WO1990000563A1 - A method of treating diseases caused by immunodeficiency states by administering human neutrophil chemotactic factor to human - Google Patents

Abstract

This invention pertains to a method for treating diseases caused by immunodeficiency states by administering human neutrophil chemotactic factor (NCF). The amino acid sequence of human NCF is represented by (I).

Description

A METHOD OF TREATING DISEASES CAUSED BY IMMUNODEFICIENCY STATES

BY ADMINISTERING HUMAN NEUTROPHIL CHEMOTACTIC FACTOR TO HUMAN

This invention relates to a method of treating diseases caused by congenital or acquired immunodeficiency states by administering human neutrophil chemotactic factor to human.

Human neutrophil chemotactic factor (abbreviated NCF hereinafter) is a physiologically active polypeptide which is produced by human mononuclear leukocytes stimulated with lipopolysaccharide. NCF has the biological activity of attracting neutrophils, and is proposed as one of the modulating factors involved in the initial stage of the inflammatory reaction (Yoshimura, T. et al., J. Immunol., 139, 788, 1987).

K. Matsushima et al. have succeeded in the isolation of a cDNA encoding the human NCF and in the efficient production of a human NCF polypeptide by recombinant DNA technology. The patent application for this process has been filed on May 2, 1988.

The base sequence of the cloned human NCF cDNA was identical in the coding region with the base sequence reported by Schmid and Weissmann (J. Immunol., 139, 250,

1987). A human NCF polypeptide was found to be a smaller polypeptide with a molecular weight of 8.4 kD based on the complete primary structure of its polypeptide as established by genetic analysis and by N-terminal amino acid sequencing of the NCF derived from human mononuclear leukocytes (Yoshimura, T. et al., Proc.Natl.Acad.Sci., USA, 84, 9233, 1987).

As for the biological activities of the NCF polypeptide, it is known that NCF has the capability to attract neutrophils and to activate neutrophils (Walz et al., Biochem. Biophys.Res.Commun., 149, 755, 1987).

The present inventors have further characterized the recombinant human NCF polypeptide produced in E.coli. Consequently, it has been surprisingly found that the human NCF in addition to attracting neutrophils, also attracts and activates T lymphocytes. These findings indicate that NCF plays an important role in developmental and homeostatic mechanism, such as migration (homing) of T lymphocytes to thymus and lymphnodes and in the modulation of immune responses.

The object of this invention is to propose treatment with an immunotherapeutic agent for patients with malignant tumors and immunodeficiency states by administering human neutrophil chemotactic factor to human.

The other object of this invention is to offer use of a composition containing human NCF for immunotherapy.

Human NCF means not only a polypeptide consisting of an amino acid sequence represented by the formula [I] shown in Table 1, but also so-called human NCF described in literatures. A typical human NCF polypeptide consisting of an amino acid sequence represented by the formula [I] shown in Table 1 can be produced by applying recombinant DNA technology.

The method of production of the human recombinant NCF polypeptide is explained as follows.

The base sequence of a DNA encoding a polypeptide consisting of an amino acid sequence represented by the formula [I], is shown as the formula [A] in Table 2.

The DNA encoding the human NCF polypeptide can be isolated, for example, according to the method as described in Referential Example 1 and the method reported by Schmid and Weissmann as mentioned above. It is also possible to perform the total synthesis of the said DNA chemically by the conventional method. As regards the extra region or deficient region of the resulting DNA, the required DNA can be produced by the methods of the digestion and/or repair such as, for example, digesting by appropriate restriction endonucleases and ligating with chemically synthesized oligodeoxyribonucleotides by DNA ligase.

By adding a translation initiation codon (ATG) to the 5'-terminus of this DNA, ligating a DNA fragment containing a termination codon to the 3'-terminus of the DNA having the initiation codon, connecting the resulting DNA with a proper promoter (e.g. trp, lac, phoS, PL, SV40 early promoter) and SD sequence, and then inserting the resulting DNA having the proper promoter and SD sequence into a proper vector (e.g. plasmid pBR322), an expression vector for production of the human NCF polypeptide is constructed.

The base sequence from the SD sequence to the translation initiation codon is preferably represented by the formula [B].

5'-AAAAGGAGGTTTAAATTATG-3' [B]

A transformant producing for human NCF can be obtained by introducing the expression vector constructed as above into a proper host cell, for example E. coli according to the method of Cohen et al.

(Proc.Natl.Acad.Sci., USA, 69, 2110, 1972).

The human NCF polypeptide can be produced by cultivating the transformant obtained as above under suitable culture conditions. The extract containing the human NCF polypeptide can be obtained from the culture after destroying the cells, for example by lysozyme digestion and freeze-thawing, sonication or by using a French press, followed by collection the extract by centrifugation or filtration.

The human NCF polypeptide can be purified from the extract, for example by combination of treatment for removing nucleic acids, salting-out, anion and/or cation exchange chromatography, ultrafiltration, gel filtration, dialysis, electrophoresis, affinity chromatography using specific antibodies, and so on.

The human NCF polypeptide represented by the formula [I] can also be chemically synthesized by using commercially available peptide synthesizer.

The chemical, physicochemical and biological properties of the recombinant human NCF polypeptide obtained by the method shown in Referential Example 2 are as follows:

(1) Molecular weight

Molecular weight of the recombinant human NCF was measured by SDS-polyacrylamide gel electrophoretic analysis. As molecular weight marker proteins, the standard protein kit (Pharmacia Fine Chemicals, Sweden) consisting of the following proteins was used: lysozyme (14.4kD), soybean trypsin inhibitor (20.1kD), carbonic anhydrase (30kD), ovalbumin (43kD), bovine serum albumin (67kD) and phosphorylase b (94kD).

As a result, the recombinant human NCF had an estimated molecular weight of approximately 8-10kD. No polymerized recombinant human NCF due to the formation of intermolecular disulfide bonds was detected.

(2) Amino acid sequence

An amino acid sequence of the recombinant human NCF was determined by the Automated Edman degradation method .

The recombinant human NCF was treated by reductive cleavage of disulfide bonds with 2-mercaptoethanol, followed by S-β-4-pyridyl-ethylation of cysteine residues with 4-vinylpyridine according to the method of Fullmer (Anal.Biochem., 142, 336, 1984). The resulting product is referred to as the pyridylethylated NCF.

Separately, several kinds of peptide fragments of the recombinant human NCF were prepared and isolated according to the following methods. The recombinant human NCF was treated with 70% formic acid by the method of Sonderegger et al. (Anal.Biochem., 122, 298, 1982) to specially cleave Asp-Pro peptide bond. From the resulting two peptide fragments, the C-terminal fragment (referred to as the FA-1 fragment) was isolated by high-performance liquid chromatography using a column of SynChropak RP-P (SynChrom, Inc., USA) under the elution condition of a linear gradient of acetonitrile concentration from 0% to 50% in 0.1% trifluoroacetic acid. The pyridylethylated human NCF was digested with a metalloendopeptidase (EC 3.4.24; Seikagaku Kogyo, Japan) and the resulting peptide fragments were isolated by high-performance liquid chromatography under the elution condition of a linear gradient of acetonitrile concentration from 0% to 30% in 0.1% trifluoroacetic acid. The two peptide fragments thus obtained are referred to as the K-1 and K-2 fragment, respectively.

N-terminal amino acid sequences of the pyridylethylated NCF and each peptide fragment were determined with a Protein Sequencer, Model 470A (Applied Biosystems, USA) and a SP8440 UV/VIS detector (Spectra- Physics, USA). The determined amino acid sequences of these peptides are shown in Table 3.

Consequently, it has been confirmed that the amino acid sequence of the recombinant human NCF polypeptide is completely identical with the sequence deduced from a base sequence encoding natural human NCF. A methionine residue due to the translation initiation codon (ATG) could not be detected at its N-terminus.

(3) Extinction coefficient

The recombinant human NCF polypeptide was lyophilized without any carrier component. Contents of water and ash in this lyophilized product were determined to be 6.01% and 0.52%, respectively.

Extinction coefficient value of the recombinant human NCF was calculated to be 8.25 at 280 nm under the conditions of 1% aqueous solution and 1cm optical path length.

(4) T lymphocyte chemotactic activity

Migration of lymphocytes was assessed using a modification of a multiwell chemotaxis chamber technique for determination leukocyte chemotaxis (Harvath,L., et al., J. Immunol.Methods, 37 , 39, 1980). The recombinant NCF diluted in Dulbecco's Modified Eagles Medium (DMEM) containing 1 % fetal calf serum was applied to the bottom of a 48 well micro chemotaxis chamber (Neuro Probe, Inc., USA, MD). T lymphocytes were purified from Buffy coat collected from healthy donors (National Institutes of Health, Blood Bank, USA) using a Nylon wool column method. Purity of T lymphocytes was checked using automated immunofluorescence analysis and was greater than 95%. T lymphocytes were applied to the upper 50 microliter chamber at a concentration of 5 × 10⁶ cells/ml. Upper and lower chambers were separated by a 5 micrometer pore-size polyvinyl pyrrolidine-free polycarbonate filter (Nucleopore Corp., USA). Before use, the filter was coated at the lower surface with type IV collagen (Bethesda Research Laboratories, USA) to accelerate the binding of migrated cells. Chambers were incubated for 2 hours at 37ºC in humidified air, and the filters were taken out. After wiping at the upper surface to remove non-migrating cells, the filter was fixed in 70% methanol, dried and stained using Giemsa solution. Lymphocyte migration was estimated on the basis of number of cells attached to the lower surface of the filter. As a result, at 0.01 ng/ml the recombinant NCF resulted in significant chemotactic migration of T lymphocytes. At 1 ng/ml recombinant NCF yielded maximal migration and adherence of migrated T lymphocytes to the collagen coated lower surface of the polycarbonate membrane. The migrated cells were identified as lymphocytes in morphology by microscopic examination of stained cells.

(5) Neutrophil chemotactic activity

Neutrophil chemotactic activity was measured in a multiwell chemotaxis Boyden chamber (Neuro Probe, Inc., USA) as reported by Harvath, L.et al. (J. Immunol. Methods, 37, 39, 1980).

The recombinant human NCF was serially diluted in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% bovine serum albumin (BSA). Normal human neutrophils were purified from Buffy coat collected from healthy donors (National Institutes of Health, Blood Bank, USA) by separating on Ficoll-Hypaque followed by lysing contaminating red blood cells with ACK-lysing buffer. The purity of neutrophils was grater than 95% by morphological analysis of cells stained with Giemsa solution. The viability was also more than 95% as judged by a trypan blue dye exclusion test. The neutrophils were incubated at a cell density of one million cells/ml in DMEM supplemented with 1% BSA for 40 minutes at 37ºC. The migrated neutrophils which adhered onto a membrane (3 μm: Nucleopore Corp., USA) were fixed with methanol and stained with Giemsa solution. As a result, considerable migration of neutrophils was observed at 0.1 ng/ml NCF by examining with a light microscope. At 10 ng/ml maximal migration was observed. The migrated cells were identified as neutrophils morphologically.

( 6 ) In vivo chemotactic activity in mice

The recombinant human NCF was administered intraperitoneally into C3H/He female mice (6-7 week old) at dose of 1 μg/mouse. Mice in the control group were administered phosphate buffered saline (pH 7.2) under the same conditions.

At certain intervals, peripheral blood was withdrawn from each mouse, and peritoneal exudate cells (PEC) were then collected by washing peritoneum with 4 ml of the phosphate buffered saline with heparin, after sacrificing. Numbers of total white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils and others in the peripheral blood and PEC were counted in the conventional manner.

As a result, the number of total WBC in peripheral blood from mice injected with the recombinant human NCF was increased to about 1.7-fold in comparison with that of the control group at 1 hour after the administration of NCF. As indicated by the differential blood count analysis, the increase in total WBC number might be mainly due to increases of neutrophils and lymphocytes. The increase in the ratios of neutrophils and lymphocytes were 2.8-fold and 1.2-fold, respectively, in the experimental over the control group.

On the other hand, the number of total WBC in PEC from mice injected with the recombinant human NCF was increased to about 2.0-fold in comparison with that of the control group at 3 hours after administration. This increase might be due to the increases of neutrophils and lymphocytes with ratios of 63-fold and 1.9-fold, respectively, in the experimental over the control group.

The human NCF polypeptide can be used as an immunotherapeutic agent for immunodeficiency states, malignant tumors and so on.

For formulating the human NCF polypeptide, it may be in the form of a solution or a lyophilized product. From the standpoint of long-term stability, it is preferable to be in the lyophilized state. It is desirable to add vehicles or stabilizers including albumin, globulin, gelatin, protamine, protamine salts, glucose, galactose, xylose, mannitol, glucuronic acid, trehalose, dextran, hydroxyethyl starch, and nonionic surface-active agents (such as polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardened castor oil, polyoxyethylene castor oil, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene polyoxypropylene block copolymer, sorbitan fatty acid esters, sucrose fatty acid esters and glycerin fatty acid esters).

Such preparations are preferably administered topically or parenterally for immunotherapy. For example, topical administration into the tumor mass is preferred for treatment of local tumor tissues. Parenteral administrations such as intravenous and intramuscular routes are used, when activation of immunological responses is intended, for example in the treatment of immunodeficiency states.

The dosage varies depending upon the conditions of the patient and the route of administration. Usually, it is 1 ng to 1 mg per kg body weight, preferably 10 ng to 0.01 mg per kg body weight.

For simplification of the description, the following abbreviations are used in the present specification and claims.

A: adenine

C: cytosine

G: guanine

T : thymine

I: Inosine

dATP: deoxyadenosine triphosphate dCTP: deoxycytidine triphosphate dGTP: deoxyguanosine triphosphate

dTTP: deoxythymidine triphosphate

ATP: adenosine triphosphate

DNA: deoxyribonucleic acid

cDNA: complementary DNA

kbp: kilobase pairs

SD sequence: Shine-Dalgarno sequence

kD: kilodaltons

SDS: sodium laurylsulfate

In the present specification and claims, the nucleotide sequence shown by a single strand is the nucleotide sequence of a sense strand, and the left end is a 5'-terminus and the right end is a 3'-terminus. In the amino acid sequence, the left end is an N-terminus, and the right end is a C-terminus.

The following Referential Examples illustrate this invention more specifically.

It should be understood however that the invention is in no way limited to these examples.

For a better understanding of the following Referential Examples, Schemata 1 to 3 are attached to the present specification.

Schema 1 shows a process of constructing an expression plasmid pHNP101 (Referential Example 2);

Schema 2 shows a process of constructing an expression vector pEP205 (Referential Example 3); Schema 3 shows a process of constructing an expression plasmid pHIPH383a (Referential Example 4).

Referential Example 1

Cloning of cDNA encoding human NCF

The cDNA library was constructed by insertion of cDNA synthesized from normal human monocyte polyadenylated mRNA obtained from the monocyte stimulated with 10 μg/ml lipopolysaccharide for 6 hours, into the EcoRI site of the phage vector λgt10. One half million individual plaques were screened for hybridization with [³²P]-labeled two kinds of chemically synthesized oligonucleotide probes represented by the following formulae, [C] and [D]:

and

In the first screening, 13 putative positive clones were obtained. From these positive clones, one clone (termed r-MDNCF 2-1) was selected in the second screening by using the another probe. The phage DNA in r- MDNCF 2-1 was subcloned into the pUC19 plasmid.

The resulting recombinant plasmid was termed pUC19-1.7-5. The cloned cDNA in a recombinant plasmid pUC19-1.7-5 contains a nucleotide sequence encoding human NCF shown in Table 4.

Referential Example 2 Production of human NCF polypeptide

Human NCF polypeptide having an amino acid sequence represented by the formula [I] in Table 1, was produced by the following methods.

(1) Construction of an expression plasmid pHNP101

From the recombinant plasmid pUC19-1.7-5 in which human NCF cDNA was inserted as mentioned in Referential Example 1 , DNA fragment encoding the polypeptide corresponding to the amino acids from the 18th position to the 97th position from the N-terminus of human NCF precursor polypeptide (corresponding to the base sequence from the base No. 52 to No. 291 in Table 4) was isolated by digestion with restriction endonucleases Pstl and EcoRI. This DNA fragment was then cloned into a phage vector M13mp18 (Takara Shuzo Co., Ltd., Japan) at a region between the restriction endonuclease cleavage site of Pstl and that of EcoRI in the polylinker sequence. By using the resultant recombinant phage DNA, a specific base sequence being 5'-TTTAAATTATG-3' was inserted between the codon corresponding to Arg at the 27th position from the N-terminus of the human NCF precursor polypeptide and the codon corresponding to Ser at the 28th position, by the technique of site-directed mutagenesis according to the method of Kunkel et al. (Methods in Enzymol., 154, 367, 1987). The site-directed mutagenesis was carried out using a Muta-Gene in vitro mutagenesis kit according to the instruction manual (Bio-Rad Labs., USA). E. coli JM105 was infected with the recombinant phage DNA, and then it was cultivated to collect the recombinant phage. Then, E. coli CJ236 was infected with the recombinant phage obtained as above and cultivated in 2×TY medium [composition; 1.6% tryptone, 1% yeast extract, 0.5% sodium chloride] supplemented with uridine (1 μg/ml) and chloramphenicol (20 μg/ml) at 37ºC for 5 hours. The single-stranded phage DNA containing uracils was isolated from the culture medium.

Separately, a mutagenic oligodeoxyribonucleotide primer consisting of 33 bases represented by the following formula [E] was chemically synthesized.

5'-GTTTTGCCAAGGTTTAAATTATGAGTGCTAAAG-3' [E]

The 5'-end of the mutagenic primer was previously phosphorylated. The phosphorylated primer was annealed with the single-stranded phage DNA containing uracils prepared as above in an annealing buffer [20 mM Tris-HCl buffer (pH 7.4) containing 2 mM magnesium chloride and 50 mM sodium chloride] by incubating at 70ºC for 10 minutes, followed by cooling down to 30ºC at a rate of 1ºC per minute. Then, the primer was extended with T4 DNA polymerase in a synthesis buffer [10 mM Tris-HCl buffer (pH 7.4) containing 0.4 mM each deoxynucleoside triphosphate (dGTP, dATP, dCTP, dTTP), 0.75 mM ATP, 3.75 mM magnesium chloride and 1.5 mM dithiothreitol] to synthesize a complementary strand and the ends were ligated with T4 DNA. ligase by sequential incubating on ice for 5 minutes, at 25ºC for 5 minutes and at 37ºC for 90 minutes. The reaction was stopped by freezing at -20ºC. The circular double-stranded DNA (heteroduplex) was introduced into E. coli JM105 cells, and they were cultivated to isolate the mutated double-stranded replicative form DNA. The nucleotide sequence of the mutated DNA was confirmed by sequencing the single-stranded DNA isolated from the culture medium.

The resultant mutated double-stranded DNA was digested with restriction endonucleases Dral and EcoRI in order to isolate a DNA fragment containing most of the coding region for human NCF polypeptide. The isolated DNA fragment is, hereinafter, referred to as the NCF (Dral-EcoRI)-fragment.

This NCF (Dral-EcoRI)-fragment was ligated by T4 DNA ligase with a chemically synthesized oligodeoxynucleotide adaptor represented by the following formula [F].

5'-AATTCATGATGAC

3'-GTACTACTGAGCT [F]

The resultant ligated DNA fragment is referred to as the NCF (Dral-Xhol)-fragment.

Separately, an expression plasmid pEP205 as mentioned in Referential Example 3 was digested with restriction endonucleases Dral and Xhol, and the resulting larger DNA fragment including an ampicillin-resistance gene and a replication origin (hereinafter referred to as the EP205 vector-DNA fragment) was isolated, and this EP205 vector-DNA fragment was ligated by T4 DNA ligase with the NCF (Dral-Xhol)-fragment previously prepared in order to construct an expression plasmid pHNP101 for producing human NCF (see Schema 1).

(2) Transformation of Escherichia coli

The resulting expression plasmid pHNP101 was introduced into E.coli HB101 by the following manner.

E. coli HB101 was inoculated in the LB broth [composition; 1% tryptone, 0.5% yeast extract, 1% sodium chloride (pH 7.5)], and cultivated overnight at 30ºC. One milliliter of the resulting culture was inoculated in 100 ml of LB broth and further cultivated at 30ºC until the turbidity at 600 nm of the culture reached approximately 0.6. After standing for 30 minutes in ice water, the cells were collected by centrifugation. They were resuspended in 50 ml of 50 mM calcium chloride, and allowed to stand for 60 minutes in ice water. Then, the cells were collected by centrifugation and again suspended in 10 ml of 50 mM calcium chloride containing 20 % glycerol.

To this cell suspension, the expression plasmid pHNP101 was added and incubated sequentially in ice water for 20 minutes and at room temperature for 60 minutes. Then, the LB broth was added to the cell suspension, and it was cultivated under shaking at 37ºC for 60 minutes. An aliquot of the resulting cell suspension was seeded on the LB agar (1.5% agar) plates containing 25 μg/ml of ampicillin. After cultivation at 37ºC overnight, ampicillin-resistant colonies were selected to obtain transformants. One of the transformants was named E.coli HB101/pHNP101 and it was used for production of the human NCF polypeptide.

(3) Production of human NCF polypeptide

E.coli HB101/pHNP101 obtained in section (2) was cultivated in the LB broth overnight at 37ºC. The culture was inoculated in 100-fold volumes of the nutrient medium [composition; 1.5% sodium phosphate, dibasic 12-water, 0.3% potassium phosphate, monobasic, 0.1% ammonium chloride, 2 mg/liter vitamin B₁, 0.5% casamino acid, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.4% glycerol] and then, 3-indoleacrylic acid was added to give a final concentration of 20 μg/ml. The cultivation v/as done at 35ºC for 28 hours. The cells were collected by centrifugation, and suspended in 50 mM Tris-HCl buffer ( pH 8.0) containing 0.1% lysozyme and 30 mM sodium chloride. The suspension was allowed to stand in ice water for 30 minutes. Further, freezing in a dry ice/ethanol bath and thawing at 37ºC were repeated to disrupt the cells. After adding 1/50 volume of 10% ethyleneimine polymer, a clarified cell-extract was obtained by centrifugation. To this cell-extact, ammonium sulfate was added to give a 70% saturation, and the formed precipitate was collected by centrifugation. The precipitate was dissolved in distilled water and it was dialyzed against 5 mM phosphate buffered saline (pH 6.5) (hereinafter referred to as PBS). The dialysate was applied onto a column of Sephacryl S-200 (Pharmacia, Sweden), and the fractions containing polypeptides having about 6 to 10 kD molecular weight were collected and pooled. The molecular sizes of polypeptides in each eluate were measured by SDS-polyacrylamide gel electrophoretic analysis. The pooled fraction was dialyzed against 20 mM phosphate buffer (pH 6.5) (hereinafter referred to as PB). Then, the dialysate was applied onto a column of CM-Sepharose CL-6B (Pharmacia, Sweden) previously equilibrated with PB. The column was washed with PB, and eluted with a linear gradient of sodium chloride molarity from 0 M to 0.5 M in PB. The fractions containing the human NCF polypeptide were collected and pooled, and concentrated by ultrafiltration. Further, the concentrate was subjected to gel filtration on Toyopearl HW-55 column (TOSOH Co., Japan) to obtain the highly purified human NCF polypeptide. By SDS -polyacrylamide gel electrophoretic analysis, any impurity was not detected in the highly purified human NCF polypeptide preparation.

This human NCF preparation was used for chemical, physicochemical and biological analyses as shown previously.

Referential Example 3

Construction of an expression vector pEP205

Plasmid pBR322 was digested with restriction endonucleases Aval and PvuII, and the resulting larger DNA fragment (about 3.7 kbp in size) was isolated. After filling-in its cohesive ends to blunt-ends with E.coli DNA polymerase I (Klenow fragment) in the presence of dGTP, dATP, dCTP and dTTP, both ends were ligated by T4 DNA ligase to construct a new plasmid vector (designated pBRS6), which was deleted a copy number regulatory gene region located near the replication origin of the plasmid pBR322.

The plasmid vector pBRS6 was digested with restriction endonucleases EcoRI and Pstl, and a smaller DNA fragment containing an upstream region of the ampicillin-resistance gene (about 0.75 kbp in size) was isolated. The resultant DNA fragment is referred to as the Amp (Pstl-EcoRI)-fragment.

This Amp (Pstl-EcoRI)-fragment was cloned in a phage vector M13mp18 as 'mentioned in Referential Example 2. By using the resultant recombinant phage DNA, one base (T) in the nucleotide sequence of the Amp (Pstl-EcoRI)-fragment was changed to another base (C) by the site-directed mutagenesis according to the method as mentioned in Referential Example 2, in order to eliminate the specific nucleotide sequence (AAATTT) recognizable with the restriction endonuclease Dral.

The single-stranded phage DNA containing uracils was isolated from the culture medium of E. coli CJ236 infected with the above recombinant phage DNA. As a mutagenic primer, the oligodeoxyribonucleotide represented by the following formula [G] was chemically synthesized.

5'-CAGAACTTTGAAAGTGCTC-3' [G]

The phosphorylated primer was annealed with the uracil-containing DNA template. According to the method described in Referential Example 2, the desired mutated double-stranded DNA was isolated.

The resultant mutated double-stranded DNA was digested with restriction endonucleases Pstl and EcoRI in order to isolate a DNA fragment corresponding to the Amp (Pstl-EcoRI)-fragment as mentioned above, but not containing the restriction endonuclease Dral cleavage recognition sequence [hereinafter referred to as the mutated Amp (Pstl-EcoRI)-fragment]. The mutated Amp (Pstl-EcoRI)-fragment was ligated with the larger DNA fragment isolated from the vector pBRS6 by digestion with restriction endonucleases EcoRI and Pstl, in order to construct a new vector which was eliminated the Dral cleavage recognition sequence in the ampicillin resistance gene of the plasmid vector pBRS6. This new vector is designated pBRS601.

Further, this new vector pBRS601 was digested with restriction endonuclease Dral, and the resulting larger DNA fragment was isolated. The larger DNA fragment was ligated with Smal linker (Takara Shuzo Co., Ltd., Japan) by T4 DNA ligase to construct a new plasmid vector. This resulting new plasmid vector is a derivative of plasmid pBRS6 and is not containing any recognition sequences for the restriction endonuclease Dral. This new plasmid vector is designated pBRS602.

The nucleotide sequence of the Smal linker is shown below.

5'-CCCGGG-3'

Furthermore, this new vector pBRS602 was digested with restriction endonuclease Aatll and SalI, and the resulting larger DNA fragment was isolated

[hereinafter referred to as the pBRS602 (Aatll-SalI)-fragment].

Separately, an expression plasmid pHIPH383a for producing human interleukin-1α as mentioned in Referential Example 4, was digested with restriction endonucleases Aatll and SalI, and the resulting DNA fragment containing E.coli tryptophan promoter sequence and the coding region for human interleukin-1α was isolated. This resulting DNA fragment is referred to as the trp promoter/IL-1 α-DNA fragment.

This trp promoter/IL-1α-DNA fragment was ligated with the pBRS602 (Aatll-SalI)-fragment by T4 DNA ligase to construct a new expression plasmid (see Schema 2).

This new expression plasmid is designated pEP205.

Referential Example 4

Construction of an expression plasmid pHIPH383a

The cloned cDNA encoding human interleukin-1α precursor polypeptide was isolated according to the method described in European Patent Publication No. 0188920.

From the recombinant plasmid pHL4 containing human interleukin-1α precursor cDNA (Furutani, Y., et al., Nucleic Acids Res., 13, 5869, 1985), the cDNA insert was isolated by digestion with restriction endonuclease Pstl, and further digested with restriction endonucleases EcoRI and BstNI, to isolate a DNA fragment (411 bp in size) containing a middle portion of the coding region for the mature human interleukin-1α. The isolated DNA fragment is corresponding to the nucleotide sequence from base No.398 to No.808 in Table 5 shown in European Patent Publication No.0188920.

This DNA fragment was sequentially ligated by T4 DNA l i ga s e with ch emi ca l l y s ynthe s i z ed oligodeoxyribonucleotide adaptors represented by the following formulae [H] and [J]. The resulting DNA fragment is referred to as the SD-IL-1-fragment.

The synthetic oligodeoxyribonucleotide adaptor [H] was prepared by sequential ligation of the following five kinds of DNA fragments represented by formulae [a]- [e].

5'-AACTAGTACGCAAGTTCAC

3'-TTGATCATGCGTTCAAGTGCATT [a]

5'-GTAAAAGGAGGTTTAAA

3'-TTCCTCCAAATTTAATAC [b]

5'-TTATGTCATCACCTTTTAG

3'-AGTAGTGGAAAATCGAAGG [c]

5'-CTTCCTGAGCAATGTGAAATACAACTTTA

3'-ACTCGTTACACTTTATGTTGAAATACTC [d] and

5'-TGAGGATCATCAAATACG

3'-CTAGTAGTTTATGCTTAA [e]

A base sequence of the formula [J] was as follows;

5'-AGGCGTGATGACTCGA

3'-CCGCACTACTGAGCTCTAG [J]

Separately, an expression vector pEP302 (Furutani, Y., et al., Nucleic Acids Res., 13, 5869, 1985) was digested with restriction endonucleases Hpal and BamHI , and the resulting larger DNA fragment containing E.coli tryptophan promoter sequence and an ampicillin resistance gene, was isolated (hereinafter referred to as the EP302 vector-DNA fragment).

The EP302 vector-DNA fragment was ligated by T4 DNA ligase with the SD-IL-1 -fragment prepared as above to construct an expression plasmid pHIPH383a for producing the mature human interleukin-1α polypeptide (see Schema 3).

Table 1

SerAlaLysGluLeuArgCysGlnCysIleLysThr TyrSerLysProPheHisProLysPhelleLysGlu LeuArgVallleGluSerGlyProHisCysAlaAsn ThrGluIlelleValLysLeuSerAspGlyArgGlu LeuCysLeuAspProLysGluAsnTrpValGlnArg ValValGluLysPheLeuLysArgAlaGluAsnSer formula [I]

Table 2 5'-AGTGCTAAAGAACTTAGATGTCAGTGCATAAAGACA

TACTCCAAACCTTTCCACCCCAAATTTATCAAAGAA CTGAGAGTGATTGAGAGTGGACCACACTGCGCCAAC ACAGAAATTATTGTAAAGCTTTCTGATGGAAGAGAG CTCTGTCTGGACCCCAAGGAAAACTGGGTGCAGAGG GTTGTGGAGAAGTTTTTGAAGAGGGCTGAGAATTCA-3' formula [A]

Table 3

Determined Amino Acid Sequence of Recombinant Human NCF Polypeptide

SerAlaLysGluLeuArgCysGlnCysIleLysThrTyrSerLysProPheHisProLys 20 ←MP - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MP

PhelleLysGluLeuArgVallleGluSerGlyProHisCysAlaAsnThrGluIlelle 40 M P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -MP→

←K-1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K-1

ValLysLeuSerAsDGlyArgGluLeuCysLeuAspProLysGluAsnTrpValGlnArg 60 K-1→←K-2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K-2→

←FA-1 - - - - - - - - - - - - - - - - FA-1

ValValGluLysPheLeuLysArgAlaGluAsnSer 72

FA-1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FA-1→

Table 4

Nucleotide Sequence of Human NCF Precursor cDNA and Its Deduced Amino Acid Sequence

MetThrSerLysLeu AlaValAlaLeuLeu 1 0

ATGACTTCCAAGCTG GCCGTGGCTCTCTTG 30

AlaAlaPheLeuI le SerAlaAlaLeuCys 20

GCAGCCTTCCTGATT TCTGCAGCTCTGTGT 60

GluGlyAlaValLeu ProArgSerAlaLys 30

GAAGGTGCAGTTTTG CCAAGGAGTGCTAAA 90

GluLeuArgCysGln CysIleLysThrTyr 40

GAACTTAGATGTCAG TGCATAAAGACATAC 1 20

SerLysProPheHis ProLysPhel leLys 50

TCCAAACCTTTCCAC CCCAAATTTATCAAA 1 50

GluLeuArgVallle GluSerGlyProHis 60

GAACTGAGAGTGATT GAGAGTGGACCACAC 1 80

CysAlaAsnThrGlu IlelleValLysLeu 70

TGCGCCAACACAGAA ATTATTGTAAAGCTT 21 0

SerAspGlyArgGlu LeuCysLeuAspPro 80

TCTGATGGAAGAGAG CTCTGTCTGGACCCC 240

LysGluAsnTrpVal GlnArgValValGlu 90

AAGGAAAACTGGGTG CAGAGGGTTGTGGAG 270

LysPheLeuLysArg AlaGluAsnSer 99

AAGTTTTTGAAGAGG GCTGAGAATTCA 297

Claims

What is claimed is:

1. A method of treating diseases caused by

immunodeficiency states by administering human neutrophil chemotactic factor to human.

2. A method according to claim 1, wherein the human neutrophil chemotactic factor is a polypeptide consisting of an amino acid sequence represented by the formula.

SerAlaLysGluLeuArgCysGlnCysIleLysThr

TyrSerLysProPheHisProLysPhelleLysGlu

LeuArgVallleGluSerGlyProHisCysAlaAsn

ThrGluIlelleValLysLeuSerAspGlyArgGlu

LeuCysLeuAspProLysGluAsnTrpValGlnArg

ValValGluLysPheLeuLysArgAlaGluAsnSer

3. A method according to claim 2, wherein the human neutrophil chemotactic factor polypeptide is a polypeptide produced in microorganisms by recombinant DNA technology.

4. A method of treating immunodeficiency states or malignant tumors by administering in an effective amount a human neutrophil chemotactic factor polypeptide consisting of an amino acid sequence represented by the formula

SerAlaLysGluLeuArgCysGlnCysIleLysThr

TyrSerLysProPheHisProLysPhelleLysGlu

LeuArgVallleGluSerGlyProHisCysAlaAsn

ThrGluIlelleValLysLeuSerAspGlyArgGlu

LeuCysLeuAspProLysGluAsnTrpValGlnArg ValValGluLysPheLeuLysArgAlaGluAsnSer

to human.

5. A method of activating T lymphocytes by administrating human neutrophil chemotactic factor to human.

6. A T iyrophocytes activating agent comprising the human neutrophil chemotactic factor polypeptide.