Monoclonal antibodies (mAbs) and mAb-derived antibody therapeutics, are currently widely used to treat human diseases, such as cancer and autoimmune diseases. Although the rabbit as a platform to producing polyclonal antibodies has been utilized for a long time, generation of rabbit monoclonal antibodies has been limited by difficulties in generating stable hybridomas. The adoption of molecular cloning of Ig genes directly from B cells followed by expression in cell culture has introduced new life in the rabbit monoclonal field. However, the efficiency of mAb production is still low and the type of mAbs available from rabbit limited. Recently, we reported that deficiency in the Transmembrane Activator and CAML Interactor (TACI), a receptor that controls plasma cell differentiation, also causes marked expansion of antigen-responsive germinal center (GC) B cells enhancing affinity maturation and facilitating production of high-affinity IgG. TACI-deficiency also increased the yield of monoclonal antibody production by the hybridoma technique. In the present application, we propose to abrogate expression of TACI in the rabbit germline with the goal of enhancing monoclonal antibody production. We have established a highly efficient rabbit genome editing platform using CRISPR/Cas9 technology. We now propose to target the rabbit TACI gene by CRISPR/Cas9 technology to produce a TACI-KO rabbit. We will use the well-established platform to first generate TACI mutant rabbit founders. These founders will be tested for germline transmission, and if so, to establish heterozygous TACI mutant animals, followed by breeding to obtain homozygous TACI mutant animals. We will test the hypothesis that TACI-deficiency in rabbits will enhance mAb production and the affinity and specificity of those antibodies. We propose to establish TACI- deficient rabbits as a superior bio-reactor for mAb production.
Due to the limitations of rabbit hybridoma technology, the type of monoclonal antibodies (mAbs) available from rabbit has been limited. Our preliminary data showing that TACI-deficiency in mouse promotes production in bursts of high affinity antibodies and increases the yield of mouse hybridoma formation by at least 10 fold offers promise to generate a better rabbit model for the production of mAbs. Here, we propose to target the rabbit TACI gene with the goal of producing a TACI-KO animal to enhance the production of rabbit mAbs.
