High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice.

Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

Rational design of an animal model for Alzheimer's disease: introduction of multiple human genomic transgenes to reproduce AD pathology in a rodent.

A major obstacle to understanding the pathogenesis of Alzheimer's disease is the lack of easily studied animal models. Our approach is to apply transgenic methods to humanize mice and rats, employing methods that introduce large genomic transgenes, because this improves the level of transgene protein expression and the tissue specificity of expression. Our plan is to reproduce AD pathology in rodents by making them transgenic for several human proteins involved in AD. This report describes transgenic animal lines that we have produced, and summarizes our current approach and future plans. Two human genes known to be involved in AD pathology are the amyloid precursor protein (APP) and the E4 isoform of apolipoprotein E (apoE4). So far, we have produced and analyzed a transgenic line carrying the entire human APP gene cloned in a yeast artificial chromosome. We have also produced but not yet analyzed a mouse carrying the human apoE4 gene. Work is in progress to produce a transgenic line carrying a disease-causing mutation in the human APP gene. As we produce these animals, we are breeding them together, and also breeding them with a mouse line that lacks endogenous apoE, to produce an animal model carrying several human proteins whose interaction is believed to be instrumental in development of AD pathology. These transgenic animals will be useful for dissecting the biochemical and physiological steps leading to AD, and for development of therapies for disease intervention.

Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM.

We have generated transgenic mice that contain human-sequence Ig miniloci and, because they are also homozygous for a targeted disruption of their endogenous heavy chain genes, must rely on the transgene sequences for B cell receptor expression. Although the human transgenes contain only a fraction of the intact human heavy chain locus, these defined sequences are able to at least partially restore the humoral immune system in the mouse. B cells expressing human heavy chains develop in the bone marrow, populate peripheral lymphoid tissue and respond specifically to antigen. Furthermore, the heavy chain transgenes contain both human mu and gamma 1 coding exons as well as the respective mu and gamma 1 switch regions. The sequences included within the transgene are sufficient to direct class switch recombination. Transgene sequences are also sufficient to direct somatic mutation of the class-switched heavy chain genes. These observations define the upper limit of the cis-acting sequences necessary to direct heavy chain class switching and somatic mutation.

Antigen-specific human antibodies from mice comprising four distinct genetic modifications.

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.