A nanobody-derived mimotope against VEGF inhibits cancer angiogenesis

Figures & data

Figure 1. CDR structures of the VEGF nanobody obtained from I-TASSER. (a) CDR1 structure. (b) CDR2 structure. (c) CDR3 structure. (d) CDR1,3 structure. (e) CDR1,2 structure.

Table 1. Results of computer-based energy minimizations on CDRs.

	Bonds	Angles	Torsion	Non bonded	Electro static	Constraint	Total
Nb	30/760	218/8	231/535	−423/64	−455/71	0	−548/62
CDR3	19/957	140/87	151/170	−333/46	−451/31	0	−438/24
CDR2	17/130	55/9	71/326	−206	−277/89	0	−277/87
CDR1	12/561	29/138	58/403	−128/15	−206/97	0	−183/54
CDR1,2	41/392	127/996	185/701	−341/80	−8/64	0	−34/93
CDR1,3	22/944	147/101	345/334	−635/45	−219/98	0	−300/12
CDR2.3	29/834	139/959	206/231	−570/65	−261/91	0	−425/22

‘Total’ indicates the most stable energy level of each structure obtained from Swiss modeller.

Table 2. Amino acid sequences of nanobody CDRs submitted to I-TASSER.

Peptides	Amino acid residues count	Amino acid sequences
CDR1	21	ASGFAYSTYSMG
CDR2	12	ATINSGTFRLWY
CDR3	19	AARAWSPYSSTVDAGDFRY
CDR1,2	21	GFAYSTYSGGGGGINSGTFRL
CDR2,3	32	INSGTFRLGGGGGAARAWSPYSSTVDAGDFRY
CDR1,3	30	GFAYTYSGGGGAARAMSPYSSTVDAGDFRY

Table 3. Results on docking simulation experiments of CDR regions and VEGF nanobody.

	E. Total (kcal/mol)	E. Shape	E. Air	Bmp (Bit maps)	Rms (Root mean square)
Nb	−342/15	−342/15	0	−1	−1
CDR3	−274/23	−274/23	0	−1	−1
CDR2	−167/2	−167/2	0	−1	−1
CDR1	−148/0	−148/0	0	−1	−1
CDR1,2	−160/5	−160/5	0	−1	−1
CDR1,3	−144/8	−144/8	0	−1	−1
CDR2,3	−162/1	−162/1	0	−1	−1

‘E. Total’ highlights the binding energy of a nanobody and its CDR3 region. The energy related to the CDR3 region is closest to that of the complete nanobody structure, as compared to other CDR regions.

Figure 2. (a) SDS-PAGE of the purified nanobody. (b) Western blotting of the purified nanobody. M; protein marker, 1; the extracted nanobody 2; wash flow through.

Figure 3. MTT assay results. (a) The effects of nanobody and peptide on the growth of HUVEC cells after 24 h and (b) 48 h. Determined IC₅₀s(24 h) were 200, 300, and 350 nM for bevacizamab, nanobody, and peptide, respectively. Moreover, calculated IC₅₀s(48 h) were 150, 170, and 200 nM for bevacizamab, nanobody, and peptide, respectively. Data are presented as mean ± SD. *p value s= .0292, **p values = .001.

Figure 4. Tube assay results. (a) Tube like structures: A; HUVEC cells with VEGF, B; without VEGF, C; Nanobody, D; Peptide, E; Bevacizumab. (b) Quantification of tube assay results. Control; HUVEC cells in the presence of VEGF and without any inhibitory factors formed complete tube-like structures. HUVEC cells in the presence of nanobody, peptide, and Bevacizumab formed 25, 35, and 20% of tube-like structures, respectively. Data are presented as mean ± SD. ***; p values = .0001.