JPH02225418A

JPH02225418A - Antineoplastic agent

Info

Publication number: JPH02225418A
Application number: JP1047594A
Authority: JP
Inventors: Takuji Kawashima; 拓司川島; Nobuya Yanagiuchi; 延也柳内; Muneo Yamada; 宗夫山田; Hajime Yokota; 横田　肇; Kunio Ujiie; 氏家　邦夫; Kazuo Yoshida; 吉田　賀津雄; Shinya Suzu; 鈴　伸也
Original assignee: Morinaga Milk Industry Co Ltd
Current assignee: Morinaga Milk Industry Co Ltd
Priority date: 1989-02-28
Filing date: 1989-02-28
Publication date: 1990-09-07

Abstract

PURPOSE:To provide an antineoplastic agent free from side effects, containing as active ingredient human monocyte-macrophage colony stimulative factor of human urine origin. CONSTITUTION:The objective antineoplastic agent prepared by conventional processes, containing as active ingredient human monocytemacrophage colony stimulative factor (abbreviated as human M-CSF) prepared from the urine of healthy man by the established method. when to be made into a pharmaceutical form, the agent is incorporated with, as stabilizer, human serum albumin and/or gelatin to improve the stability of human M-CSF. The present antineoplastic agent is prepared into a form such as an injection, which is put to non-oral administration into the vein, artery, muscle, skin, peritoneal cavity, etc., at a dose of 4X10<4>-160X10<4> units/kg. body weight/day.

Description

【発明の詳細な説明】［産業上の利用分野］本発明は、ヒト尿由来のヒト単球−マクロファージコロ
ニー刺激内Ｐ（以上ヒト尿由来のヒトＭ−Ｃ３Ｆとする
）を有効成分とする抗悪性１１！瘍剤に関する。Detailed Description of the Invention [Industrial Application Field] The present invention provides an antimicrobial agent containing human monocyte-macrophage colony-stimulating intracellular P derived from human urine (hereinafter referred to as human M-C3F derived from human urine) as an active ingredient. Malignant 11! Regarding cancer drugs.

［従来の技術１悪性腫瘍は化学療法剤に代表される薬剤の開発或は外科
的技術等の進歩により、その治療効果は改善されてきて
いる。しかし、完全に悪性［をｌＪ！除するまでには芋
っておらずしばしば再発する。[Prior Art 1] The therapeutic effects of malignant tumors have been improved due to the development of drugs such as chemotherapeutic agents and advances in surgical techniques. However, it is completely malignant [lJ! It doesn't go away by the time it's cured, and it often recurs.

また造血器又は各種臓器に対する副作用−ｂ著しく重篤
な感染症又ｔよ臓器障害を引き起こす′８の問題がある
。In addition, there is the problem of side effects on hematopoietic organs and various organs, which can cause extremely serious infections and organ damage.

コロニー刺激因子は、補乳動物の造血幹細胞の分化・増
殖を刺激する因子であり、現在までに単球−マクロファ
ージ系幹細胞に作用する因子（Ｍ−Ｃ８Ｆ）、顆粒球−
単球系幹細胞に作用する因子（ＧＭ−Ｃ８Ｆ）、顆粒球
系幹細胞に作用する因子（Ｇ−Ｃ３Ｆ）、多能性幹細胞
に作用する因子（Ｍｕｌｔｉ　−ＣＳ　Ｆ　）の４種が
知られている。ヒトマクロファージコロニー刺激因子は
純化され、その蛋白質及び遺伝子構造についても明らか
にされている（特開昭６４−２２８９９号公報）。また
顆粒球減少症に対する有用性が明らかにされ、医薬とし
ての期待が大きい（Ｈｏｔｏｙｏｓｈ　ｉにら、Ｅｘｐ
ｅｒｉｍｅｎｔａｌ　ｌｌｅｍａｔｏｌｏｏｙ　１４巻
、１０６９−１０７５．１９８６年）。Colony stimulating factor is a factor that stimulates the differentiation and proliferation of hematopoietic stem cells in supplemented mammals, and to date, it has been used as a factor that acts on monocyte-macrophage stem cells (M-C8F), granulocyte-
Four types of factors are known: a factor that acts on monocytic stem cells (GM-C8F), a factor that acts on granulocytic stem cells (G-C3F), and a factor that acts on pluripotent stem cells (Multi-CSF). . Human macrophage colony stimulating factor has been purified, and its protein and gene structure have also been clarified (Japanese Patent Laid-Open Publication No. 22899/1989). In addition, its usefulness for treating granulocytopenia has been revealed, and there are great expectations for its use as a medicine (Hotoyoshi Nira, Exp
14, 1069-1075.1986).

コロニー刺激因子の悪性腫瘍に対する作用については、
＾、　５ａｓｐｓｏｎ　−ＪＯｈａｌｌｎＯ８Ｓら（Ｊ
。Regarding the effects of colony stimulating factors on malignant tumors,
^, 5aspson-JOhallnO8S et al.
.

［ｍｍｕｎｏｌｏａｙ、　１４１　巻、３６８０−３６
８６．１９８８年）及びり、　Ｉｆ、　Ｈｕｎｎら（Ｂ
ｌｏｏｄ　、　７２巻、１２７．１９８８年）により報
告されているが、と１・尿由来のヒトＭ−Ｃ８Ｆに関す
る抗悪性ＩＩ！瘍剤への利用可・能性については未検１
のまま置かれていた。[mmunoloay, vol. 141, 3680-36
86.1988) and If, Hunn et al.
72, 127. Possibility of use as an anti-cancer agent has not been examined1
It was left as is.

［本発明が解決しようとする課題］悪性腫瘍の治療剤としてこれまでに用いられている抗悪
性腫瘍剤の中で、顕著な効果を示し且つ副作用の少ない
ものはほとんどない。特に、造血器悪性Ｍ！瘍の治療に
あっては、近年の化学療法剤の進歩に伴って治療効果が
菖しく向上したが、完全に悪性腫瘍を排除するまでには
至っておらずしばしば再発する。このため、強力な化学
療法剤施用後に骨髄移植を行ない完全治癒をめざす療法
も試みられている。骨髄移植にはＨＬＡタイプが一致す
るドナー（Ｄｏｎｏｒ　）の骨髄が使用されるが、拒絶
反応、ＧＶＨＤの発症などがあり、自家骨髄移植が望ま
しい。しかし、造血器ｌＩｍにあっては移植骨髄細胞中
に悪性腫瘍が沢在しており、悪性ｆｉ！Ｉ１ｍのパージ
（ｐｕｒａｅ　）が必須の条件となっている。[Problems to be Solved by the Invention] Among the anti-malignant tumor agents that have been used so far as therapeutic agents for malignant tumors, there are almost none that exhibit remarkable effects and have few side effects. In particular, hematopoietic malignant M! In the treatment of cancer, the therapeutic effects have improved markedly with the recent advances in chemotherapeutic agents, but the malignant tumor has not been completely eliminated and recurrence often occurs. For this reason, attempts have been made to use powerful chemotherapy drugs followed by bone marrow transplantation to achieve a complete cure. For bone marrow transplantation, bone marrow from a donor with a matching HLA type is used, but there are risks of rejection and development of GVHD, so autologous bone marrow transplantation is preferable. However, in the hematopoietic system, there are many malignant tumors in the transplanted bone marrow cells, and malignant fi! Purging (purae) of I1m is an essential condition.

そこで悪性Ｓ瘍の殺傷を目的として検討を行った結束、
本発明に係わるヒト尿由来のヒト単球−マクロファージ
コロニー刺激因子が悪性ＩＩｆ瘍殺（Ｕ作用を著しく増
強することを見いだし本発明を完成した。前述の如く、
ヒｌ−Ｍ　−ＣＳ　Ｉ＝は既に臨床試験が成されており
、その安全性が確認され副作用がほとんどないことが明
らかにされている（　ＨｏｔａｙｏｓｈｉにらＩｉｍｕ
ｎｏｂｉｏｌｇｙｌ　７２巻、２０５−２１２．１９８
６年）。それ故、本発明は、顕著な抗ａｍ活性を示し且
つ副作用のない抗悪性腫瘍剤を提供することを可能にし
た。Therefore, we investigated the idea of unity for the purpose of killing malignant S tumors.
The present invention was completed by discovering that the human monocyte-macrophage colony-stimulating factor derived from human urine according to the present invention significantly enhances the killing of malignant IIf tumors (U effect).
Clinical trials have already been conducted on Hil-M-CS I=, and its safety has been confirmed and it has been shown that there are almost no side effects (Hotayoshi et al.
nobiorgyl vol. 72, 205-212.198
6 years). Therefore, the present invention has made it possible to provide an anti-malignant tumor agent that exhibits significant anti-am activity and has no side effects.

［１題を解決するための手段〕本発明はヒト尿由来のヒトＭ　−ＣＳ　Ｉ＝を有効成分
とする抗悪性腫瘍剤を提供するものであり、また該抗悪
性ｌｌ！瘍剤を投与するに際し、悪性ｊ！瘍に対する特
異抗体を併用することを特徴とする上記抗悪性腫瘍剤の
投与方法を提供するものである。[Means for Solving the Problem] The present invention provides an anti-malignant tumor agent containing human M-CSI= derived from human urine as an active ingredient, and also provides an anti-malignant tumor agent containing human M-CSI= derived from human urine as an active ingredient. When administering anticancer agents, be sure to use malignant j! The present invention provides a method for administering the above-mentioned anti-malignant tumor agent, characterized in that a specific antibody against a tumor is used in combination.

更に本発明は長期に安定な抗悪性腫瘍剤として使用され
るために安定剤としてヒト血清アルブミン及び／又はセ
ラチンを含有させた上記抗悪性Ｍ瘍剤を提供するもので
ある。Furthermore, the present invention provides the above-mentioned anti-malignant tumor agent containing human serum albumin and/or seratin as a stabilizer in order to be used as a long-term stable anti-malignant tumor agent.

本発明に係わるヒト尿由来のヒトＭ−Ｃ３Ｆは、健常者
の尿より公知の方法（特開［１ｉ？６３−１９８７００
＠公報、特開昭６３−２５０４００号公報、特開昭６４
−２２８９９号公報）によって精製したものを凍結乾燥
して、１１製した。例えば特開昭６３−１９８７００号
公報記載の方法で次のとおり調製した。ケなわも、健康
人の尿１０００ＬをｐＨ８，５に調整後、沈澱物を濾過
除去し、分子ω５ｏ、ｏｏｏダルトンの限外濾過膜（７
ミコン社、１−１１０　Ｘ　５０　）で濃縮と脱塩を行
なった。次にｐＨを７．０に調整し密閉容器中で６０℃
で１０時間加熱殺菌した。殺菌後、遠心分離（５０００
ｘｏ３０分囚）して沈澱物を除去した後、０．０２Ｈリ
ンＭ１１１！Ｉ液（１１Ｎ７．２）で平衡化Ｌ／　タＤ
　Ｅ　Ａ　Ｅ　−セルロースと蝕合し、吸着させた。Ｄ
ＥＡＥ−セルロースを０．０５８食塩添加０．０２Ｍリ
ン！緩衝液（ｐｌｔ７．２）で洗浄した優、０．２５８
食塩添加０．０２８リンＭ緩衝液（ｐｌ！７．２）で溶
出させた。、溶出液を限外濾過膜（アミコン社Ｈ１０Ｐ
　１０　）で濃縮して、５ｅｐｈａｃｒｙｌ　Ｓ　−３
０（ファルマシア社、直径２０ｃＩＲｘ高さ８０α）を
用い、１Ｈ硫安添加緩衝液（ｐＨ７，２）でゲル濾過し
た。ゲル濾過での分子ｍ範囲７０．０００〜１５０．０
００ダルトン画分を上記１Ｍ硫安添加緩ＩＭで平衡化し
たＰｈｅｎｙｌ−ｓｅｐｈａｒｏｓｅ４　Ｂ　力５　ム
（ファルマシア社製直径１０×長さ２０ｃＩＲ）に＠着
させ、次いで０．５Ｈ４ｉｉｌｌ安添加緩衝液（ｐ１１
７．２）で溶出させた。溶出液を限外濾過膜（アミコン
社ＩＨＩＰＩＯ）で濃縮して、丁５ＫＧ３０００ＳＷ　
（東ソー製、ｉ！２．５Ｘ６０ｃｍ）で高速液体クロマ
トグラフィーにかけ、相対溶出ｍ　（Ｖｅ／Ｖｏ）１．
２〜１．５の画分を得た。Human M-C3F derived from human urine according to the present invention can be obtained by a known method (Unexamined Japanese Patent Publication [1i?63-198700]
@ Publication, JP-A-63-250400, JP-A-64
-22899) was purified and freeze-dried to produce No. 11. For example, it was prepared as follows using the method described in JP-A-63-198700. Kenawa also adjusts 1000L of urine from healthy people to pH 8.5, filters out the precipitate, and uses an ultrafiltration membrane (7
Concentration and desalting were performed using Micon Co., Ltd., 1-110×50). Next, adjust the pH to 7.0 and store at 60°C in a closed container.
It was heat sterilized for 10 hours. After sterilization, centrifugation (5000
After removing the precipitate, 0.02H phosphorus M111! Equilibrate with I solution (11N7.2) L/D
E A E - Interlocked with cellulose and adsorbed it. D
EAE-cellulose with 0.058 salt and 0.02M phosphorus! Excellent, 0.258 washed with buffer (plt7.2)
Elution was performed with 0.028 phosphorus M buffer (pl! 7.2) supplemented with saline. , the eluate was filtered through an ultrafiltration membrane (Amicon H10P
10) and concentrated with 5ephacryl S-3.
0 (Pharmacia, diameter 20cIR x height 80α) and gel filtration with 1H ammonium sulfate added buffer (pH 7,2). Molecular m range in gel filtration 70.000-150.0
The 0.00 Dalton fraction was applied to Phenyl-sepharose 4B (diameter 10 x length 20 cIR, manufactured by Pharmacia) equilibrated with the above 1M ammonium sulfate-added buffer, and then 0.5H4I ammonium sulfate added buffer (p11).
7.2). The eluate was concentrated using an ultrafiltration membrane (Amicon IHIPIO) and
(manufactured by Tosoh, i!2.5X60cm) and subjected to high performance liquid chromatography with relative elution m (Ve/Vo) 1.
Fractions from 2 to 1.5 were obtained.

この両分を再度濃縮し、Ｈｉ　−Ｐｏｒｃ２１４１Ｐ　
（バイダック社、軽２．２Ｘ長さ２５Ｇ）の逆相カラム
で０．１％トリフルＡ口酢酸を含む、アセトニトリル０
−１００％（ｐＨ２，０）の直線８１度勾配による高速
液体り０マドグラフイーにかけヒトＭ−Ｃ３Ｆ画分を集
め、凍結乾燥しヒ］・尿由来のヒトＭ−Ｃ８Ｆ４ｑを得
た。得られたヒト尿由来のヒトＭ−Ｃ８Ｆの理化学飽性
′ａは次の通りである。Both fractions were concentrated again and Hi-Porc2141P
(Vydac, light 2.2X length 25G) reversed phase column containing 0.1% triflu A-acetic acid, acetonitrile 0.
The human M-C3F fraction was collected and lyophilized by high-speed liquid chromatography using a linear 81 degree gradient of -100% (pH 2,0) to obtain human M-C8F4q derived from human urine. The physical and chemical saturation 'a of the obtained human M-C8F derived from human urine is as follows.

ａ）　分子間同−のサブユニットから成るホＥ２ｆｆｉ体であって、
ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳
動で測定した分子量が７０．０００〜９０．０００ダル
トンであり、還元剤で解離させて生物活性を洲失させた
サブユニットについてドデシル硫酸すｔ−リウムポリア
クリルアミドグル電気泳動で測定した分子量は３５，０
００〜４５゜０００ダルトンである。a) E2ffi body consisting of intermolecularly identical subunits,
Sodium dodecyl sulfate polyacrylamide gel electrophoresis for subunits with a molecular weight of 70.000 to 90.000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and whose biological activity has been lost by dissociation with a reducing agent. The molecular weight measured by electrophoresis is 35.0
00 to 45°000 Daltons.

ｂ）ザブユニットのアミノ酸配列ホモ２ｍ体を構成するサブユニット蛋白質は、次に示す
２１４乃至２３８個のアミノ酸配列を有し、１２２番目
及び１４０７ｆ！目のアスパラギンはそれぞれアスパラ
ギン（Ａｓｎ）−ｘ−スレオニン（Ｔ　ｈ　ｒ　）　／
セリン（Ｓｅｒ）で表される典型的なＮ−グリコシド結
合部位を有する。ここでＸは任意のアミノ酸を示す。b) Amino acid sequence of subunit The subunit protein constituting homo 2m has the following amino acid sequence of 214 to 238, including the 122nd and 1407f! Asparagine in the eye is asparagine (Asn)-x-threonine (T h r ) /
It has a typical N-glycosidic bonding site represented by serine (Ser). Here, X represents any amino acid.

Ｇ　ｌ　ｕ−Ｇ　Ｉｕ−Ｖａ　Ｉ　−３ｅｒ−Ｇ　ｌ　
ｕ−Ｔｙｒ−Ｃｙａ−３ｅｒ−ｔｌ　ｉ　５−Ｈｃｔ−
１１ｅ−Ｇ［ｙ−３ｅｒ−Ｇｌｙ−旧５−Ｌｅｕ−Ｇｉ
ｎ−８ｅｒ（ｃｏ−Ｇｉｎ−＾ｒｇ−ｔｅｕ−Ｉｌｅ−
＾５ｐ−３ｅｒ−Ｇｌｎ−Ｎｅｔ−Ｇｌｕ−Ｔｈｒ−３
ｅｒ−Ｃｙｓ−Ｇｉｎ−目ｅ−Ｔｈｒ−Ｐｈｅ−Ｇ　ｌ
　ｕ−Ｐｈｅ−Ｖａ　Ｉ　−Ａｓｐ−Ｇ　ｌ　ｎ−Ｇ　
１ｕ−Ｇ　Ｉ　ｎ−Ｌｅｕ−Ｌｙｓ−Ａｓｐ−Ｐｒｏ−
ＶａＩ−Ｃｙｓ−Ｔｙｒ−Ｌｅｕ−Ｌｙｓ−Ｌｙｓ−Ａ
ｉａ−Ｐｈｃ−Ｌｃｕ−ｔａｕ−シａｔ−Ｇｌｎ−Ａｓ
ｐ−１１ｅ−Ｎｅｔ−Ｇｌｕ−＾ｓｐ−丁ｈｒ−Ｎｅｔ
−＾ｒｇ−Ｐｈｃｉ−＾ｒｇ−八５ｐへＡｓｎ−Ｔｈｒ
−Ｐｒｏ−へｓｎ−へ！ａ−１１ｅ−八Ｉａ−１１０−
Ｖａ　ｌ　−Ｇ　Ｉ　ｎ−Ｌｅｕ−Ｇ　ｌ　ｎ−Ｇ　ｌ
　ｕ−Ｌｃｕ−３ｅｒ−Ｌｅｕ−Ａｒｇ−Ｌｃｕ−Ｌｙ
ｓ−３ｃｒ−Ｃｙｓ−Ｐｈｅ−Ｔｈｒ−Ｌｙｓ−Ａｓｐ
−Ｔｙｒ−Ｇｌｕ−Ｇｉｕ−Ｈｉｓ−＾５ｐ−Ｌｙｓ−
Ａｌａ−Ｃｙｓ−■ａ　Ｉ　−ＡｒＯ−Ｔｈｒ−Ｐｈｅ
−Ｔｙｒ−Ｇ　１ｕ−Ｔｈｒ−Ｐｒ。G l u-G Iu-Va I -3er-G l
u-Tyr-Cya-3er-tl i 5-Hct-
11e-G[y-3er-Gly-old 5-Leu-Gi
n-8er(co-Gin-＾rg-teu-Ile-
^5p-3er-Gln-Net-Glu-Thr-3
er-Cys-Gin-E-Thr-Phe-G l
u-Phe-Va I-Asp-G l n-G
1u-G I n-Leu-Lys-Asp-Pro-
VaI-Cys-Tyr-Leu-Lys-Lys-A
ia-Phc-Lcu-tau-at-Gln-As
p-11e-Net-Glu-^sp-dinghr-Net
-^rg-Phci-^rg-85p to Asn-Thr
-Pro- to sn-! a-11e-8Ia-110-
Va l -G I n-Leu-G l n-G l
u-Lcu-3er-Leu-Arg-Lcu-Ly
s-3cr-Cys-Phe-Thr-Lys-Asp
-Tyr-Glu-Giu-His-^5p-Lys-
Ala-Cys-■a I-ArO-Thr-Phe
-Tyr-G 1u-Thr-Pr.

−Ｌｅｕ−Ｇｔｎ−Ｌｅｕ−Ｌｅｕ−Ｇｌｕ−Ｌｙｓ−
Ｖａｔ−Ｌｙｓ−＾５ｎ−Ｖａｌ−Ｐｈｅ−Ａｓｎ−Ｇ
ｌｕ−Ｔｈｒ−Ｌｙｓ−Ａｓｎ−Ｌｅｕ−Ｌｅｕ−＾５
ｐ−ｔｙｓ−＾５ｐ−Ｔｒｐ−＾５ｎ−１１ｅ−Ｐｈａ
−８ｅｒ−Ｌｙａ−Ａｓｎ−Ｃｙｓ−＾３ｎ−＾５ｎ−
８ｅｒ−Ｐｈｅ−＾１ａ−Ｇｌｕ−Ｃｙｓ−３ｅｒ−３
ｅｒ−Ｇｌｎ−＾５ｏ−Ｖａｔ−Ｖａｔ−Ｔｈｒ−Ｌｙ
Ｓ−Ｐ「０−＾５ｐ−Ｃｙｓ−八５ｎ−Ｃｙｓ−Ｌｅｕ
−ｒｙｒ−Ｐｒｏ−Ｌｙｓ−ΔＩａ−１１ｅ−Ｐｒｏ−
３ｅｒ−３ｅｒ−Ａｓｐ−Ｐｒｏ−＾１ａ−８ｅｒ−シ
ａ　ｌ　−３ｅｒ−Ｐｒｏ−ｔｌ　＋　５−Ｇｌｎ−Ｐ
ｒｏ−Ｌｅｕ−＾１ａ−Ｐｒｏ−３ｅｒ−Ｎｅｔ−＾１
ａ−Ｐｒｏ−シミ１−へ１ａ−Ｇ　ｌｙ−Ｌｅｕ−Ｔｈ
ｒ−ｒ　ｒｐ−Ｇ　１ｕ−Ａｓｐ−３ｅｒ−Ｇ　１ｕ−
Ｇ　ｌ　ｙ−Ｔｈｒ−Ｇ　１ｕ−Ｇ　Ｉ　ｙ−３ｅｒ−
８ｅｒ−Ｌｅｕ−Ｌｅｕ−Ｐｒｏ−Ｇ　Ｉ　ｙ−Ｇ　Ｉ
　ｕ−Ｇ　ｌ　ｎ−Ｐｒｏ−Ｌｅｕ−旧５−Ｔｈｒ−Ｖ
ａ　ｌ−＾５ｐ−Ｐｒｏ−Ｇｌｙ−３ｅｒ−＾１ａ−１
−ｙｓ−Ｇｉｎ−＾ｒｇ−Ｐｒｏ−Ｐｒｏ−Ａｒｇ−８
ｅｒ−Ｔｈｒ−Ｃｙｓ−Ｇｔｎ−８ａｒ−Ｐｈａ−ＧＩ
ｕ−Ｐｒ。-Leu-Gtn-Leu-Leu-Glu-Lys-
Vat-Lys-^5n-Val-Phe-Asn-G
lu-Thr-Lys-Asn-Leu-Leu-^5
p-tys-^5p-Trp-^5n-11e-Pha
-8er-Lya-Asn-Cys-^3n-^5n-
8er-Phe-^1a-Glu-Cys-3er-3
er-Gln-^5o-Vat-Vat-Thr-Ly
S-P "0-^5p-Cys-85n-Cys-Leu
-ryr-Pro-Lys-ΔIa-11e-Pro-
3er-3er-Asp-Pro-^1a-8er-Sial -3er-Pro-tl + 5-Gln-P
ro-Leu-^1a-Pro-3er-Net-^1
a-Pro-stain 1-to 1a-G ly-Leu-Th
r-r rp-G 1u-Asp-3er-G 1u-
G ly-Thr-G 1u-G I y-3er-
8er-Leu-Leu-Pro-GI y-GI
u-G l n-Pro-Leu-old 5-Thr-V
a l-^5p-Pro-Gly-3er-^1a-1
-ys-Gin-^rg-Pro-Pro-Arg-8
er-Thr-Cys-Gtn-8ar-Pha-GI
u-Pr.

−Ｐｒｏ−Ｇｌｕ−Ｔｈｒ−Ｐｒｏ−Ｖａｌ−ｖａｌ−
Ｌｙｓ−ｃ）　　’Ｊ−重点ポリアクリルアミドゲル等電点電気泳動法及びクコクロ
ース密度勾配等電点泳動法で測定した等電点（ｐｌ）は
３．１〜３．７である。-Pro-Glu-Thr-Pro-Val-val-
Lys-c) 'The isoelectric point (pl) measured by J-weighted polyacrylamide gel isoelectric focusing method and wolfberry density gradient isoelectric focusing method is 3.1 to 3.7.

ｄ）　円二色性スペクトル円二色性分散計による遠紫外部ＣＤスペクトルは波長２
０８　ｎｌ及び２２２ｕにそれぞれ極小ピークがありα
−へリツクス構造を含んでいる。d) Circular dichroism spectrum The far ultraviolet CD spectrum measured by a circular dichroism dispersion meter has a wavelength of 2.
There are minimum peaks at 08 nl and 222u, respectively, α
- Contains a helical structure.

ｅ）熱安定性６０±０．５℃で６０分間加熱しても生物活性は失なわ
れない。e) Thermal stability No loss of biological activity when heated at 60±0.5°C for 60 minutes.

「）赤外線吸収スペクトル波数１６８０ａ　　、１２００α−１及び１１３０ＣＩ
　＋　’に強度吸収、波数１５４０ｚ−’、１４３０ｃ
ＩＩＩ−１および１０７０ｃ１１−１に中度吸収を示す
赤外線吸収スペクトラムを有する。") Infrared absorption spectrum wave number 1680a, 1200α-1 and 1130CI
Intensity absorption at +', wave number 1540z-', 1430c
It has an infrared absorption spectrum showing moderate absorption at III-1 and 1070c11-1.

ヒト尿由来のヒトＭ−Ｃ８Ｆは、ヒトＪ１球の腫瘍細胞
作用を有意に増強する作用を有し、また例えばヒト子宮
頚部癌細胞の増殖を抑制する作用を右することが明らか
にされた。従ってヒト尿由来のヒトＭ−Ｃ８Ｆは、各種
の悪性Ｎ１！、例えば骨髄性白血病、子宮頚部癌などの
造血器又は各種臓器における悪性腫瘍の治療に有効であ
る。It has been revealed that human M-C8F derived from human urine has the effect of significantly enhancing the tumor cell action of human J1 cells, and also has the effect of suppressing the proliferation of, for example, human cervical cancer cells. Therefore, human M-C8F derived from human urine has various types of malignant N1! It is effective in the treatment of malignant tumors in hematopoietic organs or various organs, such as myeloid leukemia and cervical cancer.

またヒト尿由来のヒトＭ−Ｃ３Ｆは、ヒト１１球の腫１
ｉ細胞に対する抗体依存性ｆｌ！ｓ膚胞殺傷活性（ＡＤ
ＣＣ）を有意に増強することが明らかにされた。従って
、ヒト尿由来のヒトＭ−Ｃ８Ｆは、対象とする悪性ｍ瘍
に対する特異的抗体と併用することによりその治療効果
、がより増強される。In addition, human M-C3F derived from human urine is
Antibody dependence on i cells fl! s Dermatocysticidal activity (AD
CC) was revealed to be significantly enhanced. Therefore, the therapeutic effect of human M-C8F derived from human urine is further enhanced when used in combination with a specific antibody against the target malignant tumor.

ヒト尿由来のヒトＭ−Ｃ８Ｆは、通常、静脈内、動脈内
、筋肉内、皮下、腹腔などの非経］］投与により投与す
ることができる。Human M-C8F derived from human urine can usually be administered parenterally, such as intravenously, intraarterially, intramuscularly, subcutaneously, or intraperitoneally.

投与用の製剤としては、注射剤、注入剤などが挙げられ
、これら製剤はそれ自体公知の方法によって調製するこ
とができる。例えば、ヒト尿由来のヒトＭ−Ｃ８Ｆを適
当な緩衝液に加えて無菌濾過し、ガラスバイアル中に無
菌的に充填して密封し、必要に応じて凍結乾燥して製剤
を調製することができる。Preparations for administration include injections, infusions, and the like, and these preparations can be prepared by methods known per se. For example, human M-C8F derived from human urine can be added to an appropriate buffer, filtered aseptically, filled aseptically into a glass vial, sealed, and optionally lyophilized to prepare a preparation. .

ヒト尿由来のヒトＭ−Ｃ８Ｆは、特にヒト血清アルブミ
ン及び／又はゼラチンとともに製剤化することによりそ
の安定性が著しく自重する。ヒトｒＩｎ清アルブミン、
ゼラチンの使用醋は、ヒト尿由来のヒトＭ−Ｃ８Ｆに対
して１ｏｏｔａ倍以上が望ましい。Human M-C8F derived from human urine is particularly unstable when formulated with human serum albumin and/or gelatin. human rIn clear albumin,
The amount of gelatin used is preferably at least 10 times that of human M-C8F derived from human urine.

ヒト尿出来のヒトＭ−Ｃ８Ｆの投与量は、患者の年令、
体重、症状等によって変動し得るが、通常、４Ｘ１０’
〜１６０Ｘ１０’単位／に９・体重／　［３、好ましく
は１６Ｘ１０’へ一８０Ｘ１０’単位／υ・体重７日で
ある。The dose of human M-C8F obtained from human urine depends on the age of the patient,
It may vary depending on weight, symptoms, etc., but usually 4 x 10'
~ 160 x 10' units/υ/body weight/[3, preferably 16 x 10' to -180 x 10' units/υ/body weight 7 days.

［実施例］ヒトＭ−Ｃ３Ｆを使用した本発明の実施例を次に示す。[Example] An example of the present invention using human M-C3F is shown below.

Ｃ８Ｆ活性の安定性及びヒト単球のｔ！瘍細胞殺傷作用
について検討した。ヒト尿由来のヒｌ−Ｍ−・Ｃ８Ｆの
安定性はＭ−Ｃ３Ｆ活性をマウス骨髄細胞を用いた軟寒
天法にて測定した。又と１・単球の１ｉｊｉ瘍細胞殺伽
作用についての検討は安定性試験において経時的に安定
であった抗悪性腫瘍剤にで実施した。Stability of C8F activity and t! of human monocytes. The tumor cell killing effect was investigated. The stability of human urine-derived human l-M-.C8F was determined by measuring M-C3F activity using a soft agar method using mouse bone marrow cells. The tumor cell-killing effect of Matato 1 and monocytes was investigated using anti-malignant tumor agents that were stable over time in stability tests.

抗悪性腫瘍剤の安定性は表１に示す如く、ヒト血清アル
ブミン及びゼラチンを１勺／−以上添加された製剤の生
物活性は試Ｍ開始時の７０％以上保持されており安定で
あった。As shown in Table 1, the stability of the antineoplastic agent was stable, with the biological activity of the preparation to which human serum albumin and gelatin added at least 1 g/min was maintained at 70% or more of the level at the start of test M.

ヒト尿由来のヒトＭ−Ｃ８Ｆをｐｌ＋７．２の２０１Ｉ
Ｈリン酸緩衝液に、表１に丞す蛋白質を添加すると共に
ヒト尿由来のヒトＭ−Ｃ３Ｆを１０μｇＺｔｄｔｍ度に
調製した。ニドＯセルロース系無ｉＩｉ濾過膜にて無菌
濾過しガラスバイアル中に無菌的に１ｊｌ！充填、密封
して抗悪性腫瘍剤を製造した。Human M-C8F derived from human urine was 201I at pl+7.2.
The proteins shown in Table 1 were added to H phosphate buffer, and human M-C3F derived from human urine was prepared at 10 μg Ztdtm. Filter aseptically with Nido-O cellulose-free filtration membrane and aseptically place 1 jl in a glass vial! The container was filled and sealed to produce an anti-malignant tumor agent.

これらの溶液製剤に゛ついてヒト尿由来のヒトＭ−表１即ち、ヒト尿由来のヒトＭ−Ｃ８Ｆに対する上記の蛋白
質添加紹を１００倍以上とすることで安定性が付与され
ることが知れる。It is known that stability can be imparted to these solution preparations by increasing the amount of the above-mentioned protein added to human M-C8F derived from human urine by 100 times or more.

抗悪性ｔｉｒｉ瘍剤のヒト単球の腫瘍細胞段山作用に及
ぼす作用は下記の方法にして測定した。使用した抗悪性
８ｍ剤は、ヒト血清アルブミンの添加聞を５．Ｏｑ／−
としたものを用いた。すなわち正常ヒト末梢血より、Ｆ
ｉｃｏｌｌ１層遠心法（比重１．０７７＞により単核球
を分離した後、プラスチック何者法により半球を得た。The effect of the anti-tiricancer agent on the tumor cell cascading effect of human monocytes was measured by the following method. The anti-malignant 8m agent used was added with human serum albumin at a rate of 5. Oq/-
The following was used. That is, from normal human peripheral blood, F
After separating mononuclear cells by the icoll single-layer centrifugation method (specific gravity 1.077), hemispheres were obtained by the plastic method.

（純度９０％以上）。分離した単球（１０シ／ｌ１１）
にヒｊ・尿由来のヒトＭ−Ｃ８Ｆ添加聞として２５．５
０及び１５０ｎＧ／ｄの１１１１１で抗悪性腫瘍剤を添
加し、培養液中で３７℃、２日間培養後洗浄し、ヒトＩ
ｊ球を調製し、９６穴マイクロプレートに１０６／Ｊ！
１！の［１Ｉｌｌ！数を播種した。次に５１Ｃｒ標識し
たヒト骨髄性白血病細胞（Ｋ−５６２１１胞＞１０５／
ｄを１００μ！播種し培養する。培１１１２時間後に培
養」１滴の１１１１５１Ｃｒの放射活性を測定し［１１
１１１１胞障害活性を算出１）だ。算出方法はＦ記の通
りである。(Purity 90% or more). Isolated monocytes (10 sh/l11)
25.5 as the addition of human M-C8F derived from human urine.
Antineoplastic agents were added at 0 and 150 nG/d of 11111, cultured in culture medium at 37°C for 2 days, washed, and human I
Prepare J spheres and place them in a 96-well microplate at 106/J!
1! [1Ill! A number of seeds were sown. Next, 51Cr-labeled human myeloid leukemia cells (K-56211 cells>105/
d is 100μ! Seed and culture. After 1112 hours of culture, the radioactivity of one drop of 111151Cr was measured [11
The 1111 cell-damaging activity was calculated 1). The calculation method is as described in F.

１ｆＩＩ１１５１Ｃｒ放射活性全”ＣｒＭＱｌｉ性　”　””””””障害°活性（゛
）その結果第１図に示される如く、１１腫瘍剤存在下に
て培養した単球は不存右下のものと比較し、そのｌＩ！
瘍細胞障害活性は有意に増強した。又、この効果は抗悪
性腫瘍剤の有効成分であるヒト尿由来のヒトＭ−Ｃ８Ｆ
の濃度に依存していた。1fII1151Cr radioactivity total "CrMQli activity""""""" impaired activity (゛) As a result, as shown in Figure 1, monocytes cultured in the presence of tumor agent 11 were compared with those in the lower right without tumor agent. And that lI!
Tumor cytotoxic activity was significantly enhanced. Moreover, this effect is due to human M-C8F derived from human urine, which is an active ingredient of an anti-cancer drug.
depended on the concentration of

尚、第１図は、腫瘍細胞を完全に殺傷した場合を１００
として、同時に行なった２つの実験結果の平均および標
準偏差を示した。In addition, Figure 1 shows the case where tumor cells are completely killed.
The average and standard deviation of the results of two experiments conducted simultaneously are shown.

蛋白質が添加されたと１・尿由来のヒトＭ−Ｃ３Ｆ１１
度１０μｇ／−の実施例−１と同一の溶液を：ｊＡ製し
た。ニトロセルロース系無菌濾過膜にてこの溶液を無菌
濾過しガラスバイアル中に無菌的に１−充填し、凍結乾
燥後′ｆ！封してＩＡ悪性腫瘍剤を製造した。これらの
乾燥製剤についてヒト尿由来のヒｌ−Ｍ　−ＣＳ　Ｆ活
性の経時的安定性及びヒト単球の抗体依存性Ｉ！瘍ａ胞
障害活性（ＡＤＣＧ）に及ぼす抗悪性１１１ｍ剤の効果
を検討した。安定性試醗は実施例１と同様にマウス骨髄
細胞を用いた軟寒天法にて測定した。又、ヒトｗ球の抗
体依存性Ｉｌ！瘍ｕｒｎ殺傷作用についての検討は安定
性試験において経時的に安定であった抗悪性腫瘍剤にて
実施した。1. Urine-derived human M-C3F11 with added protein
The same solution as in Example-1 with a concentration of 10 μg/- was prepared by:jA. This solution was aseptically filtered using a nitrocellulose-based sterile filtration membrane, filled aseptically into a glass vial, and after freeze-drying, 'f! The container was sealed to produce an IA malignant tumor agent. Regarding these dry preparations, the temporal stability of human urine-derived human I-M-CSF activity and the antibody dependence of human monocytes were investigated. The effect of anti-malignant 111m agent on tumor cytotoxic activity (ADCG) was investigated. The stability test was carried out in the same manner as in Example 1 by the soft agar method using mouse bone marrow cells. In addition, the antibody-dependent Il! of human W bulbs The tumor-killing effect was investigated using anti-malignant tumor agents that were stable over time in stability tests.

抗悪性腫瘍剤の安定性は表２に示す如く、ヒト面清アル
ブミン及びピラチンを１１１９／Ｉｕ１以上添加された
製剤の生物活性は試験開始時の７０％以上保持されてお
り安定であった。As shown in Table 2, the stability of the antineoplastic agent was stable, with the biological activity of the preparation to which 1119/Iu1 or more of human serum albumin and pyratin were added retained at least 70% of the level at the start of the test.

即ちヒト尿由来のヒトＭ−Ｃ８Ｆに対する上記の蛋白質
添加量を１００倍以上とすることで安定性が付与される
ことが知れる。That is, it is known that stability can be imparted by increasing the amount of the above-mentioned protein added to human M-C8F derived from human urine by 100 times or more.

表２抗悪性腫瘍剤の、ヒト単球の抗体依存性腫瘍細胞殺傷活
性（ＡＤＣＣ＞に及ぼす効果を検討した。Table 2 The effects of antineoplastic agents on antibody-dependent tumor cell killing activity (ADCC) of human monocytes were investigated.

使用した抗悪性腫瘍剤は、ゼラチンの添加ｍを５．０１
１！Ｊ／ｄとしたものである。実施例−１と同一の実施
要領でヒト単球に抗悪性ｌｌｌ１ｌｌ剤を添加し培養液
中で２日間培１１４１１洗浄しヒト単球を調製し、９６
穴マイクロプレートに１０６／−を１００μオ播種した
。次に５１Ｃｒ標識したヒトリンパ腫由来細胞（Ｒａｊ
ｉ細胞）及び該細胞特異抗体を１μ９／ａｔ及び１０μ
９／ｄ添加し培養した。培養１２時囚後に培養上清の遊
離５１ｃｒの放射活性を測定し紗ａ！細胞障害活性を算
出した。算出方法は実施例−１の通りである。The anti-malignant tumor agent used had a gelatin addition m of 5.01
1! J/d. Human monocytes were prepared by adding an anti-malignant llllll agent to human monocytes in the same manner as in Example-1, and washing the culture medium with 11411 for 2 days in a culture solution.
100 µm of 106/- were seeded in a well microplate. Next, 51Cr-labeled human lymphoma-derived cells (Raj
i cells) and the cell-specific antibodies at 1μ9/at and 10μ
9/d was added and cultured. After 12 hours of incubation, the radioactivity of free 51cr in the culture supernatant was measured. Cytotoxic activity was calculated. The calculation method is as in Example-1.

その結果第２図に示される如く、抗悪性？Ｉ！瘍剤及び
腫瘍Ｓ＋胞特異抗体存在下に培養した１１球は、不存在
下のものと比較して、ｓｓｍ胞に対するＡＤＣＣ活性が
有意に増強した。又、この増強効果は抗悪性ｍ瘍剤の有
効成分であるヒト尿由来のヒトＭ−Ｃ８Ｆ及び該ｍ胞特
異抗体の濃度に依存していた。As shown in Figure 2, the results show that it has anti-malignant properties? I! The 11 cells cultured in the presence of tumor agent and tumor S+ cell-specific antibody showed significantly enhanced ADCC activity against ssm cells compared to those in the absence. Moreover, this enhancing effect depended on the concentration of human M-C8F derived from human urine, which is an active ingredient of the anti-malignant tumor drug, and the m cell-specific antibody.

尚、第２図は、Ｒａｊ　ｉ細胞を完全に殺傷した場合を
１００として、同時に行なった２つの実験結果の平均お
よび標準偏差を示した。In addition, FIG. 2 shows the average and standard deviation of the results of two experiments conducted simultaneously, with the case where Raji cells were completely killed as 100.

実施例−３、ヒト子　頚　　の　　に　ぼす表３　抗悪
性腫瘍剤の抗腫瘍活性ヒト子宮頚部１（Ｈｅｌａ細胞）を３匹のＢａ１ｂ／ｃ
ヌードマウスの皮十に１０６個移植した。移植翌日より
実施例−１で使用した抗Ｒ竹ｌ！ｌｉ瘍剤をそれぞれの
ヌードマウスに対し４０Ｘ１０’単位／に９・体重、１
６０Ｘ１０４単位／に９・体重、６４０×１０４単位／
Ｋｇ・体重を７日間連続腹腔内に連続投与した。Ｈｅ１
ａＩＩＩＩａ移植後２８日目にＩＩ！瘍細脳細胞出し、
腫瘍重石を測定し、抗鮭瘍活性を測定した。Example 3, Table 3: Antitumor activity of antineoplastic agent on human cervix Human cervix 1 (Hela cells) was administered to three Ba1b/c mice.
106 cells were transplanted into the skin of nude mice. Anti-R bamboo l used in Example-1 from the day after transplantation! The tumor drug was added to each nude mouse at 40 x 10' units/9.body weight, 1
60 x 104 units/9・Weight, 640 x 104 units/
Kg/body weight was continuously administered intraperitoneally for 7 days. He1
II on the 28th day after aIIIa transplantation! Tumor brain cells come out,
Tumor weight was measured and anti-salmon tumor activity was determined.

表３に示される如く抗悪性！！瘍剤１６０×１０４里位
／υ・体重以上の投与において、明らかに腫瘍縮小が認
められ、抗腫瘍活性を示した。As shown in Table 3, it has anti-malignant properties! ! When the tumor drug was administered at a dose of 160 x 104 li/υ body weight or more, tumor shrinkage was clearly observed, indicating antitumor activity.

実験例−１ヒト単球上のＦｃレセプターの発現に及ぼす
ヒトＭ−Ｃ８Ｆの効果実施例−１の方法にて調製したと１〜里球を抗悪性１ｉ
ｆｔ瘍剤存在下或は不存在下にて２日間培養後、ＩＱ−
Ｇを結合させたヒツジ赤血球と混合した。Experimental Example-1 Effect of human M-C8F on the expression of Fc receptor on human monocytes.
After culturing for 2 days in the presence or absence of ft tumor agents, IQ-
Mixed with G-conjugated sheep red blood cells.

良く洗浄した後、単球に結合した赤血球数を測定し、Ｆ
ｃレセプターの発現を判定した。After thorough washing, the number of red blood cells bound to monocytes was measured, and F
c receptor expression was determined.

表４に示される如く、抗悪性腫瘍剤存在下に培養した単
球には明らかに多くの赤血球が結合していた。As shown in Table 4, many red blood cells were clearly bound to monocytes cultured in the presence of the antineoplastic agent.

実施例−２の結果より、ヒト尿由来のヒトＭ−Ｃ８Ｆが
ヒト単球のＡＤＣＣ活性を増強することが明らかにされ
たが、この機構については、本実験例の結果より抗悪性
腫瘍剤により単球上のＦｃレセプター発現がＡ進し、恕
性ｌＩ！瘍特異抗体を介した単球と標的ｉ！瘍細胞との
結合が強くなったことによると考えられた。The results of Example 2 revealed that human M-C8F derived from human urine enhances the ADCC activity of human monocytes. Fc receptor expression on monocytes progresses to A stage and becomes agonistic II! Monocytes and targets i! through tumor-specific antibodies! This was thought to be due to stronger binding to tumor cells.

表４　ヒト単球上のＦｃレヒプター発現におけるヒト尿
由来のヒトＭ−Ｃ８Ｆの効果尚、測定数は、少なくとも１００個の単球について、そ
れに結合した赤血球を数え、その平均と標準１１；ｉ差
を示した。Table 4 Effect of human M-C8F derived from human urine on Fc receptor expression on human monocytes The measured number is the difference between the average and standard 11; showed that.

［発明の効果］（１）　　ヒト尿由来のヒトＭ−Ｃ８Ｆは顕著な抗ＩＦ
Ｉｉ瘍活性を示す。従ってヒト尿由来のヒトＭ−Ｃ８Ｆ
をｈ効成分とすることにより副作用のない抗悪性腫瘍剤
を提供することができる。[Effects of the invention] (1) Human M-C8F derived from human urine has significant anti-IF
Ii indicates tumor activity. Therefore, human M-C8F derived from human urine
By using H as an active ingredient, an anti-malignant tumor agent without side effects can be provided.

■　安定剤としてヒト血清アルブミン及び／又はゼラチ
ンを添加することによりヒト尿由来のヒトＭ−Ｃ８Ｆの
安定性が向上する。従って、ヒト尿由来のヒトＭ−Ｃ８
Ｆにヒト血清アルブミン及び／又はゼラチンを加えるこ
とにより、長期間にｎつで生物活性の安定な抗悪性１ｌ
ｉＳ剤を提供することができる。(2) The stability of human M-C8F derived from human urine is improved by adding human serum albumin and/or gelatin as a stabilizer. Therefore, human M-C8 derived from human urine
By adding human serum albumin and/or gelatin to F, a stable anti-malignant agent with long-term biological activity can be obtained.
iS agents can be provided.

（３）　　ヒト尿由来のヒトＭ−Ｃ８Ｆとともに悪例腫
瘍に対する特異抗体を併用することにより効果的な抗悪
性１１１ｍ剤を提供できる。(3) An effective anti-malignant 111m agent can be provided by combining human M-C8F derived from human urine with a specific antibody against a bad tumor.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図は、ヒト単球の腫瘍細胞殺傷活性に対するヒト尿
由来のヒトＭ−Ｃ８Ｆの作用を示すグラフである。第２図は、ヒト単球の抗体依存型腫瘍細胞殺傷活性に対
するヒト尿由来のヒトＭ−Ｃ８Ｆの作用を示すグラフで
ある。FIG. 1 is a graph showing the effect of human urine-derived human M-C8F on tumor cell killing activity of human monocytes. FIG. 2 is a graph showing the effect of human M-C8F derived from human urine on the antibody-dependent tumor cell killing activity of human monocytes.

Claims

【特許請求の範囲】[Claims]

（１）ヒト尿由来のヒト単球−マクロファージコロニー
刺激因子を有効成分とする抗悪性腫瘍剤。(1) An anti-malignant tumor agent containing human monocyte-macrophage colony stimulating factor derived from human urine as an active ingredient.