Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates generally to genetically engineered variants of human GM-CSF which have GM-CSF biological activity, a process for obtaining the proteins from genetically engineered cells, and therapeutic compositions containing the proteins for therapeutic uses involving stimulating primate hematopoietic progenitor cells which are responsive to recombinant or natural human GM-CSF.
• the hematopoietic system provides a unique opportunity for the development of useful protein pharmaceuticals.
• hematopoietic progenitor cells found in the bone marrow continuously divide and differentiate ultimately resulting in the release of mature blood cells into the periphery.
• a number of different polypeptide growth factors have been identified which are capable, at least in vitro, of regulating the production of mature blood cells (reviewed in Metcalf, 1984, The Hemopoietic Colony Stimulating Factors, Amsterdam, Elsevier).
• any of these growth factors could prove valuable in the treatment of cytopenias, both naturally arising as well as those induced by chemotherapy or irradiation therapy for cancer [Gasson et al., 1983, "Lymphokines and Hematopoiesis” in Normal and Neoplastic Hematopoiesis (Golde & Marks, Eds.) Liss, NY].
• These proteins are particularly attractive as therapeutic agents because their target cells in the bone marrow are readily accessible to soluble drugs via the circulatory system.
• the hematopoietic growth factors which are also known as the colony stimulating factors were originally identified by their ability to stimulate the clonal expansion of bone marrow progenitor cells in semi-solid medium (Metcalf,
• granulocytemacrophage colony stimulating factor or GM-CSF was identified as a hematopoietic growth factor which is capable of stimulating the formation of colonies containing both granulocytes and macrophages (reviewed in Metcalf,
• GM-CSF has proved to be a potent activator of mature neutrophils, eosinophils and monocytes. (Weisbart et al., 1985, Nature 341:361; Grabstein et al., 1986, Science 232:506).
• the variants of this invention are active colony stimulating factors which may be produced in more homogeneous form and which may possess improved pharmacokinetic profiles relative to natural or recombinant GM-CSF.
• the polypeptide backbone of natural human GM-CSF includes two consensus Asn-linked glycosylation sites: Asn-Leu-Ser at positions 27-29 and Asn-Glu-Thr at positions 37-39.
• Natural human GM-CSF and the "recombinant" version thereof may be produced and recovered in somewhat heterogeneous form, in that both, either or neither of the two consensus glycosylation sites may be occupied by carbohydrate moieties.
• the numbering of amino acids begins with Ala-1 of the mature protein having an N-terminus comprising Ala-Pro-Ala-Arg- Ser-Pro... .
• This invention involves novel glycosylation site variants of human GM-CSF which contain no Asn-linked carbohydrate moieties or only one such moiety.
• the GM-CSF proteins which are characterized structurally by a reduced presence of carbo-hydrate moieties relative to fully glycosylated natural or recombinant GM-CSF are characterized biologically by an improved specific activity (up to 10-fold higher) relative to fully glycosylated natural or recombinant GM-CSF.
• This reduced presence of carbohydrate moieties results from amino acid substitution at one or both of the consensus N-linked glycosylation recognition sites present in the native human GM-CSF molecule.
• Asn-linked glycosylation recognition sites are presently believed to comprise tripeptide sequences which are specifically recognized by the appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is any amino acid, except perhaps proline. A variety of amino acid substitutions, insertions or deletions at one or more of the three positions of a glycosylation recognition site results in non- glycosylation at the modified peptide sequence.
• Asn-27 of GM-CSF has been replaced with Gln in one embodiment
• Asn-37 has been replaced with Gln in another embodiment
• both Asn's have been replaced with Gln in a third embodiment.
• the resultant glycoprotein (Gln-27, Gln-37) contains no N-linked carbohydrate moiety in contrast to native human GM-CSF which is a mixture of proteins containing zero, one or two such moieties.
• the Gln-27 and Gln-37 variants each contain zero or one carbohydrate moiety.
• analogous glycoproteins having the same degree of glycosylation or non-glycosylation may be prepared by deleting Asn-27 and/or Asn-37 and/or by substituting other amino acids at positions 27 and 37 and/or by substituting for or deleting one or more amino acids at other positions within the respective glycosylation recognitions sites, e.g.
• This invention encompasses such nonglycosylated and monoglycosylated human GM-CSF variants. These variants may be described schematically with reference to the polypeptide of formula (1) below:
• A-R 1 -B-R 2 -C (1) wherein A, B and C represent the following domains of human GM-CSF, substantially as depicted in Table 1: A comprises the polypeptide sequence Ala-1 through Leu-26, B comprises Arg-30 through Met-36 and C comprises Val-40 through Glu- 127.
• R 1 and R 2 of formula (1) above each represent a peptide bond or peptide sequence linking the aforementioned polypeptide domains A, B and C by peptide bonds.
• the compounds of this invention have a peptide sequence substantially the same as human GM-CSF (as shown in Table 1) except at one or both of R 1 and R 2 .
• R 1 and R 2 are (i) tripeptide sequences other than consensus glycosylation sites, (ii) dipeptide sequences, (iii) single amino acid residues, or (iv) peptide bonds, as described above.
• the moieties selected for R 1 and R 2 may be the same or different from one another, i.e. may be independently selected.
• Peptide sequences other than consensus glycosylation sites present in the polypeptide sequence of human GM-CSF are tabulated below in Table 2.
• X any amino acid except Asn, or a peptide bond
• Such variants are also characterized structurally (i) by being encoded by a DNA sequence capable, or capable but for the degeneracy of the genetic code, of hybridizing to a DNA sequence encoding native human GM-CSF, under stringent hybridization conditions, as is known in the art; or (ii) by having a peptide sequence at least about 90%, and preferably at least about 95%, homologous to the peptide sequence of human GM-CSF, so long as one or both of the consensus Asn-linked glycosylation sites are modified to other than a consensus Asn-linked glycosylation site.
• the proteins contain one so-called "complex carbohydrate” sugar moiety characteristic of mammalian glycoproteins.
• Such "complex carbohydrate” glycoproteins may be produced by expression of a DNA molecule encoding the desired polypeptide sequence in mammalian host cells.
• Suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See e.g. Gething and Sambrook, Nature 293:620-625 (1981), or alternatively, Kaufman et al., Molecular and Cellular Biology 5: (7):1750-1759 (185) or Howley et al., U.S. Patent No. 4,419,446.
• a further aspect of this invention involves monoglycosylated variants as defined above in which the carbohydrate moiety is a processed form of the initial dolicol-linked oligosaccharide characteristic of insect cell-produced glycoproteins, as opposed to a "complex carbohydrate" substituent characteristic of mammalian glycoproteins, including mammalian derived GM-CSF.
• Such insect cell-type glycosylation is referred to herein as "high mannose” carbohydrate for the sake of simplicity.
• complex and high mannose carbohydrates are as defined in Kornfeld et al., Ann. Rev. Biochem. 54: 631-64 (1985) .
• High mannose variants in accordance with this invention are characterized by a variant polypeptide backbone as described above and as exemplified in Table 3. Such variants may be produced by expression of a DNA sequence encoding the variant in insect host cells. Suitable insect host cells as well as methods and materials for transformation/transfection, insect cell culture, screening and product production and purification useful in practicing this aspect of the invention are known in the art.
• Glycoproteins so produced also differ from natural GM-CSF and from GM-CSF produced heretofore by recombinant engineering techniques in mammalian cells in that the variants of this aspect of the invention do not contain terminal sialic acid or galactose substituents on the carbohydrate moieties or other protein modifications characteristic of mammalian derived glycoproteins.
• the variant proteins of this invention which contain no N-linked carbohydrate moieties may also be produced by expressing a DNA molecule encoding the desired variant, e.g. compounds 2,6,7,11 and 12 of Table 3, in mammalian, insect, yeast, fungal or bacterial host cells.
• a DNA molecule encoding the desired variant e.g. compounds 2,6,7,11 and 12 of Table 3
• suitable mammalian and insect host cells and in addition, suitable yeast, fungal and bacterial host cells , as well as methods and materials for transformation/transfection, cell culture, screening and product production and purification useful in practicing this aspect of the invention are also known in the art.
• cDNAs encoding these compounds may be readily prepared and mutagenized at one or both of the codons for R 1 and R 2 and may be inserted into expression vectors and expressed in host cells by the methods disclosed herein.
• all variants of this invention are prepared by recombinant techniques using DNA sequences encoding GM-CSF analogs which contain fewer or no potential N-linked glycosylation sites relative to natural human GM-CSF.
• DNA sequences may be produced by conventional site-directed mutagenesis of DNA sequences encoding GM-CSF.
• DNA sequences encoding human GM-CSF have been cloned and characterized.
• One such clone is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 (USA) where it has been deposited in plasmid pCSF-1 (in E. coli MC1061) under accession No. 39754.
• the human GM-CSF clone may be readily excised from pCSF-1 as a ⁇ 780 basepair (bp) EcoRI fragment.
• DNA sequences encoding individual variants of this invention may be produced by conventional site-directed mutagenesis of a DNA sequence encoding human GM-CSF or analogs or variants thereof.
• Such methods of mutagenesis include the Ml3 system of Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982); Methods Enzymol. 100:468-500 (1983); and DNA 3:479-488 (1984), using single stranded DNA and the method of Morinaga et al., Bio/technology, 636-639 (July 1984), using heteroduplexed DNA.
• oligonucleotides used in accordance with such methods to convert an asparagine residue to threonine or glutamine, for example, are shown in Table 4.
• DNA encoding each of the glycoproteins of this invention may be analogously produced by one skilled in the art through site-directed mutagenesis using (an) appropriately chosen oligonucleotide(s).
• Expression of the DNA by conventional means in a mammalian, yeast, fungal, bacterial, or insect host cell system yields the desired variant. Mammalian expression systems and the variants obtained thereby are presently preferred.
• the mammalian cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art.
• the components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Bio., 159:51-521 (1982); Kaufman, Proc. Natl. Acad. Sci. USA 82:689-693 (1985).
• oligonucleotides can be readily constructed for use in deleting one or more amino acids or for inserting a different (replacement) amino acids at a desired site by deleting one or more codons or substituting the codon for the desired amino acid in the oligonucleotide, respectively.
• Other mutagenesis oligonucleotides can be designed based on an approximately 20-50 nucleotide sequence spanning the desired site, with replacement or deletion of the original codon(s) one wishes to change.
• Established cell lines, including transformed cell lines, are suitable as hosts.
• Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
• the host cells preferably will be established mammalian cell lines.
• CHO (Chinese hamster ovary) cells are presently preferred.
• the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al., Cell, 36: 391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element.
• Other usable mammalian cell lines include HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.
• Stable transformants then are screened for expression of the product by standard immunological or activity assays.
• the presence of the DNA encoding the variant proteins may be detected by standard procedures such as Southern blotting.
• Transient expression of the DNA encoding the variants during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunological assay of the proteins in the culture medium.
• the DNA encoding the variant may be further modified to contain different codons for bacterial expression as known in the art and preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permitting bacterial expression, secretion and processing of the mature variant protein, also as is known in the art.
• the compounds expressed in mammalian, insect, yeast, fungal or bacterial host cells may then be recovered, purified, and/or characterized with respect to physiochemical, biochemical and/or clinical parameters, all by known methods.
• these compounds have been found to bind to monoclonal antibodies directed to human GM-CSF and may thus be recovered and/or purified by immunoaffinity chromatography using such antibodies or by other conventional methods or methods described hereinafter. Futhermore, these compounds possess human GM-CSF-type activity, e.g. , compounds of this invention effectively stimulate the proliferation of granulocytes and macrophages, as measured in conventional assays.
• compositions for hematopoietic therapy which comprise a therapeutically effective amount of a variant described above in admixture with one or more pharmaceutically acceptable parenteral carriers and/or conventional excipients.
• Such composition can be used in the same manner as that described for human GM-CSF and should be useful in humans or other primates.
• the exact dosage and method of administration will be determined by the attending physician depending on potency and pharmacokinetic profile of the particular compound as well as on various factors which modify the actions of drugs, for example, body weight, sex, diet, time of administration, drug combination, reaction sensitivities and severity of the particular case.
• the following examples are given to illustrate embodiments of the invention. It will be understood that these examples are illustrative, and the invention is not to be considered as restricted thereto except as indicated in the appended claims.
• the growth of the Mo T-cell line has been described previously (Wong et al., 1985, Science, supra).
• the Cl0-MJ2 T-cell line was grown as described by Arya et al., 1984, Science 223:1086). These cells were induced to make GM-CSF by incubation at 5 ⁇ 10 5 cells/ml for 24 hrs in the presence of 0.3% phytohemagglutin (PHA) and 5 mg/ml phorbolmyrystic acetate (PMA) in the presence of 10% fetal calf serum.
• PHA phytohemagglutin
• PMA phorbolmyrystic acetate
• Peripheral blood lymphocyte (PBL) conditioned medium was prepared similarly using Ficoll separated PBL's at a final density of 1 ⁇ 10 6 cells/ml.
• Monkey COS-1 cell transfections were performed using the DEAE-dextran protocol with chloroquin treatment as described previously (Wong et al., 1985, Science, supra). The cells were pulse-labeled by incubation with 0.5 mC of 35 S-methionine in 0.5ml (per 10cm dish) of Dulbecco's Modified Eagle's Medium (DMEM) for 4 hours, 48 hours after the chloroquin treatment. In one transfection, 10 g/ml tuni ⁇ amycin (Sigma) was added 30 minutes prior to labeling to inhibit the addition of N-linked carbohydrate.
• DMEM Dulbecco's Modified Eagle's Medium
• the plasmid pCSF-1 which was isolated by expression cloning was introduced into Chinese Hamster Ovary cells (CHO) using standard methods (Kaufman and Sharp, 1982, J. Mol. Biol. 159:601) and the GM-CSF sequences in the resulting cells were amplified to approximately 200 copies per cell by step-wise selection with methotrexate to yield a cell line (CHO-D2) which expresses high levels of human GM-CSF.
• Confluent dishes of CHO-D2 cells were pulse-labeled for four hours with 0.5 mC of 35 S-methionine in 0.5 ml DMEM. Analysis of Natural GM-CSF's
• the linearized plasmid DNA's were mixed in equimolar amounts, denatured by base treatment and allowed to re-anneal to form heteroduplexes. These heteroduplexes were annealled with either oligonucleotide # 1585 or # 1590 then repaired by treatment with the large fragment of DNA polymerase I (Klenow fragment) in the presence of all four deoxynucleotide triphosphates, ATP and T4 DNA ligase. The products of these reactions were used to transform E. coli MC 1061 and the desired mutants identified by colony hybridization with the appropriate oligonucleotide. The double mutant in which both sites were eliminated was made beginning with the DNA from the Site 1 single mutant by an identical mutant strategy. All of the mutations were confirmed by DNA sequence analysis.
• pCSF-1 containing the mutagenized cDNA encoding a GM-CSF variant of this invention may then be used to transfect or transform desired corresponding host cells.
• Culture of the transfected of transformed host cells by conventional means yields the desired variant, which may then be recovered, and if desired, further purified, by conventional methods such as immunoaffinity chromatography using antibodies, e.g. directed to native or recombinant GM-CSF, or by the purification method described below.
• Alternative mammalian expression vectors are well known in the art and include vectors such as pMT2.
• the preparation of pMT2 from pMT2-VWF (ATCC No. 67122) and use thereof is described in published International application WO 87/07144 (see, page 23).
• Suitable bacterial, fungal and alternative mammalian expression vectors and methods for their use are des cribed in published International application WO 88/00206.
• Suitable yeast, and alternative bacterial and mammalian, expression vectors and methods for their use are described in published International application WO 86/00639.
• Conditioned medium from metabolically labeled CHO-D2 cells was diluted 1:1 with 0.02M Tris HCl, pH 7.4, 1.0mM EDTA, 0.01% Tween 80, and passed over a 1 ml column of DEAE-Trisacryl M (LKB). The column was rinsed in the same buffer containing 50mM NaCl and the GM-CSF eluted with 250mM NaCl in the same buffer.
• LLB DEAE-Trisacryl M
• the GM-CSF eluted from the DEAE column was passed over a Vydac C4 analytical reverse phase column as described previously (Wong et al., 1985, Cancer Cells, supra).
• the 2-N GM-CSF which is less hydrophobic, eluted from this column first (about 41% acetonitrile).
• the DEAE purified protein was passed over a 1 ml column of hydroxyl apatite (BioRad, DNA grade).
• the column was washed with 10mM sodium phosphate, pH 7.0 to elute the 1-N and 2-N protein and the 0-N GM-CSF (lacking N-linked carbohydrate) was eluted with 25 mM sodium phosphate, pH 7.0.
• the labeled CHO-D2 medium was passed over the anti-GM-CSF antibody column (3ml) (as described for the conditioned medium samples above).
• the extent of sialylation of the labeled GM-CSF was assessed by binding to the lectin Ricinus Communis Agglutinin I (RCA 120, agarose-bound, from Vector Laboratories) (Debray et al., 1983, in Lectins: Biology, Biochemistry, Clinical Biochemistry, 3:335 (Bog-Hausen & Spengler, eds) Walter de Gruyter, Berlin, NY). To do this, the GM-CSF was diluted 1:1 with 0.01M Tris-Ci, pH 7.4, 0.1M NaCl, 0.01% Tween 80 and passed over a 0.2 ml column of RCA 120.
• the column was washed with 10 volumes of the same buffer and the bound protein eluted with the same buffer containing 0.5 M galactose.
• the labeled GM-CSF could be bound to the RCA 120.
• Dawley rat that had previously been anesthetized with sodium pentobarbital (15 mg/250g).
• 0.2-0.3 ml aliqouts of blood were collected from the end of the tail into tubes containing 40 units of heparin.
• the rat was exsanguinated and the major body organs (lung, kidney, and liver) were collected and weighed.
• 0.05 ml aliqouts of plasma were spotted onto the glass fiber filters and washed gently with cold 5% trichlor ⁇ acetic acid then cold methanol and were finally dried and counted in anhydrous scintillation fluid (NEN). Radioactivity in each organ was determined by homogenizing 200-300 mg samples in 1ml of 1% SDS, heating to 100 C, and counting 0.1 ml in 5 ml of Aquasol (NEN).
• Administration of GM-CSF to Monkeys were determined by homogenizing 200-300 mg samples in 1ml of 1% SDS, heating to 100 C, and counting 0.1
• GM-CSF was administered to three different monkeys (M. fasicularis) by continuous infusion through catheters surgically implanted in the jugular veins as described previously (Donahue, et al. 1986).
• the GM-CSF was purified by the Genetics Institute Pilot Development Laboratory using lentil-lectin affinity chromatography and reverse phase HPLC essentially as described by Gasson et al., 1984, Science 226:1339. This GM-CSF had a specific activity of 1-2 ⁇ 10 6 units per ml in our CML proliferation assay as discussed elsewhere (Donahue et al., 1986, Nature, supra).
• the protein was found to be a mixture of about 30% 1-N and 70% 2-N GM-CSF.
• GM-CSF derived from different sources, including lectin-stimulated peripheral blood lymphocytes (PBL's), several T-cells lines and primary leukemic blast cells from a patient with acute mylogenous leukemia ranges in apparent molecular mass between 14.5 kD and about 32-34 kD. As the molecular mass predicted from the primary sequence is 14.5 kD, some forms of GM-CSF comprise more than 50% carbohydrate.
• GM-CSF produced by normal PBL's consisted largely of molecules at the high end of the range (22 to about 32-34 kD) while the AML blast cell derived protein on the average was much less glycosylated.
• GM-CSF The distribution of the different species of GM-CSF from all of the different sources was observed to fall into three general size classes, most clearly evident in the case of the AML-derived protein. Because the primary sequence of GM-CSF contains two consensus sequences (Asn-X- Thr/Ser) (Winzler, 1973, in Hormonal Proteins and Peptides 1: 1 (CH. Li, ed)., Academic Press, NY) for asparagine linked carbohydrate (Site 1 is Asn 271 Site 2 is Asn 37) we predicted that these size classes are generated by the state of occupancy of these two sites. We expected that molecules with both Site 1 and Site 2 modified would fall in the largest size class (22 to about 32-34 kD); molecules
• the expressed proteins were visualized by SDS PAGE analysis of conditioned medium from the transfected cells which had been pulse-labeled with 35 S-met. Alteration of the carbohydrate addition sites resulted in the predicted shift in the size distribution of the GM-CSF. Elimination of either Site 1 or Site 2 prevented the synthesis of any molecules falling in the largest size class while the simultaneous alteration of both Site 1 and Site 2 prevented the expres s i on o f GM-CSF in either the large or intermediate size classes.
• the double mutant directed the expression of GM-CSF with a size distribution similar in range to that observed when COS-1 cells transfected with the wild-typ e GM- CS F s equence were treated with tunicamycin , a drug which inhibits the addition of asparagine linked carbohydrate (Duskin and Mahoney, 1982 , J . Biol . Chem 257 : 3105) .
• tunicamycin a drug which inhibits the addition of asparagine linked carbohydrate
• the clearance of GM-CSF from the bloodstream of a rat follows biphasic kinetics.
• the initial phase (called the a phase) of the clearance largely reflects distribution of the protein into all extracellular fluid of the animal while true plasma clearance is represented by the second (or ⁇ phase) portion of the decay curve (Shargel, L. and A.B.C. Yu, 1985, Applied Biopharmaceutical and Pharmacokinetics, Appleton-Century-Crofts, Norwalk, Conn.).
• the extent of modification of the GM-CSF via N-linked carbohydrate had a significant effect on the a but not the ⁇ portion of the curve.
• GM-CSF having no N- linked carbohydrate more than 60% of the GM-CSF was lost from the blood stream with an apparent half life of about 2.5 minutes. The remaining 40% was cleared with an apparent half life of 16 minutes.
• Approximately 60% of the GM-CSF having one N-linked carbohydrate residue was lost from circulation with an apparent half life of 4 minutes while the remainder was cleared during the ⁇ phase having a 15 minute half life.
• the predominant site of clearance for this protein is the kidney. Because the major organs of the rat are highly vascular, their blood content would be expected to contribute significantly to the labeled GM-CSF found in each organ. If we assume for example that there is no significant clearance of GM-CSF in the lungs then it is apparent that the total level of radioactivity in the liver (which has 5-7 times the mass of the lungs) largely resulted from blood contamination and not specific clearance of the glycoprotein in this organ. The high specific activity of GM-CSF found in the kidney, however, clearly demonstrated that this organ is an important site of clearance for this protein.
• neuraminidase was used to remove the terminal sialic acid residues of metabolically labeled GM-CSF.
• the desialylated GM-CSF was isolated by binding to a column to which the lectin Ricin 120 had been covalently attached .
• This lectin has been shown to specifically bind terminal galactose residues which are exposed upon enzymatic removal of sialic acid from glycoproteins (Debray et al . , 1983 , supra) .
• lectin column Prior to the treatment with neuraminidase, none of the labeled GM-CSF was bound by the lectin column.
• GM-CSF a potent stimulator of hematopoiesis in M. fasicularis (macaque) and M. mulatta (rhesus) (Donahue et al., 1986, Nature, supra).
• M. fasicularis macaque
• M. mulatta rhesus
• GM-CSF desialylated GM-CSF (as assessed by 100% binding to a ricin 120 column) was ineffective at stimulating primate hematopoiesis (data not shown).
• GM-CSF with a much higher sialic acid content (10 moles per mole of GM- CSF) has been found to elicit a rapid leukocytosis when administered at a rate of 10 ⁇ g/kg/day.
• the observed response in the levels of circulating blood cells is dose dependent; when GM-CSF was administered at a rate of 5 ⁇ g/kg/d, a minimal response was observed.
• the dose dependency is also evident in the rate of increase in the white cell count, which increased with increasing rates of administration of the protein.
• GM-CSF erythropoietin
• CSF-1 CSF-1
• the stimulation in the levels of circulating blood cells achieved with extensively sialylated GM-CSF was much more dramatic than observed using the desialylated protein.
• the stimulation with the sialated GM-CSF was dose dependent as the higher doses resulted in a more rapid increase in the numbers of circulating blood cells.

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