SBIR-STTR Award

The goal of the program is to develop methods for the introduction of yeast artificial chromosomes (YACS) into the germline of mice, and to apply the technology toward the generation of transgenic mice expressing a broad, fully human immunoglobulin repertoire. These mice would permit the isolation of human monoclonal antibodies in an analogous manner to rodent monoclonal antibodies. The goal of Phase I is to introduce small YACs carrying V, D, J and Cmu regions of the human heavy chain immunoglobulin gene into the germline of mice to create transgenic mice carrying a human immunoglobulin minigene. The mice will be tested for expression of human IgM proteins in the serum, and for DNA rearrangement in spleen B-cells. In Phase II, the animals generated in Phase I will be bred with Human kappa light chain expressing transgenic mice, and the resultant pilot mouse tested for its capacity to express a primary repertoire of surface and secreted fully human IgMs. Also, additional constant and variable regions will be cloned in YACs to add to or replace the Phase I YACS, to permit class switching, somatic mutation and IgG production. If necessary, sequence modifications such as the introduction of additional enhancer elements will also be added. Transgenic mice will also be generated carrying large fragments of the unrearranged human kappa gene and bred to the Phase II heavy chain mice. Finally, these mice will be bred with mice lacking functional endogenous immunoglobulin genes. Hybridomas will be generated at various stages of the project from their offspring to determine if somatic mutation of the transgene can efficiently occur, leading to a broad repertoire of high affinity, fully human IgG producing B cells.Awardee's statement of the potential commercial applications of the research:Transgenic mice will be developed that express the human immunoglobulin repertoire. When presented antigen, the mice will produce human monoclonal antibodies which will be isolated using conventional hybridoma technology. Human monoclonal antibodies are particularly important for therapeutic applications such as treating cancers, cardiovascular diseases, septic shock and immunosuppressive diseases.National Institute of Allergy and Infectious Diseases (NIAID)

Our goal is to develop methods for the introduction of yeast artificial chromosomes (YACs) into the germline of mice, and to apply the technology toward the generation of transgenic mice expressing a broad, fully human immunoglobulin repertoire. These mice would permit the isolation of human monoclonal antibodies in an analogous manner to rodent monoclonal antibodies. In Phase I, methods for the introduction of YACs into mice were developed, and mice carrying a small YAC encoding a minimally competent human immunoglobulin transgene were shown to express human heavy chain IgM antibodies in the serum. In Phase II, the animals generated in Phase I will be fully characterized for transgene function and expression. Additional constant and variable regions will be cloned in YACs and added to the Phase I YAC to permit class switching somatic mutation, and IgG production. These Phase II transgenes will be introduced into mice using methodology developed in Phase I. Transgenic mice will also be generated carrying large fragments of the unrearranged human kappa gene and bred to the Phase II heavy chain mice. These mice will be bred with mice lacking functional immunoglobulin genes to produce mice demonstrating a broad repertoire of high affinity, fully human antibodies.Awardee's statement of the potential commercial applications of the research : Human immunoglobulin genes cloned in YACs will be introduced into mice. These mice will express a human Ig repertoire. Antigen challenge will elicit an immune response and will allow human monoclonal antibodies to be isolated using conventional hybridoma technology. Human monoclonal antibodies are particularly important for therapeutic applications such as treating cancers, cardiovascular disease, septic shock and immunosuppressive diseases.National Institute of Allergy and Infectious Diseases (NIAID)