JP2704214B2 - Cytokine having unpaired cysteine residue and conjugate thereof - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to cytokines and their conjugates, particularly conjugates, which have been site-specifically modified to have one unpaired cysteine residue at a location remote from the receptor binding site. The present invention relates to a conjugate in which a lipophilic group is bound to a combined cysteine residue and a conjugate of a cytokine having an unpaired cysteine residue at a position away from a receptor binding site in a native state.

BACKGROUND OF THE INVENTION Cytokines are responsible for the proliferation of functionally related neighboring cells,
It acts on cells by promoting or suppressing maturation or activity, and regulates cell growth, maturation and activity. Cytokines are produced by lymphocytes, macrophages, fibroblasts, nerve cells, or other types of cells,
Secreted.

In vivo studies of cytokines have shown that cytokines are generally latent factors that act at very low concentrations. Each cytokine has many different effects (pleiotropic effects) and acts locally on multiple types of functionally related cells. For example, sites of tissue repair, inflammation, or the immune response are generally involved in regulatory loops. Cytokines not only regulate the maturation and activity of target cells, but also the production of the cytokine itself is regulated by certain local factors. For example, in the microenvironment at the site where the immunogen was administered, macrophage was activated and interleukin 1 was activated.
Induces (IL-1) secretion. IL-1 activates adjacent antigen-specific T cells. The activated T cells then secrete interleukin-2 (IL-2), which acts as autocrine on the activated T cells, and the activated T cells also
Secretes interferon (IFN-γ), which further activates macrophages and B cell growth factors;
B cell growth factors activate adjacent antigen-specific B cells.

Let's compare the local function of cytokines with hormones of the endocrine system. Hormones are produced by local tissues and organs, are carried by the blood circulation, and act on tissues and organs throughout the body or on tissues and organs in different parts of the body.

Cytokines are generally small proteins and produce small amounts. Cytokines are not detected in the circulation using sensitive immunochemical techniques. Cytokines have a very high affinity for target cell receptors and trigger the next biological event simply by binding to a small number of receptors on the target cell surface.

Numerous cytokines have been identified and genes have been cloned and expressed in various systems. The production and purification of recombinant cytokines has been performed and has been applied in a variety of in vitro and in vivo applications for therapeutic purposes. Certain recombinant cytokines have been approved for therapeutic purposes by the U.S. Food and Drug Administration and competent authorities in other countries. For example, erythropoietin (EPO) increases granulocyte colony stimulating factor (GCSF) and granulocyte macrophage colony stimulating factor (GMCSF) to increase red blood cell count in patients undergoing renal dialysis.
Is used to increase the number of granulocytes in cancer patients undergoing chemotherapy and radiation therapy, and IL-2 is used to promote immunity in certain cancer patients, α-interferon (IFN-α)
Is licensed for patients with certain viral infections (eg, hepatitis C) or certain cancers.

Cytokines that target cells in the blood circulation or bone marrow, such as EPO and the colony stimulating factors described above (collected as hematopoietic growth factors), are safer and more potent than cytokines that target lymph nodes or other tissues. High (or at least easy to develop).
Many cytokines, such as IL-2, IL-1, tumor necrosis factor (TNF), IFN-α, and IFN-γ, are highly toxic when administered systemically, many in humans. In vivo clinical trials have shown. Furthermore, due in part to their small size, cytokines have very short pharmacological half-lives and are cleared from the circulation very early.

Cytokines that do not target cells in the blood circulation, when administered systemically, cannot provide short-term or local effects, as in vivo, or effects in the tissue microenvironment to target cells. It appears to be toxic. Cytokines are diffused by systemic administration to the circulatory system and to all sites, rather than staying at target sites.

Several cytokines have been studied for the treatment of local diseases, and some are still being studied. Epidermal growth factor (EGF), fibroblast growth factor (F
GF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) have been studied and clinically studied to promote the healing of accidental and surgical injuries. Some of these cytokines have been studied in the treatment of gastric ulcers.

The therapeutic effect would be further enhanced if the cytokines and therapeutic enzymes were delivered locally and maintained at sufficient concentrations at the target site for an appropriate period of time. In addition, since the toxic effects associated with systemic administration of cytokines are avoided, high doses of cytokines can be administered to diseased tissue sites. As an application example of local administration of cytokines, TN
Treatment of solid cancer with F, IFN-α, IFN-γ, or IL-2 is conceivable. Injured areas of the spine may be treated with nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF). IFN-α, IFN-
Administration of β, IFN-γ, or IL-2 to such tissues appears to be effective. Post-operatively, local administration of PDGF, EGF, FGF, or IGF to the injured tissue may promote healing. Inflammation of the nasal lining due to allergic reactions or infections may also be cured by using EGF, FGF, or IGF in combination with an allergy therapeutic or antibiotic. Gingival damage due to orthodontic surgery could be treated with EGF or IGF. EG
F or IGF may also be used to treat eye trauma caused by ophthalmic surgery or injury. Baldness, EGF
Alternatively, hair growth may be promoted by subcutaneously administering IGF to the site of hair loss.

It is believed that the fatty acylation of the cytokine keeps the cytokine locally, thereby avoiding toxicity associated with systemic administration. In addition, by retaining the cytokine locally for a sufficient period of time, the therapeutic effect can be increased. Proteins artificially acylated with fatty acids have been shown to have increased affinity for cell plasma membranes. For example, when ricin A chain (a toxic substance) is modified with fatty acids to form a complex, non-specific cytotoxicity to various cells is significantly increased. Kabanov, AVet e
l. Biomed . Sci. 1:33 (1990). Among cytokines, TNF
The ability to incorporate into liposomes was promoted by fatty acylation. Utsumi, et.al.Cancer Res. 51: 3362
(1991). This liposome is intravenously administered to a patient, whereby TNF is transported to reticuloendothelial cells. The bond of the aliphatic acyl group is determined by the ε of the lysine residue of the TNF polypeptide chain.
This is carried out by coupling an amino group or an α-amino group of the first amino acid residue with an N-hydroxysuccinimide ester of a fatty acid. Since the TNF polypeptide chain has six lysine residues per molecule, various types are ultimately produced depending on the number of fatty groups per molecule and with which specific lysine residue the fatty acid binds. You.

It is known that some raw proteins are modified in vivo after translation by the covalent bond of an aliphatic acyl group. The mode of binding between fatty acids and proteins can be divided into two types. Wilcox, CA and Olson, EN Bioch
emistry 26: 1029 (1987). The first acylated protein is one in which a fatty acid is bound to the polypeptide by an O-ester or thiol (SH) ester bond. Such acylated proteins are localized at the cell membrane,
It is mainly acylated by palmitic acid, a saturated fatty acid having 16 carbon residues.

The second acylated protein is one in which a fatty acid and a polypeptide are linked by an amide bond. Such proteins are acylated by binding myristic acid, a saturated fatty acid having 14 carbon residues, to an amino-terminal glycine residue. In contrast to the palmitate acylated protein, the myristic acid-containing protein is soluble and membrane-bound.

Among the cytokines, native acylated palmityl-IL-1 has been discovered. This IL-1 isoform binds to the membrane on the surface of monocytes and macrophages. Bacouche, O. et al. J. Immunol. 147: 2164 (1991). c
Within the denaturing elements of the is-Golgi and endoplasmic reticulum, the enzyme recognizes the characteristic structure of the protein to be acylated and the enzymatic attachment of O- and S-esters to aliphatic acyl groups . Only a small part of the total IL-1 is acylated, and only one of the OH groups of many serine residues or only one of the SH groups of many cysteine residues is a palmityl group. Combine with

Therefore, ricin A chain and TN
Fat acylation of F is difficult to actually acylate cytokines and promote the formation of homosexual complexes. Another difficulty is ensuring that cytokine binding sites are not aliphatically acylated. This is because such acylation may inhibit or suppress binding and / or biological activity.

SUMMARY OF THE INVENTION The present invention is a cytokine that has been site-specifically modified to include one unpaired cysteine residue at a location remote from the receptor binding site, wherein said unpaired cysteine residue comprises The cytokine is provided at a location remote from the receptor binding site such that the receptor binding activity or biological activity of the binding product is not significantly affected by the binding lipophilic substance. The present invention also provides a lipophilic cytokine, wherein the introduced SH group of the unpaired cysteine residue in the cytokine of the present invention is bound to a lipophilic substance. Furthermore, the present invention provides a pharmaceutical composition comprising the lipophilic cytokine of the present invention. In addition, the present invention relates to a position which is remote from the receptor binding site of the cytokine, and where the attachment of a lipophilic group to this position does not significantly affect the receptor binding activity or biological activity of the binding product. Is substituted with an unpaired cysteine residue, and a lipophilic substance is bound to the unpaired cysteine residue.

The present invention includes cytokines that have been site-specifically mutated to have one unpaired cysteine residue at a position remote from the receptor binding site. More particularly, cytokine molecules have been site-specifically mutated by DNA recombination techniques to produce cysteine residues that do not pair with sites distant from the receptor binding site. this is,
For example, this can be achieved by replacing other amino acid residues, such as serine, at a site distant from the receptor binding site with an unpaired cysteine. Cytokines containing unpaired cysteine residues introduced by site-directed mutagenesis, which are located away from the receptor binding site and are capable of binding lipophilic substances, are referred to as AUC cytokines (AUC is an allosteric unpaired cysteine (Alloste
ric unpaired cystein) ".

The present invention further encompasses such site-specific lipophilicization of AUC cytokines. Lipophilicity increases lipophilicity (affinity for lipids) and helps the resulting material bind to cell membranes or localize at target sites, thereby reducing the rate of clearance. The lipophilic cytokine can bind to cells at the site of administration, thereby reducing the rate of diffusion into the mucosal fluid within the intravascular space, so that the lipophilic cytokine is maintained at the target site for longer periods of time. You. However, the biological activity and binding of lipophilic cytokines is substantially identical to that of the native form.

One preferred embodiment of the present invention comprises a cytokine lipophilicized by being covalently linked to one aliphatic acyl group. Preferred aliphatic acyl groups have a carbon length of 6-18.

The invention further provides antibodies or other types of binding molecules, horseradish peroxidase, alkaline phosphatase,
Includes AUC cytokine conjugated to a fluorescent substance or biotin. Such conjugates are useful as reagents for cellular cytokine receptor analysis. IL-2,
AUC cytokines such as IL-2 and EGF can also bind cytotoxins such as ricin A chain or Pseudomonas exotoxin to target tumor cells that express the respective receptor at high density .

The invention further encompasses pharmaceutical compositions of lipophilic cytokines or other cytokine conjugates described above. Pharmaceutical compositions include suitable adjuvants, diluents and solvents.

DETAILED DESCRIPTION OF THE INVENTION A. Cytokines Suitable for Lipophilicity and Related Discussion Thereof As described above, a number of native (unmodified) cytokines have been studied to treat local disease sites. , Has been developed. These same cytokines (EGF, FGF, IGF, PDGF, TNF, IFN-α, IFN-γ, IL
-2, IL-1, NGF and CNTF) can be lipophilized by the method of the present invention, making them suitable for the above treatment. Can be inoculated by inhalation, cystic fibrosis
Certain enzymes, such as DNAases that may be useful for dissolving DNA in a patient's alveolar mucus, and superoxide dismutase that can be administered to inflamed joints in osteoarthritis patients are also provided by the methods of the present invention. It can be lipophilic and be suitable for such treatment. All of these cytokines are relatively small proteins, and the human genes that express them have also been cloned, characterized, and expressed in host cells. Therefore, the recombinant cytokine can be easily produced in an appropriate amount for the site-specific mutation, and lipophilicity is easy.

The lipophilic substance can be many, including fatty acid groups and lipophilic uncharged peptides. The lipophilic group should be non-immunogenic, non-antigenic and not large enough to affect biological activity or receptor binding. In addition, as noted above, cytokines are not lipophilic but bind to other chemical modifying groups including antibodies or other types of binding molecules, horseradish peroxidase, alkaline phosphatase, fluorophores, biotin and cytokine molecules. You can also.

Preferred lipophilic products are those linked to an aliphatic acyl group. The length of the aliphatic acyl group determines the lipophilicity of the final product, and the lipophilicity of the cytokine itself determines the equilibrium / distribution between the cellular plasma membrane and the cell space. Aliphatic acyl groups, which give the optimal pharmacokinetic properties of the end product for the intended application, are available in a wide range of carbon lengths (6, 8,
10, 12, 14, 16 and 18).

Generally, the longer the aliphatic acyl group, the greater the lipophilicity. The greater the lipophilicity, the more the final product can be distributed to a more restricted area, the less the diffusion and the longer the site of administration. Cytokines linked to acyl groups of 16 or 18 carbons bind well to the plasma membrane of cells and have low diffusivity. However, it is not necessarily the case that the greater the lipophilicity, the better. This is because some solubility and diffusivity are required to achieve maximum receptor binding and physiological effects. Aliphatic acyl groups that provide optimal pharmacokinetics for typical cytokines are saturated, linear C8-C8.
14 things.

B. Cytokines Suitable for Local Administration and Methods Thereof Cytokines are more suitable for local administration than systemic administration based on the results of human clinical trials if the cytokine has one or more of the following properties:

(1) The substance is very toxic when administered intravenously or by general systemic routes.

(2) The elimination rate of the substance is high, that is, the serum half-life is less than 3 to 4 hours.

(3) When the substance is administered systemically, the therapeutic effect on the target disease is small.

(4) The disease site targeted by the substance is appropriately localized such that local non-systemic administration facilitates distribution of the substance. For example, erythropoiesis in the bone marrow is anatomically and histologically localized, while non-metastatic solid tumors or ocular infections are well-localized examples. It is not an example.

The lipophilicity of a cytokine provides a product that has a higher affinity for the plasma membrane of the cell than the native cytokine, thereby providing a lipophilic product that stays at the target site longer than the native substance. The lipophilic substance stays attached to the cells long and does not quickly diffuse into the capillaries and be carried away by the bloodstream.
Similarly, when the lipophilic product is administered to the mucosal surface,
It can bind to cells and avoid being quickly washed away by mucosal fluids.

C. Diseases and Methods Suitable for Treatment With Lipophilic Cytokines Many diseases and conditions can be treated by topical administration of lipophilic cytokines. If the disease situation is properly localized and the dispensed drug can contact the diseased tissue,
Topical administration of lipophilic cytokines is desirable. This is because relatively large doses can be administered without the toxicity associated with systemic administration. The mode of administration should be such that the drug can be distributed to all of the diseased tissue. “High density” injection, in which a small amount of a therapeutic solution is injected at multiple sites per unit volume of tissue, is a preferred mode of administration. For example, injecting 10 μl of the treatment solution at 10 sites in 1 ml of solid tissue is a typical dosing protocol.

Solid tumors are an example of a condition suitable for treatment with a lipophilic cytokine. Lipophilic IFN-α, IFN-γ, TNF, IL-1
Alternatively, IL-2 can be injected directly into the solid tumor site. This treatment is particularly attractive when the tumor has not metastasized or has only minimal metastasis. This direct injection can also be performed in conjunction with surgery to allow more direct contact with the tumor site. Such injection near the incision site also ensures an optimal immune response to help destroy remaining tumor cells. Topical administration of lipophilic cytokines may be effective where the affected tissue is superficial, very thin or exposed, such as infected cornea, nasal mucosa, superficial trauma and bald skin .

Lesions and genital warts may be lipophilic IFN-α, IFN-β or IFN
It can be treated by direct injection of -γ. Again, direct injection results in direct contact with the disease site. Spinal cord injury can be treated by direct injection of lipophilic NGF or CNTF. Lipophilic superoxide dismutase can be administered to inflamed joints of osteoarthritis patients.

Infections affecting the lungs, such as influenza, bacterial, viral or parasitic pneumonia, are lipophilic IFN-
It can be treated by inhalation of α, IFN-β or IFN-γ. In cystic fibrosis, lipophilic DNase can be inoculated by inhalation. DNase can dissolve DNA in the alveolar mucosa and thus reduce the viscosity of the mucosal fluid. A preferred inhalation device is a graduated metered dose inhaler that can ensure that a metered amount is dispensed.

Local viral, bacterial or yeast infections of the vagina, rectum, mouth, throat, nose, eyes and / or ears are preferably treated with lipophilic IFN on mucosal surfaces using a drip or spray device.
It can be treated by administering -α, IFN-β or IFN-γ. Such topical administration of lipophilic EFG, FGF or IGF may be effective in halting wound or surgical incision or lesion healing.

Lipophilic EG to treat baldness or promote hair growth
F, FGF or IGF can be administered by intradermal injection, subcutaneous injection or topical application.

D. Preparation and Conjugation of AUC Cytokine Aliphatic acyl group is ε-
In the case of binding via an amino group, it is not possible to ensure that one aliphatic acyl group is bound to the ε-amino group of a specific lysine residue on the cytokine. Thus, the final lipophilic cytokine will be a heterogeneous mixture that differs in the number and location of aliphatic acyl groups per cytokine molecule. In such conjugates, some of the modified cytokine molecules will have lost receptor binding activity. This is because the aliphatic acyl group may bind to an amino acid group near the receptor binding site of the cytokine. In addition, such heterogeneous mixture conjugates may increase the immunogenicity of the site modified by the aliphatic acyl group. Thus, a preferred method of conjugation is to allow the lipophilic group to be site-specifically bound to a particular position rather than to another position on the cytokine molecule.
The first is to modify the cytokine in a site-specific manner.

In order to construct the lipophilic cytokine of the present invention, the cytokine gene is site-specifically mutated by a recombinant DNA method. At this time, the mutated cytokine is mutated to have an unpaired cysteine residue at a position sufficiently distant from the receptor binding site so that receptor binding or biological activity of the cytokine is not inhibited. .

Generally, a native cytokine molecule is a single polypeptide chain and contains an even number of cysteine residues. These cysteine residues form a disulfide bond between a pair of cysteine residues. The specific pairing of cysteine residues is determined by the three-dimensional folding of the polypeptide chain, which is determined by the sequence of the polypeptide. Disulfide bonds are not normally exposed on the surface of protein molecules, and their function is to maintain the protein in a rigid structure that can withstand the relatively harsh conditions present outside the cytoplasm. Proteins secreted, such as cytokines, have disulfide bonds, whereas proteins maintained in the cytoplasm or on the inner surface of the plasma membrane do not.

Cysteine residues can be introduced at specific sites on the cytokine, thereby providing a site for attachment to an aliphatic acyl group. The criteria for selecting where to place this residue are as follows.

(1) The substitution does not affect the three-dimensional folding of the cytokine molecule.

(2) This residue should be remote from the receptor binding site.

(3) This residue should be on the surface of the protein molecule and be able to contact the aliphatic acyl group.

Generally, it is most preferred to replace the serine residue in or near the highly hydrophilic peptide region with a cysteine residue. Cysteine and serine residues are structurally highly homologous. The proximity to or within the hydrophilic peptide region ensures that this residue is present on the surface of the protein molecule and thus can be subjected to a chemical bond after substitution. Other residues that can be substituted are asparagine, glutamine, tyrosine, provided that they are located in or near the hydrophilic peptide region.
Polar or charged, such as histidine, lysine, arginine, aspartic acid and glutamic acid.

Since the three-dimensional X-ray crystallographic structure of most cytokine molecules has not been determined, it is not possible to predict whether an amino acid residue is in or near the receptor binding site of a particular cytokine. A method of making this determination and identifying the appropriate residue to replace is to replace a particular residue with cysteine and, after lipophilicity, determine whether the receptor binding or biological activity of the replacement product is affected. Including determinatively.

The stepwise method of obtaining AUC cytokine is as follows.

(I) Sequencing The first step is to determine the amino acid sequence. The sequence of most cytokines has been described in the literature, and it is not necessary to determine the sequence. For others, sequencing can be performed by determining the nucleotide sequence of a cDNA clone of the mRNA for the cytokine. The deduced amino acid sequence can be confirmed by N-terminal amino acid sequence analysis and molecular weight determination of the cytokine protein.

(Ii) Hydrophilicity analysis The next step is to analyze the hydrophilicity of the cytokine polypeptide. Several software programs are available and can be used to plot hydrophilicity in quantitative figures with respect to the primary amino acid sequence. One such computer program is Hopp, TP and Woo
Developed by d, KR, Mol.Immunol.20: 483 (1983)
It is described in. Micr sold by Beckman Instruments, Palo Alto, California
The oGenie sequence analysis package provides a software program for performing hydrophilicity plots.

(Iii) Identification of candidate residues The next step is to identify hydrophilic regions in the polypeptide chain,
Identify the residue in or near the hydrophilic region that is most suitable for substitution with a cysteine residue. A suitable residue for substitution is a serine residue. However, when a serine residue is not available or is not appropriate, histidine, tyrosine, glutamic acid, aspartic acid, lysine, histidine, asparagine or glutamine can be selected in place of the serine residue. Even with only one substitution per molecule, a large number of mutants (as many as 10) can be obtained. Finally, mutants are screened to determine which substitutions are distant from the binding site by the methods described below.

(Iv) Gene synthesis The next step is to synthesize native and mutant genes. To construct a native cytokine gene, the polymerase chain reaction (PCR) can be used. Oligonucleotide primers corresponding to the 5 'and 3' ends of the cytokine mRAN and containing appropriate cloning sequences can be used. First, RNA is prepared from activated lymphocytes, leukocytes, cells whose cells produce fibers or other cell lines that produce a particular cytokine, from which cDNA is cloned. After confirming the sequence, the cloned cDNA is inserted into a plasmid such as pUC19, and subjected to the following operation. A routine laboratory method for site-directed mutagenesis is to synthesize an oligonucleotide primer containing about 25 nucleotides containing a triplet codon of a cysteine residue instead of a triplet codon of a serine (or other) residue to be replaced. Start by doing. These primers incorporating the mutation allow for the synthesis of full-length DNA genes with site-specific mutations. One convenient method is Kunkel, TAProc. Natl. Acad. Sci. USA, 82: 488 (198
Developed by 5). A step-by-step protocol using reagents is described by Kunkel, TA in Current Protocols in Molecu.
lar Bilogy, Supp. 6 § 8.2.1, Eds. Ausubel, FM et al, W
It is described in iley Intersciences (1990). PCR methods for introducing point mutations into cloned DNA are also routinely used in many molecular biology laboratories.
Cormack, B. Current Protocols in M
olecular Biology, Supp. 15 § 8.5.1 Eds. Ausubel, FM
et al, Wiley Intersciences (1991).

A preferred method of constructing the entire family of native genes and variants is to synthesize the complete gene using a DNA synthesizer. From the positive and negative chains,
Overlapping 60-80 nucleotide oligonucleotides complementary to the 3'-terminal adjacent oligonucleotide can be synthesized, for example, on one of the commercially available DNA synthesizers available from Applied Biosystems. Oligonucleotides provide both the template and primer (mutually primed synthesis), resulting in the desired sequence in a single step.
After extension with T7 DNA polymerase, the segments are ligated with ligase. Oligonucleotides at both ends of the gene are appropriately designed to include restriction enzyme sites so that the synthesized gene can be inserted into an appropriate expression vector. The reagent to be prepared and the operation method for each stage are Mo
ore, DDCerrent Protocols in Molecular Biology, Sup
p.§ 8.2.8, Eds.Ausubel, FMet al, Wiley Interscienc
es (1990). According to this method, it is easy to construct a large number of site-specific mutants required for constructing various mutants, and generally, since the cytokine gene is relatively small at less than 600 nucleotides, This method is attractive. All but one oligonucleotide with a particular mutation can be shared in constructing an individual construct. A complete synthetic interferon gene has been created. Edge,
MDet al. Interferon 7, Ed. Gresser, Ipp. 2-46 (Acade
mic Press, London, 1986).

(V) Expression The next step involves expressing a set of wild-type and mutated cDNAs in a eukaryotic or prokaryotic expression system, producing native and mutated cytokines, and producing these in sufficient quantities. It is to be. For most cytokines having disulfide bonds or being glycosylated, eukaryotic expression systems are preferred. Because
This is because formation of a disulfide bond and proper folding of the polypeptide chain are ensured. Among various eukaryotic expression systems, a preferred system for expression of a foreign cytokine gene is
Baculovirus, an insect virus (Autographa Cal
ifornica) using an expression vector derived from the nuclear polyhedral protein gene. This virus can grow in insect cells (Sprodoptera frugiperda cells, (sf9 cells)). Luckow, VA and Summers, MD, Vi
rology 167: 31 (1989). This expression system is packaged in a convenient kit (trade name "Max Bac Baculovirus Express System") and sold by Invitrogen (San Diego, CA).

Cytokines that do not contain disulfide bonds, such as IFN-γ, and cytokines that do not have a carbohydrate moiety or that do not participate in binding to cytokine receptors can be expressed in prokaryotic cells. If an E. coli expression system is used, the expressed cytokine protein is solubilized and reduced to unwind the polypeptide and then returned to normal to form the most favorable three-dimensional structure. A preferred system is the FLAG Biosystem kit marketed by International Biotechnologies of Kodak (New Haven, CT). The system also includes reagents for detection and purification of the unfused protein. Monoclonal antibodies to most cytokines have been developed and are commercially available. These monoclonal antibodies can be used for affinity purification of each cytokine.

(Vi) Conjugation The next step is to conjugate the purified native and mutant cytokine to an aliphatic acyl group, preferably about 8 to 14 carbon atoms in length. Preferred to achieve this linkage using a C10 aliphatic acyl group, Carlsson
Examples of chemical reactions modified from these methods, Biochem. J. 173: 723 (1978), are as follows.

In reaction 1, 1-bromodecane (Aldrich
(Commercially available from Milwaukee, Wis.) With 2,2'-dipyridyl disulfide (Aldrich) to form pyridyldecane disulfide. In reaction 2, the product is reacted with the AUC cytokine to form a decanoyl protein.

However, the first step before performing the binding reaction is to create a free SH group on the cytokine. The free SH group of the unpaired cysteine of the cytokine, which is often associated with other sulfhydryl group-containing metabolites during biosynthesis, is first slowly reduced to release it from the metabolite.
However, these mild reduction conditions do not reduce the disulfide bonds buried inside the cytokine molecular spine. After reduction, the reducing agent is removed by gel filtration or ion exchange chromatography. The treated cytokine is then reacted with an aliphatic acyl group previously modified with an active group.

Native cytokines are unlikely to bind to aliphatic acyl groups. Because native cytokines
It has no accessible, unpaired cysteine residues. Their receptor binding / biological activity can then be analyzed as described immediately below to determine if they are AUC cytokines. If this analysis reveals that they are not AUC cytokines, the cysteine residue is replaced with a serine residue (to ensure that the cysteine residue is not attached to an aliphatic acyl group) and other Other residues at the site can be replaced with cysteine residues. Replacing cysteine with serine will not affect receptor binding or biological activity. The coupling reaction results in aliphatic acylation at only one unpaired cysteine residue,
No aliphatic acylation takes place at any other site.

(Vii) Receptor binding / biological activity The final step is to analyze and compare the receptor binding and biological activity of native and aliphatic acyl-binding mutant cytokines. A variant cytokine molecule having a particular substituted cysteine residue and having substantially the same receptor binding and biological activity as the native cytokine is called an AUC cytokine.

2. Example: Preparation of AUC-IFN-α2 The steps described in section D. (i)-(vii) can be used, for example, for the production of the AUC cytokine IFN-α2. IFN-α2 is a member of the interferon cytokine family, and Table 1 below contains relevant important properties of this family, particularly information on its cysteine residues.

19 types of IFN-α, 1 type of IFN-β and 1 type of IFN
-Γ exists. All other IFNs except two kinds of IFN-α
-Α has four cysteine residues, residues 1 and 99
(Or 98 or 100) and between 29 and 39 form two disulfide bonds. Only IFN-α1 and IFN-αD have unpaired cysteine residues (No. 86). IFN-β has one disulfide bond (between cis residue numbers 31 and 141) and one unpaired cysteine residue (No. 1).
7). IFN-γ has no cysteine residues.

Introducing an unpaired cysteine residue into the cytokine,
The method described in section D. (i) to (vii) above should be used for most IFNs, but IFN-α
1, IFN-αD and IFN-β should not be used. Because IFN-α1, IFN-αD and IFN-β all have unpaired cysteine residues, the method described in section D. (vi) (replacement of unpaired cysteine residues with serine) was used. And then replace the other positions with cysteine).

In all IFN-α except IFN-α1 and IFN-αD, the fact that amino acid residue No. 86 is a serine or tyrosine residue means that cysteine residue No. 86 in IFN-α1 and IFN-αD is Suggests that it is not important. Therefore, for INF-α1 and IFN-αD, first, Cys86
Is linked to an aliphatic acyl group by the method of section D. (vi), and then the method of section D. (vii) is used to determine whether the conjugate has receptor binding and biological activity. If this substitution is not feasible, or if the above analysis reveals that the cytokine is not an AUC cytokine, Cys86 is replaced with a serine residue and AUC-IFN-α according to the method of section D. (vi).
1 and IFN-αD are prepared.

A similar method can be used to prepare the AUC form of IFN-β. If the native unpaired cysteine residue is replaced by a serine residue or other residue and the unpaired cysteine residue is introduced replacing another residue at a different position, the mutant cytokine will be It has two site-specific mutations, but only one site is suitable for binding to lipophilic substances.

As shown in Table 1, IFN-α2 has four cysteine residues and forms two disulfide bonds.
The nucleotide sequence and deduced amino acid sequence of the cDNA gene are known and published. Hopp, TP and Wood, K.
MicroG using the principle of R.Mol.Immunol.20: 483 (1983)
By using the hydrophilicity analysis program provided by enie, a hydrophilicity plot can be generated (not shown). This plot shows the relatively hydrophilic peptide regions. Using the criteria described above in Section D, the amino acid residues selected for site-specific mutation (ie, the residues to replace with cysteine residues)
Are serine No. 11, arginine No. 22, lysine No. 31, glutamic acid No. 42, glutamic acid No. 51, serine No. 72,
Glutamic acid No. 113, Serine No. 115, Lysine No. 133, Serine No. 160 and Serine No. 163 (11 mutants in total were created).

A preferred method for preparing the native IFN-2 gene and the 11 mutant genes is described in Section D. (i.
This is the oligonucleotide synthesis method described in v) above. Preferred coupling methods are described in section D. (vi).

The stepwise method described generally above and detailed for IFN-α2 can also be used to prepare other AUC cytokines. As mentioned above, most native cytokines have an even number of cysteine residues and no unpaired cysteine residues. For example, human
The length of an EGF polypeptide is six cysteine residues (3
The human basic FGF polypeptide has a length of 155 amino acid residues having four cysteine residues (two disulfide bonds), and has a length of 155 amino acid residues having four cysteine residues (two disulfide bonds). The peptide has a length of 70 amino acid residues with 6 cysteine residues (3 disulfide bonds) and the human TNF-α polypeptide has 2 cysteine residues (1 disulfide bond)
With a length of 157 amino acid residues, human β-NGF polypeptide has six cysteine residues (three disulfide bonds).
Has a length of 188 amino acid residues. Only a relatively small number of cytokines have an odd number of cysteine residues and thus have unpaired cysteine residues. One of them is the human IL-2 polypeptide, which has a length of 153 amino acid residues with three cysteine residues (one disulfide bond). Unpaired cysteine residue No. 125
Can be substituted for residues other than cysteine without loss of IL-2 receptor binding and biological activity. EP-A-136489. Therefore, this cysteine residue (N
o.125) will probably provide a site for binding.

E. Application of AUC Cytokines to Prepare Improved Reagents for Research and Diagnostic Assays The present invention focuses primarily on the therapeutic administration of aliphatic acyl derivatives of AUC cytokines locally or to mucosa. ing. However, these are not the only therapeutic applications of AUC cytokines. IL-2, IL-4 and
Cytokines such as EGF have been used as binding molecules and carriers for tumor cells expressing high density of each cytokine receptor for cytotoxins such as Pseudomonas exotoxin. Heterogeneous conjugates are obtained when native cytokines are conjugated to cytotoxins by conventional methods. Binding of the cytotoxin to the AUC cytokine via an unpaired cysteine residue results in an improved conjugate targeting the cytotoxin to the cell.

Therapeutic applications are not the only method of applying AUC cytokines. Provides useful research and diagnostic reagents
Several methods of application of AUC cytokines can also be developed. In studying the function of cytokines, it is important to quantify the concentration of cytokines in culture or body fluids, the expression level of cytokine receptors on target cells and / or the concentration of receptor-expressing cells in tissues. In certain diseases and conditions, the concentration of these biological parameters may vary. Thus, monitoring these concentrations may provide a means to determine or monitor disease status.

For such quantitative assays, including immunochemical and cell binding assays, binding to an indicator (eg, antibody, horseradish peroxidase, alkaline phosphatase, fluorescent, and biotin for use with an avidin-binding indicator). These cytokines are useful reagents. In conventional methods for preparing these conjugates, conjugation has been via the ε-amino group of lysine residues. However, as mentioned above, the conjugates thus formed are heterogeneous with respect to the number and position of the groups attached. Also, the activity and receptor binding of some of these binding cytokine molecules may be affected by modifying groups attached to lysine residues in or near the binding site. By linking these modifying groups via a single unpaired cysteine residue of the AUC cytokine using the methods described above, these problems can be minimized or eliminated, and therefore, research and diagnostics For improved reagents. For receptor studies, cytokines are superior to receptor-specific antibodies.
This is because cytokines usually have a higher affinity for each receptor than antibodies. Cytokines are smaller in size than antibodies, and are therefore particularly suitable for staining tissue sections where reagent penetration and diffusion is important.

The terms, expressions, and examples used herein are intended to be illustrative only and not limiting, and those of ordinary skill in the art will appreciate that the embodiments of the invention described herein need not be beyond routine experimentation. Will be able to recognize many equivalents. All such equivalents are intended to be encompassed by the following claims.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C07K 14/54 C07K 14/555 14/555 A61K 37/02 AED (56) References The Journal of Im Munology, Vol. 147, No. 7, p. 2164-2169 (1991) Cancer Research, Vol. 51, No. 13, p. 3362-3366 (1991)

Claims (10)

(57) [Claims] Claims: 1. A cytokine site-specifically modified to include one unpaired cysteine residue at a location distant from the receptor binding site, wherein said unpaired cysteine residue binds thereto. A cytokine introduced at a location remote from the receptor binding site such that the receptor binding activity or biological activity of the binding product is not significantly affected by the lipophilic substance. 2. Interferon α, γ-interferon, interleukin-1, interleukin-1 other than interferon-α1 and interferon-αD.
2. The cytokine according to claim 1, which is a tumor necrosis factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, nerve growth factor or ciliary neurotrophic factor. 3. A lipophilic cytokine, wherein the free SH group of the introduced unpaired cysteine residue in the cytokine according to claim 1 is bound to a lipophilic substance. 4. The lipophilic cytokine according to claim 3, wherein said lipophilic substance is selected from the group consisting of an antibody, biotin, a fluorescent dye, horseradish peroxidase and a lipophilic substance. 5. The lipophilic cytokine according to claim 3, wherein the lipophilic substance is an aliphatic acyl group or a lipophilic uncharged peptide. 6. An aliphatic acyl group is selected from 6, 8, 10, 12, 14,
The lipophilic cytokine according to claim 5, which has a carbon length of 16 or 18. 7. The lipophilic cytokine according to claim 6, wherein the aliphatic acyl group is a saturated linear aliphatic acyl group having 8 to 14 carbon atoms. 8. The binding cytokine is serine No. 11,
Arginine No. 22, Lysine No. 31, Glutamic acid No. 42,
Glutamic acid No.51, Serine No.72, Glutamic acid No.11
3, IFN-α having a cysteine residue replacing serine No. 115, lysine No. 133, serine No. 160 or serine No. 163 and an aliphatic acyl group bonded to the substituted cysteine residue
7. The lipophilic cytokine according to claim 6, which is 2. 9. A pharmaceutical composition comprising a lipophilic cytokine according to claim 5, suitable for administration to a patient for treating a local disease or condition. 10. A position which is distant from the receptor binding site of the cytokine and which does not significantly affect the receptor binding activity or biological activity of the binding product even when a lipophilic group is bound to this position. A method for producing a lipophilic cytokine, comprising substituting an unpaired cysteine residue and binding a lipophilic substance to the unpaired cysteine residue.