Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing
Reexamination Certificate
1996-09-25
2002-06-18
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
C424S181100, C424S182100, C424S183100, C435S007100, C435S007900, C435S029000
Reexamination Certificate
active
06406679
ABSTRACT:
TECHNICAL FIELD
The invention relates to the use of carbohydrate-binding proteins as regulators of cell differentiation and immunity. In particular, it concerns a pharmaceutical composition where the active ingredient is a soluble lectin of about 14 kD or a fragment thereof which can be isolated from human HL-60 cells or placenta tissue. Recombinant materials and methods to produce these inventive lectins are also provided. This invention is also directed to methods to treat autoimmune diseases and to prevent transplant rejection.
BACKGROUND ART
Lectins are defined as proteins which specifically bind carbohydrates of various types. Initial interest was focused on those isolated from plants such as concanavalin A and ricin agglutinin. These lectins, it was found, were useful in protein purification procedures due to the glycosylation state of a number of proteins of interest. Among the soluble lectins, there appear to be a number of varieties with varying molecular weights and/or carbohydrate specificities. Sparrow, C. P., et al.,
J. Biol. Chem
. (1987) 252:7383-7390 describe three classes of soluble lectins from human lung, one of 14 kD, one of 22 kD, and a third of 29 kD. All of these lectins are specific to &bgr;-D-galactosides. The carbohydrate specificities of the 14 kD class are for the most part similar, but the larger molecular weight species tend to have different specificities. Other species are also noted as showing more than one soluble &bgr;-D-galactoside-binding lectin, including mouse (Roff, C. F., et al.,
J. Biol. Chem
. (1983) 258:10637-10663); rat (Cerra, R. F., et al.,
J. Biol. Chem
. (1985) 260:10474-10477) and chickens (Beyer, E. C., et al.,
J. Biol. Chem
. (1980) 255:4236-4239). Among the various &bgr;-D-galactoside-specific soluble lectins, ligand specificity is considerably different, and the approximately 14 kD group appears distinct from the 22 kD and 29 kD representatives described by Sparrow, et al., supra.
Recently, however, interest has focused on a group of lactose-extractable lectins which bind specifically to certain &bgr;-D-galactoside containing moieties and are found in a wide range of mammalian, invertebrate, avian, and even microbial sources. All of the lectins in this class appear to contain subunits with molecular weights of about 12-18 kD. Furthermore, these lectins can be readily classified by virtue of a simple diagnostic test: their ability to agglutinate trypsin-treated rabbit red blood cells is specifically inhibited by certain &bgr;-D-galactose-containing moieties. Thus, although the lectins themselves agglutinate trypsinized rabbit erythrocytes, the agglutination can be inhibited by, for example, lactose, thiodigalactoside and certain other &bgr;-D-galactose containing moieties. Other common characteristics include no requirement for metal ions in effecting agglutination and the required presence of a reducing agent such as a thiol.
Gitt, M. A. et al.,
Proc. Natl. Acad. Sci. USA
(1986) 83:7603-7607 obtained two cDNA clones from immunoscreening a human hepatoma cDNA library with an antiserum specific to a human lung lectin. Gitt et al. partially sequenced the cDNAs and the lectins. Gitt compared these sequences with that of the human lung chicken lectin. Although there were marked similarities with chicken and lung lectin, Gitt et al. concluded “In contrast with lung [encoding one form of HL-14 lectin], human hepatcoma appears to express two other forms of HL-14” (page 7607). Kasai, K. et al., in Japanese Kokai 60/184020 describe a human placental lectin of approximately 14 kD. The sequence of this placental lectin was shown by the same group to be somewhat similar to that isolated from chick tissues (Ohyama, Y., et al.,
Biochem. Biophys. Res. Commun
. (1986) 134:51-56) chick-derived lectin was shown to be similar in structure to that of discoidin I, which is a lectin also observed during certain developmental stages of the cellular slime mold
Dictyostelium discoideum.
Caron, M., et al.,
Biochim. Biophys. Acta
(1987) 925:290-296 describe the purification and characterization of lectins from rat and bovine brain tissue. deCabutti, N. E. F., et al.,
FEBS Letters
(1987) 223:330-334 describe a lectin from amphibian ovary. The isolation from eel of a similar “electrolectin” had previously been described by Levi, G., et al.,
J. Biol. Chem
. (1981) 256:5735-5740. An additional analogous 14 kD lectin was produced by cloning and expression of cDNA derived from various murine fibrosarcoma cell lines by Raz, A., et al.,
Experimental Cell Research
(1987) 173:109-116. A rat lung 14 kD lectin, and the cDNA encoding it were described by Clerch, L. B., et al.,
Biochemistry
(1988) 27:692-699. Joubert, R., et al.,
Develop. Brain Res
. (1987) 36:146-150 describe the isolation of lectins from rat brain which are capable of agglutinating brain cells. Raz, A., et al.,
Cancer Res
. (1981) 41:3642-3647 describe a variety of lectins from neoplastic cells of various mammalian species.
Paroutaud, P., et al.,(
Proc. Natl. Acad. Sci. USA
(1987) 4:6345-6348) compared the amino acid sequences of several animal lectins including those from chick, eel, human placenta, human lung, and two hepatoma-derived lectins (all of these lectins described as referenced above). Only the chicken lectin contains an “N-linked” glycosylation site, which is not conjugated to saccharide. No known mammalian lectin in this family has an N-linked glycosylation site.
Although several of the above references disclose some structural similarities with the present invention, none of the references teach the same bioactivity of the unique lectin of the present invention.
The preferred lectins of the present invention are isolated from the human promyelocytic leukemia cell line HL-60 or human placenta tissue. Lectins have been isolated from the HL-60 cell line by others, but they are markedly different from the lectins of the present invention. Paietta, E., et al.,
Cancer Res
. (1988) 48:280-287 describe a membrane-bound (not soluble), 17 kd lectin which recognizes N-acetyl neuraminic acid as well as galactose terminating biantennary oligosaccharide structures. Unlike other 14 kd lectins, this 17 kd lectin is not inhibited by complex galactose saccharides such as thiodigalactoside and does not require reducing thiol groups for binding activity.
Thus, ligand specificity and biodistribution of the lectin protein described herein are an abrupt departure from the earlier disclosed lectins.
Because the activities of lectins in regulating the immune system and mediating other forms of intercellular communication are so subtle in nature and so critically tuned to the host environment, subtle changes in structure can result in a wide range of such regulators with differing therapeutic and diagnostic uses. As described above, a number of members of the class of &bgr;-D-galactose-binding soluble lectins weighing approximately 14 kD are known in the art. However, while these lectins have some similarities, they are not interchangeable therapeutically or diagnostically. In addition, it appears that for lectins which can be glycosylated, the extent and nature of the glycosylation can be manipulated to alter important lectin properties (e.g., circulating half-life, metabolism in vivo, solubility, stability, and specific activity).
Levi et al. (
Eur. J. Immunol
. (1983) 13:500-507) reported that electrolectin binds to peripheral blood and lymph node lymphocytes and is mitogenic. When Levi et al. administered electrolectin to rabbits simultaneously with acetylcholine receptor, it prevented the development of a myasthenia gravis-like condition. Administering electrolectin after development of myasthenia graves caused complete recovery, in spite of high antibody levels specific for the acetylcholine receptor. Because electrolectin did not interfere with acetylcholine interaction with its receptor, Levi et al. proposed that electrolectin had an effect on the immune system.
Prominent diseases in which there is an immune system dysfunction include autoimmune diseases such as myas
Bringman Tim
Couraud Pierre-Oliver
Nedwin Glenn
Seilhamer Jeffrey J.
Bozicevic Field & Francis LLP
Francis Carol L.
Incyte Pharmaceuticals Inc.
Nolan Patrick J.
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