JPH03501741A - Glycosylated interleukin-2-containing medicine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Pharmaceuticals containing glycosylated interleukin-2 The present invention provides a novel glycosylated interleukin-2-containing drug. - Leukin-2, a method for isolating it from the culture supernatant of protein-producing recombinant CHO and glycosylated interleukin-2.

Interleukin-2 (hereinafter sometimes simply referred to as IL-2) is an antibody or antibody. Secreted primarily by mammalian T lymphocytes in response to activation by togenic compounds It is a lymphokine. This is due to the proliferation and proliferation of various cells involved in the immune response. It performs its main functions by acting on differentiation and other cells (R, J, Log , Immnol, Today, No. 51! No. 203-2 09 pages (1984).

Human-derived glycosylated interleukin-2 has been studied in detail. This is 13 It is produced in the form of a protein that contains three amino acids and has an oligosaccharide structure at the 3rd position of threonine. produced (H, S, Conrado et al., European Journal of Biochemistry Story (Eur, J, Biochem,) Volume 153, No. 255-261 (1986)). T lymphocytes initially use this as a precursor containing 153 amino acids. It is synthesized in the body and cleaves a 20 amino acid signal peptide at the endoplasmic reticulum stage. After that, a protein with 133 amino acids was glycosylated in the Golgi apparatus. (mature glycosylated protein).

insufficient from either peripheral lymphocytes or lymphoblastoid lineages such as the Jurkat system. Given that only a small amount of interleukin-2 is available in eukaryotic and prokaryotic cells, In either case, the protein gene in the foreign host can be expressed. Replacement technology can be expected.

That is, the interleukin-2 gene was successfully cloned and Escherichia coli bacteria and various eukaryotic cells cells, and more specifically COS monkey cells and CHO Chinese hamster cells. - Expressed in ovarian cells.

Recently, the inventors have produced interleukin-2 from recombinant CHO cells, and the main yields high yields of protein in the form of glycosylated interleukin-2. (see application EP-A-307285 and P. Ferra et al., Ferra et al. Febsletters Vol. 226 (1) pp. 47-52 (1987)). Therefore, a large amount of glycosylated interleukin-2 was released. Isolation and purification of pharmaceutical proteins starting from culture supernatants poses difficult problems. There is a problem. It is present as a main component in the isolated protein contained in the production system, Contaminants such as proteins, nucleic acids, endotoxins, and viruses that deteriorate and contaminate production strains It is necessary to remove the dye. Take care not to denature the protein and maintain its activity However, it must be ensured that it does not interfere with the patient's immune response. In addition, For industrial-scale production, immunoaffinity is the preferred protein separation method. Difficult techniques such as It should be ensured that quality is obtained in high yield.

Methods for the purification of natural and recombinant interleukin-2 have been previously reported. example In particular, Katoku is an interleukin produced from cultures of activated peripheral lymphocytes. reported a purification method of 2, which involves the following consecutive steps: Thion exchange chromatography, anion exchange chromatography, exclusion chromatography chromatography and reversed-phase HPLC chromatography (K, Katoku, Bay Ochemical and Biophysical Research Communications (B iochem and B1ophys commun) Volume 127 (1) pp. 182-190 (1985)). M, P, Uniil (Uniil, M.

P, Journal of Chromatograph 4-(J, of Chromato 396, pp. 209-215 (1987)) is Escherichia ・Extraction and extraction of recombinant interleukin-2 derived from E. coli discloses a method for purification, which includes the following steps: Cell lysis, extraction with butanol, 8M guanichloride in the presence of dithiothreitol. Dissolution of Interleukin-2 Aggregates in Gin, Exclusion Chromatography, Sulfuric Acid Protein regeneration by dilution of guanidine chloride in the presence of copper, cation exchange chromatography and reverse phase HPLC chromatography.

These methods include a step of reversed-phase HPLC chromatography, which A high degree of purification can be obtained due to the hydrophobic nature of This is a harsh condition that can denature interleukin-2. this present in the culture supernatant of CHO cells in a manner that satisfies the above-mentioned criteria. Glycosylated interleukin-2 cannot be purified.

This time, surprisingly, we have demonstrated that simple and large-scale production can be achieved starting from the supernatant of recombinant CHO cells. The methods that can be used to produce glycosylated proteins that have particularly beneficial pharmaceutical properties can be It turned out that it is possible to isolate leukin-2. This method is as follows The interleukin-2-based fraction from the second supernatant containing the separation, cation exchange chromatography, hydrophobic interaction chromatography and and exclusion chromatography. The present invention therefore provides a method comprising the following steps: Glycosylated interleukin obtained from the supernatant of a culture of recombinant CHO cells by Concerning Part-2.

a) Isolation of a fraction consisting mainly of interleukin-2 from the culture supernatant b) Cation exchange chromatography C) Hydrophobic interaction chromatography d) Exclusion chromatography This glycosylated interleukin-2 is more than 15×10”U/my It has CTLL-2 activity and contains natural interleukin 2 (approximately 20 ) is close to the CTLL-2 activity of p) Glycosylated ionic acid dissolved in an aqueous solution of 16.5 After freeze-drying interleukin-2 and immediately thawing the frozen product, the thawing solution is It is clear at normal pH and has at least 80% of its original CTLL-2 activity.

Preferably, the recombinant CHO cells contain precursors of interleukin-2 and DHPR. CHOdMr cells derived from DXB 11 strain by transformation with the current vector Preferably, it is a cell. In this case, the preferred expression vector is plasmid pSV 726. Recombinant CHO cells are preferably low protein Culture in artificial medium. The culture medium can be imported from recombinant CHO cells without any disadvantage. - Contains polyvinylpyrrolidone, a compound that specifically increases the production of leukin-2 Can be done.

The present invention also provides glycosylated interleukin from the culture supernatant of recombinant CHO cells. -2, which is characterized by comprising the following steps: do.

a) Isolation of interleukin-2-rich fluxine from culture supernatant b) Cation exchange chromatography C) hydrophobic interaction chromatography and d) exclusion chromatography Recombinant CHO cells are grown in suitable media well known to those skilled in the art, preferably protein-poor. The culture is carried out in a fresh artificial medium, such as a medium containing total protein 4R9/1 (L). Preferably, it contains polyvinylpyrrolidone.

Fraxidin, which is rich in interleukin-2, is more advantageously capable of penetrating proteins. It is separated from the culture medium by double filtration between the membrane 1 and the membrane 2 that holds it. film l is, for example, a microfiltration or ultrafiltration membrane. Membrane 2 is an ultrafiltration membrane. Like Specifically, the membrane l has a stop threshold of 30 kDa or more, more specifically 50 and 150 kDa. a, the membrane 2 has a stop threshold of less than 10 kDa, preferably between 5 and 10 kDa. It has between a. Membranes 1 and 2 are preferably made of cellulose acetate and polysulfone. Manufactured from

The cation exchange step is preferably carried out at a pH between 4, 5 and 6.5, more specifically between 5.5 and 6.5. 2 and 5.7, the hydrophilic polymer, e.g. Gallose, or polyacrylamide or hydrophilic vinyl polymer, or hydrophilic poly chromatograph consisting of a rigid or semi-rigid gel based on silica coated with A roughy support with substituents grafted to give the support strong thione exchange properties. It will be implemented using a standardized system. Sulfopropyl (SP) is a preferred group of this type. Ru.

The cation exchange chromatography step is preferably an anion exchange chromatography step. A first step is performed in advance.

In this case, the anion exchange step is preferably carried out at a pH between 5.5 and 8.5, more specifically at a pH of between 5.5 and 8.5. hydrophilic polymers, such as cross-linked agarose, typically in the range between 6.5 and 8.2. or polyacrylamide or hydrophilic vinyl polymer, or hydrophilic polymer. Chromatography consisting of coated silica-based rigid or semi-rigid gels On the support, substituents are grafted to impart weak anion exchange properties to the support. It is carried out with things. Diethylaminoethyl (OEAE) is a preferred group of this type. be.

Preferably, the hydrophobic interaction step is between pH 4, 5 and 8.0, more specifically Chaotropic reagents or organic solvents or pH 4, 5 in the range between 6.0 and 8.0 Glycosylated IL- without the addition of an aqueous solvent with a 2 on a desorption chromatography support. Examples of such supports include parent Aqueous polymers, such as cross-linked agarose, or hydrophilic vinyl polymers, or It is a silica-based gel coated with an aqueous polymer. Butyl, phenyl or The propyl group is grafted onto the support.

The exclusion chromatography step is preferably carried out at pH 5, more preferably between 0 and 8.0. or in the range between 6.0 and 7.0, with a fractionation range between l and 250 kDa. Performed on a support. The support is preferably a cross-linked acrylamide Kistrin-based or agarose-based.

Glycosylated interleukin-2 isolated by the above method can be used in other purification systems. modified interleukin-2, more specifically Escherichia coli (E, co lt) are highly suitable for use as active substances in drugs compared to those derived from properties, namely improved anti-tumor activity, excellent immunostimulatory properties, greatly reduced toxicity low immunogenicity, without adding toxic lysing agents or chemically modifying the molecule. , solubility at physiological pH in an aqueous medium and after lyophilization in a pharmaceutically acceptable formulation. and its excellent stability.

Therefore, the present invention also provides that the active ingredient is a glycosylated interleukin as defined above. The present invention relates to medicines that are part-2. The substance may be used alone or with other active substances, e.g. other cytokines, such as interleukin-11 interleukin-4, α - interferon, gamma-interferon or TNF, or anti-tumor agents such as vinegar Acid flavones or cyclophosphamide or antimitotic agents or immunomodulators such as cyclophosphamide Can be used with crossporin or BCQ, or antibodies or thymus hormones.

In all known or future applications of IL-2 and in combination with other active substances. The glycosylated IL-2 according to the present invention can be obtained from Escherichia coli (E. coli). li) may be an advantageous alternative to IL-2 derived from IL-2.

These applications include:

- various conditions, such as cancer, congenital or acquired immunodeficiency, immune abnormalities (as well as autoimmunity); epidemics and inflammatory diseases) and infectious diseases (especially microbial and parasitic viral infections) treatment, - preventive measures against the aforementioned symptoms, more specifically infectious diseases, and - vaccinations ( Use of IL-2 as a juvant) with glycosylated IL-2 and other active substances In combination with glycosylated IL-2, injection into ganglia or other immune-related organs (intravenous porous, by perfusion, subcutaneously, intraperitoneally or locally (e.g. into a tumor), or alternatively As a result of in vitro action on cells collected from patients or donors. It can be used for treatment.

The invention will be more easily understood from the following examples, which are merely exemplary. It's nothing more than that.

Example 1 Vector expressing interleukin-2 precursor and DHFR Construction of Niblasmid psV720 and pSV726 plasmids Basically, starting from an existing vector, DNA fragments are created using restriction enzymes. separation of oligonucleotides, chemical synthesis of oligonucleotides - optionally modifying their ends. After - various fusions by using enzymes, e.g. DNA ligase of phage T4. Linking the fragments, after cloning (Escherichia coli) Selection of plasmids (after bacterial transformation in Richia coli) and their It consists of the purification of

Manipulation uses techniques well known to those skilled in the art.

These techniques are based on the molecular cloning near laboratory by T. Mariatis et al. Molecular Cloniig: a Labo In 1982, Cold Spring In a work published by Harbor Press, N.Y.Y. Are listed.

The set of restriction enzymes required to construct the vectors described below are: Commercially available from Nis Nis A. There is.

The DNA ligase of phage T4 was prepared using Ni-Ni England Nuclease (Ni-Ni). Available from S.A.).

a) Plasmid psV72G This substance is 1. The following seven DNA fragments were prepared using the method shown in Figure 1 and described above. It is a concatenation of components.

- Genome 5V40 (W, Fires (1978), NATCH + - (natur e) 342 base pairs obtained from Vol. 273, pp. 113-120 (1978) (hereinafter referred to as bp) and also contains the early promoter of this virus. I[-HlndI[l fragment - encodes the natural precursor of IL-2, in which It is located at the 5° end of the coding strand! AGCTTCCACAATGTACA The GG nucleotide sequence is synthesized by the synthetic sequence AGCTTCCCACCATGGCTAGG. M, Kozak is substituted at the level of the nucleotides surrounding the codon ATG. ((1984) Nucleitsk Acid Research, 12, 857-872)i : DNA having a sequence corresponding to the CCACCATGG consensus sequence described by 5°4bP HfndI[I-B amHl fragment containing the sequence- Mouse alphaglobin (Y, Nishioka and P, Rader (1979) cell , 18,875-882), and the distal intron of this gene A 305 bp BamHI-Batl fragment containing - The early polyaldenylation signal of the virus SV40 from its genome. 133 bP Hpal-B amHl fragment containing null -Plasmid pBR322 (F. Poliver (1977), Gene 22.95- 1 asbp BamHI-EcoRV fragment from 113) - Plasmid psV 2-dhf deposited with ATCC under deposit number 37146 r plasmid (Nis Subramari et al. (1981) Molecular and Cellular 2677 bP PvuI [-Ec oRl fragment and -2295 bp Eco from plasmid pBR322 RI-Pvulll fragment Plasmid psV720 contains:

Unit expressing the natural precursor of IL-2. The promoter in this unit is SV40 early promoter: below the coding sequence for the precursor of IL-2 It's in the flow. The unit contains the second intron of the mouse alphaglobin gene. sequence followed by a sequence containing the processing adenylation signal of virus SV40. . and -ahfr expressing unit. The promoter within this unit is the virus SV4 0 early promoter: Located downstream of the DHPR coding sequence, this unit is , - Using the Dubriny-Fires notation method, the genome of one virus SV40 is Md at positions 4693 and 4083.

1 site, containing sequences between the indolone and the t antigen of the virus SV40. Contains the early polyadenylation signal of the virus SV40 following the SV40 virus. This unit is plastic. PvuI[-Ec from sumid pS V 2-dhfr.

Contained in the R1 fragment.

b) Plasmid pSV726 This plasmid is shown in Figure 2 and is derived from plasmid psv720 as follows. It will be done.

Plasmid I) SV(7) located upstream of the HindlU-BamHI section (1, ■ The modified signal chain of the first amino acid of L-2 (in the body and mature 1t, -2) ptydo (that is, the substitution of a tyrosine group by adopting a sequence corresponding to the Kozak consensus sequence) In contrast, HindII [and The DNA section between the 8g1AI restriction sites is then inserted into the naturally occurring precursor of the human-produced hormone GH. precursor signal peptide (hereinafter referred to as hGH signal peptide), and mature Synthetic duplex encoding the first amino acid of IL-2 from its ninth nucleotide Replaced by oligonucleotide.

The resulting plasmid is plasmid psV726, in which HindI[[-Ba The mHI section contains the IL-2 precursor (precursor) containing the hGH signal peptide. - ps-hGH-IL-2).

Plasmid pSV726 contains:

- A unit expressing precursor (ps-hG H)-1L-2. per book The promoter used is the early promoter of virus SV40 (ps-hG H) -1L - downstream of the coding sequence for the -2 precursor, this unit is the second intron of the mouse alphaglobin gene, followed by the virus comprising a sequence containing the early polyadenylation signal of SV40, and - A unit expressing dlr. The promoter in this unit is of virus SV40. It is an early promoter; it is located downstream of the coding sequence for dhfr; The unit of is one virus SV40 using the -Dubrigny-Fires notation method. It consists of the sequence between the Mdo1 sites at genomic positions 4693 and 4083, and The intron for the t antigen of virus SV40 followed by the beginning of virus SV40. Contains early polyadenylation signals. This unit is plasmid pS V 2-dhf Contained in the Pvull-EcoRI fragment from r.

The legend below is used for Figures 1 and 2.

H-1111 DN A sequence port from plasmid pBR322 =====co DNA sequence from the genome of virus SF40; DNA sequence from the coding gene for phaglobin DNA sequence constructing the coding sequence for the natural precursor of [4]]]]Here DNA sequence encoding db4r Example 2 Cell lines that give high yields of interleukin-2, namely line 58-12 and and production of 109-27 DXB 11 strain CH0dhfr- cells (Ulllaub et al., of the National Academy of Sciences, No. CHO disclosed in Vol. 77, No. 7, pp. 4216-4220 (1980) K, D HP R- clone) into plasmid pSV726 or plasmid Transfected with psV720.

The operating instructions are from F. Graham and A. van der Niv (Pyrology, No. 54). Vol. 536-2539 (1973)).

Cells were incubated with 10% (V/V) fetal bovine serum, gentamicin 20μ9/di, and tyrosine. Alpha M mixed with 60μ9/I and L-glutamine 300μ9/I Initially grown in EM (Gibco) (hereinafter referred to as non-selective medium) .

After washing with water, cover the cells seeded the previous day with a non-selective medium and add 10 μ9 of one of the plasmids. Sperm DN was added in the presence of calcium phosphate without A. manufactured by this method Cells were incubated for 7 hours at 37°C.

Cells were incubated at 37°C for 3 hours in alpha MEM medium containing 5% (V/V) fetal bovine serum. It was cultured in After incubation, cells were placed in Gibco Minimum Essential Medium, code no. No. 041-1095 was divided into 5°10' portions per bird dish. In this case, the medium Dialysis Gibco fetal bovine serum (lO%v/v), gentamicin (20μ9/j 112), tyrosine (50μy/xc), L-glutamine (300μ9/and Q) and L-proline (150μ9/chu) were used in combination. these additives The containing medium is hereinafter referred to as a selective medium.

The cells thus produced were incubated at 37°C for 2iI1 and selective medium was added for 3 days. Replace one piece each time. All colonies observed after incubation are effective It came from a cell that had integrated a plasmid into it. Take these colonies, IL-2 cells were individually recultured on selective media to confirm their suitability for IL-2 production. It was tested by measuring type activity.

! L-2 type activity stimulates the proliferation of IL-2 murine lymphocyte T line-dependent CTLL-2. Biological activity of culture media in intensive testing (P., Baker et al., Journal of ・Isisperimental Medicine, pp. 149-173 (1979)). Ko This activity is hereinafter referred to as CTLL-2 activity, and is BRMP (Biological Research ・Modifier Program, Lymph Chukin Research, Volume 4, No. 193 227 (1984)) (hereinafter referred to as U). (omitted).

The most productive colonies were subcultured sequentially on four products of selective media, and each The product is F, Aldo et al. (Journal of Biological Chemistry, No. 25) 3, pp. 1357-1570 (1978)). Methotrex at higher concentrations (0,02,0,05,0,1 and 0.2uM) Has a cert.

The material thus maintained was CHO5 transformed with plasmid psV720. 8,12 line and 109.27 transformed with plasmid pSV726 Both lines have high interleukin-2 productivity.

Example 3 High producers of interleukin-2 i.e. CH058-12 and 109 -27 cell line culture (1) CHO58- in a culture medium with an appropriate amount of protein Production of IL-2 from 12 lines A sufficient number of cells were obtained by growing them in a roller box (rolling flask). Afterwards, about 300,000 cells/cell were inoculated into the production fermentor.

The fermentation conditions are as follows.

a) Perfuse the culture at a perfusion rate of approximately 1 volume per day, b) Transfer the supernatant to a spin filtrate. Continuously collect after passing through the router (rotating filter is an Agitator shirt) C) Denatured collagen film (Cytodex IIIR, Pharmacia d) Cultivate the microspores of cross-linked dextran covered with 100% dextran. d) Adjust the pH of the culture to approximately 7. e) Adjust the dissolved oxygen pressure to 30% (after blowing air, 100% is defined as saturated medium), f) Adjust the culture temperature to approximately 37°C. g) The basic culture medium, referred to as medium 1, is mainly a mixture (50:50) of the following: Wachi Eagle MEM (Flora) Minimum Essential Medium and F12 Ham (Gibco) Nutrition Based on medium.

The composition of medium 1 is as follows.

The fermentation process has two parts called the growth phase and the production phase. During the growth phase, the cow Fetal serum (5%) was added to the medium. During the production phase, the medium is designated FIV-1, It was mixed with protein fraction isolated from bovine serum by Cohn fractionation method. Suitable for culture medium It contained a lot of protein (400m9 of total protein/12). Production is at least 30 days It lasted for a while. The crop was cooled in line to a temperature of 6°C. Total protein concentration is 500 v: g/(1. The CTLL-2 activity of the culture supernatant was 40,000 U/next Q1 That is, about 2 R9/l! (IL-2 specific activity was 2× Assuming 10'U/x9 and IL-2 protein purity of 0.14%). This culture supernatant is hereinafter referred to as culture supernatant A.

(n) Production of IL-2 from the CHO109-27 line in protein-poor medium A sufficient number of cells were obtained by growing them in a roller box (rolling flask). Afterwards, the production fermentor was inoculated with approximately 300,000 cells/11,112 cells.

The fermentation conditions are as follows.

a) Perfuse the culture at a perfusion rate of approximately 1 volume per day, b) Transfer the supernatant to a spin filtrate. Continuously collected after passing through the router, C) Crosslinked with a film of denatured collagen Growing microspores of dextran (Itodessis II [R manufactured by Pharmacia) ), d) Adjust the temperature of the culture to about 37°C, e) Bring the dissolved oxygen tension to 100%. ・ f) the temperature of the culture is approximately 37°C, and g) the basal culture medium is as defined above. It is ground 1.

The fermentation process has two parts called the growth phase and the production phase. During the growth phase, the cow Fetal serum (2.5%) was added to the medium. During the production phase, serum was added to insulin 3JI 9/12 and lactoferrin, which is the only protein in the medium, was changed to primary 9/I2. .

For this phase, 0.5% polyvinylpyrrolidone with an average molecular weight of 40,000 was added to this medium. This polymer increases the specific output of IL-2 from recombinant CHO. It was found that IL- secreted per unit time and unit biomass 2 amount). Therefore, the culture medium was a synthetic medium with low protein content (total protein: 41!9 /(1>. Production continued for at least 30 days. Harvest was lined at 6°C, celestial degree. Cooled inside. The total protein concentration of the culture supernatant was 100 r9/Q. CTLL- 2 activity is 1200001 J/x (l or approximately 6z&/I2 concentration and 6% protein It had white purity. The culture supernatant is hereinafter referred to as culture supernatant B.

Example 4 Part of IL-2 secreted by CHO5B-12 and 109-27 cell lines segmental characteristics 1. Purification of IL-2 IL-2 was purified from culture supernatant 112.

The first step is concentration and pre-equilibration with 0.05M ammonium acetate in the upper channel. Sepharose R agarose column (S-Fast Flow Fahemacia Ke) Initial purification using ion-exchange chromatography (Mikal, Sweden) there were. Elution included NaCl (molar concentration 1f0.05M, then 0.5M) ．． 05M ammonium acetate. By measuring CTLL-2 activity Eluted fractions that show biological activity are collected and their pools are inverted. It was subjected to high performance liquid chromatography on a phase column. The selected support is C3 graph It was tosilica gel. Column dimensions are 1.0X25. It was Ocm.

Elution was from 5 to 100% (V/V) in 0.1% aqueous trifluoroacetic acid (V/V). A varying linear acetonitrile gradient was performed for 80 minutes at a flow rate of 4x (1/min).

Collect the fractionated eluted biological activities and combine the pools with column size 2.0XIO. Ocm 018 grafted silica gel under the same elution conditions as described above without interalia. It was subjected to chromatography by Doden.

The biologically active eluted fragment pool collected by chromatography is as shown by electrophoresis on polyacrylamide in the presence of sodium sulfate. The purity of IL-2 was over 95% (Raemi, Nature, Vol. 277, No. pp. 680-685 (1970)). This pool was used to characterize IL-2. This is the material used.

2. Characterization of KL-2 by determining the terminal amino acid sequence of processed samples to bromide Place on the surface of the xadimethrine (or polybrene) filter. The filter is protein with chromatography (Applied Biosystems Model 430A) White array device (Model 470A manufactured by Applied Biosystems, USA) and continuously induce phenylthiohydantoin after each degradation cycle. The body was analyzed.

The results of this determination indicate that natural products (R, Robb et al. , Proceedings of the National Academy of Sciences 81, pp. 6486-6490 (1984) ) was consistent with the published sequence. This non-detection is due to the presence of the oligosaccharide at site 3. It is explained by

The sequence is written as the first 10 amino acids.

Ala-Pro-Thr-Ser-9er-Thr-Lys-Lys-Thr Ranin is the only radical detected at the N-terminal site. This allows the precursor -(ps-hG　H)　-1L　-2 was confirmed to be properly cleaved during secretion. It was proven.

Example 5 Glycodisinfection by the method of the present invention starting from the culture supernatant of the CHO109-27 line Isolation of Interleukin-2 The water used in this example and Example 7 was It was demineralized water that had been purified and ultra-filtered with a Lee Q type device.

1. Isolation of culture medium from interleukin-2 enriched fractions As described in Example 3. Take 130 liters of the supernatant of medium B loaded on Prefiltration was performed through an 8 μl threshold filter to remove large particles.

Interleukin-2 with a molecular weight of L 5 kDa to 17 kDa was fractionated and described below. How to operate! A first membrane with a stop threshold of 00 kDa and a 10 kDa It was concentrated by a second double tangential ultrafiltration with a stop threshold of a. 1st and The second membrane is a spiral cartridge of cellulose acetate, i.e. the filtrate from the first membrane. mounted in a cascade to supply the material retained by the second membrane. The membranes were YM100 and YMIO manufactured by Amicon.

The pre-filtrate is supplied to the first membrane and is retained in the first place by the first membrane and the second membrane. The material was concentrated until the volume of material retained by the first membrane was approximately 15 liters. . Next, the retained material is removed by removing all the IL-2 from the material retained by the first membrane. 80 liters of ultra-purified water to reduce the ionic force of the material retained by the second membrane. diafiltered (constant volume wash). Next, concentrate the latter ingredient to 1.5 liters. The interleukin-2 was recovered after rinsing the membrane with water.

The resulting concentrate was passed through a 0.2 μl stop threshold to remove the precipitate formed during ultrafiltration. Filtered through a filter with a value of The purified product is a concentrated aqueous solution of IL-2 2.4 It was a little.

2. Isolation of glycosylated interleukin-2 a) Anion exchange chromatography I At this stage, the nucleic acids, exotoxins, CHO proteins and other proteins present in the supernatant culture are Some types of contaminants, such as Ta. IL-2 was not retained on the column under selective elution conditions. Chromatography used The graphic support is a gel based on weak anion exchange cross-linked agarose, i.e. DEAE cell manufactured by Pharmacia with a glass column of diameter 140 m + m height 210 + nm It was Pharos First Flora. ammonium acetate crystals at 5 mS In addition to the previously obtained concentrated IL-2, ammonium acetate at pH 7 was added to adjust the conductivity to Monium buffer was injected onto the previously equilibrated chromatography support. Then The ram was washed with the last mentioned solution.

The fixation effluent and wash solution containing IL-2 were combined. The product is rich in IL-2 The amount of an aqueous solution was 7.08 liters.

bJ & ion exchange chromatography This step is present in the culture supernatant and is not immobilized on the anionic chromatography support. Removes certain contaminants such as polyvinylpyrrolidone, CHO protein and other proteins. and to separate variants of IL-2.

The chromatography support used was a column with a diameter of 50 cm and a height of 230 mm. The gel was based on the hydrophilic resin of Merck SP Fractogel 650 (s). . The pH of the solution obtained in the previous step was adjusted to 5.5 by adding acetic acid, and the solution was colored. was injected into the tank. Next, the ionizability of IL-2 immobilized on the column was gradually increased. Elute with a 2M aqueous solution of sodium chloride and ammonium acetate in various proportions. It was obtained by mixing the solution (50mMSPH5.5). The density of the solution is 280 pages. It was m. In this way, three protein-containing fractions were separated:

Fraction 1 containing dylylated glycosylated IL-2 (N2 form X') monosialylated Fraction 2 containing glycolylated IL-2 (N, type) (1) non-sialylated glycosylated IL-2 (No type) (containing RI and non-glycosylated IL-2 (M type) (1) Picture 3(') H, Conrad (European Journal of Biochemistry) 153, pp. 255-261 (1985)). Various types, see.

Fractions 1 and 2 were combined to obtain 0.45 liters of glycosylated IL-2 aqueous solution. .

C) Hydrophobic interaction chromatography The chromatographic support used is Hydrophobic interaction gels based on aqueous polyvinyl resins, i.e. Butylph from Merck & Co. Lactogel 650 (M) was placed in a column with a diameter of 70 mm and a height of 135 mm. .

1.2M mol or more of ammonium sulfate and ammonia adjusted to pH 6.5 Before glycosylated IL-2, add the obtained solution and preliminarily (pH 6, 5, 50o Equilibrate with an aqueous solution of +M ammonium phosphate and 1.2M ammonium sulfate. The solution was injected onto a chromatographic support. Under these conditions, glycosylated IL- 2 was retained on a chromatographic support. Products that are not retained are solution, followed by (pH 6, 5, 50mM ammonium phosphate and 0.8M ammonium sulfate) was washed away with an aqueous solution of ammonium. Next, IL-2 was treated with ammonium phosphate and and eluted with 0.1M ammonium sulfate aqueous solution (pH 3, 5.50mM). Generate The amount was 2.17 liters of glycosylated IL-2 aqueous solution.

d) Exclusion chromatography The exclusion chromatography support contains cross-linked dextran and 250-5 kDa range. Gel based on fractionation of the periphery, i.e. a column with a diameter of 100 mm and a height of 900+++ m. Sephacryl 5200HR manufactured by Pharmacia was used.

The solution obtained at the end of step C) was dissolved in cellulose acetate with a stop threshold of 10 kDa. After concentration on a base spiral membrane, the solution was adjusted to pH 6 beforehand.

5 into a column equilibrated with a 50mM aqueous solution of sodium phosphate, and The compound IL-2 was eluted with the last mentioned solution. The solution is measured at an optical density of 280 nm. was detected at the column outlet. The various fractions collected according to their purity Both were analyzed by reverse phase HPLC.

The product is glycomolization of sodium phosphate! L-2 solution was 0.6 liters. .

3. Verification of product purity *Polyacrylamide electrophoresis in presence of SDS Reducing agent (dithiothreitol) presence Lower PAGE-SDS polyacrylamide (sodium dodecyl sulfate) electrophoresis is the Laemmli method (U.

K. Laemli, Annals of Biochemistry, Volume 78, Page 459 ( (1977)) and the Noryl method (C.R., Merrill, Proceedings. of the National Academy of Sciences, No. 76, p. 4335 (1979)).

The resulting electropherogram is shown in Figure 3, which shows that the purity of glycosylated IL-2 is 99%. ．． It is confirmed that the percentage is 5% or more.

*HPLC analysis of reversed phase column The chromatographic support is grafted silica gel, i.e. Société Français Nucleosil C4300A with a particle size of 5 μm manufactured by Cologne Co., Ltd. It was. Elution was with a 6 minute gradient from (70% A + 30% B) to (25% A + 75% B). carried out. A is an aqueous solution containing 0.1% trifluoroacetic acid, and B is an aqueous solution containing 0.1% trifluoroacetic acid. It was an acetonitrile solution containing fluoroacetic acid.

The resulting chromatogram is shown in Figure 4 and shows one impurity, approximately 5.5% of the total product. It will be done. The impurity has been partially characterized and it is an oxidized substance with CTLL-2 activity. It was glycosylated IL-2.

*The IPLC analytical chromatography support for the cation exchange column is Kruseau. "Polycat A5μm 300 AJ's polyaspartic acid coated silica gel Met. Elution is (85% A' + 15% B) (75% A' + 25% B) 3.5 open, and (75%A'+25%B+) to (50%A'+50%B) It was carried out with a gradient of 6°5+++m, Al was KH1PO4 (pH 5.0, molar concentration 25mM), B' is KH2PO4 (pH 5.0, molar concentration 25mM) and 1M It was sodium chloride.

The resulting chromatogram is shown in Figure 5 and shows the two types of glycosylated IL-2. presence, i.e., IL-2(N*) 48% and IL-2(N,) 52% Can be used in quantity. In other tests, N is predominant.

4. Balance At the end of each isolation step, the amount of IL-2 present in the retained product is determined by It was determined by measuring the sex. Various types of IL-2 have the same CTLL-2 activity have.

The results are shown in Table 1 below.

Table 1 Total yield = 49.4% As the table shows, the CTLL-2 activity per unit volume was higher in stage 2 than in the culture medium. -d) is 10 times more concentrated and the total yield of glycosylated IL-2 was approximately 50% of the total KL-2 present in the supernatant.

Example 6: Method of the invention starting from supernatant of CHO209,27 cell line Characteristics of glycosylated interleukin-2 isolated by Measurement of monospecific CTU-2 activity = (18±3) x 10’U/xv-terminal amino Sequence determination The product sample was passed through a hexadimethrine bromide (or polybrene) filter. did. The filter continuously analyzes the phenylthiohydantoin derivatives produced. Equipped with chromatography (Model 430A - Applied Biosystems) Protein sequencer (Model 470A - Applied Biosystems (U)) It was inserted into S A)).

The results of this measurement are based on previously decoded natural product sequences (R. Robb et al. Ding of the National Academy of Sciences, USA Volume 81 6486-6490 (1984)) and the third position where no amino acid was detected. There was an exception in this regard, but otherwise they were in agreement. No amino acid detected at position 3 This is explained by the presence of an oligosaccharide at the 3rd position.

The first six amino acids of this sequence are:

Ala -Pro -Thr -Ser -Ser -Ser -Thr -L ys-Lys-Thr-determination of primary structure The reduced and carboxymethylated product is purified by 1/30 of trypsin at room temperature. weight/weight) and allowed to decompose overnight. The obtained peptide is then Using HPLC chromatography, an acetonitrile gradient (0,1% trifluoride) was used. (in the presence of acetic acid), Journal of Immunological Methods, Volume 81, 15 -30 (1985).

Each trypsinized and purified peptide was subjected to amino acid analysis and some were Subjected to degradation.

In this way, the complete amino acid sequence of the isolated product could be analyzed.

This is based on the amino acid sequences of natural products that have already been deciphered (R. Robb et al., Proc. Journal of the National Academy of Sciences, USA Volume 81 6 486-6490 (1984)).

Tryptic peptides of the product obtained after reacting with vinyl-4-pyridine From the study shown in the figure, cysteine 58 Disulfide bridge between and cysteine 105 and cysteine! Free thio to 25 This shows that there is a metal group (S　H).

Analysis of mono-oligosaccharides According to polyacrylamide gel electrophoresis in the presence of SDS, N, type and N, Two forms of the product were shown with molecular weights of approximately 16.5 and 16.0 kDa corresponding to the (See Figure 3). Neuraminidase treatment (P, Ferrara et al., Vol. 226, No. 1, 4) 7-52 (1987)), the two detected bands disappeared and the separation Weight! A band corresponding to the 5.5 kDa No type (see Figure 3) is expressed. this This means that the difference between the two is due to the presence of sialic acid in the molecule. It shows. The product is chromatoelectrofocalization (ehr o’matoelectrorocalisat 1on) and the results was confirmed, and the isoelectric points of these two components were measured. That is, the molecular weight is 16.5 kDa The molecular weight was 7.0 for the one with a molecular weight of 16 kDa, and 7.6 for the one with a molecular weight of 16 kDa.

-Characteristics of sugar structure Substance corresponding to the 15.5 kDa band (obtained by neuraminidase treatment) ) with a specific O-glucanase (i.e. O-glucanase commercially available from Genzyme). Treatment with Lucanase (product number: 20 0-ASE) 1. and further sugar analysis. Ta. This enzyme treatment changes the molecular weight of the substance from 15.5kDa to 15.0kDa. Although it decreased, the charge of the substance did not change. Finally, purification of N1 type and N, type The N-terminal tryptic peptide was analyzed using FAB MS (fast atom bombardment mass spectrometry). analyzed. As a result, N-type N-terminal tryptic peptides contain N-acetyl Contains lugalactosamine, galan I-su and two neuraminic acids, N-type N-terminal tryptic peptides include N-acetylgalactosamine, galactosamine, It was confirmed that lactose and one neuraminic acid were attached. Main departure Glycosylation [, -2ON, type and N, type sugar structures by Conrad Tora's Neirobian Journal of Biochemistry Volume 153 255- The structure is the same as that of the N- and N-type sugars described on page 261 (1985). Ta.

Immune properties of monoglycosylated IL-2 There is a correlation between protein conformation and antigenic properties, so it is important to understand the correlation between protein conformation and antigen properties. can be studied to obtain information about the three-dimensional structure of molecules.

Glycosylated IL-2 (Nt+Nt) and natural IL-2C biote RpA) and IL-2 coli prepared as described in Example 8. The RIA (radio-immunoassay) system was used to study and compare.

Initially (RIA:A) was a natural anti-IL-2 monoclonal antibody (Sandor Regio). Powered by Nars des Transfusions Saint Jean de Besancon BG5 antibody) was used. Next (RIA:B), anti-IL-2 coli sheep anti-blood Clear (batch number PoI2-1100-09-Celtic UK) was used.

In both cases, KL-2 (Nt) labeled with iodine-125 was used as the tracer. .

Table 2 below shows the ICs obtained from the displacement curves for each of these systems. value (In other words, IL- (2 concentrations) (values and standard deviations shown are for this parameter three times, independently). (measured and calculated).

Table 2 table I.C. Although the values appear to be similar, they are significantly different from those obtained for IL-2 coli. , the results obtained with the RIA A system, in which the antibodies are monoclonal, are particularly Clear and reliable. From this knowledge, glycosylated IL-2 (N++N *) and natural! The three-dimensional structure of L-2 was shown to be similar, and glycosylation The three-dimensional structures of KL-2(N*+Nt) and [,-2 Cori are different, therefore, The antigenic determinants were shown to be different. Therefore, one of the problems with IL-2 Cori is , immunogenic (Grigel P, L, et al., Cancer Research 48-1 3875 -3881 (1988) and Alebretta M, et al., Journal of Creation. Nical Immunology, Vol. 6, No. 6, 481-490 (1986)). these questions The topic is! It depends on antigenic determinants specific to L-2 coli, and natural IL-2 has no It is very possible that it is absent. Glycosylated IL-2 Although similar to natural IL-2 in structure and method of synthesis, such antigen determination The absence of fixed groups is very plausible and confirmed by the above results. There is.

Example 7: Using the method of the invention starting from the culture supernatant of the 58.12 cell line Isolation of glycosylated interleukin 2 Isolation of interleukin-2-enriched flanks from l-culture supernatants The supernatant 20 (H) of culture A described in Example 3 was collected, separated, and the IL-2-enriched fraction first a polysulfone membrane with a cutoff limit of 100 kDa, then a polysulfone membrane with a cutoff limit of 10 kDa. Concentration was performed by double ultrafiltration using a kDa polysulfone membrane. The operation is carried out as described above. It was carried out in the same manner as in Example 5. 5.6C of a concentrated aqueous solution of TL-2 was obtained.

Isolation of 2-glycosylated IL-2 a) Cation exchange chromatography Chromatographic supports are cross-linked to agarose, with strong cationic exchange. Gel based on oxidation, that is, “S, cephalometric gel” sold by Pharmacia. "Fast Flora".

Acetic acid was added to the solution obtained in the previous step to adjust the pH to 5.5, and the conductivity was and adjusted to 5aS. The solution is then injected into the column and it is diluted with sodium chloride. Another ammonium acetate aqueous solution (pH 5.5, molar concentration 11 ! 50sM).

Glycodylation! L-2 was eluted with a 180mM solution of sodium chloride and 5.76 (!) of the IL-2 aqueous solution was obtained.

b) Hydrophobic interaction chromatography The chromatographic support is hydrophilic Hydrophobic interaction gel based on polyvinyl resin, i.e. marketed by Merck "Butyl Fractogel 650 (M)" with a diameter of 5oIII was packed into a column.

ammonium acetate to a molar concentration of 1.6 M, the previously obtained glycosylation [, -2 In addition to the solution, ammonia was added to adjust the pH to 6.5, and the solution was diluted with buffer A. In other words, it is pre-leveled with an aqueous ammonium acetate solution (pH 6.5, molar concentration 1.6M). The solution was injected into the column while being balanced. Under these conditions, glycosylation! L-2 is Retained on a chromatographic support. Wash the column with buffer A and retain Removed material that did not. The column was filled with buffer B5, i.e. ammonium acetate. Elution was carried out with a solution of NaCl (pH 6.5, molar concentration 0.65M).

1.3612 glycomolated IL-2 aqueous solution was obtained.

C) Exclusion chromatography The chromatographic support is a gel based on cross-linked dextran, i.e. This is "Sephacryl 5200HR" sold by Pharmacia.

b) The solution obtained at the end of the process is a cellulose acetate membrane, i.e. the stop threshold It was concentrated on a 10 kDa Amicon YMIO II. The solution is sodium phosphate It was first equilibrated with an aqueous solution (pH 6.5, molar concentration 0.1M) and injected into the column. . Glycosylated KL-2 eluted with this solution as described above.

3- Demonstration of product purification Laemli method (U, Laemli, Analytical Biochemistry Volume 78 ( (1977) in the presence of a reducing agent (dithiothreitol). After electrophoresis using Midgel PAGE-SDS (sodium dodecyl sulfate) gel , Silver staining by Noryl method (C,R.

Merrill, Proceedings of the National Academy of Sciences ・Of the United States of America, Volume 76, Page 4335 (19 1979)), and the purity of glycosylated IL-2 was shown to be over 99%. It was done.

Example 8 Effect of glycosylated interleukin-2 on human lymphocytes vitro activity Glycosylated interleukin-2 and LAK cell-producing Escherichia coli Comparative study of recombinant interleukin 2 derived from The experiments performed were carried out using the 109.27 line produced by the isolated 109.27 line as described in Example 5. The experiment was carried out using recombinant glycosylated IL-2. This product is typically Dilylated IL-2 or, if there is a risk of confusion, glycosylated ■L2 It is called (N r + N t).

1-Importance of in vitro LAK cell production experiments in cancer research Acquired immunotherapy is a new approach to cancer treatment that targets host tumors. The aim is to enhance the immunological defense response.

When human peripheral leukocytes are cultured in the presence of IL-2, they exhibit strong cytotoxicity against cancer cells. It becomes active. Cells that exhibit this effect are labeled with LAK (“lymphokine activity”). "Killer" XM, T., Rotsu et al., Cancer Research Vol. 41, 4420-442 6 (1981)).

The action of LAK cells affects all types of tumors, including freshly harvested tumor cells. have an influence on These do not show any activity against normal cells. Functionally defined, LAK cells are stimulated with IL-2 and then previously sensitized to antigen. Even in the absence of tumor cells that are resistant to the action of NK (“natural killer°”) cells, Restrictive valve MHC ('major histocompatibility complex') specifically selectively directed against These are cells that express cytotoxicity. By this definition, LAK cells are NK cells (i.e. i.e., it is cytotoxic as it is without being activated by IL-2) and cytotoxic. sexual T cells, whose action suppresses MHC and acquires presensitization by the target. clearly differentiated (J, E., Talmaje et al., Cancer Dourito, Rep. 70, p. 171 (1982)).

Recent studies have shown that, in fact, LAK cells are probably <IL-2-enhanced N K cells, suggesting that LAK precursors are, in fact, derived from giant granular lymphocytes. be done. (K. Ito et al., Journal of Immunology-136 3910- 3917 pages (1986)).

Interleukin-2 stimulates LAK cells not only in vitro but also in vivo. Produced. The ability of specific KL-2 to generate LAK cells in vitro is due to its Therefore, it is an important indicator of potential antitumor activity.

2- Experimental method a) Nee-glycosylated IL tested with the following types of interleukin-2: 2(N1+Nt) - Fraction 1 isolated during step 2b of Example 5 was Glycosylated IL-2 (N,) obtained by subjecting to steps C) and d) of Example 5 Fraction 3 isolated in step 2b of Example 5 was combined with C) of Example 5 and Non-glycosylated IL-2 (M) obtained by subjecting to step d) Recombinant L-2 derived from Escherichia coli prepared as described below, The gene encoding IL-2, often abbreviated as IL-2 coli, is ・Transfect into coli cells and express with high yield. Dissolve, regenerate, reverse phase HPLC chromatography Matography and the method described by Liang et al. (S, 1. Liang et al. The Biochemical Journal 2291, pp. 429-439 (1985)) When purified by exclusion chromatography, a characteristic of 5-10x 105U/z9 was obtained. Recombinant proteins with different activities are obtained.

b) Preparation of lymphocytes The medium consisted of 10% AB human serum (CTS purpa RPM11640 (Gibney BRL), 2mM Sodium Pyruvate, 5mM HEPES, 4mM L-Glutamine, I OOU/c+12 penicillin, 100 μg/mQ streptomycin and 0. 25 μg/ff1I2 amphotericin B (this medium is hereinafter referred to as complete medium). ).

Blood from healthy individuals was collected under sterile conditions.

Peripheral lymphocytes are described in “Methods in Enzymology” by A. Boyum (G. Jisabato, J., J., Lambon, H., Pan, Bunakis ed. Vol. 08, p. 88 Akademitsu Ficor was prepared using the method described in Le Press Incorporated (1984). Centrifuge using a Louheiberg (Pharmacia) gradient to remove red blood cells and granules. Separated from granulocytes.

Lymphocytes were washed three times with complete medium.

Adherent cells (monocytes and macrophages) were removed by adhering to plastic. : Cells were suspended in complete medium at a concentration of 5-10XIO' per mi2 and placed in a culture flask. The cells were seeded at a density of 1-2 x 10' cells/ml. The flask is 5% CO 1 for 1 hour at 37°C, then the non-adherent lymphocytes were placed in the culture flask. After shaking gently, it was collected by suction.

c) In vitro culture of lymphocytes in the presence of IL-2. The cells were washed once and treated with two test samples of IL-2 at various concentrations (100, 30, 10 and IU/ml) for 48 h at 37°C and in the presence of 5% co. Cultured at a concentration of lOS cells/mQ in complete medium. Cells were then washed. this These cells are hereinafter considered effective cells.

d) Demonstration of cytotoxicity The effective cell cytotoxic activity was determined by S., Z., Saratubin et al. -64 (1983) and Science 223, pp. 703-707 (1984) NK-type cytotoxicity-resistant human T lymphoid C8166-45/ Evaluation was made after contacting with C63 type target cells for 4 hours. This system is referred to as HT It is called the I-series. Target cells 6X10' cells were placed in serum-free complete medium 0,4 Cr (sodium chromate, Amersham) 20 for 1 hour at 37°C in m12 Labeled with 0 μCi and washed several times. Target cells (1 in complete medium 0, 1 m12) 04) and effective cells in complete medium 0.11, the ratio of effective cells to target cells is Round bottom microtiter plate with varying ratios (50:1, 10:1.1:l) (Falcon). The microtiter plate was centrifuged and After incubation for 4 hours at °C in the presence of 5% CO, drain the supernatant from each well. The radioactivity was measured using a gamma counter. Cytotoxicity is a dead marker. Released by target cells! It was determined from the ICr amount. Non-specific cytotoxicity Determined from the amount of radioactivity spontaneously released by target cells in the absence of cysts. Established. The highest value of cytotoxicity was in the absence of effective cells and in the presence of IN HCl. , determined from the amount of radioactivity released by target cells. Each experiment point is tripled ( 6 times) in determining non-specific and maximum cytotoxicity, and specific cytolysis. The percentage is calculated by adding or subtracting the standard deviation from the mean value according to the following formula: Percent specific cell lysis Maximum cpHl - non-specific cp+n The methods described in b), C) and d) above are applicable to glycosylated IL-2 (N,) and non-glycosylated IL-2(M). H, D, Engers (H, D. Engers et al., Methods of Engagemology-132, 437-457. (N, + N t ) is used in the case of glycosylated IL-2 and IL-2 coli, which is associated with HTI, Da Tested on target cells from Udi and Jurkat lines that are resistant to total NK lysis did.

3-Results The results are summarized in Tables 3 and 4 below.

Table 3 Healthy subjects with non-glycosylated IL-2(M) and glycosylated IL-2cN Cytolytic activity of LAK cells produced from HT, system Table 4 LAK cells produced using multiple types of interleukin-2 using lymphocytes from healthy individuals Cytolytic activity against Doughty, HTI and Jurkat systems *Results are expressed as an activity index compared to recombinant IL-2 coli and are as follows: Define.

Table 3 shows the following for target cells from the HTi system = IL-2 concentration Superiority of glycosylated IL-2 (N, ) at 1000 U/mff or less; - This dominance increases if the ratio of effective cells to target cells decreases.

- If the proportion of IL-2 is high (1000 U/if and/or standard of effective cells) If the ratio to target cells is high, dominance decreases.

In Table 4, for target cells from the Daudi, HT, and Jurkat systems, Ni indicates the following. - Glycosylation compared to IL-2 coli at a concentration of IL-2201J/mff Superiority of IL-2 - This superiority decreases if the IL-2 concentration increases.

These studies have revealed the following that ni-glycosylated IL-2 is 3N-glycosylated. It produces LAK cells that are active against tumor cell lines.

-Second LAK activity is lower in glycosylated IL-2 than in IL-2 coli. Obtained in concentration.

-LAKim cell activity produced by glycosylated IL-2 can occur in the following two cases. , greater than the activity of LAK cells produced by IL-2 coli: *Low concentration of IL-2 *Ratio of effective cells to target cells is unfavorable (10/1 or less) These results are particularly promising for clinical use of glycosylated IL-2. . Because these studies show that this form of IL-2 is more effective than IL-2 coli. This is because similar antitumor effects can be obtained using lower concentrations. For this reason, This will substantially reduce the toxicity associated with product administration.

Example 9: Immunomodulatory activity of glycosylated interleukin-2: in mice Stimulation of immune responses to humoral mediators a) Purpose of the research IL-2 is a polyclonal immunoglobulin in non-immunized mice in vivo. Phosphorus M response (hereinafter abbreviated as IgM) and specific 1gM response in immunized mice C, M. Wegand, Journal of Efperimental Methodology 492163, vol. 1607-1612 (1986)), then IL-2 coli ・Ability to induce differentiation of activated B lymphocytes in human cells in vitro (T , A. Waldman, Journal of Efperimental Medicine 160 Volume 14: +0-1466 (1984)), this study On the other hand, it was motivating. The purpose of the study was to use BALB/C mice. Glycosylation interleukin upon stimulation of immune response to humoral mediators The in vitro activity of N-2 was determined in response to immunization with a thymus-dependent antigen (ovalbumin). It is assessed by measuring the primary pancreatic response.

b) Experimental method α) Animals - environmental conditions *Species: BALB/C mouse * Acclimation: Animals were acclimatized for 1 week before the start of treatment.

*Number of animals: 90 females * Animal weight at the start of the experiment: Approximately 20 g Composite sample AO4,c was given ad libitum.

*Water: Tap water was provided ad libitum using an automatic device.

Environmental conditions - Temperature 22℃ (+2℃) -Humidity 60% (±lO%) *Housing location: Animals were housed in stainless steel cages. animals are , into cages of 5 animals each and into groups of 15 animals from the same batch.

β) Animal treatment On the first day of the experiment, the animals were treated with 98% pure egg albumin (Sigma) by electrophoresis. , product number: A-5378), 50 μg ovalbumin per mouse. , 5 If dissolved in sterile PBS buffer (phosphate buffered saline) and administered intraperitoneally immunization by Ovalbumin (molecular weight 40,000. Known antigen determination) Group 5) was selected as an antigen because it is a heterologous protein with good immunogenicity. Ta.

Glycosylated IL-2 isolated as described in Example 5 was diluted with PBS at 1 : In normal BALB/C mouse serum diluted to 80%, intraperitoneally on days 2.4.6 and 8. Administered intravenously. The presence of proteins in serum does not significantly affect the immune system. Not due to non-specific adsorption to the walls of the tube or syringe! Loss avoidance of L-2 is obtained.

The table below shows the doses administered to 15 mice for the various batches.

Animals were sacrificed on day 9.

γ) Performing measurements After measuring the body weight of the deceased animal, remove the pancreas under sterile conditions, measure the weight, Mechanically ground. The ground sample is filtered through sterile gauze to bind-fat Remove all sources, 400. Centrifuged for 10 minutes. Pancreatic cells were suspended and washed three times. Purified. After washing, count and place to calculate the total number of pancreatic cells per animal. The desired cell concentration was achieved. Pancreatic cells were grown in medium containing 10% fetal bovine serum (mesar). 2 x 10' viable spleen cells in RPMI medium 1640) d The cells were cultured for 96 hours in 51 wells of polystyrene plates containing .

1 gM of washed egg albumin was measured by the radioimmunoassay method shown below. .

* pvc play for 18 hours at +4°C (0.1M5p at 50μg/mf2 Absorption of egg albumin on H7,5 phosphate buffer solution, 100 μg per well) *After washing the wells with 0.1M 5 pH 7.5 phosphate buffer, the wells were Een 20 surfactant (vol/vol) solution in 0.1 M 5 pH 7,5 phosphate buffer Saturate at 200 μQ at 37° C. for 1 hour.

*After washing the wells, dilute the culture supernatant with 0.1M, pH 7.5 phosphate buffer. Diluted to 2, 0.1% (p/v) gelatin and 0.1% (vol/vol) tow Measured by incubation with Eene 20 detergent for 18 hours at +4°C ( 100 μI per well).

* After washing the wells, radiolabel with iodine 125 (7,5,1/2Ci/ 1/2g, 250000cpn/! 00μg, +37℃ for 3 hours) Reclonal anti-mouse IgM sheep antibody (sold by Immunotik, product number: 1) 15-0575) and 100 μQ per well.

* Fill the wells with solution (0.9% N a C + 2) (p/v), 0.1% Twi After washing with The radioactivity was measured.

The various measurements shown below for each mouse were performed for each batch of mice.

i.e. for each dose 1, calculation of average weight of pancreas relative to body weight, pancreas per animal. The average total number of cells and the average amount of anti-valve albumin IgM induced in pancreatic cells. be. The latter amount is proportional to the radioactivity.

C) Result The results are shown in Figures 6.7 and 8. The discussion is as follows: Dosage 1. If you start with 88 x 10’U/kg (i.e. 37,500 U/mouse) In this case, glycosylated IL-2 has a significant effect on pancreatic weight and total number of pancreatic cells per animal. resulting in a relative increase of more than 50%. Experiments (not shown here) show that It was also shown that the proportion of total T lymphocytes was increased (20%) compared to the others.

- When the dose is 1.88x 106U/kg or more, each plasma cell induces The amount of specific types of 18M increased significantly.

Therefore, glycosylated IL-2 has considerable immunostimulatory activity.

Example 10: Antitumor activity of glycosylated interleukin-2: in mice cancer immunotherapy a) Experimental method This experiment was performed using DBA/2 mice divided into 5 batches. On day 0, Mice were injected intraperitoneally with 0.2×104 cells of the same genotype of N. sarcoma SL2. Ta.

In a first series of experiments, the glycosylated protein isolated as described in Example 5 was used. Leukine-2 at various doses (0, 8, 40, 200, 1000 and 5000U /day) into 6 mice in 5 separate batches on days 3-7 and 10-14. did. The number of surviving mice on that day was counted for 30 days. From this first series of experiments, Antitumor activity when 5000 TJ/day of glycosylated interleukin-2 was administered. gender was shown.

In the second series of experiments, the dose was fixed at 5000 U/day and administered on days 10-14. , the date of pre-administration and the day of administration were changed.

b) Results The results of the second series of experiments are shown in Table 5 below and illustrated in FIG. 0.1, The number of mice surviving for 30 days in batches 2 and 3 is shown in Table 5.

Table 5 These results indicate that glycosylated interleukin-2 has significant antitumor activity. It is very clear that

Example! ! : Toxicity test of glycosylated interleukin-2 in mice a) Purpose of the experiment Recombinant IL-2 (produced in Escherichia coli) was administered alone to humans or mice. (M. Rosenstein et al., Journal of Immunology-137, 1735 -1742 (1986)) or together with LAK cells (S, E, etching). Hausen, Journal of National Cancer Institute 8 The main toxic effects when administered (Vol. 0, No. 3, pp. 177-188 (1988)) were: Intravascular fluid delivery, i.e. in certain organs (thymus, pancreas, lungs, liver and kidneys) This is the accumulation of liquid.

Therefore, the purpose of this experiment was that when administered intraperitoneally three times a day for 3 and 6 days, Examining intravascular fluid retention in BALB/C mice, glycosylated interleukins -2 toxicity, if any, should be evaluated.

b) Experimental method Experiments were performed using the method described by M. Rosenstein (M. Rosenstein et al., Journal). Similar to Book of Immunology, Vol. 137, pp. 1735-1742 (1986) I did it using this method.

α) Animals - environmental conditions *Species: BALB/C mouse * Appropriate age of animals at the start of the experiment: approximately 4 weeks * Acclimation = 1 week *Number of animals: 120 females * Animal weight at the start of the experiment: 201i1 * Identification method: Label the ears In response to *Water: Tap water on request * Environmental condition -Temperature = 22℃ (±2℃) -Humidity = 60% (±10℃) -Light irradiation time +12/24 hours * Breeding location: Stainless steel cage.

Animals were distributed in groups of 12 animals per batch.

β) Animal treatment Glycosylated IL-2 isolated as described in Example 5 was prepared in PBS buffer (phosphate 0.2 in normal BALB/C mouse serum diluted 1=80 with buffered saline). ．． 3.4 and 5 (3 days for Tutsi and 1, 6, 7, 8 and 9) was administered intraperitoneally three times a day (morning, afternoon, and night) for 6 days. The dose volume was 0.5πQ. It was.

The table below shows the doses administered to the 12 mice of each batch.

Animals in batches 0.2.3.4 and 5 were killed at the end of the 4th day, and Animals in batches of 8 and 8 were sacrificed on the 7th day.

γ) Tests conducted A clinical trial Animals were observed daily during the entire experiment.

The tests described below were performed only on the first 6 animals of each batch.

- Necropsy study (4 and 7 days) Mice were anesthetized with bendoparbitone.

-Blood test During necropsy, blood samples were collected from the abdominal aorta for biochemical analysis.

-Anatomical studies Removal of liver, pancreas, kidneys, thymus and lungs to detect toxicity associated with fluid retention Then, the weight was measured.

Weighing when not dry: Organs were weighed in lyophilized flasks: each Flasks (one per animal and one per organ) were weighed before containing the organs. I'll keep it.

Determination of dry weight: Organs were frozen at -80°C and lyophilized for 4 days in a freeze dryer. .

1.Special examination This test was performed on the last 6 animals of each batch. 0.2.3.4 and 5 In the case of Batsuchi, it is on the 4th, and in the case of 1.6.7.8 and 9 nokutsuchi, it is on the 7th. Bovine serum albumin radiolabeled with de-125 (specific activity 5 μci/z9) was 2μC11 mice were administered intravascularly.

Two hours after injection, mice were sacrificed and the lungs, kidneys, liver, thymus and pancreas were removed and weighed. 5dP so that the radioactivity of each organ can be measured with a gamma counter. It was installed in a VC pipe.

Radioactive iodine and bovine serum albumin should be considered as a marker indicating intravascular fluid wetting. (1). Therefore, the organs of IL-2-treated mice are the same as those of control mice. The more radioactive the IL-2, the less likely it is to affect intravascular fluid retention in the organ under test. It would be said that it would affect.

C) Result A clinical trial There was no change in animal behavior and no deaths were recorded.

-Blood test Biochemical parameters (total protein, albumin, triglycerides, urea and Based on the evaluation of creatinine), there were no problems with liver or kidney toxicity.

Undry and dry weight of monothymus, liver, lungs, pancreas and kidneys comparative study of The dry weights of the thymus and kidneys were determined by IL-2 at 3 or 6 days after treatment. did not increase regardless of the dose.

The non-dry weights of the pancreas, lungs and liver increased markedly, especially in treatment 6. The ratio of dry weight to non-dry weight after days remains unchanged. This means that The proportion of water in these organs was constant, meaning that the pharmacological activity of IL-2 Shows lymphoid tissue proliferation that occurs in

If there is any induction using radiolabeled albumin, IL- 2 Dynamic study of induced wetting Iodine-125 in the organs under test: thymus, liver, lungs, pancreas and kidneys The proportion of bovine serum albumin labeled with There were no obvious changes, and only a few changes occurred in the pancreas.

d) Conclusion B A L B/C From the toxicity test conducted on mice, the evaluation of glycosylated IL-2 was No appreciable toxic effects were shown. More specifically, M. Rosens The test method used by Tine shows that blood vessels such as those described in the case of IL-2 coli No leakage of internal fluid was observed.

Example 12: Toxicity test on glycosylated interleukin-20 a) Purpose of the experiment The purpose of this experiment was to determine the toxicity, if any, of glycosylated IL-2. More specifically, to evaluate edema formation in That's true.

b) Experimental method Glycosylated IL-2 isolated as described in Example 5 was isolated from 6 mice in each batch. rats (3 males and 3 females) were given 2 million, 10 million and 50 mm. On U　I　/97 days (i.e. 0.1,0゜5 and 2,5　z9/ kg/day) was injected intraperitoneally for 3 days. At the same time, sodium chloride ty/ Animals were orally administered 9 days/day.

On the third day, animals were given 10 zy/kg orally of water and then placed in metabolic cages. diuresis was measured for 24 hours.

Each batch included an untreated control batch and a batch that received only sodium chloride. compared to

C) Result No clinical abnormalities were observed.

Animals were weighed before the start of the experiment and on days 3 and 4. The difference between batches is I was not able to admit.

Blood tests (hematocrit, urea, creatinine, total protein, albumin, Lesterol, triglycerides, GPT (glutamic acid-virupic acid transamate) minase), LDH (lactate dehydrogenase), sodium, potassium, chlorine and and calcium) were tested on the 4th day. In effective amounts, serum albumin and protein decreased slightly.

Urine excretion test below ·capacity ・Semi-quantitative analysis using reactive strips (pH, density, protein, glucose, ketosis) ion, bilirubin, blood and urobilinogen), sodium ion, potassium , chlorine, and creatinine, no abnormality was observed.

In pancreas weight per effective dose, several organs (liver, pancreas, kidney and breast glands) showed a slight increase.

Necropsy revealed no gross injuries.

When examined by optical microscopy, IL-2 is shown to be associated with activation of the lymphatic system, as shown below. It has been shown to induce

In three animals treated with −50xl O’U1/9/day, the periarterial lymph Slight thickening of the lobes and peripheral pancreatic band – more frequently swollen forms in treated animals Plasmacytosis, but no effective dose.

d) Conclusion Toxicity studies in Sprague-Dawley rats observed by Y. Harada. Lethal dose of IL-2 coli (Y, Harada, Preclinical Safety ・Biotechnology Products Intemped for Human Co. (pages 127-142 - Alan R., Rhys, Inneeporated) Glycosylation in large amounts (2.5 m9/97 days versus 2.5 m9/97 days) ! No appreciable toxic effects of L-2 were demonstrated.

Therefore, glycosylated IL-2 is present in both mice and rats. From L-2 Cori The toxicity is also quite low.

Example 13: Experimental formulation of glycosylated interleukin-2 a) Experimental method p containing glycosylated IL-2 obtained as described in Example 5. To the sodium phosphate aqueous solution of H6,5, various excipients may be added in different proportions, or excipients may be added. Freeze-dried experimentally without added excipients.

The solute is then reconstituted, the appearance observed, the pH measured, and the glycomolated I present Quantifying the amount of L-2 by HPLC using a reverse phase column (comparison: Example 5-3) L, CTLL- 2 activities were measured. These operations were performed after storing the material at 4°C for 1 year and then at 25°C. After 3 months of storage and 3 months of storage at 37°C, it was freeze-dried and immediately thereafter.

b) Results Phosphoric acid containing glycosylated IL-280μ9/j112 without excipients A ready-to-inject preparation is made by freeze-drying an aqueous solution of sodium thorium and adding water to the solute. When reconstituted, a colorless and transparent solution containing over 80% of 0CTLL-2 activity is obtained. A liquid will be created. After storing the lyophilizate for 6 months, the reconstituted solution was initially Contains approximately 50% of CTLL-2 activity.

Hydrolyzed gelatin (Loselot, product number: DSF) 10ff? /112, Po Liethylene glycol l19, average molecular weight 6000 (Hoechst, product number: DA B8) or human serum albumin (Sigma, product number: 3782) 10rtt9 /zQ, approximately 18 x 10" specific CTLL-2 of Ulz9 during lyophilization. Phosphoric acid aqueous solution containing active glycosylated KL-2, 80μ9/ship The solute (clear and pH 6.5), the following chromatographic analysis (accuracy: 3%) is possible. ) results in - more than 90% in case of polyethylene glycol - hydrolyzed gelatin and human blood In the case of clear albumin, the specificity (of IL-2 quantified by HPLC) was observed in 100% of cases. Has heterogeneous CTLL-2 activity, approximately 18 x 10'U/9 Therefore, for the last two excipients, the 0 CTLL-2 activity per solute preference is The value was the same.

Hydrolyzed gelatin (Rozelot, product number: DSF) 10x9/x (1 and Non-pyrogenic alanine (Ajinomoto) 10 x 9/x (1) was added during freeze-drying to Glycosylated IL with a CTLL-2 activity of approximately 18×10”U/x(l) - When added to a sodium phosphate aqueous solution containing 2450μ9/Y, the frozen The moisture content of the dried product is 3% or less. After storing the freeze-dried product at 4℃ for 3 months , the reconstituted solute was clear and had a pH of approximately 6.5, and the chromatographic analysis (precision 3%), specific CTLL-2 activity (of IL-2 quantified by HPLC), ca. 100% of the initial glycosylated IL-2 with 18X10"U/zg and

From the above results, after lyophilization, glyfusylated IL-2 is acceptable as a pharmaceutical preparation. It has been shown to have excellent stability.

The lyophilizate could probably be stored for several years at 4°C. On the money side, I reconfigured it. The solute must be solubilized without adding toxic substances or chemically modifying the molecule. It is transparent even when it is cloudy and has a physiological pH.

These are the properties of glycosylated IL-2 from Escherichia coli. the latter After freeze-drying, more than 50% of the substance is lost and the solution exhibits a proteinaceous glow (i.e. (See Patent Application No. 0158487, p. 10). Therefore, to solubilize It is necessary to add toxic solubilizers such as 5DS (sodium dodecyl sulfate) to There is.

After reading this specification, you will understand the importance of glycosylated IL-2 as a drug and the Numerous advantages compared to non-glycosylated IL-2 from Escherichia coli - i.e. may exhibit the same activity at lower doses; less toxic; Well tolerated at high doses; low immunogenicity; no need to add toxic solubilizers or modify the molecule. soluble in aqueous solvents without physiological modification; and pharmaceutically acceptable. I hope you understand that it has excellent stability after freeze-drying as a convenient formulation. cormorant.

eoRI FIG.3 SDS/polyacrylamide gel electrophoresis after sample reduction Staining with silver Mixture of 14 types of IL-2 2 Product 3 obtained after completion of step 2d) in Example 1 1L2N2 A IL2N1 5 Molecular weight marker Same as 61 Same as 75 FIG.4 Reversed phase HPLC analysis of the product after completion of step 2d) in Example 5 FIG. 5 Example 5 HPLC analysis of the product after completion of step 2d) using a cation exchange column FIG.6 Critical status of pancreas per body weight FIG. 7 'Total number of tile cells per J animal F [G, 8 Analysis of anti-ovalbumin IgM Two two international search report 11 Jiichi ---^----- Shizuichi & PCT's FR89100554 International Investigation Report FR8900551 SA　32239

Claims (1)

[Claims] 1. a) Separation of a fraction mainly composed of interleukin-2 from the culture supernatant, b) Cation exchange chromatography c) hydrophobic interaction chromatography, and d) exclusion chromatography A glycosylated protein that can be isolated from recombinant CHO culture supernatant by a method that includes each step of A medicament characterized by containing interleukin-2. 2. Glycosylated interleukin-2 has specific CTLL-2 activity of approximately 15 x 10 The medicament according to claim 1, which has a concentration of 5 U/mg. 3. Glycosylated interleukin-2 was lyophilized in aqueous solution at pH 6.5; Immediately after thawing the frozen product, maintain at least 80% of the original CTLL-2 activity. The medicament according to claim 1 or 2. 4. Glycosylated interleukin-2 has a pH of 6.5 in an aqueous solution containing it. After freeze-drying and immediately thawing the frozen product, a clear thawed solution at physiological pH can be obtained. The medicament according to any one of claims 1 to 3, which is a medicament according to any one of claims 1 to 3. 5. Glycosylated interleukin-2 is a natural anti-IL-2 monoclonal antibody Any of claims 1 to 4, which has the same affinity for BG5 as natural IL-2. or 1. The medicine according to item 1. 6. Glycosylated interleukin-2 is a natural anti-IL-2 monoclonal antibody Has the same affinity for Pol-1/00-009 (Celtech) as natural IL-2. The medicament according to any one of claims 1 to 5, comprising: 7. Glycosylated interleukin-2 is present in hydrolyzed gelatin or human serum alcohol. Lyophilize at pH 6.5 in an aqueous solution supplemented with Bumin and bring the frozen product to a temperature of 4°C. After storing for one year and regenerating the frozen product, the original CTLL-2 activity is maintained. The medicament according to any one of claims 1 to 6. 8. Glycosylated interleukin-2 is loaded with hydrolyzed gelatin and alanine. The frozen product was lyophilized at pH 6.5 in an aqueous solution containing 30% of After storage and regeneration of the frozen product, the original CTLL-2 activity is maintained. The medicament according to any one of claims 1 to 7. 9. Glycosylated interleukin-2 is added with hydrolyzed gelatin or alanine. Freeze-dry the frozen product at a temperature of 37°C for 3 months at pH 6.5 in an aqueous solution containing After storage and regeneration of the frozen product, the original CTLL-2 activity is maintained. The medicament according to any one of claims 1 to 8. 10. Recombinant CHO cells are produced using IL-2 precursor and DHFR expression vectors. The cell according to any one of claims 1 to 6, which is a transformed cell derived from DXB11 strain. medicine. 11. The expression vector is a plasmid with characteristics of plasmid pSV726 , the medicament according to claim 10. 12. Claims 1 to 3, wherein the recombinant CHO cells are cultured in a low protein artificial medium. 12. The medicament according to any one of 11. 13. Claim 1, wherein the low protein artificial medium contains polyvinylpyrrolidone. The medicament according to 2. 14. Separation step a) is performed at a stop threshold of greater than 30 kDa, preferably between 50 and 15 kDa. 0 kDa and a stop threshold value smaller than 10 kDa, preferably 5 It is carried out by double filtration between membranes (2) containing ~10 kDa. , the medicament according to any one of claims 1 to 13. 15. The cation exchange step has a pH of 4.5 to 6.5, more specifically 5.2 to 5. ．． 15. The medicament according to any one of claims 1 to 14, wherein the medicament is administered for a period of 7 days. 16. The cation exchange step is performed in advance by an anion exchange chromatography step. The medicament according to any one of claims 1 to 15. 17. The anion step is carried out at a pH between 5.5 and 8.5, preferably between 6.5 and 8.2. 17. The medicament according to claim 16. 18. The hydrophobic interaction step has a pH of 4.5 to 8.0, preferably 6.0 to 8.0. 18. The medicament according to any one of claims 1 to 17, which is carried out between. 19. A hydrophobic interaction step is required for the desorption of interleukin-2 on the chromatogram. Add a chaotropic agent, or an organic solvent, or a pH of 4.5 to 8.0 Claims that the method is carried out without addition of a water-soluble solvent having a 19. The medicament according to any one of 1 to 18. 20. The exclusion chromatography step has a fractionation range between 1 and 250 kDa. The medicament according to any one of claims 1 to 18, which is applied to a support. 21. Any of claims 1 to 20, further comprising hydrolyzed gelatin and alanine. or 1. The medicine according to item 1.