The present invention provides novel transgenic nonhuman mammals capable of producing human sequence antibodies, as well as methods of producing and using these antibodies.

Transgenic transchromosomal rodents for making human antibodies

A method for replacing an endogenous immunoglobulin gene locus of the variable region as a whole against a orthologous human gene locus, or partially by replacement of one or more V and J, or V, D and J gene segments thereof to orthologous human V and J, or V, D and J gene segments in an embryonic stem (ES) cell of a mouse to generate a modified immunoglobulin locus, which may rearrange to produce a chimeric antibody, the human variable regions and contains murine constant regions, that method comprising: a) obtaining a large cloned genomic fragment greater than 20 kb, the orthologous human V and J, or V, D and J gene segments includes; b) using bacterial homologous recombination to modify the cloned genomic fragment of (a) genetically engineered to create a large targeting vector for use in the embryonic stem cell; c) introducing the large targeting vector of (b) into the embryonic stem cell to partially replace the endogenous immunoglobulin variable gene locus in the cell, in whole or; and d) detecting the modification of one allele at the endogenous immunoglobulin gene locus of the variable region in order to identify cells which have undergone homologous recombination to exchange the endogenous immunoglobulin locus of the variable region in whole or in part.

Methods of Modifying Eukaryotic Cells

Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies (mAbs). However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity mAbs for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences. This technology has now been exploited by over a dozen different pharmaceutical and biotechnology companies toward developing new therapeutic mAbs, and currently at least 33 different drugs in clinical testing--including several in pivotal trials--contain variable regions encoded by human sequences from transgenic mice. The emerging data from these trials provide an early glimpse of the safety and efficacy issues for these molecules. Nevertheless, actual product approval, the biggest challenge so far, is required to fully validate this technology as a drug discovery tool. In the future, it may be possible to extend this technology beyond rodents and use transgenic farm animals to directly generate and produce human sequence polyclonal sera.

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Human antibodies from transgenic animals.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

Cloned transchromosomic calves producing human immunoglobulin

Human antibodies as next generation therapeutics.

For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.

Terumi Yamamoto

Papers

Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.