Genetic Modification of the Rabbit for Development of Single Chain Antibodies

Project Summary With the entry into a new era of gene editingand especially the availability of CRISPR Castoolstargeted genomic modifications of the rabbitOryctolagus cuniculushave now become a scientific realityThe rabbit is the classic and primary antibody producer thanks to its distinct immune biologye ggene conversionIn recent yearsheavy chain only antibodiesHCAbsoriginally discovered in the camelidswith superior single chain based antigen binding and physico chemical propertiescompared to conventional antibodieshave emerged as the latest class of next generation biologics for diagnostic and therapeutic purposeswith the first drugs awaiting approvalIt is in this context that we proposePhase Ito genetically modify the rabbit immune system towards the endogenous production of HCAb type bindersThrough preliminary work we have generated HCAb founder rabbitsIn this projectwe will demonstrate downstream feasibility towards isolation of single chain monoclonal HCAbs in response to immunization with a standard immunogenIn combination with a confirmation of germline transmissionwe intend to achieve an initial proof of concept for our HCAb rabbit model to establish it as a breedable lineIn our Aimwe focus on Basic Validation Expansion of Recombinant Rabbits for the Production of HCAbs by verification of genome editing outcomesHCAb expression and breedingIn Aimwe focus on Basic Characterization of HCAb Model Rabbits andampDownstream Detection of HCAbs by identifying expression of common wildtype isotypes and by screening of immune libraries for HCAbs with subsequent detection of high affinity monoclonal bindersWe envision our HCAb rabbit platform to translate into the following advantagesaPredominant HCAb IgG isotype responsesin contrast to camelidsbBinding domain diversification by gene conversiontypical for rabbitscEnhanced access to HCAbs due to advantageous breeding characteristics and defined experimental proceduresas well as access to immune cells from bone marrowspleenand thymus that only a small animal can providein contrast to camelsdA more user friendly more available resource for HCAbseIn vivo affinity matured HCAbs from which monoclonal mHCAbs can be detectedfGenetic access to enable further modifications to create rabbit lines with alternative immunologicalphysiological featureschanges to HCAb binding domainsenabling of isotype switchingintroduction of transgenesetcIn Phase IIbased on the progressing interest from biotechdiagnosticand therapeutic fields and due to the intrinsic properties and adaptability of HCAbswe will work to further improve the model and to utilize this novel platform for the discovery of clinically significant HCAbs NarrativeAntibodiesthe pathogen neutralizing proteins produced by the immune cellsare critical for the humoral immune defense against bacteria and viruses and play a critical role in the immune responses to cancer and as biomedical tools and drugsThe unconventional single chain antibodies from camel species are highly attractive for therapeutic and diagnostic applications due to their streamlined format and binding behavior toward difficult antigensWe propose to genetically modify the rabbit asa known producer of good conventional antibodiesto generate antibodies of the single chain typewhich represents a preliminary stage for next generationsmall antibodies for preclinical and clinical applications