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

Formulation and delivery issues for monoclonal antibody therapeutics

Heterogeneity of Monoclonal Antibodies

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.

Germline transmission of genetically modified primordial germ cells.

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Production of human monoclonal antibody in eggs of chimeric chickens

Human antibodies are produced in transgenic chickens having functional genetic components of the human immune system stably integrated in the genome. Techniques are described to integrate unrearranged human heavy and light chain immunoglobulin loci into the genome of the transgenic chicken. The functional description of the endogenous immunoglobulin loci yields an animal whose antibody repertoire in response to antigen is fully human. In the preferred embodiment, the human immunoglobulin locus is at least as large as the native human locus and exhibits functional class switching to yield IgG isotype antibodies. In addition to monoclonal antibodies secreted from immortalized B cell populations, polyclonal antibodies may be obtained from eggs laid by the transgenic chickens of the invention.

Transgenic aves producing human polyclonal antibodies

A transgenic chicken is disclosed having disrupted endogenous immunoglobulin production. In one embodiment, a targeting construct is stably integrated into the genome of the chicken by homologous recombination in embryonic stem cells, and injection of the engineered embryonic stem cells into recipient embryos, thereby knocking out the endogenous immunoglobulin gene locus in resulting animals. The targeted disruption of the locus in embryonic stem cells is particularly useful in combination with the insertion of geneticelements encoding exogenous immunoglobulin molecules. After these chickens are cross­bred, a line of chickens is produced that has a reduction of endogenous immunoglobulin molecule production.

The functional disruption of avian immunoglobulin genes

A transgenic chicken is disclosed having disrupted endogenous immunoglobulin production In one embodiment, a targeting construct is stably integrated into the genome of the chicken by homologous recombination in embryonic stem cells, and injection of the engineered embryonic stem cells into recipient embryos, thereby knocking out the endogenous immunoglobulin gene locus in resulting animals The targeted disruption of the locus in embryonic stem cells is particularly useful in combination with the insertion of genetic elements encoding exogenous immunoglobulin molecules After these chickens are cross-bred, a line of chickens is produced that has a reduction of endogenous immunoglobulin molecule production

Targeting constructs for the functional disruption of avian immunoglobulin genes

Des anticorps humains sont produits dans des poulets transgeniques possedant des elements genetiques fonctionnels du systeme immun humain integres de facon stable dans le genome. La presente invention concerne des techniques destinees a integrer des sites actifs d'immunoglobuline de chaine legere et lourde humains non rearranges dans le genome de ce poulet transgenique. La description fonctionnelle de ces sites actifs d'immunoglobuline endogenes produit un animal dont le repertoire d'anticorps en reponse a un antigene est completement humain. Dans le mode de realisation prefere de l'invention, le site actif d'immunoglobuline humain est au moins aussi grand que le site humain endogene et il presente une classe fonctionnelle de commutation permettant d'obtenir des anticorps isotype IgG. Outre les anticorps monoclonaux secretes a partir de populations de cellules beta immortalisees, on peut obtenir des anticorps polyclonaux a partir d'oeufs portes par des poulets transgeniques de cette invention.

Robert M. Kay

Papers

Production of human monoclonal antibody in eggs of chimeric chickens

Transgenic aves producing human polyclonal antibodies

The functional disruption of avian immunoglobulin genes

Targeting constructs for the functional disruption of avian immunoglobulin genes

Oiseaux transgeniques producteurs d'anticorps polyclonaux humains