JP2697725B2 - Malignant tumor treatment kit - Google Patents

Description

【発明の詳細な説明】 【０００１】 【発明の属する技術分野】本発明はヒト顆粒球コロニー
刺激因子（以下ヒトＧ−ＣＳＦと略記する）を有効成分
とする製剤と抗悪性腫瘍血清との組み合わせからなる悪
性腫瘍治療用キットに関する。 【０００２】 【従来の技術】本発明は造血因子（ｈｅｍｏｐｏｉｅｔ
ｉｃ ｇｒｏｗｔｈ ｆａｃｔｏｒ）の１つであるヒト
Ｇ−ＣＳＦを用いて、悪性腫瘍の治療に役立てようとす
るものであって、直接これに関連する報告類の見当たら
ない新規な悪性腫瘍治療用キットを提供しようとするも
のである。ヒトＧ−ＣＳＦはｉｎ Ｖｉｔｒｏの実験系
において顆粒球の前駆細胞に働き顆粒球への分化増進を
促す機能を有している造血因子である〔例えばＭｅｔｃ
ａｌｆ．ｅｔ．ａｌ：Ｅｘｐ．Ｈｅｍａｔｏｌ．１，１
８５，（１９７３）等参照〕。ところがこのヒトＧ−Ｃ
ＳＦは今迄入手するのが極めて困難であったため、医薬
としての有用性又は有効性についての検討が充分進展せ
ず、本発明が目的とするガンの治療への可能性について
も未検討のままに置かれていた。 【０００３】 【発明が解決しようとする課題】この様な状況を打開す
べく、本出願人は研究を重ねた結果、遺伝子工学等によ
るヒトＧ−ＣＳＦの製造方法の開発に成功し、純粋均質
でしかも大量のヒトＧ−ＣＳＦが入手できるようになっ
た（特願昭５９−１５３２７３号、特願昭６０−２６９
４５５号、特願昭６０−２６９４５６号、特願昭６０−
２７０８３８号、特願昭６０−２７０８３９号等参
照）。この成果をふまえて、殺悪性腫瘍作用を有するヒ
ト成熟顆粒球に対するヒトＧ−ＣＳＦの影響等を研究
し、そこで得られた知見にもとづいて副作用の少ない優
れた悪性腫瘍治療用キットを開発しようとするのが本発
明の目的である。 【０００４】 【課題を解決するための手段】本発明者らは上記目的を
達成するため、鋭意研究を重ねた結果、悪性腫瘍細胞例
えばヒト悪性黒色腫細胞（ＨＭＶ）において、 ヒトＧ−ＣＳＦ存在下で前孵置した顆粒球は、非存在
下で前孵置した顆粒球に比べて約２〜４倍の殺ＨＭＶ効
果が認められた。 殺ＨＭＶ効果は正常ウサギ血清の存在下で影響されな
いが、抗ＨＭＶ血清の存在下ではその濃度に依存して最
高１０〜３０倍に増強した。 ヒトＧ−ＣＳＦとの前孵置による顆粒球の殺ＨＭＶ効
果の増強の程度は、ヒトＧ−ＣＳＦの濃度と明らかな相
関がある。 という事実を確認し、この結果からヒトＧ−ＣＳＦはヒ
ト成熟顆粒球（特に好中球）の殺悪性腫瘍作用を抗悪性
腫瘍血清存在下で著しく亢進させることを見出し本発明
に到達した。即ち、本発明は抗ヒト悪性腫瘍血清とヒト
顆粒球コロニー刺激因子を有効性分とする製剤の組み合
わせからなる悪性腫瘍治療用キットを提供するものであ
る。 【０００５】以下本発明を詳細に説明する。本発明のＧ
−ＣＳＦを有効性分とする製剤に含まれるヒトＧ−ＣＳ
Ｆは純度の高いヒトＧ−ＣＳＦであればその由来が制限
されるものではなく、例えば人の生体試料から抽出、分
離、精製したもの、ヒトＧ−ＣＳＦ産生細胞を培養し、
その培養上清から単離したもの、細胞融合法を用いてヒ
トＧ−ＣＳＦ産生ハイブリドーマを形成しこれから取得
したもの、遺伝子組換えによって、大腸菌、動物細胞等
の宿主を形質転換して得た形質転換体から産生せしめ単
離精製したもの、又はそれを化学修飾したもの等のいず
れも使用することができる。 【０００６】しかし、それらの中でも純度よく均質大量
に入手できる本出願人が製造した次の（１）及び（２）
で示すヒトＧ−ＣＳＦが特に好ましいものである。 （１）次の理化学的性質を有するヒトＧ−ＣＳＦ。 分子量：ドデシル硫酸ナトリウム−ポリアクリルアミ
ドゲル電気泳動法による測定で約１９，０００±１，０
００。 等電点：ｐＩ＝５．５±０．１，ｐＩ＝５．８±０．
１，ｐＩ＝６．１±０．１の三つの等電点のうち少なく
とも１つを有する。 紫外部吸収：２８０ｎｍに極大吸収を有し、２５０ｎ
ｍに極小値をもつ。 Ｎ末端から２１残基目迄のアミノ酸配列が次の如くで
ある。 Ｈ₂ Ｎ−Ｔｈｒ−Ｐｒｏ−Ｌｅｕ−Ｇｌｙ−Ｐｒｏ−Ａ
ｌａ−Ｓｅｒ−Ｓｅｒ−Ｌｅｕ−Ｐｒｏ−Ｇｌｎ−Ｓｅ
ｒ−Ｐｈｅ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｃｙｓ−Ｌｅｕ
−Ｇｌｕ−Ｇｌｎ−Ｖａｌ− 【０００７】（２）配列番号１、２、３又は４のアミノ
酸配列またはその一部で表わされるヒト顆粒球コロニー
刺激因子活性を有するポリペプチド又はこれと糖鎖部を
有する糖蛋白質を含有するヒトＧ−ＣＳＦ。上記のヒト
Ｇ−ＣＳＦは例えば後述する参考例に示す方法によって
製造することができる。即ち、上記（１）のヒトＧ−Ｃ
ＳＦは参考例１によって、又（２）のヒトＧ−ＣＳＦは
参考例２に示す方法により得ることができる。なおこれ
らの方法の詳細な製造条件については、本出願人が先に
出願した特願昭５９−１５３２７３号，特願昭６０−２
６９４５５号，特願昭６０−２６９４５６号，特願昭６
０−２７０８３８号，特願昭６０−２７０８３９号の各
明細書を参照されたい。 【０００８】又、その他の方法としてＧ−ＣＳＦ産生細
胞と自己増殖能を有する悪性腫瘍細胞とを細胞融合して
得られるハイブリドーマをマイトジェンの存在または非
存在下で培養することによって得ることもできる。これ
等の方法で得たヒトＧ−ＣＳＦは全て本発明に含まれ
る。得られたヒトＧ−ＣＳＦ含有液は必要により公知の
手段でさらに精製、濃縮した後凍結保存とするかまたは
凍結乾燥、真空乾燥などの手段により水分を除去して保
存することができる。また所望によりヒトＧ−ＣＳＦを
適当な緩衝液に溶解した後、ミリポアフィルター等で無
菌濾過して注射剤とすることもできる。ヒトＧ−ＣＳＦ
を有効性分とする製剤はヒトまたは動物医薬用に適した
医薬製剤としての形態をとるために必要な製薬担体や賦
形剤を、さらには安定化剤、吸着防止剤を含むことがで
きる。 【０００９】次に本発明の悪性腫瘍治療用キットについ
て説明する。後述する実験例（薬理効果）から明らかな
通り、ヒトＧ−ＣＳＦは抗悪性腫瘍血清の存在下で著し
く顆粒球の殺悪性腫瘍作用を増大する。これは、該抗血
清が顆粒球の腫瘍細胞認識力を高め、それへのとりつき
を容易にする結果、顆粒球（特に好中球）の殺悪性腫瘍
作用の効果が一段と向上をするためと推定される。この
作用効果をガン治療に活用すべく開発したのが抗悪性腫
瘍血清とヒトＧ−ＣＳＦを有効成分とする製剤の組み合
わせからなる本発明の悪性腫瘍治療用キットである。 【００１０】ヒトＧ−ＣＳＦを有効成分とする製剤に含
まれるヒトＧ−ＣＳＦの投与量、投与回数は対象の疾患
患者の病状を配慮して決めることができるが、通常成人
一人当たり０．１〜５００μｇ、好ましくは０．５〜２
００μｇのヒトＧ−ＣＳＦを含有する製剤を１週間に１
〜７回投与することができる。しかし本発明はヒトＧ−
ＣＳＦの含有量によって限定されるものではない。本キ
ットで用いられる抗悪性腫瘍血清は公知の方法で調製さ
れたものでよく、その投与量は通常成人一人当たり０．
１〜５ｃｃである。又、該抗悪性腫瘍血清の投与方法と
しては、本発明の抗悪性腫瘍剤投与により顆粒球が増加
し、それがピークとなる約１時間前頃に静脈注射等の注
射によって投与するのがよい。 【００１１】 【発明の実施の形態】以下本発明を参考例（ヒトＧ−Ｃ
ＳＦの製造例）実験例（薬理効果）、実施例（製剤例）
をあげて説明するが、本発明はこれらに限定されるもの
ではない。なお、抗腫瘍血清不存在下の結果を参考とし
て併記した。 【００１２】参考例１ （Ｇ−ＣＳＦ産生細胞の培養に
よるヒトＧ−ＣＳＦの製造例） 特願昭５９−１５３２７３号の実施例１に示す方法で樹
立したヒト口腔底癌細胞由来のＧ−ＣＳＦ産生細胞株Ｃ
ＨＵ−１（Ｃ．Ｎ．Ｃ．Ｍ受託番号「Ｉ−３１５」）ま
たは同様の方法で樹立した細胞株ＣＨＵ−２（Ｃ．Ｎ．
Ｃ．Ｍ受託番号「Ｉ−４８３」）をウシ胎児血清を含有
するＲＰＭＩ １６４０培養液に浮遊した後、ローラー
ボトルにいれて回転培養を行った。細胞がローラーボト
ル内壁に密に増殖したところで培養液を血清不含ＲＰＭ
Ｉ １６４０にかえ、４日間培養後上清を回収。次いで
血清含有ＲＰＭＩ １６４０を加えて３日間培養した
後、培養液を血清不含ＲＰＭＩ １６４０に液替し、４
日間培養後上清を回収する。以下これを繰返し培養上清
を回収した。得られた血清不含培養上清を限界ろ過で約
１０００倍に濃縮し精製、次いで検定を行った。精製及
び検定は前記特願昭５９−１５３２７３号明細書の実施
例と同じ方法で行った。 【００１３】参考例２ （遺伝子組換えによるヒトＧ−
ＣＳＦの製造例） 本出願人によって微工研に寄託されているエシエリヒア
・コリ（Ｅ．Ｃｏｌｉ）χ１７７６Ｒ−２株（ＦＥＲＭ
ＢＰ−９５５）から切り出してきたヒトＧ−ＣＳＦ遺
伝子を有するｃＤＮＡ断片をベクターｐｄＫＣＲに組み
込みｐＨＧＶ２プラスミドとした後これをＳａｌＩで処
理し、次いでＤＮＡポリメラーゼ−Ｋｌｅｎｏｗ断片を
反応させる。このＤＮＡにＥｃｏＲＩリンカーを付加
し、再びＥｃｏＲＩで部分消化した後、アガロースゲル
電気泳動にて約２．７Ｋｂのフラグメントを回収する。 【００１４】一方、ｐＡｄＤ２６ＳＶｐＡプラスミド
（Ｋａｕｆｍａｎ，Ｒ．Ｇ．＆ Ｓｈａｒｐ，Ｐ，Ａ．
（１９８２）Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ，２巻１３０
４〜１３１９）をＥｃｏＲＩで処理し、ＢＡＰ処理し、
脱リン酸する。次でフェノール処理後電気泳動でｐＡｄ
Ｄ２６ＳＶｐＡのＥｃｏＲＩ断片を回収した。上記の
２．７ｋｂ断片とｐＡｄＤ２６ＳＶｐＡ断片をアニール
し、Ｅ．ｃｏｌｉ ＤＨＩ株に塩化ルビジウム法により
形質転換してｐＨＧＶ２−ｄｈｆｒプラスミドを得た。 【００１５】つぎにＣＨＯ細胞（ｄｈｆｒ⁻株、コロン
ビア大学Ｄｒ．Ｌ．Ｃｈａｓｉｎより入手）を９ｃｍ径
のプレート（Ｎｕｎｃ社製）中１０％仔牛血清を含むα
最小必須培地（α−ＭＥＭ，アデノシン、デオキシアデ
ノシン、チミジン添加）で培養増殖し、これをリン酸−
カルシウム法（Ｗｉｇｌｅｒ等、Ｃｅｌｌ１４巻７２５
頁（１９７８））によって形質転換した。即ち、前記の
ｐＨＧＶ２−ｄｈｆｒプラスミド１μｇにキヤリア−Ｄ
ＮＡ（子牛胸線ＤＮＡ）を適量加えて、ＴＥ溶液３７５
μｌに溶解し１Ｍ ＣａＣｌ₂ １２５μｌを加える。３
〜５分氷上で冷やし５００μｌの２×ＨＢＳ（５０ｍＭ
Ｈｅｐｅｓ、２８０ｍＭ ＮａＣｌ、１．５ｍＭリン酸
緩衝液）を加え再び氷冷後、上記のＣＨＯ細胞培養液１
ｍｌと混合し、プレートに移し、ＣＯ₂ インキュベータ
ー中で９時間培養する。 【００１６】以下洗浄、２０％グリセロール含有ＴＢＳ
（Ｔｒｉｓ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ）添加、
再び洗浄した後非選択培地（前出α−ＭＥＭ培地、ヌク
レオシド添加）を添加して２日間インキュベートし、選
択培地で１：１０に細胞を分割した。次いで２日毎に選
択培地（ヌクレオシド無添加）にて培地交換を行いなが
ら培養を続行し生じた集塊（ｆｏｃｉ）を選別して新し
いプレートに移した。新しいプレートでは０．０２μＭ
メトトレキセート（ＭＴＸ）存在下で増殖し再び０．１
μＭ ＭＴＸ存在下で増殖させてクローニングを行っ
た。更にクローニングを続けた結果１０ｍｇ／ｌ以上の
ヒトＧ−ＣＳＦの生産を確認した。なお、精製、検定は
特願昭６０−２６９４５６号明細書の実施例２及び１５
の記載の方法によって行った。 【００１７】実験例１ 〔顆粒球の殺黒色腫細胞（ＨＭＶ）効果と添加抗ＨＭＶ
血清及びヒトＧ−ＣＳＦ濃度変動の影響〕ｄｅｘｔｒａ
ｎ沈降法、Ｆｉｃｏｌｌ−Ｈｙｐａｑｕｅ（１．０７
７）重層遠心法、低張ショック赤血球除去法を用いて、
健常者末梢血顆粒球を分離した（純度及び生細胞率９０
％以上）。分離された細胞に種々の濃度のヒトＧ−ＣＳ
Ｆを添加し培養液中で３７℃、１時間インキュベートし
た後、洗浄し、ヒト成熟顆粒球の調製をした。 【００１８】次に一定数のヒト悪性黒色腫細胞株（ＨＭ
Ｖ）をマイクロプレート中で培養し、生細胞が充分にウ
エル底に付着したところで表１に示す各濃度のウサギ正
常血清又は抗ＨＭＶ血清と上記の調製済みの一定数の成
熟顆粒球を添加し、３７℃，２４時間培養した。その後
各ウエルを培養液で洗浄し、付着して生存している細胞
の数を染色後算定した。結果を表１に示す。 【００１９】表１から明らかな通り、ヒトＧ−ＣＳＦを
添加し調製した顆粒球は無添加の場合に比べ約２〜４倍
の殺ＨＭＶ効果が認められ、且つ抗ＨＭＶ血清を添加す
るとその傾向がいっそう顕著となる。又、ヒトＧ−ＣＳ
Ｆとのインキュベートによる顆粒球の殺ＨＭＶ効果の増
強の程度にはヒトＧ−ＣＳＦの濃度と明らかな相関がみ
られる。これらの結果はヒトＧ−ＣＳＦがヒト成熟顆粒
球の殺ＨＭＶ作用を著しく亢進することを明らかにして
いる。 【００２０】 【表１】【００２１】実験例２ 〔顆粒球の殺黒色腫細胞効果と添加ヒトＧ−ＣＳＦ及び
抗ＨＭＶ血清濃度変動の影響〕一定数のＨＭＶをマイク
ロプレート中で培養し、生細胞が充分にウエル底に付着
したところで表２に示す各濃度の抗ＨＭＶ血清又は正常
血清を添加し、次で実験例１と同様にして調製した活性
化顆粒球又は非活性化顆粒球を加え、３７℃，２４時間
インキュベートした。その後各ウエルを培養液で洗浄し
付着生存している細胞の数を染色後算定した。結果を表
２に示す。 【００２２】表２より明らかな通り、殺ＨＭＶ効果は正
常血清の存在下では影響されないが、抗ＨＭＶ血清の存
在下ではその濃度に依存して最高約30倍に増強される。
又、実験例１の結果と同様にヒトＧ−ＣＳＦで活性化さ
れた顆粒球はヒトＧ−ＣＳＦを用いた非活性の顆粒球に
比べ殺ＨＭＶ効果が正常血清添加下でも２倍以上優れて
おり、この差は抗ＨＭＶ血清存在下ではより顕著になる
ことがわかる。これらの結果はヒトＧ−ＣＳＦが抗ＨＭ
Ｖ血清存在下でヒト成熟顆粒球の殺ＨＭＶ作用を顕著に
亢進せしめる働きがあることを示している。 【００２３】 【表２】 【００２４】実験例３ 〔ヒトＧ−ＣＳＦの有無と顆粒球又はリンパ球の各種腫
瘍細胞に対する殺効果〕実験例１と同様にして健常者末
梢血顆粒球及びリンパ球を分離し、ヒトＧ−ＣＳＦを添
加後インキュベートし、活性化させた。コントロールと
してはヒトＧ−ＣＳＦを添加せずに同様にインキュベー
トしたものを用意した。一方、表３に示す各腫瘍細胞に
上記の活性化顆粒球又は非活性化顆粒球もしくはリンパ
球を加え、インキュベートし、各種腫瘍細胞のＤＮＡ合
成を［ ³Ｈ］サイミジンの取り込み率でみた。結果を表
３に示す。表３より明らかな通り、ＨＭＶだけでなく他
の腫瘍細胞に対してもヒトＧ−ＣＳＦは成熟顆粒球の殺
腫瘍細胞作用を亢進させることが認められる。一方リン
パ球には顆粒球のような効果は認められない。 【００２５】 【表３】 【００２６】上記の実験の結果をまとめると前述した通
り、 ヒトＧ−ＣＳＦ存在下で前孵置した顆粒球は、非存在
下で孵置した顆粒球に比べて約２〜４倍の殺ＨＭＶ効果
が認められた。 殺ＨＭＶ効果は正常ウサギ血清の存在下で影響されな
いが、抗ＨＭＶ血清の存在下ではその濃度に依存して最
高１０〜３０倍に増強した。 ヒトＧ−ＣＳＦとの前孵置による顆粒球の殺ＨＭＶ効
果の増強の程度は、ヒトＧ−ＣＳＦの濃度と明らかな相
関があった。 上記の傾向はＨＭＶ以外の腫瘍細胞に対しても同様に
認められた。したがって、ヒトＧ−ＣＳＦはヒト成熟顆
粒球の殺腫瘍細胞作用を抗腫瘍血清存在下で著しく亢進
させることが確認された。 【００２７】 【実施例】本発明は以下の実施例（製剤例）で得られた
ヒトＧ−ＣＳＦを含有する製剤と抗ヒト悪性腫瘍血清の
製剤を組み合わせてキットとするものであるが、本発明
はこれらの組み合わせに限定されるものではない。 【００２８】実施例１（製剤例） 参考例１によって得られ且つ精製されたヒトＧ−ＣＳＦ
を無菌処理した後−２０℃で凍結された凍結物を用いて
注射剤とした。 【００２９】実施例２（製剤例） 参考例２によって得られ且つ精製されたヒトＧ−ＣＳＦ
を無菌操作で１０ｍｌバイアル瓶に５ｍｌ充填し、−２
０℃で凍結乾燥後ゴム栓にて施栓した凍結乾燥物を用い
て注射剤とした。 【００３０】実施例３（製剤例） ＨＭＶ細胞をウサギに免疫して得られた抗ＨＭＶ血清を
フィルター滅菌処理した後−２０℃で凍結させた凍結物
を用いて注射剤とした。 【００３１】 【発明の効果】本発明の悪性腫瘍治療用キットは人の体
にもともと存在しているヒト成熟顆粒球の抗腫瘍作用を
純化されたヒトＧ−ＣＳＦによって増強し、且つ抗腫瘍
細胞血清を併存せしめることによって、さらに一段と増
強させ、この作用にもとづいて悪性腫瘍を治療しようと
するものであり、副作用の少ないガン治療剤となる期待
が大きい。 【００３２】 【配列表】 配列番号：１ 配列の長さ：１７４ 配列の形：アミノ酸 鎖の数： トロポジー：直鎖状 配列の種類：ペプチド 配列 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 48 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 64 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 96 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 112 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 128 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 144 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 174 【００３３】配列番号：２ 配列の長さ：１７５ 配列の形：アミノ酸 鎖の数： トロポジー：直鎖状 配列の種類：ペプチド 配列 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 16 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 32 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 48 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 64 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 96 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 112 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 128 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 144 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 175 【００３４】配列番号：３ 配列の長さ：１７７ 配列の形：アミノ酸 鎖の数： トロポジー：直鎖状 配列の種類：ペプチド 配列 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32 Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu 48 Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu 64 Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln 80 Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu 96 Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp 112 Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly 128 Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala 144 Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 160 Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln 176 Pro 177 【００３５】配列番号：４ 配列の長さ：１７８ 配列の形：アミノ酸 鎖の数： トロポジー：直鎖状 配列の種類：ペプチド 配列 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 16 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 32 Gln Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro 48 Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro 64 Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser 80 Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu 96 Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu 112 Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu 128 Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe 144 Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His 160 Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala 176 Gln Pro 178Description: TECHNICAL FIELD [0001] The present invention relates to a combination of a preparation containing human granulocyte colony stimulating factor (hereinafter abbreviated as human G-CSF) as an active ingredient and an anti-malignant tumor serum. And malignant tumor treatment kits. [0002] The present invention relates to hematopoietic factors.
A novel kit for treating malignant tumors, which is intended to be useful for treating malignant tumors by using human G-CSF, which is one of the ic growth factors, and for which there are no reports directly related thereto. What you want to do. Human G-CSF is a hematopoietic factor having a function of acting on progenitor cells of granulocytes in an in vitro experimental system and promoting the promotion of differentiation into granulocytes [for example, Metec
alf. et. al: Exp. Hematol. 1,1
85, (1973) etc.]. However, this human GC
Since SF has been extremely difficult to obtain until now, studies on its usefulness or efficacy as a medicine have not been sufficiently advanced, and the possibility of the present invention for the treatment of cancer remains unexamined. Was placed in [0003] To overcome such a situation, the present applicant has made repeated studies and as a result, has succeeded in developing a method for producing human G-CSF by genetic engineering or the like, and has succeeded in developing a pure homogenous method. In addition, a large amount of human G-CSF has become available (Japanese Patent Application Nos. 59-153273 and 60-269).
No. 455, Japanese Patent Application No. 60-269456, Japanese Patent Application No. 60-269
No. 270,838, Japanese Patent Application No. 60-270839). Based on this result, we studied the effects of human G-CSF on human mature granulocytes having a malignant tumor-suppressing action, and developed an excellent kit for treating malignant tumors with few side effects based on the findings. It is the purpose of the present invention. Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, the presence of human G-CSF in malignant tumor cells, for example, human malignant melanoma cells (HMV). The granulocytes pre-hatched below had about 2 to 4 times the HMV killing effect compared to the granulocytes pre-hatched in the absence. The HMcidal effect was not affected in the presence of normal rabbit serum, but in the presence of anti-HMV serum it was enhanced up to 10-30 fold depending on its concentration. The degree of enhancement of the granulocytic HMV killing effect by pre-incubation with human G-CSF has a clear correlation with the concentration of human G-CSF. From the results, the present inventors have found that human G-CSF markedly enhances the malignant killing effect of human mature granulocytes (particularly neutrophils) in the presence of anti-malignant tumor serum, and reached the present invention. That is, the present invention provides a kit for treating a malignant tumor, comprising a combination of an anti-human malignant tumor serum and a preparation containing human granulocyte colony stimulating factor as an active ingredient. Hereinafter, the present invention will be described in detail. G of the present invention
-Human G-CS contained in a preparation containing CSF as an active ingredient
The origin of F is not limited as long as it is high-purity human G-CSF. For example, extracted, separated and purified from a human biological sample, and cultured human G-CSF-producing cells,
A product isolated from the culture supernatant, a product obtained by forming a human G-CSF-producing hybridoma using a cell fusion method, and a product obtained by transforming a host such as Escherichia coli or an animal cell by genetic recombination. Any of those produced from the transformant, isolated and purified, or those chemically modified can be used. [0006] However, among them, the following (1) and (2) produced by the present applicant, which can be obtained in a large quantity with good purity and uniformity,
Are particularly preferred. (1) Human G-CSF having the following physicochemical properties. Molecular weight: about 19,000 ± 1,0 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
00. Isoelectric point: pI = 5.5 ± 0.1, pI = 5.8 ± 0.
It has at least one of three isoelectric points of 1, pI = 6.1 ± 0.1. Ultraviolet absorption: maximum absorption at 280 nm, 250 n
m has a minimum value. The amino acid sequence from the N-terminal to the 21st residue is as follows. H ₂ N-Thr-Pro-Leu-Gly-Pro-A
la-Ser-Ser-Leu-Pro-Gln-Se
r-Phe-Leu-Leu-Lys-Cys-Leu
-Glu-Gln-Val- (2) a polypeptide having the activity of human granulocyte colony-stimulating factor represented by the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or a part thereof, A human G-CSF containing a glycoprotein. The above-mentioned human G-CSF can be produced, for example, by a method shown in Reference Examples described later. That is, the human GC of the above (1)
SF can be obtained according to Reference Example 1, and human G-CSF of (2) can be obtained by the method described in Reference Example 2. The detailed production conditions of these methods are described in Japanese Patent Application No. 59-153273 and Japanese Patent Application No. 60-2, filed earlier by the present applicant.
No. 69455, Japanese Patent Application No. 60-269456, Japanese Patent Application No. 6
See Japanese Patent Application Nos. 0-270838 and 60-270839. [0008] Alternatively, it can be obtained by culturing a hybridoma obtained by cell fusion of a G-CSF producing cell and a malignant tumor cell having self-proliferation ability in the presence or absence of mitogen. All human G-CSF obtained by these methods are included in the present invention. If necessary, the obtained human G-CSF-containing liquid can be further purified and concentrated by known means and then stored frozen, or can be stored by removing moisture by means such as freeze-drying or 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. Human G-CSF
Can contain a pharmaceutical carrier and excipients necessary for taking a form as a pharmaceutical preparation suitable for human or veterinary medicine, as well as a stabilizer and an anti-adsorption agent. Next, the kit for treating malignant tumors of the present invention will be described. As is clear from the experimental examples (pharmacological effects) described later, human G-CSF significantly increases the activity of granulocytes in killing tumors in the presence of anti-malignant tumor serum. This is presumed to be because the antiserum enhances the ability of granulocytes to recognize tumor cells and facilitates their attachment to the cells, and as a result, the effect of granulocytes (particularly neutrophils) on malignant tumors is further improved. Is done. A kit for treating a malignant tumor of the present invention, which comprises a combination of an anti-malignant tumor serum and a preparation containing human G-CSF as an active ingredient, has been developed in order to utilize this effect in treating cancer. [0010] The dose and frequency of administration of human G-CSF contained in a preparation containing human G-CSF as an active ingredient can be determined in consideration of the disease state of the target disease patient. ~ 500 μg, preferably 0.5-2
A formulation containing 00 μg of human G-CSF is administered once a week.
It can be administered up to 7 times. However, the present invention relates to human G-
It is not limited by the content of CSF. The anti-malignant tumor serum used in this kit may be prepared by a known method, and its dosage is usually 0.1 to 0.1 per adult.
1 to 5 cc. As an administration method of the anti-malignant tumor serum, it is preferable to administer the anti-malignant tumor agent of the present invention by injection such as intravenous injection about one hour before the administration of the anti-malignant tumor agent increases granulocytes and peaks. . DETAILED DESCRIPTION OF THE INVENTION The present invention is described below with reference to a reference example (human GC).
Production example of SF) Experimental example (pharmacological effect), Example (formulation example)
However, the present invention is not limited to these. The results in the absence of antitumor serum are also shown for reference. Reference Example 1 (Example of production of human G-CSF by culturing G-CSF-producing cells) G-CSF derived from human oral floor cancer cells established by the method shown in Example 1 of Japanese Patent Application No. 59-153273. Producing cell line C
HU-1 (CNCM accession number "I-315") or a cell line CHU-2 (CN.
C. M accession number "I-483") was suspended in RPMI 1640 culture solution containing fetal calf serum, and then placed in a roller bottle for spin-culture. When the cells have grown densely on the inner wall of the roller bottle, the culture solution is subjected to serum-free RPM.
After 1 day of culture, the supernatant was recovered in place of I 1640. Next, after adding serum-containing RPMI 1640 and culturing for 3 days, the culture solution was changed to serum-free RPMI 1640,
After culturing for one day, collect the supernatant. Hereinafter, this 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 specification of Japanese Patent Application No. 153273/1984. Reference Example 2 (Human G-
Example of Production of CSF) E. coli strain # 1776R-2 (FERM) deposited by the present applicant with the microfabrication laboratory.
A cDNA fragment having the human G-CSF gene cut out from BP-955) is incorporated into a vector pdKCR to form 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, the pAdD26SVpA plasmid (Kaufman, RG & Sharp, P, A .;
(1982) Mol. Cell. Biol, Vol. 130
4 to 1319) with EcoRI, BAP processing,
Dephosphorylate. Next, after phenol treatment, pAd by electrophoresis
The EcoRI fragment of D26SVpA was recovered. above
The 2.7 kb fragment and the pAdD26SVpA fragment were annealed, The E. coli DHI strain was transformed by the rubidium chloride method to obtain a pHGV2-dhfr plasmid. Next, CHO cells (dhfr ^- strain, obtained from Dr. L. Chasin, Columbia University) were placed in a 9 cm-diameter plate (manufactured by Nunc) containing α containing 10% calf serum.
Cultivated and grown in a minimum essential medium (α-MEM, adenosine, deoxyadenosine, thymidine added), and
Calcium method (Wigler et al., Cell 14, 725)
(1978). That is, Carrier-D was added to 1 μg of the above pHGV2-dhfr plasmid.
Add an appropriate amount of NA (calf chestline DNA) and add TE solution 375
Dissolve in μl and add 125 μl of 1M CaCl ₂ . 3
Cool on ice for 〜5 minutes and 500 μl of 2 × HBS (50 mM
Hepes, 280 mM NaCl, 1.5 mM phosphate buffer) and ice-cooled again.
mix with the plate, transfer to a plate and incubate for 9 hours in a CO ₂ incubator. Washing, TBS containing 20% glycerol
(Tris-buffered saline),
After washing again, a non-selective medium (α-MEM medium, with nucleosides) was added and incubated for 2 days, and the cells were split 1:10 in the selective medium. Subsequently, the culture was continued while exchanging the medium with a selection medium (no nucleoside added) every two days, and the resulting clumps (foci) were selected and transferred to a new plate. 0.02 μM for new plate
Grow in the presence of methotrexate (MTX) and regrow
Cloning was performed by growing in the presence of μM MTX. As a result of continuing cloning, production of human G-CSF of 10 mg / l or more was confirmed. The purification and assay were performed according to Examples 2 and 15 of Japanese Patent Application No. 60-269456.
Was performed according to the method described in the above section. Experimental Example 1 [Melanoma cell (HMV) effect of granulocytes and added anti-HMV
Effect of Fluctuations in Serum and Human G-CSF Concentration] dextra
n sedimentation method, Ficoll-Hypaque (1.07
7) Using a multilayer centrifugation method and a hypotonic shock red blood cell removal method,
Normal human peripheral blood granulocytes were separated (purity and viable cell ratio 90
%that's all). Various concentrations of human G-CS were added to the separated cells.
After adding F and incubating at 37 ° C. for 1 hour in the culture solution, washing was performed to prepare human mature granulocytes. Next, a certain number of human malignant melanoma cell lines (HM
V) was cultured in a microplate, and when the living cells had sufficiently adhered to the well bottom, rabbit normal serum or anti-HMV serum at each concentration shown in Table 1 and a certain number of the prepared mature granulocytes prepared above were added. And incubated 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 clear from Table 1, the granulocytes prepared by adding human G-CSF showed about 2 to 4 times the HMV killing effect as compared with the case where no G-CSF was added, and the tendency was observed when anti-HMV serum was added. Becomes more pronounced. Also, human G-CS
The degree of enhancement of the granulocytic HMV killing effect by incubation with F has a clear correlation with the concentration of human G-CSF. These results demonstrate that human G-CSF significantly enhances the HMV killing effect of mature human granulocytes. [Table 1] Experimental Example 2 [Melanoma Cell Effect of Granulocytes and Effect of Fluctuations in Serum Concentration of Added Human G-CSF and Anti-HMV] A certain number of HMVs were cultured in a microplate, and the viable cells were sufficiently placed on the well bottom. When adhered, anti-HMV serum or normal serum at each concentration shown in Table 2 was added, and then activated or non-activated granulocytes prepared as in Experimental Example 1 were added, and incubated at 37 ° C. for 24 hours. did. 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.
In addition, similarly to the result of Experimental Example 1, the granulocytes activated with human G-CSF are more than twice as effective in inactivating HMV even in the presence of normal serum as compared with inactive granulocytes using human G-CSF. It can be seen that this difference becomes more pronounced in the presence of anti-HMV serum. These results indicate that human G-CSF has anti-HM
This shows that the presence of V serum has a function to markedly enhance the HMV killing effect of human mature granulocytes. [Table 2] 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 isolated. After addition of CSF, it was incubated and activated. As a control, one that was similarly incubated without adding human G-CSF was prepared. On the other hand, the above-mentioned activated granulocytes or non-activated granulocytes or lymphocytes were added to each tumor cell shown in Table 3, and incubated, and the DNA synthesis of various tumor cells was examined by the incorporation rate of [ ³ H] thymidine. Table 3 shows the results. As is evident 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. [Table 3] To summarize the results of the above experiments, as described above, granulocytes pre-incubated in the presence of human G-CSF are about 2 to 4 times more HMV killed than granulocytes hatched in the absence of human G-CSF. The effect was recognized. The HMcidal effect was not affected in the presence of normal rabbit serum, but in the presence of anti-HMV serum it was enhanced up to 10-30 fold depending on its concentration. The degree of enhancement of the granulocytic HMV killing effect by pre-incubation with human G-CSF was clearly correlated 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 markedly enhances the tumoricidal cell effect of human mature granulocytes in the presence of antitumor serum. The present invention relates to a kit comprising a combination of the preparation containing human G-CSF obtained in the following examples (preparation examples) and a preparation of anti-human malignant tumor serum. The invention is not limited to these combinations. 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
Is filled into a 10 ml vial by aseptic operation in an amount of 5 ml.
After freeze-drying at 0 ° C., the freeze-dried product sealed with a rubber stopper was used as an injection. Example 3 (Preparation Example) An anti-HMV serum obtained by immunizing rabbits with HMV cells was subjected to filter sterilization, and then frozen at -20 ° C to prepare an injection. The kit for treating malignant tumors of the present invention enhances the antitumor activity of human mature granulocytes originally present in the human body by purified human G-CSF, and provides antitumor cells. By coexisting serum, it is further enhanced, and it is intended to treat malignant tumors based on this effect, and is expected to be a therapeutic agent for cancer with few side effects. [Sequence List] SEQ ID NO: 1 Sequence length: 174 Sequence form: Number of amino acid chains: Tropoie: Type of linear sequence: Peptide sequence Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 48 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 64 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 96 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 112 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 128 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 144 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 174 [SEQ ID NO: 2] Sequence length: 175 Sequence form: Number of amino acid chains: Troposi: Type of linear sequence: Peptide sequence Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 16 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 32 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 48 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 64 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 96 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 112 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 128 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 144 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 175 [SEQ ID NO: 3] Sequence length: 177 Sequence form: Number of amino acid chains: Tropoie: Type of linear sequence: Peptide sequence Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32 Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu 48 Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu 64 Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln 80 Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu 96 Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp 112 Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly 128 Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala 144 Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 160 Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln 176 Pro 177 SEQ ID NO: 4 Sequence length: 178 Sequence form: Number of amino acid chains: Topoposite: Type of linear sequence: Peptide sequence Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 16 Lys Cys Leu Glu Gln Va l Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 32 Gln Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro 48 Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro 64 Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser 80 Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu 96 Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu 112 Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu 128 Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe 144 Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His 160 Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala 176 Gln Pro 178

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Claims (1)

(57)【特許請求の範囲】 １．抗ヒト悪性腫瘍血清とヒト顆粒球コロニー刺激因子
を有効成分とする製剤の組み合わせからなる悪性腫瘍治
療用キット。 ２．悪性腫瘍が悪性黒色腫であることを特徴とする請求
項１記載の悪性腫瘍治療用キット。 ３．ヒト顆粒球コロニー刺激因子が好中球コロニー刺激
因子であることを特徴とする請求項１記載の悪性腫瘍治
療用キット。 ４．ヒト顆粒球コロニー刺激因子がヒト顆粒球コロニー
刺激因子産生細胞の培養上清から得られたものであるこ
とを特徴とする請求項１記載の悪性腫瘍治療用キット。 ５．ヒト顆粒球コロニー刺激因子が次の理化学的性質を
有するものであることを特徴とする請求項１記載の悪性
腫瘍治療用キット。 「理化学的性質」 分子量：ドデシル硫酸ナトリウム−ポリアクリルアミ
ドゲル電気泳動法による測定で１９，０００±１，００
０。 等電点：ｐＩ＝５．５±０．１，ｐＩ＝５．８±０．
１，ｐＩ＝６．１±０．１の三つの等電点のうち少なく
とも１つを有する。 紫外部吸収：２８０ｎｍに極大吸収を有し、２５０ｎ
ｍに極少値をもつ。 Ｎ末端から２１残基目迄のアミノ酸配列が次の如くで
ある。 Ｈ₂ Ｎ−Ｔｈｒ−Ｐｒｏ−Ｌｅｕ−Ｇｌｙ−Ｐｒｏ−Ａ
ｌａ−Ｓｅｒ−Ｓｅｒ−Ｌｅｕ−Ｐｒｏ−Ｇｌｎ−Ｓｅ
ｒ−Ｐｈｅ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｃｙｓ−Ｌｅｕ
−Ｇｌｕ−Ｇｌｎ−Ｖａｌ− ６．ヒト顆粒球コロニー刺激因子がヒト顆粒球コロニー
刺激因子活性を有するポリペプチドをコードする遺伝子
を含む組換えベクターを含有する形質転換体から産生さ
れたヒト顆粒球コロニー刺激因子活性を有するポリペプ
チドまたは糖蛋白質であることを特徴とする請求項１記
載の悪性腫瘍治療用キット。 ７．ヒト顆粒球コロニー刺激因子活性を有するポリペプ
チドが下記のアミノ酸配列またはその一部で表わされる
請求項１項記載の悪性腫瘍治療用キット。 (Met)_n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 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 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro （但しｍは０又は１を表わし、ｎは０又は１を表わす） ８． ヒト顆粒球コロニー刺激因子活性を有する糖蛋白
質が下記のアミノ酸配列またはその一部で表わされるポ
リペプチドと糖鎖部とを有するものである請求項１項記
載の悪性腫瘍治療用キット。 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 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 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro （ただし ｍは０または１を表わす）。(57) [Claims] A malignant tumor treatment kit comprising a combination of an anti-human malignant tumor serum and a preparation containing human granulocyte colony-stimulating factor as an active ingredient. 2. The malignant tumor treatment kit according to claim 1, wherein the malignant tumor is malignant melanoma. 3. The kit for treating a malignant tumor according to claim 1, wherein the human granulocyte colony stimulating factor is a neutrophil colony stimulating factor. 4. The kit for treating a malignant tumor 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 kit for treating a malignant tumor 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.
0. Isoelectric point: pI = 5.5 ± 0.1, pI = 5.8 ± 0.
It has at least one of three isoelectric points of 1, pI = 6.1 ± 0.1. Ultraviolet absorption: maximum absorption at 280 nm, 250 n
m has a minimal value. The amino acid sequence from the N-terminal to the 21st residue is as follows. H ₂ N-Thr-Pro-Leu-Gly-Pro-A
la-Ser-Ser-Leu-Pro-Gln-Se
r-Phe-Leu-Leu-Lys-Cys-Leu
-Glu-Gln-Val- 6. Polypeptide or sugar having human granulocyte colony stimulating factor activity produced from a transformant containing a recombinant vector containing a gene encoding a polypeptide having human granulocyte colony stimulating factor having human granulocyte colony stimulating factor activity The kit for treating a malignant tumor according to claim 1, which is a protein. 7. The kit for treating a malignant tumor 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. (Met) _n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 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 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (where m represents 0 or 1 and n represents 0 or 1) 7) The kit for treating a malignant tumor 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. Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 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 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (where m represents 0 or 1).