Recombinant VEGF Antibody
Description
Definition and Mechanism of Action
Recombinant VEGF antibodies are humanized or fully human antibodies that mimic the structure and function of natural immunoglobulins. They target VEGF-A, preventing its interaction with receptors like VEGFR1 and VEGFR2, thereby inhibiting endothelial cell proliferation, migration, and vascular permeability .
Key Features:
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Structure: Typically IgG1 isotype; some are Fab fragments (e.g., ranibizumab) .
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Binding: High affinity for VEGF-A isoforms (e.g., VEGF165, VEGF121) .
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Therapeutic Use: Approved drugs include bevacizumab (Avastin), ranibizumab (Lucentis), and aflibercept (VEGF Trap) .
Production and Characteristics
Recombinant VEGF antibodies are produced in mammalian expression systems (e.g., CHO cells) using recombinant DNA technology.
Research Applications
Recombinant VEGF antibodies are used in:
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ELISA: Quantifying VEGF-A levels in serum or tumor lysates .
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Western Blot: Detecting VEGF-A isoforms (e.g., VEGF165 vs. VEGF165b) .
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Flow Cytometry: Analyzing VEGF-A expression on cell surfaces .
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In Vitro Assays: Inhibiting endothelial cell proliferation (e.g., HUVEC cells) .
Example Data:
Recombinant VEGF165 (293-VE) stimulates HUVEC proliferation with an ED₅₀ of 1.5–12 ng/mL . Neutralization assays show IC₅₀ values of 0.02–0.12 µg/mL for anti-VEGF antibodies .
Clinical Therapeutic Use
Key Clinical Findings:
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Tumor Suppression: Murine anti-VEGF antibodies (2.5 mg/kg) inhibit tumor growth in nude mice, maintaining plasma trough levels >10 µg/mL .
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Phase I Safety: Recombinant humanized VEGF antibodies (rhuMAb VEGF) show a half-life of 21 days, with no dose-limiting toxicity up to 10 mg/kg .
Pharmacokinetics and Biodistribution
| Parameter | Value | Source |
|---|---|---|
| Half-life | 21 days (human) | |
| Plasma Clearance | Dose-dependent | |
| Tumor Uptake | Significant retention (24+ hours) |
Mechanistic Insights:
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VEGF165b Isoform: Anti-angiogenic VEGF165b binds VEGFR2 but inhibits downstream signaling, reducing tumor vessel density in colon carcinoma models .
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VEGF Trap: Binds VEGF-A, VEGF-B, and PlGF with subpicomolar affinity, surpassing bevacizumab and ranibizumab in blocking endothelial cell migration .
Future Directions
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Combination Therapies: Pairing anti-VEGF agents with immune checkpoint inhibitors to overcome immunosuppression .
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Biomarker-Driven Selection: Identifying patients with high VEGF165/165b ratios for tailored therapies .
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Next-Generation Constructs: Engineered antibodies with dual specificity (e.g., targeting VEGF and PD-L1) .
Product Specs
DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN
RGEC.
HEAVY CHAIN:
EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL
LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
PGK.
Q&A
Here’s a structured collection of FAQs for researchers working with recombinant VEGF antibodies, organized by complexity and grounded in methodological rigor:
Advanced Research Questions
What structural analysis methods resolve discrepancies in VEGF antibody-antigen binding predictions?
Advanced approaches combine:
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Homology modeling: Use tools like PyMOL or Rosetta to compare 3D structures of humanized vs. parental antibodies, focusing on FR residues influencing CDR orientation .
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Functional validation: Mutate residues flagged by modeling (e.g., FR residues near CDRs) and test binding via SPR or competitive ELISA .
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Cross-species reactivity screens: Test antibody binding against VEGF orthologs (e.g., murine, primate) to identify species-specific epitopes .
How are conflicting data on VEGF antibody efficacy in preclinical models reconciled?
Strategies include:
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Tumor microenvironment (TME) profiling: Analyze VEGF isoform expression (e.g., VEGF-A165 vs. VEGF-A121) via qPCR or LC-MS, as antibody efficacy varies by isoform .
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Orthogonal assays: Pair in vitro bioassays (e.g., Promega’s VEGF Bioassay ) with in vivo imaging of vascular permeability or tumor burden .
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Mechanistic studies: Assess downstream signaling (e.g., ERK phosphorylation) to confirm on-target effects despite phenotypic variability .
What methodologies identify off-target effects of VEGF antibodies in complex biological systems?
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Proteome-wide profiling: Use HuProt™ microarrays to screen for interactions with unrelated receptors (e.g., EGFR, PDGFR) .
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High-content imaging: Quantify endothelial cell migration/proliferation in co-culture systems with fibroblasts or immune cells .
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Transcriptomic analysis: Single-cell RNA sequencing of treated tissues to detect unintended pathway activation (e.g., inflammatory cytokines) .
Table 1: Key Validation Parameters for VEGF Antibody Bioassays
| Parameter | Requirement | Example Data (Bevacizumab) |
|---|---|---|
| Precision (CV%) | ≤20% | 12.3% |
| Linearity (R²) | ≥0.95 | 0.98 |
| Accuracy (% recovery) | 80–120% | 94% |
Table 2: Anti-VEGF Antibody Classes and Mechanisms
| Class | Example | Target | Mechanism |
|---|---|---|---|
| Humanized IgG1 | Bevacizumab | VEGF-A | Blocks VEGF-A binding to VEGFR2 |
| Recombinant Fab | Ranibizumab | VEGF-A | Higher affinity than full IgG |
| Decoy receptor | Aflibercept | VEGF-A/B, PlGF | Traps multiple ligands |
Technical Notes for Experimental Design
Product Science Overview
Recombinant human anti-vascular endothelial growth factor (VEGF) is a biotechnologically engineered monoclonal antibody designed to inhibit the activity of VEGF, a key molecule involved in angiogenesis. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a critical process in both normal physiological conditions and pathological states, such as cancer.
VEGF is a signal protein that stimulates the growth of blood vessels. It plays a pivotal role in both normal and pathological angiogenesis. VEGF is produced by cells that are deprived of oxygen, a condition known as hypoxia. It binds to VEGF receptors on the surface of endothelial cells, triggering a cascade of events that lead to the formation of new blood vessels. VEGF is crucial for wound healing and the formation of the circulatory system during embryonic development. However, its overexpression is associated with diseases such as cancer, where it promotes tumor growth by supplying nutrients and oxygen through the blood vessels .
The development of anti-VEGF therapy began with the understanding that inhibiting VEGF could potentially halt the growth of tumors by cutting off their blood supply. This led to the creation of monoclonal antibodies that specifically target VEGF. One of the first and most well-known anti-VEGF therapies is bevacizumab, a humanized monoclonal antibody that binds to VEGF and prevents it from interacting with its receptors on endothelial cells .
Recombinant human anti-VEGF antibodies are produced using recombinant DNA technology. This involves inserting the gene that encodes the anti-VEGF antibody into a host cell, such as a Chinese hamster ovary (CHO) cell, which then produces the antibody. The antibody is then purified and formulated for therapeutic use. These antibodies are designed to have high affinity for VEGF, ensuring that they effectively neutralize the protein and inhibit angiogenesis .
The primary mechanism of action of recombinant human anti-VEGF antibodies is the inhibition of VEGF binding to its receptors, VEGFR-1 and VEGFR-2, on the surface of endothelial cells. By blocking this interaction, the antibodies prevent the downstream signaling pathways that lead to endothelial cell proliferation, migration, and new blood vessel formation. This results in the inhibition of angiogenesis, which is particularly beneficial in the treatment of cancers and other diseases characterized by excessive blood vessel growth .
Recombinant human anti-VEGF therapies have been approved for the treatment of various cancers, including metastatic colorectal cancer, non-small cell lung cancer, and glioblastoma. They are also used in the treatment of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, where abnormal blood vessel growth in the retina leads to vision loss .
