KR20060108375A - Human granulocyte-colony stimulating factor isoform and composition thereof - Google Patents

Abstract

The present invention relates to a human granulocyte colony forming factor homolog having an amino acid sequence modified, in particular, a human granulocyte colony forming factor having an amino acid sequence of SEQ ID NO: 1 having a sustained in vivo by modifying an amino acid sequence of a specific site, Lys40, Human granulocyte colony forming factor homologues modified with amino acids of any of His43, Glu45, Glu46, Val48, Leu49, and Phe144, pharmaceutical compositions comprising the same, genes encoding the homologues, expression vectors comprising the genes , Oligodeoxynucleotides used as primers to modify the amino acid sequence of human granulocyte colony forming factor.

hG-CSF, isoform, human granulocyte colony forming factor homologue, human granulocyte colony forming factor receptor, in vivo stability

Description

Human Granulocyte-Colony Stimulating Factor Isoform and Composition Equivalent

Figure 1 shows the sequence of human granulocyte colony forming factor gene and amino acid.

Figure 2 shows the amino acid modification position according to the present invention in the amino acid sequence of human granulocyte colony forming factor.

Figure 3 shows a schematic diagram of the expression vector pCJKM-GCSF isoform.

Figure 4 shows the expression results of transformants of human granulocyte colony forming factor homologues.

Figure 5 shows the results of electrophoresis of purified human granulocyte colony forming factor.

Figure 6 shows the effect of human granulocyte colony forming factor in the cell line.

The present invention relates to human granulocyte colony forming factor homologues in which at least one amino acid is modified. More specifically, the present invention is directed to binding an immunoglobulin-like region of a human granulocyte colony forming factor receptor by modifying the amino acid sequence for the site of binding of the human granulocyte colony forming factor to its receptor in order to increase the in vivo stability of the protein. The present invention relates to human granulocyte colony forming factors having increased in vivo persistence by making human granulocyte colony forming factors homologues in which the amino acid sequence of a specific site known as an amino acid in a related or adjacent site is modified to another amino acid.

Nicolae et al. (J. Biol. Chem., 1983, 258, 9017-9023) is a glycoprotein group that acts on the proliferation, differentiation and activation of hematopoietic cells in granulocyte-colony stimulating factor (G-CSF). In addition, it has been reported that it promotes the proliferation and differentiation of neutrophil progenitor cells outside the bone marrow to increase neutrophil release, which accounts for more than 70% of white blood cells.

When the number of neutrophils is generated and present by the granulocyte colony forming factor above the normal level, the extra granulocyte colony forming factor has a mechanism of binding to and removing the granulocyte colony forming factor receptor present in the neutrophils, thereby reducing the number of neutrophils in the blood. Adjust

According to Jerkman et al. (Eeu. J. Haematol., 2002, 69, 265-274), the stability of physiologically active proteins in vivo is determined by the degradation of glycoproteins degraded by proteases and glycolytic enzymes in the liver. Hepatocytic galactose receptor-mediated uptake, renal filtration through glomerular filtration in the kidney, and receptor-mediated uptake by receptor binding . However, in the case of granulocyte colony forming factors produced from E. coli, there is no glycosylation, so glycolytic enzymes or glycoprotein breakdown in the liver should not be considered, whereas filtration in the kidneys may be necessary to remove granulocyte colony forming factor. It is an important factor. In the presence of neutrophils in the blood, relatively low concentrations of granulocyte colony forming factor are also important factors in vivo persistence of human granulocyte colony forming factor.

The structures of granulocyte colony forming factors and receptors include immunoglobulin-like domains (Ig-like domains), cytokine receptor homology (CRH), and three fibronectin type III sites at the N-terminus. domain), the transmembrane domain, and the cytoplasmic domain at the C-terminus, Fukunaga et al. (Proc. Naltl. Acad. Sci. USA, 1990, 87, 8702-8706; Cell, 1990, 61, 341-350.

The function of the immunoglobulin-like domain is known to promote signal transduction by forming polymers between granulocyte colony forming factors and receptors after granulocyte colony forming factors contact with their receptors. Cytokine Receptor Homology (CRH) has a conserved cysteine site and WSXWS sequence and is known to be involved in signal transduction of granulocyte colony forming factors.

Rythhal-Olson et al. (Biochemistry, 1996, 35, 9034-9041) reported that amino acids Lys40, Val48, Leu49, and Phe144 are involved in binding to human granulocyte colony forming factor receptors among the amino acid sequences of human granulocyte colony forming factors. Raton et al. (J. Biol. Chem., 2001, 276, 36779-36787) reported that Glu46, Leu49, and Phe144 are immunoglobulin-like regions of human granulocyte colony forming factor (hG-CSF) receptors. Lys40 and Val49 are located nearby, so contact with the receptor is not clear. Aritomy et al. (Nature, 1999, 401, 713-717) describe Gly4 of human granulocyte colony forming factor protein in the X-ray structure of the complex of human granulocyte colony forming factor and the BN-BC site (CRH domain) of its receptor. , Pro5, Ala6, Ser7, Ser8, Leu9, Pro10, Gln11, Ser12, Leu15, Lys16, Glu19, Gln20, Leu108, Asp109, Asp112, Thr115, Thr116, Gln119, Glu122, Glu123, and Leu124 , Laton et al. (J. Biol. Chem., 1999, 274, 17445-17451), said that Glu19 of human granulocyte colony forming factor functions to transmit signals of human granulocyte colony forming factor by interacting with Arg288 of the receptor. Said.

Saka et al. (Nature Biotechnology, 2002, 20, 908-913) described the position of the D110 and D113 positions of human granulocyte colony forming factors to reduce the binding affinity with receptors on endosomes inside cells of human granulocyte colony forming factors. The replacement of aspartic acid amino acid with histidine reported that the human granulocyte colony forming factor was released from the endosome and reused to increase its persistence.

Bishop et al. (J. Biol. Chem., 2001, 276, 33465-33470) showed increased stability when Gly26, Gly28, Gly149, Gly150 was replaced with alanine.

United States Patent Nos. 5,214,132; 5,399,345; 5,790,421; 5,581,476; 4,999,291; 4,810,643; 4,833,127; 5,218,092; 5,362,853; 5,830,705; 5,580,755; 5,399,345; 5,416,195; 6,627,186; 6,790,628; U.S. Patent Publication 20030171559 et al. Disclose variants of G-CSF to increase the activity or half-life of hG-CSF. In addition, U.S. Patent Nos. 6,831,158; 6,790628; 6,646,110; 6,555,660 and the like describe conjugates of G-CSF wherein one or more polypeptides and one or more non-polypeptide moieties are formed by covalent bonds.

As described above, various efforts have been made to increase the activity and in vitro stability of hG-CSF, but there are no specific research results with increased in vivo persistence.

Granulocyte colony forming factor gene was cloned in human bladder cancer cell 5637. The use of these cDNAs to produce recombinant human G-CSF in Escherichia coli and animal cells has been actively conducted, and the granulocyte colony forming factor produced by E. coli is activated by human granulocyte colony forming factor produced from natural or animal cells. Was equivalent. However, commercially available human granulocyte colony forming factors have low in vivo stability, and thus, excessive and frequent administration of the human granulocyte colony forming factors required to stimulate neutrophil production must be achieved. As a result, the human granulocyte colony forming factor has been actively developed to increase the in vivo stability that can alleviate the pain and discomfort of the patient.

In the United States, a drug has recently been approved in which the N-terminus is linked to polyethylene glycol and human granulocyte colony forming factors. However, this method is accompanied by the hassle of having to perform the addition reaction after the production and purification of the protein from the microorganisms in the first place, there may be a problem in the homogeneity of the final product because cross-linking may occur at an unwanted position. In addition, there is a method of using glycosylation, but proteins produced by eukaryotic cells have a disadvantage in that the production efficiency of glycosylated proteins is lowered since sugar chain modification is easily removed during production.

Accordingly, an object of the present invention is to provide a G-CSF having excellent in vivo sustainability without complicated production process. Specifically, in the present invention, an amino acid present at or adjacent to an immunoglobulin-like site that does not participate in signal transduction and only provides efficiency in signal transduction in the binding of the sarcophage colony forming factor to its receptor The aim is to increase the persistence in vivo by lowering the affinity with the receptor by modifying the sequence. More specifically, in the human granulocyte colony forming factor having the amino acid sequence of SEQ ID NO: 1, human granulocyte colony formation in which the amino acid at any one of Lys40, His43, Glu45, Glu46, Val48, Leu49, and Phe144 is modified Provide factor homologues. Met residue may be present at the N-terminus of the -1 position in the sequence. Preferably, the modification is any one of K40R, H43L, E45D, E46D, E46Q, E46G, V48L, L49I, L49V, and F144Y, even more preferably the modification is H43L or E46G.

In another aspect, the present invention provides a pharmaceutically acceptable human homolog of the human granulocyte colony forming factor homolog having a modified amino acid sequence region of the human granulocyte colony forming factor, which has an effect on the specific site and does not affect signaling. It provides a pharmaceutical composition comprising a carrier.

In another aspect, the present invention encodes (encodes) a human granulocyte colony forming factor homolog in which an amino acid sequence region of a human granulocyte colony forming factor has a function with a receptor at the specific site and does not affect signaling. To provide genes.

In another aspect, the present invention provides a gene for encoding a human granulocyte colony forming factor homologue in which the amino acid sequence region of the human granulocyte colony forming factor has a function with a receptor at the specific site and does not affect signaling. To provide an oligodeoxynucleotide used as a primer for. Preferably, the oligodeoxynucleotide is an oligodeoxynucleotide represented by SEQ ID NOs: 2 to 21.

As used herein, the term isoform of human granulocyte colony forming factor has been modified from at least one of the native amino acid sequence residues of the native human granulocyte colony forming factor to another amino acid residue but retains its unique activity. By analog or variant.

As used herein, the three letter alphabet of amino acids means the following amino acids in accordance with standard abbreviation provisions in the field of biochemistry:

Ala (A): alanine; Asx (B): asparagine or aspartic acid; Cys (C): cysteine;

Asp (D): aspartic acid; Glu (E): glutamic acid; Phe (F): phenylalanine;

Gly (G): glycine; His (H): histidine; IIe (I): isoleucine; Lys (K): lysine;

Leu (L): Leucine; Met (M): methionine; Asn (N): asparagine; Pro (P): proline;

ln (Q): glutamine; Arg (R): arginine; Ser (S): serine; Thr (T): threonine;

Val (V): valine; Trp (W): tryptophan; Tyr (Y): tyrosine; Glx (Z): Glutamine

Or glutamic acid.

As used herein, "(amino acid letter) (amino acid position) (amino acid letter)" means that the amino acid previously indicated at the corresponding amino acid position of the human granulocyte colony forming factor is replaced by the amino acid indicated later. For example, K40R indicates that lysine corresponding to No. 40 of the native human granulocyte colony forming factor is substituted with arginine.

In the present specification, for substitution of the corresponding amino acid, the primer is designated as "(amino acid letter) (amino acid position) (amino acid letter) 1 or 2", where 1 is a 5 '→ 3' direction of the double stranded template. A primer complementary to a single stranded template and 2 refers to a primer complementary to a single stranded template that runs in the 3 '→ 5' direction in the double strand.

In an embodiment of the present invention, the gene encoding human granulocyte colony forming factor gene can be secured through human granulocyte forming factor producing strain for animal cell expression as described in the references cited above. Normally, cloning and isolation of genes can employ methods known in the art. In addition, the DNA sequence encoding the human granulocyte colony forming factor or its homologue according to the present invention may be prepared by a standard method known in the art, for example, using an automated DNA synthesizer (eg, BioSearch, Applied Biosystem ^TM ). You can also synthesize.

Human granulocyte colony forming factor genes obtained through this process can be modified in one or more selected codons. Modification in the context of the present invention can be defined as causing a change in the amino acid sequence of human granulocyte colony forming factor by substituting one or more codons on the gene encoding human granulocyte colony forming factor. More specifically, it refers to the substitution of an amino acid with another amino acid for modification of the K40, H43, E45, E46, V48, L49, F144 sequences on the human granulocyte colony forming factor amino acid sequence.

As an example of the present invention, K40R indicates that lysine corresponding to No. 40 of the natural human granulocyte colony forming factor is substituted with arginine. In an aspect of the present invention, synthetic oligonucleotides are prepared comprising codons encoding the desired amino acid modifications in human granulocyte colony forming factors. Generally about 27-32 nucleotides of oligonucleotides in length are used. Although shorter oligonucleotides may be introduced, the optimal oligonucleotide is one having 12 to 15 nucleotides that are complementary to the template on both sides of the nucleotide encoding the modification. Such oligonucleotides can be sufficiently hybridized to template DNA. Synthetic oligonucleotides used for the production of amino acid modifications in the present invention are listed in Table 1. Such oligonucleotides can be synthesized by techniques known in the art.

In an aspect of the present invention, human granulocyte colony forming factor homolog DNA is modified with one amino acid. PCR is performed using human granulocyte colony forming factor DNA as a template and synthetic oligonucleotides encoding modifications as primers. When the double stranded template is separated in the heating step of PCR, primers complementary to each single stranded template are hybridized. DNA polymerase connects the nucleotides complementary to the template in the 5 ′ → 3 ′ direction from the —OH group of the primer encoding the modification. As a result, the second strand will contain a primer that encodes the modification, thus coding for the desired modification on the gene. The second strand will act as template DNA in the repeated replication steps of PCR, so the gene encoding the modification will continue to be amplified.

For example, in the embodiment of the present invention, K40R has a primer type N-term seq using a natural granulocyte colony forming factor DNA as a template to replace lysine corresponding to No. 40 of natural human granulocyte colony-type adult with arginine. PCR was performed by pairing K40R2, K40R1, and C-term seq, respectively. As a result, two DNA fragments modified with codons corresponding to arginine instead of lysine at the 40th amino acid position were obtained. Put two DNA fragments obtained in this way and perform a second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony-forming homolog K40R modified with arginine instead of lysine at the 40th amino acid position. Was able to obtain a modified gene.

Homologs according to the present invention generally comprise one or more expression control sequences that ⒜ operatively linked the DNA sequence encoding the human granulocyte colony forming factor homolog to the DNA sequence. inserted into a vector comprising the expression control sequence, ⒝ transforms or transfects the host with a recombinant expression vector formed therefrom, and ⒞ expresses the resulting transform or transfectant human granulocyte colony forming factor homolog DNA sequence. Prepared by isolating human granulocyte colony forming factor homologues by cultivation under appropriate media and conditions whenever possible.

Affinity with the receptors of cultured and purified human granulocyte colony forming factor homologues and their in vivo persistence were investigated. Analysis of Human Granulocyte Colony Forming Factors The growth curves of the human granulocyte colony forming factors were added to the culture medium of the cell line (M-NFS60) and compared to the growth curves of the recombinant human granulocyte colony forming factors which did not cause modifications in amino acid sequences. . At this time, homologues having low affinity with the receptor are degraded in the efficiency of absorption, degradation, ie, receptor mediated degradation by receptor binding into cells, and remain in the culture medium for a relatively long time.

In a related aspect, the present invention provides recombinant expression vectors comprising DNA sequences encoding human granulocyte colony forming factor homologues and host cells transformed or transfected with such expression vectors.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Also in selecting a host, compatibility with the selected vector, toxicity of the products encoded by the nucleotide sequence, their secretion properties, the ability to correctly fold the polypeptide, fermentation or culture requirements, and nucleotides Consideration should be given to the ease of purification of the product encoded by the sequence.

As used herein, the term "vector" refers to a DNA molecule as a carrier that can stably transport a foreign gene into a host cell. To be a useful vector, it must be replicable, have a way to enter the host cell, and have the means to detect its presence. The term "recombinant expression vector" also generally refers to a circular DNA molecule formed operably linked to the vector so that the foreign gene can be expressed in the host cell. Recombinant expression vectors can generate several copies and inserted heterologous DNA. As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operatively linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequences and the corresponding genes are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host cell.

The construction of a suitable vector comprising the above components (ie, regulatory sequences) as well as genes encoding human granulocyte colony forming factor homologues can be prepared using basic recombinant DNA techniques. Each DNA fragment isolated to form the desired vector must first be cleaved with a restriction enzyme and then linked in consideration of the specific order and orientation.

DNA can be cleaved using specific restriction enzymes in an appropriate buffer. Typically, about 0.2-1 ug of plasmid or DNA fragment is used with about 1 to 2 units of the corresponding restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, incubation times and temperatures are specified by the manufacturer of the restriction enzyme. In general, it is suitable to incubate at 37 ° C. for about 1 to 2 hours, but some enzymes require higher temperatures. After incubation, the enzyme and other impurities are removed by extracting the digestion solution with a mixture of phenol and chloroform and the DNA can be precipitated with ethanol and recovered from the aqueous layer. In this case, the ends of the DNA fragments must be compatible with each other in order for the DNA fragments to be connected to form a functional vector.

Cleaved DNA fragments should be sorted and selected by size using electrophoresis. DNA can be electrophoresed through agarose or polyacrylamide matrices. The choice of matrix depends on the size of the DNA fragment to be separated. After electrophoresis, the DNA is extracted from the matrix by electroelution, extracted from the matrix, or if low melting agarose is used, the agarose is melted and the DNA is extracted therefrom.

DNA fragments to be linked must be added to the solution in the same molar amount. The solution contains ligase, such as ATP, a ligase buffer, and about 10 units of T4 ligase per 0.5 ug of DNA. In order for a DNA fragment to be linked to a vector, the vector must first be cleaved and linearized with a suitable restriction enzyme. The linearized vector should be treated with alkaline phosphatase or bovine visceral enzyme. This phosphatase treatment prevents self-ligation of the vector during the ligation step. The recombinant expression vector produced through such a method is now transformed or transfected with a host cell.

Polynucleotides are described in basic experimental guidelines such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook, J., et al. (1989) “Molecular Cloning” A Laboratory Manual 2nd edition. Can be introduced into the host cell by a conventional method. Preferred methods for introducing polynucleotides into host cells include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection. Cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection, etc. do.

In the production method of the present invention, host cells are cultured in a nutrient medium suitable for the production of polypeptides using known techniques. For example, the cells can be cultured by small or large scale fermentation, shake flask culture in a laboratory or industrial fermenter carried out under conditions that allow the appropriate medium and polypeptide to be expressed and / or secreted. Cultivation takes place in a suitable nutrient medium, including carbon, nitrogen sources and inorganic salts, using known techniques. The medium is well known to those skilled in the art and may be used commercially or manufactured by yourself. If the polypeptide is secreted directly into the nutrient medium, the polypeptide can be separated directly from the medium. If the polypeptide is not secreted, it can be separated from the cell's lysate.

Polypeptides can be separated by methods known in the art. For example, it may be separated from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Further polypeptides are commonly known, including chromatography (eg, ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction It can be purified through various methods.

Pharmaceutically acceptable carriers, excipients, or stabilizers should be nontoxic to the recipient at the dosages and concentrations introduced and compatible with the other ingredients of the formulation. For example, the agent should not contain oxidants or other substances known to be harmful to the polypeptide.

Suitable carriers include buffers such as phosphoric acid, citric acid, and other organic acids; Antioxidants including ascorbic acid; Low molecular weight polypeptides; Proteins such as plasma albumin, gelatin, and immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelate factors such as EDTA; Metal ions such as zinc, cobalt, or copper; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as Tween, Pluronic, or Polyethylene Glycol (PEG).

Human granulocyte colony forming factor homologues must be sterile in order to be used for administration for treatment. Sterilization can be readily accomplished through filtration through sterile filtration membranes.

Therapeutic human granulocyte colony forming factor allomeric compositions generally have a container with a sterile access port, for example an intravenous bag or vial with a stopper that can be penetrated by a hypodermic needle. should be stored in a vial.

Human granulocyte-forming factor homologues according to the invention may be administered directly to the animal by appropriate techniques, including parenteral administration, and may be administered locally or systemically. The particular route of administration will be determined according to the patient's history, including, for example, side effects recognized or expected using human granulocyte forming factors. Examples of parenteral administration include subcutaneous tissue, intramuscular, intravenous, intraarterial, intraperitoneal administration. Most preferably, the administration is by continuous infusion (eg a mini pump such as an osmotic pump) or by injection, for example via an intravenous or subcutaneous tissue route. In the case of human granulocyte-forming factor homologues, the preferred mode of administration is in the subcutaneous tissue.

The human granulocyte forming factor homologue of the present invention will be administered to a patient in an amount effective for treatment. "Effective for treatment" can be defined as an amount sufficient to achieve the desired therapeutic effect in a given condition and mode of administration. The human granulocyte-forming factor composition to be used for treatment may include the specific condition to be treated, the clinical condition of the individual patient (especially side effects of treatment with human granulocyte-forming factor alone), the location of delivery of the human granulocyte-forming factor homologue composition, the method of administration, the dosing schedule, It should be prepared and taken consistent with the desired medical practice taking into account other requirements known to those skilled in the art. The amount effective for the treatment of human granulocyte forming factors homologues is determined by consideration of such matters. The daily dosage of the human granulocyte forming factor homologue of the present invention is generally about 1 μg / kg to 100 μg / kg, preferably 1 μg / kg to 10 μg / kg.

The invention will be more specifically illustrated by the following examples. However, these examples are merely embodiments of the invention and do not limit the scope of the invention.

<Example 1>

Securing Human Granulocyte Formation Factor Gene

Genes of human granulocyte colony forming factor used as templates for the production of native human granulocyte colony forming factor homologues were synthesized using an automated DNA synthesizer (BioSearch, Applied Biosystems ^TM ). The nucleotide sequence and amino acid sequence of the synthetic gene are shown in FIG. The amino acid sequence shown in FIG. 1 is the same as having the Met in 1-position with the amino acid sequence shown in SEQ ID NO: 1.

<Example 2>

Selection of Modified Sites in Human Granulocyte Colony Forming Genes

The structural analysis of J. Biol. Chem., 1992, 5, 8770 was used to select a site to modify the amino acid of human granulocyte colony forming factor. The site of modification was determined by considering the glutamic acid residue at position 46 which binds to the receptor of human granulocyte colony forming factor. The deformation site is shown in FIG.

<Example 3>

Construction of Human Granulocyte Colony Forming Factor Homolog

Genes encoding human granulocyte-forming factors in which one or more amino acids were modified could be prepared by performing PCR using synthetic oligodeoxynucleotides as primers. Synthetic oligodeoxynucleotides used herein are shown in Table 1. The oligodeoxynucleotide sequences of K40R to F144Y described in Table 1 correspond to the oligodeoxynucleotide sequences of SEQ ID NOs: 2 to 21, respectively.

1) Manufacture of K40R

In this example, K40R is a primer of N-term seq and K40R2 and K40R1 and C-, using DNA of natural granulocyte colony forming factor as a template to replace lysine corresponding to No. 40 of natural human granulocyte colony forming factor with arginine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to arginine instead of lysine at the 40th amino acid position were obtained. Put two DNA fragments obtained in this way and perform a second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony-forming homolog K40R modified with arginine instead of lysine at the 40th amino acid position. Was able to obtain a modified gene.

2) H43L production

In this example, H43L is a primer of N-term seq, H43L2, and H43L1 and C- with a template of natural granulocyte colony forming factor DNA in order to replace histidine corresponding to No. 43 of natural human granulocyte colony forming factor with leucine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to leucine instead of histidine at the 43rd amino acid position were obtained. Put two DNA fragments thus obtained and perform a second PCR with N-term seq and C-term seq as primer pairs. The human granulocyte colony forming factor isoform H43L modified with leucine instead of histidine at the 43rd amino acid position. Was able to obtain a modified gene.

3) Fabrication of E45D

In the present embodiment, E45D is a primer of N-term seq and E45D2 and E45D1 and C using a template of natural granulocyte colony forming factor DNA to replace glutamic acid corresponding to No. 45 of natural human granulocyte colony forming factor with aspartic acid. PCR was performed with each pair of -term seq. As a result, two DNA fragments modified with codons corresponding to aspartic acid instead of glutamic acid at the 45th amino acid position were obtained. Put two DNA fragments thus obtained and perform the second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony forming factor homologue modified with aspartic acid instead of glutamic acid at the 45th amino acid position. A variant gene of E45D was obtained.

4) Fabrication of E46D

In the present Example, E46D is a primer of N-term seq, E46D2 and E46D1 and C using a template of natural granulocyte colony forming factor DNA to replace glutamic acid corresponding to No. 46 of natural human granulocyte colony forming factor with aspartic acid. PCR was performed with each pair of -term seq. As a result, two DNA fragments modified with codons corresponding to aspartic acid instead of glutamic acid at the 46th amino acid position were obtained. Put two DNA fragments obtained in this way and perform the second PCR with N-term seq and C-term seq as primer pairs. The human granulocyte colony forming factor homologue transformed into aspartic acid instead of glutamic acid at the 46th amino acid position. A variant gene of E46D was obtained.

5) Fabrication of E46G

In the present embodiment, E46G is a primer of N-term seq, E46G2, E46G1, and C- with a template of natural granulocyte colony forming factor DNA in order to replace glutamic acid corresponding to No. 46 of the native human granulocyte colony forming factor with glycine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to glycine instead of glutamic acid at the 46th amino acid position were obtained. Put two DNA fragments obtained in this way and perform the second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony forming factor homolog E46G modified with glycine instead of glutamic acid at the 46th amino acid position. Was able to obtain a modified gene.

6) Fabrication of E46Q

In the present Example, E46Q is a primer of N-term seq and E46Q2 and E46Q1 and C- with a template of natural granulocyte colony forming factor DNA to replace glutamic acid corresponding to No. 46 of native human granulocyte colony forming factor with glutamine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to glutamine instead of glutamic acid at the 46th amino acid position were obtained. Put two DNA fragments thus obtained and perform the second PCR with N-term seq and C-term seq as primer pairs. The human granulocyte colony forming factor homolog E46Q modified with glutamine instead of glutamic acid at the 46th amino acid position. Was able to obtain a modified gene.

7) Fabrication of V48L

In this example, V48L is a primer of N-term seq, V48L2, and V48L1 and C- with a template of natural granulocyte colony forming factor DNA to replace valine corresponding to No. 48 of natural human granulocyte colony forming factor with leucine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to leucine were obtained instead of valine at the 48th amino acid position. Two DNA fragments thus obtained were inserted and a second PCR was performed using primer pairs of N-term seq and C-term seq. The human granulocyte colony forming factor homolog V48L modified with leucine instead of valine at the 48th amino acid position. Was able to obtain a modified gene.

8) Fabrication of L49I

In this embodiment, L49I is a primer of N-term seq, L49I2, and L49I1 and C- using a natural granulocyte colony forming factor DNA as a template to replace leucine corresponding to No. 49 of the natural human granulocyte colony forming factor with isoleucine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to isoleucine instead of leucine at the 43rd amino acid position were obtained. Put two DNA fragments thus obtained and perform the second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony forming factor homolog L49I modified with isoleucine instead of leucine at the 49th amino acid position. Was able to obtain a modified gene.

9) Fabrication of L49V

In this embodiment, L49V is a primer of N-term seq and L49V2 and L49V1 and C- with a template of natural granulocyte colony forming factor DNA to replace leucine corresponding to No. 49 of natural human granulocyte colony forming factor with valine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to valine at the 49th amino acid position were obtained. Put two DNA fragments thus obtained and perform a second PCR with primer pairs of N-term seq and C-term seq. Human granulocyte colony forming factor homolog L49V modified with valine instead of leucine at the 49th amino acid position. Was able to obtain a modified gene.

10) Fabrication of F144Y

In the present embodiment, F144Y is a primer of N-term seq and F144Y2 and F144Y1 and C-, using native granulocyte colony forming factor DNA as a template to replace phenylalanine corresponding to No. 144 of native human granulocyte colony forming factor with tyrosine. PCR was performed using pairs of term seqs. As a result, two DNA fragments modified with codons corresponding to tyrosine instead of phenylalanine at the 43rd amino acid position were obtained. Put two DNA fragments thus obtained and perform a second PCR with N-term seq and C-term seq as primer pairs. Human granulocyte colony forming factor homolog F144Y modified with tyrosine instead of phenylalanine at the 144th amino acid position. Was able to obtain a modified gene.

Synthetic Oligodeoxynucleotides Primer Name Primer sequence K40R1 (SEQ ID NO: 2)  5'TGCGCTACTT ACCGTCTGTG CCATCCG3 ' K40R2 (SEQ ID NO: 3)  5'CGGATGGCAC AGACGGTAAG TAGCGCA3 ' H43L1 (SEQ ID NO: 4)  5'TACAAACTGT GCCTGCCGGA AGAGCTG3 ' H43L2 (SEQ ID NO: 5)  5'CAGCTCTTCC GGCAGGCACA GTTTGTA3 ' E45D1 (SEQ ID NO: 6)  5'CTGTGCCATC CGGACGAGCT GGTACTG3 ' E45D2 (SEQ ID NO: 7)  5'CAGTACCAGC TCGTCCGGAT GGCACAG3 ' E46D1 (SEQ ID NO: 8)  5'TGCCATCCGG AAGACCTGGT ACTGCTG3 ' E46D2 (SEQ ID NO: 9)  5'CAGCAGTACC AGGTCTTCCG GATGGCA3 ' E46Q1 (SEQ ID NO: 10)  5'TGCCATCCGG AACAACTGGT ACTGCTG3 ' E46Q2 (SEQ ID NO: 11)  5'CAGCAGTACC AGTTGTTCCG GATGGCA3 ' E46G1 (SEQ ID NO: 12)  5'TGCCATCCGG AAGGTCTGGT ACTGCTG3 ' E46G2 (SEQ ID NO: 13)  5'CAGCAGTACC AGACCTTCCG GATGGCA3 ' V48L1 (SEQ ID NO: 14)  5'CCGGAAGAGC TGCTGCTGCT GGGTCAT3 ' V48L2 (SEQ ID NO: 15)  5'ATGACCCAGC AGCAGCAGCT CTTCCGG3 ' L49I1 (SEQ ID NO: 16)  5'GAAGAGCTGG TAATCCTGGG TCATTCT3 ' L49I2 (SEQ ID NO: 17)  5'AGAATGACCC AGGATTACCA GCTCTTC3 ' L49V1 (SEQ ID NO: 18)  5'GAAGAGCTGG TAGTACTGGG TCATTCT3 ' L49V2 (SEQ ID NO: 19)  5'AGAATGACCC AGTACTACCA GCTCTTC3 ' F144Y1 (SEQ ID NO: 20)  5'TTCGCTTCTG CATACCAGCG TCGTGCA3 ' F144Y2 (SEQ ID NO: 21)  5'TGCACGACGC TGGTATGCAG AAGCGAA3 ' N-Term seq  5'TGCTCTAGAA AAAACCAAGG AGGTAATAAA TA3 ' C-Term seq  5'TTACTGGACC GGATCCGAAT TCTATTA3 '

<Example 4>

Plasmid Preparation and E. Coli Transformant Preparation of Human Granulocyte Colony Forming Factor Homologs

The pCJKM plasmid vector shown in FIG. 3 was cut with restriction enzymes XbaI and BamHI, and the human granulocyte colony forming factor homolog gene prepared in Example 3 was cut with the same restriction enzyme and inserted using T4 DNA ligase to express the expression vector. To complete. A schematic diagram of the expression vector pCJKM-isoform is shown in FIG. 3.

The completed recombinant plasmid was transformed into E. coli pop2136 (ATCC Cat.47049, Nucleic Acids Res. 17 (1989)) to prepare a transformant.

Example 5

Expression and Purification of Human Granulocyte Colony Forming Factor Homologs

The E. coli transformant prepared in Example 4 was inoculated in 2X YT medium containing 20ug / ml kanamycin and incubated for 2 hours at 30 ° C, followed by 6 hours at 42 ° C to induce the expression of G-CSF. To confirm this, the culture medium was partially crushed to confirm that the protein expressed by SDS-PAGE was G-CSF. (Figure 4)

In order to purify G-CSF from the culture solution, first, the cultured cells were centrifuged (10,000 g, 30 minutes), followed by crushing the cells with a homogenizer and centrifugation of the lysate to remove the supernatant. Was recovered. The collected inclusion body was suspended in distilled water and then washed by centrifugation to recover the precipitate. The inclusion body is completely dissolved in 8M urea and 50mM glycine buffer (pH11), followed by centrifugation to take the supernatant. 50 mM glycine solution was added to the inclusion body solution so that the concentration of urea was 2M, and the pH was adjusted to 9.0 with sodium hydroxide solution. The pH-adjusted lysate was slowly stirred at room temperature for 15 hours to perform refolding of the protein. The protein reconstituted solution was adjusted to pH5.5 and then centrifuged to remove the precipitate. The supernatant was taken and adjusted to pH 3.0 with hydrochloric acid, and then concentrated to a protein concentration of 1 to 5 mg / ml using a filter for fractionating molecular weight 10 kD. The concentrated solution was purified using cation exchange resin. The eluted protein was collected into one sample using reverse phase HPLC and then adjusted to pH3.0 with hydrochloric acid.

Protein purified with ion exchange resin was purified using reverse phase HPLC. The fractionated protein was analyzed by reverse phase HPLC, and then collected into one sample and concentrated to a final protein concentration of 5-10 mg / ml using a filter that fractionates molecular weight 10 kD. The concentrated protein was finally purified by gel filtration chromatography. The purified final protein was subjected to protein quantitation (absorbance at 280 nm) by reversed phase HPLC and absorbance analyzer. (Figure 5)

<Example 6>

Analysis of Human Granulocyte Colony Forming Factor Homologs Investigating Effects within Cell Lines

M-NFS60 (Biochem. J. 253 (1988) 213-218. Exp. Hematol. 17 (1989)), a known G-CSF dependent cell line, was added to medium containing 1 ng / ml G-CSF (RPMI 1640, 10%). Passage in FBS). In order to remove the G-CSF added to the culture the next day, the culture was centrifuged (600rpm, 10 minutes) to remove the supernatant, suspended in PBS and centrifuged again. After repeating this twice, make a cell suspension at 1 × 10 ⁴ cells / ml and dispense into T75 flask. At this time, the FBS added to the medium to minimize the proteolytic enzyme action by serum to 5%, using a medium containing 0.1X protease inhibitor (protease inhibitor cocktail tablet, EDTA-free: Roche). Each of the G-CSF homologs purified in Example 5 and the natural (wt) G-CSF protein (leucocaine manufactured by CJ Corp.) were added at a concentration of 2.5 ng / ml, followed by incubation in a 37 ° C CO ₂ incubator, and The cell number is measured every hour using a cell viability analyzer (Beckman coulter).

As a result, as shown in FIG. 6, the medium to which the natural (wt) G-CSF protein was added has a large number of initially proliferated cells, but no cell proliferation occurred after 10 days. The medium to which each of the G-CSF homologs purified in 5 was added showed a slow growth rate but increased cell proliferation for a long time and increased in vivo persistence. In particular, with H43L E46G and in vivo persistence can be seen to increase significantly.

Such an increase in in vivo persistence is found for the first time in the present invention. In the case of analogues in the prior art, only the results on the biological activity of G-CSF are most often known. For example, in the US20030171559, US6555660, US6358505 described above, the activity of the analogues is rarely observed or lower than the natural G-CSF protein, or the presence or absence of activity is mostly observed.

The human granulocyte colony forming factor homologue according to the present invention can increase the half-life in vivo by lowering the affinity of the cell receptor while simplifying the preparation method, and consequently can reduce the dose and the frequency of administration in the clinical.

<110> CJ Corp. <120> Human Granulocyte-Colony Stimulating Factor Isoform and          Composition <130> 0448 <160> 21 <170> KopatentIn 1.71 <210> 1 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> G-CSF <400> 1 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys   1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln              20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val          35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys      50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser  65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser                  85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp             100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro         115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe     130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro                 165 170 <210> 2 <211> 27 <212> DNA <213> PRIMER <400> 2 tgcgctactt accgtctgtg ccatccg 27 <210> 3 <211> 27 <212> DNA <213> PRIMER <400> 3 cggatggcac agacggtaag tagcgca 27 <210> 4 <211> 27 <212> DNA <213> PRIMER <400> 4 tacaaactgt gcctgccgga agagctg 27 <210> 5 <211> 27 <212> DNA <213> PRIMER <400> 5 cagctcttcc ggcaggcaca gtttgta 27 <210> 6 <211> 27 <212> DNA <213> PRIMER <400> 6 ctgtgccatc cggacgagct ggtactg 27 <210> 7 <211> 27 <212> DNA <213> PRIMER <400> 7 cagtaccagc tcgtccggat ggcacag 27 <210> 8 <211> 27 <212> DNA <213> PRIMER <400> 8 tgccatccgg aagacctggt actgctg 27 <210> 9 <211> 27 <212> DNA <213> PRIMER <400> 9 cagcagtacc aggtcttccg gatggca 27 <210> 10 <211> 27 <212> DNA <213> PRIMER <400> 10 tgccatccgg aacaactggt actgctg 27 <210> 11 <211> 27 <212> DNA <213> PRIMER <400> 11 cagcagtacc agttgttccg gatggca 27 <210> 12 <211> 27 <212> DNA <213> PRIMER <400> 12 tgccatccgg aaggtctggt actgctg 27 <210> 13 <211> 27 <212> DNA <213> PRIMER <400> 13 cagcagtacc agaccttccg gatggca 27 <210> 14 <211> 27 <212> DNA <213> PRIMER <400> 14 ccggaagagc tgctgctgct gggtcat 27 <210> 15 <211> 27 <212> DNA <213> PRIMER <400> 15 atgacccagc agcagcagct cttccgg 27 <210> 16 <211> 27 <212> DNA <213> PRIMER <400> 16 gaagagctgg taatcctggg tcattct 27 <210> 17 <211> 27 <212> DNA <213> PRIMER <400> 17 agaatgaccc aggattacca gctcttc 27 <210> 18 <211> 27 <212> DNA <213> PRIMER <400> 18 gaagagctgg tagtactggg tcattct 27 <210> 19 <211> 27 <212> DNA <213> PRIMER <400> 19 agaatgaccc agtactacca gctcttc 27 <210> 20 <211> 27 <212> DNA <213> PRIMER <400> 20 ttcgcttctg cataccagcg tcgtgca 27 <210> 21 <211> 27 <212> DNA <213> PRIMER <400> 21 tgcacgacgc tggtatgcag aagcgaa 27  

Claims (7)

The human granulocyte colony forming factor having the amino acid sequence of SEQ ID NO: 1, wherein the human granulocyte colony forming factor homologue in which the amino acid at any one of Lys40, His43, Glu45, Glu46, Val48, Leu49 and Phe144 is modified. The human granulocyte colony forming according to claim 1, wherein in the human granulocyte colony forming factor amino acid sequence, the modification is any one of K40R, H43L, E45D, E46D, E46Q, E46G, V48L, L49I, L49V, and F144Y. Factor homologues. The human granulocyte colony forming factor homologue of claim 1, wherein the modification is H43L or E46G. A pharmaceutical composition comprising the human granulocyte colony forming factor homologue according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. The gene encoding the human granulocyte colony forming factor homologue according to any one of claims 1 to 3. The oligodeoxynucleotide of the human granulocyte colony forming factor homolog of any one of Claims 1-3 used as a primer for modifying the amino acid sequence of human granulocyte colony forming factor. The oligodeoxynucleotide of claim 6, wherein the primer is an oligodeoxynucleotide of SEQ ID NO: 2 to 21. 8.

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