Human lactoferrin (hLF) is an highly cationic, iron-binding glycoprotein(Mr 77,000) of the transferrin family. Human LF was first isolated from milk in which it is present at about 1.5 g/L, but has also been found in many other external secretions such as tears and saliva, as well as in the specific granules of neutrophils. Extensive in vitro and some in vivo evidence indicates that hLF participates in host defense against infection, in iron metabolism and in modulation of inflammatory and immune responses, most notably at mucosal surfaces such as those of the gastrointestinal tract. Most anti-infective and anti-inflammatory activities of hLF are mediated through sequestration of iron and/or interaction with microbial cell wall components and specific receptors on intestinal epithelial cells and lymphocytes, through its highly positively charged N-terminus.

We have chosen to produce recombinant hLF in milk of transgenic cows. Mammary gland specific expression vectors based on regulatory elements from the bovine αS1 casein gene and either hLF cDNA (Krimpenfort et al, 1991, Bio/Technology 9:844-847) of the genomic hLF sequences were introduced into the bovine germline by pronuclear injection of one-cell stage embryos. The latter genomic hLF constructs were selected after a process of construct optimization and evaluation in transgenic mice, which expressed hLF in a mammary gland-specific and lactation-restricted fashion in milk (Platenburg et al, 1994, Transgenic Res. 3:99-108; Nuijens et al 1997, J. Biol. Chem., in press). Human LF was found at relatively low levels in the milk of hLF cDNA transgenic cows, but at levels in the g/L range in the milk of the first lactating genomic hLF founder cow. Based on the correlation of transgene copy number and hLF expression level in transgenic mice, we expect that the hLF expression levels from the other founder animals harbouring a higher copy number will even be higher. The expression of hLF did not affect milk quantity or composition, nor was associated with changes in health or welfare of the lactating cows. Comparison of recombinant hLF with hLF from human milk revealed the proteins to be virtually identical by immunological (double antibody assays; hyperimmunization experiments), functional (iron-binding and -release; binding to LPS, glycosaminoglycans, DNA, human lysozyme; binding to cultured human enterocytes) and structural criteria (analytical chromatography; protein sequencing; spectroscopy; utilization of glycosylation sites; resistance to tryptic digestion).

Based on these observations, we anticipate that recombinant hLF will exert similar if not identical atimicrobial and anti-inflammatory actions in vivo. Currently, we are currently generating herds for the large-scale production of hLF and focusing on the pharmaceutical, preclinical and clinical development of the protein to evaluate its therapeutic and prophylactic potential in several human pathological conditions.