian Cancer

VSIG4 expression levels were analyzed in the blood and tissues of ovarian cancer patients, separately, researchers found that VSIG4 was overexpressed in ovarian cancer tissues and inhibited the activation of recruited T lymphocytes compared to benign tumors. Moreover, plasma levels of VSIG4 were higher than in ovarian cancer tissues.

The researchers concluded that increased soluble VSIG4 in ovarian cancer gives rise to increased C3b deposition, which promotes the formation of ovarian cancer neovascularization and the progression of ovarian cancer. Besides, VSIG4 in cancer tissue and VSIG4 in plasma were also associated with disease-free survival (DFS) ^{[6, 18]}. Therefore, VSIG4 may be a potential biomarker to predict the prognosis of ovarian cancer or a therapeutic target for ovarian cancer immunotherapy.

4.3 VSIG4 and Breast Cancer

A research based on bioinformatics database reported that VSIG4 was significantly associated with prognosis and abundance of M2-type macrophage infiltration in breast cancer patients. Further experiments implied that VSIG4 was regulated by miR-29a-3p. Specifically, tumor immune characteristics was performed using TIMER and TISIDB. The results suggested a significantly correlation between M2-type macrophages and VSIG4. Prognostic analysis indicated that the higher the VSIG4 expression level, the higher the abundance of M2-type macrophage infiltration, the worse the patient prognosis.

VSIG4-related signaling pathway enrichment analysis showed that VSIG4 was significantly associated with endocytosis, cytokine production and release, Toll-like receptors (TLRs), and cell adhesion pathways. Based on miRmap, miRanda, and PITA prediction tools, miR-29a-3p was found to regulate VSIG4 expression and had a great influence on the prognosis of breast cancer patients ^[19-20]. Therefore, VSIG4 may be used as a potent prognostic marker for breast cancer.

4.4 VSIG4 and Glioma

VSIG4 expression in malignant glioma and normal brain was detected by immunohistochemistry assays. It was found that VSIG4 expression was remarkably elevated in patients with high-grade glioma compared with normal brain tissue. Further studies suggest that overexpression of VSIG4 induces epithelial-mesenchymal transition (EMT) process and notably stimulates cell invasion and migration. As a result, it contributes to the formation of glioma stem cell phenotype. Another study identified that VSIG4 could cause immune escape of glioma cells through T lymphocyte dysfunction ^[21-22].

4.5 VSIG4 and Multiple Myeloma

In some cases, bone marrow biopsy was conducted in multiple specimens, overall survival (OS) was obviously lower in the VSIG4 high expression than in the VSIG4 low expression group. Stratification of high-risk multiple myeloma (MM) patients suggests that VSIG4 expression might serve as an independent predictor of poor OS prognosis or a candidate target for MM immunotherapy ^[23]. Another study found that protein kinase Cα (PKCα) could control VSIG4 expression. In addition, PKC signaling pathway is associated with the proliferation of multiple myeloma cells. Therefore, the PKC signaling pathway may be responsible for VSIG4 overexpression in multiple myeloma, but its specific mechanism needs to be further investigated ^[24]. Taken together, VSIG4 expression may not only help to predict the prognosis and stratification of patients, but also function as a new target for multiple myeloma.

4.6 VSIG4 and Other Diseases

Not just in tumors, VSIG4 also has a close association with inflammatory diseases like multiple sclerosis (MS), inflammatory bowel disease (IBD), kidney disease, hepatitis, and arthritis ^[25-26]. For example, in experimental autoimmune encephalomyelitis (EAE) animal model for studying MS, the research team confirmed that VG11 mAb treatment induces activation of JAK2-STAT3-A20 cascades, which further results in nuclear factor κB (NF-κB) suppression and NLRP3 and IL-1β repression ^[27].

Existing evidence shown that inhibition of NF-κB, NOD-like receptor protein 3 (NLRP3), and IL-1β expression exerted a significant therapeutic effect on MS in mice ^[28-29]. Thus, VSIG4 is considered as a novel target for MS treatment. Epstein-Barr virus (EBV) is a herpes tumor virus associated with chronic kidney disease. It has been shown that VSIG4 expression is able to inhibit MS through the LMP1/NF-κB signaling pathway. In brief, VSIG4 is a potential target for patients with chronic kidney disease and LMP1-positive EBV infection ^[30-31].

5. VSIG4 Clinical Research Prospects

Currently, specific targeting of VSIG4 is considered to be a novel, efficacious strategy for the cancer treatment. A succession of pharmaceutical companies are conducting VSIG4-targeted clinical trials, like Verseau Therapeutics and Sensei Biotherapeutics Inc in the US, Kanaph Therapeutics Inc in South Korea, and 3SBio Group in China (Table 1). According to the Pharnexcloud database, there is one anti-VSIG-4 antibody (VTX-1218) is in Preclinical studies. VTX-1218 is a mAb that binds to VSIG4. Verseau Therapeutics Inc. announced that VTX-1218 repolarizes macrophages to a pro-inflammatory state, activates T-cells and attracts other immune cells to generate a coordinated and powerful antitumor response.

In addition, a number of targeted drug studies for VSIG4 are in the drug discovery stage for diseases such as ophthalmology, immunomodulation, endocrine and metabolism, and gastrointestinal system, etc. VSIG4, as a novel B7 family co-stimulatory molecule and C3 complement receptor, becomes a novel target for immunotherapy. It is believed that VSIG4 will bring new strategies for the treatment in tumors or inflammatory diseases!

Table 1: VSIG4 clinical research progress

To assist researchers or pharmaceutical companies in their research on VSIG4 in tumor and inflammatory diseases, CUSABIO offers the VSIG4 active protein product (Code: CSB-MP896869HU) to support your research on the mechanism of VSIG4 or its potential clinical value.

Recombinant Human V-set and immunoglobulin domain-containing protein 4(VSIG4), partial (Active)

High Purity Validated by SDS-PAGE

The purity was greater than 95% as determined by SDS-PAGE.(Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel.

Excellent Bioactivity Validated by Functional ELISA

Immobilized Human VSIG4 at 2 μg/ml can bind Anti-VSIG4 recombinant antibody (CSB-RA896869MA1HU), the EC₅₀ is 51.14-68.73 ng/mL.