Part:BBa_K1694003

Single-chain variable fragment (Anti-VEGF)

Introduction: scFv(Bevacizumab)

ScFv (Single-Chain Variable Fragment)

ScFv (single-chain variable fragment) is a fusion protein containing light (VL) and heavy (VH) variable domains connected by a short peptide linker (Fig. 1). The peptide linker (GGSSRSSSSGGGGSGGGG) is rich in glycine and serine which makes it flexible.

Features of scFv:

1. Specific：Though remove of the constant regions , scFv still maintain the specificity of the original immunoglobulin.

2. Efficient：ScFv is smaller than the entire antibody, so it place little stress for E. coli to express it

Vascular endothelial growth factor

1. VEGF (Vascular endothelial growth factor), a protein that can stimulates vasculogenesis and angiogenesis. Some cancers can overexpress VEGF, which will cause some vascular disease. Drug bevacizumab can inhibit VEGF and control or slow those diseases.

2. VEGF is a sub-family of growth factors, which comprises：VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D.

3. There are three types of VEGF receptors on the cell surface, and VEGFR-2 is one type of receptor which can mediate almost all of the known cellular responses to VEGF.

Bevacizumab

We selected the single chain variable fragments (scFv) of monoclonal antibodies Bevacizumab and named it Anti-VEGF. Bevacizumab is a monoclonal antibody which blocks angiogenesis by inhibiting vascular endothelial growth factor A (VEGF-A). VEGF-A stimulates angiogenesis in a variety of diseases, especially in cancer.

Mechanism:

When VEGF-A binds to VEGFR-2, it causes two VEGFR-2 to combine to form a dimer. This allows for signaling molecules to enter to the cell, bind to the receptor, and become activated. Then start the angiogenesis.
Bevacizumab can bind with VEGF released from tumor cell, block VEGFR to inhibit tumor angiogenesis, thereby cutting off the tumor's supplies and prevent tumor growth.

Reference:

Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotný, J., Margolies, M. N., … Crea, R. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 85(16), 5879–5883.
Los, M.; Roodhart, J. M. L.; Voest, E. E. (2007). "Target Practice: Lessons from Phase III Trials with Bevacizumab and Vatalanib in the Treatment of Advanced Colorectal Cancer". The Oncologist 12 (4): 443–50.
Dougher-Vermazen M, Hulmes JD, Böhlen P, Terman BI (November 1994). "Biological activity and phosphorylation sites of the bacterially expressed cytosolic domain of the KDR VEGF-receptor". Biochem. Biophys. Res. Commun. 205 (1): 728–38.

The experiment of scFv

Fig. The PCR result of the scFv-VEGF. The DNA sequence length of scFv-VEGF are around 700~800 bp, so the PCR products should appear at 900～1000 bp.

After receiving the DNA sequences from the gene synthesis company, we recombined each scFv gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the scFvs. The DNA sequence length of the scFvs are around 600~800 bp. In this PCR experiment, the scFv products size should be near at 850~1050 bp. The Fig. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.</p>

Application of the part

Modeling

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]