Development and Evaluation of a Cetuximab-based Humanized Single Chain Antibody Against EGFR-overexpressing Tumors

Abstract

Production of humanized single chain antibodies (hscFv) can potentially be a powerful solution to limitations imposed by large size and murine nature of cetuximab. The present study describes generation of a cetuximab-based hscFv using CDR-grafting method. Cetuximab CDRs were grafted on frameworks selected from human germline antibody sequence repertoire. The strategy employed in selecting human sequences was the highest sequence similarity of variable domains between human and parental antibodies as well as similarity in the CDRs canonical structures. To maintain the binding affinity, the parental vernier zone residues were retained murine in hscFv. Recombinant hscFv was expressed in E. coli and affinity purified by Ni-NTA column. The potency of hscFv in targeting EGFR was evaluated using A431, a cell line over-expressing EGFR. Dot blot and ELISA tests were used to assess the reactivity and MTT assay to evaluate the growth inhibition of hscFv on A431 cell line. The humanization of cetuximab variable regions resulted in 22.2% increase in humanness of hscFv. Reactivity analyses of hscFv on A431 cells showed better binding affinity and higher growth inhibition effect (2.6 times) comparing to murine counterpart. In conclusion, hscFv produced in this study displayed reduced potential immunogenicity as well as enhanced cytotoxic effect on EGFR- overexpressing tumor cells.

Key words

EGFR - single chain antibody - humanization - CDR grafting

• References

• 1 Kuramochi H, Hayashi K, Nakajima G et al. Epidermal growth factor receptor (EGFR) mRNA levels and protein expression levels in primary colorectal cancer and corresponding liver metastases. Cancer Chemother Pharmacol 2010; 65: 825-831
• 2 Nie F, Yang J, Wen S et al. Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis. Cancer 2012; 118: 5198-5209
• 3 Zhang H, Yun S, Batuwangala TD et al. A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment. Int J Cancer 2012; 131: 956-969
• 4 Hirsch FR, Varella-Garcia M, Bunn Jr PA et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003; 21: 3798-3807
• 5 Di LG, Tortora G, D’Armiento FP et al. Expression of epidermal growth factor receptor correlates with disease relapse and progression to androgen-independence in human prostate cancer. Clin Cancer Res 2002; 8: 3438-3444
• 6 Bardelli A, Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J Clin Oncol 2010; 28: 1254-1261
• 7 Esteva FJ. Monoclonal antibodies, small molecules, and vaccines in the treatment of breast cancer. Oncologist 2004; 9 (Suppl. 03) 4-9
• 8 Imai K, Takaoka A. Comparing antibody and small-molecule therapies for cancer. Nat Rev Cancer 2006; 6: 714-727
• 9 Li S, Schmitz KR, Jeffrey PD et al. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell 2005; 7: 301-311
• 10 Quesnelle KM, Grandis JR. Dual kinase inhibition of EGFR and HER2 overcomes resistance to cetuximab in a novel in vivo model of acquired cetuximab resistance. Clin Cancer Res 2011; 17: 5935-5944
• 11 Chames P, Van RM, Weiss E et al. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 2009; 157: 220-233
• 12 Goto M, Kuribayashi K, Umemori Y et al. High prevalence of human anti-mouse antibodies in the serum of colorectal cancer patients. Anticancer Res 2010; 30: 4353-4356
• 13 Pluen A, Boucher Y, Ramanujan S et al. Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci USA 2001; 98: 4628-4633
• 14 Steinitz M. Three decades of human monoclonal antibodies: past, present and future developments. Hum Antibodies 2009; 18: 1-10
• 15 Batra SK, Jain M, Wittel UA et al. Pharmacokinetics and biodistribution of genetically engineered antibodies. Curr Opin Biotechnol 2002; 13: 603-608
• 16 Huston JS, McCartney J, Tai MS et al. Medical applications of single-chain antibodies. Int Rev Immunol 1993; 10: 195-217
• 17 Foote J, Winter G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol 1992; 224: 487-499
• 18 Hwang WY, Almagro JC, Buss TN et al. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods 2005; 36: 35-42
• 19 Makabe K, Nakanishi T, Tsumoto K et al. Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528. J Biol Chem 2008; 283: 1156-1166
• 20 Safdari Y, Farajnia S, Asgharzadeh M et al. Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot. Monoclonal Antibodiesin Immunodiagnosis and Immunotherapy 2014; 33: 1
• 21 Safdari Y, Farajnia S, Asgharzadeh M et al. Protein L: a robust enzyme-conjugated molecule for detection of humanized single chain antibodies. Monoclon Antib Immunodiagn Immunother 2013; 32: 409-412
• 22 Abhinandan KR, Martin AC. Analyzing the “degree of humanness” of antibody sequences. J Mol Biol 2007; 369: 852-862
• 23 Magdelaine-Beuzelin C, Kaas Q, Wehbi V et al. Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment. Crit Rev Oncol Hematol 2007; 64: 210-225
• 24 Guo JQ, You SY, Li L et al. Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli. J Biotechnol 2003; 102: 177-189
• 25 Yu Y, Lee P, Ke Y et al. A humanized anti-VEGF rabbit monoclonal antibody inhibits angiogenesis and blocks tumor growth in xenograft models. PLoS One 2010; 5: e9072
• 26 Uchiyama S, Suzuki Y, Otake K et al. Development of novel humanized anti-CD20 antibodies based on affinity constant and epitope. Cancer Sci 2010; 101: 201-209
• 27 Torres M, Casadevall A. The immunoglobulin constant region contributes to affinity and specificity. Trends Immunol 2008; 29: 91-97