The introduction of human immunoglobulin gene segments in their unrearranged configuration into the germ line of mice might allow the production of a repertoire of human antibodies. Such transgenic mice could be used for the production of human monoclonal antibodies against human antigens. To test the feasibility of this approach, mice were created that carry a human heavy-chain minilocus comprising unrearranged immunoglobulin variable, diversity, and joining elements linked to a human I-chain gene. The gene segments of this minilocus are rearranged in a large proportion of cells in thymus and spleen but not in nonlymphoid tissue. Some 4% of the B lymphocytes synthesize human ,l chains resulting in a serum titer of about 50 jig of transgenic IgM antibody per ml.Hybridomas were established from the transgenic mice that stably secreted several micrograms of antibodies containing human ,L heavy chains per milliliter.The preparation of monoclonal antibodies of rodent origin is an established and widely used procedure (1). Their therapeutic application would be greatly assisted by the availability of monoclonal antibodies of human origin as these are likely to exhibit greatly reduced immunogenicity in humans. However, two types of problem beset the preparation of such antibodies (2). (i) Most immortalized human cell lines or hybridomas do not stably express large amounts of antibody.(ii) In vivo immunization of humans is not feasible for many antigens; in vitro priming is usually inefficient.Antibodies are composed of variable (V) and constant (C) domains with the V domains defining the antigen specificity. Recently, a considerable advance has been achieved by making chimeric antibodies composed of mouse V domains linked to human C domains; these retain the antigen-binding specificity of the original mouse antibody but over two-thirds of the sequence of the human chimeric antibody is of human origin (3-5). Further improvements have been made by exploiting the fact that the hypervariable regions of the V domains are-at least in several cases-sufficient to determine antigen specificity; antibodies have been prepared in which only the hypervariable regions are of rodent origin (6, 7).The genes encoding the V domains of the immunoglobulin heavy (IgH) chain are assembled during lymphoid differentiation by rearrangement of constituent VH, diversity (D), and joining heavy-chain (JH) MATERIALS AND METHODSConstruction of the Minilocus and of Transgenic Mice. The minilocus was assembled in a pUC12 derivative that contained an Sph I linker in the Nar I site. The mouse C, membrane exons were inserted as a HindIII-Sph I fragment and the Sph I site then converted to a Bgl II site by linker insertion. The C,. region (9)
IgE antibodies are best known for their pathological role in allergy. The class-specific effector sites are located in the epsilon chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite C epsilon 2 domains. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is unresolved. For structural and functional analysis site-specific mutations were introduced into the C epsilon 2 domain of recombinant human IgE. The introduction of an additional cyanogen bromide cleavage site (His246----Met) facilitated the identification of parallel disulfide bond pairing. This linkage was also confirmed for myeloma IgE PS by sequence determination of disulfide-linked C epsilon 2 dimers. Substitution of Cys241 and Cys328 by Ser does not destroy receptor binding, but reductive alkylation, or the replacement of Cys328 by Met, leads to loss of activity. This shows that covalent dimerization is not essential for IgE/receptor interaction and points to the importance of the structural integrity of the site surrounding Cys328, visualized in a new model of human Fc epsilon.
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