Marianne Brüggemann

Production of human antibody repertoires in transgenic mice

Transgenic mice have been created that carry human immunoglobulin heavy and light chain genes in germline configuration and that have the corresponding endogenous genes silenced. The transgenes are either minigene constructs or large, almost authentic, transloci on yeast artificial chromosomes and undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Monoclonal antibodies with good affinities for human antigens have been obtained after immunisation. These mice may be a future source of human antibodies for therapy.

Multiple sequences from downstream of the Jκ cluster can combine to recruit somatic hypermutation to a heterologous, upstream mutation domain

Recruitment of somatic hypermutation to the Igκ locus has previously been shown to depend on the enhancer elements, Ei/MAR and E3 ′ . Here we show that these elements are not sufficient to confer mutability. However, hypermutation is effectively targeted to a chimeric β‐globin/Igκ transgene whose 5 ′ end is composed of the human β‐globin gene (promoter and first two exons) and whose 3 ′ end consists of selected sequences derived from downstream of the Jκ cluster (Ei/MAR, Cκ + flank and E3 ′ ). Thus, multiple downstream Igκ sequences (all derived from 3 ′ of the Jκ cluster) can combine to recruit mutation to a heterologous mutation domain. The location of this hypermutation domain is defined by the position of the transcription start site and this applies even if the Igκ Ei/MAR is positioned upstream of the promoter. Hotspots within the mutation domain are, however, defined by local DNA sequence as evidenced by a new hotspot being created within the β‐globin domain by a mutation within the transgene. We propose that multiple, moveable Igκ sequences (that are normally located downstream of the transcription start site) cooperate to bring a hypermutation priming factor to the transcription initiation complex; a mutation domain is thereby created downstream of the promoter but the local sequence defines the detailed pattern of mutation within that domain.

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human VHs, all human D and JH segments in natural configuration linked to the rat CH locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production.

Expression studies of multigene families, such as the immunoglobulin (Ig) loci, are difficult because of their large size and the necessity to introduce germline configured regions into an animal. Antibody diversity from Ig gene miniloci is limited by the number of variable (V) region genes and the need for distal regulatory elements to control expression. Here, we show germline transfer into mice of a 1300 kb human Igκ light chain locus on a yeast artificial chromosome that resulted in early DNA rearrangement and highly efficient human light chain expression. The human locus was assembled from a 300 kb authentic region using contig extension by addition of cosmid multimers to supplement the variable gene cluster. This resulted in the addition of about 100 V region genes in germline configuration from different families. In transgenic animals with Igκ disruption, this large human κ locus replaced the endogenous locus, and subsequent down‐regulation of Igλ light chain contribution led to a dominant expression of the rearranged human genes. Contrary to expectation, rather than providing a solely selective advantage for ensuring repertoire formation controlled by the sheer number of introduced genes, the λ/κ ratio in serum appears to be the result of competition for early surface Ig expression maintained in the developing B cell.—Zou, X., Xian, J., Davies, N. P., Popov, A. V., Brüggemann, M. Dominant expression of a 1.3 Mb human Igκ locus replacing mouse light chain production. FASEB J. 10, 1227‐1232 (1996)

Characterization of immunoglobulin heavy chain knockout rats.

The rat is a species frequently used in immunological studies but, until now, there were no models with introduced gene‐specific mutations. In a recent study, we described for the first time the generation of novel rat lines with targeted mutations using zinc‐finger nucleases. In this study, we compare immune development in two Ig heavy‐chain KO lines; one with truncated Cμ and a new line with removed JH segments. Rats homozygous for IgM mutation generate truncated Cμ mRNA with a de novo stop codon and no Cγ mRNA. JH‐deletion rats showed undetectable mRNA for all H‐chain transcripts. No serum IgM, IgG, IgA and IgE were detected in these rat lines. In both lines, lymphoid B‐cell numbers were reduced >95% versus WT animals. In rats homozygous for IgM mutation, no Ab‐mediated hyperacute allograft rejection was encountered. Similarities in B‐cell differentiation seen in Ig KO rats and ES cell‐derived Ig KO mice are discussed. These Ig and B‐cell‐deficient rats obtained using zinc‐finger nucleases‐technology should be useful as biomedical research models and a powerful platform for transgenic animals expressing a human Ab repertoire.

Human Antibody Production in Transgenic Animals

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

Heavy-chain only antibodies derived from dromedary are secreted and displayed by mouse B cells

Whereas functional heavy (H)‐chain antibodies devoid of light (L)‐ chains account for about half of the circulating immunoglobulins in Camelidae, H‐chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen‐recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H‐chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen‐egg lysozyme (HEL). This IgG2a H‐chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H‐ or L‐chains. Unexpectedly the mouse cells processed and expressed the introduced H‐chain as naturally occurring dromedary antibody. For this the first constant (C) region exon was proficiently removed from the recombinant H‐chain transcript. This resulted in specific H‐chain antibodies of the correct molecular weight (2 × 50 000 MW) secreted as disulfide‐linked homodimers and displayed on the mouse cell surface as glycosyl‐phosphatidyl‐inositol‐linked B‐cell receptor. The results indicate that antibody expression and maturation without immunoglobulin L‐chain is feasible and paves the way for the generation of transgenic single chain antibody repertoires.

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a ‘logic’ for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ‐line human V3–23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3–23 gene in combination with different mouse Vκ genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human μ‐chains and mouse light chains, were multimeric in structure and expressed the human V3–23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non‐CDR3 sequences intrinsic to the V3–23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

Block in Development at the Pre-B-II to Immature B Cell Stage in Mice Without Igκ and Igλ Light Chain

Silencing individual C (constant region) λ genes in a κ−/− background reduces mature B cell levels, and L chain-deficient (λ−/−κ−/−) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.

Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: Requirements for antibody-mediated host cell-target cell interaction

Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction.