JPH0655153B2 - Method for producing human interleukin-2 crystal - Google Patents

Description

TECHNICAL FIELD The present invention relates to a recombinant human interleukin useful as a medicine for the prevention and treatment of tumors and the treatment of diseases with impaired immune function.
2 relates to a method for producing crystals.

As used herein, recombinant human interleukin-
2 may be abbreviated as “rIL-2”.

2. Description of the Related Art Human interleukin-2 is a kind of lymphokine produced by T lymphocytes stimulated by lectins and alloantigens. Human interleukin-2 has T in vitro
In addition to promoting long-term cell proliferation, induction of cytotoxic T cells,
Biological activities such as induction of antibody production of B cells in splenocytes and activation of NK cells have been reported, and it has been clarified that they play a major role in controlling the immune response of the living body.

Recent rapid progress in recombinant DNA technology has enabled large-scale supply of human interleukin-2, which was conventionally obtained only in a very small amount from the living body.

Problems to be Solved by the Invention By crystallizing rIL-2, it is expected that the purity and stability can be significantly improved.

Means for Solving the Problems The present inventors have attempted to crystallize rIL-2 by various methods. As a result, a salt or polyethylene glycol was added to a high-concentration rIL-2 solution containing water as a main component to add rIL-2. The present invention was completed as a result of further research based on the finding that rIL-2 crystals could be precipitated by decreasing the solubility of -2. The present invention relates to a high-concentration ammonium human acetate solution of recombinant human interleukin-2 having a final concentration of about 5 to 25% (W / V) or a final concentration of about 0.5 to 30%. Recombinant human interleukin by adding (W / V) polyethylene glycol
A method for producing a recombinant human interleukin-2 crystal, which comprises precipitating a recombinant human interleukin-2 crystal by decreasing the solubility of 2. Specific examples of the rIL-2 of the present invention include, for example, a polypeptide having the amino acid sequence shown in FIG. 1 (X in FIG. 1).
Represents a hydrogen atom or a methionine residue. ) Or a fragment consisting of a partial amino acid sequence necessary for its biological or immunological activity, for example, one amino acid from the amino terminus of the polypeptide represented by X in FIG. 1 is a hydrogen atom ( EPC Publication 91539) or a fragment lacking 4 amino acids (JP-A-60-1)
No. 26088) and fragments lacking several amino acids at the carboxyl-terminal portion. Furthermore, some of the constituent amino acids of the polypeptide represented by X in FIG. It may be substituted with an amino acid, for example, the cystine residue at the 125-position may be substituted with a serine residue (JP-A-59-93093).

These may be substances having 100 or more amino acid residues and having a human interleukin-2 activity, that is, an action capable of substituting and maintaining T cells while maintaining their functions.

In the present specification, in the polypeptide having the amino acid sequence shown in FIG. 1, one in which X is a hydrogen atom is IL-2 and one in which X is a methionine residue is Met-.
It may be abbreviated as IL-2.

Further, in FIG. 1, when amino acids are represented by abbreviations, IUPAC-IUB Commission on Biocheical N
It is based on the abbreviations according to omenclature or the abbreviations commonly used in the art. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. Examples of the ammonium acetate solution containing water as the main component used in the method of the present invention include ammonium acetate buffer. RIL-2 in the present invention
The concentration of ammonium acetate in the ammonium acetate solution is, for example, 0.05% (w / v) or higher, and more preferably 0.
1% (w / v) or more is good. The polyethylene glycol preferably has an average molecular weight of about 1,000 to 15,000, and more preferably about 2,000 to 8,000.

Regarding the concentration of addition of the above-mentioned salt, the final concentration is about 5 to 25% (w
/ v), more preferably about 8-15% (w / v).

The final concentration of polyethylene glycol added was about 0.
It is 5 to 30% (w / v), preferably about 5 to 10% (w / v).

The pH of the solution used for carrying out the method of the present invention should be kept at about 2 to 10. More preferably, the pH should be maintained at about 4-9. The temperature of the solution is preferably kept between about 0 ° C. and 50 ° C., and the time required for crystallization is 1 minute to 2 months.

The rIL-2 crystals thus precipitated are filtered using, for example, a glass filter, washed with a saturated food solution (eg, saline solution, ammonium sulfate solution, sodium citrate solution, sodium phosphate solution), and then washed with water. It is collected by being washed with and dried.

The rIL-2 crystals produced by the present invention all have the same biological or immunological activity as the corresponding natural protein, are purified to extremely high purity, and are extremely free from contaminating proteins and pyrogens. Since it is few, it can be safely used as an injection drug substance.

The rIL-2 crystal obtained by the present invention has an activity of proliferating normal T cells and natural killer cells while retaining their functions. Therefore, the rIL-2 crystals obtained according to the present invention can be used to proliferate, passage or clone T cells and natural killer cells in vitro for a long period of time, respectively. Note that the activity of human interleukin-2 can be measured by utilizing this property.

Furthermore, the rIL-2 crystals obtained according to the present invention, for example, are antigen-specific killer Ts that recognize and destroy tumor antigens.
Natural killer cells capable of killing tumors can be selectively proliferated in vitro regardless of whether or not they have undergone cell or antigen sensitization, and when the killer T cells are transferred to a living body, the present invention RIL-2 obtained by
Inoculation with crystals simultaneously enhances its antitumor effect, and thus prevents tumors in warm-blooded animals (eg, mice, rats, rabbits, dogs, cats, pigs, horses, sheep, cows, humans, etc.), It can be used for treatment and treatment of immunocompromised diseases.

The rIL-2 crystals obtained according to the present invention have no antigenicity due to contaminant proteins and have low toxicity.

To use the rIL-2 crystals obtained by the present invention as a prophylactic / therapeutic agent for tumors, the substance is mixed and diluted with a carrier, an excipient, a diluent and the like known per se to give, for example, an injection or a capsule. It can be administered parenterally or orally. Furthermore, it can be used together with killer T cells or natural killer cells expanded in vitro as described above or alone.

The rIL-2 crystal of the present invention has substantially the same biological activity as that of known human interleukin-2 isolated from nature, and thus can be used in the same manner as the human interleukin-2 receptor of cells. Since the dissociation constant with and is extremely small, an extremely small dose may be administered.

For the purpose of expanding T cells in vitro, the rIL-2 crystal of the present invention is added to the medium at a concentration of about 0.01 to 1 unit / m, preferably about 0.1 to 0.5 unit / m. It can be used by adding to.

The biological activity of human interleukin-2 is measured by a method using human interleukin-2 dependent cells [Biochemical Biophysical Research Communications (Biochemical Biophysical Research Co
mmunications), 109 , 363 (1982)].

Specific examples of the use for the purpose of expanding T cells in vitro include, for example, RPM containing 20% fetal bovine serum.
I 1640 medium containing T cells (1
× 10 ⁶ cells / m) and X-ray (1500 rad) irradiated B cell transformants (1 × 10 ⁶ cells / m)
) Is added to the cell suspension containing alloantigen-sensitized T cells obtained by performing mixed lymphocyte culture in the presence of 5% CO _{2 at} 37 ° C. for 0.1 to 0 times the rIL-2 crystals of the present invention. ．
Examples include a method in which the concentration is 5 units / m and the culture is continued for about 1 month while changing the medium about every 1 week.

The crystallization of the present invention can significantly improve the purity and stability of rIL-2. For example, pyrogens expected to be present in ultrafine particles and contaminant proteins can be removed by crystals, and the improved stability will allow rIL-2 to be stored at room temperature.

Further, by crystallization, rIL by X-ray analysis method
It is expected that the three-dimensional structure of -2 can be elucidated, and that it will greatly contribute to academic fields such as a clue to elucidation of its action machine.

Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples, but these do not limit the scope of the present invention.

Example 1 Production of IL-2 Crystals A 5 mM ammonium acetate solution of IL-2 obtained in Reference Example 1 described later (pH 5.0, protein mass, 0.5 to 1.5 mg /
m) 10 m was concentrated to about 1 m by a centrifugal concentrator (Servant Co., USA). The solution was ice-cooled, saturated aqueous sodium chloride solution was added to the solution until the solution became slightly cloudy, and the solution was allowed to stand at 4 ° C. for 3 weeks to precipitate crystals. The crystals were collected by centrifugation and washed with saturated saline and then with a small amount of cold distilled water to obtain 0.5 to 4.5 mg of IL-2 crystals.

[Α] ²⁴ _D -23 ° C (c = 1.0, 5 mM ammonium acetate buffer, pH 5.0) A photograph of this crystal is shown in Fig. 2.

The crystals had a specific activity comparable to that of uncrystallized IL-2, and the mobility on SDS-polyacrylamide gel electrophoresis was the same as that of uncrystallized IL-2. The amino acid analysis value of the acid decomposition product (6N HCl, 110 ° C., 24 hours) was also in good agreement with the theoretical value.

Example 2 Production of Met-IL-2 A 5 mM ammonium acetate solution of Met-IL-2 obtained in Reference Example 1 described later (pH 5.0, protein mass, 0.5-3.
5 mg / m) and 10 m were treated in the same manner as in Example 1 to obtain 0.5 to 11 mg of Met-IL-2 crystals.

[Α] ²⁴ _D -24 ° C. (c = 1.0, 5 mM ammonium acetate buffer, pH 5.0) A photograph of this crystal is shown in FIG.

This crystal had the same specific activity as uncrystallized Met-IL-2, and the mobility in SDS-polyacrylamide gel electrophoresis was the same as that of uncrystallized Met-IL-2. . The amino acid analysis value of the acid decomposition product (6N HCl, 110 ° C., 24 hours) was also in good agreement with the theoretical value.

Example 3 Production of IL-2 Crystals A 5 mM ammonium acetate solution of IL-2 obtained in Reference Example 1 described later (pH 5.0, protein mass, 0.5 to 1.5 mg /
m) 10 m was concentrated to about 1 m by a centrifugal concentrator (Servant). 20 mg of polyethylene glycol 6000 (average molecular weight 6000) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to and dissolved in this solution, and crystals were deposited when left at room temperature for 3 weeks. Crystals were collected by centrifugation and 0.5% (w / v) polyethylene glycol 600
Wash with 0 solution. This crystal is not crystallized I
It has the same specific activity as L-2 and the mobility in SDS-polyacrylamide gel electrophoresis is not crystallized.
It was the same as that of L-2. Acid decomposition product (6N hydrochloric acid, 1
The acid analysis value at 10 ° C. for 24 hours was also in good agreement with the theoretical value.

Example 2 Production of Met-IL-2 A 5 mM ammonium acetate solution of Met-IL-2 obtained in Reference Example 1 described later (pH 5 mM 5.0, protein amount, 0.5).
~ 3.5 mg / m) 10m and treated in the same manner as in Example 3 to obtain Met-IL-2 crystals. The obtained crystals had the same specific activity as uncrystallized Met-IL-2, and the mobility in SDS-polyacrylamide gel electrophoresis was the same as that of uncrystallized Met-IL-2. . The amino acid analysis value of the acid decomposition product (6N HCl, 110 ° C., 24 hours) was also in good agreement with the theoretical value.

Reference Example 1 Separation of IL-2 and Met-IL-2 by FPLC Non-glycosyl which is a mixture of IL-2 and Met-IL-2 obtained in Examples 4 to 6 of JP-A-60-115528. 5 mM ammonium acetate buffer containing denatured human interleukin-2 (pH 5 mM 5.0 (protein concentration, 1.
18 mg / m) 5 m was equilibrated with 0.025 M diethanolamine-hydrochloric acid buffer solution (pH 9.4) for FPLC (Fast
Place on a Mono P column (0.5 x 20 cm, Pharmacia) for Protein Liquid Choromatography, and then 1% (v / v) Pharmalite (8-10.5) -5.2%
(v / v) Polybuffer 96-hydrochloric acid buffer solution (pH 8.0) was used to elute the protein adsorbed on the Mono P column.
The FPLC was performed at room temperature at a flow rate of 30 m / h. As a result, it was separated into a peak 1 which was dissolved at pH 8.0 and a peak 2 which was eluted at pH 7.9. Therefore, after collecting these, in order to remove the polybuffer used in FPLC, high performance liquid chromatography was performed using a trifluoroacetic acid-acetonitrile system as an elution solvent.

Column, Ultrapore RPSC (1.0 x 25 cm, Altex, USA column temperature, 30 ° C; elution solvent A,
0.1% trifluoroacetic acid-99.9% water; elution solvent B,
0.1% trifluoroacetic acid-99.9% acetonitrile, elution program, 0 min (55% A + 45% B) -4 min (5
5% A + 45% B) -28 minutes (42% A + 58% B)-
38 minutes (34% A + 66% B) -43 minutes (20% A + 8)
0% B) -44 minutes (55% A + 45% B), elution rate 3.0 m / min. The solutions obtained by this chromatography were dried and dried to obtain white powder. Those obtained from peak 1 and peak 2 in FPLC were designated as P1 and P2, respectively. P1
Yield of 1.12 mg (19.0%), P2 yield of 3.
It was 01 mg (51.0%).

From the results of amino terminal amino acid sequence analysis and amino acid composition analysis, P1 is a molecular species (IL-2) shown by X = hydrogen atom in FIG. 1, and P2 is a molecular species shown by X = methionine residue in FIG. It was confirmed to contain (Met-IL-2) at a purity of 98% and 99% or higher, respectively.

EFFECTS OF THE INVENTION According to the method of the present invention, rIL-2 can be crystallized, whereby the purity of rIL-2 and its stability can be markedly improved, and thus can be advantageously used as a medicine.

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of IL-2 (X = hydrogen atom in the figure) and the amino acid sequence of Met-IL-2 (X = X in the figure).
Methionine residue) is shown respectively. FIG. 2 shows a photograph of the IL-2 crystal obtained in Example 1. FIG. 3 shows a photograph of the Met-IL-2 crystal obtained in Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-115528 (JP, A), Koichi Anan (4 persons), “Basic Biochemistry Experimental Method 2 Extraction / Separation / Purification” (Maruzen) (SHO) 49-9-30) p. 57-67 Methods Enzymology, 1979 [55] P. 800

Claims (1)

[Claims] 1. A final concentration of about 5 in an ammonium acetate solution of high-concentration recombinant human interleukin-2, which is mainly water.
~ 25% (W / V) salt or final concentration about 0.5-30
% (W / V) polyethylene glycol is added to reduce the solubility of recombinant human interleukin-2 to precipitate recombinant human interleukin-2 crystals. -2
Crystal manufacturing method.