Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-21
2003-05-27
Low, Christopher S. F. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S008100, C530S350000, C536S023100, C536S024100, C435S069100, C435S320100, C435S252300, C435S410000, C435S419000, C800S287000, C800S288000, C800S295000
Reexamination Certificate
active
06569831
ABSTRACT:
The present invention relates to recombinant lactoferrins (rLf), their production from plants, and uses thereof.
The use of certain proteins found in mammals has been questioned due to the possibility of contamination by non conventional infectious agents, particularly of the prion type. The impact on marketing and regulations is great. The development of proteins free of all animal contamination is, therefore, a new possibility but one which faces new difficulties depending on the protein and the plant matter chosen.
Prior art includes the publication by Mitra et al. (1994), which describes the transformation of tobacco cells with the DNA sequence which codes for human lactoferrin. However, this publication is limited to plant cells, and does not allow for the production of regenerated plants from these cells. Furthermore, the protein expressed is not purified, and seems, in any case, imperfect as only a part (48 kDa) is detected, and not the entire protein.
In the prior art, application WO 9637094 is also known; this describes the production of plants which are resistant to viruses, by means of transforming them with a gene which codes for lactoferrin. However, in this document as well, the protein is not purified and the only indication of its presence is a Weston blot test.
As a result of the complexity of lactoferrin and the characteristics of the plant matter used, the extraction and purification procedures also represent major obstacles in the production of lactoferrin from plants. The difficulties posed by these two procedures can represent a new set of problems for each protein which one wishes to produce from a plant.
Lactoferrin is a glycoprotein of the transferrin family. Following its discovery in human milk, lactoferrin was shown to be present in many other species such as cows, goats, pigs, mice and guinea pigs, but a highly variable concentrations. In human milk, the concentration of lactoferrin is in the range of 1 to 2 g/l; the concentration is particularly high in colostrum and diminishes over the course of lactation. In milk, lactoferrin is present primarily in the apo form, that is to say, unsaturated in iron. Lactoferrin has also been found in many other secretions, such as the saliva, bile, pancreatic fluid, and secretions of the small intestine. It is found in most mucus, such as bronchial, vaginal, nasal and intestinal secretions.
Lactoferrin is also present in polymorphonuclear neutrophilic leucocytes, where it is localized in the secondary granules of cells that do not contain myeloperoxidase. Leucocytic lactoferrin is synthesized during granulopoiesis of the promyelocyte stage of the metamyelocyte stage. When the neutrophils degranulate, lactoferrin is released into the plasma at a relatively low concentration (0.4 to 2 mg/l) as compared with the level of transferrin found in the blood (2 to 3 g/l).
The peptide sequence for human lactoferrin (hLf) was determined in 1984 by Metz-Boutigue et al. This sequence of 692 amino acids was confirmed by way of cloning of the cDNA for lactoferrin of the human mammary gland (Powell and Ogden, 1990, Rey et al., 1990). Lactoferrin and serotransferrin have very similar primary structures and spatial configurations. Their polypeptide chains are formed of two lobes (N terminal lobe and C terminal lobe) joined by a small alpha helix peptide. Sequence similarities between the N and C terminal halves of human lactoferrin reach 37%. Trypsic hydrolysis of human lactoferrin allowed Legrand et al. (1984) to produce the 30 kDa N trypsic (N-t) fragment (residues of amino acids 4 to 283), and the 50 kDa C trypsic (C-t) fragment (residues of amino acids 284 to 692). At equimolar proportions, these fragments can reunite to form a non-covalent N-t/C-t complex which has electrophoretic and spectroscopic characteristics similar to those of human lactoferrin (Legrand et al, 1986).
Lactoferrin can reversibly bind two ferric ions which results in a salmon-pink coloration, the maximum absorption of which is centered at 465 nm. The binding of each ferric ion requires the same of a carbonate ion. At a pH of 6.4, the association constant of the complex [Fe3+]2-Lf is in the range of 10
24
M
−1
, which decreases with pH. X-ray diffraction study of human lactoferrin at 3.2 Å to 2.8 Å and at 2.2 Å show that each ferrous ion is coordinated with 2 tyrosine residues, a histidine, an aspartic acid and a carbonate ion. These amino acids are the same in both lobes. Both lactoferrin iron binding sites have a strong affinity for this metal, but they release it at different pH levels. The N-t lobe releases its iron at pH 5.8 (acid labile lobe), while the C-t lobe (acid stable lobe) releases its iron at pH 4. Other ions may bind to the protein, in particular gallium. Researchers interested in the use of a
67
Ga-Lf complex as a tracer in cancer diagnostics have shown that following injection with
67
Ga, the complex is preferentially found in the mammary tissues, in physiological and pathological secretions and in Burkitt and Hodgkin lymphomas.
In terms of glycosylation, lactoferrin isolated from human milk has three glycosylation sites, Asn
138
, ASN
479
AND Asn
624
, the first located on the N-t lobe and the other two on the C-t lobe. Glycosylation occurs preferentially at two sites (Asn
138
and ASN
479
) in 85% of molecules, while glycosylation of one site (Asn
479
) and of the three sites simultaneously happens in 5% and 9% of cases, respectively. Lactoferrin glycans are of the mono or disialylated and fucosylated N-acetyllactosamine type (Spik et al, 1982). The fucose residues are a (1,6) branched on the N-acetylglucosamine 5′ of the attachment point, or are &agr; (1,3) branched on the N-acetylglucosamine 5′ of the antenna. Leucocytic lactoferrin differs from that above by the total absence of fucose.
While serotransferrin is unquestionably the principal transporter of iron in all the cells of the organism, the roles played by lactoferrin appear to be essentially linked to defense of the organism and inflammatory mechanisms, working either directly on pathogenic micro-organisms, or indirectly on the effector immune cells.
Lactoferrin is an anti-microbial agent which works by way of several mechanisms. The first of these is the bacteriostatic effect of lactoferrin by means of iron deprivation (Spik et al., 1978). By taking up iron from its surroundings, lactoferrin inhibits bacteria division, as iron is an indispensable element in the biosynthesis of DNA. Furthermore, a more complex mechanism which causes antibodies to act has been shown. The bacteriostatic activity of lactoferrin increases in the presence of the specific IgA and IgG of the pathogenic bacterium. At the same time, lysozyme can associate its lytic activity on the walls of Gram+ bacteria with the action of lactoferrin. Thus, in milk, lactoferrin, lysozyme and antibodies can act synergistically in case of microbe attack.
The second anti-bacterial effect of lactoferrin is linked to it bactericidal capacity. Lactoferrin appears to bind to the walls of Gram− bacteria, which appears to destabilize them and to provoke the release of lipopolysaccharides (LPS). Thus it would seem that the walls become more fragile and more susceptible to the effects of hydrophobic antibiotics. These theories have been confirmed by use of electron microscopy which shows the destabilizing effects of lactoferrin on Gram− bacteria, including
E. coli
. A hypothesis has been made to the effect that the binding of lactoferrin occurs on the A lipid of the LPS, and that this is followed by the extraction of these LPS from the external membranes of the bacteria, irreparably damaging them. The bactericide regions of human lactoferrin (lactoferrin A) and bovine lactoferrin (lactoferrin B) have recently been identified. They are both found in an N-terminal lobe loop comprising 18 amino acids. This loop is formed by a disulphur bridge between the residues Cys 30 and 37 for human lactoferrin, and 19 and 36 for bovine lactoferrin. In addition, the impor
Bournat Philippe
Gruber Veronique
Legrand Dominique
Merot Bertrand
Salmon Valerie
Feit Irving N.
Hoffman & Baron LLP
Kam Chih-Min
Low Christopher S. F.
Meristem Therapeutics S.A.
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