Development of a Type IIs cloning strategy for the RUBYTM format & PCR optimization and development of a Type IIs cloning strategy for pool cloning
Abstract: For Alligator Bioscience AB, a company developing immunotherapeutic antibodies, it is crucial to efficiently evaluate a large number of potential drug candidates at an early stage. The in-house developed RUBYTM concept, combining monoclonal antibodies into bispecific antibodies without lead optimization, requires implementation of high-throughput technologies. Restriction enzyme digestion, used for generating expression vectors through cloning of antibody fragments, is not suitable for high-throughput implementation. Instead, this thesis project established Type IIs cloning strategies for the RUBYTM format. Developed using a newly designed pcDNA3.4 expression vector, before utilizing the procedure for cloning of antibody fragments in pool. The pcDNA3.4 vector system was evaluated, against a previously used vector system, based on antibody production and protein characteristics. The pcDNA3.4 expression vector considerably improved antibody yield and decreased aggregation. Type IIs cloning was proven to be a viable and versatile strategy, as both PCR fragments and a donor vector could be used to achieve a high cloning efficiency. A donor vector will provide greater flexibility to future antibody development and does not require repeated amplification. The high cloning efficiency was maintained during pool cloning as well, illustrating that it is now possible to efficiently generate a greater number of antibodies in a desired format for future screenings.
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