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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (2) : 204-211     DOI: 10.1007/s11684-016-0443-1
RESEARCH ARTICLE |
CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes
Jiangnan Liu1,2,Bin Yi1,2,Zhe Zhang1,2,Yi Cao1,*()
1. Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
2. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650223, China
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Abstract  

CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate epitope (glycotope) functionally involved in blood spread and liver metastasis of cancer cells by mediating the adhesion of cancer cells to endothelial cells and hepatocytes, respectively. CD176 could be a promising target for antitumor immunotherapy. We applied B lymphocytes obtained from mice immunized with CD176 antigen and constructed a phage display library. A positive clone of CD176 single-chain variable antibody fragment (scFv) was successfully screened from this library. The CD176 scFv was expressed in Escherichia coli and purified. The purified scFv can bind to the natural CD176 expressed on the surface of cancer cells. Furthermore, the CD176 scFv inhibits the adhesion of CD176+ cancer cells to endothelial cells and hepatocytes. This CD176 scFv provides a basis for future development of recombinant CD176-specific antibodies that can be used in therapeutic application.

Keywords CD176      Thomsen-Friedenreich antigen      scFv      cancer      therapy      adhesion      metastasis     
Corresponding Authors: Yi Cao   
Just Accepted Date: 29 March 2016   Online First Date: 18 April 2016    Issue Date: 27 May 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0443-1     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I2/204
Fig.1  The binding ability of phage display CD176 scFv to antigens. The displayed CD176 scFv against CD176 (TF-PAA) (A) and aGP (B) was detected by ELISA. CD176 (TF-PAA) and aGP were coated on 96-plates (200 ng/well). M13KO7 helper phage was used as a negative control and CD176 mAb (IgM) as a positive control (mean±SD, n = 3).
Fig.2  Construction, expression, and purification of the CD176 scFv. (A) Expression of the CD176 scFv fusion protein was detected by SDS-PAGE (lines 1?4). Lines 1 and 2 were the suspense and sediment of lysis after induction with IPTG, respectively. Lines 3 and 4 were the suspense and sediment of lysis without IPTG induction, respectively. PET32a(+) was used as the control (lines 5?8). Lines 5 (suspense) and 6 (sediment) showed expressed results of PET32a(+) vector with IPTG induction, whereas lines 7 (suspense) and 8 (sediment) showed expressed results without IPTG induction. (B) Line 1 showed the CD176 scFv fusion protein after nickel-affinity purification; lines 2?5 showed the CD176 scFv fusion protein digested by enterokinase in 16, 24, 36, and 48 h, respectively. (C) The digested protein sample (scFv) was purified by IEC and yielded 11 protein fractions. All fractions were subjected to SDS-PAGE. (D) The result of the highest fraction, as demonstrated by SDS-PAGE. Line 1 is the fusion protein before purification and line 2 is the digested protein after purification.
Fig.3  Biological characteristics of the CD176 scFv. The binding of the purified CD176 scFv to the natural CD176 on the surfaces of cancer cells was shown (A and B). (A) Flow cytometry analysis showed that the scFv bound to KG1 cells (middle) compared with the controls [incubation with BSA (left) and irrelevant scFv (right)]. (B) The scFv reacted with CD176+ cell lines by immunofluorescence staining, and a CD176 mAb (IgM) was used as the positive control. The adhesion of CD176+ cancer cells to HUVEC and HL-7702 was inhibited by the CD176 scFv (C?E). (C) The adhesion of FITC-labeled SGC7901 cells to HUVEC was suppressed by the CD176 scFv (scFv) at different incubation times compared with the negative control (control). The inhibition effects of the CD176 scFv were semiquantitatively analyzed by luciferase assay (D, E). The adhesion of SW480 and SGC7901 to HUVEC (D) and HL-7702 (E) were inhibited significantly by the CD176 scFv compared with negative controls (control scFv and no treatment). y-axis is mean intensity fluorescence (mean±SD, n = 4).
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