VEGFR2 Fc Human

What is the molecular structure of VEGFR2 and its physiological role?

VEGFR2 (also known as KDR/Flk-1) belongs to the class III subfamily of receptor tyrosine kinases (RTKs), alongside VEGFR1 (Flt-1) and VEGFR3 (Flt-4). The VEGFR2 cDNA encodes a 1356 amino acid (aa) precursor protein containing a 19 aa signal peptide . Mature VEGFR2 comprises a 745 aa extracellular domain, a 25 aa transmembrane domain, and a 567 aa cytoplasmic domain . The extracellular portion contains seven immunoglobulin-like repeats crucial for ligand binding, while the cytoplasmic region contains kinase insert domains essential for signal transduction .

VEGFR2 serves as the primary mediator of VEGF-induced angiogenic responses. While VEGFR1 binds both VEGF and PlGF with high affinity, VEGFR2 binds VEGF but not PlGF . Expression of VEGFR2 is almost exclusively restricted to endothelial cells under normal conditions, where it plays essential roles in vasculogenesis and angiogenesis . Upon ligand binding, VEGFR2 undergoes dimerization and tyrosine phosphorylation, activating downstream signaling pathways that promote endothelial cell proliferation, migration, and survival.

How does VEGFR2 Fc fusion protein differ from native VEGFR2?

VEGFR2 Fc fusion protein represents an engineered version of the receptor that combines the extracellular domain of VEGFR2 with an immunoglobulin Fc fragment. This design creates significant structural and functional differences from the native receptor:

• Recombinant human VEGFR2 Fc typically contains only the extracellular domain (Ala20-Glu764) fused to an Fc tag at the C-terminus . In contrast, native VEGFR2 is a transmembrane protein with extracellular, transmembrane, and cytoplasmic domains.

• Native VEGFR2 functions as a signaling receptor that transduces intracellular responses upon VEGF binding, whereas VEGFR2 Fc acts as a decoy receptor that binds and sequesters VEGF, preventing it from interacting with cell-surface receptors .

• The molecular weight profile differs significantly. While native VEGFR2 has a predicted molecular weight based on its amino acid sequence, recombinant VEGFR2 Fc exhibits variable apparent molecular weights due to glycosylation. For example, human VEGFR2 with mFc tag has an approximate molecular weight of 110 kDa but migrates to 150-200 kDa in Tris-Bis PAGE due to glycosylation .

• VEGFR2 Fc fusion protein retains the high-affinity binding to VEGF characteristic of the native receptor's extracellular domain but serves as a potent VEGF antagonist rather than a signal transducer .

What expression systems are optimal for producing functional VEGFR2 Fc fusion proteins?

The production of functional VEGFR2 Fc fusion proteins requires expression systems that enable proper protein folding and post-translational modifications. Based on the literature, mammalian expression systems generally yield the most functionally relevant products:

Human Embryonic Kidney (HEK293) cells represent the primary system of choice for VEGFR2 Fc production. Multiple sources confirm that recombinant human VEGFR2 Fc protein is successfully expressed in HEK293 cells with functional activity . This system provides proper glycosylation patterns similar to native human proteins, which is critical for maintaining the protein's structure and function.

Critical factors affecting expression quality include vector design with appropriate signal peptides, codon optimization for the expression host, culture conditions optimization, and purification strategies that maintain protein integrity. The expression system choice should align with the intended application, with HEK293 or other mammalian cell systems preferred for research requiring high biological activity and human-like glycosylation patterns.

What are the recommended storage and reconstitution procedures for VEGFR2 Fc proteins?

Proper storage and reconstitution of VEGFR2 Fc proteins are essential for maintaining biological activity. The recommended protocols vary depending on the protein format and intended timeframe:

For lyophilized VEGFR2 Fc protein:

• Long-term storage should be at -20°C to -80°C for up to 1 year from the date of receipt .

• Before reconstitution, centrifuge tubes to ensure all material is at the bottom .

• Reconstitution to a concentration greater than 100 μg/mL is recommended (typical laboratory practice uses 1 mg/mL solution) .

• Dissolve the lyophilized protein in distilled water under sterile conditions .

For reconstituted VEGFR2 Fc protein:

• Short-term storage (2-7 days): Store at 2-8°C under sterile conditions .

• Medium-term storage (3-6 months): Store at -20°C to -80°C under sterile conditions .

• It is strongly recommended to aliquot the reconstituted protein into smaller quantities to avoid repeated freeze-thaw cycles .

For fluorescein-conjugated VEGFR2 antibodies (which follow similar principles):

• These should be protected from light to maintain fluorescence activity .

• Do not freeze fluorescein-conjugated antibodies, as this can damage the fluorophore .

• Store at 2-8°C for up to 12 months from date of receipt .

These storage and reconstitution guidelines help maintain the structural integrity and functional activity of VEGFR2 Fc proteins, ensuring reliable experimental results.

How can researchers effectively measure VEGFR2 Fc binding activity?

Multiple complementary assays can be employed to effectively measure VEGFR2 Fc binding activity, each providing unique insights:

ELISA-based binding assays represent a primary approach for quantitative assessment. Immobilized human VEGF165 (typically at 1 μg/mL, 100 μL/well) can be used to determine binding of VEGFR2 Fc proteins through dose-response curves . This method allows determination of EC50 values, with human VEGFR2 mFc demonstrating an EC50 of 36.1 ng/mL in such assays .

Flow cytometry provides another valuable approach, particularly for assessing binding to cells expressing native VEGFR2. Human umbilical vein endothelial cells (HUVECs) represent an excellent cellular model for this purpose, as demonstrated in protocols using fluorescein-conjugated anti-VEGFR2 antibodies . This approach can detect binding to cell-surface VEGFR2 and evaluate competitive interactions.

Functional cell-based assays offer insights into the biological consequences of binding. These include measuring inhibition of VEGF-induced VEGFR2 phosphorylation in endothelial cells, assessment of downstream signaling pathway modulation, and evaluation of cellular responses like proliferation, migration, or tube formation .

For more detailed binding kinetics, biophysical methods such as Surface Plasmon Resonance (SPR) can determine association and dissociation rates, while isothermal titration calorimetry provides thermodynamic parameters of the interaction.

What strategies exist for validating VEGFR2 Fc specificity in experimental systems?

Establishing the specificity of VEGFR2 Fc in experimental systems requires multiple validation approaches:

Competitive binding assays represent a key validation strategy. Researchers can test the ability of VEGFR2 Fc to compete with unlabeled VEGF for binding to cell-surface VEGFR2, comparing IC50 values with other receptor constructs . In reported studies, effective anti-VEGFR2 constructs demonstrated IC50 values of 3-7 nM for competitive inhibition of VEGF-A binding .

Appropriate controls are essential for specificity validation. These should include Fc-only controls to rule out Fc-mediated effects, non-relevant receptor-Fc fusion proteins as negative controls, and established VEGFR2 antagonists (such as ramucirumab) as positive controls .

Functional validation provides compelling evidence of specificity. This includes demonstrating inhibition of VEGF-A-induced VEGFR2 phosphorylation while showing no effect on PlGF-induced signaling (which acts through VEGFR1) . Researchers have successfully used this approach to identify potent VEGFR2-specific inhibitors that block VEGF-A-mediated VEGFR2 phosphorylation in HUVECs .

Domain-specific binding analysis offers additional specificity confirmation. Mapping interactions with specific domains of VEGFR2 (particularly Ig-like domain 3) can provide mechanistic insights into how the construct disrupts VEGF-VEGFR2 interactions .

The combination of these validation approaches provides robust evidence for VEGFR2 Fc specificity, strengthening the reliability of experimental findings.

How can VEGFR2 Fc be utilized in angiogenesis inhibition studies?

VEGFR2 Fc serves as a valuable tool in angiogenesis research through its ability to sequester VEGF and prevent receptor activation. Several experimental approaches leverage this mechanism:

In vitro angiogenesis assays provide the foundation for VEGFR2 Fc application. These include inhibition of endothelial cell tube formation on Matrigel, prevention of endothelial cell sprouting from spheroids, and disruption of capillary structure formation . Studies have demonstrated that anti-VEGFR2 constructs can effectively inhibit these processes, providing direct evidence of their anti-angiogenic potential.

More complex ex vivo and in vivo models offer insights into tissue-level effects. These include suppression of vessel outgrowth in aortic ring assays, Matrigel plug assays with co-administered VEGFR2 Fc, and corneal pocket assays to assess anti-angiogenic effects .

In disease-specific models, VEGFR2 Fc and related anti-VEGFR2 approaches have shown significant therapeutic potential. For example, VEGFR2 blockade using the kinase inhibitor SU5416 improved renal function and reduced glomerular damage in diabetic nephropathy models . Similarly, anti-VEGFR2 antibodies have demonstrated efficacy in cancer xenograft models, including prostate cancer and leukemia .

The mechanism of action involves VEGFR2 Fc acting as a decoy receptor, sequestering VEGF and preventing its interaction with endogenous receptors. This approach is particularly useful for dissecting the specific contribution of VEGF/VEGFR2 signaling in various angiogenic processes.

What approaches exist for developing high-affinity anti-VEGFR2 antibodies?

The development of high-affinity anti-VEGFR2 antibodies employs sophisticated methodologies:

Phage display technology represents a powerful approach for antibody development. This method involves using phage-displayed human naïve scFv libraries to isolate phages that bind to recombinant VEGFR2 extracellular domain protein . After multiple rounds of affinity selection (biopanning), the titer of bound phage can increase by as much as 3455-fold . ELISA screening and DNA sequencing identify distinct phage clones that bind tightly to VEGFR2-Fc but not to Fc control protein .

Affinity maturation enhances binding properties of initial antibody candidates. This process typically involves constructing synthetic phage-displayed scFv libraries based on promising initial clones, with random mutations introduced at specific amino acid residues (particularly in CDR regions) . After stringent in vitro biopanning against VEGFR2-immobilized beads, superior binding clones can be identified through comparative affinity studies .

Functional screening ensures therapeutic potential. Candidate antibodies should be evaluated for their ability to competitively inhibit VEGF-A binding and antagonize VEGF-A-mediated activation of VEGFR2 . In reported studies, effective clones demonstrated the ability to inhibit tyrosine phosphorylation of VEGFR2 induced by VEGF-A in endothelial cells .

These approaches have successfully yielded antibodies with therapeutic potential. For example, the anti-VEGFR2-AF antibody developed through these methods showed efficacy comparable to FDA-approved ramucirumab in prostate cancer models and superior efficacy in leukemia models .

How does VEGFR2 blockade affect tumor microenvironments?

VEGFR2 blockade exerts multifaceted effects on tumor microenvironments:

Direct effects on endothelial cells represent the primary mechanism. By binding to Ig-like domain 3 of VEGFR2's extracellular region, anti-VEGFR2 constructs disrupt the interaction between VEGF-A and VEGFR2, neutralizing downstream signaling . This leads to inhibition of endothelial cell proliferation, migration, and tube formation, reducing tumor angiogenesis.

Effects on VEGFR2-expressing tumor cells provide an additional therapeutic mechanism. Research has shown that VEGFR2 is expressed in certain cancer cell types, including the PC-3 human prostate cancer cell line, and is associated with malignancy and metastasis . Anti-VEGFR2 treatment can exert antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against these cells .

In vivo studies confirm significant anti-tumor effects. In a PC-3 xenograft mouse model, treatment with anti-VEGFR2 antibodies repressed tumor growth and angiogenesis as effectively as FDA-approved ramucirumab . In HL-60 human leukemia-xenografted mice, anti-VEGFR2 treatment prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes .

Combination approaches enhance therapeutic efficacy. Studies have demonstrated that anti-VEGFR2 antibodies can enhance the efficacy of chemotherapeutic agents like docetaxel in prostate cancer models . This represents an important finding suggesting that VEGFR2 targeting can be effectively integrated into combination treatment strategies.

What evidence supports VEGFR2 as a therapeutic target in cancer?

Substantial evidence supports VEGFR2 as a therapeutic target across multiple cancer types:

In prostate cancer, VEGFR2 expression is associated with malignancy and metastasis . Preclinical studies using PC-3 xenograft mouse models demonstrated that anti-VEGFR2 antibody treatment repressed tumor growth and angiogenesis . Notably, combination of anti-VEGFR2 antibody with docetaxel enhanced efficacy compared to either agent alone, providing support for combination approaches in this cancer type .

For hematological malignancies, particularly leukemia, VEGFR2 targeting has shown significant promise. In HL-60 human leukemia-xenografted mice, anti-VEGFR2 antibody treatment demonstrated superior efficacy compared to ramucirumab, with prolonged survival and reduced metastasis to ovaries and lymph nodes . This suggests that novel anti-VEGFR2 therapies may offer advantages over existing approved agents in certain contexts.

The dual mechanism of action enhances therapeutic potential. Anti-VEGFR2 agents can simultaneously target tumor vasculature (interrupting angiogenesis) and directly affect VEGFR2-expressing tumor cells . This dual targeting strategy may provide advantages over approaches that exclusively target either component.

The molecular mechanisms have been well-characterized. Anti-VEGFR2 antibodies bind to specific domains (such as Ig-like domain 3) of VEGFR2's extracellular region, disrupting VEGF-A/VEGFR2 interaction and neutralizing downstream signaling . Additionally, these antibodies can exert immune-mediated effects including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity in vitro .

What role does VEGFR2 play in non-oncological conditions?

VEGFR2 has significant implications in non-oncological conditions, particularly diabetic nephropathy:

In diabetic kidney disease, the VEGFR2 pathway becomes activated in both experimental models and human patients . This activation contributes to pathological changes in the kidney, including alterations in the glomerular filtration barrier and vascular permeability.

Therapeutic VEGFR2 blockade shows promising renoprotective effects. Studies using the VEGFR2 kinase inhibitor SU5416 in BTBR ob/ob mice (a model that mimics key features of advanced human diabetic nephropathy) demonstrated significant improvements in renal function . Importantly, these benefits were observed even when treatment was initiated after kidney disease had already developed, suggesting therapeutic rather than merely preventive potential .

The renoprotective effects manifest through multiple mechanisms. VEGFR2 blockade improved glomerular damage (reducing mesangial matrix expansion and basement membrane thickening) and attenuated tubulointerstitial inflammation and tubular atrophy compared to untreated diabetic mice . These findings suggest that VEGFR2 inhibition addresses multiple aspects of diabetic kidney injury.

These observations highlight VEGFR2 as a potential therapeutic target for diabetic nephropathy, a significant complication of diabetes with limited treatment options. The effectiveness of VEGFR2 blockade in established disease models suggests particular relevance for clinical applications where intervention typically occurs after disease onset.

How do different VEGFR2-targeting strategies compare in experimental models?

Different VEGFR2-targeting strategies demonstrate distinct efficacy profiles across experimental models:

Antibody-based approaches offer specificity and multiple mechanisms of action. The novel fully human antibody anti-VEGFR2-AF showed effectiveness comparable to FDA-approved ramucirumab in PC-3 prostate cancer xenograft models . Interestingly, in HL-60 leukemia xenografts, anti-VEGFR2-AF demonstrated superior efficacy to ramucirumab, with prolonged survival and reduced metastasis . These antibodies can exert effects through ligand blocking and immune effector functions (ADCC and CDC).

Small molecule tyrosine kinase inhibitors provide an alternative approach. The VEGFR2 kinase inhibitor SU5416 effectively improved renal function and reduced glomerular damage in diabetic nephropathy models . These inhibitors typically have broader specificity than antibodies, often targeting multiple receptor tyrosine kinases, and may have different tissue penetration properties.

Decoy receptor approaches using VEGFR2-Fc fusion proteins target the ligand rather than the receptor directly. Recombinant soluble VEGFR2-Fc chimeras bind VEGF with high affinity and act as potent VEGF antagonists . These constructs may have different pharmacokinetic properties compared to antibodies due to the Fc portion.

Each approach demonstrates context-dependent efficacy across disease models. Response rates may vary based on factors such as VEGFR2 expression levels, pathway activation status, and tissue-specific factors. Understanding these comparative profiles is essential for selecting optimal VEGFR2-targeting strategies for specific disease contexts and patient populations.