Novel antibody library design: super stable single chain fragment variable (scFv) antibody library with disulfide bridge stabilized loop

Abstract

The smallest antibody fragment that can bind to an antigen is the single chain variable fragment (scFv). Its size can be advantageous in many biotechnological and clinical applications. However, compared to other antibody fragments, its pharmacokinetic properties are unsatisfactory as it has lower thermostability and shorter serum life. One of the methods to improve its function is to introduce an extra disulfide bond between two complementary determining regions (CDR-H3 and L1). A novel single-chain variable fragment (scFv) antibody library with an unnatural disulfide bond was generated to provide scFv fragments with improved stability. In addition, this library consists of five sub-libraries with different CDR-H3 loop lengths. The framework for the library is Trastuzumab, a commercial antibody against human epidermal growth factor receptor 2. To pursue this, the CDR-H3, CDRL1, and L3 regions of the paratope were diversified using Trim Oligo technology. The CDR H1 and H2 were amplified from an existing fragment antigen binding library. After panning the light chain library against protein L to select the functional clones, all fragments were combined through simultaneous digestion and seamless cloning. The resulting fragment was cloned in pEB3v3 and then by the transformation in E. coli XL1-Blue the final library was generated. Library size after transformation was 2.13x109 for all sub-libraries combined which covers 7x10-25 times the diversity design. Considering the diversity design of the primers, in theory, the final library diversity would be 3x1035. However, due to practical limitations, this is not achievable.