Daniel Pollock

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.

Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice.

Antibodies fused to human enzymes offer an alternative to specifically targeting tumors with antibodies linked to plant or bacterial toxins. Since large amounts of these reagents can be administered without eliciting non-specific toxicities, efficient methods of production are needed. The goal of this work was to express a complex immunoenzyme fusion protein (immunotoxin) in the mammary gland of transgenic mice. A chimeric mouse/human antibody directed against the human transferrin receptor (E6) was fused at its CH2 domain to the gene for a human angiogenic ribonuclease, angiogenin (Ang). It was expressed in the mammary gland of mice and secreted into mouse milk. Expression levels in milk were approximately 0.8 g/l. The chimeric protein retained antibody binding activity and protein synthesis inhibitory activity equivalent to that of free Ang. It was specifically cytotoxic to human tumor cells in vitro.

Expression of a Bovine κ-CN cDNA in the Mammary Gland of Transgenic Mice Utilizing a Genomic Milk Protein Gene as an Expression Cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine κ-casein (κ-CN) cDNA under the control of the goat β-CN 5′ promoter elements and 3′ flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine κ-CN RNA to the mammary gland and secretion of bovine κ-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine κ-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine κ-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine κ-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did κ-CN from bovine milk. A high degree of variation in the production of bovine κ-CN within each of the transgenic lines was observed.

Neutralization of HIV by milk expressed antibody.

Background:In some areas of the world, mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding, which is essential because of scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained adequate levels of protective antibodies in breast milk will prevent transmission of HIV. Methods:The HIV-neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk-specific expression vectors using the GTC goat &bgr;-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. Results:When milk-derived b12 IgA2 was compared with CHO-derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk-derived b12 IgA2 was at least comparable to CHO-derived antibody and in some cases, superior to CHO-derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody-dependent cell killing. Conclusions:These results suggest that it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice, and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV-infected women.