JP2589094B2 - Antineoplastic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to human granulocyte colony stimulating factor (hereinafter referred to as human G-
(Abbreviated as CSF) as an active ingredient.

[Conventional technology]

The present invention relates to one of the hematopoietic growth factors.
Another object of the present invention is to provide a novel antineoplastic agent which is useful for the treatment of malignant tumors using human G-CSF, and for which there are no reports directly related thereto.

Human G-CSF is a hematopoietic factor having a function of acting on granulocyte precursor cells in an in vitro experimental system and capturing the enhancement of differentiation into granulocytes (for example, Metcalf.et.al: Exp.Hematol.
1,185 (1973) etc.].

However, since it has been extremely difficult to obtain human G-CSF until now, studies on its usefulness or efficacy as a medicine have not sufficiently progressed, and the potential of the present invention for the treatment of cancer, which is the object of the present invention, has been investigated. Was also left unconsidered.

[Problems to be solved by the invention]

In order to overcome such a situation, the present applicant has conducted extensive research, and as a result, succeeded in developing a method for producing human G-CSF by genetic engineering and the like, so that pure and homogeneous and a large amount of human G-CSF can be obtained. (Japanese Patent Application No. 59-153273, Japanese Patent Application No. 60-153273)
No. -269455, Japanese Patent Application No. 60-269456, Japanese Patent Application No. 60-270838
No., Japanese Patent Application No. 60-270839).

Based on these results, we will study the effects of human G-CSF on human mature granulocytes with malignant tumor activity, and based on the findings, we will develop an excellent antineoplastic agent with few side effects That is the object of the present invention.

[Means for solving the problem]

The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that malignant tumor cells such as human melanoma cells (HM
In V), granulocytes pre-hatched in the presence of human G-CSF exhibited about 2 to 4 times the HMV killing effect as compared to granulocytes pre-hatched in the absence.

HMV killing effect is not affected in the presence of normal rabbit serum, but up to 10 in the presence of anti-HMV serum, depending on its concentration
Up to 30 times.

The degree of enhancement of the HMV killing effect of granulocytes by pre-incubation with human G-CSF is clearly correlated with the concentration of human G-CSF.

From the above results, it was found that human G-CSF markedly enhances the malignant killing effect of human mature granulocytes (especially neutrophils) (especially in the presence of anti-malignant tumor serum), and reached the present invention. did.

That is, the present invention provides an anti-neoplastic agent comprising human granulocyte colony-stimulating factor as an active ingredient.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The origin is not limited as long as it is high-purity human G-CSF, for example, extraction from human biological samples, separation,
Purified and human G-CSF-producing cells were cultured and isolated from the culture supernatant, and human G-CSF-producing cells were isolated by cell fusion.
A CSF-producing hybridoma is formed and obtained therefrom, a gene is obtained from a transformant obtained by transforming a host such as Escherichia coli, animal cells, etc. by genetic recombination, isolated and purified, or a chemically modified product thereof Can be used.

However, among them, human G-CSF shown in the following (1) and (2) manufactured by the present applicant, which can be obtained in a homogeneous and large amount with high purity, is particularly preferable.

(1) Human G-CSF having the following physicochemical properties.

Molecular weight: about 19,000 ± 1,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Isoelectric point: has at least one of three isoelectric points of pI = 5.5 ± 0.1, pI = 5.8 ± 0.1, pI = 6.1 ± 0.1.

Ultraviolet absorption: having a maximum absorption at 280 nm and a minimum at 250 nm.

The amino acid sequence from the N terminal to the 21st residue is as follows.

_{H 2 N-Thr-Pro-} Leu-Gly-Pro-Ala-Ser-Ser-Leu-
Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-
Gln-Val- (2) a polypeptide having human granulocyte colony-stimulating factor activity represented by the following amino acid sequence or a part thereof, or a human G- containing a glycoprotein having a polypeptide having a sugar chain portion:
CSF.

(Met) _n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro G
ln Ser Phe Leu Leu Lys Cys Lue Glu Gln Val Arg Lys
Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu
(Val Ser Glu) _m Cys Ala Thr Tyr Lys Leu Cys His P
ro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala L
eu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu G
ly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp G
ln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln
Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala P
he Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu A
rg His Leu Ala Gln Pro (where m represents 0 or 1,
n represents 0 or 1).

The above-mentioned human G-CSF can be produced, for example, by the method shown in Reference Examples described later. That is, the human G-CSF of the above (1) was prepared according to Reference Example 1 and the human G-C
SF can be obtained by the method shown in Reference Example 2.

The detailed manufacturing conditions of these methods are described in Japanese Patent Application No. 59-153273 and Japanese Patent Application No. 60-26 filed earlier by the present applicant.
No. 9455, Japanese Patent Application No. 60-269456, Japanese Patent Application No. 60-270838, and Japanese Patent Application No. 60-270839.

In addition, as another method, it can be obtained by culturing a hybridoma obtained by cell fusion of a G-CSF producing cell and a malignant tumor cell capable of self-proliferation in the presence or absence of mitogen. All human G-CSFs obtained by these methods are included in the present invention.

If necessary, the obtained human G-CSF-containing solution can be further purified and concentrated by known means and then stored frozen, or can be stored after removing water by means such as freeze-drying and vacuum drying.

If desired, human G-CSF can be dissolved in an appropriate buffer, and then sterile filtered with a Millipore filter or the like to prepare an injection.

Further, the antineoplastic agent of the present invention can contain a pharmaceutical carrier or excipient necessary for taking a form as a pharmaceutical preparation suitable for human or veterinary medicine, and can further contain a stabilizer and an anti-adsorption agent. .

The dosage of human G-CSF contained in the antineoplastic agent of the present invention, the number of administration can be determined in consideration of the disease state of the target disease patient, usually 0.1 to 500 μg per adult, preferably 0.5 to 200 μg. Can be administered 1 to 7 times a week. However, the present invention is not limited by the content of human G-CSF.

〔Example〕

Hereinafter, the present invention will be described with reference to Reference Examples (manufacturing examples of human G-CSF) Examples (pharmacological effects) and Examples (preparation examples)
The present invention is not limited to these.

The results in the presence of anti-tumor serum are also shown for reference.

Reference Example 1 (Human G-CSF obtained by culturing G-CSF-producing cells)
Production Example) G-CSF-producing cell line CHU-1 derived from human oral floor cancer cells established by the method shown in Example 1 of Japanese Patent Application No. 59-153273 (C.
NCM accession number "I-315") or a cell line CHU-2 (CNCM accession number "I-483") established in a similar manner was suspended in RPMI 1640 culture medium containing fetal calf serum.
Rotary culture was performed in a roller bottle. When the cells grew densely on the inner wall of the roller bottle, the culture solution was replaced with serum-free RPMI 1640, and the supernatant was collected after culturing for 4 days. Then, after adding serum-containing RPMI 1640 and culturing for 3 days, the culture solution is replaced with serum-free RPMI 1640, and after 4 days of culture, the supernatant is recovered. The above procedure was repeated to collect the culture supernatant. The obtained serum-free culture supernatant was concentrated and purified about 1000-fold by ultrafiltration, and then assayed.

Purification and assay were carried out in the same manner as in the examples of the aforementioned Japanese Patent Application No. 59-153273.

Reference Example 2 (Production example of human G-CSF by genetic modification) Human isolated from E. coli x1776R-2 strain (FERM BP-955), which has been deposited by the present applicant with the National Institute of Fine Arts and Sciences. A cDNA fragment having the G-CSF gene is incorporated into a vector pdKCR to prepare a pHGV2 plasmid, which is then treated with SalI, and then reacted with a DNA polymerase klenow fragment.

After adding an EcoRI linker to the DNA and partially digesting it again with EcoRI, a fragment of about 2.7 kb is recovered by agarose gel electrophoresis.

On the other hand, pAdD26SVpA plasmid (kaufman, RG & Sharp,
P, A. (1982) Mol. Cell. Biol, Vol. 2, 1304-1319) is treated with EcoRI, BAP-treated, and dephosphorylated. Next, after treatment with phenol, the EcoRI fragment of pAdD26SVpA was recovered by electrophoresis.

Anneal the above 2.7 kb fragment and the pAdD26SVpA fragment, Ec
oli DHI strain by the rubidium chloride method to transform pHGV
The 2-dhfr plasmid was obtained.

Next, CHO cells (dhfr ^- strain, Columbia University Dr.L.chasi
n) (available from Nunc) in a 9 cm-diameter plate (Nunc) and cultured on α-minimal essential medium (α-MEM, adenosine, deoxyadenosine, thymidine added) containing 10% calf serum. (Wigle et al., Cell Vol. 14, p. 725 (197
8)).

That is, an appropriate amount of carrier DNA (calf chestline DNA) was added to 1 μg of the aforementioned pHGV2-dhfr plasmid, and 375 μl of TE solution was added.
And add 1 μM CaCl ₂ 125 μl. Cool on ice for 3-5 minutes and 500μ of 2 × HBS (20 mM Hepes, 280 mM NaCl, 1.5 mM
(Phosphate buffer) was added, and after ice-cooling again, the mixture was mixed with 1 ml of the above CHO cell culture solution, transferred to a plate, and cultured in a CO ₂ incubator for 9 hours.

Washing, TBS containing 20% glycerol (Tris-buffere
d saline), and after washing again, a non-selective medium (α-
(MEM medium, nucleosides were added), and the mixture was incubated for 2 days, and the cells were split 1:10 in the selective medium. Subsequently, the culture was continued while changing the medium with a selection medium (without adding nucleosides) every two days, and the resulting foci were selected and transferred to a new plate.

New plates contain 0.02 μM methotrexate (MT
X) The cells were grown in the presence and again grown in the presence of 0.1 μMMTX for cloning.

As a result of continuing cloning, human G
-The production of CSF was confirmed.

Purification and assay were performed by the methods described in Examples 2 and 15 of Japanese Patent Application No. 60-269456.

Experimental Example 1 [Addition of melanoma cell (HMV) effect of granulocytes on the effect of fluctuation of anti-HMV serum and human G-CSF concentration] dextran sedimentation method, Ficoll-Hypaque (1.077) double layer centrifugation method, hypotonic shock erythrocyte removal method Was used to separate peripheral blood granulocytes from healthy subjects (purity and viable cell rate of 90% or more). Various concentrations of human G-CSF were added to the separated cells, incubated at 37 ° C. for 1 hour in a culture solution, and then washed to prepare human mature granulocytes.

Next, a certain number of human malignant melanoma cell lines (HMV) were cultured in a microplate, and when the viable cells were sufficiently attached to the well bottom, rabbit normal serum or anti-rabbit at each concentration shown in Table 1 was obtained.
HMV serum and the above-prepared fixed number of mature granulocytes were added and cultured at 37 ° C. for 24 hours. Thereafter, each well was washed with a culture solution, and the number of adherent and surviving cells was calculated after staining. Table 1 shows the results.

As is evident from Table 1, granulocytes prepared by adding human G-CSF exhibited about 2 to 4 times the HMV killing effect as compared to the case without the addition, and the tendency was more remarkable when anti-HMV serum was added. Becomes

Further, the degree of enhancement of the HMV killing effect of granulocytes by incubation with human G-CSF has a clear correlation with the concentration of human G-CSF.

These results indicate that human G-CSF kills human mature granulocytes
It has been shown to significantly enhance the action.

Experimental Example 2 [Melanoma cell effect of granulocytes and added human G-CSF and anti-H
Effect of MV Serum Concentration Fluctuation] A certain number of HMVs were cultured in a microplate, and when viable cells sufficiently adhered to the well bottoms, anti-HMV serum or normal serum at each concentration shown in Table 2 was added, and then an experimental example Activated or non-activated granulocytes prepared in the same manner as in 1 were added and incubated at 37 ° C. for 24 hours. Thereafter, each well was washed with a culture solution, and the number of cells surviving adherence was calculated after staining. Table 2 shows the results.

As is clear from Table 2, the HMV killing effect is not affected in the presence of normal serum, but is enhanced up to about 30-fold in the presence of anti-HMV serum depending on its concentration.

Further, similarly to the results of Experimental Example 1, the granulocytes activated with human G-CSF had an HMV killing effect that was at least twice as high as that of inactive granulocytes using human G-CSF even under normal serum addition. This indicates that this difference becomes more pronounced in the presence of anti-HMV serum.

These results indicate that human G-CSF has a function of significantly enhancing the HMV killing effect of human mature granulocytes in the presence of anti-HMV serum.

Experimental Example 3 [Presence / absence of human G-CSF and killing effect of granulocytes or lymphocytes on various tumor cells] Peripheral blood granulocytes and lymphocytes of healthy subjects were separated in the same manner as in Experimental Example 1, and human G-CSF was added. Incubate after
Activated. As a control, a human G-CSF was prepared in the same manner without incubation.

On the other hand, the activated granulocytes, non-activated granulocytes or lymphocytes described above were added to each of the tumor cells shown in Table 3, and incubated, and the DNA synthesis of various tumor cells was examined based on the incorporation rate of [ ³ H] thymidine. Table 3 shows the results.

As is clear from Table 3, it can be seen that human G-CSF enhances the tumoricidal cell effect of mature granulocytes not only on HMV but also on other tumor cells.

On the other hand, lymphocytes do not have the same effect as granulocytes.

Summarizing the results of the above experiments, as described above, granulocytes pre-incubated in the presence of human G-CSF have about 2 to 4 times the HMV killing effect compared to granulocytes pre-incubated in the absence of human G-CSF. Admitted.

HMV killing effect is not affected in the presence of normal rabbit serum, but up to 10 in the presence of anti-HMV serum, depending on its concentration
Up to 30 times.

The degree of enhancement of the HMV killing effect of granulocytes by pre-incubation with human G-CSF had a clear correlation with the concentration of human G-CSF.

The above tendency was similarly observed for tumor cells other than HMV.

Therefore, it was confirmed that human G-CSF significantly enhances the tumoricidal cell action of human mature granulocytes (particularly in the presence of antitumor serum).

Example 1 (Formulation Example) Human G-CSF obtained and purified according to Reference Example 1
Was aseptically treated and used as an injection using a frozen product frozen at -20 ° C.

Example 2 (Formulation Example) Human G-CSF obtained and purified according to Reference Example 2
Was filled in a 10 ml vial via aseptic operation, lyophilized at −20 ° C., and used as an injection using a lyophilized material sealed with a rubber stopper.

〔The invention's effect〕

The anti-neoplastic agent of the present invention is a human G-CS purified from the antitumor effect of human mature granulocytes originally present in the human body.
It is intended to further enhance malignant tumors based on this effect by enhancing with F and coexisting with antitumor cell serum, and is expected to be a cancer therapeutic agent with few side effects.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:91)

Claims (8)

(57) [Claims] (1) An antineoplastic agent comprising human granulocyte colony stimulating factor as an active ingredient. 2. The anti-malignant tumor agent according to claim 1, wherein the malignant tumor is malignant melanoma. 3. The antineoplastic agent according to claim 1, wherein the human granulocyte colony stimulating factor is a neutrophil colony stimulating factor. 4. The antineoplastic agent according to claim 1, wherein the human granulocyte colony stimulating factor is obtained from a culture supernatant of a human granulocyte colony stimulating factor producing cell. 5. The antineoplastic agent according to claim 1, wherein the human granulocyte colony stimulating factor has the following physicochemical properties. "Physicochemical properties" Molecular weight: 19,000 ± 1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point: has at least one of three isoelectric points of pI = 5.5 ± 0.1, pI = 5.8 ± 0.1, and pI = 6.1 ± 0.1. Ultraviolet absorption: having a maximum absorption at 280 nm and a minimum at 250 nm. The amino acid sequence from the N terminal to the 21st residue is as follows. _{H 2 N-Thr-Pro-} Leu-Gly-Pro-Ala-Ser-Ser-Leu-
Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-
Gln−Val− 6. The human granulocyte colony stimulating factor activity of a human granulocyte colony stimulating factor produced from a transformant containing a recombinant vector containing a gene encoding a polypeptide having human granulocyte colony stimulating factor activity. 2. The antineoplastic agent according to claim 1, which is a polypeptide or glycoprotein having the same. 7. The antineoplastic agent according to claim 1, wherein the polypeptide having human granulocyte colony stimulating factor activity is represented by the following amino acid sequence or a part thereof. (However, m represents 0 or 1, n represents 0 or 1) 8. The method according to claim 1, wherein the glycoprotein having human granulocyte colony stimulating factor activity has a polypeptide represented by the following amino acid sequence or a part thereof and a sugar chain portion. Antineoplastic agent. (Where m represents 0 or 1).