| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 11.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 28.11.00 |
| PATENT TITLE |
Method of delivering drugs to cells specific for an HL-60 lectin |
| PATENT ABSTRACT |
Pharmaceutical compositions useful in the treatment of autoimmune conditions include as an active ingredient a soluble lectin having a molecular weight of about 14 kilodaltons or a fragment thereof. The lectin or fragment binds .beta.-galactoside-containing moieties independent of the presence or absence of Ca.sup.+2, stimulates hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays wherein the stimulation is inhibited by lactose or thiogalactoside, has an amino acid sequence containing at least one N-glycosylation site and is at least 90% homologous to the amino acid sequence shown in positions 2-135 of FIG. 1 or the relevant portions thereof. The composition is used for treatment of autoimmune conditions such as rheumatoid arthritis, myasthenia gravis, and multiple sclerosis, as well as modulating the immune response in an allergic reactions or to organ or tissue transplant rejection. The inventive composition can be combined with general immunosuppressants. Drugs can be delivered by coupling the lectin to a drug to form a lectin-drug complex and administering the lectin-drug complex to a subject. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 25.09.96 |
| PATENT REFERENCES CITED |
Tufveson et al. Immunol. Reviews 136:99-109, 1993. Dijkstra, C. et al. TIPS 14: 124-129, Apr. 1993. Rudinger, J. in Peptide Hormones, Parsons, J. A. (Ed.), University Park Press, Baltimore, MD, pp. 1-7, Jun. 1976. Rabinovich, A. Diabetes 43: 613-621, May 1994. Gilliland, D. G. et al. J. Biol. Chem. 256 (24): 12731-12739, Dec. 25, 1981. Hafler, D. A. et al. Neurology 36: 777-784, Jun. 1986. Rawlins, E.A., Bentley's Textbook of Pharmaceutics, pp. 186, 192-194 (1978) Bailliere Tindall (London). Sharon, "Lectins: An overview" Vertebrate Lectins Olden, K., et al., eds., pp. 27-32 (1987) Van Nostrand Reinhold Advanced Cell Biology Series (NY). Liener, et al., The Lectins:Properties, Functions and Applications in Biology and Medicine, p. 353 (Mar. 2, 1986) Academic Press (NY). Goding, J.W., Monoclonal Antibodies: Principles and Practice, pp. 250-261 (1983) Academic Press (NY). Sparrow et al., "Multiple soluble .beta.-galactoside-binding lectins from human lung", J. Biol. Chem. (1987) 262(15):7383-7390. Roff et al., "Endogenous lectins from cultured cells", J. Biol. Chem. (1983) 258(17):10657-10663. Cerra et al., "Three soluble rat .beta.-galactoside-binding lectins", J. Biol. Chem. (1985) 260(19):10474-10477. Beyer et al., "Two lactose binding lectins from chicken tissues", J. Biol. Chem. (1980) 255(9):4236-4239. Gitt et al., "Evidence that a human soluble .beta.-galactoside-binding lectin is encoded by a family of genes", Proc. Natl. Acad. Sci. (1986) 83:7603-7607. Ohyama et al., "Nucleotide sequence of chick 14K .beta.-galactoside-binding lectin mRNA", Biochem. Biophys. Res. Comm. (1986) 134(1):51-56. Caron et al., "Purification and characterization of a .beta.-galactoside-binding soluble lectin from rat and bovine brain", Biochim. Biophys. Acta (1987) 925:290-296. Fink de Cabutti et al., "Purification and some characteristics of a .beta.-galactoside binding soluble lectin from amphibian ovary", Federation of European Biochemical Societies Letters (1987) 223(2):330-334. Levi et al., "Isolation and physiochemical characterization of electrolectin, a .beta.-D-galactoside binding lectin from the electric organ of electrophorus electricus", J. Biol. Chem. (1981) 256(11):5735-5740. Raz et al., "Cloning and expression of cDNA for two endogenous UV-2237 fibrosarcoma lectin genes", Experimental Cell Research (1987) 173:109-116. Clerch et al., "Sequence of a full-length cDNA for rat lung .beta.-galactoside-binding protein: primary and secondary structure of the lectin", Biochem. (1988) 27:692-699. Joubert et al., "Brain lectin-mediated agglutinability of dissociated cells from embryonic and postnatal mouse brain", Developmental Brain Research (1987) 36:146-150. Raz et al., "Lectin-line Activities Associated with Human and Murine Neoplastic Cells", Cancer Research (1981) 41:3642-3647. Paroutaud et al., "Extensive amino acid sequence homologies between animal lectins", Proc. Natl. Acad. Sci. (1987) 84:6345-6348. Paietta et al., "A membrane-bound lectin responsive to monocytic maturation in the promyelocytic leukemia cell line HL-60", Cancer Research (1988) 48:280-287. Levi et al., "Prevention and therapy with electrolectin of experimental autoimmune myastenia gravis in rabbits", Eur. J. Immunol. (1983) 13:500-507. Wraith et al., "T cell recognition as the target for immune intervention in autoimmune disease", Cell (1989) 57:709-715. Drickamer, "Two distinct classes of carbohydrate-recognition domains in animal lectins", J. Biol. Chem. (1988) 263(20):9557-9560. Hirabayashi et al., "Complete amino acid sequence of a .beta.-galactoside-binding lectin from human placenta", J. Biochem. (1988) 104:1-4. Hirabayashi et al., "Complete amino acid sequence of 14 kDa .beta.-galactoside-binding lectin of chick embryo", J. Biochem. (1987) 101:775-787. Leffler et al., "Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian .beta.-galactosides", J. Biol. Chem. (1986) 261(22):10119-10126. Powell et al., "Purification and properties of lung lectin: Rat lung and human lung .beta.-galactoside-binding proteins", Biochem. J. (1980) 187:123-129. Hirabayashi et al., "Further characterization and structural studies on human placenta lectin", J. Biochem. (1987) 101 (4):987-995. Lempfrid et al., "Lectinreceptor-mediated endocytosis of helix pomatia lectin by zajdela hepatoma cells", in Lectins: Biology, Biochemistry, Clinical Biochemistry, Volume 3, (Proceedings of the Fifth Lectin Meeting, Bern, Bog-Hansen and Spengler, eds., Walter de Gruyter, Berlin, New York, 1983) pp. 73-84. Fudenberg et al., "Chapter 39: Experimental Immunotherapy", in Basic & Clinical Immunology, (5th Edition, Stites et al., eds., Lange Medical Publications, Los Altos, California, 1984) p. 756. Hirabayashi et al., "Human placenta .beta.-galactoside-binding lectin. Purification and some properties", Biochem. Biophys. Res. Comm. (1984) 122(3):938-944. Couraud et al., "Molecular cloning, characterization, and expression of a human 14-kDa lectin", J. Biol. Chem. (1989) 264(2):1310-1316. Weber et al., "The reliability of molecular weight determinations by dodecyl sulfate-polyarcylamide gel electrophoresis", J. Biol. Chem. (1969) 244(16):4406-4412. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of delivering drugs to cells of a mammalian subject, said cells having chemical moieties specific for an HL-60 lectin, the method comprising: coupling a lectin consisting of an amino acid sequence of positions 2-135 of FIG. 1 to a drug to form a lectin-drug complex; and administering said lectin-drug complex to the mammalian subject; wherein the lectin-drug complex selectively binds to cells having chemical moieties specific for the HL-60 lectin. 2. The method of claim 1, wherein the mammalian subject is a human. |
| PATENT DESCRIPTION |
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 .beta.-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 .beta.-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 .beta.-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 .beta.-D-galactoside containing moieties and are found in a wide range of mammalian, in-vertebrate, 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 .beta.-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 .beta.-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. U.S.A. (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 hepatoma 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). The 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. U.S.A. (1987) 84:6345-6348) 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 .beta.-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). Levy et al. (Eur. J. Immunol. (1983) 13:500-507) reported that electrolectin binds to peripheral blood and lymph node lymphocytes and is mitogenic. When Levy 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 gravis 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, Levy 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 myasthenia gravis (MG), rheumatoid arthritis (RA) systemic lupus erythematosus (SLE), multiple sclerosis (MS) and juvenile arthritis. Typically MG, RA, SLE and MS are treated first with corticosteroids. Steroidal drugs have been used for decades and their adverse effects are well known. Adverse effects that can be anticipated in all patients on prolonged steroid therapy include osteoporosis, truncal obesity, impaired wound healing, infections and growth arrest in children. Less frequently occurring adverse effects include myopathy, hypertension, hyperlipidemia, diabetes mellitus and cataracts. Severe side effects may develop and require patient monitoring. These include glaucoma, intracranial hypertension, intestinal perforation, and ulcers. If MG, RA, SLE or MS become refractory to steroids, then increasingly toxic drugs are employed, including azathioprine, methotrexate and cyclophosphamide. The primary effect of azathioprine is inhibiting DNA synthesis, thus lowering numbers of T and B lymphocytes. In addition, azathioprine inhibits the mixed lymphocyte reaction and immunoglobulin production, but does not consistently affect delayed-type hypersensitivity. The major adverse effect of azathioprine is pancytopenia, particularly lymphopenia and granulocytopenia. Consequently, there are increased risks of viral, fungal, mycobacterial and protozoal infections. An increased rate of lymphoreticular malignancies has been reported in kidney transplant patients, but not in patients with RA. Methotrexate inhibits folic acid synthesis and is cytotoxic, suppressing bone marrow. At the low doses used for RA, methotrexate should not decrease the numbers of lymphocytes; but IgM and IgG are reduced. Side effects include pneumonia, nausea, stomach upsets, mouth ulcers, leukopenia, thrombocytopenia, and a form of hepatic fibrosis, which can only be diagnosed by liver biopsy. Cyclophosphamide is also used in RA therapy. It is metabolized in the liver to a compound which cross-links DNA. Cyclophosphamide is cytotoxic, with severe toxicity seen even at low doses. It affects RA by reducing numbers of B- and T-lymphocytes, decreasing the immunoglobulin concentrations and diminishing B-cell responsiveness to mitogenic stimuli. Hair loss, infections, and powerful nausea are common. With prolonged administration, patients develop malignancies at an increased rate. Cyclosporin does not suppress white cells, but it is a powerful immunomodulatory drug and is effective in treating rheumatoid arthritis. However, an important side effect is renal toxicity. Monoclonal antibodies to CD4 have been used in autoimmune diseases, but they cause nonspecific immunosuppression. It has been recommended that new therapies interfere with the initial presentation of specific inciting antigens to T-lymphocytes. (Wraith et al., Cell (1989) 57:709-715). Other drugs have been used specifically in RA, including gold salts, antimalarials, sulfasalazine and penicillamine. Gold salts are given intramuscularly and their effect may not be seen for months. Adverse effects of gold treatment include bone marrow aplasia, glomerulonephritis, pulmonary toxicity, vasomotor reactions and inflammatory flare. Antimalarials exert several effects on the immune system without decreasing the numbers of lymphocytes. The most serious side effects of antimalarials include retinal pigment deposition, rash and gastrointestinal upset. Sulfasalazine has several effects which contribute to its effect on RA; however, it has numerous side effects. Penicillamine has been successfully used in RA; however, its numerous side effects have limited its use. Penicillamine has been reported to cause other autoimmune diseases, including myasthenia gravis and SLE. When patients receive allografts (transplanted tissue from other humans or other sources), their immune systems can destroy the allografts in short order were it not for the administration of immunosuppressant drugs. A number of different organs and tissues are now transplanted, including the kidneys, heart, lungs, skin, bone marrow, cornea, and liver. Drugs frequently used in transplant patients include cyclosporin, azathioprine, rapamycin, other macrolides such as FK506, prednisone, methylprednisolone, CD4 antibodies and cyclophosphamide. Frequently these drugs must be given in higher doses and for longer periods to transplant patients than to patients with autoimmune diseases. Hence, side effects from these drugs (discussed above) may be more common and severe in transplant patients. What was needed before the present invention is a drug that would selectively treat autoimmune diseases and transplant rejection without the severe side effects of the previously known therapies. SUMMARY OF THE INVENTION The present invention is directed to a pharmaceutical composition. The active agent is a soluble lectin of MW of about 14 kD or a fragment thereof, wherein the lectin or fragment (1) binds .beta.-galactoside-containing moieties whether Ca.sup.+2 is present or not, (2) stimulates hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays which is inhibited by lactose or thiodigalactoside, (3) provides an amino acid sequence containing at least one N-glycosylation site and at least 90% homologous to the amino acid sequence shown in positions 2-135 of FIG. 1 or the relevant portions thereof, and wherein the active ingredient is mixed with a carbohydrate and at least one pharmaceutically acceptable excipient. The composition can also contain one or more general immune system suppressants such as cyclosporin. The invention also provides recombinant materials and methods to produce these new lectins. In other aspects, the invention is directed to methods to treat autoimmune diseases and to prevent transplant rejection. The efficacy of these methods results from the surprising ability of the inventive lectin to suppress the host immune response to both autoimmunogens and foreign tissue. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the cDNA sequence and deduced amino acid sequence of both the HL-60 and placenta lectin. Superscript numbers correlate to the corresponding nucleotides and boxes show start and stop codons. The asterisk indicates the possible N-linked glycosylation site. FIG. 2 shows immunostaining of thymic epithelium by anti-lectin and anti-cytokeratin antibodies. FIG. 3 shows an electrophoresis gel with staining in a location indicative of mRNA for the inventive lectin in thymic epithelial cells. FIG. 4 shows the percentage of ARR T-cells that adhered to thymic epithelial cells with and without enzyme treatment. FIG. 5 shows results of hemagglutinin bioassay of purified lectins from human placenta, HL-60 cells and E. coli cells transfected with vectors containing lectin cDNA and an operably linked secretion signal, and inhibition of agglutination by sugars. FIG. 6 shows the effect of several doses of the inventive lectin on the primary immune response to tetanus toxoid. FIG. 7 shows the effect of several doses of the inventive lectin on the primary immune response to pneumococcal polysaccharide. FIG. 8 shows the effect of several doses of the inventive lectin on the primary immune response against the Torpedo receptor. FIG. 9 shows the effect of several doses of the inventive lectin on the primary immune response against mouse skeletal muscle receptor in animals immunized with the Torpedo receptor. FIGS. 10 and 11 show the results of the treatment with the inventive lectin on clinical symptoms in a rat model for multiple sclerosis. FIG. 12 is a graphical representation of the effect of recombinantly produced lectin in a rat model for multiple sclerosis. FIG. 13 shows the effect of the recombinantly produced lectin on a relapse murine model for multiple sclerosis. FIG. 14 is a graphical comparison of the survival of skin allografts when rats received lectin and placebo. BEST MODES OF CARRYING OUT THE INVENTION The Inventive Compositions The invention is directed to pharmaceutical compositions wherein the active ingredient is a soluble, 14 kD lectin or fragment thereof. The preferred lectin of the invention is isolatable from HL-60 cells as well as from human placental tissue. The lectins of this invention are soluble, capable of binding .beta.-galactoside-containing moieties independent of the presence or absence of calcium ion, capable of stimulating hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays which can be inhibited by lactose or thiogalactoside, and at least 90% homologous to the relevant portion of positions 2-135 of FIG. 1. One embodiment consists essentially of the amino acids shown in FIG. 1 numbered from 2-135 which contain at least one tripeptide sequence, Asn-X-Thr or Asn-X-Ser. Preferably, this tripeptide sequence provides a glycosylation site. In native production by HL-60 cells and placental tissue, the N-terminal methionine of the HL-60 lectin appears to be cleaved, and the remaining protein can be acetylated. Various forms of post-translational processing can be expected depending on the cell producing the protein. The processed proteins resulting therefrom are, of course, included within the scope of the invention. In particular, it should be noted that unlike the prior art mammalian lectins whose protein sequences are known, a preferred embodiment of this invention provides a lectin with at least one glycosylation site, exemplified by Asn-Leu-Thr. Most preferably, the glycosylated tripeptide starts at position 96 of the illustrated HL-60 lectin. The native material, at least in part, apparently is not glycosylated at this site; however, when the lectin is appropriately produced in certain recombinant hosts, the site can be glycosylated. Accordingly, the invention provides for glycosylated forms, so long as glycosylation does not destroy activity. The preferred lectins include the peptide comprising positions 2-135 in FIG. 1 or the naturally occurring mutants or allelic variations thereof. It is well understood that proteins produced by organisms do not necessarily remain stable in the form studied, but that the genes encoding them are subject to occasional natural mutations and variations that change one or a few amino acids; therefore, the proteins resulting from natural mutation are included in the invention, so long as the mutation does not destroy activity. An allelic mutation changing as many as 15 amino acids is not expected to destroy the activity of the lectin. Preferably, the allelic mutation would change no more than about 10 amino acids. Most preferably, only about five amino acids would be changed by an allelic mutation. Also included within the invention are fragments of the lectins typified by that shown in FIG. 1. The fragments, in order to be included within the scope of the invention, must exhibit the specific properties characterizing the class, i.e., they must be soluble, capable of binding .beta.-galactoside-containing moieties independent of the presence or absence of calcium ion, capable of stimulating hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays which can be inhibited by lactose or thiogalactoside, and at least 90% homologous to the relevant portion of positions 2-135 of FIG. 1. While a specific minimum fragment length cannot now be identified, it is within ordinary routine experimentation to assay candidate fragments for these activities. It is well understood that only portions or fragments of a particular protein can have the activity of the complete natively produced protein. Deletion of one or several amino acids from either the N- or C-terminus generally does not destroy activity; in addition, internal regions not required to maintain conformation or associated with the active sites can be deleted. Whether or not a protein fragment falls within the scope of the invention is readily determined by means of the assay systems described below for determination of ability to effect hemagglutination both in the presence and absence of lactose or thiodigalactoside. HL-60 cells and placenta tissue produce the preferred 14 kD .beta.-gal binding lectin of the invention; this lectin is representative of the inventive class of 14-.beta.-gal soluble lectins, one embodiment of which has at least one glycosylation site. As used herein, the phrase "14-.beta.-gal mammalian lectin containing at least one glycosylation site" refers to a class of peptides having the characteristics of the group of lectins exemplified by the HL-60 lectin of FIG. 1 which contain at least one tripeptide sequence, Asn-X-Thr or Asn-X-Ser, which provides a glycosylation site. To be included in the inventive class of 14-.beta.-gal lectins or fragments containing at least one glycosylation site, a peptide must exhibit the following biological properties: For the forms which are not fragments, a molecular weight of the nonglycosylated protein is approximately 14 kD. As a practical matter, the molecular weight ranges from about 12-18 kD when it is measured by various techniques. The lectin or fragment is capable of binding .beta.-D-galactoside containing moieties (e.g., lactose). Specifically, the inventive lectin causes hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays, in which the stimulation of agglutination is inhibited by moieties containing the .beta.-galactoside linkage, such as lactose and thiogalactoside. Hemagglutination can occur without a reducing agent, which is capable of maintaining thiol groups in the reduced form, but hemagglutination occurs without metal ions, in particular calcium ions. The inventive lectins and fragments have at least 40% homology with the HL-60 lectin of FIG. 1, preferably at least 75% homology, more preferably over 90% homology and most preferably over 95% homology. The preferred location of the glycosylation site is at residues 96-99, as is the case for the lectin of FIG. 1. However, the glycosylation site can be within, at most, a four-amino acid spacing upstream or three-amino acid spacing downstream, i.e., between residues 92 and 101 inclusive. Other preferred locations include those which contain Asn, X (any amino acid), and Ser/Thr residues in any of the animal lectins at nonconserved regions. The most preferred embodiment of the 14-.beta.-gal lectins containing glycosylation sites is that of the HL-60 lectin of FIG. 1, particularly the lectin from HL-60 cell or placental tissue sources or a lectin from the naturally occurring mutants and allelic variants thereof. Glycosylated forms of this unique lectin having a molecular weight in the range of approximately 12-18 kD are also within the scope of this invention. The glycosylation of these inventive lectins can be manipulated to provide different properties for therapeutic and diagnostic uses. It is known, in general, that proteins exist in a variety of essentially equivalent forms including the acidic and basic salts thereof, forms which are derivatized at side-chain functional groups, forms associated with lipids and membranes, and other modifications made through post-translational processing of the cell expressing the DNA encoding the desired lectin. All of the proteins defined above are inclusive of these various forms. Preparation of the Lectins and Fragments The lectins of the invention can be isolated from native sources, synthesized, or produced by recombinant methods. The isolation of these lectins from native sources, such as HL-60 cells or placental cells, is described in detail in European Publication No. 337,799, published Oct. 18, 1989, and is incorporated herein by reference. This publication also describes the retrieval of cDNA encoding HL-60 lectin. The structure of the full length cDNA clone is shown herein in FIG. 1. The lectins and fragments of the invention can be prepared, if desired, by standard solid phase or other peptide synthesis methods This mode of preparation is generally considered most suited for smaller peptide fragments. Although this method is clearly within the skill of the art with respect to the full-length lectin sequences, such as that shown in FIG. 1, part or all of the molecule can more conveniently be synthesized using recombinant techniques. For recombinant production, the DNA which encodes the inventive lectin or fragment is mobilized by ligating the appropriate sequence to control sequences regulating expression, transfecting the resulting expression systems into appropriate hosts, and culturing the transformed or transfected hosts under conditions favorable for the expression of the DNA. For procaryotic systems, an intronless DNA is required; however, in eucaryotes a genomic DNA can also be used. Genomic DNA encoding the HL-60 and placental lectin and its naturally occurring mutants and allelic variants can be recovered from the HL-60 or placental genome using the cDNA of FIG. 1 as a probe. The lectin-encoding sequence can be ligated into the expression system preceded by an ATG to obtain the lectin as a mature protein. Alternatively, signal sequences known to be operable in the intended host, such as the penicillinase or alkaline phosphatase system in bacteria, the alpha-factor system in yeast, or various hormone signal sequences in mammalian cells can be used to effect secretion by constructing the expression system with the DNA encoding signal in reading phase with the lectin DNA. The lectin could also be produced as a fusion protein by ligating the coding sequence into reading frame with an additional coding sequence if desired. A variety of host systems with appropriate controls are by now well known in the art. For example, among procaryotic hosts, E. coli are preferred, although other bacterial strains, such as Bacillis and Pseudomonas, could be used. Suitable control systems include, but are not limited to, promoters associated with bacterial proteins such as .beta.-lactamase and lactose (lac) promoter systems (Chang et al., Nature (1977) 198:1056; the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8:4057); and the lambda-derived P.sup.L promoter and N-gene ribosome binding site system (Shimatake et al., Nature (1981) 292:128). Similarly, a variety of vectors and promoters is known for yeast systems and includes, but is not limited to, the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. (1980) 255:2073), the enolase gene promoter (Holland, M. J., et al., J. Biol. Chem. (1981) 256:1385), and the leu2 gene obtained from YEp 13 (Broach, J., et al., Gene (1978) 8:121). For expression in cells of higher organisms, promoters operable in such cells include, but are not limited to, viral promoters such as the SV40 promoter (Fiers et al., Nature (1978) 273:113) and promoters derived from adenovirus, bovine papilloma virus, Rous sarcoma virus, and so forth. Also usable are regulatable promoters such as the metallothionein I or metallothionein II promoters. Control sequences for retroregulation are also available such as that associated with the crystal protein gene of Bacillus thuringiensis. Currently available also are systems for production of recombinant proteins in insect cells and in plant cells, although plant cell systems are currently less convenient. Their inconvenience, however, is a result of the current state of the art, and not of an inherent incompatibility between this host cell system and the gene encoding the proteins of the invention. The appropriate coding sequences are ligated to the control sequences in operable configuration and in suitable vectors for transfection into the intended host. The vectors include, but are not limited to, plasmids, virus particles and phages depending on the intended host and the mode of transformation. "Transformation", as used herein, includes all forms of causing uptake of foreign DNA by a host cell including viral infection, transduction, conjugation or, probably most common, induction of uptake in vitro by transfection using transfecting agents such as calcium chloride or DEAE/dextran, depending on the host. The transformed cells are then screened for those which contain the desired DNA and the successful transformants are cultured under conditions which affect the expression of the coding sequences. The lectin produced is then purified from the medium (if the construction results in secretion) or from the lysed cells (if the construction results in an intracellular protein). Whether the lectin is isolated from natural sources, synthesized, or produced by recombinant methods, the lectin can be purified by standard methods, including extraction in lactose solution followed by chromatographic procedures. Convenient chromatographic procedures includes chromatography on lactose sepharose gels, a sephadex S-200 HR column, or a lactose-HEMA column. After using any of these chromatography procedures, the presence of the protein in the active fractions can be easily detected by the ability of the fraction, after removal of the lactose, to cause hemagglutination of trypsinized rabbit erythrocytes, wherein the hemagglutination is inhibited by millimolar concentrations of lactose or thiodigalactoside. Antibodies Reactive with the Inventive Lectins The lectins of the invention can be used in conventional ways to raise antisera reactive with, and specific for, these lectins. An antibody "specific for" the referenced lectin means an antibody which is immunoreactive with this lectin or, in some cases, with other lectins of the invention, but not immunoreactive with non-galactose binding lectins. Because of the extensive homology of the FIG. 1 HL-60 lectin with other lectins of the inventive class, polyclonal antibodies raised against this lectin are likely to cross-react with other inventive lectins. However, by producing monoclonal antibodies with respect to this lectin, antibodies specific to one particular embodiment or to a selected group of inventive lectins can be generated. In addition, antibodies specific for various glycosylated forms can also be prepared. Antibodies can be prepared using known techniques with specificities for any particular member of the inventive 14-.beta.-gal lectin class, including those with at least one glycosylation site and in nonglycosylated and especially glycosylated forms. In short, the antibodies within the scope of the invention are those which are reactive with one or more members of the lectins of the invention, but the antibodies are not cross-reactive with the lectins presently known in the art. Also included in the scope of the invention are antisera raised by any of the lectins of the invention, since these antisera are unique to these lectins even if they contain antibodies which are cross-reactive in some measure with known lectins. Uses of the Inventive Lectins The lectins and fragments of the invention and their compositions are useful in a range of therapeutic and diagnostic applications. In general, these peptides and proteins are particularly useful as immunosuppressants. The inventive lectins and fragments can be used in the treatment of autoimmune diseases such as myasthenia gravis. Other autoimmune diseases which are subject to treatment by these lectins include rheumatoid arthritis, systemic lupus erythematosus, juvenile diabetes, and multiple sclerosis. The inventive lectins can also be useful in controlling allergic reactions. Since these proteins are immune system regulators, they are also useful in the prevention of graft-versus-host disease and inhibition of rejection of transplants in general. Thus, the inventive lectins and fragments can be administered in conjunction with various surgical transplantations including skin allografts, bone marrow transplants, and organ transplants such as kidney, heart, liver or lung transplants. When used as an immunosuppressant, either in treating autoimmune conditions or in preventing transplant rejection, the lectins and fragments of the present invention can be administered along with amounts of known general immunosuppressants that enhance their effects. Such suitable general immunosuppressants include, for example, cyclophosphamide, prednisone, cyclosporin, rapamycin, other macrolide derivatives such as FK506, azathioprine, mycophenolic acid, anti-Tac, lymphocyte immune globulin, and OKT3 antibodies. The inventive lectins and fragments thereof can be administered simultaneously or sequentially with general immunosuppressants. The inventive lectins are also useful in drug delivery and diagnostic applications by carrying the chemical entity to suitable targets. Suitable targets for lectins are those cells of mammalian subjects with galactose-terminating ligands. These lectins are coupled to a drug, for example, cytotoxic or therapeutic agents, or to a label, by methods in the art. Okuda et al., Infect. Immun. (1980) 27:690-92; Samoszuk et al., Antibody Immunoconjugates Radiopharm. (1989) 2:37-46; and Knowles et al., J. Clin. Invest. (1973) 52: 1443-52. In addition, antibodies specific for the inventive lectins are useful in targeting drugs or labels to tumors, since the level of certain lectins increases on the cell surface in metastatic cancer. Anti-lectin antibodies are coupled to the drug, for example, a cytotoxic or therapeutic agent, or label. For diagnostic purposes, the label coupled to the lectin or anti-lectin can be administered to a living mammalian subjects (in vivo use) or used in in vitro tests, for example, as part of a test kit. While not wishing to be bound by any theory, the Inventors propose that the inventive lectins behave as immunomodulating agents and regulate the immune system by binding activated lymphocytes to other activated lymphocytes and to endothelial cells, for example on the inside of blood vessels. Surface glycoproteins on resting lymphocytes contain terminal sialic acid residues, but activated lymphocyte glycoproteins are desialylated to expose galactose. Hence the inventive lectin is specific for activated T-cells and causes them to agglutinate or to adhere to endothelial cells. It is believed that these interactions may inhibit or modify T-cell migration, e.g. extravasation from the circulation, during inflammation. The inventive lectin may affect the immune response by another route. Antibodies specific for the inventive lectin have been observed reacting with human thymic tissue, particularly thymic cortical epithelial cells whose interaction with immature cortical thymocytes is crucial in deleting auto-reactive T-cells. Although the thymus typically atrophies early in life and is not known to play an active role in adult autoimmune pathology, administration of the inventive lectin may possibly increase thymic deletion of autoreactive T-cells. Formulation For use in therapeutic applications, the lectins and fragments are formulated in a manner suitable for the desired mode of administration using formulation technology known in the art as described, for example, in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Philadelphia, Pa. Typical formulations for injection include admixture with physiological buffer for injection such as Hank's solution or Ringer's solution, encapsulation in liposomes or other emulsifying agents suited for drug delivery, and the like. A particularly preferred method of formulation provides for long term storage of the soluble lectin of this invention in the lyophilized, or freeze-dried, form. Lyophilization is preferably conducted in the presence of a concentration of a carbohydrate which is effective to stabilize the lectin during the lyophilization process and at a relatively low pH of about 5. This pH appears to minimize oxidation. Also preferred is addition of a low ionic strength buffer. Suitable protective carbohydrates include, but are not limited to, monosaccharides such as galactose; disaccharides such as lactose, maltose and sucrose; and oligosaccharides containing galactose moieties. The preferred carbohydrates are lactose and maltose. The most preferred protective carbohydrate is lactose. Because the lyophilized product is used in a pharmaceutical composition, the protective carbohydrate must be physiologically and pharmaceutically acceptable at the concentrations used. The effective concentration of the protective carbohydrate can be 1-40% wt./volume but is preferably around 5-15%, and even more preferably around 10%. The maintenance of the pH at about 4 to 8, preferably at about 4.5 to 6, and more preferably at about 5 discourages oxidation of cysteine residues. It is further preferable to maintain this pH in a buffer of relatively low ionic strength, since the freezing point of the mixture is then not lowered significantly. Preferably, the buffer is bicarbonate, gluconate, lactate, acetate or phosphate. Most preferably, the buffer is citrate. It is preferred that the buffer concentration be about 5-20 mM, more preferably 7-12 mM, and most preferably about 10 mM. Other conditions of lyophilization can also be used; however, it has been found that the presence of about 10% lactose in about 10 mM citrate and a pH of 5 are particularly favorable conditions. Administration and Dosage The dosage level and manner of administration of the lectins and fragments of the invention depends on the indication and the subject, as well as the severity of the condition to be treated. When the full lectin is administered, a higher dose (mg/day) is required than when active fragments of the lectin are administered. Some indications for use (such as transplantation rejection, particularly full-blown rejections) require higher doses than to others (such as rheumatoid arthritis, particularly between flare-ups of the disease). The subject who receives the inventive lectin can be a mammal, bird or other vertebrate, because the lectins of mammals, birds, eels, and fish have been found to be related. For purposes of this invention, the term subjects refers to mammalian subjects, including humans, farm animals, sport animals and pets. Farm animals include, but are not limited to, cows, hogs and sheep. Sport animals include, but are not limited to, dogs and horses. The category pets includes, but is not limited to, cats and dogs. Smaller animals generally require somewhat higher doses per kilogram. Preferred dose levels range from about 0.004 mg/kg/day to about 2 mg/kg/day. When the inventive lectins are used in conjunction with general immunosuppressants, however, lower dosages are generally preferred. In general, the inventive lectins or fragments are administered in a manner suitable for peptides or proteins--i.e., by injection or by other parenteral routes including transmembrane or transmucosal transitions. Formulations suitable for these modes of administration are well understood in the art. Oral administration is always desirable, provided the formulation permits the substantially intact lectin or fragment thereof to survive the digestive tract and enter the |
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