Transgenic milk as a method for the production of recombinant antibodies

Abstract

Recombinant antibodies and their derivatives are increasingly being used as therapeutic agents. Clinical applications of antibodies often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential to the full exploitation of the antibody technology. Production of recombinant protein in the milk of transgenic dairy animals is currently being tested as an alternative to plasma fractionation for the manufacture of a number of blood factors (human antithrombin, human alpha-1-antitrypsin, human serum albumin, factor IX). The ability to routinely yield mg/ml levels of antibodies and the scale-up flexibility make transgenic production an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. The following review examines the potential of transgenic expression for the production of recombinant therapeutic antibodies.

Fig. 1

Schematic representation of the transgenic production process. The coding region of the protein to be expressed is linked to mammary gland specific regulatory elements. The resulting transgene is introduced by pronuclear microinjection into embryos of the selected species (alternatively, somatic cell nuclear transfer using cell lines transfected with the transgene can be used as method to create transgenic sheeps, goats or cattle). Embryos are then transferred to the oviduct (or the uterus) of a surrogate mother and carried to term. Transgenic offspring are identified and, when mature, are either bred or hormonally induced to lactate. Expression level of the target protein in the milk of transgenic animals is determined and a suitable founder line is chosen for the generation of the production herd.

Fig. 2

Transgenes for milk expression of antibodies. The gene of interest replaces the coding region of caprine β-casein, a milk specific gene. The promoter region (6.2 kb) linked to the coding regions of either light or heavy chains immunoglobulin, followed by untranslated caprine beta casein 3′ sequences and downstream elements (7.2 kb). Black boxes indicate the exons of the light and heavy chain of hBR96-2 (IgG1). Striped boxes indicate genomic introns. Arrows indicate direction of transcription.

Fig. 3

Fluorescence in situ hybridization (FISH) of blood metaphase spread form a transgenic goat carrying heavy and light chain antibody transgenes co integrated in the same chromosome (A) 150×; (B) 300×. The heavy chain-specific signal is red, the light chain-specific signal is green.

Fig. 4

SDS-PAGE and Western analysis of several antibodies produced in mice and goats. Two 10–18% SDS-PAGE gels were electrophoresed in parallel under reducing conditions. (A) Lanes were loaded with 0.2 μl of either mouse or goat milk and stained with Coomassie Brilliant Blue R250 (Sigma). (B) Lanes were loaded with 0.1 μl of either mouse or goat milk. Antibodies were detected with affinity purified HRP-goat anti-human IgG (Cappel) and an Enhanced Chemiluminescence Kit (Amersham). This antibody recognizes the heavy chain more efficiently than the light chain. Lane 1. 10 mg/ml of standard antibody (in water). Lane 2. Pre-stained molecular weight markers (Biorad low-range, lot 77813). Lane 3. Negative mouse milk. Lane 4. Negative mouse milk spiked with 10 mg/ml of antibody standard. Lane 5. hBR96-2 a humanized IgG1 produced in mouse milk. Lane 6. Human IgG1 produced in mouse milk. Lane 7. Humanized IgG4 produced in mouse milk. Lane 8. Negative goat milk. Lane 9. Negative goat milk spiked with 10 mg/ml of standard antibody. Lane 10. hBR96-2 a humanized IgG1 produced in goat milk. Lane 11. Human IgG1 produced in goat milk, hormonally induced lactation. Lane 12. Human IgG1 produced in goat milk, natural lactation.

Fig. 5

Milk and antibody production during the first natural lactation of a transgenic goat.

Fig. 6

Silver stained SDS-PAGE of transgenic goat milk samples at different stages of an antibody purification process: Lane 1. Milk sample containing a human IgG1. Lane 2. Protein A eluate. Lane 3. CM HyperD column eluate (BioSepra Inc., Marlboro, MA). Lane 4. Methyl HyperD column eluate (BioSepra Inc., Marlboro, MA).