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

Figures & data

Figure 1. (A) Titration results of c-Met-specific phages after bio-panning of the Tomlinson library. (B) Polyclonal phage and (C) Monoclonal Phage ELISA of selected scFvs against c-MET peptides. From 56 clones, 26 presented with positive reactivity to c-Met peptide. Data shown are means [± SD] of OD values from duplicate experiments. An arbitrary cutoff 2.5 times greater than the negative control was considered positive. Positive control: anti BSA scFv, negative control: BSA.

Figure 2. Production of soluble scFv. Binding of soluble scFv to c-Met and BSA wells was assessed by ELISA. Data are presented as means [± SD] of OD values from duplicate experiments. An arbitrary cutoff 2.5 times greater than the negative control was considered positive. Positive control: anti BSA scFv, negative control: BSA.

Figure 3. (A) Amplification of scFv fragments by PCR (V_H and Vκ insert, ≈ 935 bp). Lane M: 100 bp +3K DNA ladder. Lanes 1–9: amplified scFv gene in 9 clones. (B) Mva-I DNA finger-printing of scFv clones, scFv genes were amplified by PCR then digested with Mva-I (BstNI) and separated over an agarose gel. Lane M: 100 bp +3K DNA ladder. Lanes 1–9: single clones digested by Mva-I. Two common patterns were seen in lanes 1, 3, and 7, and in lanes 2, 4, 8 and 9. (C) Western blot analysis of expressed scFv. Lane 1, 2, and 3: expressed products of ES1-scFv, ES2-scFv, and ES3-scFv, respectively. Lane 4: total lysate from non-induced E. coli Rosetta-Gami 2. A representative blot is shown.

Table 1. VDJ genes and CDR3 sequences of anti c-Met scFv.

	V-GENE	D-GENE	J-GENE	CDR3
VH domains
ES1	IGHV3-23*02 F	IGHD2-21*02F	IGHJ4*02 F	QQSYSTPNT
ES2	IGHV3-23*01 F	IGHD1-1*01 F	IGHJ4*02 F	QQTSAYPDT
ES3	IGHV3-23*01 F	IGHD3-9*01 F	IGHJ4*02 F	QQGSDTPTT
	V-GENE	J-GENE	CDR3
VL domains
ES1	IGKV1-39*01 F	IGKJ1*01 F	QNGLLLT
ES2	IGKV1-39*01 F	IGKJ1*01 F	AKCTSAFDY
ES3	IGKV1-39*01 F	IGKJ1*01 F	AKACYYFDY

The VDJ genes and CDR3 amino acid sequences were deduced from the IMGT database (www.imgt.org).

Table 2. Specificity of soluble scFv.

	Antigen
Clone	c-Met	IGF-1R	CD137	ROR1	PD-1	skimmed milk	BSA
ES1	1.70 ± 0.12	0.78 ± 0.12	0.61 ± 0.13	0.33 ± 0.04	0.45 ± 0.07	0.21 ± 0.04	0.47 ± 0.06
ES2	1.70 ± 0.07	0.72 ± 0.13	0.64 ± 0.11	0.32 ± 0.13	0.33 ± 0.05	0.17 ± 0.06	0.27 ± 0.03
ES3	1.86 ± 0.07	0.83 ± 0.05	0.57 ± 0.04	0.26 ± 0.04	0.28 ± 0.08	0.28 ± 0.07	0.19 ± 0.07

Binding to a variety of immobilized peptides and proteins was determined by ELISA.

Data are presented as means [± SD] of OD from triplicate experiments.

c-Met: Mesenchymal Epithelial Transition factor; IGF-1R: insulin-like growth factor 1 receptor; CD137: 4–1BB receptor; ROR1: receptor tyrosine kinase-like orphan receptor 1; PD-1: programmed cell death protein 1; BSA: bovine serum albumin; skimmed milk.

Figure 4. Functional inhibition assay. (A) Proliferation of HCT-116 cells in presence of HGF (100 ng/ml) (p = 0.05 vs. untreated cells). (B) Proliferation inhibited by ES1, ES2 and ES3 (77%, 72%, and 66%, respectively. (p = 0.001, p = 0.003, p = 0.002, respectively, vs. effect from an unrelated scFv). Data shown are means ± SD of OD of three independent experiments.

Figure 5. Annexin V/PI apoptosis assay in HCT-116 cells treated with anti-c-Met scFv. Apop-totic death was analyzed in cells treated for 24 h with unrelated scFv (negative control), HGF, ES1, ES2, or ES3. Apoptotic cell death was characterized by Annexin V⁺/PI^- and Annexin V⁺/PI⁺staining status using FACS. (A) Untreated cells. Cells treated with (B) unrelated scFv, (C) ES1scFv, (D) ES2 scFv, or (E) ES3 scFv.

Figure 6. Annexin V/PI apoptosis assay in HCT-116 cells treated with HGF (100 ng/ml) and anti-c-Met scFv. Data shown are means [± SD] of three independent experiments.