Transgenic goats producing an improved version of cetuximab in milk.

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10 g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

Passive Immunity to Vibrio cholerae O1 Afforded by a Human Monoclonal IgA1 Antibody Expressed in Milk.

BACKGROUND: In cholera epidemics, the spread of disease can easily outpace vaccine control measures. The advent of technologies enabling the expression of recombinant proteins, including antibodies, in the milk of transgenic animals raises the prospect of developing a self-administered and cost-effective monoclonal antibody (MAb)-based prophylactic to reduce the incidence of Vibrio cholerae infection. METHODS: We generated a transgenic mouse line in which the heavy and light chain variable regions (Fv) specific for a conserved epitope in the core/lipid A of V. cholerae O1 lipopolysaccharide were expressed as a full-length human dimeric IgA1 (ZAC-3) and secreted into the milk of lactating dams. Milk containing ZAC-3 IgA1 was assessed for the ability to passively protect against experimental cholera infection in a newborn mouse model and to impact bacterial swimming behavior. RESULTS: Newborn mice that were passively administered ZAC-3 IgA1 containing milk, or that suckled on dams expressing ZAC-3 IgA1, were immune to experimental cholera infection, as measured by a reduction of V. cholerae O1 colony forming units recovered from intestinal lysates 12 hours after oral challenge. In vitro analysis revealed that ZAC-3 hIgA1-containing milk arrested V. cholerae motility in soft agar and liquid media and was effective at promoting bacterial agglutination, possibly accounting for the observed reduction in bacterial colonization in vivo. CONCLUSIONS: These results demonstrate that consumption of milk-derived antibodies may serve as a strategy to passively protect against cholera and possibly other enteric pathogens.

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 ß-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.

Production of recombinant albumin by a herd of cloned transgenic cattle.

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats.

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.

Viable transgenic goats derived from skin cells.

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.

A recombinant vaccine expressed in the milk of transgenic mice protects Aotus monkeys from a lethal challenge with Plasmodium falciparum.

Two strains of transgenic mice have been generated that secrete into their milk a malaria vaccine candidate, the 42-kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP1(42)). One strain secretes an MSP1(42) with an amino acid sequence homologous to that of the FVO parasite line, the other an MSP1(42) where two putative N-linked glycosylation sites in the FVO sequence have been removed. Both forms of MSP1(42) were purified from whole milk to greater than 91% homogeneity at high yields. Both proteins are recognized by a panel of monoclonal antibodies and have identical N termini, but are clearly distinguishable by some biochemical properties. These two antigens were each emulsified with Freund's adjuvant and used to vaccinate Aotus nancymai monkeys, before challenge with the homologous P. falciparum FVO parasite line. Vaccination with a positive control molecule, a glycosylated form of MSP1(42) produced in the baculovirus expression system, successfully protected five of six monkeys. By contrast, vaccination with the glycosylated version of milk-derived MSP1(42) conferred no protection compared with an adjuvant control. Vaccination with the nonglycosylated, milk-derived MSP1(42) successfully protected the monkeys, with 4/5 animals able to control an otherwise lethal infection with P. falciparum compared with 1/7 control animals. Analysis of the different vaccines used suggested that the differing nature of the glycosylation patterns may have played a critical role in determining efficacy. This study demonstrates the potential for producing efficacious malarial vaccines in transgenic animals.