Study on the relationship between the structure and functions of anti-human cervical cancer single-chain antibody and the lengths of linkers

Background: This study aimed to find the linker with minimal impact among chains to fight against the structure and function of cervical cancer (CC) single-chain antibody. Materials and Methods: The original variable region of heavy chain (VH) and variable region of light chain (VL), and the single-chain antibody with linkers of different lengths (n = 1~8) were modeling by homologous modeling, while the peptide chain structure of (Gly₄Ser)_n was utilized by the linkers. Comparison of the similarity of original VH/VL and VHn/VLn was carried out by applying the algorithm of spatial hierarchical alignment based on the spherical coordinates. The fore and aft distance and diffusion radius of alpha (α) were also calculated. The stability of antibody with different linker length was then compared. ELISA method was adopted to evaluate the immunological activity of single-chain antibody with different linkers. MTT assay was used to analyze the inhibition effect of ScFv-n on CC cells. Results: When n = 4, the structures were the most similar between ScFv and the original VH/VL. When n = 3, the influence of adding connecting peptide on the stability of single-chain antibody was the least. The result of ELISA and MTT methods indicated that when n = 3, single-chain antibody gained the highest activity. Conclusion: The optimum length of linker of anti-human CC single-chain antibody was n = 3 from the point of mathematical modeling and biology experiments. This study provided new ideas for the design and constructions of single-chain antibody, and theoretical basis for the treatment of CC.

Cervical Cancer; Single-chain Antibody; Linker; Homologous Modeling; Similarity Algorithm.

[1] Chang W.C., Li C.H., Huang S.C., Chang D.Y., Chou L.Y., Sheu

B. C.: “Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma”. Cancer, 2010, 116, 5777.

[2] Ferlay J., Parkin D.M., Steliarova-Foucher E.: “Estimates of cancer incidence and mortality in Europe in 2008”. Eur. J. Cancer, 2010, 46, 765.

[3] Karimi Z.M., Behatash N., Chiti Z., Karqar S.: “Cervical cancer and HPV vaccines in developing countries”. Asian Pac. J. Cancer Prev., 2009, 10, 969.

[4] Foldert M.R., Shih K.K., Abu-Rustum N.R., Jewell E., Kollmeier M. A., Makker V., et al.: “Postoperative pelvic intensity-modulated radiotherapy and concurrent chemotherapy in intermediate and high-risk cervical cancer”. Gynecol. Oncol., 2013, 128, 288.

[5] Kesic V.: “Management of cervical cancer”. Eur. J. Surg. Oncol., 2006, 32, 832.

[6] Shu X.Y., Hu J.: “Tumor inhibition of recombined superantigen SEB-scFv fusion protein in vivo”. Chin. J. Exp. Surg., 2009, 26, 1743.

[7] Wang Y., Li X., Chen M.: “Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancer”. J. South Med. Univ., 2006, 26, 16.

[8] Huston J.S., Levinson D., Mudgett-Hunter M., Tai M.S., Novotný J., Margolies M.N., et al.: “Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fvanalogue produced in Escherichia coli”. Proc. Natl. Acad. Sci. U S A., 1988, 85, 5879.

[9] Sandee D., Tungpradabkul S., Tsukio M., Imanaka T., Takagi M.: “Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma”. BMC Biotechnol., 2002, 2, 16.

[10] Huhalov A., Chester K.A.: “Engineered single chain antibody fragments for radioimmunotherapy”. Nucl. Med. Mol. Imaging, 2004, 8, 279.

[11] Mukherjee S., Zhang Y.: “Proteinprotein complex structure predictions by multimeric threading and template recombination”. Structure, 2011, 19, 955.

[12] Zhang J., Chen Z.: “Analyzing influence on the conformation of single-chain antibody with the differential length of linkers”. Afr. J. Microbiol. Res., 2011, 5, 5737.

[13] Zitzmann S., Mier W., Schad A., Kinscherf R., Askoxylakis V., Krämer S., et al.: “A new prostate carcinoma binding peptide (DUP21) for tumor imaging and therapy”. Clin. Cancer Res., 2005, 11, 139.

[14] DeNardo S.J.: “Radioimmuno detection and therapy of breas cancer”. Semin. Nucl. Med., 2005, 35, 143.

[15] Perk J.W., Hong K., Kirpotin D.B., Colbern G., Shalaby R., Baselga J., et al.: “Anti-HER-2 immunoliposomes: enhanced efficacy attributable to targeted delivery”. Clin. Cancer Res., 2002, 8, 1172.

[16] Kim M.K., Jeong H.J., Kao C.H., Yao Z., Paik D.S., Pie J.E., et al.: “Improved renal clearance and tumor targeting of 99mTc-labeled anti-Tac monoclonal antibody Fab by chemical modifications”. Nucl. Med. Bio., 2002, 29, 139.

[17] Hu Y.C., Liu Q.Z.: “The research process of technology and application of monoclone antibody”. Anhui. J. Preventive Med., 2008, 14, 116.

[18] Qin H.Y., Mao X.Y., Qiao Y.L., Li X.J., Zhao H.: “Progress in single- chain antibody”. Progress Mod. Biomed., 2001, 11, 795.

[19] Gustavsson M., Lehtio J., Denman S., Teeri T.T., Hult K., Martinelle M.: “Stable linker peptide for a cellulose binding domain-lipase fusion protein expressed in Pichiapastori”. Protein Eng., 2001, 14, 711.

[20] Le Gall F., Reusch U., Little M., Kipriyanov S.M.: “Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody”. Protein Eng. Des. Sel., 2004, 17, 357.

[21] Weisser N.E., Hall J.C.: “Applications of single-chain variable fragment antibodies in therapeutics and diagnostics”. Biotechnol. Adv., 2009, 27, 502.