Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies

Abstract

The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response

“…To investigate the effect of PI.12, PI.42, PII.16 and PII.29 scFvs on T-cell activation, CD69 upregulation was measured via flow cytometry. In brief, after a 24-h treatment with anti-4-1BB and an unrelated scFv (anti-c-Met scFv named ES1) [33], one million cells were washed with washing buffer, labeled with PE-conjugated anti-CD69 antibody mAb (eBioscience), and analyzed using flow cytometry. Untreated T cells were used as a negative control.…”

A phase I study of LY3164530, a bispecific antibody targeting MET and EGFR, in patients with advanced or metastatic cancer

“…Epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition factor (MET; also known as hepatocyte growth factor [HGF] receptor) are receptor tyrosine kinases that are coexpressed in many tumors, including non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and head and neck cancers (HNSCC) [ 1 – 3 ]. EGFR signaling plays a crucial role in tumor biology by modulating cellular proliferation, angiogenesis, metastasis, and survival of cancer cells.…”

Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil

“…Subsequently, we extracted the total RNA of tonsil lymphocytes screened by immunomagnetic bead sorting technique, based on the genetic theory of antibody diversity [18][19][20], a specific primer was designed, and the coding sequence of the restriction fragment and restriction site was designed in the primer, RT-PCR technology was used to amplify VH, Vλ, and Vκ genes. Then the overlap extension splicing method was used, and the 15-peptide sequence (Gly4 Ser) 3 (Linker) which was designed by Huston [9] was used to connect VH and Vλ, VH and V κ, then the scfv was constructed. The renaturation peptide (Gly4Ser) three refolding process appeared high efficiency, it maybe because its construction is long and soft.…”

“…The detection results of the P/N ratio was more than or equal to 2.1 were considered as positive. As shown in Figure 4(a), except for samples 6,8,9,17,27,32,37, 49, 50, 53, 55, 55, and 55(total of 13, accounting for 19.4%), the rest were positive, indicating that scfv had good binding activity.…”

“…The single-chain antibody is a kind of genetic engineering antibody and has many advantages like lower molecular weight, stronger tissue penetration, and faster body clearance [7,8]. It has the potential value for clinical diagnosis and treatment [9][10][11][12][13].…”