Lately, Xencor, Inc. has launched a clinical drug called AGS-16M8F12 (ENPP3 x CD3) to target ENPP3 in patients with metastatic renal cell carcinoma (RCC). This drug is being evaluated for its safety, pharmacokinetics, and recommended dosage. ENPP3 is an extracellular enzyme belonging to the ENPP family, which has gained attention from researchers. While ENPP1 has been extensively studied, research on ENPP3 has been limited. However, recent studies have revealed its significance in processes like energy metabolism, especially in tumor therapy. Therefore, exploring ENPP3 further may uncover new mechanisms for diagnosing and treating related diseases.
1. What is the Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase (ENPP) Family?
5. The Clinical Potential of ENPP3 Targeted Therapy
6. CUSABIO ENPP3 Recombinant Proteins & Antibodies for Research Use
Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase (ENPPs or E-NPPs) family is a group of similar membrane proteins found in mammals, with seven members ENPP1-ENPP7 identified. ENPP1, ENPP2, and ENPP3 can hydrolyze pyrophosphate/phosphodiester bonds in nucleotides and exhibit some phospholipase activities. Despite the unknown functions of ENPP4, ENPP5, and ENPP6, they are part of this family due to structural similarities. Research has revealed their roles in various physiological and pathological processes, especially in diseases related to their extracellular functions, making them potential treatment targets [1-3].
NPP3, or CD203c, is part of the Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase (ENPPs) family. Initially named Gp130RB13-6, it was first recognized for its interaction with certain proteins in rat glioblastoma cells. The ENPP3 gene is found on human chromosome 6q23.2-q23.3. ENPPs share similar amino acid sequences. NPP1, NPP2, and the well-studied NPP3 are part of a type II membrane protein subfamily. These three proteins have common structural traits, including a short intracellular N-terminus, a single membrane-penetrating section, and an extended extracellular region. This extracellular portion includes two structural domains resembling growth-regulating hormones, a conserved catalytic domain similar to phosphodiesterases, a nuclease-like sequence, and a Ca2+-binding motif essential for the EF-hand (Figure 1) [5-6].
ENPP3 is found in various human tissues, including the prostate, uterus, colon, basophils, mast cells, and progenitor cells, as well as certain tumors. Interestingly, while ENPP3 isn't detected in the human liver, pancreas, or intestine, it is present in these tissues in rats. ENPP3 and other ENPPs enzymes process nucleotide substrates, which can activate P2X ion channel receptors (eg. P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7) and G protein-coupled receptors (eg. P2RY1, P2RY2, P2RY3, P2RY4, P2RY5, P2RY6, P2RY7, P2RY8). These receptor families play crucial roles in various physiological and pathological processes [7-10].
One of ENPP3's vital roles as an extracellular enzyme is to regulate purine metabolism, inhibiting mast cell and basophil activity during chronic inflammation and allergic reactions. Furthermore, studies have hinted at a potential link between ENPP3 and various human cancers, particularly renal cell carcinoma. Although there's limited research on this topic in China, international studies have consistently shown ENPP3's association with allergies and tumor development. As a result, ENPP3 holds promise as a diagnostic marker for allergies and a significant target for cancer therapy [7-10].
Figure 1. Structure of ENPP3 [6]
ENPPs are enzymes that break down extracellular nucleotides, and they play crucial roles in energy metabolism and extracellular signaling. A recent study discovered that ENPP3 has an inhibitory effect on GnT-IX (GnT-Vb), a glycosyltransferase involved in glycosylation processes. This enzyme utilizes UDP-GlcNAc as a substrate for glycosylation, providing sugar molecules (GlcNAc) that it transfers to other molecules, forming complex glycan chain structures. These glycan chains influence various biological processes like protein stability, cell recognition, signaling, and cell adhesion [12].
However, the study revealed that ENPP3 breaks down UDP-GlcNAc into UMP (uridylate monophosphate). When UMP competes with UDP-GlcNAc for binding to GnT-IX (GnT-Vb), it occupies the enzyme's substrate site, preventing efficient binding of GnT-IX (GnT-Vb) to UDP-GlcNAc. As a result, ENPP3 reduces the activity of the GnT-IX (GnT-Vb) enzyme by breaking down UDP-GlcNAc (Figure 2) [12].
Figure 2. ENPP3-mediated UDP-GlcNAc hydrolysis [12]
FcεRI is an immune receptor that plays a crucial role in allergic reactions. It's mainly found on specific immune cells like basophils and mast cells and interacts with IgE molecules [13]. When FcεRI is crosslinked, it can trigger allergic inflammation, though the exact mechanism isn't fully understood .
Studies suggest that ENPP3, activated by FcεRI cross-linking, plays a key role in chronic allergic inflammation. In mice lacking ENPP3, basophils and mast cells increase, and ATP levels in the blood rise, making them more susceptible to chronic allergies. When FcεRI is cross-linked, it releases ATP from these cells. However, in ENPP3-deficient cells, ATP removal is impaired, causing ATP to build up inside cells and trigger allergic inflammation. ENPP3, in this context, helps reduce ATP levels and dampen basophil and mast cell activity, thus suppressing allergic inflammation (Figure 3) [14].
Figure 3. ENPP3 is responsible for suppression of ATP-dependent allergic inflammation [14]
Eosinophils and mast cells play vital roles in allergic reactions. Recently, basophils have gained prominence in metabolic reaction studies due to their presence in circulation, offering a breakthrough for in vitro research. CD63 and ENPP3 (CD203c) serve as surface activation markers with high sensitivity and specificity for basophils [15-16].
ENPP3 (CD203c) levels rise significantly in activated basophils, offering superior diagnostic accuracy to CD63. In allergic asthma, dust mite stimulation leads to increased CD203c and CD63 fluorescence in eosinophils [17]. CD203c matches CD63 in diagnosing latex and insect venom allergies, but it's more sensitive in bee and wasp allergies [18-20]. Omalizumab treatment reduces CD203c expression in basophils among nut allergy patients, hinting at its ability to inhibit basophil activation via FcεRI [21].
In another study, using CRTH2-FlTC, CD203c-PE, and CD3-Per-CP antibodies, researchers gauged ENPP3 (CD203c) levels on eosinophil surfaces, assessing serum effects on eosinophil activation in urticaria patients [22]. These insights deepen our understanding of chronic urticaria's origins. In summary, ENPP3 (CD203c) holds significant promise in diagnosing and treating various allergic diseases.
Recent discoveries about mast cells expressing ENPP3 (CD203c) have piqued interest [23-24]. Studies have found ENPP3 (CD203c) in mast cells cultured from human umbilical cord blood-derived CD34+ hematopoietic stem cells and mast cells in the human gastrointestinal tract [23-24]. Furthermore, mast cells in mastocytosis patients exhibit upregulated CD203c expression [23-24]. This suggests that ENPP3 (CD203c) could serve as a marker for diagnosing mast cell-related diseases.
Additionally, research indicates that the antitumor drug midostaurin can inhibit mediator release from mast cells and basophils, but it doesn't affect IgE-dependent upregulation of ENPP3 (CD203c) on basophils. IgE cross-linking, on the other hand, upregulates CD203e on mast cells in mastocytosis patients without affecting stem cell factor (SCF) levels [25-27]. Moreover, the carbon monoxide-releasing molecule CORM-3 exhibits potent anti-inflammatory effects while promoting histamine release and ENPP3 (CD203c) expression in mast cells [28-29].
ENPP3 is a crucial tumor marker, especially in renal cell carcinoma (RCC) [30]. In RCC, ENPP3 levels significantly rise, making it a potential target for advanced antibody-drug conjugates (ADCs) like AGS-16M8F and AGS-16C3F. These ADCs, combining an anti-ENPP3 antibody with the potent MMAF toxin, selectively identify and eliminate RCC cells, delivering long-lasting anti-tumor effects with manageable side effects. This points to the promise of exploring new-gen anti-ENPP3 antibody ADC drugs for metastatic renal cell carcinoma.
In ovarian cancer, certain enzymes like ADA, ALP, ENPP1, and ENPP3 in ovarian endometrioma may serve as biomarkers for endometriosis [31]. In colon cancer, ENPP3 is highly expressed, linked with tumor cell growth and spread [32-33]. Despite its importance in various cancers, research on ENPP3-inhibiting drugs is limited. Most current inhibitors, mostly nucleotide derivatives, lack ideal drug properties within the human body, breaking down into multiple metabolites that can affect purine receptors instead of effectively inhibiting ENPP3 enzyme activity. Further research into ENPP3's role in tumors is vital to finding more potent inhibitors [34-35].
Abnormal ENPP expression can impact bone mineralization and metabolic health. Elevated 1,25(OH)2D levels may affect bone mineralization via the Vitamin D Receptor (VDR) on mature osteoblasts and osteoclasts, increasing local bone mineralization inhibitors like Enpp1, Enpp3, and Ank [36]. ENPP3 interacts directly with GRIA2, potentially affecting calcium signaling in Vascular Smooth Muscle Cells (VSMCs) and MAPK signaling [37].
Furthermore, ENPP3 and GRIA2 levels increase significantly in arteries post-Percutaneous Transluminal Angioplasty (PTA), correlating with restenosis severity [37]. ENPP3 plays a role in regulating fluid balance in respiratory epithelium and may influence bile formation and cerebrospinal fluid secretion in the liver. These liver functions have important implications for lipid metabolism, insulin sensitivity, and potentially diabetes prevention [38].
ENPP3 is involved in various physiological processes like nucleotide cycling, cell motility, and cell proliferation. Its role in metaplastic diseases and tumors is gaining attention. AGS-16M8F, an ADC antibody drug targeting ENPP3, is in phase I clinical trials for metastatic renal cell carcinoma. Clinical results are pending. The CD203c-labeled basophil stimulation assay, like CD63, may help diagnose chronic urticaria and allergies. Taken together, research on ENPP3 in allergic diseases and tumors, like renal cancer, holds promise for new therapies and opportunities for patients.
ENPP3, or CD203c, is a multifunctional molecule crucial in cancer therapy and allergy diagnosis. AGS-16M8F, an antibody drug targeting ENPP3, is being tested for treating metastatic renal cell carcinoma, potentially benefiting cancer patients. Meanwhile, ENPP3 plays a pivotal role in diagnosing allergic conditions, including chronic urticaria and various allergies, making it a valuable tool for allergy diagnostics. These dual roles highlight ENPP3's potential for advancing cancer treatment and improving allergy diagnosis.
To fully support researchers and pharmaceutical companies in their research on ENPP3 in energy metabolism, allergies, and tumors, CUSABIO presents ENPP3 active proteins & antibodies to support your research on the mechanism of ENPP3 or its potential clinical value.
CUSABIO ENPP3 Protein
● Recombinant Macaca fascicularis ENPP3, partial (Active) (Code: CSB-MP4278MOV)
The high purity is greater than 95% as determined by SDS-PAGE.
Immobilized Macaca fascicularis ENPP3 at 2 μg/ml can bind Anti-ENPP3 recombinant antibody (CSB-RA007681MA1HU), the EC50 is 3.313-4.724 ng/mL.
● Recombinant Human ENPP3, partial (Active) (Code: CSB-MP007681HU)
The high purity is greater than 95% as determined by SDS-PAGE.
Immobilized Human ENPP3 at 2 μg/ml can bind anti-ENPP3 recombinant antibody (CSB-RA007681MA1HU), the EC50 is 2.151-2.492 ng/mL.
CUSABIO ENPP3 antibody
ENPP3 Recombinant Monoclonal Antibody (Code: CSB-RA007681MA1HU)
Untransfected and transfected HEK293T cells were stained with anti-ENPP3 antibody and analyzed using flow cytometry with FITC-labeled anti-Human IgG Fc antibody.
CUSABIO ENPP3 Stable Culture Cell Line
HEK293T/Human ENPP3 Stable Cell Line (Code: CSB-SC007681HU)
Untransfected and transfected HEK293T cells were stained with anti-ENPP3 antibody and analyzed using flow cytometry with FITC-labeled anti-Human IgG Fc antibody.
References
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