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Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.