Identification of single chain variable fragment (ScFv) antibodies specific to immune checkpoints by phage display

Abstract

The inhibition of immune checkpoints is a significant emerging strategy in immunotherapy for the treatment of several malignancies, with much potential for further applications. However, currently there are many pitfalls and drawbacks to the widespread use of this therapy in a clinical setting. One such drawback is the lack of diverse options in the choice for immune checkpoints targeted. This study therefore aimed to identify, isolate, and validate high affinity single chain variable fragment (scFv) antibodies specific to three known immune checkpoints (CD47, TIGIT, and GITR), in an effort to further drive the research and development of new immunotherapeutic strategies for the treatment and management of various cancers. This was accomplished in collaboration with the International Centre for Cancer Vaccine Science in Gdansk, as well as the Roslin Institute, University of Edinburgh, using the phage display method. Numerous rounds of biopanning were performed, beginning from a naïve canine scFv bacteriophage library to create enriched libraries of phages carrying scFv with high affinity to the target proteins. The affinity of the generated scFv antibodies was verified using ELISA, after which, specific phage clones were selected from the biopanning round which showed highest affinity to the target, which were then further amplified and validated with ELISA. The protein sequences of these high affinity phage clones were determined using Sanger sequencing and manually compared. Of the three targets, high affinity scFv clones were only successfully produced against GITR His-tag, isolating 60 scFv phage clones, 43 of which showed high affinity and specificity against GITR protein. All but three clones were successfully sequenced, showing a relatively high amount of diversity of scFv sequences. Further research is required to fully explore the drawbacks of this methodology, as well as successfully identify high affinity scFvs against CD47 and TIGIT.