SK97999A3 - Dna sequence, a polypeptide having hematopoietic biological property, the use thereof and pharmaceutical composition - Google Patents

Abstract

An isolated DNA sequence for use in securing expression in a procaryotic or eucaryotic host cell of a polypeptide product having an amino acid sequence sufficiently duplicative of that of naturally occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occur ring stem cell factor, said DNA sequence selected from among: (a) DNA sequences set out in Figure 44A-C, or their complementary strands; (b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b). Purified and isolated polypeptide, optionally with a methionyl residue at N-end position. Use of the polypeptide for the production of a drug useful in the hematopoietic therapy of a mammal. Pharmaceutical composition containing an effective amount of said polypeptide and a pharmaceutically acceptable solvent, additive or carrier.

Description

DNA sequence, polypeptide composition its use and pharmaceutical

Technical field

The invention relates to a DNA sequence ^- the expression of a polypeptide having the hematopoietic biological property of naturally occurring stem cell factor, said polypeptide in purified and isolated form, the use for the manufacture of medicaments and pharmaceutical compositions based on the peptide.

BACKGROUND OF THE INVENTION

The human blood-producing (haemopoietic) system is composed of variants of basophils, blood cells (megakaryocytes, platelets).

from white blood cells (including neutrophils, macrophages, eosinophils, T and B cells), red and clot-forming cells, ointment cells, (erythrocytes)

It is believed that a small amount of certain haemopoietic growth factors is responsible for differentiating a small number of stem cells in different blood cell progenitors for tremendous proliferation of these cells and irreversible differentiation of mature cells from these lines. Under normal conditions, the haemopoietic regeneration system works well. Once stressed by radiation chemotherapy or natural myelodysplastic impairment, the patient experiences a period of time during which patients are affected by severe leukopenia, anemia or thrombocytopenia. The development and use of haemopoietic factors accelerates bone marrow regeneration during this dangerous phase.

In certain virus-induced disorders, such as Acquired Immunodeficiency Syndrome (AIDS), blood elements such as

T cells, specifically destroyed. Increasing T-cell production may be therapeutic in such cases.

Because haemopoietic growth factors are present in extremely small amounts, their detection and identification is carried out by many assays that have so far only made a distinction between different factors, based on stimulatory effects on cultured cells under artificial conditions.

The application of recombinant genetic techniques has been clarified by understanding the biological activities of individual growth factors. For example, the amino acid and DNA sequence for human erythropoietin (EPO), which simulates erythrocyte production, has been obtained (see Lin, U.S. Pat. No. 4,703,008, incorporated herein by reference). Recombinant methods have also been applied to isolate cDNA for human granulocyte cell stimulating factor, G-CSF (see Souza, U.S. Patent No. 4,810,663), herein incorporated by reference, and human granulocyte macrophage cell stimulating factor (GM-CSP) (Lee et al. USA Proc. 82, 4360-4364 (1985), Wong et al., Science, 228, 810-814 (1985), mouse G- and GM-CSF (Yokota et al., Proc. Natl Acad Sci (USA) 81, 1070 (1984), Fung et al., Nature, 307, 233 (1984), Gough et al., Nature, 309, 763 (1984), and human macrophage colonies. stimulating factor (CSF-1) (Kawasaki et al. Science, 230, 291 (1985)).

The High Proliferative Potential Colony Forming Cell (HPP-CFC) assay system tests the activity of factors in early haemopoietic progenitors (Zont., J. Exp. Med. 159, 679-690 (1984)). There are numerous reports in the literature of factors that are active in the HPP-CFC assay. The sources of these factors are shown in Table 1. The best characterized factors are listed below.

Activity in human spleen conditioned media was named synergistic factor (SF). Several human tissues and human and murine cell lines produce SF designated SF-1, which is synergistic with CSF-1 to stimulate early HPP-CFC. SF-1 was detected in conditioned media of human spleen cells, human placenta, 5637 cells (bladder cancer cells) and EMT-6 cells (mouse mammary carcinoma cell line). The identity of SF-1 is being determined. Initial results showed the activity of SF-1 from cell line 5637 above that of interleukin (Zsebo et al., Blood, 71, 962-968 (1988)). Further results showed that the combination of interleukin-1 (IL-1) plus CSF-1 cannot stimulate the same colony formation that can be obtained. with CSF-1 plus partially purified formulations of 5637 conditioned media (McNiece, Blood, 73,919 (1989)).

The synergistic factor present in the uterine mouse uterine extract is CFS-1. WEHI-3 cells (a murine myelomonocytic leukemia cell line) produce a synergistic factor that appears to be identical to IL-3. Both CSF-1 and IL-3 stimulate hemopoietic progenitors that are more mature than targeted SF-1.

Another class of synergistic factors has been shown to be present in the medium from TC-1 cells (cells derived from bone tissue trees). This cell line produces a factor that stimulates early myeloid and lymphoid cell types. It was named as haemolymphopoietic growth factor 1 (HLGF-1). It has an apparent molecular weight of 120000 (McNiece et al., Exp. Hematol., 16, 383 (1988)).

Of the known interleukins and CSF, IL-1, IL-3 and CSF-1 were identified as being active in the HPP-CFC assay. Other sources of synergistic activity listed in Table 1 have not been structurally identified. Based on the polypeptide sequence and biological activity profile, the present invention relates to molecules that differ from IL-1, IL-3, CSF-1 and SF-1.

Table 1

Preparations containing factors active in the HPP-CFC assay

Source ¹ link CM of human spleen (Kriegler, Blood, 60, 503 (1982)) CM of mouse spleen (Bradley, Exp. Hematol. Today Baum. Ed., 285 (1980)) CM rat spleen (Bradley, Supra, (1980)) CM of mouse lungs (Bradley, Supra, (1980)) CM of human placenta (Kriegler, Supra, (1982)) pregnant mouse uterus (Bradley, Supra, (1980)) GTC-C CM (Bradley, Supra, (1980)) RH3 CM (Bradley, Supra, (1980)) PHA PBL (Bradley, Supra, (1980)) WEHI-3B CM (McNiece, Cell Ciol. Int. Rep. 6,243 (1982)) EMT-6 CM (McNiece, Exp. Hematol., 15, 854 (1987) CM L-cells (Kriegler, Exp. Hematol., 12, 844 (1984) 5637 CM (Stanley, Cell, 45,667 (1986)) TC-1 CM (Song, Blood, 66,273 (1985)) ^ CM = edited media

On parenteral administration, proteins are frequently and rapidly removed from the circulation and may thus induce relatively short pharmacological activity. Thus, frequent injections of relatively large doses of bioactive proteins may be required to maintain therapeutic efficacy. Proteins modified by covalent association with water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit a substantially longer half-life in the blood after intravenous injection, et al., in: Enzyme as Drugs, Holcenberg et al., eds. Wiley-Interscience, New York, NY, 367-383 (1981); Newmark et al., J. Appl. Biochmc. 4: 185-189 (1982) and Katre et al., Proc. Natl. Acad. Sci. USA 84: 1487-1491 (1987)). Such modifications may also increase the solubility of the protein in aqueous solutions, eliminate aggregation, increase the physical and chemical stability of the protein, and greatly reduce the immunogenicity and antigenicity of the protein. As a result, the desired biological activities in vivo can be achieved by administering such a polymer-protein adduct at a lower frequency or at lower dosages than the unmodified protein.

The attachment of polyethylene glycol (PEG) to the protein is very advantageous because PEG has very low mammalian toxicity (Carpenter et al., Toxicol Appl. Pharmacol., 18, 35-40 (1971)). For example, the PEG adduct of adenosine deaminase has been approved in the United States for use in humans in the treatment of severe combined immunodeficiency syndromes. A second advantage associated with conjugation with PEG is an effective reduction in the immunogenicity and antigenicity of heterologous proteins. For example, the PEG adduct of a human protein may be useful in treating diseases in other mammalian species without the risk of eliciting a severe immunological response.

Polymers such as PEG may be suitably linked to one or more reactive amino acid residues in the protein, such as the amino amino terminus of the amino acid, the epsilon amino amino group of the lysine, the sulfhydryl groups of the cysteine side chain, the carboxyl groups of the aspartyl side chain and glutamyl. an alpha-carboxyl group of a carboxyl-terminal amino acid, tyrosine side chains, or activated glycosyl chain derivatives, by attachment to certain asparagine, serine, or threonine residues.

A number of activated forms of PEG suitable for direct reaction with proteins have been described. Suitable PEG reactants for reaction with protein amino groups include active carboxylic acid esters or carbonate derivatives, preferably those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are suitable reactants for modifying sulfhydryl-free proteins. Similarly, PEG reactants containing amine, hydrazine or hydrazide groups are suitable for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.

It is an object of the present invention to provide a factor which causes the growth of early hernopoietic progenitor cells.

SUMMARY OF THE INVENTION

The present invention provides a DNA sequence for use in expressing a polypeptide product exhibiting a hepatopoietic biological property of a naturally occurring stem cell factor in a prokaryotic or eukaryotic host cell, wherein said DNA sequence is selected from:

(a) the DNA sequences shown in Figure 44A-C or their complementary strands, (b) DNA sequences that hybridize under stringent conditions to the DNA sequences defined in (a) or fragments thereof, and (c) DNA sequences that would not be degenerate coding sequence, they would hybridize to the DNA sequences defined in (a) and (b), which sequences encode a polypeptide having the same amino acid sequence.

An object of the present invention is to provide a preferred embodiment of the DNA sequence of the above-mentioned basic embodiment, comprising:

a) DNA of a preferred sequence comprising one or for expression in non-mammalian cells;

multiple codons

b) DNA of a preferred sequence comprising one or cells for expression in E. coli;

multiple codons

c) DNA preferred with one or a sequence comprising for expression in yeast cells;

multiple codons

d) a DNA sequence according to the basic embodiment or according to embodiments a) to c), which encodes the expression of a methionyl residue at the N-terminal position of the mature version of said polypeptide;

e) a DNA sequence according to the basic embodiment or according to the embodiments referred to in a) to d), covalently linked to a detectable label; and

f) The DNA sequence of paragraph e), wherein the label is a radiolabel.

The invention also provides a purified and isolated polypeptide having all or part of the primary structure of the sequence shown in FIG. 44A-C, optionally with a methionine residue at the N-terminus, exhibiting a hematopoietic biological property of a naturally occurring stem cell factor.

The present invention further provides preferred embodiments of the purified and isolated polypeptide of the aforementioned basic embodiment, comprising:

a) a purified and isolated polypeptide comprising the amino acid sequence of FIG. 44A-C: 1-220, 1-161, 1-160, 1-157 and 1-152, optionally with a methionine residue at the N-terminus; and

b) a purified and isolated polypeptide having hematopoietic activity stimulating the growth of early hematopoietic progenitor cells.

The present invention further provides a pharmaceutical composition comprising an effective amount of any of the above-described polypeptides of the invention and a pharmaceutically acceptable diluent, excipient or carrier.

The invention further provides the use of any of the above-described polypeptides of the invention for the manufacture of a medicament for the hematopoietic therapy of a mammal.

In particular, it may be a drug for

(a) treating leukopenia, treating thrombocytopenia, treating anemia, increasing bone marrow transplant intake, increasing bone marrow recovery in radiation therapy, chemically or chemotherapeutically induced bone marrow aplasia or myelosuppression, and increasing the sensitivity of cells to chemotherapy;

b) increasing the number of cells capable of transplantation after bone marrow aspiration or peripheral blood leukopheresis;

c) treatment of AIDS, myelofibrosis, myelosclerosis, osteoporosis of metastatic carcinoma, acute leukemia, multiple myeloma, Hodgkin's lymphoma disease, Geucher disease, Niemann-Pick disease, Letter-Siw disease, resistant erythroblastic anemia, Di Guglielmest's syndrome, Di Guglielmest's syndrome, sarcoidosis, primary splenic pancytopenia, million tuberculosis, sowing fungal disease, severe septicemia, malaria, vitamin B ₁₂ deficiency, folic acid deficiency, and pyridoxine deficiency in mammals;

d) treating nerve damage, infertility or intestinal damage in a mammal; and

e) treating a hypopigmentation disorder.

The invention also provides such use wherein the medicament to be manufactured further comprises at least one additional hematopoietic factor selected from

a) EPO, G-CSF, GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IGF-1, and M- GDF;

b) IL-8, IL-9 and IL-10; and

c) IL-11 and LIF.

The above DNA sequences may be introduced into suitable vectors, such vectors may be transformed or transfected with suitable host cells, which may then be used to produce the peptides of the invention.

Prolonged in vivo half-life and increased efficacy in mammals can be achieved by converting the peptides of the invention to conjugates with water-soluble polymers. The polymer is preferably selected from polyethylene glycol and copolymers of polyethylene glycol and propylene glycol. These polymers may be unsubstituted or substituted at one end with an alkyl group. The preparation of such adducts comprises reacting SCF with a water-soluble polymer having at least one terminal reactive group and purifying the resulting adduct. The products obtained have an increased half-life in the circulatory system and increased biological activity.

Stem cell factor (SCF) can be obtained from the material containing it using ion exchange chromatography techniques and / or reverse phase liquid chromatography separation.

An overview of the drawings

Fig. 1 is an anion exchange chromatogram from purification of mammalian SCF.

Fig. 2 is a gel filtration chromatogram of purification of mammalian SCF.

Fig. 3 is a wheat germ-agarose agglutinin chromatogram from purification of mammalian SCF.

Fig. 4 represents cation exchange chromatogram from cleaning mammalian SCF. Fig. 5 is C ₄ chromatogram z cleaning mammalian SCF. Fig. 6 is electrophoresis on sodium dodecyl sulfate

(SDS) -polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) fraction from the _C4 column of FIG. 5th

Fig. 7 is an analytical C ₄ chromatogram of mammalian SCF.

Fig. 8 shows SDS-PAGE C of the ₄ column fractions of FIG. 7th

Fig. 9 shows SDS-PAGE of purified mammalian SCF and deglycosylated mammalian SCF.

Fig. 10 is an analytical chromatogram of purified mammalian SCF.

Fig. 11 represents the amino acid sequence of mammalian SCF derived from protein sequencing.

Fig. 12 presents

A. rat SCF cDNA oligonucleotides

B. human SCF DNA oligonucleotides

C. universal oligonucleotides.

Fig. 13 presents

A. Scheme of polymerase chain building (PCS) amplification of rat SCF cDNA

B. PCR amplification scheme of human SCF cDNA.

Fig. 14 presents

A. Sequence strategy for rat genomic DNA

B. the nucleic acid sequence of rat genomic DNA

C. the nucleic acid sequence of the rat SCF cDNA and the amino acid sequence of the rat SCF protein.

Fig. 15 presents

A. strategy of sequencing human genomic DNA

B. human genomic DNA nucleic acid sequence

C. composite nucleic acid sequence of human SCF cDNA and amino acid sequence of SCF protein.

Fig. 16 shows the aligned amino acid sequences of human, monkey, canine, mouse and rat SCF protein.

Fig. 17 shows the structure of the mammalian cell SCF expression vector V19.8.

Fig. 18 depicts the structure of the VDSVE.1 mammalian CHO cell expression vector.

Fig. 19 shows the structure of the E. coli expression vector pCFM1156.

Fig. 20 presents

A. radioimmunoassay of mammalian SCF

B. SDS-PAGE of immune precipitated mammalian SCF.

Fig. 21 represents Western analysis of recombinant human SCF.

Fig. 22 is a Western analysis of recombinant rat SCF.

Fig. 23 is a bar graph of the effect of COS-1 cell-produced recombinant rat SCF on bone marrow transplantation.

Fig. 24 shows the effect of recombinant rat SCF on the treatment of Steel macrocytic anemia.

Fig. 25 shows peripheral white blood cells (WBC) in recombinant rat SCF treated Steel mice.

Fig. 26 shows the number of platelets of steel mice treated with recombinant rat SCF.

Fig. 27 represents the difference in WBC count in Steel mice treated with recombinant SCF ^1_164 PEG25.

Fig. 28 depicts the lymphocyte subsets of Steel mice treated with recombinant rat SCF ^1-116 PEG25.

Fig. 29 shows the effect of recombinant human SCF sequence in the treatment of normal primates with an increased number of peripheral WBCs.

Fig. 30 shows the effect of recombinant human SCF sequence in the treatment of normal primates on increasing the number of hematocrits and platelets.

Fig. 31 presents photographs

A. human bone marrow colonies stimulated with recombinant human SCF ^1-162

B. Wright-Giemse stained cells from the colonies in FIG. 31A.

Fig. 32 is a SDS-PAGE of the fractions from the chromatogram on the S-Sepharose column shown in FIG. 33

A. with a reducing agent

B. without reducing agent.

Fig. 33 is a chromatogram of recombinant E. coli derived on an S-Sepharose column.

Fig. 34 is an SDS-PAGE of the chromatogram fractions shown in FIG. 35 human SCF

C ₄ columns from

A. with a reducing agent

B. without reducing agent.

Fig. 35 is a chromatogram of recombinant human SCF derived from E. coli on a C ₄ column.

Fig. 36 is a chromatogram on a Q-Sepharose column from CHO-derived recombinant rat SCF.

Fig. 37 is a chromatogram of a C ₄ column of recombinant rat SCF derived from CHO.

Fig. 38 is a SDS-PAGE of the C ₄ column fractions from the chromatogram shown in FIG. 37th

Fig. 39 shows SDS-PAGE of purified CHO-derived recombinant rat SCF before and after deglycosylation.

Fig. 40 presents

A. gel filtration chromatography of recombinant rat pegylated SCF ¹ ” ¹⁶⁴ reaction mixture

B. gel filtration chromatography of recombinant rat SCF ^1-164 , unmodified.

Fig. 41 represents radiolabeled SCF bound to fresh leukemic blasts.

Fig. 42 represents the human SCF cDNA sequence obtained from the HT1080 fibrosarcoma cell line.

Fig. 43 is an autoradiograph of COS-7 cells expressing human SCF ^3--24 ® and CHO cells expressing human SCF ^1-164 .

Fig. 44 represents the cDNA sequence of human SCF derived from bladder cancer cell line 5637.

Fig. 45 represents an increase in survival of irradiated mice after SCF treatment.

Fig. 46 represents increased survival of irradiated mice after bone marrow transplantation with 5% femur and treatment

SCF.

Fig. 47 represents an increase in survival of irradiated mice following bone marrow transplantation of 0.1 and 20% femur and SCF treatment.

Numerous aspects and advantages of the present invention will be apparent to those skilled in the art from the following detailed description, which serves to illustrate the practice of the invention in its particularly preferred embodiments.

A detailed description of the invention follows.

In accordance with the present invention, novel stem cell factors and DNA sequences encoding all portions of such SCFs are provided. The term stem cell factor or SCF as used herein refers to naturally occurring SCF (i.e., natural human SCF) as well as to non-naturally occurring (i.e., different from naturally occurring) polypeptides having amino acid sequences and glycosylation sufficiently consistent with these characteristics naturally. the occurring stem cell factor so as to provide the ability of the hematopoietic biological activity of the naturally occurring stem cell factor. The stem cell factor has the ability to stimulate the growth of early hematopoietic progenitors that are capable of maturing into erythrocytes, megakaryocytes, granulocytes, lymphocytes and macrophages. Treatment of mammals with SCF produced results in an absolute increase in the number of hematopoietic cells of both the myeloid and lymphoid lineages. One of the main characteristics of stem cells is their ability to differentiate between myeloid and lymphoid cells ((Weissman Science, 241, 58-62 (1988)). Treatment of Steel mice (Example 8B) with recombinant SCF rats resulted in increased granulocytes, monocytes, erythrocytes, lymphocytes Treatment of normal primates with recombinant human SCF resulted in an increase in the number of myeloid and lymphoid cells (Example 8C).

The embryonic expression of SCF occurs in the cells in a migratory manner and at the maternal sites of melanoblasts, germ cells, hematopoietic cells, brain and spinal cord.

Early hematopoietic progenitor cells were expanded in mammalian bone marrow treated with 5-fluorouracil (5-FU). The chemotherapeutic drug 5-FU selectively depletes late hematopoietic progenitors. SCF is active in bone marrow at 5-FU sites.

The biological activity and tissue distribution model of SCF shows its central role in embryogenesis and hematopoiesis, as well as its ability to treat various stem cell deficiencies.

The present invention provides DNA sequences comprising: incorporation of codons preferred for expression by selected non-mammalian hosts; providing restriction endonuclease enzyme cleavage sites; and providing additional start, end, or intermediate DNA sequences which allow the construction of readily expressible vectors. The present invention also provides DNA sequences encoding polypeptide analogs or SCF derivatives that differ from naturally occurring forms in the identity or position of one or more amino acid residues (i.e., deletion analogs containing less than all SCF-specific residues, substitution analogs, wherein one or more specific residues are replaced by other residues and addition analogs where one or more amino acid residues are added to the terminal or middle portion of the polypeptide) and which have some or all of the properties of naturally occurring forms. Specifically, the present invention provides DNA sequences encoding the full-length unprocessed amino acid sequence as well as DNA sequences encoding the processed form of SCF.

The disclosed new DNA sequences include sequences suitable for providing expression in prokaryotic and eukaryotic host cells of polypeptide products having at least a portion of the primary structural conformation and one or more biological properties of naturally occurring SCF. Such sequences specifically include: a) The DNA sequences shown in FIG. B) DNA sequences that hybridize (under the hybridization conditions described in Example 3, or under more stringent conditions) to the DNA sequences of FIGS. 44A-C, or fragments thereof; and c) DNA sequences that hybridize to the DNA sequences of FIGS. 44A-C if there was no degeneracy of the genetic code. Specifically, those sequences set forth in parts b) and c) are genomic DNA sequences encoding allelic variants of SCF and / or encoding SCF from other mammalian species, and produced DNA sequences encoding SCF, SCF fragments and SCF analogs. The DNA sequences may incorporate codons facilitating the transcription and translation of messenger RNA in microbial hosts. Such sequences can be readily constructed by the methods of Alton et al., Published PCT application WO 83/04053.

In accordance with another aspect of the present invention, the DNA sequences described herein that encode polypeptides having SCF activity are valuable for providing information regarding the amino acid sequence of mammalian proteins that have previously been worthless. DNA sequences are also valuable as products useful in carrying out a wide variety of SCF syntheses

Using another method, they are useful in generating by various recombinant techniques. DNA sequences provided by the invention of novel and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacterial and yeast cells and cultured mammalian cells) and novel and appropriate methods of culturing such host cells capable of SCF expression and products derived therefrom.

The DNA sequences of the invention are also suitable material for labeled probes in the isolation of human genomic DNA encoding SCF and other and other genomes for these proteins, as well as cNDA and genomic DNA sequences of other mammalian species. DNA sequences can also be used in various alternative methods of protein synthesis (e.g., in insect cells) or in genetic therapy of a human or other mammal. The DNA sequences of the invention are believed to be useful in the development of transgenic mammalian species that can serve as eukaryotic hosts for the production of SCF and SCF products, see Palmiter et al., Science 222, 809-814 (1983).

The present invention provides a purified and isolated naturally occurring SCF (i.e., purified from natural or manufactured such that the primary, secondary and tertiary conformation and glycosidation model are identical and naturally occurring material) as well as a naturally occurring polypeptide having a structural conformation ( i.e., a continuous sequence of amino acid residues) and glycosylation sufficiently duplicate with the same naturally occurring stem cell factor and thus permitting hematopoietic biological activity of naturally occurring SCF. Such polypeptides include derivatives and analogs.

In a preferred embodiment, the SCF is characterized as an expression product in a prokaryotic or eukaryotic host (i.e., cultured bacteria, yeast, higher plant cells, insect or mammalian cells) of exogenous DNA sequences obtained by genomic or cDNA cloning or gene synthesis. In a preferred embodiment, the SCF is a recombinant SCF. Expression products usually in yeast (e.g., Saccharomycees cerevisiae) or prokaryotic (e.g., E. coli) host cells are free of association with other mammalian proteins. Expression products in vertebrate cells (e.g., non-human mammals (e.g., COS or CHO)) and birds are free of association with any human proteins. Depending on the host used, the polypeptides of the invention may be glycosylated by mammalian or other eukaryotic carbohydrates or may be non-glycosylated. The host cell may be altered using techniques such as those described by Lee et al., J. Biol. Chem. 264, 13848 (1989), the disclosure of which is incorporated herein by reference. The polypeptides of the invention may also contain an initiating methionine amino acid residue (at position 1).

In addition to the naturally occurring allelic forms of SCF, the present invention also includes other SCF products, such as SCF polypeptide analogs. Such analogs include SCF fragments. The procedures of the aforementioned published application of Alton et al. (WO 83/04053), it is possible to readily shape and produce coding genes for microbial conformations that match and position expression of polypeptides having primarily different from those specified herein at a ratio of one or more residues (e.g.

substitution by terminal and intermediate addition and deletion).

Alternatively, modification of cDNA and genomic genes can be readily achieved by well-known site-directed mutagenesis techniques and used to generate SCF analogs and derivatives.

Such products have at least one of the biological properties of SCF, but may differ in others. By way of example, the products of the invention include those that are truncated e.g. deletions, or those that are more stable to hydrolysis (and may therefore have more pronounced or longer lasting effects than naturally occurring); or which are altered by the deletion or addition of one or more potential sites for O-glycosylation and / or N-glycosylation, or which have one or more cysteine residues deleted or replaced, for example, by alanine or serine residues and potentially more readily isolated in active form from microbial systems ; or having a single phenylalanine protein or polypeptide or multiple tyrosine residues replaced and bind more or less readily to the target cell receptors. Also included are fragments duplicating only a portion of the contiguous amino acid sequence or secondary SCF conformation;

fragments may have one SCF property (e.g., receptor binding) but not further (e.g., early hematopoietic cell growth activity). Remarkably, activity is not necessary for one or more of the products of the invention to have therapeutic ability (see Weiland et al., Blut, 44, 173-175 (1982)) or ability in a context other than the assay. SCF antagonism. Competitive antagonists can be fully utilized, for example in cases of SCF overproduction or in cases of human leukemia where malignant cells express too many SCF receptors, as indicated by overexpression of SCF receptor in leukoplasts (Example 13).

For the utility of the polypeptide analogs of the invention, their immunological properties are disclosed for synthetic peptides that substantially duplicate the amino acid sequence existing in naturally occurring proteins, glycoproteins and nucleoproteins. More specifically, the relatively small molecular weight polypeptides may participate in an immune response that is similar to the course and extent of immune responses of physiologically important proteins, such as viral antigens, polypeptide hormones, and the like. A number of immune responses of such polypeptides stimulate the formation of specific antibodies in immunologically active animals (Lerner et al. Cell, 23, 309-310 (1981); Ross et al., Nature, 294, 654-656 (1981); Walter et al., Proc Natl Acad Sci USA 77: 5197-5200 (1980); Lerner et al., Proc Natl Acad Sci USA,

78, 3403-3407 (1981); Walter et al., Proc. Natl. Acad. Sci. US

78, 4882-4886 (1981); Wong et al., Proc. Natl. Acad. Sci. US

79, 5322-5326 (1982); Baron et al., Cell, 28, 395-404 (1982);

Dressman et al., Nature, 295, 185-160 (1982); and Lerner,

Scientific American, 248, 66-74 (1983)). See also Kaiser et al., Science, 223, 249.255 (1984)) for the biological and immunological properties of synthetic peptides that approximately have secondary structures of peptide hormones but do not have their primary structural conformations.

The present invention also encompasses herein a class of polypeptides encoded by portions of DNA complementary to a protein coding strand of human cDNA or genomic DNA sequence SCF, i.e. complementary inverted proteins, as described by Tramontan et al. (Nucleic Acid. Res., 12, 5049-5059 (1984)).

The SCF can be purified by techniques known in the art. The present invention includes a method of purifying SCF from SCF material, such as conditioned media or human urine, serum. The method comprises one or more of the following steps: subjecting a material containing SCF ion-exchange chromatography (either cation exchange or anion exchange chromatography), the processing, the SCF containing material separation by liquid chromatography, reverse-phase, for example comprising the immobilized C ₄ or C _g resin; immobilized lactin chromatography, e.g. by binding SCF to immobilized lactin and eluting with the sugar that is candidate for such binding. The details of these methods will be apparent from the descriptions given in Examples 1, 10 and 11 for SCF purification. The techniques described in Example 2 of Lai et al. U.S. Pat. No. 4,667,016, which is incorporated herein by reference, is also suitable for purifying stem cell factor.

SCF isoforms are isolated using standard techniques, such as those disclosed in U.S. Pat. no. No. 421444, entitled Erythropoietin Isoforms, filed October 13, 1989, which is incorporated herein by reference.

The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the polypeptide product of the invention together with suitable diluents, preservatives, solubilizers, emulsifiers and excipients and / or carriers useful in SCF therapy. A therapeutically effective amount is an amount that provides conditions and route of administration, or lyophilized or otherwise diluents or various phosphate buffers, pH and ionic strength, gelatin to prevent adsorption of Tween 20, Tween 80, Pluronic solubilizing agents (e.g., antioxidants (e.g. e.g. acid a;

Such compositions are liquid-dried formulations and comprise (e.g., Tris-HCl, acetate, additives such as albumin or on surfaces, detergents (e.g., F68, bile salts), glycerol, polyethylene glycol), cork, sodium metabisulfite ), preservatives (e.g. Thimerosal, benzyl alcohol, parabens), swelling agents or tonicity modifiers (e.g. lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein (described in Example 12 below), metal ion complexation or incorporation material into or for particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or into liposomes, microemulsions, micelles, monolayer or multilayer vehicles, erythrocyte hosts or spheroplasts Such compositions will affect rate, physical stability, in vivo release and rate secretion of SCF in vivo. The choice of compound will depend on the physical and chemical properties of the protein having SCF activity. For example, a product derived from a membrane-bound form of SCF may require a detergent-containing composition. Controlled or sustained release formulations include lipophilic depot formulations (e.g., fatty acids, waxes, oils). Also included within the scope of the invention are particulate formulations coated with polymers (e.g., poloxamers or poloxamines) and SCF coupled to antibodies to tissue-specific receptors, ligands or antigens, or coupled to tissue-specific receptor ligands. Other embodiments of the compositions of the invention include particulate forms, a protective coating, proteinase inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral.

The invention also encompasses compositions comprising one or more hematopoietic factors such as EPO, G-CSF, GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IGF-I, or LIF (leukemia inhibiting factor).

The polypeptides of the invention may be labeled and connected to the detectable marker-substance (e.g. radio-labeled L ²⁵ I or biotinylated) in order to provide reagents useful in detection and quantification of SCF or its receptor bearing cells in solid tissue and fluid sample such as blood or urine.

Biotinylated SCF is useful in conjunction with immobilized streptavidin for the purification of leukemic bone marrow blasts in autologous bone marrow transplantation. Biotinylated SCF is useful in conjunction with immobilized streptavidin to enrich stem cells in autologous or allogeneic bone marrow transplants. SCF-linked toxin such as ricin (Uhr, Pro. Clin. Biol. Res. 288, 403-412 (1989)), diphtheria toxin (Moolten, J. Natl. Con. Inst., 55, 473-477 ( 1975)) and radioisotopes are suitable for antineoplastic therapy (Example 13) or a bone marrow transplantation conditioning regimen.

The nucleic acid products of the invention are useful when they are labeled with detectable markers, such as radioactive markers and non-isotopic markers such as biotin, and used in the hybridization process to locate the human SCF gene position and / or the position of any such gene family on the chromosome map. They are also useful for identifying DNA-level disruption of the human SCF gene and for use as gene markers to identify adjacent genes and their disruptions. The human SCF gene is encoded by chromosome 12 and the murine SCF gene is mapped to chromosome 10 at the SI locus.

SCF is useful alone or in combination with other therapy in the treatment of a number of haematopoiesis disorders. SCF can be used alone or with one or more other hematopoietic factors such as Drawbar G-CSF, GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IGF-I, or LIF in the treatment of hematopoietic diseases.

There is a group of stem cell disorders that is characterized by a reduction in bone marrow function caused by toxic, radiation or immunological damage and which can be treatable by SCF. Aplastic anemia is stem cell damage in which hematopoietic tissue is replaced by fat and pancytopenia. SCF increases hematopoietic proliferation and is useful in the treatment of aplastic anemia (Example 8B). Steel mice were used as models of human aplastic anemia (Jones, Lxp. Hematol., 11, 571-580 (1983)). promising results have been obtained using the cytokine-related, GM-CSF in the treatment of aplastic anemia * (Antin et al., Blood 70, 129a (1987)). Paroxysmal noctural hemoglobinuria is a stem cell disorder characterized by the formation of defective platelets and granulocytes as well as abnormal erythrocytes. There are many diseases. which are treatable by SCF. These include: myelofibrosis, myelosclerosis, osteoporosis, metastatic carcinoma, acute leukemia, multiple myeloma, Hodkin's disease, lymphoma, Gaucher disease, Niemen-pick disease, Letterer-Siwe disease, stubborn erythroblastic anemia, Hodgkin's congestive syndrome, Di Guglenegeal disease, Kala azar, sarcoidosis, primary spleen pancytopenia, million tuberculosis, fungal diseases, septicemia, malaria, vitamin B12 and folic acid deficiency, pyridoxine deficiency, Diamond-Blackfan anemia, hypopigmentation diseases such as vitiligo and piebaldism.

Erythroids, megacaroycytes and granulocytes stimulating the properties of SCF are shown in Examples 8B and 8C.

Growth growth in non-hematopoietic stem cells, such as primordial germ cells, germline-derived melanocytes, spinal cord commissural axons, intestinal crypt cells, mesonephric and metanephric kidney tubules, and the olfactory sites, are useful in these situations, and are useful in these situations. tissue damage. SCF is useful in the treatment of neurological damage and is a growth factor for nerve cells. SCF is useful during the in vitro fertilization process or in the treatment of infertility. SCF is useful in the treatment of intestinal damage by radiation or chemotherapy.

There are myeoloproliferative lesions of stem cells such as polycythemia vera, chronic myelogenous leukemia, myeloid metaplasia, primary thrombocythemia and acute leukemia that can be treated with SCF, anti-SCF antibodies or SCF-toxin conjugates.

There are a number of cases that document the increase in leukemia cell proliferation according to hematopoietic growth factors G-CSF, GM-CSF and IL-3 (Delwel et al., Blood, 72, 1944-1949 (1988)). Because the success of many chemotherapeutic drugs depends on the factor that neoplastic cells cycle more actively than normal cells, SCF acts alone or in combination with other factors as a growth factor for neoplastic cells and sensitizes them to the toxic effect of chemotherapeutic drugs. Overproduction of SCF receptors on leukoblasts is shown in Example 13.

A number of recombinant hematopoietic factors are under investigation for their ability to lower the leukocyte boundary as a result of chemotherapy and radiation treatment. Although these factors are very useful in this assembly, there is an early hematopoietic comportment that is damaged, especially by radiation, and is to be recreated before these late acting growth factors can exhibit optimal action. The use of SCF alone or in combination with these factors promotes the reduction or elimination of leukocyte platelet levels from chemotherapy or radiation therapy. In addition, SCF allows dose intensification in antineoplastic or radiation treatment (Example 19).

SCF is useful for the expression of early hematopoietic progenitors in asyngeneic, allogeneic or autologous bone marrow transplants. The use of hematopoietic growth factors shows a reduction in transplant replacement time (Donahue and Welte et al., J. Bone Scenario Transplantation alone or a combination of neutrophils after 872-873 (1986) (1987)). For the following three, treated with SCF alone or in a factor prior to bone marrow aspiration by leukoporesis to enhance et al., Nature, 321, Exp. Med., 165, 941-948 Marrows are used in combination: the donor is another hematopoietic s

or peripheral blood levels of cells suitable for transplantation; bone marrow is treated in vitro due to activation or increase in cell number prior to transplantation; ultimately, the recipient is treated to increase the ability to receive the donor marrow.

SCF is useful for enhancing the ability of gene therapy based on transfection (or retroviral vector infection) of hematopoietic stem cells. SCF allows the cultivation and multiplication of early hematopoietic stem cells to be transfected. Cultivation may be performed with SCF alone or in combination with IL-6, IL-3, or both. After transfection, these cells are then introduced into a bone marrow transplanted into a patient suffering from a genetic disorder (Lim, Proc. Natl. Acad. Sci., 86, 8892-8896 (1989)). Examples of genes that are useful in the treatment of genetic damage are adenosine deaminase, glucocerebrosidase, hemoglobin and cystic fibrosis.

SCF is useful in the treatment of Acquired Immunodeficiency Syndrome (AIDS), or several combined immunodeficiency states (SCIDs) alone or in combination with other factors such as IL-7 (see Example 7). An illustration of these effects is the ability of SCF treatment to increase the absolute level of circulating T-helper (CD4 ⁺ , CKT ₄ ⁺ ) lymphocytes. These cells are the primary cellular target of human immunodeficiency virus (HIV), leading to the immunodeficiency status of AIDS patients (Montagnier,

Human T-Cell Leukemia / Lymphoma Spring Harbor, New York, 369-379 in suppressing myelosuppressive AZT (Gogu Life Sciences, 45, No. 4)

Virus, ed. R.C. Gallo, Cold (1984)). In addition, SCF is useful for the effect of anti-HIV drugs such as (1989)).

SCF is useful for acute blood loss.

increase hematopoietic replacement after

In in vivo treatment, SCF is useful for stimulating the immune system to combat neoplasia (cancer). An example of therapeutic ability to directly enhance immune function by a newly cloned cytokine (IL-2) is described by Rosenberg et al., N. Eng. J. Med., 313, 1485 (1987).

Administration of SCF with agents other than one or more hematopoietic factors occurs at certain time intervals or is co-administered. Previous treatment with SCF expands the progenitor population that corresponds to end-functional hematopoietic factors such as G-CSF or EPO. The route of administration may be intravenous, intraperitoneal, subcutaneous or intramuscular.

The present invention also relates to antibodies that specifically bind the stem cell factor. Example 7, below, describes the production of polyclonal antibodies. Another application of the invention is monoclonal antibodies that specifically bind SCF (see Example 20). In contrast to conventional antibody (polyclonal) preparations, which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies are useful for improving the selectivity and specificity of diagnostic and analytical assay methods using antigen-antibody binding. They are also used to neutralize or replace SCF from serum. A second advantage of monoclonal antibodies is that they can be synthesized by hybrid cells in a culture uncontaminated by other immunoglobulins. Monoclonal antibodies can be prepared from the supernatant of cultured hybridoma cells or from ascites induced by seeding hybridoma cells intraperitoneally with the mouse. The hybridoma technique originally described by Kohler and Milstein (Eur. J. Immunol. 6, 511-519 (1976)) has been applied extensively to produce hybrid cell lines that produce high levels of monoclonal antibodies against specific antigens.

Examples of the invention

The following examples serve to illustrate the present invention and do not limit it in any way.

Example 1

Purification / characteristics of stem cell factor from media obtained from Buffalo rat liver cells

A. In vitro biological assays

1. HPP-CFC Assay

Various biological activities may be intrinsic to mammalian rat SCF as well as recombinant SCF protein. One such activity is its action on early hematopoietic cells. This activity can be measured in the High Proliferative Potential Colony Forming Cell Assay (HPP-CFC) / Zsebo et al., Supra (1988)). To assess the effect of the factor on early hematopoietic cells, the HPP-CFC assay system utilizes mouse bone marrow obtained from animals 2 days after treatment with 5-fluorouracil (5-FU). The chemotherapeutic drug 5-FU selectively depletes late hematopoietic progenitors with subsequent detection of early progenitor cells and therefore factors affecting such cells. Rat CFC is plated in the presence of CSF-1 or IL-6 in semi-solid agar culture. The agar culture contains complete McCoys medium (GIBCO), 20% fetal bovine serum, 0.3% agar, and 2 x 10 ⁵ bone marrow cells / ml. McCoy's complete medium contains the following components: 1 x McCoy medium supplemented with 0.1 mM pyruvate, 0.24 x essential amino acids, 0.24 x non-essential amino acids, 0.027% sodium bicarbonate, 0.24 x vitamins, 0.72 mM glutamine 25 μg / ml of L-serine and 12 µg / ml of L-asparagine. Bone marrow cells were obtained from Balb / c mice injected iv with 150 mg / kg 5-FU. Femurs are harvested 2 days after treatment with 5-FU mice and bone marrow is removed. The red blood cells are lysed with the red cell cell lysing agent (Becton Dickenson) prior to plating. The test substances are placed in plates with the above mixture in 30 mm dishes. Fourteen days later, colonies (> 1 mm in diameter) are counted that contain thousands of cells. This assay is used everywhere to purify SCF from rat natural cells.

In a conventional rat SCF assay, approximately 50 HPP-CFCs proliferate from 20,000 cells per plate. Rat SCF has synergistic activity on mouse bone marrow cells treated with 5-FU; HPP-CFC colonies are. do not create in the presence of themselves

factors, but in this assay or SCF and IL-6. is an active combination SCF and CSF-1 2. MC / 9 exam Other useful biological activity naturally and recombinant rat SCF is ability make proliferation from IL-4 dependent murine ointment cell line, MC / 9

(ATCC CRL 8306). MC / 9 cells are cultured with a source of IL-4 according to the ATCC CRL 8306 protocol. The medium used in the bioassay is RPMI 1640, 4% fetal bovine serum, 5 x 10 ^-5 M 2-mercaptoethanol and 1 x glutamine-pen-STREP: MC / 9 cells proliferate in response to SCF without the need for other growth factors. This proliferation is measured by first culturing the cells for 24 hours without growth factors, placing 5,000 cells in each well of a 96 well plate with a test sample for 48 hours, processing for 4 hours with 0.5 μθί ³ H-thymidine (specific activity 20 Ci / min). mmol), obtaining a solution on glass fiber filters and then measuring the specifically bound radioactivity. The assay was used to purify rat SCF-derived mammalian cells after the ACA goal filtration step, section C2 of this example. SCF causes a 4-10 fold increase in CPM from baseline.

3. CFU-GM

Action of pure mammalian SCF, both naturally obtained and recombinant, without interfering colony stimulating factors (CSFs), was found on normally unspent bone marrow. CFU-GM Assay / Broxmeyer et al., Exp. Hematol., 5, 87 (1977)) is used to assess the effect of SCF on normal marrow. Briefly, all bone marrow cells after lysis of red blood cells are plated in semi-solid agar culture containing the test substance. After 10 days colonies containing groups of more than 40 cells are counted. The agar culture can be dried on glass supports and cell morphology can be determined by specific histological staining.

In normal mouse bone marrow, mammalian SCF rats were purified with pluripotent CSF, where it stimulates colony growth, containing immature cells, neutrophils, macrophages, eosinophils and megakaryocytes, without the need for other factors. Of the 200,000 cells plated, over 100 colonies grew over 10 days. Both rat and human recombinant CF stimulate erythroid cell production in combination with EPO, see Example 9.

B. Conditioned medium

Buffalo rat (BRL) liver cells from the American Type Culture Collection (ATCC CRL 1442) were grown on microcarriers in a 20 liter perfusion culture system for SCF production. This system uses a Biolafitte fermenter (Model ICC-20) except for the sieves used to contain microcarriers and oxidation tubes. The 75-micrometer sieves are secured from clogging by microcarriers by periodic backwashing achieved by a controlled valve system and computer controlled pumps. Alternatively, each screen operates as a media feed and a starting screen. The oscillating induced flow thus ensures that the sieves are not clogged. The oxygenation is provided by winding silicone leads (50 feet length, 0.25 inch inner diameter, 0.03 inch wall). The growth medium used for BRL 3A cell culture was at least baseline medium (with Earl soles) (GIBCO), 2 mM glutamine, 3 g / l glucose, tryptose phosphate (2.95 g / l), 5% fetal bovine serum and 5% fetal calf serum. The medium obtained was identical except for the omission of serum. A 5 g / l Cytodex 2 microcarrier reactor (Pharmacia) was seeded with 3 x 10 ⁹ BRL CA cells that were grown in cylindrical vessels and transferred by trypsinization. Cells were allowed for eight days to attach and grow on microcarriers. The growth medium was perfused by the reactor depending on glucose consumption. The glucose concentration was maintained at about 1.5 g / L. After eight days, the reactor was perfused with six volumes of serum-free medium to remove most of the serum (protein concentration <50 µg / ml). The reactor was then batch operated until the glucose concentration fell below 2 g / L. From this point on, the reactor was operated at a continuous perfusion rate of about 10 L / day. The pH of the culture was maintained at 6.9 ± 0.3 by adjusting the CO ₂ flow rate. The dissolved oxygen was maintained at more than 20% by air saturation and, if necessary, by refilling with pure oxygen. The temperature was maintained at 37 ± 0.5 ° C.

Approximately 336 liters of serum-free conditioned medium was obtained from the above system and was used as a starting material for purification of native mammalian cell derived rat SCF.

C. cleaning

All purification work was performed at 4 ° C unless otherwise noted.

Anion exchange chromatography on DEAE-cellulose

Conditioned medium obtained from serum free of BRL 3A cell cultures was purified by filtration by filtration through 0.45 μτη Sartocapsules (Sartorius). Several different portions (41 L, 27 L, 39 L, 30.2 L, 37.5 L and 161 L) were separately concentrated, treated by diafiltration / buffer exchange and anion exchange chromatography on DEAE-cellulose, following the same procedure for each batch. The DEAE-cellulose pools were then combined and processed further as one portion in section C2-5 of this example. By way of illustration, processing 41 L was as follows. The filtered conditioned medium was concentrated to -700 ml using a Millipore Pellicon tangential flow ultrafiltration apparatus with a 10000 mol polysulfone membrane cassette. weight (20 ft ² total membrane area; flow rate -1095 ml / min and filtration rate 250-315 ml / min). Diafiltration / buffer exchange in preparation for anion exchange chromatography was performed by adding 500 mL of 50 mM tris-HCl, pH

7.8 to the concentrate, again concentrated to 580 ml using a tangential flow ultrafiltration device and this was repeated six times. The concentrated / diafiltered preparation was finally obtained in a volume of 700 ml. The preparation was applied to a column of anion exchange DEAE-cellulose (5 x 20.4 cm, Wahtman DE-52 resin) which was equilibrated with 50 mM tris-HCl, pH-7.8 buffer. After sample application, the column was washed with 2050 mL of tris-HCl buffer and with a salt gradient (0-300 mM NaCl in tris-HCl buffer, 4 L total volume). Fractions were collected at a flow rate of 167 ml / h. Chromatography is shown in FIG. 1. HPP-CFC colony count corresponds to biological activity in HPP-CFC assay; 100 μΐ of the indicated fractions were investigated. The fractions collected during sample application and eluted are not shown in the figure, no biological activity was detected in these fractions.

The behavior of all aliquots of the conditioned medium subjected to concentration, diafiltration / filter exchange and anion exchange chromatography was the same. Protein concentrations in all proportions, as determined by Bradford (Anal. Biochem. 72, 248-254 (1976)) with bovine serum albumin as standard, ranged from 30 to 50 µg / ml. The total volume of conditioned medium used in this preparation was 336 L.

2. ACA 54 gel filtration chromatography

Fractions having biological activity from processing on DEAE-cellulose columns for every six aliquots of conditioned medium listed above (e.g., fractions 87-114 shown in FIG.

1) were pooled (total volume 2900 mL) and concentrated to a total volume of 74 mL using shuffled Amicon and YM10 membranes. This material was applied to an ACA 54 (LKB) gel filtration column (Fig. 2) equilibrated in 50 mM tris-HCl, 50 mN NaCl, pH 7.4. Fractions of 14 ml were collected at a flow rate of 70 ml / h. Due to the inhibitory factors co-eluted with the active fractions, the peak of activity (number of HPP-CFC column) appears cleaved; however, with respect to the previous chromatograms, the activity co-elutes with the major protein peak and therefore one pool of fractions was prepared.

3. Agarose agarose chromatography with wheat germ agglutinin

The gel filtration fractions 70-112 on the ACA 54 column were combined (500 mL). The pool was split in half and each half was chromatographed on agglutin-column with wheat germ agglutinin (5 x 24.5 cm, resin from EY Laboratories, San Matco, CA; wheat germ agglutinin recognizes certain carbohydrate structures) equilibrated in 20 mM tris-HCl, pH 7.4. After sample application, the column was washed with about 2200 ml of column buffer, and the elution of bound material was then performed with a solution of 350 mM N-acetyl-D-glucosamine dissolved in column buffer, starting at fraction -210 in Fig. 2. 3. Fractions of 13.25 ml were collected at a flow rate of 122 ml / h. The course of one chromatography is shown in FIG. 3. Fractions of fractions to be assayed were dialyzed against phosphate buffered saline; 5 μΐ of the dialysed materials were placed in the MC / 9 assay (cpm values in Fig. 3) and 10 μΐ in the HPP-CFC assay (numerical values in Fig. 3). Obviously, the active material bound to the column was then eluted with N-acetyl-D-glucosamine, passing the contaminating material through the column during sample application and washing.

4. Cation-exchange fast flow S-Sepharose chromatography

Fractions 211 to 125 from wheat germ agglutinin chromatography shown in FIG. 3 and equivalent fractions from the second experiment were pooled (375 mL) and dialyzed against 25 mM sodium formate, pH 4.3. To minimize exposure to low pH, dialysis was performed over a period of 8 h, against 5 L of buffer, with four changes over an 8-hour period. At the end of this dialysis period, the sample volume was 480 ml and the pH and conductivity of the sample were close to these dialysis buffer values. During the dialysis, a precipitated product appeared in the sample. This was removed by centrifugation at 22000 g for 30 minutes and the supernatant from the centrifuged sample was applied to a S-Sepharose cation exchange column at a rapid flow rate (3.3 x 10.25 cm; Pharmacia resin) which was equilibrated in buffer - sodium formate. The flow rate was 465 ml / h and 14.2 ml fractions were collected. After application of the sample, the column was washed with 240 ml of buffer and elution of bound material was performed by applying a gradient of 0-750 mM NaCl: NaCl dissolved in column buffer (total gradient volume 2200 ml), starting at fraction -45 in Fig. 2. 4. The elution profile is shown in FIG. 4. The pooled fractions were adjusted to pH 7-7.4 by adding 200 μΐ of 0.97 M tris base, cpm in FIG. 4 refers to the results obtained in the MC / 9 bioassay; proportions of indicated fractions were dialyzed against phosphate buffered saline and were used in an amount of 20 μΐ for this assay. FIG. 4, it is evident that the bulk of the biologically active material passed through the column unbound, while the contaminating material was bound and eluted in a salt gradient.

5. Chromatography using silicon dioxide bound hydrocarbon resins

Fractions 4-40 from the S-Sepharose column of FIG. 4 were combined (540 mL). 450 ml of this pool was combined with an equal volume of buffer B (100 mM ammonium acetate, pH 6: isopropanol) and applied at a flow rate of 540 ml / h to a C ₄ column (Vydac Proteins C ₄ , 2.4 x 2 cm) equilibrated with buffer A (60 mM ammonium acetate, pH 6: isopropanol, 62.5: 37.5). After application of the sample, the flow rate was reduced to 154 ml / h and the column was washed with 200 ml of buffer A. Then a linear gradient from buffer A to buffer B (total volume 140 ml) was applied and the 9.1 ml fractions were collected. Pool proportions from S-Sepharose chromatography, starting sample from column C ₄ , pool pool and washed pool were adjusted to 40 µg / ml bovine serum albumin by adding a suitable volume of 1 mg / ml stock solution and dialyzed against phosphate buffered saline for preparation of a biological study. Similarly, 40 μΐ fractions of gradient fractions were combined with 360 μό phosphate buffered saline containing 16 μ9 bovine serum albumin and these were then dialyzed against phosphate buffered saline to prepare a biological study. These different fractions were tested by the MC / 9 assay (6.3 µΐ fractions of prepared gradient fractions, cpm in Fig. 5). The test results also show that about 75% of the activity obtained was in pooled and washed fractions and 25% gradient fractions as shown in FIG. 5 SDS-PAGE (Laemmli, 680-685 (1970)); the storage gels contain 4% acrylamide and the separation gels contain 12.5

Nature, 227, (w / v) (w / v) acrylamide; the proportions of the different fractions are shown in FIG. 6. For gel processing, the aliquots of the samples were vacuum dried and then redissolved using a 20 μenej sample treated with a buffer (non-reducing, i.e. without 2-mercaptoethanol) and boiled 5 minutes before loading onto the gel. Lanes A and B represent the starting column material (75 μΐ of 890 ml) and the material that passed through the column (75 μΐ of 880 ml), numbered marks on the left side of Fig. represent the migration positions (reduced) of markers having a molecular weight 10 ³ times greater than indicated, where the markers are phosphorylase B (M _r 97400), bovine serum albumin (M _r 66200), ovalbumin (M _r 42700), carbon anhydrase ( M _r 31000), soybean trypsin inhibitor (M _r 21500) and lysozyme (M _r 14400), lanes 4-9 represent the corresponding fractions collected during the application of the gradient (60 μΐ of 9.1 ml). The gel was silvered (Morrissey, Anal. Biochem., 117, 307-310 (1981)). A comparison of lanes A and B shows that the bulk of the dyed material passed through the column. The stained material in fractions 4-6 in the region above and below the M _r 31000 standard position corresponds to the biological activity detected in the gradient fractions (Fig. 5) and represents the biologically active material. It should be noted that this material is visualized in lanes 4-6 but not in lanes A and / or B because it was volume (0.66% of the total volume for lanes A and a much larger fraction of 4-6 versus total fractions). 0.0084% B) used for processing.

Fractions 4-6 from this column were pooled.

As mentioned above, approximately 75% of the activity obtained was passed through a C ₄ column; 5. This material was rechromatographed using a C ₄ resin substantially as described above with tir. except that larger columns (1.4 x 7.8 cm) and a lower flow rate (50-60 ml / h) were used. Approximately 50% of the activity obtained was in the flow rate and 50% in the gradient fractions showing a similar course on SDS-PAGE as the active gradient fractions in FIG. 6. Combine the active fractions (29 mL).

An analytical C ₄ column was run essentially as above and the fractions were tested by both assays. As shown in FIG. 7, the fractions from the analytical column co-eluted with both MC / 9 and HPP-CFC bioactivities. SDS-PAGE analysis (Fig. 8) reveals the presence of M _r -31000 protein in fractions from the column that contain biological activity in both assays.

The active material in the second (relatively small) activity peak, as seen in the S-Sepharose chromatography (i.e., Figure 4, fractions 62-72, earlier fractions in the salt gradient) was also purified by C ₄ chromatography. It exhibited the same SDS-PAGE behavior and had the same N-terminal amino acid sequences (see Example 20) as the material obtained by C ₄ chromatography on the fractions which passed through S-Sepharose.

6. Summary of cleaning

A summary of the purification steps described in steps 1-5 above is given in Table 2.

Table 2

Summary of purified mammalian SCF

degree volume overall protein (Ml) (mg) ⁵ conditioned medium 335.700 13,475 DEAE cellulose ¹ 2,900 2,164 ACA-54 550 1,513 wheat agglutinin 375 431 germ-agarose ² S-Sepharose 540 ⁴ 10 C ₄ resin ³ 57.3 0,25-0,40 ⁶

¹ The values given are a summary of values from the various batches described in the text.

² As described above, in the example, the precipitated material, which occurs during dialysis of the sample in the preparation of the S-Sepharose chromatography was removed by centrifugation. The centrifuged sample (480 ml) contained 264 mg of total protein.

³ Combination of the active gradient fractions from the two experiments

C ₄ columns in sequence as described.

⁴ Only 450 ml of this material was used for the next step (this example, above).

^{5 As} determined by the method of Bradford (supra, 1976) unless otherwise indicated.

⁶ Evaluated by silver coating intensity after SDS-PAGE and amino acid analysis as described in Section K of the Example

Second

D. SDS-PAGE and glycosidase treatment

The SDS-PAGE of the combined fraction gradient from two large-scale experiments on the C ₄ column is shown in FIG. 9. Sixty µΐ pool from the first C ₄ column was used (lane 1) and 40 µΐ pool from the second C column (lane 2) in the experiment. These gel pathways were silvered. The molecular weight markers were as described in Figure 6. As noted, the material migrating above and below the M _r 31000 marker position represents a biologically active material; the apparent heterogeneity is mostly due to heterogeneity in glycosylation.

To characterize glycosylation, the purified material was iodinated with ¹²⁵ L, treated with various glycosidases, and analyzed by SDS-PAGE (reducing conditions) by autoradiography. The results are shown in FIG. 9. Lanes 3 and 9, ¹²⁵ I-labeled material without any glycosidase treatment. Lanes 4-8 are ¹²⁵ I-labeled glycosidase-treated material as follows. Lane 5 by neuraminidase and O-glycanase. Lane 6 with N-glycanase. Lane 7 by neuraminidase and N-glycanase. Lane 8 with neuraminidase, O-glycanase and N-glycanase. Conditions were 5 mM 3 - [(cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 33 mM 2-mercaptoethanol, 10 mM tris-HCl, pH 7-7.2 for 3 h at 37 ° C. Neuraminidase (from Arthrobacter ureafaciens, Calbiochem) was used at a final concentration of 0.23 units / ml. O-Glycanase (Genzym, endo-alpha-N-acetyl-galactosaminidase) was used at a concentration of 45 milliunits / ml. N-Glycanase (Genzym, peptide; N-glycosidase F; peptide-N ⁴ - (N-acetyl-beta-glucosaminyl) asparagine amidase) was used at a concentration of 10 units / ml.

The same results as in FIG. 9 were obtained by treating unlabeled purified SCF glycosidase and visualizing the product by silvering after SDS-PAGE.

Occasionally, various control incubations were performed. These include: incubation in a suitable buffer, but without glycosidase, to verify that the results were obtained by treatment with the added glycosidase preparation; incubation with a glycosylated protein (i.e., a glycosylated recombinant human erythropoietin) known as a substrate for glycosidase, to verify that the glycosidase enzyme used was active; and incubating with glycosidase but without substrate, to verify that the glycosidase itself does not form bands on the visualized gel.

Glycosidase treatment was also performed with endo-beta-N-acetylglucosamidase (endo F; NEN Dupont) and endo-beta-N-acetylglucosaminidase H (endo H, NEN Dupont), each with appropriate control incubations. Endo F treatment conditions were: var 3 in in the presence of 1% (w / v) SDS, 100 mM 2-mercaptoethanol, 100 mM EDTA, 320 mM sodium phosphate, pH 6, followed by a 3-fold dilution with Nonidet P- 40 (1.17%, v / v, final concentration), sodium phosphate (200 mM, final concentration) and endo F (7 units / ml, final concentration). Endo H treatment conditions were the same except that the SDS concentration was 0.5% (w / v) and endo H was used at 1 µg / ml. The results with endo F were the same as with N-glycanase, whereas endo H was ineffective on purified CF material.

The conclusions that can be drawn from the results are described below for glycosidase experiments. Various treatments with N-glycanase (which removes both complex and highly mannose N-cross-linked carbohydrate (Tarentino et al., Biochemistry 24, 4665-4671 (1985)), endo F (acts like N-glycanase (Elder and Alexander Sci. USA 79: 4540-4544 (1982)), endo H (removes both high mannose and certain hybrid type N-crosslinked carbohydrate (Tarentino et al., Methods Enzymol.

50C, 574-580 (1978)), neuraminidase (removes sialic acid residues) and O-glycanase (removes certain O-crosslinked carbohydrates (Lambin et al., Biochem. Soc. Trans. 12, 599-600 (1984)) confirm that: both N-crosslinked and O-crosslinked carbohydrates are present, the largest proportion of N-crosslinked carbohydrate is of a complex type and sialic acid is present, at least a part of which is in the form of O-crosslinked groups. The crosslinking can be obtained from the amino acid sequence data (Example 2) The fact that N-glycanase, endo F and N-glycanase / neuraminidase treatment can convert heterogeneous material, as evident by SDS-PAGE, to rapidly migrating forms that are more homogeneously, is consistent with the conclusion that all material represents the same polypeptide, with heterogeneity due to heterogeneity in glycosylation. treatments with various glycosylases are in the range M _r 18000-20000 relative to the molecular weight gauges used in SDS-PAGE.

Confirmation that the diffuse migrating material around plate M _r 31000 on SDS-PAGE is a biologically active material having the same polypeptide chains is due to the fact that amino acid sequence data obtained from material migrating in this region (e.g. after electrophoretic transfer and cyanogen bromide treatment) , Example 2) correspond to those demonstrated for the isolated gene that expression of recombinant DNA leads to biologically active material (Example 4).

Example 2

Amino acid sequence analysis of mammalian SCF

A. High Performance Reverse Phase Liquid Chromatography (HPLC) of purified protein

Approximately 5 µg of the SCF purified as in Example 1 (concentration = 0.117 mg / ml) was subjected to reverse phase HPLC using a C ₄ narrow column (Vydac, 300 A bore, 2 mm x 15 cm). The protein was eluted with a linear gradient of 97% mobile phase A (0.1% acid · trifluoroacetic) / 3% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) to 30% mobile phase A / 70% mobile phase B over 70 min followed by isocratic elution for an additional 10 min at a flow rate of 0.2 mL per min. After reading the blank chromatogram of the buffer, the SCF appears as a single symmetrical band (peak) with a retention time of 70.05 min as shown in FIG. 10. Under these conditions, no major peak of the contaminating protein was detected.

B. Sequencing of electrophoretically transferred protein bands

SCF purified as in Example 1 (0.5-1.0 nmol) was treated as follows with N-glycanase, an enzyme that specifically cleaves Asn-crosslinked carbohydrate groups covalently attached to proteins (see Example 1D). Six ml of pooled material from fractions 4-6 of the C ₄ column in FIG. 5 is dried under vacuum. Then, 150 μΐ 14.25 mM CHAPS, 100 mM 2-mercaptoethanol, 335 mM sodium phosphate, pH 8.6 are added and incubated for 95 minutes at 37 ° C. Add 300 μΐ 74 mM sodium phosphate, 15 units / ml N-glycanase, pH 8.6 and continue incubation for 19 h. The sample is then run on a 9-18% SDS-polyacrylamide gradient gel (0.7 mm thickness, 20x20 cm). The protein bands in the gel are electrophoretically transferred to polyvinyldifluoride (PVDF, Milipore Corp.) using 10 mM Caps buffer (pH 10.5) at a constant current of 0.5 Amp for 1 h (Matsudaira, J. Biol. Chem., 261, 10035-10038 (1987)). The transferred protein bands are visualized by Coomasie Blue. The bands are present at M _r -29000-33000 and M _r -26000, i.e. deglycosylation was only partial (Example 1D, Fig. 9); the former represents uncleaved material and the latter represents material from which the N-crosslinked carbohydrate is removed. The bands were cut and directly inserted (40% for M _r 29000-33000 protein and 80% for M _r 26000 protein) per protein sequencer (Applied Biosystems Inc., Model 477). Protein sequence analysis was performed using the manufacturer's recommended programs (Hewick et al., J. Biol. Chem.,

256 7990-7997)) and the released fenyltiohydantionylaminokyseliny analyzed on-line using a micro-drilling _Ig C by reverse phase HPLC. Both bars do not give signals for 20-28 sequence cycles, confirming that they are both non-sequential by the Edman chemistry method. The baseline level of each sequencing was between 1-7 pmol, being far lower than the amount of protein present in the band. These data confirm that the protein in the bands was N-terminally blocked.

C. In situ CNBr digestion of electrophoretically transferred protein and sequencing

To confirm that the protein was actually blocked, the membrane was removed from the sequencer (part B) and digestion of the cyanogen bromide in situ (CNBr 5%, w / v) in 70% formic acid for 1 hour at 45 ° C (followed by drying and sequence analysis). Strong sequence signals representing internal peptides obtained from CNBr cleavage of the methionyl peptide bond were detected.

Both elements provide identical mixed sequence signals listed below in the first five cycles.

Identified amino acids

cycle 1: Asp, Glu, Val, Ile, Leu cycle 2: Asp, Thr, Glu, Ala. for, cycle 3: Asn, Ser. His, for, Leu cycle 4: Asp, Asn, Ala. for, Leu cycle 5: Ser. Tyr, for

Both lanes also provide similar signals for up to 20 cycles. Initial yields were 4C-115 pmol for the M _r 26000 lane and 40-150 pmol for the M _r 29000-33000 lane. These values are comparable to the original molar amount of protein inserted into the sequencer. The results confirm that the protein bands corresponding to SCF contain a blocked N-terminus. The procedures used to obtain the necessary sequence information for N-terminally blocked proteins include: a) unblocking the N-terminus (see section D) and b) recovering peptides by internal digestion with CNBr (see section E), trypsin (see section F) and Staphylococcus aureus (strain V-8) protease (Glu-C) (see section G). Sequence analysis can be performed after removal of the blocked N-terminal amino acid or after isolation of peptide fragments. The examples are described in detail below.

D. Sequence analysis of stem cell factor BRL after treatment with pyroglutamic acid aminopeptidase

It was difficult to predict the chemical nature of the blocking group present at the amino terminus of the SCF. Blocking may be post-translational in vivo (F. Wold, Ann. Rev. Biochem., 50, 783-814 (1981)) or may arise in vitro during purification. Usually, two post-translational modifications are most commonly observed. Acetylation of certain N-terminal amino acids such as Ala, Ser, etc., catalyzed by Ν-α-acetyltransferase, may occur. This can be confirmed by isolation and analysis by mass spectrometry of the N-terminally blocked peptide. If the amino terminus of the peptide is glutamine, deamidation of its gamma-amide may take place. Cyclization involving gamma carboxylate and the free N-terminus may then proceed to form pyroglutamate. The enzyme pyroglutamate aminopeptidase can be used to detect pyroglutamate. This enzyme removes the pyroglutamate residue cleaving the free amino terminus of the second amino acid. Edman chemistry may be used for sequencing.

SCF (purified as in Example 1, 400 pmol) in 50 mM sodium phosphate buffer (pH 7.6, containing dithiothreitol and EDTA) was incubated with 1.5 units pyroglutamic acid aminopeptidase veal liver (pE-AP) for 16 h at 37 ° C. C. After the reaction, the mixture was directly inserted into a protein sequencer. Major sequences were identified after 46 cycles. The initial yield was about 40% and the repeated yield was 94.2%. The N-terminal SCF sequences comprising the N-terminal pyroglutamic acid is: PE-AP pyroGlu-Glu-Ile-Cys-Arg-Asn-Pro-Vla-Thr-Asp-Asn-Val-Lys-Asp20 cleavage site

Ile-Thr-Lys-Leu-Val-Ala-Asn-Leu-Pro-Asn-Asp-Tyr-Met-Ile-Thr

Leu-Asn-Tyr-Val-Ala-Gly-Met-Asp-Val-Leu-Pro-Ser-His-xxx-TrpLeu-Arg-Asp -......

xxx, at position 43 not determined

These results demonstrate that the SCF contains pyroglutamic acid at its N-terminus.

E. Isolation and sequence analysis of CNBr peptides

The SCF purified as in Example 1 (20-28 µg), (1.0-1.5 nmol) was treated with N-glycanase as described in Example 1. In this case, the conversion to M _r 26000 was complete. The sample was dried and digested with CnBr in 70% formic acid (5% for 18 h at room temperature). After digestion, the material was diluted with water, dried and redissolved in 0.1% trifluoroacetic acid. CNBr peptides were separated in reverse phase HPLC using a C ₄ narrowbore column under elution conditions as described in section A of this example. Some major peptide fractions were isolated and sequenced and the results are summarized as follows:

Peptide Retention Sequence Time (min)

CB-4

CB-6 ¹

15.5

22.1

L-P-P - a. I-T-L-N-Y-V-A-G- (M)

CB-B

CB-10

28.0

30.1

b. V-A-S-D-T-S-D-C-V-L-S-_-L-GP-E-K-D-S-R-V-S-V - (_) - K --- D-V-L-P-S-H-C-W-L-R-D

CB-15 ² 43.0 • EE — NA — P — K — N — VKESLKK — PT- R- (N) -FTPEEFFSIFD ³ -RS- IDA ------ CB-14 37.3 and CB-16 both peptides contain the sequence CB-15

^{1 The} amino acids at positions 12, 13 and 25 were not detected. Peptide b was not sequenced at the end.

² (N) in CB-15 was not detected, was estimated as a potential N-glycosylation site. The peptide was not sequenced at the end.

³ Marked site where Asn can be converted to Asp by N-glycanase removal of N-linked sugar.

⁴ The following letter code was used: A Ala, C Cys, D Asp, E

Glu, F Phe, G Gly, H His, I il, K Lys, L Leu, M Met, N Asn,

P Pro, Q Gln, R Arg, S Ser, T Thr, V Val, W Trp, and Y Tyr.

F. Isolation and sequencing of stem cell factor BRL as tryptic fragments

The SCF purified as in Example 1 (20 μg in 150 μΐ 0.1 M ammonium bicarbonate) was digested with 1 μg trypsin at 37 ° C for 3.5 h. The material was immediately transferred to narrowbore c ₄ HPLC using elution conditions as described in Section A of this Example. All eluted peptide peaks had retention times from uncleaved SCF (section A). Sequence analysis of isolated peptides is provided below:

peptide retention time (min) sequence T-L 7.1 E-S-L-K-K-P-E-T-R T-2 ¹ 28.1 V-S-V - (_) - K T-3 32.4 I-V-D-D-L-V-A-A-M-E-E-N-A-P-K T-4 ² 40.0 N-F-T-P-E-E-F-F-S-I-F - (_) - R T-5 ³ 46,4 a. L-W-A-N-L-P-N-D-Y-M-I-T-L-N-Y-V-A-G-

M-D-W-L-P-S-H-C-W-L-R

b. SIDAFKDFMVASDTSDC-VL-S - (_) - (_) - LC --- T-7 ⁴ 72.8 'ESLKKPETR- (N) -FTPEEF-FS-IF - (_) - R

T-8 73.6 ESLKKPETRNFTPEEFFS-IF-DR ¹ Amino acid at position 4 not labeled ² Amino acid at position 12 not determined ³ Amino acids at positions 20 and 21 in 6 peptide T-5 were not identified, were estimated as sugar O-attachment sites .

⁴ The amino acid at position 10 was not detected, was considered to be Asn with respect to the potential N-linked glycosylation site. The amino acid at position 21 was not detected.

G. Isolation and sequencing of stem cell factor BRL peptides after cleavage of S. aureus Glu-C protease

The SCF purified as in Example 1 (20 μg in 150 μΐ 0.1 M ammonium bicarbonate) was subjected to Glu-c protease digestion at a protease-substrate ratio of 1:20. The digestion was carried out at 37 ° C for 18 hours. The material was immediately separated by marrowbore C ₄ reverse phase HPLC. Five major peptide fractions were separated and sequenced:

Peptide Retention Sequence ⁴ Time (min)

5.1

27.7

46.3

71.0

N-A-P-A-N-V-K-E

S-R-V-S- (_) -K-P-F-M-L-P-P-V-A- (A) No Sequence Detected S-L-K-K-P-E-T-R-N-F-T-P-E-E

72.6 (N) - R - S - I - D - A - F - K - D - F - M - V - A - S - D

SLKKPETRNFTPEEFFSIF (N) -RSIDAFKDFMVASDTSD ¹ The amino acid at position 6 of the S-2 peptide has not been determined, this could be the point of attachment of the O-linked sugar. However, at position 16, the S-2 peptide was detected in low yield.

² The peptide S-3 may be an N-terminally blocked peptide derived from the N-terminus of SCF.

³ N in brackets indicates the potential attachment point of N-linked sugar.

H. Sequence analysis of stem cell factor BRL after BNPS-scatol digestion

4Ί

SCF (2 μ9) in 10 mM ammonium bicarbonate was completely dried by vacuum centrifugation and then redissolved in 100 μΐ glacial acetic acid. 10- to 20-fold molar excess

BNPS-skatol was added to the solution and the mixture was incubated at 50 ° C for 60 min. The reaction mixture was then dried by vacuum centrifugation. The dried residue was extracted with 100 μΐ of water and again with 50 μΐ of water. The combined extracts were then subjected to the sequence analysis described above. The following sequence was detected:

10 Leu-Arg-Asp-Met-Val-Thr-His-Leu-Ser-Val-Ser-Leu-Thr-Thr-Leu-Leu

Asp-Lys-Phe-Ser-Asn-Ser-Ile-Glu-Gly-Leu-Ser- (Asn) -Tyr-Ser-Ile-Ile

Asp-Lys-Leu-Gly-Ile-Val-Asp --- Position 28+ was not positively determined, was determined to be Asn as a potential N-linked glycosylation site.

An aliquot of the SCF protein (500 pmol) was transferred to 10 mM sodium acetate, pH 4.0 (total volume 90 μΐ), and Brij-35 was added at a concentration of 0.05% (w / v). The 5 μΐ fraction was collected for protein quantification. Forty μΐ of the sample was diluted to 100 μΐ with the buffer described above. Carboxypeptidase P (from Penicillium janthinellum) was added at an enzyme-substrate ratio of 1: 200. Cleavage was performed at 25 ° C and 20 µL aliquots were taken at 0, 15, 30, 60 and 120 min. cleavage was terminated at each time by the addition of trifluoroacetic acid at a total concentration of 5%. The samples were dried and the liberated amino acids were derivatized by reaction with Dabsyl chloride (dimethylaminoazobenzenesulfonyl chloride) in 0.2 M NaHCO ₃ (pH 9.0) at 70 ° C for 12 min (Chan et al., Methods Enzymol., 90, 41-48 ( 1983)). Derivatized amino acids (one-sixth of each sample) were analyzed by reversed phase narrowbore HPLC with modification of the procedure of Chang et al., (Techniques in Crotein).

Chemistry, T. Hugli Ed. , Acad. Press, NY (1989) p. 305-311). A quantitative composition corresponding to a certain time was obtained by comparing the derivatized amino acid standards (1 pmol). At time 0, contaminating glycine was detected. Alanine was the only amino acid that increased with incubation time. After 2 hours incubation, Ala was detected in a total amount of 25 pmol, equivalent to 0.66 moles of Ala released per mole of protein. The result indicates that the native mammalian SCF molecules of Ala at its carboxyl terminus are consistent with the sequence analysis of the C-terminal peptide, S-2, which contains the C-terminal Ala. This conclusion is also consistent with the known specificity of carboxypeptidase P (Lu et al., J. Chromatog. 447, 351-364 (1988)). For example, cleavage occurs when the Pro-Val sequence is predicted. Peptide S-2 had the sequence S-R-V-S-V- (T) -K-P-P-M-L-P-P-V-A- (A) and was thought to be the C-terminal peptide of SCF (see section J of this example). The C-terminal sequence -P-V-A- (A) limits protease cleavage to alanine only. The amino acid composition of peptide S-2 indicates the presence of 1 Thr, 2 Ser, 3 Pro, 2 Ala, 3 Val, 1 Met, 1 Leu, 1 Phe, 1 Lys, and 1 Arg, a total of 16 residues. Detection of 2 Ala residues indicates that there may be two Ala residues at the C-terminus of this peptide (see table in section G). Thus, the BRL CSF is terminated with Ala 164 or Ala 165.

J. SCF Sequence

Combining the results obtained from sequence analysis of (1) intact stem cell factor after removal of its N-terminal pyroglutamic acid, (2) CNBr peptides, (3) trypsin peptides and (4) Glu-C peptidase fragments, N-terminal sequences and C- the terminal sequences were deduced (FIG. 11). The N-terminal sequence starts at pyroglutamic acid and ends at Met-48. The C-terminal sequence comprises 84/85 amino acids (positions 82 to 164/165). The sequence from position 49 to 81 was not detected in any of the isolated peptides. However, the sequence was detected in the large peptide after BNPS-scattering of BRL SCF as described in Section K of this Example. From these additional data, as well as DNA sequences obtained from rat SCF (Example

3) the N- and C-terminal sequences can be determined and the overall sequence assembled; as shown in FIG. 11. The N-terminus of the molecule is pyroglutamic acid and the C-terminus is alanine, as confirmed by digestion with pyroglutamate aminopeptidase and carboxypeptidase P.

It is apparent from the sequence data that Asn-72 is glycosylated, Asn-109 and Asn-120 are probably glycosylated in some molecules, but not in others. Asn-65 could be detected during sequence analysis and therefore can only be partially glycosylated, if any. Ser-142, Thr-143 and Thr-155, predicted from the DNA sequence, were not detected during amino acid sequence analysis and therefore could be O-linked carbohydrate attachment sites. These potential carbohydrate attachment sites are shown in FIG. 11; N-bound carbohydrate is indicated by bold letters in parentheses, O-bound carbohydrate by loose bold letters.

K. Analysis of stem cell factor BRL amino acid composition

The C ₄ column material in FIG. 7 was prepared for amino acid composition analysis by concentration and buffer exchange into 50 mM ammonium bicarbonate.

The two 70 μΐ samples were separately hydrolysed in 6 N HCl containing 0.1% phenol and 0.05% 2-mercaptoethanol at 110 ° C under vacuum for 24 hours. The hydrolysates were dried, reconstituted in sodium citrate buffer and analyzed using ion exchange chromatography (Beckman Model 6300 amino acid analyzer). The results are shown in Table 3. Using 164 amino acids (from protein sequence data) to calculate amino acid composition provides a better prediction of values than using 193 amino acids (as deduced from PCR-derived DNA sequence values, Fig. 14C).

Table 3

I

Quantitative composition of amino acids in mammalian SCF

Aminokyselny composition of amino acids mole per mole of protein ¹ predicted residues per molecule of ² (A) (B) pok. no. 1 pok. no. 2 Asx 24.46 24.26 25 28 Thr 10.37 10.43 11 12 Ser 14.52 14,30 16 24 Glx 11.44 11.37 10 10 for 10.90 10.85 9 10 Gly 5.81 6.20 4 5 Ala 8.62 8.35 7/5 8 Cys th th 4 5 wall 14.03 13.96 15 15 Met 4.05 3.99 6 7 Ile 8.31 8.33 9 10 Leu 17,02 16.97 16 19 Tyr 2.86 2.84 3 7 Phe 7.96 7.92 8 8 His 2.11 2.11 2 3 Lys 10.35 11.28 1 1 Trp th th 1 1 Arg 4.93 4.99 5 6 · overall 158 158 164/165 193 calculated molecular mass 18 424 ³

¹ Based on 158 residues from protein sequence analysis (except Cys and Trp).

² Theoretical values calculated from protein sequence data (A) or DNA sequence data (B).

³ Based on 1-164 sequences.

The inclusion of a known amount of internal standard in the amino acid composition analysis also allows quantification of the protein in the sample, obtaining a value of 0.117 mg / ml for the sample analyzed.

Example 3

Cloning genes for rat and human SCF

A. Amplification and sequencing of rat SCF cDNA fragments

Determination of the amino acid sequence of rat SCF protein fragments made it possible to label a mixed sequence of rat SCF specific oligonucleotides. Oligonucleotides have been used as hybridization probes to detect rat cDNA and genomic libraries and as primers in attempts to amplify portions of cDNA using a polymerase chain reaction (PCR) strategy (Mullis et al., Methods in Enzymol. 155, 335-350 (1987)). Oligodeoxynucleotides were synthesized by the phosphoramidite method (Beaucage et al., Tetrahedron Lett., 22, 1859-1862 (1981), McBride et al., Tetrahedron Lett., 24, 245-248 (1983)), the sequences of which are shown in FIG. 12A. The letters mean: A adenine, T thymine, C cytosine, G guanine, I inosine, * in FIG. 12A represents oligonucleotides that contain sequences detectable by restriction endonucleases. The sequences are written 5 '----> 3'.

The rat genomic library, rat liver genomic library and two BRL cDNA libraries were detected using ³² P-labeled mixed oligonucleotide probes, 219-21 and 219-22 (Fig. 21A), whose sequences were based on the amino acid sequences obtained in Example 2. None SCF clones were not obtained using standard cDNA cloning methods in this experiment (Maniatis et al., Molecular Cloning, Cold Spring Harbor 212-246 (1982)).

In an alternative experiment which resulted in the isolation of the SCF nucleic acid sequence, a PCR technique was used. In these methods, the DNA region delimited by the two primers is amplified in vitro by multiplying replication cycles catalyzed by a suitable DNA polymerase (such as Taql DNA polymerase) in the presence of deoxynucleoside triphosphates in a thermal cycle apparatus. The specificity of PCR amplification rests on two oligonucleotide primers that flank the segment to be amplified and hybridized to the upstream strand. PCR with bilateral specificity for mixtures with sufficient primer specificity, individual DNA regions in the complex are achieved using two primers with a region-specific sequence. One-sided PCR uses a region-specific primer and the other that can start at the deletion sites present on many or all of the DNA molecules in the mixture (Loh et al.,

Science, 243, 217-220 (1989)).

The DNA products of a successful PCR amplification reaction are a source of DNA sequence information (Gyllesten, Biotechniques, 7, 700-708 (1989)) and can be used to produce labeled hybrid probes having greater length and higher specificity than oligonucleotide probes. PCR products can also be labeled, with a suitable primer sequence, for cloning into plasmid vectors that allow expression of the encoded peptide product.

The basic strategy of obtaining the DNA sequence of the rat SCF cDNA is outlined in FIG. 13A. Small arrows indicate DNA sequence fractions. PCRs 90.6 and 96.2, in conjunction with DNA sequencing, were used to obtain a partial nucleic acid sequence for rat SCF cDNA. The primers used in these PCRs were mixed oligonucleotides based on the amino acid sequences shown in FIG. 11. Using sequence information obtained from PCR 90.6 a

96.2, unique sequence primers (224-27 and 224-28, Fig. 12A) were produced and used in subsequent amplification and sequence reactions. DNA containing the 5 'end of the cDNA was obtained by PCR

90.3, 96.6 and 625.1 using one-sided specific PCR. An additional DNA sequence near the C-terminus of the SCF protein was obtained by PCR

90.4. The DNA sequence for the remainder of the coding region of the rat SCF cDNA was obtained from PCR products 630.1, 630.2, 84.1 and 84.2 as described further in section C of this example. The techniques used to obtain rat SCF cDNA are described below.

RNA was prepared from BRL cells as described by Okayama et al. (Methods Enzymol. 154: 3-28 (1987)). PolyA + RNA was isolated using an oligo (dT) cellulose column as described by Jacobson in (Methods in Enzymology, Vol. 152, 254-261 (1987)).

First strand cDNA was synthesized using BRL 1 mg of poly A + RNA as template and (dT) ₁₂ _ ₁₈ as a primer according to the protocol supplied with the enzyme, Mo-MLV reverse transcriptase (Bethesda Research Laboratories). RNA chain degradation was performed using 0.14 M NaOH at 84 ° C for 10 minutes or incubation in a boiling water bath for 5 minutes. Excess ammonium acetate was added to neutralize the solution and the cDNA was first extracted with phenol / chloroform, then extracted with chloroform / z-amyl alcohol and then precipitated with ethanol. To allow the use of oligo (dC) primers in PCR with unilateral specificity, the poly (dG) end was added to the 3 'end of a portion of the first strand cDNA by terminal calf thymus transferase (Boehringer Mannheim) as previously described (Deng et al. Methods Enzymol., 100, 96-103 (1983)).

Unless indicated in the further descriptions that follow, the denaturation step in each PCR cycle was performed at 94 ° C for 1 minute and elongation was at 72 ° C for 3 or 4 minutes. The temperature and duration of the cooling varied, according to the PCR, often represent a compromise related to the estimation of the need for several different PCRs performed simultaneously. If the primer concentrations were reduced to reduce the accumulation of primary artifacts (Watson, Amplifications, 2, 56 (1989)), a longer annealing time is indicated when the PCR product concentration was high, a shorter time and a higher primer concentration was used to increase yield. An important factor in determining annealing temperature was the estimation of T _d primer-target site association (Suggs et al.,

Developmental Biology Using Purified Genes, eds. Brown D.D. and Fox

CF. (Academic, New York), p. 683-693 (1981)). The enzymes used in the amplification were obtained from one of three manufacturers: Stratagene, Promega or Perkin-Elmer Cetus. The reactants were used according to the manufacturer's recommendations. Amplification was performed in a Coy Tempcycle or Perkin-Elmer Cetus DNA thermal cycle apparatus.

Amplification of SCF cDNA fragments was usually assayed by agarose gel electrophoresis in the presence of ethidium bromide and visualized by fluorescence of the DNA strips stimulated by ultraviolet radiation. In some cases where small fragments were anticipated, PCR products were analyzed by polyacrylamide gel electrophoresis. Verification that the observed bands represent SCF cDNA fragments was obtained by observing the respective DNA bands following amplifications with one or more internally located primers. Final verification was performed by dideoxy sequencing (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)) of PCR products and comparing the putative translation products with the sequence information of the SCF peptide.

In the initial PCR oligonucleotides based Proc. Natl. Acad. Sci.

experiments were used mixed on the SCF protein sequence (Gould, USA, 86, 1934-1938 (1989)). Further described are PCR amplifications that were used to obtain DNA sequence information for the rat cDNA encoding amino acids -25 to 162.

In PCR 90.6, BRL cDNA was amplified with 4 pmol 222-11 and 223-6 in a reaction volume of 20 μΐ. A portion of the PCR product 90.6 was electrophoresed on an agarose gel, and bands of approximately the expected size were observed. One μΐ PCR of 90.6 product was amplified further with 20 pmol of primers 222-11 and 223-6 at 50 μΐ for 15 cycles, annealed at 45 ° C. A portion of this product was then subjected to 25 cycles of amplification in the presence of primers 222-11 and 219-25 (PCR 96.2), then striped to one major product by agarose gel electrophoresis. Asymmetric amplification of PCR product 96.2 with the same two primers produced templates, which were then successfully sequenced. Further selective amplification of the SCF sequences in 96.2 was performed by PCR amplification of the product in the presence of 222-11 and the fused primer 219-21. The product of this PCR was used as a template for asymmetric amplification and production of radiolabeled probes (PCR2).

To isolate the 5 'end of the rat SCF cDNA, primers containing (dC) _n sequences complementary to the poly (dG) ends of the cDNA were used as non-specific primers. PCR 90.3 contains (dC) ₁₂ (10 pmol) and 223-6 (4 pmol) as primers and BRL cDNA as template. The reaction products form very high molecular weight aggregates while remaining in the wells of agarose gel electrophoresis. One μΐ of the produced solution was further amplified in the presence of 25 pmol (dc) ₁₂ and 10 pmol 223-6 in a volume of 25 μΐ in 15 cycles, annealed at 45 ° C. Half μΐ of this product was then amplified for 25 cycles with internally primed primers 219-25 and 201-7 (PCR 96.6). The sequence 201-7 is shown in FIG. 12C. No strips were observed in agarose gel electrophoresis. 25 other PCR cycles were performed at 40 ° C, after which one prominent band was observed. Southern blotting was performed and one distinct hybridized band was then observed. An additional 20 cycles of PCR (625.1), at 45 ° C, were performed using 201-7 and the primed primer 224-27. Sequencing was performed after asymmetric amplification by PCR to obtain sequences that contained the putative signal peptide coding for the pre-SCF sequence after the apparent amino terminus. This sequence is used to designate oligonucleotide primer 227-29 containing the 5 'end of the cDNA coding region of rat SCF. Similarly, the 3'DNA sequence ending at amino acid 162 was obtained by PCR sequencing of 90.4 (see Fig. 13.A).

B. Cloning of Rat Cell Stem Factor Genomic DNA

A probe obtained from a PCR amplifying cDNA that encodes rat SCF, as described in section A above, was used to screen a library containing the rat genomic sequence (obtained from CLONTECH Laboratories, Inc., catalog number RL1022 j). The library was constructed in the bacteriophage lambda vector EMBL-3 SP6 / T7 using DNA obtained from an adult male Sprague-Dowley rat. The library, as characterized by the vendor, contains 2.3x10 ⁶ independent clones with an average insert size of 16 kb.

PCR was used to generate ³² P-labeled probes used in genomic library screening. PCR1 probes (Figure 13A) were prepared in a reaction containing 16.7 μΜ ³² P (alpha) dATP, 200 μΜ dCTP, 200 μΜ dGTP, 200 μΜ dTTP, reaction buffer recommended by Perkin Elmer Cetus, Taq polymerase (Perkin Elmer Cetus ,) at 0.05 units / ml, 0.5 μΜ 219-26, 0.05 μΜ 223-6 and 1 μΜ template 90.1, containing the target site for the two primers. The PCR 2 probe was created using the same reaction conditions, only the primer and template were altered. PCR 2 was made using 0.5 μΜ 222-11, 0.05 μΜ 219-21 and 1 μΐ PCR-derived template 96.2.

Approximately 10 ⁶ bacteriophages were plated per plate as described by Maniatis et al. (supra, 1982). The plaques were transferred to GeneScreen Plus ™ filters (22 cm x 22 cm, NEN / DuPont), which were denatured, neutralized and dried as described in the manufacturer's protocol. Two filter transfers were performed from each plate.

The filters were prehybridized in 1M NaCl, 1% SDS, 0.1% bovine serum albumin, 0.1% ficol, 0.1% polyvinylpyrrolidone (hybridization solution) for approximately 16 h at 65 ° C and stored at -20 ° C. The filters were transferred to fresh hybridization solution containing ³² P-labeled PCR 1 probe V1, 2x10 ⁵ cpm / ml and hybridized for 14 h at 65 ° C. The filters were washed in 0.9 M NaCl, 0.09 M sodium citrate, 0.1% SDS, pH 7.2 (wash solution) for 2 hours at room temperature, followed by a second wash with fresh wash solution at 65 ° C for 30 minutes. Bacteriophage clones from the plate regions corresponding to the radioactive points were interspersed from the plates and again screened by PCR 1 and PCR 2 probes.

DNA from positive clones was digested with BamHI, SphI or SstI restriction endonucleases and the resulting fragments were subcloned into pUC119 and then sequenced. The sequencing strategy of rat genomic SCF DNA is shown schematically in FIG. 14A. In this figure, the curve drawn above represents the region of rat genomic DNA that encodes SCF. Gaps in the curve indicate areas that are not sequenced. The large boxes represent the exons of the coding region of the SCF gene with the corresponding encoded amino acid above each box. The arrows represent individual regions that have been sequenced and used to assemble a consensus sequence for the rat SCF gene. The sequence for the rat SCF gene is shown in FIG. 14B.

Using the PCR 1 probe for screening the rat genomic library, clones corresponding to exons encoding amino acids 19-176 SCF were isolated. In order to obtain clones for other exons encoding regions for amino acid 19, the library was screened using oligonucleotide probes 228-30. The same filter set as previously used with PCR probe 1 was prehybridized as before and hybridized in a hybridization solution containing ³² P-labeled oligonucleotide 228-30 (0.03 picomol / ml) at 80 ° C for 16 hours. The filters were washed in the wash solution at room temperature for 30 minutes and then washed again in fresh wash solution at 45 ° C for 15 minutes. Bacteriophage clones from the plate regions corresponding to the radioactive points on the autoradiograms were removed from the plates and rescreened with a 228-30 probe. DNA from the positive clones was digested with restriction endonucleases and subcloned as described. Using the 228-30 probe, clones corresponding to exons encoding amino acids -2e to 18 were obtained.

Several attempts have been made to isolate clones corresponding to an exon containing a 5 ^1- untranslated region for amino acids -25 to -21. No clones were isolated for this region of the rat SCF gene.

C. Cloning of rat cDNA for expression in mammalian cells

Mammalian cell expression systems were devised to determine whether the active rat SCF polypeptide products are capable of being expressed and cererated by mammalian cells. Expression systems were devised to express truncated versions of rat SCF (SCF ^1-162 and SCF ^1-164 ) and protein (SCF ^1-193 ), predicted from translation of the gene sequence in FIG. 14C.

The expression vector used in these studies was a shuttle vector containing pUC119, SV40 and HTLVI sequences. The vector was labeled to allow autonomous replication in both E. coli and mammalian cells and to express the inserted exogenous DNA under the control of viral DNA sequences. This vector, designated V19.8 in E. coli DH5, is deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. (ATCC No. 68124). This vector is a derivative of pSVDM19 described in U.S. Pat. No. 4,810,663, which is incorporated herein by reference.

cDNA for rat SCF ⁹ · "^ ® ² was inserted into a plasmid vector V19.8. The cDNA sequence is shown in FIG. 14C. The cDNA used in this construct was synthesized in PCR reactions 630-1 and 630-2, as shown in FIG. 13A. These PCRs represent independent amplifications and utilize synthetic oligonucleotide primers 227-29 and 227-30. The sequence for these primers was obtained from PCR generated cDNA as described in Section A of this example. The reaction mixture, 50 μΐ volume, contains 1x reaction buffer (from Perkin Elmer Cetus kit), 250 μΜ dATP, 250 μΜ dCTP, 250 μΜ dGTP and 250 μΜ dTTP, 200 ng oligo (dT) -pressed cDNA, 1 pmol 227-29 , 1 pmol 227-30 and 2.5 Taq polymerase units (Perkin Elmer Cetus). The cDNA was amplified for 10 cycles using a denaturation temperature of 94 ° C for 1 minute and cooling for 2 minutes at 37 ° C and an elongation temperature of 72 ° C for 1 minute. After these first rounds of PCR amplification, pmol 227-29 and 10 pmol 227-30 were added. Amplifications were continued for 30 cycles under the same conditions except that the cooling temperature was changed to 55 ° C. The PCR products were digested with restriction endonucleases HindIII and SstIII. V19.8 was identical to HindIII and SstIII, and in one case the digested plasmid vector was treated with calf intestinal alkaline phosphatase, in another case the long fragment from the digestion was isolated from an agar gel. The cDNA was ligated to V19.8 using a T4 polynucleotide ligase. The ligation products were transformed into the competent E. coli strain DH5 as described (Okayama et al., Supra (1987)). DNA prepared from individual bacterial clones was sequenced by the Sanger dideoxy method. Fig. 17 shows the construction of the V19.8 SCF. These plasmids were used for transfection of mammalian cells as described in Example 4 and Example 5.

The expression vector for rat SCF ^{1 "164} was constructed using a strategy similar to that was used for the SCF ³ · · ® ^{-3 2,} in which the cDNA was synthesized using PCR amplification and subsequently inserted into V19.8. The cDNA used in the constructs was synthesized in PCR amplifications with V19.8, containing SCF ^1-162 cDNA (V19.8: SCF ^1-162 ) as template, 227-29 as a primer for the 5'-end of the gene, and 237-19 as a primer for the 3'-end of the gene. Next reaction (50 μΐ), content: 1 x reaction buffer, 250 μΜ each of dATP, dCTP, dGTP and dTTP, 2.5 units of Taq polymerase, 20 ng V19.8: SCF ^1-162 and 20 pmol of each primer. The cDNA was amplified for 35 cycles using a denaturation temperature of 94 ° C for 1 minute, a cooling temperature of 55 ° C for 2 minutes, and an elongation temperature of 72 ° C for 2 minutes. The amplification products were digested with restriction endonucleases HindIII and SstIII and inserted into V19.8. The resulting vector contains a coding region for amino acids -25 to 164 of SCF followed by a stop codon.

The 193 amino acid cDNA generated by rat SCF (rat SCF ³ · ^-3 · ^ ³ is predicted from translation of the DNA sequence in Figure 14C) was also inserted into plasmid vector V19.8 using a procedure identical to that used for rat SCF ^1-162 . The cDNA used in this combination was synthesized in the PCR reactions of 84.1 and 84.2 (FIG. 13A) using oligonucleotides 227-29 and 230-25. The two reactions represent independent amplifications starting from different RNA preparations. The sequence 227-29 was obtained by PCR reactions as described in section A of this example and the sequence for primer 230-25 was obtained from rat genomic DNA (Fig. 14B). The reaction mixture, 50 μΐ volume, contained 1x reaction buffer (from Perkin Elmer Cetus kit), 250 μΜ dATP, 250 μΜ dCTP, 250 μΜ dGTP and 250 μΜ dTTP, 200 mg oligo (dT) -pressed cDNA, 10 pmol 227-29 , 10 pmol 230-25 and 2m5 Taq polymerase units (Perkin Elmer Cetus). The cDNA was amplified in 5 cycles using a denaturation temperature of 94 ° C for 1 1/2 minutes with a cooling temperature of 50 ° C for 2 minutes and an elongation temperature of 72 ° C for 2 minutes. After these initial cycles, amplification continued under the same conditions, except that the cooling temperature was changed to 60 ° C. PCR amplification products were digested with restriction endonucleases HindIII and SstII. V19.8 DNA was digested with HindIII and SstIII, and long fragments from the digest were isolated from an agarose gel. The cDNA was ligated to V19.8 using a polynucleotide ligase. The ligation products were transformed into competitive E. coli strain DH5 and the DNA prepared from individual bacterial clones was sequenced. These plasmids were used to transfect mammalian cells in Example 4.

D. Amplification and sequencing of PCR products of human SF cDNA

Human SCF cDNA was obtained from the hepatomic HepG2 cell line (ATCC HB 8065) using PCR amplification as shown in FIG. 13B. The basic strategy was to amplify human cDNA PCR with primers whose sequences were obtained from rat SCF cDNA.

RNA was prepared as described by Maniatis et al. (supra, 1982). PolyA + RNA was prepared using oligo dT cellulose according to the manufacturer's instructions (Collaborative Research Inc.).

The first strand cDNA was prepared as described above for BRL-cDNA, except that the synthesis was initiated with the 2 μΜ oligonucleotide 228-28 shown in FIG. 12C, which contained a short random sequence at the 3 'end linked to a longer unique sequence. The unique sequence of 228-28 provides a PCR amplification target site with primer 228-29 as a non-specific primer. The human cDNA sequence related at least partially to the rat SCF sequence was amplified from the Hep62 cDNA PCR using 227-29 and 228-29 (PCR 22.7, see Figure 13B, 15 cooling cycles at 60 ° C, followed by 15 cooling cycles at 55 ° C). C). Agarose gel electrophoresis showed faint bands, only spots of apparently heterogeneous DNA. Another preferred amplification of rat SCF cDNA sequences was tested by performing a PCR with 1 μ PCR of PCR 22.7 using internally deposited rat SCF primer 222-11 and primer 228-29 (PCR 24.3, 20 cooling cycles at 55 ° C). Again, only heterogeneous spots of DNA products were observed on agarose gels. Bilateral specific amplification of PCR product 24.3 with primers 222-11 and 227-30 (PCR 25.10, 10 cycles) yielded a single band of majority product of the same size as the respective rat SCF cDNA PCR product. Sequencing of the asymmetric PCR product (PCR 33.1) by DNA using 224-24 as a sequencing primer yielded about 70 bases of human SCF sequences.

Similarly, amplification of 1 μΐ of PCR product 22.7, first with primers 224-25 and 228-29 (PCR 24.7, 20 cycles), then with primers 224-25 and 227-30 (PCR 41.11) elicited one major band with the same size as corresponding to the rat SCF product and after asymmetric amplification (PCR 42.3) gave a sequence that was highly homologous to rat SCF when 224-24 was used as a sequence primer. The unique sequences of oligonucleotides targeted to human SCF cDNA were synthesized and their sequence is shown in FIG. 12B.

To obtain the human counterpart of the rat SCF PCR generated coding sequence that was used in the expression and activity study, PCR was performed with primers 227-29 and 227-30 at 1 μΐ

PCR of 22.7 product in a reaction volume of 50 μΐ (PCR 39.1). Amplification was performed on a Coy-Tempcycler. Since the degree of difference between the human SCF cDNA and the rat SCF unique primer 227-30 was unknown, significant cooling (37 ° C) was used for the first three cycles, then cooled to 50 ° C. A prominent band of the same size (about 590 bp) as the rat homologue appeared and was further amplified by diluting a small dose of PCR product 39.1 and PCR with the same primers (PCR).

41.1). Since more than one band was observed in PCR products 41.1, another internally primed PCR was performed to determine at least a portion of its sequence before cloning. After 23 PCR cycles with primers 231-27 and 227-29 (PCR

51.2), a separate intense band was evident. Asymmetric PCR with primers 227-29 and 231-27 and sequencing confirmed the presence of the human SCF cDNA sequence. The cloning of PCR 41.1 SCF DNA into the V19.8 expression vector was performed as described for rat SCF 1-162 PCR fragments in section c. DNA from individual bacterial clones was sequenced by Sanger's didoxymethod.

E. Cloning of Genomic DNA of Human Stem Cell Factors

PCR 7 probe obtained from PCR amplification of cDNA, see FIG. 13B, was used to screen a library containing a human genomic sequence. Ribbon probe complementary to a portion of the human SCF cDNA, see above, the probe used was prepared with the material (see Fig. Amplified with primers fragment eluted from the same primers (PCR 58.1). PCR 58.1 products were for screening positive plaques.

13B). PCR product

227-29 and 227-30. agarose gel and as starting material

41.1 was on

The resulting 590 bp reamplified with diluted

1000 x 50 μΐ reaction mixture, containing 10 pmol 233-13 and amplified in 10 cycles. After addition of 10 pmol 227-30 to the reaction mixture, PCR is performed for 20 cycles. An additional 80 pmol 233-13 is added and the reaction volume is increased to 90 μΐ and PCR is continued for a further 15 cycles. The reaction products were diluted 200-fold in a 50 μΐ reaction mixture, 20 pmol 231-27 and 20 pmol 233-13 were added and PCR was performed for 35 cycles using a cooling temperature of 48 ° C in reaction 96.1. For preparation of ³² P-labeled PCR7, the same reaction conditions were used as for PCR1 production with the following differences: in a reaction volume of 50 μΐ, PCR 96.1 was diluted 100-fold, 5 pmol 231-27 was used as single primer and 45 PCR cycles was performed with denaturation at 94 ° C for 1 minute, cooled for 2 minutes at 48 ° C, and elongated for 2 minutes at 72 ° C.

Ribon probe, ribon probe 1, was a ³² P-labeled single-stranded RNA complementary to nucleotides 2-436 of the hSCF DNA sequence shown in FIG. 15B. To construct a vector for the production of this probe, PCR 41.1 (FIG. 13B) was a DNA product digested with HindIII and EcoRI and cloned into the polylinker of plasmid vector pGEM3: hSCF (Promega Madison, Wisconsin). Recombinant pGEM3: hSCF plasmid DNA was then linearized by digestion with HindIII. ³² P-labeled ribosome 1 was prepared from linearized plasmid DNA by funoff transcription with T7 RNA polymerase according to the instructions provided by Promega. The reaction mixture (3 μΐ) contained 250 ng of linearized plasmid DNA and 20 μΜ ³² p-rCTP (Catalog No: NEG-008H New England Nuclear (NEN) with no further unlabeled CTP).

The human genomic library was obtained from Stratagene (La Jolla, CA, Catalog # 946203). The library was constructed in the Fix II vector of bacteriophage Lambda, using DNA prepared from Caucasian male placenta. The library, as characterized by the supplier, contained 2.10® primary plaques ^- an average insert size greater than 15 kilobytes Approximately 10 ⁶ bacteriophages were plated per plate, as reported by Maniatis et al. (supra (1982)). The plaques were transferred to Gene Screen Plus ™ filters (22 cm ² , NFN / DuPont) according to the manufacturer's protocol. Two filter transfers were made for each plate.

The filters were prehybridized in 6XSSC (0.9 M NaCl, 0.09 M sodium citrate, pH 7.5), 1% SDS at 60 ° C. The filters were hybridized in fresh 6XSSC, 10 SDS solution containing ³² P-labeled PCR 7 probe at 2x10 ⁵ cpm / ml and hybridized for 20 h at 6262 ° C. The filters were washed in 6XSSC, 1% SDSD for 16 h at 62 ° C. Bacteriophage sections were removed from the area of the plate corresponding to the radioactive points on the autoradiograms and screened by PCR 7 and ribosome 1. The PCR screen was performed under conditions identical to the initial screening conditions. Ribosonde 1 screening was performed as follows: filters were prehybridized in 6XSSC, 1% SDS and hybridized at 62 ° C for 18 h in 0.25 M NaPO ₄ , (pH 7.5), 0.25 M NaCl, 0.001 M EDTA, 15 % formamide, 7% SDS and ribosome at 1X10® cpm / ml. The filters were washed in 6XSSC, 1% SDS for 30 min at 62 ° C followed by 1XSSC, 1% SDS for 30 min at 62 ° C. DNA from positive clones was digested with Bam HI, SphI or SstI restriction endonucleases and the resulting fragments were subcloned into pUC119 and then sequenced.

Using PCR probe 7, clones were obtained containing the exons encoding amino acids 40-176, and these clones are deposited in the ATCC (Deposit No. 40681). In order to obtain clones for other SCF exons, the human genomic library was screened with ribosome 2 and oligonucleotide probe 235-29. The library was screened to some extent as before, except that the 235-29 probe was hydrated at 37 ° C and the wash for this hybridization was 1 hour at 37 ° C and 1 hour at 44 ° C. Positive clones were re-screened with ribosome 2, ribosome 3, and oligonucleotide probes 235-29 and 236-31. Ribosondes 2 and 3 were produced according to the same procedure used for the production of ribosonde 1, with the following exceptions: a) recombinant pGEM3: hSCF plasmid DNA was linearized by restriction endonuclease PvuII (ribosonde 2) or Pstl (ribosonde 3) and b) for ribosonde synthesis 3, SP6 RNA polymerase (Promega) was used.

Figure 15A represents the strategy used for sequencing human genomic DNA. In this figure, the curve above represents the region of human genomic DNA that encodes SCF. Gaps in the curve indicate areas that were not sequenced. The large boxes represent the exons coding for a region of the SCF gene with the corresponding encoded amino acids above each box. The sequence of the human SCF gene is shown in FIG. 15B. The sequence of the human SCF cDNA obtained by the PCR technique is shown in FIG. 15C.

F. Sequence of human SCF cDNA 5 'region

Sequencing of PCR products primed by two gene-specific primers revealed the sequence of the region flanked by the 3 'end of the two primers. Unilateral PCR, as shown in Example 3A, can yield a sequence of adjacent regions. Single-PCR was used to provide the sequence of the 5'-untranslated region of human SCF cDNA.

The first strand cDNA was prepared from polyA + RNA from human bladder cancer cell line 5637 (ATCC HTB 9) using oligonucleotides 228-28 (Fig. 12C) as primers as described in Example 3D. The end of this cDNA with dG residues followed by unilateral PCR amplification using primers containing (dC) _n sequence in combination with SCF-specific primers did not yield cDNA fragments providing known sequences in the (5 ') direction.

A small amount of sequence information was obtained from PCR amplification of second strand synthesis products primed with oligonucleotide 228-28. The unprepared 5637 first strand cDNA described above (about 50 ng) and 2 pmol 228-28 were incubated with Klenow polymerase and 0.6 mM each of dATP, dCTP, dGTP and dTTP at 10-12 ° C for 30 minutes at 10 μΐ lxNick translation buffer (Maniatis et al., Molecular Cloning and Laboratory Manual, Cold Spring Harbor Laboratory (1982)). Amplification of the resulting cDNA by sequential one-sided PCR with primers 228-29 in combination with fused SCF primers (in order of use: 235-3e, 233-14, 236-31, and finally 235-29) yielded a complex mixture of products that appeared as spots on agarose gel. A significant increase in SCF cDNA fragments was indicated by an increase in band intensity, a specific product observed when comparable volumes of successful single-site PCR products were amplified with two SCF primers (227-29 and 235-29, e.g., yielded products with 150 bp). Attempts to select a preferred product size range by digesting the stains on an agarose gel and reamplifying the PCR in most cases did not provide well-defined bands containing SCF-related sequences.

One reaction, PCR 16.17, which contained only 235-29 primers, yielded a band that appears to have originated from priming 235-29 at an unknown site of the 5 'coding region next to the expected site, as shown by PvuII and PstI restriction enzyme mapping and PCR primed analysis . These products were gel purified and reamplified with primers 235-29 and sequencing was tested by the Sanger dideoxy method using the ³² P labeled primer 228-30. The resulting base sequence to label oligonucleotide 254-9 (FIG. 12B). When this 3 'directed primer was used in subsequent PCRs in combination with 5' directed SCF primers, bands of the expected size were obtained. Sanger direct sequencing of such PCR products yielded nucleotides 180-204 of the human SCF cDNA sequence; 15C. To obtain multiple sequences at the 5 'end of the hSCF cDNA, the first strand cDNA was prepared from 5637 polaA + RNA (about 300 µg), using a SCF specific primer (2 pmol 233-14) of a 16 µΐ reaction mixture containing 0.2 µl. MMLV reverse transcriptase (derived from BRL) and 500 μΜ of each dNTP. After standard phenol-chloroform and chloroform extraction and ethanol precipitation (from 1 M ammonium acetate), the nucleic acids were resuspended in 20 μΐ of water, placed in a boiling bath for 5 minutes, then quenched with terminal transferase in the presence of 8 μΜ dATP in CoCl-containing buffer. ₂ (Deng and Wu, Methods in Enzymology, 100, pp. 96-103). The product, (dA) _n end with the first strand cDNA was purified by phenol-chloroform extraction and ethanol precipitation and resuspended in 20 μΐ 10 mM Tris, pH 8.0 and 1 mM EDTA.

Enrichment and amplification of human SCF with respect to the 5 'end fragments of the cDNA of about 20 μ9 (dA) -functioned 5637 cDNA were performed as follows: initial 26 cycles of one-sided

PCR was performed in the presence of SCF-specific primer 236-31 and a primer or primer mix containing (dT) _n sequences at or near. 3 'ends, for example primer 221-12 or a mixture of primers 220-3, 220-4 and 220-11 (FIG. 12C). The products (1 μ1) ί of these PCRs were primed at approximately 370 bp and then amplified in a second PCR kit, with 221-12 and 235-29. The main product band was observed in each case by agarose gel analysis. The gel slice containing a portion of this strip was pierced from the gel with the tip of a Pasteur pipette and transferred to a dry microfuge tube. 10 μΐ of water was added and the cut-out was melted at 84 ° C in the melting block. PCR containing primers

221-12 and 235-29 (8 pmol each) at 40 μΐ are inoculated with 2 μΐ of molten, diluted gel cut. After 15 cycles, a slight diffusion band of approximately 370 bp was visible when analyzed on an agarose gel. The gel slices containing portions of this strip were removed from the gel with the tip of a Paster pipette and transferred to a small microfuge tube. 10 μΐ of water was added and the cut-out was melted at 84 ° C in the heating block. A PCR containing primers 221-12 and 235-29 (8 pmol each) at 40 μΐ was inoculated with 2 μΐ of molten, diluted gel slice. After 15 cycles, a slight diffusion band of approximately 370 bp was observed when analyzed on an agarose gel. Asymmetric PCRs were performed to generate the upper and lower ends of the chain of the sequenced templates: for each reaction, 4 μΐ of PCR reaction product and 40 pmol of either primer 221-12 or primer 235-29 in a total reaction volume of 10 μΐ were subjected to 25 PCR cycles ( 1 minute, 95 ° C, 30 seconds, 55 ° C, 40 seconds, 72 ° C). Direct sequencing of the 221-12 mixture of primed PCR products (after standard extraction and ethanol precipitation) with ³² P-labeled primer 262-13 (Fig. 12B) yielded a 5 'sequence of nucleotides 1-179 (Fig. 15C).

6. Amplification and sequencing of human genomic DNA at the site of the first stem cell factor exon coding

Screening of the human genome library with SCF by oligonucleotide probes revealed no clones containing a known portion of the first coding exon. The experiment was then initiated using a one-sided PCR technique to amplify and clone the genomic sequences flanking this exon.

Primer extension of heat-denatured human placental DNA (obtained from Sigma) was performed by DNA polymerase (Klen enzyme, long fragment, Boehringer Mannheim) using a non-SCF primer such as 228-28 or 221-11 under low conditions (low temperature) such as is 12 ° C, to facilitate priming at a wide variety of sites. Each reaction mixture was then diluted 5x into Taql DNA polymerase buffer containing Taql polymerase and 100 μΜ of each of the dNTPs and elongation of the DNA strands was performed at 72 ° C for 10 minutes. The product was then enriched in the first stem cell factor exon sequence by PCR in the presence of SCF of the first exon oligonucleotide (such as 254-9) and a suitable non-SCF primer (228-29 or 221-11). Agarose gel electrophoresis revealed that most products were short (less than 300 bp). To enrich longer species, a portion of each agarose goal strip corresponding to a length greater than 300 bp was excised and eluted electrophoretically. After ethanol precipitation and redissolution in water, the gel-purified PCR products were cloned into pGEM4 derivatives containing the SfiT site as a HindIII to Sfil fragment.

Colonies were screened with ³² P labeled first SCF exon oligonucleotide. Several positive colonies were identified and the sequences of the inserts were obtained by the Sanger method. The resulting sequence, which is upstream of the first exon across the agreed exon-intron border, to the nearest intron, is shown in FIG. 15B.

H. Amplification and sequencing of SCF cDNA coding regions from mouse, monkey and dog

The first strand cDNA was prepared from total RNA or polyA + RNA from monkey livers (obtained from Clontech) and from NIH-3T3 (mouse, ATCC CRL 1658) and D17 (dog, ATCC CCL 183) cell lines. The primer used in first strand cDNA synthesis was either a non-specific primer 228-28 or an SCF primer (227-30, 237-19, 237-20, 230-25 or 241-6). PCR amplification with primer 227-29 and one of the primers

227-30, 237-19 or 237-20 gave a fragment of the expected size that was sequenced either immediately or after cloning into a V19.8 or pGEM vector.

Additional sequences near the 5 'end of the SCF cDNA were obtained by PCR amplifications using SCF-specific primers in combination with either 254-9 or 228-9. Additional sequences at the 3 'end of the SCF coding region were obtained after PCR amplification of 230-25 primed (in this case murine) or 2416 primed (in this case monkey) cDNA with either 230-25 or 241-6 as appropriate, and 3 'by a controlled SCF primer. No bands of SCF PCR product were obtained in the same attempts to amplify the D17 cDNA. Non-specific primer 228-28 was used to primate first strand synthesis from D17 total RNA and the resulting complex product mixture was enriched in SCF related sequences by PCR and 3 'directed SCF primers such as 227-29 or 225-31 in combination with

The product mixture was digested with Sfil and cloned into pGEM4 derivatives (Promega, Madison, Wisconsin) containing the Sfil site, such as a fragment from the Sfil cleavage site to the blunt end of the fragment. The resulting heterogeneous library was screened with radiolabeled 237-20 and several positive clones were sequenced to yield the 3 'end sequence of canine SCF. The aligned amino acid sequences of the human (FIG. 42), monkey, canine, mouse, and rat SCF mature protein are shown in FIG. 16th

Known SCF amino acid sequences are highly homologous over most of their length. Identical signal peptide consensus sequences are present in the coding region of all five species. The amino acid expected at the amino terminus of the mature protein by analogy to the rat SCF is indicated in this figure by number 1. The canine cDNA sequence contains ambiguity that is manifested by the ambiguity of valine / leucine in the amino acid sequence at codon 12a. The human, monkey, rat and mouse amino acid sequence co-ligated without insertions and deletions. The canine sequence has one particular residue at position 120 as compared to other species. Human and simian differ only in one position by conservatively replacing valine (human) with alanine (simian) at position 130. The predicted SCF sequence immediately before and after the putative treatment of a site near residue 164 is highly conserved between species.

Example 4

Expression of recombinant SCF in COS-1 cells

For transient expression in COS-1 cells (ATCC CRL 1650), the vector V19.8 (Example 3C), containing rat SCF ^{1 162} and SCF ^{1 193 193} genes, was duplicated on 60mm plates / Wigler et al., Cell, 14, 725-731 (1979)). Plasmid V19.8 SCF is shown in FIG. 17. The vector without insert was also transformed for control. The tissue culture of the supernatant was harvested at various times after transfection and its biological activity was examined. Table 4 summarizes the results of the HPP-CFC bioassay and Table 5 summarizes the MC / 9 ³ H-thymidine data obtained from a typical transfection experiment. Bioassay results of supernatants from COS-1 cells transfected with the following plasmids are shown in Table 4 and 5: C-terminal truncated shape of rat C-terminal SCF at amino acid position 162 (V19.8 rats SCF ¹ " ¹⁶² ), SCF ¹ " ¹⁶² s containing a glutamic acid at position 81 (V19.8 rat SCF ^1-162 (Glu81)), and SCF ^1-162, with an alanine at position 16 (V19.8 rat SCF ^{1 "162} (Alal9)). The amino acid substitutions were the product of PCR amplification regimens carried out in the rat SCF ^1-162, as shown in Example 3. Individual clones of V19.8 rat SCF ^{1 '162,} and two were sequenced clone was found to amino acid substitution. As shown in Tables 4 and 5, recombinant rat SCF is active in the bioassays used to purify the native mammalian SCF in Example 1.

Table 4 «

HPP-CFC assay of COS-1 supernatants from rats transfected with SCF DNA

Sample colony volume of test CM (μΐ) No / 200000 cells

V19.8 (no insert 100 0

0

0

0

V19.8 rat SCF ^{1 * 3 * * 6 162} 100>50>50>50> 50

30

8

V19.8 rat SCF ^{1 162} 100 26 (G1U81) 50 10

2

0

V19.8 rat SCF ¹ “ ¹⁶² 100 41 (Ala1) 50 18

5

0

0

0

Table 5.

MC / 9 ³ H-thymidine absorption in COS-1 supernatants from rats transfected with SCF DNA

sample volume of test culture. media (μΐ) cpm V19.8 (no insert) 25 1,936 12 2,252 6 2,182 3 1,682 V19.8 SCF ¹ “ ¹⁶² 25 11,648 12 11,322 6 11,482 3 9,638 ^V19.9 SCF ^1-162 (G1U81) 25 6,220 12 5,384 6 3,692 3 1,980 V19.8 SCF ¹¹⁶² (Alal9) 25 8,396 12 6,646 6 4,566 3 3,182

Recombinant rat SCF and other factors were tested individually in human CFU-GM (Broxmeyer et al., Supra (1977)) assay that measures normal bone marrow cell proliferation and data are shown in Table 6. Results for COS-1 culture supernatants 4 days after transfection of V19.8 SCF ^{1 162} in combination with other factors are also shown in Table 6. The number of colonies is the average number of three cultures.

Recombinant rat SCF primarily has synergistic activity on normal human bone marrow in the CFU-GM assay. In the experiment in Table 6, SCF was synergist 1 of human GM-CSF, human IL-3, and human CSF-1. In another assay, synergism with G-CSF was also observed. Some proliferation of human bone marrow was observed after 14 days with rat SCF; cell groups consisted of <40 cells. The same results were obtained in natural mammalian SCF.

Table 6 Human CFU-GM assay of COS-1 supernatants from rats transfected with SCF DNA

sample colony No / 100000 cells (+ SEM) saline 0 GM-SCF 7 ± 1 G-CSF 24 ± 1 IL-3 5 ± 1 CSF-1 0 SCF ¹ “ ¹⁶² 0 GM-CSF + SCF ¹¹⁶² 29 + 6 G-CSF + SCF ¹ “ ¹⁶² 20 ± l IL-3 + SCF ¹¹⁶² 11 ± 1 CSF-1 + SCF ^1-162 4 ± 0

Example 5

Expression of recombinant SCF in Chinese Hamster Ovary cells

This example relates to stable mammalian expression systems for secretion of SCF from CHO cells (ATCC CCL 61 selected by DHFR-).

A. Recombinant rat SCF

Expression vector used for production

SCF was V19.8 (fig.

17). The selectable transformant marker was the pDSVE.1 gene.

The plasmid pDSVE.1 used on the dihydrofolate (Fig. 18) provides stable reductases in the plasmid as a pDSVE derivative constructed by digestion of pDSVE with the restriction enzyme SalI and ligation to an oligonucleotide fragment containing two oligonucleotides.

5'TCGAC CCGGA TCCCC 3 '3' G GGCCT AGGGG AGCT 5 '.

The pDSVE vector is described in US application Ser. 025344 and 152045, incorporated herein by reference. The portion of vector VI9.8 and pDSVE.1 contains long regions that are homologous, including the bacterial ColE1 origin of replication and the gene for ampicillin resistance and the SV40 origin of replication. These overlapping regions may contribute to homologous recombination during the transformation process, thus facilitating co-transformation.

Calcium phosphate co-precipitates of V19.8 SCF constructs and pDSVE.1 were prepared in the presence or absence of 10 µ9 transient mouse DNA using 1.0 or 0.1 µg pDSVE.1, which was linearized with Pvul restriction endonuclease and 10 µg V19.8 SCF, as described by Wigler et al., supra (1978). Colonies were selected for expression of the DHFR gene from pDSVE.1. Colonies capable of growth without the addition of hypoxanthine and thymidine were selected using cloning cylinders and expanded as independent cell lines. Cell supernatants from individual cell lines were tested in the MC / 9 ³ H-thymidine absorption assay. The results of the characteristic experiment are shown in Table 7.

Table 7

Assay of ³ H-thymidine uptake from stable CHO cells

Transfected DNA volume of conditioned medium to be tested cpm V19.8 SCF ¹ “ ¹⁶² 25 22,926 12 34,973 6 30,657 3 14,714 1.5 7,160 no 25 694 12 1,082 6 880 3 672 1 1,354

B. Recombinant human SCF

Expression of SCF in CHO cells was also achieved using the expression vector pDSVE a 2, which is described in US application no. No. 501904 filed Mar. 29, 1990, which is incorporated herein by reference. This vector contains a gene for the selection and amplification of clones based on the expression of the DHFR gene. The pDSRα2 SCF clone was obtained in a two-step method. V19.8 SCF was digested with a restriction enzyme and BamHI and the SCF insert was ligated into the BamHI site of pGEM3. DNA from pGEM3 SCF was digested with HindIII and SalI and ligated into HindIII and SalI digested pDSR 2. The same procedure was repeated for the human genes encoding the COOH-terminus of amino acid positions 162, 164, and 183 of the sequence shown in FIG. 15C and position 248 of the sequence shown in FIG. 42. Stabilized cell lines were stimulated with methotrexate (Shimke, in Methods in Enzymology, 151 85-104 (1987)) at 10 mM to increase expression levels of the DHFR gene and the adjacent SCF gene. Expression levels of recombinant human SCF were assayed by radioimmunoassay as in Example 7 and / or by inducing colony formation in vitro using human peripheral blood leukocytes. This assay is performed as described in Example 9 (Table 11 ') except that peripheral blood is used instead of bone marrow and incubation is performed at 20%.

0 _2, 5% C0 ₂ and 75%

Results z.typických

N ₂ in the presence of human EPO (10 J / ml). The CHO clone expressing human SCF ^1-164 was deposited

September 25, 1990

ATCC (CRL 10557) and designated HU164SCF17.

Table 8

Assay of hPBL colonies of conditioned media in stable CHO cell lines transfected with human SCF DNA

Transfected DNA Test medium (μΐ) colony count / 10 ⁵ pDSRα2 hSCF ^1-164 50 53 25 45 12.5 27 6.25 13 pDSRa2 hSCF ¹ “ ¹⁶² 10 43 5 44 2.5 31 1.25 17 0,625 21 none (CHO control) 50 4

Example 6

Expression of recombinant SCF in E. coli

A. Recombinant rat SCF

This example relates to expression in E. coli SCF polypeptides using the DNA sequence encoding (Met ^-1 ) rat SCF ^1-193 (FIG. 140). Although any suitable protein expression vector can be used using this DNA, the plasmid used was pCFM1156 (FIG. 19). This plasmid can be easily constructed from pCFM 836 (see U.S. Patent No. 4,710,473, which is incorporated herein by reference) by disruption at two endogenous NdeI restriction sites by filling the end with a T4 polymerase enzyme followed by blunt end ligation and substituting a small DNA sequence between unique Clal and KpnI restriction sites by the small oligonucleotide set forth below.

'CGATTTGATTCATGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3' 3 'TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5 ¹

Control of protein expression in the pCFM1156 plasmid is by means of a synthetic lambda P _L promoter which is itself under the control of the temperature sensitive lambda CI857 repressor gene (as provided in E. coli FMS strains (ATCC dep. No. 53911 or K12 Htrp). such that it has a DNA sequence containing optimal ribosome binding sites and an initiation codon directly of the 3 'synthetic PL promoter The unique NdeI restriction site that contains the AT6 initiation codon precedes the multi-restriction site relative to the cloning groups followed by a lambda t-oop transcriptional stop sequence.

Plasmid V19.8 SCF ^{1 '193} having ^one rat SCF ^"193 gene cloned from PCR amplified cDNA (FIG. 14C), as described in Example 3 was digested with BglII and SstII 603 bp DNA fragment was isolated. In order to provide a Met initiation codon and to restore codons for the first three amino acid residues (Gln, Glu, and ile) of the rat SCF polypeptide, a synthetic oligonucleotide linker was made

5 'TATGCAGCA

3 'ACGTCCTCTAG

5 'with NdeI and BglII sticky ends. The small oligonucleotide and rat SCF ^{1 "193} gene fragment were inserted by ligation into pCFM1156 at the unique NdeI and SstII sites of the plasmid shown in FIG. 19. The product of this reaction is an expression plasmid, pCFM1156 rat SCF ^{1 "193rd}

pCFM1156 rat SCF ³ · ^"3 · ⁹³ plasmid was transformed into competent E. coli FMS host cells. Selection of plasmid-containing cells was based on the antibiotic resistance (kanamycin) marker gene carried by the pCFM1156 vector. Plasmid DNA was isolated from cultured cells and the DNA sequence of the synthetic oligonucleotide and its association with the rat SCF gene was confirmed by DNA sequencing.

To construct the plasmid pCFM1156 rat SCF ^{1 "162} encoding (Met ^-1) rat SCF ^{1" 162} polypeptide is isolated restriction fragment from the EcoRI to SstII of V19.8 rat SCF, and introduced into the plasmid pCFM rat SCF ³ · ^"193 the unique restriction EcoRI and SstIII sites, and thus replace the coding region for the carboxyl terminus of the rat SCF gene.

227-29, and 237-19 in the plasmids pCFM1156 rat SCF ^1-165 and ³¹⁶⁴ encoding (Met ^-1) rat SCF ^1-164 were isolated by Eco RI and the amplified DNA encoding the 3 'to introduce site-directed carboxyl terminus of the SCF gene. DNA using rat pCFM1156 oligonucleotide constructs

For the construction of pCFM1156 rat SCF and (Met ^-3 ·) rat SCF ³ · ³ · ⁶⁵ , SstIII restriction fragments from the PCR end of the SCF gene and were labeled with DNA changes in the coding region: primers were performed by primers

SCF ^1-164 and 227-29 and 237-20 SCF ^1-165 ..

in pCFM1156 rat construction

B. Recombinant human SCF refers to

DNA human SCF ¹ "using the example of a ¹⁸³ (Met ^-1) SCF ^1-162, SCF containing the human template

The SCF polypeptide ^{1 "164} of the human as a template to generate the restriction oligonucleotide codon (Glu, a synthetic oligonucleotide linker

PCR primers PCR DNA that endonucleases. and codon renewal for Gly, ile, expression in the coding sequence (FIG. 15C).

^{The 1-162} gene, the amplification of human 227-29 and 237-19 were then cleaved to obtain the first four amino acid residues of the Cys) human SCF polypeptide, was prepared

E. coli human SCF (Met ^-1 ) human Plasmid V19.8 used the SCF gene used for PstI and SstII

Met initiation

5 'TATGGAAGGTATCTGCA' ACCTTCCATAG

5 'with Ndel and PstI sticky ends. A small gene fragment of the human SCF gene of the expression plasmid pCFM1156 (as both the NdeI and SstIII sites in the plasmid, as an oligolinker, and was inserted was described)

PCR derived by ligation into the unique FIG. 19th

pCFM human competent FMS E. plasmid containing marker gene bases transferred to cultured cells human SCF sequence

SCF ^{1 "164} plasmid was transformed coli host cells. The selection of cell hosts was performed for antibiotic resistance (kanamycin) was isolated from the DNA was confirmed to the na pCFM1156 vector. Plasmid DNA and DNA sequencing.

of plasmid pCFM1156 human SCF ^1-183 (FIG.

of human SCF ^1-183 15C) was isolated

To construct encoding increased anti- (Met ^"1) EcoRI to HindIII restriction fragment encoding the carboxyl terminus of the human SCF gene of pCFM human SCF ^{114" 183} (described below), a SstI to EcoRI restriction fragment encoding the amino terminus of the human SCF gene from pCFM1156 human SCF ¹¹⁶⁴ and the longer restriction fragment from pCFM1156 was also isolated. The three DNA fragments were ligated together to form the pCFM1156 human SCF ^{1 "183} plasmid which was then transformed into FM5 E. coli host cells. After selection of colonies using kanamycin resistance, plasmid DNA was isolated and the repaired DNA sequence confirmed by DNA sequencing. The pGEM human SCF ^114-183 plasmid is derived from pGEM3, which contains an EcoRI-Sph1 fragment that comprises nucleotides 609-820 of the human SCF cDNA sequence shown in FIG. 15C. The EcoRI-SphI insert in this plasmid was isolated from PCR using oligonucleotide primers 225-31 and 241-6 (Figure 12B) and PCR 22.7 (Figure 13B) as template. The sequence of primer 241-6 was based on the human genomic sequence on the 3 'side of the exon containing the codon for amino acid 176.

C. Fermentation of E. coli producing human SCF ^1-164

Fermentations for the preparation of SCF 1-164 were performed in 16-liter fermenters using an E. coli 'kl2 host containing plasmid pCFM 1156 human SCF ^1-164 . The stock culture stock was maintained at -80 ° C in 17% glycerol in Luria soil. For inoculum production, 100 μΐ thawed stock material transferred to a 500 μΐ Luria land in a 2 L Erlenmeyer flask and allowed to grow overnight at 30 ° C on a rotary shaker (250 rpm ^"1).

To produce E. coli cell paste used as a starting material for the purification of human SCF ³ · ^-3 · ⁴ in this example, the following fermentation conditions were used.

The inoculum culture was transferred aseptically to a 16-liter fermenter containing an 8-liter batch of medium (see Table 9). The culture was cultured in a batch manner until the OD-600 culture was 3.5. At this time, a sterile nutrient medium (nutrient medium 1, Table 10) was introduced into the fermenter using a peristaltic pump to control the nutrient rate. The nutrient medium velocity was increased exponentially with time to achieve a growth rate of 0.15 h ^-1 . The temperature was maintained at 30 ° C during the growth phase. The dissolved oxygen concentration in the fermenter was automatically maintained at 50% saturation using air flow rate, mixing rate, vessel pressure and oxygen replenishment. The pH in the fermenter was automatically controlled to 7.0 using phosphoric acid and ammonium hydroxide. At OD-600 of about 30, the production phase of the fermentation was induced by raising the fermentation temperature to 42 ° C. At this time is complete feed rate 1, and were brought the addition of nutrients 2 (Table 11) at a rate of 200 ml (h. Approximately six hours after the temperature fermentor, the fermentor contents cooled to 15 ° C. The yield of SCF ¹¹⁶⁴ yielded the 30 mg The cell pellets were then collected by centrifugation in a Beckman J6-B rotor at 3000 xg for one hour, and the cell paste was stored frozen at -70 ° C.

The preferred method of producing SCF ^{1 164} is similar to the method described above except that the following modifications have been made:

1) Nutrient 1 does not start until the OD-600 culture reaches 5-6.

2) Nutrient addition rate 1 is increased more slowly, resulting in a slower growth rate (approximately 0.08).

3) Culture is induced at OD-600 20.

4) Nutrients 2 are introduced into the fermenter at a rate of 300 ml / h.

All other operations are the same as above, including media.

Using these methods the yields have been drawn SCF ^{1 "164} of approximately 35-40 mg / OD-L at OD = 25th

Table 9

Feed medium composition yeast extract glucose k ₂ hpo ₄ kh ₂ after ₄

MgSO ₄ .7H ₂ 0

NaCl

Dow P-2000, anti-foaming vitamin solution ^ trace metal solution ^c

10 ^and g / l

3.5

0.625 additive 5 ml / 8 1 ml / 1 ml / 1 ^a Unless otherwise stated, all components are intended to be g / l. trace metal solution: FeCl ₃ .6H ₂ O 27 g / l, ZnCl ₂ .4H ₂ O 2 g / l,

CaCl ₂ .6H ₂ O 2 g / l, Na ₂ MoO ₄ .2H ₂ 0 2 g / l, CuSO ₄ .5H ₂ 0 1.9 g / l, concentrated HCl, 100 mL / l.

^c Vitamin solution: riboflavin 0.42 g / l, pantothenic acid 5.4 g / l, niacin 6 g / l, pyridoxine 1.4 g / l, biotin 0.06 g / l, folic acid 0.04 g / l first Table 10 Nutrient composition yeast extract 50 ^a glucose 450 MgSO ₄ .7H ₂ O 8.6 trace metal solution ^b 10 ml / l vitamin solution ^c 10 ml / l

^and Unless otherwise stated, all components are intended as g / l. trace metal solution: FeCl ₃ .6H ₂ O 27 g / l, ZnCl ₂ .4H ₂ O 2 g / l,

CaCl ₂ .6H ₂ O 2 g / l, Na ₂ MoO ₄ .2H ₂ 0 2 g / l, CuSO ₄ .5H ₂ O 1.9 g / l, concentrated HCl, 100 mL / l.

^c Vitamin solution: riboflavin 0.42 g / l, pantothenic acid 5.4 g / l, niacin 6 g / l, pyridoxine 1.4 g / l, biotin 0.06 g / l, folic acid 0.04 g / l first

Table 11

Composition of the nutrient medium

Tryptone172 yeast extract86 glucose258 ^and All components are reported in g / l.

Example 7

SCF detection immunoassay

Radioimmunoassays (RIAs) were used to quantitatively detect SCF in the samples and were performed as follows.

The SCF preparation from BRL 3A cells purified as in Example 1 was incubated with the antiserum for two hours at 37 ° C. after two hours of incubation, the sample tubes were then cooled on ice, ¹²⁵ I-SCF was added, and the tubes were incubated at 4 ° C for at least 20 h. Each tube contained 500 μΐ of incubation mixture, containing 50 μΐ of diluted antiserum, -600000 cpm ¹²⁵ I-SCF (3.8 x ΙΟ ⁷ σρπ / μς), 5 μΐ of Trasylol and 0.400 μΐ of SCF standard, with buffer (phosphate buffered saline) 0.1% bovine serum albumin 0.05 Triton X-100, 0.025% azide) constituting the rest of the volume. Antiserum for the second assay was obtained from the blood of rabbits immunized with 50% pure natural SCF preparation from BRL 3A conditioned medium. The final dilution of antiserum in the assay was 1: 2000

The antibody bound ¹²⁵ I-SCF was precipitated by the addition of 150 μΐ Staph. A (Calbiochem). After 1 hour incubation at room temperature, the samples were centrifuged and the pellets were washed twice with 0.75 ml of 10 mM Tris-HCl pH 8.2, containing 0.15M NaCl, 2 mM EDTA and 0.05% Triton X-100. The washed pellets were measured in a gamma counter to determine the percentage of ¹²⁵ I-SCF binding. Binding counts in tubes lacking serum were subtracted from all final values for non-specific clotting correction. The characteristic RIA is shown in FIG. The percentage of inhibition of ¹²⁵ I-SCF binding that is produced by the unlabeled standard is dose dependent (FIG. 20A) and as shown in FIG. 20B, in SDS-PAGE and autoradographically assaying pellets that have been immunoprecipitated, the ¹²⁵ I-SCF protein band is competing. In FIG. 20B is lane 1 ¹²⁵ I-SCF and lanes 2, 3, 4 and 5 are immunoprecipitated ¹²⁵ I-SCF compared to 0, 2, 100 and 200 ng of SCF standard. As determined by both the decrease in antibody precipitation in the cpm observed in the RIA tubes and the decrease in the band of immunoprecipitated ¹²⁵ I-SCF protein (migrating at about M _r 31000), the polyclonal antibody recognizes an SCF standard that was purified as in Example 1.

Western methods were also applied to determine recombinant SCF expressed in E. coli, COS-1 and CHO cells. Partially purified E. coli epxrimovaný rat SCF ^{1 "162} and SCF ^1-193, SCF and human ¹¹⁶² (Examples 4 and 9), and CHO cell expressed rat SCF ^{1 '162} (Example 5), were subjected to SDS-PAGE. On subsequent electrophoresis, protein bands were transferred to 0.2 μη by nitrocellulose using a Bio-Rad Transblot at 60 V for 5 hours. Nitrocellulose filters were blocked for 4 h in PBS, pH 7.6, containing 10% goat serum and further incubated for 14 h at room temperature with a 1: 200 dilution of either rabbit pre-immune or immune serum (immunization described above). Antibody-antiserum complexes visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector laboratories) and 4-chloro-1-naphthol reagents as coloring agents.

Examples of two Western analyzes are shown in FIG. 21 and 22. On bor. 21, lanes 3 and 5 are 200 μΐ COS-1 cells produced by human SCF ¹ ” ¹⁶² , lanes 1 and 7 are 200 μΐ COS-1 cells produced by human EPO (COS-1 cells transfected with V19.8 EPO) and lane 8 is lane molecular weight markers. Lanes 1-4 were incubated with pre-immune serum and lanes 5-8 were incubated with immune serum. Immune serum specifically recognizes a diffuse band with a Mr of about 30,000 daltons from COS-1 cells producing human SCF ^{1 '162,} but not from COS-1 cells producing human EPO.

In a Western analysis of μ9 partially purified produced in E. coli, shown in FIG. 22 lanes 1 and 7 are 1 rat SCF ¹ 'preparation ¹⁹³ lanes 2 and 8 are rats SCF ^1-3 · ⁹³ produced by a COS-1 cell and purified on wheat germ agglutinin agarose. Lanes 4 and 9, the rat SCF ^{1 "162} produced by COS-1 cell-agarose purified wheat germ agglutinin, lanes 5 and 10 of the rat SCF ^1-162 produced in a CHO cell, and agarose purified wheat germ agglutinin, and lane 6 is colored molecular weight markers. Lanes 1-5 and lanes

6-10 were incubated with rabbit pre-immune and immune serum. ¼. coli produced rat SCF ^{1 "193} (lanes 1 and 7) migrates at an apparent M ^r -24,000 daltons while the COS-1 cell produced rat SCF ^1-193 (lanes 2 8) migrates in the M _r 24-36000 Daltons. This difference in molecular weight is expected because mammalian cells, but not bacteria, are capable of glycosylation. Transfection of the sequence encoding rat SCF ^{1 '162} in the COS-1 (lanes 4 and 9), or CHO cells (lanes 5 and 10), results in expression of SCF with a lower average molecular weight than that produced by transfection with SCF ^1-193 (lanes 2 and 8).

Rat SCF ^1-162 expression products from COS-1- and CHO cells are a series of bands in the apparent mole range. mass M _r between 24-36000 dalton. The heterogeneity of the expressed SCF is likely due to changes in carbohydrate content since the SCF polypeptide is glycosylated to varying degrees.

In summary, Western analyzes indicate that immune serum from rabbits immunized with natural mammalian SCF recognizes recombinant SCFs produced in E. coli, COS-1 and CHO cells, but does not recognize any bands in the control sample containing COS-1 cells produced by EPO. . As a further boost to the specificity of the SCF antiserum, preimmune serum from the same rabbit does not react with any rat or human SCF expression products.

Example 8

In vivo activity of recombinant SCF

A. Rat SCF in bone marrow transplantation

COS-1 cells transfected with V19.8 SCF ^{1 '162} in Test large scale (T175 cm ² of the container instead of the plates 60 m) as described in Example 4. Approximately 270 ml of supernatant. This supernatant was chromatographed on wheat germ agglutinin agarose and S-Sepharose in essentially the same manner as described in Example 1. Recombinant SCF was evaluated in a bone marrow transplant model based on W / W ^v mice.

Mouse W / W ^to be defective strain of macrocytic anemia (a transplant of bone marrow from radiation recipients (Russel (1964)). Stem cell beeches, leading among other things to the cells) and allows animals without the need to Science, 144, 844-846 are rising transplant red normal and co., normal donor faster than defective recipient cells.

In the following example, each group contains six mice of appropriate age. Bone marrow was obtained from normal donor mice and transplanted with W / W ⁱⁿ mice. The blood profile of the recipient is monitored at various times after transplantation, and bone marrow engraftment is determined by changes in peripheral blood cells from the recipient phenotype to donors. Conversion from recipient to donor phenotype is detected by monitoring the anterior scatter profile (FASCAN, Becton Dickenson) of red blood cells. The profile for each transplant for each animal was compared to that of the control animals of both donor and recipient at the same time. Comparison was performed using a computer program based on Kolmogorov-Smirn statistics for histogram analysis from flow systems (Young, J. Histochem. And Cytochem., 25, 935-941 (1977)). An independent qualitative indicator of engraftment is hemoglobin, detected by electrophoresis of recipient blood hemoglobin (Wong et al., Mol. And Cell. Biol., 9, 798-808 (1989)), consistent with the conclusions of Kolmogorov-Smirnov statistics.

Approximately 3 x 10 ⁵ cells were transplanted without SCF treatment (control group in Figure 23) from C56BL / 6J W / W donors ^{in the} recipient. The second group received 3 x 10 ⁵ donor cells that were treated with SCF (600 µl / ml) at 37 ° C for 20 min and injected together (pre-treated group in Fig. 23). (One SCF unit is defined as the amount that results in half the maximum stimulation in the MC / 9 bioassay). In the third group, recipient mice were injected subcutaneously (sub-Q) of approximately 400 µ SCF / day, 3 days after transplantation of 3 x 10 ⁵ donor cells (sub-Q in the group in Fig. 23). As shown in FIG. 23, in both SCF-treated groups, bone marrow is engrafted more rapidly in the untreated control. 29 days after transplantation, the SCF pre-treated group was converted to donor phenotype. These examples illustrate the suitability of SCF therapy in bone marrow transplantation.

B. In vivo Activity of Rat SCF in Steel Mice

Mutations in the SI locus cause deficiencies in hematopoietic cells, pigment cells, and germ cells. The hematopoietic defect is manifested as reduced red blood cell count (Russell, in: Al Gordon, Regulation of Hematopoiesis, Vol. I, 649-675 Appleton-Centruy-Crafts, New York (1970)), neutrophils (Ruscetti, Proc. Soc. Exp. Biol. Med., 152, 398 (1976)), monocytes (Shibata, J. Immunol. 135, 3905 (1985)), megakaryocytes (Ebbe, Exp. Hematol., 6, 201 (1978)), natural killer cells. (Clark, Immunogenetics, 12, 601, (1981)) and cell ointment (Hayashi, Dev. Biol., 109, 234 (1985)). Steel mice are pure bone marrow transplant recipients for their ability to provide stem cells (Bannerman, Prog. Hematol., 8, 131 (1973)). The SCF gene is deleted in Steel mice (S1 / S1).

Steel mice provide a sensitive in vivo model for SCF activity. Various recombinant SCF proteins were tested in Steel-Dickie (S1 / S1) mice for a long time. Six to ten week old Steel mice (WCB671-Sl / Sl ^d ) were obtained from Jackson Labs, Ber Harbor, ME. Peripheral blood was monitored by SYSMEX F-800 microcell counter (Baxter, Irvine, CA) for red blood cells, hemoglobin and platelets. Coulter Channalyzer 256 (Coulter Electronis, Marietta, GA) was used to count peripheral white blood cells (WBC).

In the experiment of FIG. 24, Steel-Dickie mice were treated with SCF 1-164 obtained from E. coli, purified as in Example 10, at a dose of 100 gg / kg / day for 30 days, followed by an additional 20 days at a dose of 30 µg / kg / day. The protein was formulated in saline for injection (Abbott Labs, North Chicago, IL) + 0.1% fetal bovine serum. Injections were given daily subcutaneously. Peripheral blood was monitored at the tail by playing 50 μΐ at the times shown in Figs. 24. Blood was collected in 3% EDTA coated syringes and transferred to EDTA powder microfuge tubes (Brinkmann, Westburry, NY). There is a significant correction of macrocyte anemia in treated animals relative to control animals, at the end of treatment the treated animals returned to the initial state of macrocyte anemia.

In the experiment shown in FIG. 25 and 26, Steel-Dickie mice were treated with various recombinant forms of SCF as described above at a dose of 100 µg / kg / day for 2 days. Two forms were obtained from E. coli obtained rat SCF, SCF ^1-1 ® ⁴ and SCF ¹¹⁹³ , as described in Example 10. Furthermore, E. coli SCF ^{1 164} , modified by the addition of polyethylene glycol (SCF ^1_164 PEG25), was also tested. ) to Example 12 also was tested on CHO derived SCF ^{1 "162} is produced as in Example 5 and purified as in Example 11. the animals were subjected to cardiac puncture, the blood being performed syringes with a 3% EDTA, which was placed in tubes with EDTA powder. Peripheral blood profiles 20 days after treatment are shown in FIG. 23 for white blood cells (WBC); and FIG. 26 for inserts. The WBC differences for the SCF group ^1-164 PEG25 are shown in FIG. 27. Absolutely increased neutrophils, monocytes, lymphocytes and platelets. The most dramatic effect is seen with SCF ¹ '' ^{I '64} PEG25.

Independent lymphocyte measurements were also performed and the data are shown in FIG. 28. The mouse equivalent of human CD4 or T helper cell marker is L3T4 (Dialynas, J. Immunol., 131, 2445 (1983)). Lyt-2 is a murine cytotoxic T cell antigen (Ledbetter, J. exp. Med., 153, 1503 (1981)). Monoclonal antibodies to these antigens were used to assess T cell subgroups in treated animals.

Blood was evaluated for T lymphocytes as follows. Two hundred microliter blood was collected from individual animals into EDTA-treated tubes. Each blood sample was lysed with sterile deionized water for 60 seconds and then treated isotonic with 10X Dulbecco's phosphate buffered saline PBS (Gibco, Grand Island, NY). The lysed blood was washed twice with 1X PBS (Gibco, Grand Island, NY) supplemented with 0.1 L fetal bovine serum (Flow Laboratory, McLean, VA) and 0.1% sodium azide. Each blood sample was placed in a 96-well round bottom dish and centrifuged. A cell pellet (containing 2-10 x 10 ⁵ cells) was resuspended with 20 microliters of L3T4 (rat anti-mouse) conjugated to phycoerythrin (PE) (Becton Dickinson, Mountain View, CA) and 20 microliters of Lyt-2 (rat anti-mouse) ) conjugated to fluorescein isothiocyanate, incubated on ice (4 ° C) for 30 minutes (Becton Dickinson). Subsequent cell incubations were washed twice in 1X PBS supplemented as above. Each blood sample was then analyzed on a FACScan cell analysis system (Becton Dickinson, Mountain View, CA). This system was standardized using standard auto-compensation procedures and Calibrite Beads (Becton

Dickinson, Mountain View, CA). These data indicate an increase in both helper T cells and cytotoxic T cells.

C. In vivo SCF activity in primates

Human SCF 1-164 expressed in E. coli (Example 6B) and purified to homogeneity as in Example 10 was tested for in vivo biological activity in normal primates. Adult baboons (Papio sp.) Were studied in three groups: untreated, n = 3, SCF 100 gg / kg / day, n = 6, and SCF 30 μg / kg / day n = 6. Treated animals received SCF subcutaneous injections once daily. Test blood samples were obtained from animals under ketamine. Whole blood, reticulocyte and platelet samples were obtained

1, 6, 11, 15, 20 and 25 days after treatment.

All animals survive and have no negative response to SCF therapy. White blood cell counts increased in animals treated with 100 µg / kg as shown in FIG. 29. A different number, obtained manually from Wright Giemsa stained peripheral blood spots, is also shown in FIG. There was an absolute increase in neutrophils, lymphocytes and monocytes. As shown in FIG. 30 at 100 μ9 / Π <9 also increased hematocytes and platelets.

Human SCF (hSCF ^1-1 ® ⁴ modified by the addition of polyethylene glycol, as in Example 12) was also tested on normal baboons at a dose of 200 µg / kg / day administered continuously by intravenous infusion and compared to unmodified protein. The animals started to receive SCF on day 0 and were treated for 28 days. The results of peripheral WBC are shown in the following table. PEG modified SCF induces an earlier increase in peripheral WBC than modified SCF.

Treatment, 200 µg / kg / day hSCF ¹ " ¹⁶⁴

Animal no. M88320 Animal no. M88129 day WBC day WBC 0 5880 0 6800 +7 10700 +7 7400 +14 12600 +14 20900 +16 22000 +21 18400 +22 31100 +23 24900 +24 28100 +29 13000 +29 9600 +30 23000 +36 6600 +37 12100 +43 5600 +44 10700 +51 7800

Treatment with 200 μ9 / λ9 / β-PEG-hSCF 1-164. • Animal no. M88350 Animal no. M89116 day WBC day WBC -7 12400 -5 7900 -2 11600 0 7400 +4 24700 +6 16400 +7 20400 +9 17100 +11 24700 +13 18700 +14 32600 +16 19400 +18 33600 + 20 27800 +21 26400 +23 20700 +25 16600 + 27 20200 +28 26900 +29 18600 +32 9200 +33 7600

Example 9

In vitro activity of recombinant human SCF cDNA of human SCF corresponding to amino acids 1-162 obtained by PCR reactions as in Example 3D was expressed in COS cells as described for rat SCF in Example 4. COS-1 supernatants were assayed on human bone as well as in mouse HPP-CFC and MC / 9 assays. Human protein was not active at the concentrations tested in both mouse assays and was active in human bone marrow. The culture conditions were as follows: human bone marrow from healthy volunteers was centrifuged over Picoli-hypaque gradients (Pharmacia) and cultured in 2.1% methylcellulose, 30% fetal calf serum, 6 x 10 ^-5 M 2-mercaptoethanol, 2 mM glutamine, ISCOVE medium (GIBCO), 20 U / ml EPO and 1 x 10 ⁵ cells / ml for 14 days in a humidified atmosphere containing 7% O ₂ , 10% CO ₂ and 83% N ₂ . The number of colonies formed with recombinant human and rat SCF CSO-1 supernatants is shown in Table 1. Only those colonies having a size of 0.2 mm or greater are counted.

Table 12

Human bone marrow colonies growth in response to SCF

Transfected plasmid CM volume colony num / 100,000 cells + st. Try. (Μΐ) V19.8 (no insert) 100 0 50 0 V19.8 human SCF ^{1 162} 100 33 ± 7 50 22 + 3 V19.8 rat SCF ¹ “ ¹⁶² 100 13 ± 1 50 10

Colonies that grew during the biweekly period are _{uve e} d in Fig. 31A (magnified 12x). The arrow indicates a typical colony. Colonies resemble mouse HPP-CFC colonies in size (0.5 mm diameter). Due to the presence of EPO, some colonies were hemoglobinized. After colony isolation and centrifugation on glass plates using Cytospin (Shandon) followed by Wright-Giemsy staining, the predominant cell type was the large nucleus cell type: cytoplasm ratio as shown in FIG. 13B (400x magnification). The arrows in FIG. 31B represent: arrow 1 - cytoplasm, arrow 2 - nucleus, arrow 3 - vacuole. Immature cells as a class are large and the cells obtained are smaller than mature (Dis et al., The Morphology of Human Blood Cells, Abbott Labs, 3 (1978)). The nuclei of early cells from the hematopoietin mature sequence are relatively large with respect to the cytoplasm. Further, the cytoplasm of the immature cells is stained with Wright-Giemsa darker than the nucleus. As the cells mature, the nucleus becomes darker than the cytoplasm. The morphology of human bone marrow cells resulting from culture with recombinant human SCF is consistent with the conclusion that the final product and intermediate of SCF action is a relatively matured hematopoietic progenitor.

Recombinant human SCF was tested by colony assay on human bone marrow agar in combination with the other growth factors described above. The results are shown in Table 13. SCF synergizes with G-CSF, GM-CSF, IL-3, and EPO to increase bone marrow proliferation for individual CSF.

Table 13

Synergism of recombinant human SCF with other factors stimulating human colonies

colony count / 10 ⁵ cells (14 days) blind attempt 0 hG-CSF 32 ± 3 hG-CSF + hSCF 74 + 1 hGM-CSF 14 ± 2 hGN-CSF + hSCF 108 ± 5 hIL-3 23 ± 1 hIL-3 + hSCF 108 ± 3 hEPO 10 ± 5 hEPO + IL-3 17 ± 1 hEPO + hSCF 86 ± 10 hSCF 0

Another activity of recombinant human SCF is the ability to induce proliferation of the human acute myelogenous leukemia (AML) cell line, KG-1 (ATCC CCL 246) in soft agar. COS-1 supernatants from transfected cells were tested in KG-1 agar cloning assay (Koeffler et al. Science, 200, 1153-1154 (1978)) essentially as described, except that cells were plated in concentration 3000 / ml. Data from three cultures are shown in Table 14.

Table 14

KG-1 cloning assay on soft agar

Transfected plasmid test volume (μΐ) colony count / 3000 cells + SD (standard offsets) V19.8 (no insert) 25 2 ± 1 V19.8 human SCF ¹ “ ¹⁶² 25 14 ± 0 12 8 ± 0 6 9 + 5 3 6 ± 4 1.5 6 ± 6 V19.8 rat SCF ^{1 162} 25 6 ± 1 human GM-CSF 50 (5 ng / L) 14 ± 5

Example 10

Purification of recombinant SCF products expressed in E. coli

Fermentation of E. coli human SCF ^{1 "3-64} was performed according to Example 6C. The obtained cells (912 g wet weight) were suspended in water in a volume of 4.6 L total and allowed to disrupt by triple pass through a laboratory homogenizer (Gaulin Model 15MR-8TBA) at 8000 psi. The pelleted cell pellets were obtained by centrifugation (17700 xg, 30 min, 4 ° C), washed once with water (resuspended and recentrifuged) and finally suspended in water to a volume of 400 ml.

A fraction of the pellets containing insoluble SCF (approximately 10-12 g SCF) was added to 3950 ml of the appropriate mixture so that the final concentration of the ingredients in the mixture was 8 M urea (ultrapure), 0.1 mM EDTA, 50 m sodium acetate, pH 6 -7; The SCF concentration was evaluated as 1.5 mg / ml. Incubation was performed at room temperature for 4 hours to dissolve the SCF. The remaining insoluble material was removed by centrifugation (17700 xg, 30 min, room temperature) to form / reoxidize the dissolved SCF, the supernatant fractions were slowly added with stirring, to 39.15 L of the appropriate mixture so that the final concentrations of the ingredients in the mixture were 2, 5 M urea (ultrapure), 0.01 mM EDTA, 5 mM sodium acetate, 50 mM Tris-HCl, pH

8.5, 1 mM glutathione, 0.02% (w / v) sodium azide. The SCF concentration was evaluated as 150 µg / ml. After 60 h at room temperature (shorter time (e.g. -20 h) is also suitable), the mixture was concentrated by passing twice through a Millipore Pellion ultrafiltration apparatus with a polysulfone mole cassette. weight 10,000 (three cassettes) (15 ft ² total surface) and then diafiltered against 7 volumes of 20 mM Tris-HCl, pH

8. The temperature during the concentration / ultrafiltration was 4 ° C, the pumping rate was 5 l / min and the filtration rate was 600 ml / min. The final volume of the obtained retentate was 26.5 L. Using SDS-PAGE, performed with or without sample reduction, it is apparent that at most (> 80%) the SCF fraction pellets are solubilized by incubation with 8 M urea and after composition / oxidation multiple SCF species (forms) are present, as seen from SDS-PAGE of non-reduced samples. The major form, which represents a properly oxidized SCF (see below), migrates with an apparent M _{r of} about 17000 (unreduced) relative to the molecular weight markers (reduced) described in FIG. 9. Other forms include material migrating with an apparent M _{r of} about 18-20000 (unreduced), believed to represent SCFs with incorrect disulfide bonds within the chain; and bands migrating with an apparent M _r in the range of 37,000 (unreduced) or greater, which are believed to represent different SCF forms having internal disulfide bonds formed in SCF polypeptide chains that are covalently linked to form dimers or long oligomers. The following steps of fractionation consist in removing the remaining E. coli contaminants and unexpected SCF homogeneity in the forum, so that SCF is purified to an obvious biologically active conformation.

The pH of the ultrafiltration retentate was adjusted to 4.5 by adding 375 mL of 10% (v / v) acetic acid, resulting in precipitation of the material. After 60 minutes, that is to say, when the most material settles on the bottom of the container, the upper 24 L is decanted and filtered through a Cuno ™ 30SP coarse filter at 500 ml / min to completely clarify the solution. The filtrate was then diluted 1.5 times with water and applied at 4 ° C to a S-Sepharose Fast Flow (Pharmacia) column (9 x 18.5 cm) equilibrated in 25 mM sodium acetate, pH 4.5. Material flowed through the column at 5 L / h at 4 ° C. After sample application, the column was washed with five column volumes (6 L) of column buffer, and the SCF material bound to the column was eluted with a gradient from 0 to 0.35 M NaCl by column chromatography. The total volume of the gradient was 20 L and 200 ml fractions were collected. The elution profile is shown in FIG. 33. Fractions (10 μΐ) of the fractions collected from the S-Sepharose column were analyzed by SDS-PAGE; performed both with (Fig. 32A) and without (Fig. 32B) sample reduction. From these analyzes it is evident that probably all absorbances at 280 nm (Figs. 32 and 33) are due to SCF material.

The correctly oxidized form predominates in the main absorbance peak (fractions 22-38, Fig. 33). Smaller species (forms) that can be visualized in the fractions include an incorrectly oxidized material with an apparent M _{r of} 18-20000 on SDS-PAGE (not reduced), present in the ascending arm of the main absorbance peak (fractions 10-21, Fig. 32B); and a disulfide-bound dimer material present in the absorbance region (fractions 10-38, bor. 32B).

Fractions 22-38 from the S-Sepharose column were combined and adjusted to pH 2.2 by adding about 11 mL of 6n HCl and applied to a Vydac C ₄ column (8.4 cm high, 9 cm diameter) equilibrated with 50% ( v / v) ethanol, 12.5 mM HCl (solution A) and the operation was performed at 4 ° C. The column resin was prepared by suspending the dry resin in 80% (v / v) ethanol,

1Ϊ, 5 mM HCl (solution B) and then equilibrated in solution A. Before application of the sample, a blank gradient was applied from solution A to solution B (6 L of total volume) and the column was then re-equilibrated with solution A. The 5 L solution A and the column bound SCF material was eluted with a gradient from solution A to solution B (18 L total volume) at a flow rate of 2670 ml / h. 286 fractions of 50 ml were collected and aliquots analyzed by absorbance at 280 nm (Fig. 35) and SDS-PAGE (25 μΐ per fraction) as described above (Fig. 34A, reducing conditions, Fig. 34B non-reducing conditions). . Fractions 62-161, containing properly oxidized SCF in high purity, were pooled (a relatively small amount of incorrectly oxidized monomer with an M _{r of} about 18-20000 (not reduced), eluting later in the gradient (about fractions 166-211) and the disulfide dimer material also eluted later (about fractions 199-235) (Fig. 35)).

The following procedure using Q-Sepharose Fast Flow (Pharmacia) ion exchange resin was used to remove ethanol from pool of fractions 62-161 and to concentrate SCF. Pool (5 L) was diluted with water to a volume of 16.625 L, to an ethanol concentration of about% (v / v). Then, 1 M Tris base (135 mL) was added to adjust the pH to 8, followed by 1 M Tris-HCl, pH 8 (23.6 mL) to adjust the total Tris concentration, to 10 mM. An additional 10 mM Tris-HCl, pH 8 (15.5 L) was added to a total volume of about 10% (v / v). The material was then applied at 4 ° C to a Q-Sepharose Fast Flow column (6.5 cm high, 7 cm diameter) equilibrated with mM Tris-HCl followed by a 2.5 L column buffer wash.

washing was about

200 mM NaCl, 10

Sample flow rate at application a

5.5 1 / h. For the elution of bound SCF, mM Tris-HCl, pH 8 was pumped in the opposite direction through the column at a rate of about 200 ml / h. Fractions of about 12 ml were collected and analyzed by absorbance at 280 nm and SDS-PAGE as above. Fractions 16-28 were combined (157 mL).

The pool containing SCF was then applied in two separate chromatographic experiments (78.5 ml each) on a Sephacryl S-200 HR (Pharmacia) gel filtration column (5 x 138 cm) equilibrated with phosphate buffered saline at 4 ° C. Fractions of about 15 ml were collected at a flow rate of about 75 ml / h. In any case, the major peak of the 280 nm absorbance materials eluting in the fractions corresponds approximately to an elution volume ranging from 1370 to 1635 ml. Fractions representing absorption peaks from two column experiments were pooled to a volume of 525 ml containing about 2.3 g SCF. This material was sterilized by filtration using a Millipore Millipak 20 membrane insert.

Alternatively, the material from column C ₄ may be concentrated by ultrafiltration and the buffer exchanged by diafiltration prior to sterile filtration.

The isolated recombinant human SCF ^{1 '164} material is highly pure (> 98% by SDS-PAGE with silver-induction), and are considered to be pharmaceutically pure. Using the methods of Example 2, the material was found to have an amino acid composition corresponding to the SCF gene analysis and has an N-terminal amino acid sequence of Met-Glu-Gly-Ile ..., as expected, keeping Met encoded by the initiation codon.

Similar to that described for human SCF ^1-164 expressed in E. coli, it can also be derived rat SCF ^{1 "164} (also present in insoluble form inside the cell after fermentation) in the pure state with high biological specific activity. Also human CF ^1-183 and rat SCF ^1-193 can be obtained. Rat SCF ^1-193 during folding / oxidation tends to form various oxidized species and it is more difficult to remove these unexpected species by chromatography.

Rat SCF ^{1 193} and human SCF ^1-183 are prone to proteolytic degradation during the initial recovery stages, i.e. solubilization and folding / oxidation. The primary site of proteolysis is located between residues 160 and 170. Proteolysis can be minimized by appropriate adjustment of conditions (e.g.

100

SCF, pH change, addition of EDTA to 2-5 mM, or other protease inhibitors) and expected degraded forms can be removed by appropriate separation steps.

While the use of urea for solubilization and during folding / oxidation as mentioned is a preferred embodiment, other solubilizing agents such as guanidine.HCl (e.g., 6 M during solubilization and 1.25 M during folding / oxidation) may also be used. and sodium N-lauryl sarcosine. After removal of the reagents after folding / oxidation, purified SCF can be obtained. as determined by SDS-PAGE, using appropriate separation steps.

Furthermore, a preferred embodiment is the use of glutathione at a concentration of 1 mM during folding / oxidation, but other conditions with the same or instead of 0.2 mM oxidized 0.4 mM oxidized or other thiol may be used.

for example, glutathione glutathione

2-mercaptoethanol include use plus plus proximity effects.

mM glutathione glutathione, glutathione

These mM or mM mM

In addition to the chromatographic procedures described, »

other methods of obtaining SCF in purified active form which include hydrophobic interaction chromatography (e.g. using phenyl-Sepharose (Pharmacia) using a sample at neutral pH in the presence of 1.7 M ammonium sulfate and eluting with a gradient of decreasing ammonium sulfate concentration); immobilized metal affinity chromatography (e.g., using chelated Sepharose (Pharmacia) with Cu ²⁺ ions, application of the sample at near neutral pH in the presence of 1 mM imidazole and elution with increasing imidazole concentration gradient); hydroxylapatite chromatography (application of a sample at neutral pH in the presence of 1 mM phosphate and eluting with an increasing phosphate gradient) and other procedures known to those skilled in the art.

Other forms of human SCF corresponding to all or part of the open reading frame encoding amino acids 1-248 of FIG. 42, or corresponding to an open reading frame

101 encoded by alternatively linked mRNAs that may exist (as represented by the cDNA sequence in Figure 44) can also be expressed in E. coli and obtained in purified form by procedures similar to those described above in this example and by other procedures that are obvious to experts.

Purification and preparation of forms involving the so-called transmembrane region (Example 16) may employ detergents including nonionic detergents and lipids, including phospholipids containing a liposome structure.

Example 11

Recombinant SCF from mammalian cells

A. Fermentation of SCF producing CHO cells

Recombinant Chinese Hamster Ovary (CHO) cells (CHO strain pDSRa hSCF ^{1 162} ) were cultured on microcarriers in a 20 liter perfusion culture system to produce human SCF ^{1 162} . The fermenter system was similar to that used in BRL 3A cell culture, Example 1B, except for the following conditions: the culture medium used for CHO cell culture was a mixture of Dulbecco's Modified Eagle Medium (DMEM) and Ham * with F-12 nutrient mixture at a 1: 1 (GIBCO), supplemented with 2 mM glutamine, non-essential amino acids (to double the existing concentration using a 1: 100 dilution of Gibco # 320-1140) and 5% fetal bovine serum. The medium obtained was identical except for the omission of serum. The reactor was inoculated with 5.6 x 10 ⁹ CHO cells cultured in two 3-liter flasks. Cells were allowed to grow to a concentration of 4 x 10 ⁵ cells / ml. At this point, 100 grams of pasteurized cytodex-2 microcarrier (Pharmacia) was added to the reactor as a suspension in 3 liters of phosphate buffered saline. Cells were allowed to mount and grow on microcarriers for 4 days. The growth medium was perfused by the reactor as needed with respect to glucose consumption. The glucose concentration was maintained at approximately 2.0

102 g /. After four days, the reactor was perfused with six volumes of serum-free medium to remove most of the serum (protein concentration <50 µg / ml). The reactor was confirmed batchwise until the glucose concentration fell below 2 g / L. From this point on, the reactor was operated at a continuous perfusion rate of about 20 L / day. The pH of the culture was maintained at 6.9 + 0.3 by adjusting the CO ₂ flow rate. The dissolved oxygen was maintained above 20% by air saturation and supplemented with pure oxygen if necessary. The temperature was maintained at 37 ± 0.5 ° C.

About 450 liters of conditioned medium was obtained from the above system and this was used as a starting material for the purification of recombinant human SCF ^1-162 . Approximately 589 liters of serum-free conditioned medium was also obtained by the same procedure, but using the strain CHO pDSRα2 rSCF · ^-3 · ⁸² was used as the starting material for the rat SCF ¹ “ ¹⁶² .

B. Purification of Recombinant Mammalian Expressed Rat SCF ^1-162

All purification procedures were performed at 4 ° C unless otherwise noted.

1. Concentration and diafiltration

Conditioned medium obtained from serum free of I- ¹⁶² as clarified by filtration through 0.45 µm Several different batches (36 L, 150 L) were separately concentrated and diafiltration / buffer. To illustrate, the charge was as follows. Filtered was concentrated to 500 ml using the culture strain of the CHO pDSRα2 rat SCF cell strain described in section A above, Sartocapsules 101 1, 102 performed conditioned (Sartorius).

1, sa

200 L and Millipore Pellion tangential flow ultrafiltration medium 1 with cellulose acetate membrane cartridges (three cartridges) separating 10000 molecular weight (15 ft ² total area)

103 membranes, pumping rate 2200 µl / min and filtration rate 750 ml / min). The diafiltration / buffer exchange for anion exchange chromatography was then performed by adding 1000 ml of 10 mM Tris-HCl, pH 6.7-6.8 for the concentrate, _from refolding to 500 ml using a tangential flow ultrafiltration device and repeating this addition. 5 times. The concentrated / diafiltered preparation was finally obtained in a volume of 1000 ml. The procedure for all conditioned medium batches subjected to concentration and buffer diafiltration / exchange was the same. Protein concentrations in the batches, determined by the method of Bradford (Anal. Biochem. 72, 248-254 (1976)) with bovine serum> J albumin as standard, were in the range of 70-90 gg / ml. The total volume of conditioned medium used for this preparation was - about 589 L.

2. Anion exchange chromatography on Q-Sepharose fast flow

The concentrated / diafiltered formulations from each of the five conditioned medium batches listed above were pooled (total volume 5000 mL). The pH was adjusted to 6.75 by addition of 1 M HCl. 2000 ml of 10 mM Tris-HCl, pH 6.7 was used to adjust the conductivity to about 0.700 mmho. The preparation was applied to a Q-Sepharose anion exchange high-speed column (36 x 14 cm, Q-Sepharose Fast Flow Pharmacia resin) which was equilibrated with 10 mM Tris-HCl, pH 6.7 buffer. After application of the sample, the column was washed with 28700 ml of Tris buffer. After this washing, the column was washed with 23000 ml of 5 M acetic acid / 1 mM glycine / 6 M urea / 20 μΜ CuSO ₄ with a pH of about

4.5. The column was then washed with 10 mM Tris-HCl, 20 µM CuSO ₄ , pH

6.7, buffer to restore neutral pH and remove urea and saline (0-700 mM NaCl, in 10 mM Tris-HCl, 20 μΜ CuSO ₄ , pH 6.7 buffer; 40 L total volume). Fractions of about 490 ml were collected at a flow rate of 3250 ml / h. The chromatogram is shown in FIG. 36. MC / 9 cpm reports biological activity in MC / 9 assay; 5 μΐ of each labeled fraction was tested. Eluates collected during sample application and not washed

104 are shown in the figure, there was no biological activity in these fractions.

3. Chromatography using silica-bonded hydrocarbon resin

Fractions 44-66 of the experiment shown in FIG. 36 were pooled (11200 mL) and EDTA was added to a final concentration of 1 mM. This material was applied at a flow rate of about 2000 ml / h to a C ₄ column (Vydac Proteins C ₄ , 7 x 8 cm) equilibrated with buffer A (10 mM Tris pH 6.7 / 20% ethanol). After applying the sample to the column, the column was washed with 1000 ml of Buffer A. Then a linear gradient from Buffer A to Buffer B (10 mM Tris pH 6.7 / 94% ethanol) was applied (total volume 6000 ml) and fractions 30-50 were collected. ml. Aliquots from sample start on column C ₄ , pool pool and wash pool added to 0.5 mL aliquots of gradient fractions were dialyzed against phosphate buffered saline in preparation for the bioassay. These different fractions were tested in the MC / 9 assay (5 μΐ fractions of prepared gradient fractions; cpm in Fig. 37). SDS-PAGE (Laemli, Nature 227, 680-685 (1970); stock gels contain 4% (w / v) acrylamide and separation gels contain 12.5% (w / v) acrylamide) fractions of different fractions is shown in FIG. 38. For gel use, aliquot samples (100 μΐ) were vacuum dried and then redissolved using 20 μΐ sample processing buffer (reducing, i.e., 2-mercaptoethanol) and cooked for 5 min before loading onto the gel. The numbered markings on the left side of the figure represent the migration positions of the molecular weight markers (reduced) as in FIG. 6. Numbered lanes represent the corresponding fractions collected during the application of the last part of the gradient. The gels were silver-induced (Morrisey, Ana. Bioch. 117, 307-310 (1981)).

4. Fast flow anion exchange chromatography on Q-Sepharose

105

Fractions 98-124 of the C ₄ column are shown in FIG. 37, these were combined (1050 mL). The pool was diluted 1: 1 with 10 mM Tris, pH 6.7 buffer to reduce ethanol concentration. The diluted pool was then applied to the anion exchange flow (3.2 x 3 cm, resin which was equilibrated with 10 mM

Q-Sepharose column with Pharmacia Q-Sepharose Fast Flow, Tris-HCl, pH 6.7 buffer. The flow rate was 463 ml / h. After application of 135 ml of column buffer and elution by washing with 10 mM Tris-HCl, the flow through the column was inverted, the sample was washed with bound material was 350 mM NaCl, pH 6.7. Direction to minimize the volume of eluted material and 7.8 ml fractions were collected during elution.

5. SephacrylS-200 HR gel filtration chromatography

Fractions containing the eluted protein from the column wash with anionic Sepharose Fast Flow salt were pooled (31 mL). 30 ml was applied to a Sephacryl S-200 gel filtration column (Pharmacia) (5 x 55.5 cm) equilibrated in phosphate buffered saline. Fractions of 6.8 ml were collected at a flow rate of 68 ml / h. Fractions corresponding to the absorbance peak at 280 nm were pooled and represent the final purified material.

Table 15 is a summary of the purifications.

Table 15

A summary overview of the purification of mammalian-expressed rat SCF ¹¹⁶²

degree volume total (ml) protein (mg) ^x conditioned medium 7000 28420 (Concentrated) Q-Sepharose Fast Flow 11200 974 C ₄ resin 1050 19 Q-Sepharose Fast Flow 31 20 Sephacryl S-200 HR 82 19 ^xx

^x Determined by the Bradford method (supra, 1976)

^106xx Determined as 47.3 mg by quantitative amino acid analysis using a method similar to that described in Example 2.

N-terminal amino acid sequence of purified rat SCF ^{1 "162} is approximately half Gln-Glu-Ile ... and half Pyr-Glu-Glu-Ile ..., as determined by the methods of Example 2. The results show that rat SCF ¹ ' ¹⁶² is the product of proteolytic cleavages between residues designated as (-1) (Thr) and (+1) (Gln) in Figs. 14C. Similarly, purified human SCF ^1-162 from conditioned medium of transfected CHO cells (below) had the N-terminal amino acid sequence of Glu-Gly-Ile, indicating that it was a cleavage product between residues designated as (-1) (Thr) and (+) 1) (Glu) in FIG. 15C.

Using the above procedure, purified human SCF protein was obtained, either recombinantly produced by expression in CHO cells or naturally obtained.

Other purification methods suitable for purification from recombinant SCF-derived mammalian cells include those described in Examples 1 and 10 and other methods readily apparent to those skilled in the art.

Other forms of human SCF, corresponding to all or part of the open reading frame encoding amino acids 1-248 shown in FIG. 42, or corresponding to an open reading frame encoding alternatively linked mRNAs that may exist (as the cDNA sequence in Figure 44) may also be expressed in mammalian cells and obtained in purified form by procedures similar to those described in this Example and other procedures apparent to those skilled in the art.

C. SDS-PAGE and glycosidase treatment

The SDS-PAGE of the pooled fractions from a Sephacryl S-200 HR gel filtration column is shown in FIG. 39. 2.5 μΐ of the pooled fraction was loaded onto the gel (lane 1). The track was triggered by silver.

107

Molecular weight markers are shown in Figure 6 (lane 6).

The various material migrating along and above the M _r 31000 marker position represents a biologically active material; the apparent heterogeneity is largely due to heterogeneity in glycosylation.

To characterize glycosylation, the purified material was treated with various glycosidases, analyzed by SDS-PAGE (reducing conditions) and was visualized by silvering. The results are shown in FIG. 39. Lane 2 - neuraminidase. Lane 3 neuraminidase and O-glycanase. Lane 4 - neuraminidase, O-glycanase and N-glycanase. Lane 5 - neuraminidase and N-glycanase. Lane 7 - N-glycanase. Lane 9 - N-glycanase without substrate. Lane 9 - O-glycanase without substrate. Conditions were 10 mM

3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 66.6 mM 2-mercaptoethanol, 0.04% (w / v) sodium azide, phosphate buffered saline, 30 minutes at 37 ° c, followed by incubation at half the indicated concentrations in the presence of glycosidases for 18 h at 37 ° C. Neuraminidase (from Arthrobacter ureafaciens, supplied by Calbiochem) was used at a final concentration of 0.5 unit / ml. O-Glycanase (Genzyme; endo-alpha-N-acetylgalactosaminidase) was used in concentration

7.5 milliunits / ml. N-Glycanase (Genzyme; peptide: N-glycosidase F; peptide-N ⁴ (N-acetyl-beta-glucosaminyl) asparaginamidase) was used at a concentration of 10 units / ml.

Where appropriate, various control incubations were performed. These include: incubation without glycosidases, to verify that the results are due to added glycosidase preparations; incubation with glycosylated proteins (e.g., glycosylated recombinant human erythropoietin), known as substrates for glycosylase, to verify that the glycosidase enzymes used were active; and incubations with glycosidases, but without substrate, to assess whether glycosidase preparations contribute or obscure the visualization of gel bands (Figures 8 and 9).

Numerous assessments can be made from the above experiments. Various treatments with N-glycanase (which removes as

108 complex, as well as high mannose N-linked carbohydrate (Tarentino et al., Biochemistry 24, 4665-4671 (1988)), neuraminidase (which removes sialic acid residues) and O-glycanase removes certain O-linked carbohydrates (Lambin and

Biochem. Soc. Trans. 12, 599-600 (1984)), confirm that (both of which are present both N-linked and O-linked carbohydrate), sialic acid is present, at least a portion of which is O-linked groups. N-glycanase can convert a heterogeneous material according to SDS-PAGE to a faster migrating form, which is much more homogeneous, indicating that all the material represents the same polypeptide, with heterogeneity which is mainly due to heterogeneity in glycosylation.

Example 12

Preparation of recombinant SCF ¹¹¹⁶ PEG

The rat SCF ^{1 "164,} purified from a recombinant E.coli expression system according to Examples 6A and 10, was used as starting material for modification with polyethylene glycol, as described below.

Methoxypolyethylene glycol succinimidyl succinate (18.1 mg = 3.63 μπιοί, SS-MPEG = Sigma Chemical Co. No M3152, approximate molecular weight = 5000) in 0.327 ml deionized water was added to 13.3 mg (0.727 μπιοί) recombinant rat SCF ^{1 "164} in 1.0 ml of 138 mM sodium phosphate, 62 mM NaCl, 0.62 mM sodium acetate, pH 8.0. The solution was shaken gently (100 ^rpm-1) at room temperature for 30 minutes. 1.0 ml aliquots of the final reaction mixture (10 mg protein) were then applied to a Pharmacia Superdex 75 gel filtration column (1.6 x 50 cm) and eluted with 100 mM sodium phosphate, pH 6.9, at a rate of 0.25 ml / min at room temperature. The first 10 ml of the column was discarded and then 1.0 ml of fraction was collected. The UV absorbance at 280 nm was monitored for the eluent from the column and is shown in FIG. 40A. Fraction # 25-27 were pooled and sterilized by ultrafiltration through a 0.2 μπι polysulfone membrane (Gelman

109

Sciences no. 4454) and the resulting pool was designated PEG-25. Similarly, fractions no. 28 to 32 are combined, sterilized by ultrafiltration and designated PEG-25. The PEG-25 pool fractions contain 3.06 mg protein and the PEG-32 pool fractions contain 3.55 mg protein as calculated from A280 measurement using a calibration given by absorbance of a 1.0 ml / mL solution of unmodified rat SCF ^1-164 at 0.66. Unreacted rat SCF ¹ " ¹⁶⁴ , representing

11.8% of the total protein in the reaction mixture was eluted in fractions # ^34-37 . Under the same chromatographic conditions, unmodified rat SCF ^1-164 eluted as the main peak with a retention volume of 45.6 ml; 40B. Fraction # 77 to 80 in FIG. 40A, include N-hydroxysuccinimide, a by-product of the reaction of rat SCF ^{1 "164} with SS-MPEG.

Potentially reactive amino groups in rat SCF ¹ · ⁴ → ⁴ include 12 lysine residues and an alpha-amino group at the N-terminus of the glutamine residue. PEG-25 pooled fractions contain 9.3 moles of reactive amino groups per mole of protein, as determined by spectroscopic titration of trinitrobenzenesulfonic acid (TNBS) using the method described by Habeeb, Anal. Biochem.

14: 328-336 (1966). Similarly, the pooled PEG-32 fractions contain 10.4 moles and the unmodified rat SCF ^{1 164} contains 13.7 moles of reactive amino groups minus 10.4 µmoles per mole of protein. An average of 3.3 (13.7 rat SCF ¹ ” ¹⁶⁴ in the pooled fraction)

PEG-32 was modified by reaction with SS-MPEG. Similarly, on average

The 4.4 amino group of rat SCF ^1-164 in the PEG-25 ^pool fraction was modified. Human SCF (hSCF ^{1 "164)} produced in Example 10 was also modified as described above,. Specifically, 714 mg (38.5 μπιοί) hSCF ^{1 164 164} reacted with 962.5 mg (192.5 μπιοί) SS-MPEG in 75 ml 0.1 M sodium phosphate buffer, pH 8.0 for 30 minutes at temperature rooms. The reaction mixture was applied to a Sephacryl S-200HR column (5 x 134 cm) and eluted with PBS (Gibco Dulbecco phosphate buffered saline without CaCl ₂ and MgCl ₂ ) at a rate of 102 mL / h and 14.3 mL fractions were collected. Fraction # 39-53 analogous to the PEG-25 pool described above and in FIG. 40A were combined and found to contain a total of 354

110 mg protein. The in vivo activity of such modified SCF in primates is described in Example 8C.

Example 13

SCF receptor expression on leukemic blasts

Leukoblasts were obtained from the peripheral blood of a patient with mixed leukemia. Cells were purified by gradient density centrifugation and suppression of adhesion. Human SCF ^{1 "164} has been iodinated as described in Example 7. The cells were incubated with different concentrations of iodinated SCF as described (Broudy, 75 1622-1626 (1990)). The results of the receptor binding experiment are shown in FIG. 41. The density of the receptor is approximately 70000 receptors / cell.

Example 14

Rat SCF activity on early lymphoid precursors

The ability of recombinant rat SCF ^1-164 (rrSCF ^1-164 ) to act synergistically with IL-7 to increase lymphoid cell proliferation was studied in mouse bone marrow agar cultures. In this assay, the colonies formed by the very rrSCF ^1-164 include monocytes, neutrophils, and blast cells, while the colonies stimulated by IL-7 alone or in combination with rrSCF ^{1 "164} containing primarily pre-B cells. Pre-B cells, characterized as B220 ⁺ , slg ”, σμ ⁺ , were identified by FACS analysis of pooled cells using fluorescently labeled antibodies to the B220 antigen (Coffman, Immunol. Rev., 69, 5 (1982)) and surface

Ig (FITC-goat anti-K, Southern Biotechnology Assoc., Birmingham,

AL) and cytospin slides for cytoplasmic μεχρΓβΞΪη using fluorescently labeled antibodies (TRITC-goat anti-μ, Southern Recombinant Human IL-7 (rhIL-7) Internationol (Westlake Village, CA).

Biotechnology Assoc.). was obtained from Biosource When rrSCF ^1-164 was added in combination with the pre-B IL-7 cell growth factor, it was observed

111 synergistic increase in colony formation (Table 16), indicating a role in stimulating rrSCF ^{1 "164} on early B cell progenitors.

Table 16

Stimulation of Pre-B cell colony formation with rrSCF ^1_164 in combination with hIL-7

Growth factors colony count ¹

saline solution 0 13 ± 2 7 ± 4 4 ± 2 rrSCF ^{1 * 164} 200 ng 100 50 ng ng rhIL-7 200 ng 21 ± 6 100 ng 18 ± 6 50 ng 13 ± 6 25 ng 4 ± 2 rhIL-7 200 ng + rrSCF ¹ “ ¹⁶⁴ 200 ng 60 ± 0 100 ng + 200 ng 48 + 8 50 ng + 200 ng 24 ± 10 25 ng + 200 ng 21 ± 2

¹ Number of colonies per 5 x 10 ⁴ mouse bone marrow cells that were plated.

Each value is the average of three plates ± standard. deviation.

Example 15

Identification of SCF receptor

A c-kit is a receptor for SCF ¹¹⁶⁴

112

To test whether SCF ^{1 "164} is the ligand for c-kit, the cDNA for the entire murine c-kit (Qiu et al., EMBO J., 7, 1003-1011 (1988) is amplified using PCR from the SCF ^{1- 164} sensitive ointment of cell line MC / 9 (Nabel et al., Nature, 291, 332-334 (1981)) with primers according to the published sequence Ligand binding and transmembrane domain of human c-kit encoded by amino acids 1-549 (Yarden et al. , EMBO J., 6, 3341-3351 (1987)), were cloned using the same techniques from the human erythroleukemic cell line, HEL (Martin and Papaynnopoulou, Science, 216, 1233-1235 (1982)). were inserted into mammalian V19.8 expression vector transfected into COS-1 cells and membrane fractions prepared for binding assay using either rat or human ¹²⁵ I-SCF ^1-164 according to the methods described in sections B and C. Table 17 presents data from a typical There are no specific ¹²⁵ I binding of human S CF ^{1 '164} to COS-1 cells transfected with V19.8 alone. However, COS-1 cells expressing human recombinant c-kit ligand binding plus transmembrane domains (hCKI-tl) bound ¹²⁵ I-hSCF ^{1 "164} (Table 17). Addition of 200-fold molar excess of unlabelled human SCF ^{1 "164} reduces binding to baseline. Similarly, COS-1 cells transfected with the full-length mouse c-kit (mckit-L1) bind rat ¹²⁵ I-SCF ^1-164 . A small amount of rat ¹²⁵ I-SCF ^{1-164 binding} was detected in COS-1 cells transfected with V19.8 alone and was also observed in non-transfected cells (not shown), indicating that COS-1 cells express endogenous c-kit. This finding is consistent with the broad molecular distribution of the c-kit. Rat ^I2 5i-SCF ^1-1 ® ⁴ binds to both human and mouse c-kit, whereas human ¹² ®-SCF ^1-1 ® ⁴ binds with lower activity to mouse c-kit (Table 2). 17). These data are consistent with the cross-reactivity model SCF ¹ " ¹ ® ⁴ between species. The rat SCF ^{1 '1} ® ⁴ induces proliferation of human bone marrow with a specific activity as well as human SCF ^1-164, SCF as human ^{1 "1} ® ⁴ induced proliferation of murine mast cells with a specific activity 800-fold less than the rat protein.

113

Taken together, these data are consistent with the phenotypic abnormalities expressed by the W or SI mutant mouse resulting from primary defects in the c-kit by receptor / ligand interaction, which are critical for the development of various cell types.

Table 17

SCF ^{1 "164} binding to recombinant c-kit expressed in COS-1 cells

Transfected plasmid CPM binding ³ human SCF ¹ “ ^{164 125} I-SCF ^{b 125} I-SCF + unk. ^C rat SCF ^{1-164 125} I-SCF ^{d 125} I-SCF + unk. ^e V19.8 2,160 2,150 1,100 550 V19.8: 59,350 2,380 70,000 1,100 hCKI-ltl V19.8:, 9500 1,100 52,700 600 McKie-LI

^and is the mean of two measurements; the experiment was performed independently with the same results three times.

^b 1.6 nM human ¹²⁵ I-SCF ^{1-164 c} human nm human ¹²⁵ I-SCF ^1-164 + 320 nM unlabeled human

SCF ^{1-64 d} 1.6 nM rat ¹²⁵ I-SCF ^{1-164 e} 1.6 nM rat ¹²⁵ I-SCF ^1-164 + 320 nM unlabeled rat

¹¹⁶⁴ SCF

B. Expression of recombinant c-kit in COS-1 cells

Human and mouse c-kit cNDA clones were obtained using PCR techniques (Saiki et al. Science, 239, 487-491 (1988)) from total RNA isolated by an acid phenol / chloroform extraction procedure (Chomczynsky and Sachi, Anal. Biochem., 162). , 156-159 (1987)) from the human erythroleukemic cell line HEL and MC / 9

114 cells. Unique oligonucleotide sequences were designed from published human and mouse c-kit sequences. The first strand cDNA was synthesized from total RNA according to the protocol obtained with the enzyme, Mo-MLV reverse transcription (Bethesda Research Laboratories, Bethesda, MD), using opposite c-kit oligonucleotides as primers. Amplification of overlapping regions of c-kit ligand binding and tyrosine kinase domains was performed using appropriate pairs of c-kit primers. These regions were cloned into the mammalian expression vector V19.8 (Fig. 17) for expression in COS-1 cells. DNA sequencing of some clones reveals independent mutations, predominantly generated during PCR amplification, at each step. A clone lacking these mutations was constructed by rebuilding mutations lacking restriction fragments from separate clones. Some deviations from the published sequences occurred in all or about half of the clones; these were considered to be actual sequences present in the cell line used and may represent allelic differences of the published sequences: V19.8: mckit-LT1, whole mouse c-kit; and V19.8: kckit-L1, comprising a ligand bond plus the transmembrane region (amino acids 1-549) of the human c-kit.

Plasmids were transfected into COS-1 cells essentially as described in Example 4.

12 _ C _{5 R SCF} 1-164 _{in the} mining of the COS-1 cells expressing the recombinant c-kit

Days days after transfection, COS-1 cells were scraped from the plate, washed in PBS and frozen until use. After thawing, the cells were resuspended in 10 mM Tris-HCl, 1 mM MgCl ₂ containing 1 mM PMSF, 100 µg / ml aprotinin, 15 µg / ml leupeptin, 2 µg / ml pepstatin and 200 µg / ml TLCK-HCl. The suspension was dispersed by suction and drain from the pipette five times, incubated on ice for 15 minutes, and the cells were homogenized with the homogenizer. Sucrose (250 mM) was the core fraction and the remaining undisturbed

15-20 Dounce added to the homogenate and cells were pelleted

115 by centrifugation at 500 xg for 5 minutes. The supernatant was centrifuged at 25000 g for 30 minutes at 4 ° C to pellet the remaining cell pieces. Human and rat SCF ^{1 164} were labeled with radioactive iodine using chloramine-Z (Hunter and Greenwood,

Nature, 194, 495-496 (1962). COS-1 membrane fractions were incubated with either human or rat ¹²⁵ I-SCF ^1-164 (1.6 nM) with or without 200-fold excess of unlabeled SCF ^{1 164} in binding buffer containing RPMI supplemented with 1% bovine serum albumin and 50 µl. mM HEPES (pH 7.4) for 1 hour at 22 ° C. At the end of the binding incubation, the membrane preparations were gently stratified to 150 μΐ phthalate oil and centrifuged for 20 minutes in a Beckman Microfuge 11 to separate the membrane bound ¹²⁵ I-SCF ^{1 164} from free ¹²⁵ I-SCF ^1-164 . The pellets were separated and membrane- ^{bound 125} I-SCF ^1-164 was quantified.

Example 16

Ä. Isolation of human SCF cDNA

Total RNA was isolated from the HT-1080 human fibrosarcoma cell line (ATCC CCL 121) by extraction using an acid guanidinium thiocyanate-phenol-chloroform mixture (Chomczynski et al., Anal. Biochem. 162, 156 (1987)) and poly (A).

RNAs were obtained using an oligo (dT) column obtained from Clontech. Double stranded cDNA was prepared from 2 µg of poly (A) RNA with BRL (Bethesda Research Laboratory) cDNA synthesis kit under conditions recommended by the manufacturer. Approximately 100 ng on a 2kb double stranded cDNA column was ligated to 300 ng SalI / NotI digested vector pSPORT 1 (D'Alessio et al., Focus, 12, 47-50 (1990)) and transformed into DH5α (BRL, Bethesda, MD) cells by electroporation (Dower et al., Nucl. Acids Res., 16, 6127-6145 (1988)).

B. Screening cDNA library

Approximately 2.2 x 10 ⁵ primary transformants were divided into 44 pools, each containing 5000 individual

116 clones. Plasmid DNA was prepared from each pool of CTAB-DNA by the clotting method as described (Del Sal et al., Biotechniques, 7, 514-519 (1989). Two micrograms of each pool of plasmid DNA were digested with the restriction enzyme Note and separated by gel electrophoresis. Linerized DNA was transferred to a GeneScreen Plus membrane (DuPont) and hybridized with ³² P-labeled PCR generated by human SCF cNDA (Example 3) under conditions previously described (Lin et al., Proc. Natl. Acad. Sci. USA, 82, 7580). Three pools containing a positive signal were identified from hybridization, and these colony pools were re-screened by a hybridization colony procedure (Lin et al., Gene 44, 201-209 (1986)) until one pool was obtained from each pool. The cDNA size of the three isolated clones was between 5.0 and 5.4 kb. Restriction enzyme digestion and nucleotide sequence determination at the 5 'end indicated that two of the three clones are both identical (10-1a and 21-7a), both containing a coding region and approximately 200 bp of the 5 'untranslated region (5'UTR). The third clone (26-la) is approximately 400 bp shorter at the 5 'end than the other two clones. The human SCF cDNA sequence is shown in FIG. 42. A hydrophobic transmembrane domain sequence beginning at amino acid region 186-190 and ending at amino acid 212 is particularly detailed.

C. Construction of pDSRa2 hSCF ¹ "pDSR.alpha.2 hSCF ²⁴⁸ · ³ '' ²⁴⁸ was generated using plasmids 10-la (as described in Example 16B) and pGEM3 hSCF ^{1 '164} as follows: HindIII insert from pGEM3 hSCF ^1-164 was transferred to M13pml8. Nucleotides immediately upstream of the ATG initiation codon were replaced by proper mutagenesis from tttccttATG to gccgccgccATG using the opposite oligonucleotide.

5'-TCT TCT TCA TGG CGG CGG CAA GCT T 3 'and oligonucleotide driven in vitro kit mutagenesis system and Amersham Corp. protocols. to generate M13mpl8 hSCF ^K1 ' ¹⁶⁴ . This DNA was digested with HindIII and inserted into

117 pDSRα2, which had been digested with HindIII. This clone is designated pDSRα2 hSCF ^{K1 164} . PDSR.alpha.2 hSCF DNA ^{K1 '164} was digested with XbaI and the DNA made blunt by the addition provided with Klenow enzyme and four dNTPs. Upon completion of this reaction, the DNA was further digested with Spel. Clone 10-1a was digested with DraI to create a blunt end 3 'for the open reading frame in the insert and with SpeI, which cleaves at the same site in the gene as in pDSRα2 hSCF ¹ → ^1-164 and ^10-1a . This DNA was joined together to form a pDSR.alpha.2 hSCF ^{K1 "248th}

D. Transfection and immunoprecipitation of COS cells with pDSRa2 hSCF ^{K1 "248} DNA

COS-7 (ATCC CRL 1651) cells were transfected with DNA constructed as described above. 4xl0 ⁶ cells in 0.8 ml DMEM + 5% FBS were electroporated at 1600 V with either 10 mg pDSR.alpha.2 hSCF ^ ^{1 "248} DNA or 10 g pDSR 2 vector DNA (vector control). After electroporation, the cells were placed in two 60 mm dishes. After 24 hours, the medium was replaced with fresh complete medium.

hours after transfection, each plate was marked ³ ^ S-medium according to modification of the protocol Yarden et al. (PNAS 87, 2569-2573, 1990). Cells were washed once with PBS and then incubated with non-methionine-free DMEM, non-cysteine (met ”cys” DMEM) for 30 minutes. The medium was discarded and 1 ml of met 'cys' DMEM containing 100 μϋί / ηΐ Tran ³⁵ S-labeled (ICN) was added to each plate. The cells were incubated at 37 ° C for 8 hours. The medium was collected, clarified by centrifugation to remove cell debris and frozen at -20 ° C.

Aliquots of labeled conditioned medium of COS / pDSR 2 hSCF ^{K1 '248} and COS / pDSRa2 vector control were immunoprecipitated along with medium samples of ^35S-labeled CH0 / pDSR.alpha.2 hSCF ^{1 "164} clone 17 cells (see example 5) according to a modification of the protocol Yardena et al., (EMBO, J., 6, 3341-3351, 1987). One ml of each conditioned media sample was processed

118 with 10 μΐ pre-immune rabbit serum (# 1379 PI). The samples were incubated for 5 hours at 4 ° C. One hundred microliters of a 10% suspension of Staphylococcus aureus (Pansorbin, Calbiochem) in 0.15 M NaCl, 20 mM Tris pH 7.5, 0.2% Tri Ton X-100 was added to each tube. The samples were incubated for an additional hour at 4 ° C. Immune complexes were pelleted by centrifugation at 13,000 xg for 5 minutes. The supernatants were transferred to new tubes and incubated with a rabbit polyclonal antiserum 5μ1 (no. 1381 TB4), purified as in Example 1, the hSCF ^{1 "162} CHO overnight at 4 ° C. 100 μorb of Pansorbine was added over 1 hour and the immune complexes were pelleted as before. The pellets were washed with 1x lysis buffer (0.5% N-deoxycholate, 0.5% NP-40, 50 mM NaCl, 25 mM Tris pH 8), 3x with wash buffer (0.5 M NaCl, 20 mM Tris pH 7.5, 0.2% Triton X-100) and 1 x 20 mM Tris pH 7.5. The pellets were resuspended in 50 μΐ 10 mM Tris pH 7.5, 0.1% SDS, 0.1 M β-mercaptoethanol. The SCF protein was eluted by boiling for 5 min. The samples were centrifuged at 13,000 xg for 5 minutes and the supernatants discarded.

Glycosidase treatment was performed as follows: three microliters of 75 mM CHAPS containing 1.6 mU O-glycanase, 0.5 U N-glycanase and 0.02 U neuraminidase were added to 25 μΐ of the immune complex and incubated for 3 hours at 37 ° C . An equal volume of 2xPAGE sample buffer was added and the samples were boiled for 3 minutes. The digested and uncleaved samples were electrophoresed on a 15% SDS-polyacrylamide reducing gel overnight at 8 mA. The gel was fixed in methanol-acetic acid, treated with NEN for 30 minutes, dried and exposed at -70 ° C to Kodak XAR-5 film.

Figure 43 is an autoradiograph of results. Lanes 1 and 2 are samples from control COS / pDSRα2 cultures, lanes 3 and 4 from COS / pDSRα2 hSCF ^{K1 248} , lanes 5 and 6 from CHO / pDSRα2 hSCF ¹ " ¹⁶⁴ . Lanes 1, 3 and 5 are uncleaved immune clots, lanes 2, 4 and 6 were cleaved by glycanases as described above. The positions of the molecular weight markers are indicated on the left. The preparation of the SCF in COS transfected with pDSR.alpha.2 hSCF ^{K1 "248} in accordance with the hSCF ^{1" 164} secreted by transfected CHO pDSR 2 hSCF ^1-164 (Example 11).

119

This strongly suggests that the natural proteolytic site releasing SCF from the cell lies adjacent to amino acid 164.

Example 17

Quaternary structural analysis of human SCF

When calibrating the gel filtration column (ACA 5) described in Example 1 for purifying SCF from BRL cell medium with molecular weight standards and eluting purified SCF from other calibrated gel filter columns, it is apparent that SCF purified from BRL cell medium exhibits a clear molecular weight of about 70000 to 90000 based on the molecular weight of the standards. In contrast, the apparent molecular weight by SDS-PAGE is about 28000-35000. Although it is found that glycosylated proteins may behave anomalously in such assays, the results confirm that rat SCF-derived SCF may exist as a non-covalently associated dimer under non-denaturing conditions. Similar results apply to recombinant SCF forms (e.g., rats and human SCF ¹ " ¹⁶⁴ obtained from E. coli, rats and human SCF ¹ " ¹⁶² obtained from CHO cells) whose molecular size determined by gel filtration under non-denaturing conditions is approximately twice as large as determined by gel filtration under denaturing conditions (i.e. in the presence of SDS), or by SDS-PAGE, in each individual case. Further analysis of the sedimentation speed, which provides an accurate determination of molecular weight in solution, gives a value of about 36,000 for molecular weight of recombinant human SCF ^{1 "164} derived from E. coli. Again, this value is approximately twice that provided by SDS-PAGE ('18000-19000). Therefore, while it may be recognized that multiple oligomeric states (including monomeric state) may exist, it is apparent that the dimeric state in solution prevails under certain circumstances.

120

Example 18

Isolation of human SCF cDNA clones from cell line 5637

A. Construction of a 5637 cDNA library

Total RNA isolated from human blood carcinoma cell line 5637 (ATCC HTB-9) using an acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski et al., Anal. Biochem, 162, 156 (1987)) and poly (A) RNA were obtained using an oligo (dT) column obtained from Clontech. Double stranded cDNA was prepared from a 2 µg pole (A) RNA with a BRL cDNA synthesis kit under conditions recommended by the supplier. Approximately 80 ng on a 2 kb average double stranded cDNA column was ligated to 300 ng SalI / NotI digested vector pSPORT 1 (D'Alessio et al., Focus, 12, 47-50 (1990)) and transformed into DH5α cells by electroporation (Dower et al., Nucl. Acids Res., 16, 6127-6145 (1988)).

B. Screening of cDNA library

Approximately 1.5 x 10 ⁵ primary transformants were divided into 30 pools, each containing approximately 5,000 individual clones. Plasmid DNA was prepared from each pool of CTAB-DNA by precipitation as described (Del Sal et al., Biotechniques, 7, 514-519 (1989)). Two micrograms of each plasmid DNA from the pool were digested with the restriction enzyme Note and separated by gel electrophoresis. Linearized DNA was transferred hybridized to GeneScreen Plus membrane ³² P-labeled full-length cDNA (DuPont) and human SCF isolated from

HT1080 cell line (example

16) under conditions already signed (Lin et al., Proc. Natl. Acad. Sci. USA,

82, 7580-7584 (1985)). Seven pools containing a positive signal were identified by hybridization. Colony pools were re-screened with ³² P-labeled PCR obtained human SCF cDNA (Example 3) by a colony hybridization procedure (Lin et al., Gene, 44, 201-209 (1986)) until one of four colonies was obtained.

121 pools. The insert sizes of the four isolated clones are approximately 5.3 kb. Restriction enzyme digestion and nucleotide analysis of the 5'-end of the clones showed that the four clones were identical. The sequence of this human cDNA is shown in FIG. 44. The cDNA of FIG. 44 encodes a polypeptide wherein amino acids 149-177 of the sequence of FIG. 42 are replaced by a Gly residue.

Example 19

Effect of SCF on survival after lethal irradiation

A. Effect of SCF in vivo survival after lethal irradiation

The effect of SCF on mouse survival after lethal iridation was tested. The mice used were 10 to 12 weeks old female Balb / c. Groups of 5 mice were used in all experiments and the mice were weighed before each experiment. Mice were irradiated at 850 rad or 950 rad per dose. Mice were injected with factors alone or factors plus normal Balb / c bone marrow cells. In the first case, mice were injected intravenously 24 hours after irradiation with rat PEG-SCF ^{1 "164} (20 gg / kg), purified from E. coli and modified by the addition of polyethylene glycol as in Example 12, or with saline in terms of control animals. For the transplant model, mice were injected iv at different doses of normal Balb / c bone marrow cells 4 hours after irradiation. Treatment with rat PEG-SCF ^{1 "164} was made by the addition of 200 ug / kg of rat PEG-SCF ^{1" 164} to the cell suspension 1 hour prior to injection and given as a single IV injection of factor plus cells.

After irradiation at 850 rad, mice were injected with rat PEG-SCF ^1-164 or saline. The results are shown in FIG. 45. Injection of rat PEG-SCF ^{1 "164} significantly increases the survival of mice compared to controls (p 0.0001). Mice injected with saline survive on average 7.7, while PEG-SCF ^{1 "164} treated mice surviving at approximately 9.4

122 (FIG. 45). The results shown in FIG. 45 are a summary of 4 separate experiments with 30 mice in each treatment group.

Increased survival of mice treated with rat PEG-SCF ^{1 "164} confirms the effect of SCF on the bone marrow cells of the irradiated animals. A preliminary hematological study of these animals shows a slight increase in platelet levels compared to control animals 5 days after irradiation, but 7 days after irradiation, platelet levels are not significantly different from control animals. No differences in RBC or WBC levels or bone marrow cell structure were detected.

I

B. Survival of transplanted SCF treated mice

Doses of 10% of the bone marrow cells of femur of normal Balb / c mice transplanted into mice irradiated at 850 rad can save 90% or more of the animals (data not shown). Therefore, the applied radiation dose of 850 rad transplant dose of 5% femur to study the effects of rat PEG-SCF ^{1 '164} of survival. At this cell dose, it was assumed that a large percentage of mice not receiving SCF would not survive; if rat PEG-SCF ^{1 "164} could stimulate the survival of transplanted cells could be increased. As shown in FIG. 46, approximately 30% of control mice survived 8 days after irradiation. Treatment with rat PEG-SCF ^{1 "164} result in a significant increase of survival with greater than 90% of mice that survive for more than 30 days (FIG. 46). The results shown in FIG. 46 are a compilation of results from 4 separate experiments, which were used 20 mice, both in terms of control mice as well as mice treated with rat PEG-SCF ^{1 '164.} At higher doses of radiation treatment of mice rat PEG-SCF ^{1 "164} in connection with transplantation pulp also results in increased survival of the animals (FIG. 7). Control mice irradiated at 950 rad transplantation and 10% died on day 8 of the femur, whereas 40% of mice treated with rat PEG-SCF ¹¹⁶⁴ survived 20 days or more. 20% of control mice transplanted with 20% of the femur survived for 20 days, and 80% of the animals survived in rSCF treatment (Fig. 47).

123

Example 20

The formation, monoclonal antibodies to the SCF week-old female Balb / c mice (Charles River, Wilmington, MA) were subcutaneously injected with 20 micrograms of human SCF ¹¹⁶⁴ expressed in E. coli in complete Freund's adjuvant (H37-Ra; Difco Laboratories, Detroit, MI) . Further immunizations of 50 μ9 of the same antigen in incomplete Freud's adjuvant were subsequently administered on days 14, 38 and 57. Three days after the last injection, 2 mice were sacrificed and their spleen cells fused to the sp 2/0 myeloma line by the procedure described by Nowinsky et al., (Virology 93, 111-116 (1979)).

The medium used for sp 2/0 cell culture and hybridomas was Dulbecco's Modified Eagle's Medium (DMEM), (Gibco, Chagrin Falls, Ohio) supplemented with 20% heat-inactivated fetal bovine serum (Phibro Chem., Fort Lee, NJ), 110 mg / ml. ml sodium pyruvate, -100 U / ml penicillin and 100 mcg / ml streptomycin (Gibco). After cell fusion, the hybridomas were selected in HAT medium containing 10 ⁴ M hypoxanthine, 4x10 ^-7 aminopterin α1, 6x10 ^-5 thymidine for two weeks, then cultured in medium containing hypocantin and thymidine for two weeks.

Hybridomas were screened as follows: polystyrene wells (Costar, Cambridge, MA) were synthesized with 0.25 μg human SCF ^1-164 (E. coli) in 50 μΐ 50 mM bicarbonate buffer pH

9.2 for two hours at room temperature, then overnight at 4 ° C. The plates were then blocked with 5% BSA in PBS for 30 minutes at room temperature, then incubated with the hybridoma culture supernatant for one hour at 37 ° C. The solution was decanted and bound antibodies incubated with a 1: 500 dilution of horseradish peroxidase-conjugated goat-anti-mouse IgG (Boehringer Mannheim Biochemicals, Indianapolis, IN) for one hour at 37 ° C. The plates were washed with wash solution (KPL,

124

Gaitherburg, MD) then developed with a mixture of H ₂ O ₂ and ABTS (KBL).

Colorimetry was performed at 405 nm.

Cultures of hybridoma cells secreting antibody specific for the human SCF ^{1 "164} (E. coli) were tested by ELISA and for screening hybridoma methods, the cross-reactivity of the human SCF ^{1" 162} (CHO). Hybridomas were subcloned by the limiting dilution method. 55 wells of hybridoma supernatant was strongly positive in the assay to the human SCF ^{1 "164} (E.coli); 9 of which are cross-reactive to human SCF ¹¹⁶² (CHO).

Some hybridoma cells were cloned as follows:

monoclonal IgG isotype Reactivity to human SCF ¹¹⁶² (CHO) 4G12-13 IgG not 6C9A IgG not 8H7A IgG Yes

Hybridomas 4G12-13 and 8H7A were deposited at the ATCC on September 26, 1990.

While the present invention has been described by preferred embodiments, it is to be understood that other modifications and variations of this embodiment will be apparent to those skilled in the art. The present claims cover all such possible variations that fall within the scope of the present invention.

Claims (20)

PATENT REQUIREMENTS A DNA sequence for use in expressing a polypeptide product exhibiting a hematopoietic biological property of a naturally occurring stem cell factor in a prokaryotic or eukaryotic host cell, wherein said DNA sequence is selected from: (a) the DNA sequence shown in FIG. 44A-C or their complementary chains, p (b) DNA sequences which hybridize to the DNA sequence -k ^'0 definovanýmtpod (a) or fragments thereof, and (c) DNA sequences that if there was no degeneracy of the genetic code, would hybridize ^ to the DNA sequence defined in (a) and (b) wherein the sequences encode a polypeptide having the same amino acid sequence. and A DNA sequence according to claim 1 comprising one or more codons preferred for expression in non-mammalian cells. The DNA sequence of claim 1 comprising one or more codons preferred for expression in E. coli cells. 4. The DNA sequence of claim 1 comprising one or more codons preferred for expression in yeast cells. The DNA sequence of any one of claims 1 to 4, which encodes the expression of a methionyl residue at the N-terminal position of the mature version of said polypeptide. DNA sequence according to any one of claims 1 to 4, covalently linked to a detectable label. 7. The DNA sequence of claim 1, radiolabeling. 6, where the marking is 126 A purified and isolated polypeptide having all or part of the primary structure of the sequence shown in FIG. 44A-C, optionally with a methionine residue at the N-terminus, exhibiting a hematopoietic biological property of a naturally occurring stem cell factor. The purified and isolated polypeptide of claim 8, comprising the amino acid sequence of FIG. 44A-C: 1-220, 1-161, 1-160 1-157 and 1-152, optionally with a methionine residue at the N-terminus. The purified and isolated polypeptide of claim 1, having hematopoietic activity stimulating the growth of early hematopoietic progenitor cells. A pharmaceutical composition comprising an effective amount of the polypeptide of any one of claims 8 to 10 and a pharmaceutically acceptable diluent, excipient or carrier. Use of a polypeptide according to any one of claims 8 to 10 for the manufacture of a medicament for pre-hematopoietic therapy of a mammal. Use according to claim 12 for the manufacture of a medicament for treating leukopenia, treating thrombocytopenia, treating anemia, increasing bone marrow transplant uptake, increasing bone marrow recovery in radiation therapy, chemically or chemotherapeutically induced bone marrow aplasia or myelosuppression, and for increasing cell sensitivity to chemotherapy. Use according to claim 12 for the manufacture of a medicament for increasing the number of cells capable of transplantation following bone marrow aspiration or peripheral blood leukopheresis. Use of a polypeptide according to any one of claims 8 to 10 for the manufacture of a medicament for the treatment of AIDS, myelofibrosis, myelosclerosis, osteoporosis, metastatic cancer, acute leukemia, multiple myeloma, Hodgkin's lymphoma disease, 127 disease, resistant to congestive splenic fungal vitamin Niemann-Pick disease, erythroblastic anemia, Di splenomegaly, Kala azar, pancytopenia, million-dollar disease, severe septicemia, B ₁₂ acid deficiency Gaucher disease, as well as syndrome, primary sowed pyridoxine deficiency in mammals. Letter-Siw Guglielm sarcoidosis, tuberculosis, malaria, foliar and Use of a polypeptide according to any one of claims 8 to 10 for the manufacture of a medicament for the treatment of nerve damage, mfertility or intestinal damage to a mammal. Use of a polypeptide according to any one of W 10 for the manufacture of a medicament for the treatment of hypopigmentation claims of a disorder. 8 to 4 ^ 7 for more The use according to any one of claims 1 to 2, wherein the medicament further comprises at least a hematopoietic factor selected from EPO, G-CSF, GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5. IL-6, IL-7, IGF-1 12 to 17 and M-GDF. The use according to any of the manufacture of a medicament further comprising a hematopoietic factor selected from IL-8, IL-9 of claims 12 to at least one, and IL-10. 17 -for the next Use according to any of the manufacture of a medicament further comprising a hernopoietic factor selected from IL-11 and LIF. claims 12 to at least one 17 for next Fig. 1