JPS63190830A - Anti-malignant tumor agent and kit for treating malignant tumor - Google Patents

Abstract

PURPOSE:To obtain an anti-malignant tumor agent comprising a human granulocyte colony stimulating factor as an active ingredient. CONSTITUTION:An anti-malignant tumor agent containing a human granulocyte colony stimulating factor (G-CSF) (e.g. one obtained by extraction, a separation and purification from human organism test specimen, one obtained by cultivating human G-CSF, followed by isolating a supernatant liquid of culture thereof, one obtained by forming human G-CSF producing hybridoma by using cell fusion method and collecting from the hybridoma, etc.; especially preferably having physical and chemical properties shown by the table) as an active ingredient. A preparation containing 0.1-500mug, preferably 0.5-200mug human G-CSF may be administered once - seven times a week. Since human G-CSF extremely enlarges killing action of granulocyte on malignant tumor in the presence of anti-malignant tumor serum, both the components are combined as active ingredients to give a kit for treating malignant tumor. A dose thereof is 0.1-5 cc/person.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to human granulocyte colony stimulating factor (hereinafter referred to as human GC).
An anti-malignant drug containing 3F (abbreviated as 3F) as an active ingredient! 'I!
The present invention relates to a malignant tumor treatment kit comprising a tumor agent and a combination thereof with an anti-malignant tumor serum.

[Conventional technology]

The present invention relates to hemopoietic factors (hemopoietic grow).
The aim is to use human G-C8F, which is one of the thfactors, in the treatment of malignant tumors,
The present invention aims to provide a novel anti-malignant tumor agent for which no reports directly related to this have been found.

Human G-C3F is a hematopoietic factor that acts on granulocyte precursor cells and promotes their differentiation into granulocytes in an in VitrO experimental system [e.g., Hctcalf,
et, al:Fxn,) lematol-1-185,
r1973) Nezu Zhuzha].

However, since it has been extremely difficult to obtain this human G-C3F until now, sufficient progress has been made in studying its usefulness or effectiveness as a medicine! First, the possibility of the present invention in treating cancer remained unexamined.

[Problem that the invention seeks to solve]

In order to overcome this situation, the applicant has conducted repeated research and succeeded in developing a method for producing human G-C3F using genetic engineering, etc., and has made it possible to obtain pure, homogeneous, and ceiling-suspended human G-C3F. (Japanese Patent Application No. 59-153273, Patent Application No. 60-269455 @, Patent Application No. 60-269
456 @, Patent Application No. 1983-270838, Patent Application No. 1983
-270839 etc.).

Based on this result, we will study the effects of human G-C3F on human mature granulocytes, which have malignant tumoricidal effects, and based on the knowledge obtained, we will try to develop excellent anti-malignant tumor agents with fewer side effects. This is the object of the present invention.

[Means for solving problems]

In order to achieve the above object, the present inventors have conducted intensive research and found that malignant tumor cells such as human malignant melanoma cells (HM
In V), granulocytes pre-assembled in the presence of human G-C3F have approximately 2 to 4 times more ftf1M than granulocytes pre-assembled in the absence of human G-C3F.
V effect was observed.

(2) The HMV killing effect was not affected in the presence of normal rabbit serum, but was enhanced up to 10 to 30 times in the presence of anti-HMV serum, depending on the concentration.

■ Killing of granulocytes by pre-positioning with human G-C3F 1-I
The degree of enhancement of the MV effect is 1, which clearly correlates with the concentration of human G-C8F.

We confirmed this fact, and based on this result, we found that human G-C8F significantly enhances the malignant tumoricidal effect (particularly in the presence of anti-malignant tumor serum) of human mature granulocytes (especially neutrophils), which led to the present invention. Reached.

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

A kit for treating malignant tumors comprising a combination of preparations containing colony stimulating factors as an effective ingredient is also provided.

Human G-C3F, which is an active ingredient of the anti-malignant tumor agent of the present invention, will be described in detail below.
There are no restrictions on the origin of human G-C3F as long as it is of high purity; for example, human G-C3F extracted, separated, and purified from human biological samples, human G-csr: produced by culturing production cells,
Those isolated from the culture supernatant, those obtained by forming human G-C3F-producing hybridomas using the cell fusion method, and those obtained by transforming hosts such as E. coli and animal cells through genetic recombination. Keshime monoli produced from transformants! Either purified i11 or chemically modified i11 can be used.

However, among these, the human G-C3F shown in the following [1) and (2) manufactured by the present applicant, which is available in large quantities and homogeneous with good tJi, is particularly preferred.

(1) Human G-C8F0 with the following physicochemical properties ■ Molecular weight approximately 19,000 ± 1.0000 as measured by sodium nidodecyl sulfate-polyacrylamide gel electrophoresis ■ Isoelectric point: pI = 5.5 ± 0. 1, pI=5.8±
It has at least one of three isoelectric points of 0.1°pI=6.1±0.1.

■Ultraviolet absorption: Maximum absorption at 280 nm, 250 nm
It has a minimum value at nm.

■The amino acid sequence from the N-terminus to the 21st remaining base is as follows.

t12N-Tllr-PrO-LeLl-G IV-
PrO-A 1a-3er-ser-teu-pro-
G l n-3er-Phe-Leu-Leu-Lys
-Cys-Lcu-Glu-Gln-Val
-(2) Human G-C8F containing a polypeptide having human granulocyte colony-stimulating factor activity expressed by the following amino acid sequence or a part thereof, or a glycoprotein having a sugar chain moiety thereof.

(G1et) nThr Pro Leu Gly Pr
o Ala scr Ser LetjPro Gi
n Ser Phc Leu Leu Ly
s Cys Leu Glu GlnVat
Herg Lys Ile Gln Gly Asp
Gly Eightla Ala LeuGln Glu
Lys Leu (Vat Ser Gl
u ) IlI Cys Ala ThrTyr
Lys Leu Cys His Pro Glu G
lu Leu Val LeuLeu Gly tli
s Ser Leu Gly Ile Pro Trp
Ala Pr.

Leu Ser Ser Cys Pro
Ser Gln Ala Leu Gln
LeuAla Gly Cys Leu Se
r Gln Leu tlis Ser G
ly LeuPhe teu Tyr Gln Gl
ytell Leu Gin Ala teu Glu
Gly Ile Ser Pro Glu Leu G
ly Pro Thr Lcu AspThr Le
u Gln Leu Asp Vat Al
a Asp Phe Ala DinghrThr
lie Trp Gln Gln Het Gl(J
GltJ Ieu c+y )f(!LA)a Pr
o Ala Leu Gln Pro 'r
hr G! n Gly Ala NetPro
Ala Phe Ala Ser Ala Phe
Gln Arg Arg AlaGly Gly Va
l Leu Vat Ala Ser 1lis Le
u Gln 5erPhe Leu Glu V
at Ser Dir Arg Val L
eu 8rg tlisLcu^la Gln P
ro (where m represents O or 1, and n represents O or 1).

The above-mentioned human G-C3F can be produced, for example, by the method shown in Reference Examples described below. That is, the human G-C8F in (1) above was prepared according to Reference Example 1, and the human G-C8F in (2) was prepared according to Reference Example 2. Regarding manufacturing article A1, the present applicant previously filed Japanese Patent Application No. 153273/1980, Patent Application No. 269455/1982, Patent Application No. 269456/1982, Patent Application No. 270838/1983, and Special Application IM60- 2708
39Q(7) Please refer to each specification m.

Alternatively, hybridomas obtained by cell fusion of G-C3F-producing cells and malignant tumor cells capable of self-proliferation can also be obtained by culturing in the presence or absence of mitogens. All human G-C3F obtained by these methods are included in the present invention.

The 17-G-C3F-containing solution can be further purified and concentrated by known means if necessary, and then stored frozen, or can be stored after removing water by freeze-drying, vacuum drying, or the like.

Furthermore, if desired, human G-C3F can be dissolved in an appropriate buffer and then sterile filtered using a Millipore filter or the like to prepare an injection.

Furthermore, the anti-malignant tumor agent of the present invention can contain an anti-adsorption agent in order to take the form of a pharmaceutical preparation suitable for human or veterinary medicine.

The dose and frequency of administration of human G-C3F contained in the anti-cancer agent of the present invention can be determined taking into account the condition of the target disease patient, but is usually 0.1 to 500μ per adult.
A formulation containing human G-C3F, preferably 0.5 to 200 μm, can be administered 1 to 7 times per week. However, the present invention is not limited by the content of human G-C3F.

Next, the kit for treating malignant tumors of the present invention will be explained.

As is clear from the experimental examples (pharmacological effects) described later, human G
-C3F significantly enhances the malignant tumoricidal effect of granulocytes in the presence of antineoplastic serum.

This is presumed to be because the antiserum enhances the ability of granulocytes to recognize tumor cells and makes it easier to attach to them, thereby further improving the effectiveness of the malignant tumor-killing action of granulocytes (especially neutrophils). be done.

The kit for treating malignant tumors of the present invention, which consists of a combination of anti-malignant tumor serum and a preparation containing human G-C8F as an active ingredient, was developed to utilize this action and effect in cancer treatment.

The anti-malignant tumor serum used in this kit may be prepared by a known method, and the dose is usually 0.1 to 5 CC per adult.

The anti-malignant tumor serum is preferably administered by intravenous injection or the like approximately one hour before the increase in granulocytes reaches its peak due to the administration of the anti-malignant tumor agent of the present invention.

(Examples) The present invention will be explained below with reference examples (manufacturing examples of human G-C8F), experimental examples (pharmacological effects), and examples (formulation examples).
The present invention is not limited to these.

Reference Example 1 (Human G produced by culturing G-C8F producing cells)
- Production example of C8F) G-C3F producing cells Jt derived from hydrocavitary carcinoma cells established by the method shown in Example 1 of Japanese Patent Application No. 59-153273@
+n1l11 ”+ 11% kl r%Lm, t
ltt! "#CI ry-ri1"1λ or the cell line c Hu-2 (C, N, C,
MI 164C) @ After floating in a nutrient solution, the cells were placed in a roller bottle and subjected to rotational culture. When the cells have grown densely on the inner wall of the roller bottle, the culture medium is diluted with serum-free RPMI.
1640 and collect the supernatant after culturing for 4 days. Next, serum-containing RPMI 1640 was added and cultured for 3 days, and then the culture solution was incubated with serum-free RPMI 1640.
After culturing for one day, collect the supernatant. This process was then repeated to collect the culture supernatant. The obtained serum-free culture supernatant was concentrated and purified approximately 1000 times by ultrafiltration, and then assayed.

Purification and assay were carried out in the same manner as in the example of the aforementioned Japanese Patent Application No. 1i59-153273.

Reference Example 2 (Production example of human G-C3F by genetic recombination) Escherichia coli (E, Co11) χ1776R-2 strain (FERM B
The CDNA fragment containing the human G-C3F gene excised from P-955> is inserted into the vector pdKCR to form a pf-IGV2 plasmid, which is then treated with 5alI, and then the lenow fragment is reacted with DNA polymerase.

Add this DNAk:EcoRI linker and re-EC
After partial digestion with 0RI, a fragment of about 2.7 Kb is recovered by agarose gel electrophoresis.

On the other hand, pAdD26SVpA plasmid (Kau fm
an, R, G, & 5harp, P, A, (
1982) Ho1. Cell 1. Biol, Volume 2, Volume 1
304-1319) were treated with EC0RI, 8AP treated, and dephosphorylated. Next, after treatment with phenol, the EC0RI fragment of pAdD26SVpA was recovered by electrophoresis.

The above 2.7 kb fragment and the pAdD26SVpA fragment were annealed and transformed into the E,co I i DHI strain by the rubidium chloride method to obtain the p)-IGV2-dhfr plasmid; (obtained from Yoh, L. Chasin, Columbia University) on a 9rm diameter plate (
The cells were cultured and grown in α minimal diluted medium containing 10% calf serum (manufactured by Nunc, Inc., supplemented with thymidine), and transformed by the calcium phosphate method (Wi (Ller et al., p. 141725 (1978)).

That is, an appropriate amount of carrier DNA (calf thymus DNA) was added to 1 μ9 of the pf-IGV2-d h fr plasmid, dissolved in 375 μg of TE solution, and 1 M CaCρ2 was added.
Add 125 μg. Chill on ice for 3-5 minutes 500μJ
) of 2X HBS (50mM HepeS, 280 m
After adding HNaCN (11.5 mM phosphate buffer) and cooling with water again, the mixture was mixed with 1rrIl of the above CHO cell culture solution, transferred to a plate, and cultured in a CO2 incubator for 9 hours.

Following washing, TBS containing 20% glycerol (Tris-
After washing again, a non-selective medium (alpha-MEM medium mentioned above, nucleoside added) was added and incubated for 2 days.
: Cells were split into 10. Next, culture was continued while replacing the medium with a selective medium (nucleoside-free) every two days, and the resulting clumps (fOc i ) were selected and transferred to a new plate.

0.02 μM methotrexate (M
-rX) and again in the presence of 0.1 μM -rX for cloning.

As a result of further cloning, production of human G-C8F of 10 my/J or more was confirmed.

The purification and assay were carried out according to the methods described in Examples 2 and 15 of the specification of Japanese Patent Application No. 60-269456.

Experimental Example 1 (Melanoma cell (HMV) effect of granulocytes and added anti-+1M
Effect of V serum and human G-C3F concentration fluctuation] deXtr
An precipitation method, Ficol 1-HVpaQ(Je(1,
077) Separate peripheral blood granulocytes from healthy subjects using multilayer centrifugation and hypotonic shock red blood cell removal (purity and viable cell rate of 90% or higher). Different concentrations of human G-
After adding C3F and incubating in a culture medium at 31°C for 1 hour, the cells were washed to prepare human mature granulocytes.

Next, a certain number of human malignant melanoma cell lines (1-IMV) were incubated in a microplate with Urigi normal serum at each concentration shown in Table 1. Alternatively, anti-HMV serum and a certain number of mature granulocytes prepared above were added and cultured at 37°C for 24 hours. Each well was then washed with culture medium and stained to determine the number of adherent and living cells. Calculated later.

The results are shown in Table 1.

As is clear from Table 1, the granulocytes prepared with the addition of human G-C3F have approximately 2 to 4 times the HMV killing effect compared to the case without the addition, and the addition of anti-IMV inhibitors also tends to increase the HMV killing effect. becomes even more prominent.

Furthermore, there is a clear correlation between the degree of enhancement of the killing effect of IMV on granulocytes by incubation with human G-C3F and the concentration of human G-C3F.

These results indicate that human G-C3F kills human mature granulocytes).
- It has been revealed that it significantly enhances IMV action.

(Leaving space below) Experimental Example 2 [Melanoma cell killing effect of granulocytes and the effect of added human G-C3 and variation in anti-Japanese MV serum depth] A certain number of l-IMV was cultured in a microplate, and the When the cells were sufficiently attached to the bottom of the well, anti-)IMV serum or normal serum of each ta shown in Table 2 was added, and then activated granulocytes or non-activated granulocytes prepared in the same manner as in Experimental Example 1 were added. The wells were then incubated at 37° C. for 24 hours. Thereafter, each well was washed with culture medium, and the number of adherent and surviving cells was determined after staining.

The results are shown in Table 2.

As is clear from Table 2, the killing +1 MV effect is not affected in the presence of normal serum, but is enhanced up to about 30 times in the presence of anti-HMV serum, depending on its concentration.

In addition, similar to the results of Experimental Example 1, granulocytes activated with human G-C3f' have a HMV killing effect of 2 even when normal serum is added, compared to granulocytes with inactivated f't using human G-C8F. It can be seen that this difference is more significant in the presence of anti-)-IMV hemolyte.

These results indicate that human G-C3F has a tendency to significantly enhance the HMV killing effect of human mature granulocytes in the presence of anti-IMV blood.

(Left below) Experimental Example 3 [Presence or absence of human G-C3r-' and killing effect of granulocytes or lymphocytes against various tumor cells] Granulocytes and lymphocytes in the peripheral blood of nail technicians were separated in the same manner as in Experimental Example 1. , incubate after adding human G-C3F,
Activated. As a control, a sample incubated in the same manner without the addition of human G-C3F was prepared.

17'J, the above activated granulocytes or non-activated granulocytes or lymphocytes were added to each tumor cell shown in Table 3, incubated, and various 11! I! DNA synthesis in tumor cells [3H
The uptake rate of l-thymidine was measured. The results are shown in Table 3.

As is clear from Table 3, it is recognized that human a-csr enhances the tumor-killing action of mature granulocytes not only against IIMV but also against other tumor cells.

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

(Margins below) Table 3. Effect of presence or absence of G-C3F on killing of various tumor cells such as granulocytes and lymphocytes Unit: cpm To summarize the results of the above experiments, as mentioned above,
The 1-IMV killing effect of the granulocytes placed in the presence of -C3F was approximately 2 to 4 times greater than that of the granulocytes placed in W in the absence of -C3F.

(2) The HMV killing effect was unaffected in the presence of normal Urigi blood, but was enhanced up to 10 to 30 times in the presence of anti-IMV serum, depending on the concentration.

■ Killing of granulocytes by a pre-apparatus with -〇-C3F +-I
The degree of enhancement of the MV effect was clearly correlated with the concentration of human G-C3F.

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

Therefore, it was confirmed that human G-C3F promotes the tumoricidal cell action of human mature granulocytes (particularly in the presence of antitumor serum).

Example 1 (Formulation Example) Human G-C8F obtained and purified according to Reference Example 1
After sterilization, the frozen product was frozen at -20°C to prepare an injection.

Example 2 (Formulation Example) Human G-C3F obtained and purified according to Reference Example 2
Fill a 10ttdl vial with aseptic technique for 5d, and -
After drying at 20° C. for 211 hours, the freeze-dried product was sealed with a rubber stopper and used to prepare an injection.

(Effects of the Invention) The anti-tumor agent of the present invention enhances the anti-JET' tumor action of mature granulocytes that originally exist in the human body by purified human G-C3F, and has an anti-tumor effect. The aim is to further enhance cellular blood FFI (Jf) and treat malignant tumors based on this effect, and there are great expectations that it will become a cancer treatment agent with few side effects.

Claims (1)

[Claims] 1. An anti-malignant tumor agent containing 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 anti-malignant tumor agent according to claim 1, wherein the human granulocyte colony-stimulating factor is a neutrophil colony-stimulating factor. 4. The anti-malignant tumor agent according to claim 1, wherein the human granulocyte colony-stimulating factor is obtained from a culture supernatant of human granulocyte colony-stimulating factor-producing cells. 5 Claim 1 characterized in that the human granulocyte colony stimulating factor has the following physical and chemical properties.
The anti-malignant tumor agent described in Section 1. "Physical and chemical properties" (1) Molecular weight: 19,000±1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (2) Isoelectric point: pI=5.5±0.1, pI=5.8±
0.1, pI=6.1±0.1. (3) Ultraviolet absorption: maximum absorption at 280 nm, 25
It has a minimum value at 0 nm. (4) The amino acid sequence from the N-terminus to the 21st residue is as follows. [Amino acid sequence is available] 6. 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. The anti-malignant tumor agent according to claim 1, which is an active polypeptide or glycoprotein. 7. The anti-malignant tumor 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. [There is an amino acid sequence] (where m represents 0 or 1, and n represents 0 or 1) 8. A polypeptide whose glycoprotein having human granulocyte colony stimulating factor activity is represented by the following amino acid sequence or a part thereof. The anti-malignant tumor agent according to claim 1, which has a sugar chain moiety. [There is an amino acid sequence] [There is an amino acid sequence] (where m represents 0 or 1). 9. A malignant tumor treatment kit comprising a combination of a preparation containing anti-human malignant tumor serum and human granulocyte colony-stimulating factor as active ingredients. 10. The kit for treating malignant tumor according to claim 9, wherein the malignant tumor is malignant melanoma. 11. The kit for treating malignant tumors according to claim 9, wherein the human granulocyte colony-stimulating factor is a neutrophil colony-stimulating factor. 12. The kit for treating malignant tumors according to claim 9, wherein the human granulocyte colony-stimulating factor is obtained from a culture supernatant of human granulocyte colony-stimulating factor-producing cells. 13. The kit for treating malignant tumors according to claim 9, wherein the human granulocyte colony stimulating factor has the following physical and chemical properties. "Physical and chemical properties" (1) Molecular weight: 19,000±1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (2) Isoelectric point: pI=5.5±0.1, pI=5.8±
0.1, pI=6.1±0.1. (3) Ultraviolet absorption: maximum absorption at 280 nm, 25
It has a minimum value at 0 nm. (4) The amino acid sequence from the N-terminus to the 21st residue is as follows. [Amino acid sequence is available] 14. 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. The kit for treating malignant tumors according to claim 9, which is a polypeptide or glycoprotein having activity. 15. The kit for treating malignant tumors according to claim 9, wherein the polypeptide having human granulocyte colony stimulating factor activity is represented by the following amino acid sequence or a part thereof. [There is an amino acid sequence] [There is an amino acid sequence] (where m represents 0 or 1, n represents 0 or 1) 16 A glycoprotein with human granulocyte colony stimulating factor activity has the following amino acid sequence or one of them The kit for treating malignant tumors according to claim 9, which has a polypeptide represented by the following formula and a sugar chain moiety. [There is an amino acid sequence] [There is an amino acid sequence] (where m represents 0 or 1).