Vascular endothelial growth factor antagonists and methods of making
Inventors
Bottaro, Donald P. • Cecchi, Fabiola
Assignees
US Department of Health and Human Services
Publication Number
US-10035833-B2
Publication Date
2018-07-31
Expiration Date
2033-04-26
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Abstract
The present invention provides variant VEGF polypeptides which have been altered in their C-terminal heparin binding region to lower their heparin binding affinity. These variants have been found to act as receptor antagonists for VEGF receptors and antagonize angiogenesis. These variants are useful to treat diseases characterized by pathological angiogenesis.
Core Innovation
The invention provides variant vascular endothelial growth factor (VEGF) polypeptides altered in their C-terminal heparin binding domain to lower their heparin binding affinity. These variant polypeptides retain native receptor tyrosine kinase binding domains but have one or more amino acid substitutions designed to occupy VEGF receptors and reduce or repel binding to heparin and heparan sulfate proteoglycans, resulting in antagonism of VEGF receptor activation and angiogenesis.
The problem solved by the invention concerns the need for effective VEGF antagonists to treat diseases characterized by pathological angiogenesis such as cancer and ocular diseases. Prior VEGF antagonists, including neutralizing antibodies like Bevacizumab and receptor fusion proteins like Aflibercept, have limitations such as high production costs requiring mammalian expression systems. Additionally, existing approaches targeting VEGF signaling either lack selectivity or are expensive to manufacture. The invention addresses these issues by providing modified VEGF variants that competitively antagonize VEGF receptor activation through disruption of heparin binding, offering a potentially more cost-effective and selective therapeutic alternative.
Claims Coverage
The patent contains multiple independent claims relating to VEGF variant polypeptides, their encoding polynucleotides, recombinant vectors, host cells, and methods for creating antagonistic polypeptides. The main inventive features focus on VEGF variants with specific acidic amino acid substitutions in the heparin binding domain to antagonize VEGF-mediated angiogenesis.
VEGF variant polypeptides with specified acidic substitutions
Variant VEGF polypeptides having the ability to antagonize VEGF-mediated angiogenesis comprising polypeptides selected from SEQ ID NO:25 with positions 149, 150, and 185 substituted with glutamic or aspartic acid, or SEQ ID NO:26 with positions 123, 124, and 159 substituted with glutamic or aspartic acid, or polypeptides having at least 95% identity to these sequences with the specified substitutions.
Maintenance of receptor affinity in VEGF variants
The affinity of the variant polypeptides for both VEGFR-1 (FLT-1) and VEGFR-2 (KDR/FLK-1) is substantially maintained compared to native VEGF, ensuring receptor binding despite heparin binding alterations.
Specific variant polypeptides with multiple acidic substitutions
Variant polypeptides comprising SEQ ID NO:6 or SEQ ID NO:7, or polypeptides with at least 95% identity to these sequences, containing R to E substitutions at specified positions for antagonizing VEGF angiogenesis.
Pharmaceutical compositions comprising VEGF variants
Pharmaceutically acceptable compositions including the variant VEGF polypeptides described.
Polynucleotides encoding VEGF variant polypeptides
Polynucleotides encoding the variant VEGF polypeptides with the specified acidic substitutions, including those encoding polypeptides of SEQ ID NO:6, SEQ ID NO:7, or variants with at least 95% identity.
Recombinant expression vectors and host cells
Recombinant expression vectors containing polynucleotides encoding the VEGF variants and host cells comprising these vectors for expression of the variant polypeptides.
Method for creating angiogenesis-inhibiting VEGF polypeptides
A method involving providing native VEGF with a C-terminal heparin binding domain and modifying it by introducing acidic amino acid substitutions at defined positions to form variant VEGF polypeptides that antagonize VEGF-mediated angiogenesis.
The claims cover VEGF variant polypeptides with specific acidic substitutions in the heparin binding domain, maintaining receptor affinity while antagonizing VEGF angiogenic signaling, along with respective encoding polynucleotides, expression systems, compositions, and methods of production.
Stated Advantages
Variant polypeptides derived from native VEGF can be more cost-effective to produce than larger antibody molecules while retaining comparable target selectivity.
Receptor antagonists that bind the receptor and repel heparin can inhibit VEGF signaling even when receptor activation is independent of VEGF binding.
Charge-based repulsion of heparan sulfate proteoglycans from the ligand/receptor complex is an effective and general strategy for antagonizing VEGF signaling.
Documented Applications
Treatment of diseases characterized by pathological angiogenesis, including cancer and ocular diseases such as macular degeneration and diabetic retinopathy.
The variant VEGF polypeptides can be used to inhibit tumor growth and metastasis by antagonizing VEGF receptor-mediated angiogenesis.
The polypeptides may be used as molecular imaging agents for VEGF receptor detection in pathological angiogenesis.
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