EP0355093A1 - Interleucine-3 humaine et mutations de celle-ci - Google Patents

Interleucine-3 humaine et mutations de celle-ci

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Publication number
EP0355093A1
EP0355093A1 EP88902307A EP88902307A EP0355093A1 EP 0355093 A1 EP0355093 A1 EP 0355093A1 EP 88902307 A EP88902307 A EP 88902307A EP 88902307 A EP88902307 A EP 88902307A EP 0355093 A1 EP0355093 A1 EP 0355093A1
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European Patent Office
Prior art keywords
leu
ile
asn
ala
pro
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EP88902307A
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German (de)
English (en)
Inventor
Takeshi Otsuka
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Schering Biotech Corp
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Schering Biotech Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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

  • the invention generally relates to protein mediators of hematopoietic system development, and to nucleic acids coding therefor. More particularly, the invention relates to a variant of human interleukin-3, to muteins thereof, and to their encoding nucleic acids.
  • Circulating blood cells are constantly replaced by newly developed cells.
  • Replacement blood cells are formed in a process termed hematopoiesis which involves the production of at least eight mature blood cell lineages: red blood cells (erythrocytes), macrophages (monocytes), eosinophilic granulocytes, megakaryocytes (platelets), neutrophilic granulocytes, basophilic granulocytes (mast cells), T lymphocytes, and B lymphocytes (Burgess and Nicola, Growth Factors and Stem Cells (Academic Press, New York, 1983)).
  • CSFs colony stimulating factors
  • glycoproteins are so named because of the in vivo and in vitro assays used to detect their presence.
  • Techniques for the clonal culture of hematopoietic cells in semisolid culture medium have been especially important in the development of in vitro assays.
  • individual progenitor cells i.e., cells developmentally committed to a particular lineage, but still capable of proliferation
  • proliferate to form a colony of maturing progeny in a manner which is believed to be essentially identical to the comparable process in vivo.
  • the role of CSFs in hematopoiesis is the subject of many recent reviews, e.g.
  • CSFs As more CSFs become available, primarily through molecular cloning, interest has heightened in finding clinical applications for them. Because of physiological similarities to hormones (e.g., soluble factors, growth mediators, action via cell receptors), potential uses of CSFs have been analogized to the current uses of hormones, e.g. Dexter, Nature, Vol. 321, pg. 198 (1986). Their use has been suggested for several clinical situations where the stimulation of blood cell generation would be desirable, such as for rehabilitative therapy after chemotherapy or radiation therapy of tumors, treatment of myeloid hypoplasias, treatment of neutrophil deficiency, treatment to enhance hematopoietic regeneration following bone marrow transplantation, and treatment to increase host resistance to established infections, e.g. Dexter (cited above), and Metcalf, Science (cited above).
  • hormones e.g., soluble factors, growth mediators, action via cell receptors
  • IL-3 interleukin-3
  • Murine IL-3 exhibits a broad spectrum of CSF activities, being capable of stimulating proliferation and development of the progenitor cells of all the recognized myeloid lineages, e.g. Dexter, Nature, Vol. 309, pgs. 746-747 (1984); Ihle et al., Lymphokines, Vol. 9, pgs. 153-200 (1984). Because of the broad stimulatory effects of the factor, there has been a great deal of interest in isolating the analogous factors in other mammals, particularly man.
  • the present invention is directed to a human interleukin-3 (IL-3) and genetically engineered muteins thereof. It includes polypeptides which comprise a sequence of amino acids selected from the set defined below by Formula I, and which possess a colony stimulating factor (CSF) activity as defined by standard assays.
  • the invention also includes nucleic acids capable of encoding the polypeptides defined by Formula I, methods for making such polypeptides by use of the nucleic acids of the invention, and methods of using the polypeptides of the invention either alone or with other CSFs to treat disorders involving myeloid hypoplasia or to enhance hematopoietic system regeneration following bone marrow transplantation or following certain cancer therapies.
  • W(Xaa) and X(Xaa) represent groups of synonymous amino acids to the amino acid Xaa located in a hydrophilic or a hydrophobic region of the protein, respectively, as determined by Hopp-Woods analysis.
  • Synonymous amino acids within a group have sufficiently similar physicochemical properties for substitution between members of the group to preserve the biological function of the molecule: Grantham, Science, vol. 185, pgs. 862-864 (1974); and Dayhoff et al., "A Model of Evolutionary Change in Proteins", Atlas of Protein Sequence and Structure 1972, Vol. 5, pgs. 89-99.
  • insertions and deletions of amino acids may also be made in the above-defined sequence without altering biological function, particularly if the insertions or deletions only involve a few amino acids, e.g. under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g. cysteine residues: Anfinsen, "Principles That Govern The Folding of Protein Chains", Science, Vol. 181, pgs. 223-230 (1973). Proteins and muteins produced by such deletions and/or insertions come within the purview of the present invention. Whenever amino acid residues of the protein of Formula I are referred to herein by number, such number or numbers are in reference to the N- terminus of the protein.
  • human IL-3 polypeptide represented by the actual amino acids Xaa in formula I contains a non-conservative amino acid substitution of proline for serine at position 7 with respect to sequences for human IL-3 previously reported by Yang et al. in Cell, Vol. 47, pgs. 3-10 (1986).
  • the synonymous amino acid groups are those defined in Tables I and II. More preferably, the synonymous amino acid groups are -those listed before the second slash in each line in Tables I and II; and most preferably the synonymous amino acid groups are those listed before the first slash in each line in Tables I and II (each most preferred W(Xaa) group consisting of just Xaa itself). Table I. Preferred, More Preferred and Most Preferred Groups of Synonymous Amino Acids X(Xaa)
  • the invention includes the polypeptides of Formula I with amino acid substitutions (between an amino acid of the polypeptide defined by Formula II (the putative native sequence) and a synonymous amino acid) at a single position or at multiple positions.
  • N-fold substituted is used to describe a subset of polypeptides defined by Formula I wherein synonymous amino acids have been substituted for 0-N amino acids of the native sequence.
  • the group of 1- fold substituted polypeptides of Formula I consists of 381 polypeptides for the preferred groups of synonymous amino acids, 245 for the more preferred groups of synonymous amino acids, and 38 for the most preferred groups of synonymous amino acids.
  • the group of human IL-3 polypeptides consists of the 10-fold substituted polypeptides of Formula I wherein the amino acids in the regions defined by residues 10-32 inclusive, 47-60 inclusive, 71-86 inclusive, and 95-113 inclusive are not more than 3-fold substituted.
  • the amino acid sequences in these regions are more highly conserved relative to the rest of the molecule when compared to mouse, rat, and gibbon IL-3 sequences.
  • the group of human IL-3 polypeptides consists of the 5-fold substituted polypeptides of Formula I wherein the amino acids in the regions defined by residues 10-32 inclusive, 47-60 inclusive, 71-86 inclusive, and 95-113 inclusive are not more than 1-fold substituted. Still more preferably, the group of human IL-3 polypeptides consists of the 2-fold substituted polypeptides of Formula I wherein the amino acids in the regions defined by residues 10-32 inclusive, 47-60 inclusive, 71-86 inclusive, and 95-113 inclusive undergo no substitutions.
  • the polypeptide of the invention comprises the native amino acid sequence defined by the following formula:
  • N-fold inserted in reference to the polypeptides of Formula I is used to describe a set of polypeptides wherein from 1 to N amino acids have been inserted into the sequence defined by
  • the inserted amino acids are selected from the preferred groups of synonymous amino acids of the amino acids flanking the insertion; more preferably they are selected from the more preferred groups of synonymous amino acids of the amino acids flanking the insertion, and most preferably from the most preferred groups of synonymous amino acids of the amino acids flanking the insertion (see Tables I and II).
  • one subgroup of the group of 1-fold inserted peptides comprises an amino acid inserted between the N-terminal X(Pro) and the adjacent X(Met).
  • the insertions defining the members of this subgroup are preferably selected from Pro, Ala, Gly, Thr, Met, Phe, Ile, Val, and Leu; more preferably they are selected from Ala, Pro, Met, Leu, Phe, Ile, and Val, and most preferably they are selected from Pro, Met, Ile, and Leu. Insertions can be made between any adjacent amino acids of Formula I and also at the beginning and end of the amino acid sequence. Since there are 133 possible insertion locations, and since multiple insertions can be made at the same location, a 2-fold inserted peptide of Formula I gives rise to 17,689 subgroups of peptides, and the size of each subgroup depends on the sizes of the synonymous amino acid groups of the amino acids flanking the insertions.
  • N-fold deleted in reference to the polypeptides of Formula I is used to describe a set of peptides having from 1 to N amino acids deleted from the sequence defined by Formula I.
  • the set of 1-fold deleted polypeptides of Formula I consists of 132 subgroups of polypeptides each 131 amino acids in length (131-mers).
  • Each of the subgroups in turn consists of all the 131-mers defined by the preferred, more preferred, and most preferred synonymous amino acid groups depending on the multiplicity of substitutions.
  • a set of polypeptides is defined in terms of both deletions with respect to the sequence of Formula I and insertions with respect to the sequence of Formula I, first the set of polypeptides defined by the insertions is formed and then for each member of that set a set of polypeptides defined by deletions is formed. The sum of the members of all the latter sets forms the set of polypeptides defined by both insertions and deletions.
  • Polypeptides of the invention may be part of a longer polypeptide which comprises an additional peptide linked to the N-terminus of the polypeptide of the invention.
  • the additional peptide may be a signal sequence for directing secretion of the protein from the host organism, e.g. MFalphal signal sequence in yeast (Kurjan et al., Cell, Vol. 30, pgs. 933-943 (1982)).
  • the additional peptide may be used to facilitate the purification of the desired polypeptide, e.g. Sassenfeld et al., "A Polypeptide Fusion Designed for the Purification of Recombinant Proteins", Biotechnology, pgs. 76-81 (January 1984).
  • the additional peptide contains a selective cleavage site adjacent to the N-terminus of the desired polypeptide for the later splitting of the superfluous (with respect to biological activity) amino acid sequence by either enzymatic or chemical means.
  • the above preferred embodiment of the invention further includes nucleotide sequences capable of encoding the polypeptides of Formula I for the preferred, more preferred, and most preferred groups of synonymous amino acids.
  • the invention includes nucleic acids having the sequence of the cDNA insert of clone pcD-huIL3-22-1 illustrated in Figure 2.
  • pcD-huIL3-22-1 has been deposited with the American Type Culture Collection, Rockville, Maryland, under accession number 67318.
  • standard abbreviations are used to designate amino acids, nucleotides, restriction endonucleases, and the like, e.g. Cohn, "Nomenclature and Symbolism of ⁇ -Amino Acids, "Methods in Enzymology, Vol.
  • the present invention is addressed to problems associated with the application of immunoregulatory agents to treat medical and/or veterinary disorders.
  • it provides compounds capable of stimulating the regeneration of blood cells and which may be useful in cancer therapy, the treatment of certain blood diseases, and the treatment of persistent infections.
  • Figure 1 illustrates restriction sites and major coding regions of plasmid pcDVl used in constructing the pcD expression and cloning vector
  • Figure 2 illustrates restriction sites and major coding regions of plasmid pLl used in constructing the pcD expression and cloning vector
  • Figure 3 illustrates the nucleotide sequence and predicted amino acid sequence for a human IL-3 cDNA of the invention.
  • Figure 4 illustrates the modified pcD vector, pSP6, used for generating mRNA for injection into oocytes.
  • the present invention includes glycosylated or unglycosylated polypeptides which exhibit human IL-3 activity, and which are derivable from the IL-3 polypeptides disclosed herein using standard protein engineering techniques.
  • the invention also includes nucleic acids having sequences capable of coding for the polypeptides of the invention.
  • the invention includes methods of making the glycosylated or unglycosylated polypeptides of the invention which utilize the nucleotide sequences disclosed herein, and methods of using the polypeptides of the invention.
  • cDNAs of the present invention can be obtained by constructing a cDNA probe from the cDNA sequence disclosed in Figure 2. The probe is then used to identify a complementary cDNA clone in a cDNA library.
  • the cDNA library is constructed from a population of mitogen-induced T-cell clones or peripheral blood lymphocytes (PBLs). Construction of cDNA libraries from cells induced to grow and/or to synthesize certain cellular products is a well-known technique in molecular biology: e.g. recent reviews are given by Doherty, "Cloning and Expression of cDNA” , Chapter 10 in Gottesman, Ed.
  • a preferred source of mRNA encoding the desired polypeptides is cells whose supernatants contain an activity associated with the polypeptides of the present invention.
  • suitable T-cells can be obtained from a variety of sources, such as human spleen, tonsils and peripheral blood. T-cell clones, such as those isolated from peripheral blood T-lymphocytes, may also be used (see Research Monographs in Immunology, eds. von Doehmer, H. and Haaf, V.; Section D: "Human T-Cell Clones", vol.8, pgs. 243-333; Elsevier Science Publishers, N.Y. [1985]).
  • Probes for the cDNA library are constructed using standard techniques, e.g. nick-translation of fulllength or nearly full-length cDNA clones of the present invention, or by labeled synthetic oligonucleotides.
  • Maniatis et al. (cited above) provide detailed instructions for the nick-translation technique, and labeled synthetic oligonucleotides of any desired base sequence can be constructed using commercially available DNA synthesizers, e.g. Applied Biosystems, Inc. (Foster City, CA) model 381A, or the like.
  • the cDNA library is then screened by the probe using standard techniques, e.g. Beltz et al., "Isolation of Multigene Families and Determination of Homologies by Filter Hybridization Methods," Methods in Enzymology, Vol. 100, pgs. 266-285 (1983); Callahan et al., "Detection and Cloning of Human DNA Sequences Related to the Mouse Mammary Tumor Virus Genome," Proc. Natl. Acad. Sci., Vol. 79, pgs. 5503-5507 (1982), Grunstein et al., Proc. Natl. Acad. Sci., Vol. 72, pgs. 3961-3965 (1975); or Benton et al., Science, Vol. 196, pgs. 180-183 (1977).
  • Muteins of the natural polypeptide may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain muteins, particularly if the side effect activity is associated with a different part of the polypeptide from that of the desired activity.
  • the native polypeptide may be susceptible to degradation by proteases; then selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields: e.g. British patent application 2173-804- A where Arg at position 275 of human tissue plasminogen activator is replaced by Gly or Glu.
  • Muteins may also increase yields in purification procedures and/or Increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications.
  • methionine readily undergoes oxidation to form a sulfoxide, which in many proteins causes loss of biological activity: e.g., Brot and Weissbach, Arch. Biochem. Biophys., Vol. 223, pg. 271 (1983).
  • methionines can be replaced by more inert amino acids with little or no loss of biological activity: e.g. Australian patent application AU-A-52451/86.
  • yields can sometimes be Increased by eliminating or replacing conformationally inessential cysteine residues: e.g. Mark et al., U.S. Patent 4,518,584. In sections below the notation used by Estell et al.
  • human IL-3 mutein Leu 13 indicates a polypeptide whose amino acid sequence is identical to that of the native protein except for position 13 with respect to the N-terminus. At that position Leu has been substituted for Val. More than one substitution can be similarly indicated; e.g. a mutein having Leu substituted for Val at position 13 and Phe for Met at position 18 is referred to as human IL-3 mutein (Leu 13 , Phe 18 ). Deletions are indicated by " ⁇ 's”.
  • human IL-3 mutein ⁇ 4 a mutein lacking Gin at position 4 is referred to as human IL-3 mutein ⁇ 4 .
  • An insertion is indicated by "IN(Xaa)".
  • a mutein with a Leu inserted after Gin at position 4 is referred to as human IL-3 mutein IN4(Leu).
  • human IL-3 mutein (Ser 10 , ⁇ 4 , IN 6 (Gly)) represents the native human IL-3 sequence which has been modified by replacing Thr by Ser at position 10, deleting Gin at position 4, and inserting
  • cassette mutagenesis is employed to generate human IL-3 muteins.
  • a synthetic human IL-3 gene is constructed with a sequence of unique restriction endonuclease sites spaced approximately uniformly along the gene. The uniqueness of the restriction sites should be retained when the gene is inserted into an appropriate vector, so that segments of the gene can be conveniently excised and replaced with synthetic oligonucleotides (i.e. "cassettes”) which code for desired mutations.
  • Determination of the number and distribution of unique restriction sites entails the consideration of several factors including (1) preexisting restriction sites in the vector to be employed in expression, (2) whether species or genera-specific codon usage is desired, (3) the number of different non-vector-cutting restriction endonucleases available (and their multiplicities within the synthetic gene), and (4) the convenience and reliability of synthesizing and/or sequencing the segments between the unique restriction sites.
  • Formula I defines sets of polypeptides with conservative amino acid substitutions, insertions, and/or deletions with respect to the putative native human IL-3 sequence.
  • Constant as used herein means (i) that the alterations are as conformationally neutral as possible, that is, designed to produce minimal changes in the tertiary structure of the mutant polypeptides as compared to the native human
  • alterations are as antigenically neutral as possible, that is, designed to produce minimal changes in the antigenic determinants of the mutant polypeptides as compared to the native human IL-3.
  • Conformational neutrality is desirable for preserving biological activity, and antigenic neutrality is desirable for avoiding the triggering of immunogenic responses in patients or animals treated with the compounds of the invention.
  • rules exist which can guide those skilled in the art to make alterations that have high probabilities of being conformationally and antigenically neutral e.g. Anfisen (cited above), and 3erzofsky, Science, Vol. 229, pgs. 932-940 (1985).
  • Some of the more important rules are (1) substitution of hydrophobic residues are less likely to produce changes in antigenicity because they are likely to be located in the protein's interior, e.g.
  • assays are available to confirm the biological activity and conformation of the engineered molecules.
  • Biological assays for the polypeptides of the invention are described more fully below. Changes in conformation can be tested by at least two well known assays: the microcomplement fixation method, e.g. Wasserman et al., J. Immunol., Vol. 87, pgs. 290-295 (1961), or Levine et al. Methods in Enzymology, Vol. 11, pgs. 928-936 (1967), used widely in evolutionary studies of the tertiary structures of proteins; and affinities to sets of conformation-specific monoclonal antibodies, e.g. Lewis et al., Biochemistry, Vol. 22, pgs. 948-954 (1983).
  • hemopoie t ic cells e.g. bone marrow cells or fetal cord blood cells
  • the individual cells are then "immobilized" in a semi-solid (agar) or viscous (methylcellulose) medium containing nutrients and usually fetal calf serum.
  • a semi-solid (agar) or viscous (methylcellulose) medium containing nutrients and usually fetal calf serum.
  • an appropriate stimulating factor individual cells will proliferate and differentiate. Since the initial cells are immobilized, colonies develop as the cells proliferate and mature. These colonies can be scored after 7-14 days.
  • Detailed instructions for conducting CSF assays are given by Burgess, A., Growth Factors and Stem Cells, pgs.
  • Bone marrow cells collected from patients with nonhematologic disease are layered over Ficoll (type 400, Sigma Chemical Co., St. Louis, MO) and centrifuged (600 x g, 20 min), and the cells at the interface are removed. These cells are washed twice in Iscove's Modified Dulbecco's Medium containing 10% fetal calf serum (FCS) and resuspended in the same medium, and the adherent cells are removed by adherence to plastic Petri dishes (adherent cells are frequently GM-CSF producing cells (Metcalf, cited above)).
  • Ficoll type 400, Sigma Chemical Co., St. Louis, MO
  • FCS fetal calf serum
  • the nonadherent cells are added at 10 5 cells/ml to Iscove's Medium containing 20% FCS, 50 iM 2-mercaptoethanol, 0.9% methylcellulose and varied concentrations of either supernatants known to contain colony stimulating activity or test supernatants.
  • One ml aliquots are plated in 35 mm petri dishes and cultured at 37°C in a fully humidified atmosphere of 6% CO 2 in air.
  • 3 days after the initiation of the culture 1 unit of erythropoietin is added to each plate.
  • Granulocyte macrophage colonies and erythroid bursts are scored at 10-14 days using an inverted microscope.
  • Cord blood cells collected in heparin are spun at 600 x g for 6 min.
  • the white blood cells at the interface between the plasma and red blood cell peak are transferred to a tube containing 0.17 N ammonium chloride and 6% FCS.
  • the suspension is underlaid with 4 ml FCS and centrifuged for 6 minutes at 600 x g.
  • the cell pellet is washed with Dulbecco's phosphate buffered saline and put through the Ficoll and plastic adherence steps as described above for bone marrow cells.
  • the low-density nonadherent cells are collected and placed at 10 cells/culture in the semisolid culture medium as described above.
  • the cellular composition is determined after applying the individual colonies to glass slides and staining with Wright- Geimsa. Eosinophils are determined by staining with Luxol Fast Blue, e.g. Johnson, G. and Metcalf, D. , Exp. Hematol., Vol. 8, pgs. 549-561 (1980).
  • the quantity of active polypeptide in a unit dose according to the present invention can be from 1 ⁇ g to 100 mg , according to the potency and application.
  • compositions can selectively stimulate various components of the immune system, either alone or with other agents well known to those skilled in the art.
  • the compositions may include other immune-reactive agents, such as lymphokines (e.g. IL-1, IL-2, IL-4, GM- CSF, or the like).
  • Any appropriate expression system can be chosen; its nature is not critical to the present invention, given that it is appropriate. Suitable expression systems are discussed in Section VI, Expression Systems, on pages 45-50 of Application PCT/US 86-02464.
  • Example I Preparation of Human IL-3 cDNA by Screening a pcD cDNA Library with a Synthetic Probe and Expression in COS 7 Monkey Cells and Xenopus Oocytes.
  • a pcD cDNA library was constructed from mRNA isolated from a human T-cell clone (6D11) induced to synthesize mRNA by exposure to concanvalin A (conA) (2-10 microgram/ml for 4-12 hours) using the techniques disclosed by Okayama and Berg, Mol . Cell . Biol., Vol. 2, pgs. 161-170 (1982) and Vol. 3, pgs. 280-289 (1983).
  • Other induction compounds such as phytohemagglutinin (PHA), poke weed mitogen, Calcium ionophore, ant ⁇ -T3 antigen, or the like, may be more effective for some cell types and under some conditions.
  • PHA phytohemagglutinin
  • pcDVl and pLl are commercially available from Pharmacia, Inc. (Piscataway, NJ).
  • the cDNA clones are collected, and random pools checked for the presence of the desired cDNAs by standard procedures, e.g. hybrid selection, detection of antigenic determinants on expressed products, and/or functional assays.
  • Total cellular RNA was isolated from 6D11 cells using the guanidine isothiocyanate procedure of Chirgwin et al., Biochemistry, Vol. 18, pgs. 5294-5299 (1979). The procedure is also disclosed by Maniatis et al. (cited above). Washed and pelleted conA-stimulated 6D11 cells were suspended in guanidine isothiocyanate lysis solution (50 g guanidinium thiocyanate with 0.5 g of sodium N-lauroyl sarcosine (final concentration 0.5%), 2.5 mL of 1 M sodium citrate, pH 7. 0 (25 mM ) , 0 .
  • guanidine isothiocyanate lysis solution 50 g guanidinium thiocyanate with 0.5 g of sodium N-lauroyl sarcosine (final concentration 0.5%), 2.5 mL of 1 M sodium citrate, pH 7. 0 (25 mM ) , 0
  • a modified pcDVl plasmid which contained an Nsil site at the previous location of the Kpnl site
  • a modified pLl was used which contained an insertion at the Xhol site consisting of a portion of the long terminal repeat (LTR) of a HTLV(I) retro virus.
  • LTR long terminal repeat
  • the presence of the LTR segment has been found to generally enhance expression in mammalian cell hosts by a factor of 20-50.
  • the modified constructions are illustrated in Figures 1 and 2.
  • Retroviral LTRs have been shown to enhance expression in a number of systems; see e.g. Chen et al., Proc. Natl. Acad. Sci., Vol. 82, pgs. 7285-7288 (1985); Gorman et al., Proc. Natl. Acad. Sci., Vol. 79, pgs. 6777-6781 (1982); Temin, Cell, Vol. 27, pgs. 1-3 (1981); and Luciw et al., Cell, Vol. 33, pgs. 705-716 (1983).
  • the portion of the HTLV(I) LTR inserted at the Xhol site of pLl consists of a 267 base segment including the entire R region and part of the U5 region (designated U5 ' in Figure 2) extending from the R/U5 boundary to the first downstream TagI site (39 bases in all).
  • the sequence of the HTLV(I) LTR is disclosed by Yoshida et al. in Current Topics in Microbiology and Immunology, Vol. 115, pgs. 157-175 (1985).
  • the HTLV(I) LTR segment is inserted into pLl in four steps utilizing procedures disclosed in Example II.
  • pLl is digested with Bgll and Xhol, and the large fragment is isolated.
  • the large fragment is then mixed with synthetics 1A/B and 2A/B (defined below) in a standard l igation mixture .
  • Synthetic 1A/B and 2A/B together consist of, in sequence 5'-->3", the sequence of the small Bgll/Xhol fragment, the 26 base 5' piece of the LTR R region, and a polylinker consisting of Smal, SacI, Nael, and Xhol sites in the stated order.
  • Synthetics 2A/B After ligation the resulting plasmid of step 1 is propagated, isolated, and digested with Smal and SacI, and the large fragment is isolated. The large fragment is then mixed with synthetics 3A/B and 4A/B (defined below) in a standard ligation mixture.
  • step 2 After ligation the resulting plasmid of step 2 is propagated, isolated, and digested with SacI and Nael, and the large fragment is isolated. The large fragment is then mixed with synthetics 5A/B (defined below) in a standard ligation mixture.
  • Synthetics 5A/B After ligation, the resulting plasmid of step 3 is propagated, isolated, and digested with Nael and Xhol, and the large fragment is isolated. The large fragment is then mixed with synthetics 6A/B and 7A/B (defined below) in a standard ligation mixture to form the modified pLl plasmid, designated pLl'.
  • phenol-CHCl 3 water-saturated 1:1 phenol-CHCl 3 (hereafter referred to as phenol-CHCl 3 ) and precipitation with ethanol.
  • dT deoxythymidylate residues per end were added to the Nsi I endonuclease-generated termini with calf thymus terminal transferase as follows:
  • the reaction mixture (38 ⁇ l) contained sodium cacodylate-30 mM Tris.HCI pH 6.3 as buffer, with 1 mM CoCl 2 , 0.1 mM dithiothreitol, 0.25 mM dTTP, the Nsil endonucleasedigested DNA, and 68 U of the terminal deoxynucleotidyl transferase (P-L Biochemicals, Inc., Milwaukee, WI ) . After 30 min.
  • the reaction was stopped with 20 ⁇ l of 0.25 M EDTA (pH 8. 0 ) and 10 ⁇ l of 10% SDS , and af ter several extractions with phenol-CHCl 3 the DNA was recovered by precipitation with ethanol.
  • the DNA was then digested with 15 U of EcoRI endonuclease in 50 ⁇ l containing 10 mM Tris.HCI pH 7.4, 10 mM MgCl 2 r 1 mM dithiothreitol, and 0.1 mg of BSA per ml for 5 hr at 37°C.
  • the large fragment containing the SV40 polyadenylation site and the pBR322 origin of replication and ampicillin-resistance gene, was purified by agarose (1%) gel electrophoresis and recovered from the gel by a modification of the glass powder method (Vogelstein, B. & Gillespie, D., Proc. Natl. Acad. Sci. 76: 615-619 [1979]).
  • the dT-tailed DNA was further purified by absorption and elution from an oligo (dA) -cellulose column as follows: The DNA was dissolved in 1 ml of 10 mM Tris.HCI pH 7.3 buffer containing 1 mM EDTA and 1 M NaCl, cooled at 0°C, and applied to an oligo (dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same buffer at 0°C and eluted with water at room temperature. The eluted DNA was precipitated with ethanol and dissolved in 10 mM Tris.HCI pH 7.3 with 1 mM EDTA.
  • the oligo (dG) tailed linked DNA was prepared by digesting 75 ⁇ g of pLl' DNA with 20 U of PstI endonuclease in 450 ⁇ l containing 6 mM Tris.HCI pH 7.4, 6 mM MgCl 2 , 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per ml. After 16 hr at 30°C the reaction mixture was extracted with phenol-CHCl 3 and the DNA was precipitated with alcohol.
  • Tails of 10 to 15 deoxyguanylate (dG) residues were then added per end with 46 U of terminal deoxynucleotidyl transferase in the same reaction mixture (38 ⁇ l ) as described above, except that 0.1 mM dGTP replaced dTTP.
  • the mixture was extracted with phenol-CHCl 3 , and the DNA was precipitated with ethanol and then digested with 35 U of Hindlll endonuclease in 50 ⁇ l containing 20 mM Tris.HCI pH 7.4, 7 mM MgCl 2 , 60 mM NaCl, and 0.1 mg of BSA at 37°C for 4 hr.
  • the small oligo (dG)-tailed linker DNA was purified by agarose gel (1.8%) electrophoresis and recovered as described above.
  • Radioactively labeled probes for the above colonies were constructed as follows: (1) using the human IL-3 sequence data disclosed by Yang et al.. Cell, Vol. 47, pgs. 3-10 (1986), a synthetic oligonucleotide was synthesized comprising the first 55 nucleotides of the coding strand of the reported human IL-3 sequence (starting with ATG ) ; (2) using the same sequence data an overlapping antisense-strand oligonucleotide was synthesized comprising bases 45-100 of the reported sequence; (3) the two synthetic strands were annealed and used as primers for DNA polymerase I Klenow fragment in the presence of ATP, GTP, TTP, and radioactively labeled CTP; (4) finally the reaction was "cold chased" with unlabeled precursors to ensure maximal strand extension.
  • Cold chased means that the radioactive precursor CTP is replaced by its non-radioactive form.
  • the 20 plates prepared above were screened for clones using the synthetic probes by a standard colony hybridization protocol, e.g. Maniatis et al. (cited above). Two positive colonies were detected.
  • the cDNA sequence is illustrated in Figure 3 along with the predicted amino acid sequence of the largest open reading frame.
  • the adjacent Ala may be the correct N-terminus of the native protein in view of the discovery that the mouse IL-3 N-terminus is Ala.
  • the signal sequence is not a critical portion of the active molecule, and may comprise a wide variety of sequences with no detrimental effect on secretion or activity, e.g. Kaiser et al., Science, Vol. 235, pgs. 312-317 (1987). Potential N-glycosylation sites are enclosed by boxes in Figure 3.
  • the isolated human IL-3 cDNA contained a significant amino acid difference from the sequence reported by Yang et al. (cited above): the isolated cDNA encodes a proline at position 7 (with respect to the N-terminus of Formula II) instead of a serine as reported by Yang et al. (cited above). Such a change should correspond to significant conformational differences in the two polypeptides given the dissimilarity of proline and serine.
  • the same clone was separately amplified; the pcD cloning vectors were isolated using standard techniques, e.g. Maniatis et al .
  • the pcD vectors were used to transfect COS 7 cells as follows. One day prior to transfection, approximately 10° COS 7 monkey cells were seeded onto individual 100 mm plates in Dulbecco's modified Eagle's medium (DME) containing 10% fetal calf serum and 2 mM glutamine. To perform the transfection, the medium was aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCI pH 7.4, 400 ⁇ g/ml DEAE-Dextran and 50 ⁇ g of the plasmid DNAs to be tested.
  • DME Dulbecco's modified Eagle's medium
  • the plates were incubated for four hours at 37 °C, then the DNA-containing medium was removed, and the plates were washed twice with 5 ml of serum-free DME.
  • DME containing 150 ⁇ M Chloroquine was added back to the plates which were then incubated for an additional 3 hrs at 37°C.
  • the plates were washed once with DME, and then DME containing 4% fetal calf serum, 2 mM glutamine, penicillin and streptomycin was added.
  • the cells were then incubated for 72 hrs at 37°C.
  • the growth medium was collected and evaluated for CSF activity using the fetalcord-blood assay described above. A variety of colony types were observed.
  • the modified pcD vector also contained a disabled PstI site in the ampicillin resistance gene (indicated by ⁇ PstI in Figure 3) and a polylinker region inserted at the Xhol site of the polyA region of pcD.
  • the polylinker region contained EcoRI, SacI, Smal, BamHI , Xbal, Sail, SstI, and Hindlll sites.
  • the disabled PstI site permits the SV40 ori region to be excised by digestion (prior to insertion of the polylinker) with Hindlll and PstI.
  • the appropriate SP6 promoter insert (approximately 60 bases in length) can be synthesized using standard techniques from the sequence disclosed by Melton et al. (cited above). After insertion of the SP6 promoter, the resulting plasmid was amplified, isolated, and digested with Xhol. The polylinker synthesized using standard techniques is inserted at the Xhol site. Alternatively, commercially available plasmids containing the SP6 promoter and polylinker regions can be used, e.g. pSP62-PL available from NEN Research Products (Boston, MA), or pSP18 or pSPl9 available from BRL Life Technologies (Gaithersburg, MD) .
  • pSP62-PL available from NEN Research Products (Boston, MA)
  • pSP18 or pSPl9 available from BRL Life Technologies (Gaithersburg, MD) .
  • Human IL-3 cDNA was excised from pcD-huIL3-22-1 by digesting with PstI and BamHI, isolated, and ligated with Pstl/BamHI-diqested pSP6 to form pSP6-huIL3.
  • pSP6- huIL3 was amplified in E. coli MC1061, isolated, and linearized with Xbal.
  • RNA for oocyte injection was made by transcribing the linearized pSP6-huIL3 with SP6 RNA polymerase (available from BRL Life Technologies), following the protocol of Kreig et al. (cited above) and Melton et al. (cited above).
  • RNAse e.g., RNAsin and RNAse-free DNAse at 1 unit/microliter and 20 micrograms/ml, respectively
  • DNAse e.g., RNAsin and RNAse-free DNAse at 1 unit/microliter and 20 micrograms/ml, respectively
  • RNAsin and RNAse-free DNAse at 1 unit/microliter and 20 micrograms/ml, respectively
  • the synthetic human IL-3 gene is assembled from a plurality of chemically synthesized double-stranded DNA fragments. Base sequences of the synthetic gene are selec ted so that the assembled synthetic gene conta ins a series of unique restriction sites with respect to the vector carrying the gene.
  • the series of unique restriction sites defines a series of segments which can be readily excised and replaced with segments having altered base sequences.
  • the synthetic fragments are inserted either directly or after ligation with other fragments into a suitable vector, such as a pcD plasmid, or the like.
  • the above-mentioned segments correspond to the "cassettes" of Wells et al. (cited above) .
  • the synthetic fragments are synthesized using standard techniques, e.g. Gait, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984).
  • an automated synthesizer is employed, such as an Applied Biosystems, Inc. (Foster City, CA) model 380A.
  • pcD plasmids and other suitable vectors are commercially available, e.g. Pharmacia.
  • cloning can be carried out in E. coli MC1061 or HB101, and expression can be carried out in COS monkey cells or Chinese hamster ovary cells.
  • a synthetic human IL-3 gene is constructed for insertion Into a pcD plasmid.
  • the pcD vector is constructed by first ligating four fragments together with T4 DNA ligase: the large HindllI/BamHI fragment from pcDVl containing the pBR322 ori and Ampr, the HindlII/Pstl fragment from pLl containing the SV40 early region promoter and late region introns, and synthetics 11A/B and 12A/B, described below.
  • T4 DNA ligase the large HindllI/BamHI fragment from pcDVl containing the pBR322 ori and Ampr
  • the HindlII/Pstl fragment from pLl containing the SV40 early region promoter and late region introns
  • synthetics 11A/B and 12A/B described below.
  • the alkaline method (Maniatis et al., cited above) is used for small-scale plasmid preparations.
  • a modification of the alkaline method is used in which an equal volume of isopropanol is used to precipitate nucleic acids from the cleared lysate.
  • Precipitation with cold 2.5 M ammonium acetate is used to remove RNA prior to cesium chloride equilibrium density centrifugation and detection with ethidium bromide.
  • Complementary strands of synthetic DNAs to be cloned are mixed and phosphorylated with polynucleotide kinase in a reaction volume of 50 ⁇ l.
  • This DNA is ligated with lug of vector DNA digested with appropriate restriction enzymes, and ligations are in a volume of 50 ⁇ l at room temperature for 4 to 12 hours.
  • Conditions for phosphorylation, restriction enzyme digestions, polymerase reactions, ligations, and bacterial transfections have been described (Maniatis et al., cited above).
  • pcDVl is amplified, isolated, and digested with HindIII and BamHI.
  • the fragment containing the pBR322 or i and Ampr regions is isolated by gel electrophoresis using standard techniques, e.g. Maniatis et al. (cited above).
  • pLl is amplified, isolated and digested with HindIII and PstI.
  • the fragment containing the SV40 early region promoter is isolated by gel electrophoresis.
  • the two isolated pcDVl and pLl fragments are then mixed with synthetics 11A/B and 12A/B (illustrated below) in a standard T4 DNA ligase solution. Synthetics 11A/B and 12A/B anneal to form an insert to the newly created pcD vector, which is amplified in E. coli and then isolated.
  • step 2 the isolated pcD vector of step 1 is digested with Spel and Saul. The large fragment is separated and mixed with synthetics 13A/B and 14A/B in a standard T4 DNA ligase solution to form the pcD vector of step 2, which is amplified in E. coli MC1061 and isolated.
  • step 3 the isolated pcD vector of step 2 is digested with Mlul and Saul. The large fragment is separated and mixed with synthetics 15A/B and 16A/B in a standard T4 DNA ligase solution to form the pcD vector of step 3, which is amplified in E. coli MC1061 and isolated.
  • step 4 the isolated pcD vector of step 3 is digested with EcoRI and Saul. The large fragment is separated and mixed with synthetics 17A/B and 18A/B in a standard T4 DNA ligase solution to form the complete synthetic IL-3 gene in pcD.
  • the pcD vector of step 4 is amplified in E. coli MC1061, isolated, and transfected into COS 7 cells as described in Example I. The culture supernatants are harvested and assayed for CSF activity as described above.
  • the sequences of synthetics 11A/B through 18A/B are listed below. Locations and types of unique restriction sites are indicated above the sequences.
  • Ile at position 131 is changed to Leu to form human IL-3 mutein Leu 131 .
  • the pcD plasmid of Example II containing the complete human IL-3 gene is digested with Saul and Nhel. The large fragment is isolated and combined with the following synthetic in a standard T4 DNA ligase solution:
  • the resulting pcD vector is amplified in E. coli MC1061, isolated, and transfected Into COS 7 monkey cells in accordance with the protocols described above. After incubation for 3-4 days COS 7 culture supernatants are harvested and assayed for CSF activity.
  • the resulting pcD vector is amplified in E. coli MC1061, isolated, and transfected into COS 7 monkey cells in accordance with the protocols described above. After incubation for 3-4 days COS 7 culture supernatants are harvested and assayed for CSF activity.
  • accession number 67318 Applicants have deposited pcd-huIL3-22-l with the American Type Culture Collection, Rockville, MD, USA (ATCC), under accession number 67318.

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Abstract

De nouvelles protéines interleucine-3 humaines contiennent une substitution non traditionnelle d'acides aminés (Ser --> Pro en position 7) par rapport à des séquences antérieurement décrites d'IL-3 humaine. Des procédés de synthèse de protéines mutantes à conformation et antigénicité neutres sont également décrits. Cette protéine IL-3 humaine favorise la croissance et le développement d'un vaste spectre de lignées hématopoïétiques et peut être utile pour traiter des états caractérisés par une régénération réduite de cellules sanguines et/ou des populations cellulaires, tels que l'hypoplasie de la moelle, des infections chroniques et des thérapies utilisant des transplants de moelle osseuse. La séquence de l'IL-3 décrite est illustrée dans la Figure (I).
EP88902307A 1987-02-18 1988-02-18 Interleucine-3 humaine et mutations de celle-ci Pending EP0355093A1 (fr)

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US5304637A (en) * 1987-07-13 1994-04-19 Gist-Brocades N.V. Expression and purification of human interleukin-3 and muteins thereof
US6384194B1 (en) 1987-12-16 2002-05-07 Dsm N.V. Expression and purification of human interleukin-3 and muteins thereof
EP0425536A4 (en) * 1988-07-20 1992-01-02 Immunex Corporation Nonglycosylated human interleukin-3 compositions
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US5516512A (en) * 1989-08-14 1996-05-14 Gist-Brocades, N.V. N- and C-terminal truncation and deletion mutants of human interleukin-3
DE69030937T2 (de) * 1989-08-14 1997-10-09 Gist Brocades Nv Mutanten von Interleukin-3
US5108910A (en) * 1989-08-22 1992-04-28 Immunex Corporation DNA sequences encoding fusion proteins comprising GM-CSF and IL-3
ES2055445T3 (es) * 1989-08-22 1994-08-16 Immunex Corp Proteinas de fusion que comprenden gm-csf e il-3.
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US7049102B1 (en) 1989-09-22 2006-05-23 Board Of Trustees Of Leland Stanford University Multi-gene expression profile
US5376367A (en) * 1991-11-22 1994-12-27 Immunex Corporation Fusion proteins comprising MGF and IL-3
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US6153183A (en) * 1992-11-24 2000-11-28 G. D. Searle & Company Co-administration of interleukin-3 mutant polypeptides with CSF's or cytokines for multi-lineage hematopoietic cell production
EP0672145B1 (fr) * 1992-11-24 2003-04-23 G.D. Searle & Co. Polypeptides a mutations multiples d'interleukine-3
US6361976B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Co-administration of interleukin-3 mutant polypeptides with CSF'S for multi-lineage hematopoietic cell production
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