Selection of antibody fragments – scFv – against triple-negative breast cancer target

Introduction: Phage Display is a molecular biology technique that involves the expression of peptides, proteins, or antibody fragments on the surface of phage particles (WILLATS, 2002).This technique was first described in 1985 by George Smith, considered innovative for physically linking a genotype and phenotype in a single viral particle and amplifying them billions of times in bacteria (SMITH, 1985).This exposure allows for interaction with a broad diversity of external target molecules, and later the isolation of these ligands displayed on the surface of the phage (SCOTT; SMITH, 1990).Objectives: To develop a library of human antibody fragments (scFv) and select against a triple-negative breast cancer target. Methodology:The scFv library was constructed by isolating lymphocytes from 10 healthy donors.Degenerate primers established by Barbas et al. (2001) were used in PCRs.To construct the scFv, we performed six amplifications using sense primers for variable chains (heavy and light) combined with antisense primers for the kappa region, finishing with a set of primers for scFv PCR overlap.Then, the fragments and the pComb 3XSS vector were digested, ligated, and transformed into the library.The selected target for triple-negative breast cancer (TNBC) was commercially acquired and used at four different concentrations (50, 25, 10, 1nM) during the scFv selection phase. Results:The scFv library reached a diversity of 1.70x10^9 with a colony insertion percentage of approximately 70%, leading to a final diversity of 1.19x10^9.Three selection cycles were performed, resulting in a 16-fold enrichment in the first cycle, a 10-fold enrichment in the second cycle, and no enrichment in the third cycle, which led to the selection of fragments from the second selection cycle. Conclusion:The technology of constructing antibody fragment libraries via Phage Display is the starting point for generating antibodies against specific targets to aid in the diagnosis and treatment of various diseases and was able to positively select against a TNBC target.