KR20090103209A - Human Interleukin-2 Isoforms - Google Patents

Abstract

PURPOSE: A human interleukin-2 congener is provided to largely increase activity and resistance in the human body and lower the administeration frequency clinically. CONSTITUTION: A human interleukin-2 congener has one or more amino acids in front Ala at N-terminal site of human interleukin-2 of sequence number 1 or after Thr at C-terminal site. The one or more amino acid is cystein. An amino acid added to each terminal site of the human interleukin-2 is cystein and 1-12.

Description

Human Interleukin-2 Isoforms

The present invention relates to a new homolog of human human interleukin-2 that binds a polymer to a human interleukin-2 (rhIL-2) variant to enhance biological utility such as persistence in the body. In particular, the present invention relates to a homolog in which copolyimide of polyethylene glycol as a nonprotein part is covalently linked to a homolog of cysteine added to human interleukin-2.

Human IL-2 is produced from 153 amino acid precursors, followed by cleavage to form a naturally occurring IL-2 protein consisting of 133 amino acids.

There are three cysteines in the naturally occurring IL-2 protein, of which two cysteines participate in disulfide bonds, but the cysteine present at the 125 th is free.

In addition, the natural human IL-2 synthesized in vivo exists in the O-type sugar chain bound to the third amino acid threonine.

The biological function of human interleukin-2 in vivo induces the growth and differentiation of lymphocytes as T-cell growth factors, resulting in the production of mature cytotoxic T-cells (Ezard. Et. Al., J. Immunol. 134, 1644-1652, 1985). In addition, it is involved in the growth and differentiation of B cells (Mingari, MC, et. Al., Nature (1984), 312, 641; Mittler, R., et. Al., J. Immunol. (1985), 134, 2393-2399; Muraguchi, A. et.al., J. Exp. Med. (1985), 161, 181-197)), produce lymphokine-activated killer cells, increase natural killer cells, and specific neoplastic cells It has several functions such as impeding the growth of neoplastic cells.

Various biological functions of IL-2 are accomplished by various signaling systems through IL-2 receptors expressed in various cell types.

Because of these characteristics, IL-2 can activate or suppress the host's immune system by manipulating IL-2's action system during antigen-specific T cell clonal expansion process. Studies are underway to inhibit the use of antibodies against the two receptors or a method capable of inhibiting their function (IL-2 linked diphtheria toxin) to inhibit taggraft rejection.

In addition, IL-2 is used in cancer treatment because it can produce a large amount of tumor specific lymphocytes, and it can be used as an adjuvant in a vaccine by using an immune response-enhancing property. Therefore, many researches are being conducted medically (US patent 5,800,810). Sep. 1. 1998; Chiron Corp; Kawamura et. Al., J. Exp. Med. (1985), 162, 381-386; McFeeters and Nadler.Fed.Proc. 45 (3), 633 (presented Apr. 13-18, (1986)).

However, such use may cause problems in the immune response due to the hydrophobic nature of binding between IL-2 proteins (R. Illig (1970) J. Clin Endrocr, 31, 679-688; W. Moore (1978) J Clin Endrocrinol. Metab. 46, 20-27; W. Moore and P. Leppert (1980) J. Clin Endrocrinol. Metab. 51, 691-697).

To address this, US Pat. No. 5,206,344 (Apr. 27. 1993) proposes attaching PEG polymers to IL-2 variants. A report that the binding of PEG polymers can mitigate the immune response has been reported in US Pat. No. 4,261,973, which maintains its inherent activity in other therapeutic proteins, in particular by binding PEG or PPG to insulin and therapeutic enzymes. Immunity has been reduced (USP 4,179, 337).

Variants for human IL-2 have been reported in US Pat. No. 4,518,584 replacing the native 125th amino acid cysteine with serine or alanine. This variant was found to have the same activity as the native type IL-2 (Wang A., Lu S. -D., And Mark D. F. (1984) Science, 224, 1431-1433).

Another IL-2 variant is reported in a US patent application, in which the native 104th amino acid methionine is replaced with alanine (US application Ser. No. 810,656 filed Dec. 17. 1985).

In addition, it is possible to develop a variant to form a PEG polymer, a region containing four amino acids at the end of IL-2 D helix, five sites at the AB loop, BC loop, and CD loop amino terminus, and the first amino acid position alanine. The last amino acid, 133th threonine, has been proposed as a cysteine substitution site.

US Pat. No. 5,206,344 suggests the attachment of PEG-polymers by replacing the thionine at the 20 amino acid position at the amino terminus of IL-2, in particular the threonine at position 3, with cysteine.

Goodson and Katre ((1990) Biotechnology, 8, 343-346) confirmed that even after replacing threonine, an amino acid of the O-glycosylation position at position 3 of IL-2, with cysteine, it showed complete activity. There are reports of experiments on PEG binding.

Methods for increasing the in vivo persistence of IL-2 include polymers such as polyethylene glycol (PEG) or polyoxyethylated polyol (US patented application Ser.No. 866,459, filed May 21. 1986) and succinated IL-2 (US patent). Application Ser.No. 903,668 filed Sep. 4. 1986) has been proposed to release slowly.

Many therapeutics have been published since the publication of a report that increased PEG half-life and reduced antigenicity by binding PEG to asparaginase isolated from E. coli (Abuchowski et. Al., Cancer Biochem Biophys (1984), 7, 175-186). The development of drugs with increased function applied to medicines has been actively developed and is currently being applied to human interferon alpha and human granulocyte colony forming factors.

European patents (EP 154,316 published Sep. 11. 1985 to Takeda Chemical Industries, Ltd.,) report PEG-linking of at least one amino group to a protein including IL-2. PCT W087 / 00056 (published Jan 15. 1987 (Cetus Corporation)) also discloses binding of PEG or polyoxyethylated polyols to protein lysine or cysteine to IL-2, IFN-β and immunotoxins. This modification showed the effect of inhibiting the reduction of antigenicity and the generation of aggregates by masking the physiological half-life increase and antigenic part of IL-2.

Recently, US Pat. No. 6,608,183 B1 (Aug. 19. 2003; Bolder Biotechnology) described an IL-2 variant in which threonine, located at position 3 of the naturally occurring IL-2 (O-glycosylation site), was replaced with cysteine. Produced by making a PEG bond here.

The amino acid sequence of human interleukin-2 is described by (Taniguchi T., Matsui H., Fujita T., Takaoka C., Kushima N., Yoshimoto R., and Hamuro J. (1983) Nature, 302, 305-310; Devos R ., Plaetinck G., Cheroutre H., Simons G., Dcgrave W., Tavcrnicr J., Rcmaut E., and Ficrs W. (1983) Nucleic Acid Res. 11, 4307).

Removal in the body of a polypeptide or polymer thereof is a protein degradation with or without substrate specificity that acts on a particular protein as removal from the kidney, spleen or liver and receptor mediated degradation. By the action of enzymes. In general, the removal of proteins from the body is large enough to avoid filtration in the glomerular filtration, the degree of charge on the protein molecules, the degree of glycosylation adhesion, the receptor on the cell surface of the protein, etc. Depends on.

In particular, the elimination that occurs in the kidneys depends on physical properties such as the size (diameter of the material), symmetry, form / rigidity, charge of the protein or polymer thereof.

Methods for increasing the half-life of protein in the blood should reduce the breakdown of the protein by elimination and receptor binding that occurs in the kidneys, which has become a major factor. This can reduce the elimination in the kidneys by binding polymers to proteins that can increase the size of the apparent molecule, and achieve an increase in half-life in vivo.

In addition, the attachment of polymers to proteins can effectively block proteolytic enzymes and thus prevent the action of nonspecific proteases.

Polyethylene glycol (PEG) is one of the polymers widely used in the preparation of therapeutic protein products. Combining protein drugs with synthetic polymers to alter the surface properties of protein molecules can increase their solubility in water or organic solvents. It can also increase biocompatibility to reduce immune responsiveness, increase stability in vivo, and allow slower clearance by the intestinal system, kidney, spleen or liver. If the molecular weight of the protein is small, it is lost by filtration in the intestinal system or kidneys, which is prevented by binding the polymer PEG (Knauf, M. J. et al, J. Biol. Chem. 263: 15064, 1988). Therefore, modification of the polymer polyethylene glycol (PEG) in the protein drug can increase the stability of the protein molecule in solution. In addition, it is possible to effectively protect the intrinsic surface properties of the protein to prevent non-specific protein adsorption. In this regard, studies have been conducted on the effect of binding PEG to biologically active peptides or proteins to prolong half-life in the body, increase solubility, and reduce the immune response in the body, which was first reported in US Pat. No. 4,179,337. There is a bar. However, it is true that the side effects are reduced due to the above advantages when the protein and PEG are combined, but at the same time, after the binding with PEG, the site required for the protein activity is blocked by PEG, which greatly reduces the bioactivity. Indicates.

PEG, which is widely used to date, covalently bonds with one or more free lysine residues on the surface of the protein, so that PEG is attached to the surface of the protein. When bound, their activity is reduced. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEGylated protein binders exist in a mixture, resulting in a complicated and difficult process of purely separating the desired binder.

However, in the above-mentioned invention, the polyethyleneglycol molecule does not bind certain predictable residues, and PEG is non-selectively bound to any reactor or N-terminus located inside a protein such as a lysine residue, resulting in a heterogeneous population. Is generated. US Pat. No. 5,766,897 and WO 00/42175 disclose that PEG reacts with the active site of human growth hormone in binding PEG with human growth hormone to bind only a specific portion of the protein to PEG. To avoid this, PEG-maleimide was used to allow PEG to selectively react with cysteine. However, this type of PEG requires free cysteine residues in human growth hormone that are not involved in disulfide bonds, but since all four cysteine residues in human growth hormones participate in disulfide bonds, they are artificially present in human growth hormone. Pegylation reactions were performed using residue-inserted human growth hormone derivatives.

On the other hand, Bowen et al. (Experimental Hematology, 27, 425-432, 1999) found an association between the molecular weight and the persistence of polyethylene in polyethylene polymerized human granulocyte formation factors. In vitro experiments showed an inverse relationship between the potency of the protein and the molecular weight of the polyethylene moiety bound to the protein. In vivo activity, however, increased with increasing molecular weight. It is assumed that the polymer of the bioactive protein increases the half-life of low affinity in the degradation of the protein by receptor binding. Therefore, the polymers of these human granulocyte forming factors have been shown to be inferior to the human granulocyte forming factors which do not form polymers in the activity in vitro despite the recovery of neutrophils in vivo.

The prior art still suffers from problems such as poor activity of human interleukin-2 with altered amino acid sequence, in vivo half-life, and no uniform product obtained when binding PEG to human interleukin-2. Remains.

Therefore, the present invention solves the problems of the prior art, human interleukin-2 homologous with excellent activity and significantly increased in vivo half-life compared to the conventional by binding polyethylene glycol to human interleukin-2 modified amino acid sequence The present invention relates to a human sieve, which is modified by substitution or addition of an amino acid sequence with cysteine at a specific site in order to facilitate binding of polyethylene glycol to human interleukin-2, and a human interleukin-2 homolog in which some polyethylene glycol is bound to the modified site. To provide. Conventionally, human interleukin-2 has been enhanced in vivo activity or persistence by modifying the sequence inside the polypeptide and pegylation to a suitable amino acid at a specific site. By adding amino acids and PEGylating them, the inventors have found that activity or persistence in vivo is further increased than conventional homologs.

In particular, one or more amino acids are added before Ala of the N terminal portion of human interleukin-2 represented by amino acid sequence number 1 or after Thr of the C terminal portion, and at least one of the added amino acids is cysteine, and any of the cysteines Provided are human interleukin-2 homologues in which at least one polyethylene glycol is bound.

It is known to those skilled in the art that the N-terminus may include Met at SEQ ID NO: 1, and the cysteine added when Met is included may be added between the Met and alanine at the N-terminus, and these homologs are also present. Naturally, it belongs to the scope of the invention.

The amino acids added to each terminal portion may be added from 1 to 12, respectively, it is preferred that one cysteine is added, it is more preferred that the cysteine is 134C added to the C terminal.

In addition, the polyethylene glycol is covalently bound to cysteine added to human interleukin-2, and the molecular weight thereof is preferably 20 kDa to 40 kDa, and more preferably has a branched branch.

In addition, the human interleukin-2 is preferably one or more amino acid is substituted with another amino acid before the polyethylene glycol is bonded.

According to another embodiment of the present invention, there is provided a pharmaceutical composition comprising the human interleukin-2 homologues and a pharmaceutically acceptable carrier.

According to another embodiment of the present invention, a gene encoding a human interleukin-2 homologue protein having cystein added to the N or C terminus is provided.

According to still another embodiment of the present invention, an oligodeoxynucleotide is used, which is used as a primer for modifying the amino acid sequence of human interleukin-2, as the human interleukin-2 homolog. The primer is preferably an oligodeoxynucleotide of SEQ ID NO: 2 to 7,

Hereinafter, the present invention will be described in detail.

1 shows the sequence of human interleukin-2 genes and proteins. Amino acid sequence number 1 differs only in Met from -1 position compared with the amino acid sequence shown in FIG. 1, and the other sequences are identical.

Figure 2 shows the amino acid sequence of the homologue that caused the amino acid modification at the N-terminal portion of human interleukin-2. AA denotes amino acids and n denotes the number of amino acids. n is an integer from 1 to 12. It also includes at least one cysteine.

Figure 3 shows the amino acid sequence of the homologue that caused the amino acid modification to the C-terminal portion of human interleukin-2. AA denotes amino acids and n denotes the number of amino acids. n is an integer from 1 to 12. It also includes at least one cysteine.

4 shows the structure of a plasmid vector expressing human interleukin-2 isoforms. The promoter used bacteriophage T7.

FIG. 5 shows the persistence of the body in rats of human interleukin-2 isoforms. The test substance was prepared by diluting the carrier (PBS), the interleukin-2 variant without PEG covalent bond, and the native interleukin-2 by diluting with PBS, respectively, at a dose of 100 ug / kg based on the weight measured on the day of administration. The tail was administered to the tail vein. Frequency of administration and duration of administration were administered once the test start date.

The inventors conducted a study combining the protein structure of human interleukin-2 and the effects of amino acids constituting it on activity. The basic data for the present invention is the GOR4 protein secondary structure analysis prediction method (http: // http: //www.gor4.com/) to determine the information on the activity of the structure and constituent amino acids of human interleukin-2, and further on the protein structure after amino acid modification. The results of the GOR program in the secondary structure prediction of http://us.expasy.org are referred to.

As used herein, the isoform of human interleukin-2 has been modified from native amino acid sequence residues of native human interleukin-2 to analogous amino acids residues that retain their intrinsic activity, It refers to variants (mutes, muteins), conjugates (conjugates).

As used herein, the three letter (amino acid) amino acid refers to the following amino acids according to standard abbreviations 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; Gln (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 human interleukin-2 is replaced by the amino acid indicated later. For example, C125S indicates that the cysteine corresponding to 125 of the native human interleukin-2 is substituted with serine.

In order to cause amino acid modification of a specific site herein, the primer is designated as "(amino acid letter) (amino acid position) (amino acid letter) 1 or 2", where 1 is in the 5 '→ 3' direction in the double stranded template. Primer for a single stranded template that proceeds and 2 refers to a primer for a single stranded template that progresses in the 3 '→ 5' direction among the double strands.

The human interleukin-2 gene 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 interleukin-2 by replacing or adding one or more codons on the gene encoding human interleukin-2. More specifically, the addition of cysteine before the first amino acid alanine on the human interleukin-2 amino acid sequence, the addition of cysteine after the threonine at the C-terminus, and further modification of these terminal portions Sequence modification at an intermediate site;

In one embodiment of the present invention, synthetic oligonucleotides are prepared comprising codons encoding the desired amino acid modifications in human interleukin-2. Generally about 27-36 nucleotides of oligonucleotides in length are used. Shorter oligonucleotides may be introduced but 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 embodiment of the present invention, human interleukin-2 homolog DNA is prepared in which one amino acid is modified. In this case, PCR is performed using human interleukin-2 DNA (FIG. 1) 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 encoding the modification, thus encoding the desired modification on the gene. The second strand acts as template DNA in the repeated replication step of PCR, so the gene encoding the modification will continue to be amplified.

Modification of the amino acid sequence in the middle of a polypeptide of human interleukin-2 will be described taking C125S as an example. C125S uses primers IL2 Nde and C125S2, and C125S1 and IL2 C-term, respectively, using a template of native interleukin-2 DNA (FIG. 1) to replace cysteine corresponding to 125 of the native human interleukin-2 with serine. PCR was performed in pairs. As a result, two DNA fragments modified with codons corresponding to cysteine instead of threonine at the 125th amino acid position were obtained. The two DNA fragments thus obtained were inserted and a second PCR was performed using primer pairs of IL2 Nde and IL2 C-term, and the modified gene of human interleukin-2 homolog C125S modified with serine instead of cysteine at the 125th amino acid position. Can be obtained.

According to one embodiment of the invention, amino acids at the N- or C- terminus are added. Construction of human interleukin-2 with one or more amino acid modifications can be made at the N-terminus and C-terminus, with modifications to this site being met- (AA) n- for the N-terminus as shown in FIG. It becomes the same sequence as Ala-, and has the sequence of -Gln-Thr- (AA) n for the C-terminal as shown in FIG. AA represents an arbitrary amino acid, and glycine serine alanine, etc. may be used as an amino acid mainly having a small molecular weight and not exhibiting a specific function. AA also includes at least one cysteine amino acid. In addition, n represents the number of amino acids and n may have a number from 1 to 12 amino acids. This is the minimum number of amino acids needed to be recognized in order to form antibodies by being recognized as heterologous proteins in vivo by additionally added amino acids, and the maximum possible amount of amino acids to avoid this range is required. Means number.

Therefore, the DNA base of ATG- (NNN) n-GCA- is produced by having the amino acid sequence of Met- (AA) n-Ala- in the case of N-terminus in order to construct a gene of human interleukin-2 having such additional amino acid. In the case of the C-terminus, the sequence has the amino acid sequence of -Leu-Thr- (AA) n to include the DNA base sequence of -CTG-ACT- (NNN) n-TAA. Where NNN represents a DNA sequence encoding the amino acid.

The primer containing the DNA sequence thus prepared can be used to produce a human interleukin-2 gene in which one or more amino acids are modified using the human interleukin-2 gene shown in FIG. 1.

For example, when one glycine and cysteine are added to the N-terminus, the amino acid sequence of the N-terminal region may be Met- (Gly-Cys-) Ala-. At this time, the design of the primer to be used may be GTAATAAATA CAT-ATG- (GGT-TGT-) GCA-, which is combined with the IL2 C-term primer shown in Table 1 using the human interleukin-2 gene template of FIG. 1. Can be used to construct a modified human interleukin-2 gene having a sequence of Met- (Gly-Cys-) Ala-.

The human interleukin-2 gene prepared above is subjected to PCR again using IL2 Nde and IL2 C-term primers.

In addition, the amino acid may be added to the N- or C-terminal of the present invention, and further, polyethylene glycol may be bound after substituting one or more amino acids of the intermediate region of the human interleukin-2 polypeptide with another amino acid. For example, -1C / C125S is a primer prepared by using -1C human interleukin-2 homolog DNA prepared as an example to add cysteine at the N-terminal and to replace cysteine at position 125 in the human interleukin-2 polypeptide with serine. PCR is performed by pairing IL2 Nde and C125S2 and C125S1 and IL2 C-term, respectively. As a result, two DNA fragments modified with codons corresponding to cysteine instead of threonine at the 125th amino acid position can be obtained. Put the two DNA fragments thus obtained and perform a second PCR using primer pairs of IL2 Nde and IL2 C-term, and the cysteine is added to the N-terminal alanine at the 125th amino acid position. Modified genes of human interleukin-2 allogene-1C / C125S can be obtained. The preparation of homologues other than the 125 position can also be prepared using the same method as the preparation method of the -1C / C125S homologue using the corresponding DNA primers.

According to the present invention, the amino acid modification position in the intermediate region of the polypeptide of the homologue to which the amino acid including at least one or more cysteines at the N- or C-terminus is added can be a known position. As described above, a conventional technique of substituting one or more amino acids in the intermediate region of a human interleukin-2 polypeptide with another amino acid

In the US patent US 4,518,584, the 125th amino acid cysteine is serine or alanine, and in US patent US 5,206,344 the third amino acid threonine is cysteine, US patent application US application Ser. The methionine at position 810,656 is alanine, and is used at the amino terminus and C-terminus of the amino terminus of the human interleukin-2 protein, including the replacement of threonine, which is also the third amino acid, in the US patent US 6,608,183B1 with cysteine. There are reports of replacing leonin with cysteine.

However, no additional cysteine has been added to the amino and C-terminal amino acid sequences in human interleukin-2.

The contents described in these documents are incorporated herein by reference of the present invention. In addition, methods for producing human interleukin-2 homologs described in these, pegylation methods, and the like are also incorporated herein by reference of the present invention.

DNA sequences encoding human interleukin-2 homologues according to the invention can also be synthesized by standard methods known in the art, for example using automated DNA synthesizers (e.g., biosearch, Applied Biosystems ^TM ). .

In a related aspect, the present invention provides recombinant expression vectors comprising DNA sequences encoding human interleukin-2 homologues and host cells transformed or transfected with such expression vectors.

The term "polypeptide" as used herein may be used as the term protein or bioactive protein.

As used herein, "homogen" refers to a bioactive protein that has the main function of the original bioactive protein but has been modified through genetic or other manipulations. Modification herein refers to the substitution, addition and deletion of amino acid sequences, including modifications or additions to cysteine for the binding of polyethylene glycol. In addition, it may mean that the polymer of polyethylene glycol is covalently bonded to the cysteine by a chemical reaction.

As used herein, "persistence in the body" means that when the bioactive protein is administered to humans or animals, the total amount and activity of the initially administered bioactive protein is reduced through proteolytic enzymes and removal from the kidney, The function will be lost. Therefore, it means the retention time of the protein to the extent that the physiologically active protein can function in the body, and is represented by the half-life of the physiologically active protein in the body.

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.

In the recombinant expression vector, a DNA vector into which human interleukin-2 is inserted may be generated. In the construction of recombinant expression vectors, as is well known in the art, in order to increase the expression level of transfected genes in host cells, the genes act on transcriptional and translational expression control sequences that function in the selected expression host. Must be connected as Preferably, the expression control sequences and the corresponding genes are included in one expression vector including the bacterial selection marker and the replication origin.

The construction of a suitable vector containing the above constituents (ie, regulatory sequences) as well as genes encoding human interleukin-2 isoforms can be produced 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, reaction times and temperatures are specified by the manufacturer of the restriction enzyme. In general, it is appropriate to react at 37 ° C. for about an hour or two, but some enzymes require higher temperatures. After the reaction, 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, in order for the DNA fragments to be linked to form a functional vector, the ends of the DNA fragments must be compatible with each other.

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 is determined by the size of the DNA fragment to be separated. After electrophoresis, 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 can be used by treating with alkaline phosphatase or bovine hydrolases. This phosphatase treatment prevents self-ligation of the vector during the ligation step. The host cell is transformed or transfected with the recombinant expression vector produced by the above method.

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 calcium chloride transformation, electroporation, and the like.

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 lysate of the cell.

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.

Purified human interleukin-2 homologues are PEGylated in the following manner.

As the "non-protein moiety" suitable for the present invention for use in PEGylation, polyethylene glycol (PEG) is preferred, especially branched polyethylene glycol. Other examples include, but are not limited to, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and Derivatives thereof, poly (amino acids), poly (vinyl alcohol), polyurethanes, polyphosphazenes, poly (L-lysine), polyalkylene oxides, PAO), water-soluble polymers such as polysaccharides, and ratios such as dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacryl amide, and the like. One or more selected from the group consisting of immunological polymers can be used. In addition, the molecular weight of the biocompatible polymer used in the synthesis of the polymer derivative of the present invention is preferably about 2,000 to 100,000, more preferably 20,000 to 40,000.

The biocompatible polymer must be activated to bind to Sara Interleukin-2 via the thiol group of human interleukin-2, and can be coupled to a reactive functional group for this purpose. A "reactive functional group" refers to a group or moiety that activates a biocompatible polymer to bind to a biologically active substance of interest. The process of converting one of the end groups to a reactive functional group to bind the biocompatible polymer to the biologically active material is called "activation". For example, one can convert one of the hydroxyl end groups to a reactive functional group, such as a carbonate, to bind the poly (alkylene oxide) to the biologically active protein, so that the product obtained is a modified poly (alkylene oxide) do.

In the present invention, a reactive functional group for activating the biocompatible non-protein partial polymer is maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, iso It may be selected from the group consisting of isothiocyanate, cyanuric chloride, 1,4-benzoquinone, disulfides and the like.

The method of binding PEG through the thiol group of the protein can be carried out by generally known methods.

Examples of known methods are as follows. For example, there is a method in which a thiol group is bonded to a double bond activated by Michael reaction using PEG-maleimide [Ishii et al., Biophys J. 1986, 50: 75-80 ]. There is also a method using PEG-iodoacetamide reagent, as is well known in protein chemistry. This modification method has the advantage of making carboxymethylcysteine, a stable form of PEG bound cysteine derivative that can be identified and quantified by standard amino acid analysis by strong acidic hydrolysis [Gard FRN. Carboxymethylation. Method Enzymol 1972; B25: 424-49. In addition, a method of obtaining stable symmetric disulfide using PEG-ortho-pyrimidyl-disulfide [Woghiren et al. Bioconjgate Chem 1993, 50: 75-80; A method of covalently binding sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate active-PEG, which is activated PEG, to a thiol group of cysteine [USP 5,166,322]; Maleimido-6-aminocaproyl ester-activated PEG4000, which is activated PEG, is conjugated to a cysteine residue by reacting an IL-2 variant having a cysteine substituted at a specific site [USP 5,206,344]; Chemically modifying thiol groups of proteins using gamma-maleimidobutyl acid and beta-maleimidopropionic acid [Rich et al., J. med. Chem. 18, 1004, 1975].

The PEGylation method carried out in the embodiment of the present invention was used branched mPEG40,000 of NOF and branched mPEG40,000 of SunBio, was used in 20 times the molar ratio of human interleukin-2, the reaction is 0.1M phosphate buffer Stirring was performed at room temperature in a solution (pH 7-8) for 2 hours.

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).

Glycosylated human interleukin-2 homologues must be sterile in order to be used for therapeutic administration. Sterilization can be accomplished through filtration through sterile filtration membranes.

Therapeutic glycated human interleukin-2 allomeric composition generally comprises a container with a sterile access port, for example an intravenous bag with a stopper that can be penetrated by a hypodermic needle. Or in a vial.

Human interleukin-2 isoform 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 interleukin-2. 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 interleukin-2 homologues, the preferred mode of administration is in the subcutaneous tissue.

The human interleukin-2 homologue of the invention will be administered to the 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 interleukin-2 composition to be used for treatment may include the particular condition to be treated, the clinical condition of the individual patient (especially the side effects of treating human interleukin-2 alone), the location of delivery of the human interleukin-2 allomeric 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 interleukin-2 homologues is determined by consideration of such considerations.

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

As described above, the homologue of human interleukin-2 according to the present invention has an effect of greatly increasing the activity or persistence in the body and thus reducing the number of administrations in the clinic.

Figure 1 shows the sequence of the template human interleukin-2 gene and protein used in the present invention.

Figure 2 shows the amino acid sequence of the homologue that caused the amino acid modification at the N-terminal portion of human interleukin-2.

Figure 3 shows the amino acid sequence of the homologue that caused the amino acid modification to the C-terminal portion of human interleukin-2.

4 shows the structure of a plasmid vector expressing human interleukin-2 isoforms.

FIG. 5 shows SDS-PAGE results of homologs that bind PEG 40 to human interleukin-2.

Example 1 Preparation of Human Interleukin-2 Homolog

The gene of human interleukin-2 used as a template for the production of native human interleukin-2 homologues was synthesized by PCR using primers (IL2-1 and IL2-2) from the cDNA library. The base sequence of the synthetic gene is shown in Table 1, and the DNA sequence and amino acid sequence of human interleukin-2 used as a template are shown in FIG.

T7 promoter 5'TAA TAC GAC TCA CTA TAG GG3 T7 term 5'GCT AGT TAT TGC TCA GCG G3 IL2-1 5'ATG GCA CCT ACT TCA AGT TCT ACA3 IL2-2 5'TCA AGT CAG TGT TGA GAT GAT GCT3 IL2 Nde 5'AGA TAT ACA TAT GGC ACC TAC TTC3 IL2 Nde (-1C) 5'AGATAT ACA TAT GTG TGC ACC TAC3 mIL2 Nde 5'AGA TAT ACA TAT GCC TAC TTC AAG TTC3 IL2 C + 5'TTC GGA TCC TCA ACA AGT CAG TGT TGA3 C125S1 5'AGA TGG ATT ACC TTT TCT CAG AGC ATC ATC3 C125S2 5'GAT GCT CTG AGA AAA GGT AAT CCA TCT GTT3 IL2 C-term 5'TTC GGA TCC TCA AGT CAG TGT TGA3

1. Fabrication of the C125S Human Interleukin-2 Homolog

C125S is constructed as an example of amino acid modification at the intermediate site of a human interleukin-2 polypeptide or as a control as a conventionally known homologue in the present invention. In order to replace the cysteine corresponding to No. 125 of native human interleukin-2 with serine, C125S was PCR with a pair of primers IL2 Nde and C125S2 and C125S1 and IL2 C-term, respectively. Was done. As a result, two DNA fragments modified with codons corresponding to serine instead of cysteine at the 125th amino acid position were obtained. The two DNA fragments thus obtained were inserted and a second PCR was performed using primer pairs of IL2 Nde and IL2 C-term, and the modified gene of human interleukin-2 homolog C125S modified with serine instead of cysteine at the 125th amino acid position. Could get

2. Fabrication of the -1C Human Interleukin-2 Homolog

 -1C is a pair of primers IL2 Nde (-1C) primers and IL2 C-term primers in order to produce homologues in which the DNA of natural human interleukin-2 was used as a template and a cysteine amino acid was added to the alanine at the N-terminus. PCR was performed. Human interleukin-2 homolog gene wherein cysteine amino acid was added to alanine at the N-terminal end by cleaving the amplified DNA fragment with restriction enzymes (Nde I and BamH I) and introducing into the expression vector (pET12a) digested with the same restriction enzyme. Could get

The N-terminal amino acid sequence is in the order of methionine-cysteine-alanine-proline-threonine-serine.

Here, the expression vector, pET12a, is used for the supply of an operator that regulates expression by lac repressor in the plasmid vector and for supplying sufficient lac repressor (since there is a limit to the lac repressor present in the chromosome of the host cell). There are no two factors necessary for precise adjustment. Therefore, the gene introduced into pET12a was further cut into Xba I and BamH I and introduced into pET28a (+) vector digested with the same restriction enzyme.

In the case where the Nde I and BamH I cleavage sites of the pET28a (+) vector are used, the human interleukin-2 gene introduced from pET12a is converted to Xba I because the target protein human interleukin-2 also includes His-Tag and thrombin cleavage sites. And BamH I restriction enzymes are cut and introduced into pET28a (+).

3. Fabrication of the -1C / C125S Human Interleukin-2 Homolog

As an example of modification of an amino acid added to the N-terminus and an amino acid in a human interleukin-2 polypeptide, -1C / C125S is further substituted with threonine for the cysteine corresponding to No. 125 in the produced -1C human interleukin-2 homologue. To this end, PCR was performed using primers IL2 Nde (-1C) and IL2 C-term, using the prepared C125S human interleukin-2 homolog DNA as a template. As a result, a modified gene of human interleukin-2 homologue -1C / C125S was obtained from a DNA fragment that was modified with codons corresponding to threonine instead of cysteine at the 125th amino acid position and cysteine was introduced between methionine and alanine at the N-terminus. Could.

The procedure of introducing into an expression vector is the same as that of the above -1C human interleukin-2 homologue.

4. Fabrication of 134C Human Interleukin-2 Homolog

134C carried out PCR using a pair of IL2 Nde primer and IL2 C + primer as a template of DNA of C125S human interleukin-2. Amplified DNA fragments were digested with restriction enzymes (Nde I and BamH I), introduced into pET28a (+) digested with the same restriction enzymes, and introduced into the expression vector to homologous human interleukin-2 with cysteine amino acid added to the C-terminus. Sieve genes were obtained.

The procedure of introducing into an expression vector is the same as that of the above -1C human interleukin-2 homologue.

The C-terminal amino acid sequence is in the order of -alanine-glutamine-proline-cysteine.

5. Fabrication of 134C / C125S Human Interleukin-2 Homolog

The 134C / C125S was subjected to PCR by pairing the prepared C125S human interleukin-2 homolog with IL2 Nde primer and IL2 C + primer.

The procedure of introducing into an expression vector is the same as that of the above -1C human interleukin-2 homologue.

The gene of human interleukin-2 homologue thus prepared was inserted into an expression vector to be expressed by the T7 promoter.

6. Fabrication of des1Ala / 134C / C125S Human Interleukin-2 Homolog

PCR was performed using a pair of the 134C / C125S human interleukin-2 homologue prepared above as a template and mIL2 Nde primer and IL2 C + primer.

The procedure of introducing into an expression vector is the same as that of the above -1C human interleukin-2 homologue.

The gene of human interleukin-2 homologue thus prepared was inserted into an expression vector to be expressed by the T7 promoter.

7. Introduction of Methionine Aminopeptides

Forward primer ECOR1MAPf (5'-GGA ATT CAC GTA TCC CAT ATT ACC GAC CCC AAA GG-3 ') with methionine aminopeptidase gene as a template from E. coli K12, reverse primer MAPrBAMH1 (5'-CGG GAT CCA TCT TAT TCG PCR was performed in pairs TCG TGC GAG ATT GC-3 ').

The E. coli methionine aminopeptides gene thus prepared was cut and inserted into the expression vector pSTV29 (TAKARA, Cat. No. 3332) with restriction enzymes EcoR1 and BamH1.

8. Des1Ala / 134C / C125S Human Interleukin-2 Homologous Expression Strains

A strain producing the des1Ala / 134C / C125S human interleukin-2 isoform by sequentially transforming the des1Ala / 134C / C125S human interleukin-2 homolog expression vector and the Escherichia coli methionine aminopeptides expression vector into Escherichia coli BL21. Produced.

Example 2 Expression and Purification of Human Interleukin-2 Homologs

A plasmid vector containing a gene of human interleukin-2 isoform is transformed into E. coli to produce a strain that produces a human interleukin-2 homologue protein.

The transformation method used a commonly known calcium chloride method, and E. coli, which is used as a host cell, also polymerized T7 RNA into chromosomes of host cells of the commonly used Escherichia coli (BL21 (DE3), HMS174 (DE3) strains. Strains containing enzymes, etc.) can be used.

Transformed E. coli was inoculated in 5 ml of Luria Broth (LB) medium in a 15 ml tube and incubated overnight in a 30 ° C. incubator. This strain was inoculated in 500 ml of Luria Broth (LB) medium in a 3,000 ml Erlenmeyer flask and incubated at 30 ° C. When the absorbance at 600 nm reached 0.8-1.2, IPTG was finally added to 1 mM, and then cultured for 4 hours to induce the expression of human interleukin-2 homologue.

The culture was completed, the culture was centrifuged to recover the cells and washed with distilled water. The washed cells were crushed four times at 12,000 psi with a Microfluidizer (Microfluidics), and the inclusion body was recovered by centrifugation and washed three times with distilled water.

Collect the inclusion body mass with 0.01% Tween80, 10mM Phosphate buffer, pH 7.5, and then collect the inclusion body mass by high-speed centrifugation at 6,000 rpm, 4 ℃ for 20 minutes. The collected inclusion body is dissolved in 50mM Phosphate buffer, 5mM EDTA, pH 7.5 to about 50mg / mL protein. Dilute with the same solution to a final protein concentration of 12 mg / mL and dissolve the inclusion body for 5 hours. The dissolved inclusion body liquid is centrifuged at 4000 rpm for 10 minutes to collect only the supernatant. DTT is added to the collected inclusion body solution to a final concentration of 100 mM, and then the IL-2 protein is reduced by slowly stirring the inclusion body solution. IL-2 protein is collected by passing the reduced IL-2 solution through Sephacryl S-200 Gel chomatography. Reducing IL-2 is loaded at 2% of the total resin volume by volume. This process removes heterologous proteins with high DNA and high molecular weight. CuCl ₂ is added to the IL-2 reducing protein collected by primary gel chromatography, followed by stirring at room temperature. The oxidized IL-2 protein is purified by Reverse Phase HPLC. The column used is a C4 column, and purification is separated by a linear gradient of two mobile phases, collecting only IL-2 protein. This process aims at removing proteins and pyrogenic substances of similar molecular weight to IL-2. The IL-2 solution collected by reverse phase HPLC chromatography was passed through Sephacryl S-200 Gel chomatography to collect the IL-2 protein. Load IL-2 corresponding to 2% of the total resin volume by volume. In this process, IL-2 proteins with a purity of 98% or higher are collected and the IL-2 polymer and molecular weight less than IL-2 are removed. The collected IL-2 protein is 10mM Phosphate, pH 7.5, and Diafiltration using 10Kda Ultramembrane. Only pure monomer of Interleukin-2 thus obtained was used for the polyethylene glycol addition reaction.

Example 3 Binding of Polyethylene Glycol of Human Interleukin-2 Homolog

1.Manufacture of Branched mPEG (40,000) -maleimide-IL-2 homologue

10 mg of each human interleukin-2 isoform prepared in Example 2 was added to 0.1 M phosphate buffer (pH 7.0-8.0) and 400 mg of branched mPEG (40,000) -maleimide (manufactured by NOF) was added. The reaction was carried out with stirring at room temperature for 2 hours and the reaction was stopped by lowering the pH to 3.0 with 1 N hydrochloric acid solution.

Example 4 Purification of Polymer of Human Interleukin-2 Homolog and Polyethylene Glycol

The PEG-IL-2 polymer prepared in Example 3 was purified purely by the following procedure.

1.CM Sepharose FF Chromatography

10 ml of CM Sepharose FF (Amersham Biosciences), equilibrated with 2 mM HCl of at least 3 column volumes, after diluting each mPEG-IL-2 polymer to pH 3.0 with dilution of 10 times or more with distilled water The packed column was loaded. The loaded column was equilibrated with at least 3 column volumes of 2 mM HCl and then washed with 3 column volumes of 50 mM sodium acetate buffer (pH 5.0). 50 mM sodium acetate buffer solution (pH 5.4) containing at least 12 column volumes of 40 mM sodium chloride for mPEG (20,000) -maleimide-IL-2 isoform and branched mPEG (20,000) -maleimide-IL-2 isoform MPEG-IL-2 polymer was eluted. For branched mPEG (40,000) -maleimide-IL-2 isoform, the mPEG-IL-2 polymer was eluted with 50 mM sodium acetate buffer solution (pH 5.4) containing more than 12 column volumes of 20 mM sodium chloride. The eluate was fractionated by 5 ml and the eluted fractions were collected after analysis by HPLC.

2. Gel Permeation Chromatography

The mPEG-IL-2 polymer purified by CM Sepharose FF was run at 3000 rpm for 25 min at 10,000 Da molecular weight Centricon Plus-80 to concentrate the protein concentration of the final concentrate to about 5 mg / ml.

The concentrated protein was loaded onto a column packed with 300 ml of Sephacryl S-200 (Amersham Biosciences) equilibrated with at least 3 column volumes of 10 mM sodium acetate buffer (pH 5.4). The buffer was developed at a rate of 1 ml per minute to purely separate the mPEG-IL-2 polymer contained in the protein solution. At this time, the protein was eluted in the order of IL-2 multimer, PEG-IL-2, IL-2 dimmer, IL-2 monomer.

Claims (10)

At least one amino acid is added before Ala of the N terminal portion of human interleukin-2 represented by amino acid sequence number 1 or after Thr of the C terminal portion, at least one of the added amino acids is cysteine, and at least one of the cysteines Human interleukin-2 homologue with polyethylene glycol bonded to it. The human interleukin-2 homologue according to claim 1, wherein 1 to 12 amino acids are added to each terminal portion of human interleukin-2. The human interleukin-2 homologue according to claim 1, wherein the added amino acid is cysteine. 4. The human interleukin-2 homolog of claim 3, wherein the polyethylene glycol has a branched branch and has a molecular weight of 20 kDa to 40 kDa. The human interleukin-2 homologue of claim 4, wherein the cysteine is 134C added to the C terminus. The human interleukin-2 homologue of claim 1, wherein the human interleukin-2 is substituted with one or more amino acids by another amino acid before the polyethylene glycol is bound. A pharmaceutical composition comprising the human interleukin-2 homologue according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The gene encoding the human interleukin-2 homolog protein according to any one of claims 1 to 6, wherein cysteine is added to the N- or C- terminus. The oligodeoxynucleotide of any one of Claims 1-6 used as a primer for modifying the amino acid sequence of human interleukin-2. 10. The oligodeoxynucleotide of claim 9, wherein the primer is an oligodeoxynucleotide of SEQ ID NOs: 2-7.