NT5E

Detailed Information

The NT5E gene (also known as CD73) is located on the q14.3 region of human chromosome 6 and consists of 11 exons, encoding a 574-amino-acid type II transmembrane glycoprotein. The protein is made up of three domains: an intracellular N-terminal tail (amino acids 1-5), a single transmembrane helix (amino acids 6-26), and an extracellular C-terminal catalytic domain (amino acids 27-574). The extracellular domain has a typical 5'-nucleotidase fold, including two zinc-binding sites (His-267, His-317) and a substrate-binding pocket (Arg-354, Asp-362). Post-translational modification of the NT5E protein results in a homodimer with a molecular weight ranging from approximately 70 kDa (non-glycosylated form) to 100 kDa (fully glycosylated form). This enzyme catalyzes the hydrolysis of extracellular nucleotide monophosphates to membrane-permeable nucleosides, with a substrate preference of AMP > IMP > UMP > GMP > CMP. It also exhibits significant activity against dAMP and dCMP, but no activity towards cyclic nucleotides (e.g., cAMP).

Kinetic studies of NT5E indicate that its optimal pH is between 7.0 and 7.5, with a maximum reaction rate (Vmax) of 8.3 μmol/min/mg and a Michaelis constant (Km) for AMP of 12.5 μM. Notably, enzyme activity is regulated by several ions: Mg²⁺ or Mn²⁺ are essential cofactors (EC50 ≈ 50 μM), while Ca²⁺ concentrations above 100 μM lead to competitive inhibition. Under pathological conditions such as the tumor microenvironment, hypoxia-inducible factor-1α (HIF-1α) directly binds to the hypoxia response element (HRE) in the NT5E gene promoter, upregulating enzyme expression by 2-3 times. Additionally, the inflammatory factor TNF-α enhances NT5E transcription through the NF-κB pathway, forming a positive feedback loop.

Figure 1. Structure and function of CD73/NT5E. (Gao ZW, et al., 2014)

Tumor Immune Microenvironment Regulation

Adenosine-Mediated Immune Suppression

The core role of NT5E in tumor immune evasion is mediated through the catalysis of extracellular adenosine. In the tumor microenvironment, CD39 (encoded by the ENTPD1 gene) first hydrolyzes ATP to AMP, after which NT5E converts AMP to adenosine. The accumulated adenosine (at concentrations of 1-10 μM) binds to the A2A adenosine receptor (A2AR)on immune cells, activating the Gs protein-cAMP-PKA signaling axis, resulting in multiple immune-suppressive effects: in T cells, increased cAMP inhibits T-cell receptor (TCR) signaling, reducing IL-2 secretion and proliferation; in natural killer (NK) cells, PKA phosphorylation inhibits the DAP12-Syk-ZAP70 pathway, weakening cytotoxicity; in dendritic cells, adenosine signaling downregulates the expression of co-stimulatory molecules CD80/CD86, inducing a tolerant phenotype.

Evidence from gallbladder cancer studies directly supports NT5E's involvement in tumor progression. In a TGF-β1-induced epithelial-to-mesenchymal transition (EMT) model, NT5E mRNA expression was upregulated by 2.8 times (P<0.01). Analysis of tissue microarrays from 108 gallbladder adenocarcinoma samples showed that 54.6% of tumors had high NT5E expression, which negatively correlated with differentiation grade (positive rate of 82.3% in poorly differentiated vs 35.7% in well-differentiated). Clinical pathological analysis further revealed that NT5E-positive patients had significantly higher lymph node metastasis rates (68.2% vs 32.7%) and nerve invasion rates (59.3% vs 24.5%) compared to negative patients (P<0.01), with a median overall survival reduced by 11.3 months (16.7 vs 28.0 months, P<0.05).

Expansion of Immunosuppressive Cells

In addition to directly suppressing immune cells, NT5E also contributes to the expansion of tumor-associated immunosuppressive cell populations. In myeloid-derived suppressor cells (MDSCs), adenosine generated by NT5E catalysis promotes STAT3 phosphorylation via the A2BR receptor, enhancing its immunosuppressive function and prolonging survival. In regulatory T cells (Tregs), adenosine signaling induces FoxP3 expression through cAMP, promoting Treg proliferation and enhancing their suppressive activity. Clinical studies have shown that in non-small cell lung cancer patients, the proportion of NT5E⁺ Tregs in tumor-infiltrating lymphocytes was as high as 45.2% of CD4⁺ T cells, and their number was inversely correlated with response to PD-1 inhibitors (the proportion of NT5E⁺ Tregs in the non-responding group was 2.3 times that in the responding group).

Table: NT5E Expression in Various Tumor Types and Its Clinical Significance

Targeted Therapy Strategies

CD73 Monoclonal Antibodies

Monoclonal antibodies targeting NT5E are an important strategy to overcome immune suppression. Traditional anti-CD73 antibodies (such as mAb 2C5) block the enzyme's active site via steric hindrance, reducing adenosine production by 80%. However, clinical studies have shown that some antibodies may induce CD73 dimerization, thereby enhancing enzyme activity. To address this, next-generation allosteric-lock antibodies adopt an allosteric inhibition mechanism, stabilizing CD73 in an "open conformation" that loses its catalytic function. Phase II clinical trials (NCT02754141) have shown that BMS-986179, combined with PD-1 inhibitors for treating advanced solid tumors achieved an objective response rate (ORR) of 32.1%, with significantly higher response rates in the high CD73 expression group compared to the low expression group (12.3%, P<0.01).

Photoimmunotherapy Technology

Photoimmunotherapy technology developed by the Ru Yong team represents an innovative breakthrough in targeting NT5E. This technology conjugates anti-CD73 monoclonal antibodies with the near-infrared dye IR-700 (αCD73-Dye conjugates), enabling specific binding to CD73⁺ cells. Upon exposure to 690 nm near-infrared light, the conjugate generates reactive oxygen species, causing necrotic death of the target cells, with no damage to surrounding cells. In a tumor-bearing mouse model, αCD73-Dye combined with PD-1 antibodies led to complete regression of primary tumors without recurrence, and a reduction of 87.5% in pulmonary metastases, extending the mice's survival by 2.3 times. This strategy not only eliminates CD73-high expressing tumor cells but also effectively removes MDSCs, TAMs, and other CD73⁺ immunosuppressive cells, reshaping the tumor immune microenvironment.

Small Molecule Inhibitors

Small-molecule inhibitors are another key direction in targeting NT5E. AB680, the first competitive CD73 inhibitor to enter clinical trials, has an IC50 of 0.6 nM and an oral bioavailability of 78%. Phase Ib trials (NCT04104672) have shown that AB680 combined with chemotherapy and PD-1 inhibitors in pancreatic ductal adenocarcinoma achieved a disease control rate (DCR) of 65.4%, significantly reducing chemotherapy-induced CD8⁺ T-cell depletion. Another class of allosteric inhibitors (e.g., OP-5244) binds at the enzyme dimer interface, blocking substrate-induced conformational changes and offering greater resistance to high ATP concentrations in the tumor microenvironment.