Abstract
A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.
Highlights
Recombinant antibodies represent the fastest growing class of new medicines, and generation of antibodies that meet specific criteria is increasingly important for therapeutic applications
Immunoglobulin heavy chain D(J) regions were amplified from cDNA derived from spleen and lymph nodes of immunized mice, and cloned into an episomal vector containing the IgG1 constant region linked to a transmembrane sequence for cell surface display and a small repertoire of nine fully human germline V-regions, combined into four sub-libraries (Figure 1)
Each heavy chain (HC) sub-library was transfected and stably selected together with a light chain (LC) library composed of five human germline kappa V-regions fused to J-region sequences isolated from pooled peripheral blood mononuclear cells (PBMCs) from normal human donors as described previously [21]
Summary
Recombinant antibodies represent the fastest growing class of new medicines, and generation of antibodies that meet specific criteria is increasingly important for therapeutic applications. The majority of therapeutic antibodies available were derived from rodent immunization and the subsequent generation of a panel of hybridomas [1,2]. Immunization of wild-type animals was used to produce the first therapeutic antibody, the anti-CD3 monoclonal antibody (mAb) muromonab [3]. Immunization of transgenic mice in which endogenous immunoglobulin (Ig) loci have been replaced by a repertoire of human heavy and light chain germline transgenes, followed by the generation of human Ig-producing hybridomas, has recently emerged as an effective way of generating human antibodies to many antigens [5,6,7,8]. More than 40 fully human antibodies produced in transgenic mice have advanced to clinical evaluation (reviewed in [9])
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