JP2544146B2 - Nasopharyngeal cancer treatment - Google Patents
Nasopharyngeal cancer treatmentInfo
- Publication number
- JP2544146B2 JP2544146B2 JP62164780A JP16478087A JP2544146B2 JP 2544146 B2 JP2544146 B2 JP 2544146B2 JP 62164780 A JP62164780 A JP 62164780A JP 16478087 A JP16478087 A JP 16478087A JP 2544146 B2 JP2544146 B2 JP 2544146B2
- Authority
- JP
- Japan
- Prior art keywords
- npc
- monoclonal anti
- idiotype antibody
- antibody
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は癌の治療剤に関し、更に詳細にはモノクロー
ナル抗イデイオタイプ抗体を有効成分とする上咽頭癌治
療剤に関する。TECHNICAL FIELD The present invention relates to a therapeutic agent for cancer, and more particularly to a therapeutic agent for nasopharyngeal cancer containing a monoclonal anti-idiotype antibody as an active ingredient.
リンパ球および抗体分子の免疫応答がイデイオタイプ
によつて調節されていることは知られている。そしてこ
のイデイオタイプに対する抗体、すなわち抗イデイオタ
イプ抗体についての研究も種々行なわれている。例えば
R.C.Kennedyらは、Ｂ型肝炎ウイルスの表面抗原に対す
る抗イデイオタイプ抗体がチンパジーのＢ型肝炎をワク
チン様作用により予防すると報告している（Science,23
2,220〜223,1968および特開昭59−116230号）。また、
M.S.Kaminskiらは、ネズミにおけるＢ細胞性リンパ腫に
対し、同じＢ細胞性リンパ種に対応するモノクローナル
抗イデイオタイプ抗体の投与が治療効果を示したことを
（J.Immunology,136（３）,1123〜1130,1986）、J.Maho
nyらは、骨髄腫蛋白MOPC315IgAに対するモノクローナル
抗イデイオタイプ抗体がin vitroでMOPC315骨髄腫細胞
に対し細胞毒性を発揮したと報告している（J.Immunolo
gy,126（１）,113〜117,1981）。It is known that the immune response of lymphocytes and antibody molecules is regulated by the idiotype. Various studies have been conducted on antibodies against this idiotype, that is, anti-idiotype antibodies. For example
RCKennedy et al. Reported that an anti-idiotype antibody against the surface antigen of hepatitis B virus prevents chimp hepatitis B by a vaccine-like action (Science, 23
2, Nos. 220～223,1968 and JP 59-116230). Also,
MS Kaminski et al. Showed that administration of a monoclonal anti-idiotype antibody corresponding to the same B-cell lymphoma showed a therapeutic effect on B-cell lymphoma in a mouse (J. Immunology, 136 (3) , 1123-1130, 1986), J. Maho
ny et al. reported that a monoclonal anti-idiotype antibody against the myeloma protein MOPC315IgA exerted cytotoxicity against MOPC315 myeloma cells in vitro (J. Immunololo).
gy, 126 (1) , 113-117,1981).
ところで癌は生体の種々の部位に発生し、癌の種類に
よつて性質、薬剤に対する反応性等が異なることから、
種々の癌にそれぞれの治療薬を開発することは極めて重
要である。By the way, cancer occurs in various parts of the living body, and properties, reactivity to drugs, etc. differ depending on the type of cancer,
Developing different therapeutic agents for different cancers is extremely important.
斯かる実状において本発明者らは上咽頭癌由来細胞の
膜表面抗原に対するモノクローナル抗イデイオタイプ抗
体を、細胞融合技術により作製し、これを利用すれば癌
の診断が早期かつ適確にできることを見い出し、先に特
許出願した（特願昭62−54920号，同62−54921号）。さ
らに、研究を続けた結果、このモノクローナル抗イデイ
オタイプ抗体を生体に投与すれば、上咽頭癌患者におけ
る癌細胞の増殖を抑制できることを見い出し、本発明を
完成した。In such an actual situation, the present inventors have found that a monoclonal anti-idiotype antibody against the membrane surface antigen of nasopharyngeal cancer-derived cells is produced by a cell fusion technique, and if this is used, the diagnosis of cancer can be performed early and appropriately, I applied for a patent first (Japanese Patent Application Nos. 62-54920 and 62-54921). Further, as a result of continuing the study, they found that the administration of this monoclonal anti-idiotype antibody to the living body could suppress the growth of cancer cells in patients with nasopharyngeal cancer, and completed the present invention.
すなわち、本発明は上咽頭癌由来細胞の膜表面抗原に
対するモノクローナル抗イデイオタイプ抗体を有効成分
とする上咽頭癌治療剤を提供するものである。That is, the present invention provides a therapeutic agent for nasopharyngeal cancer containing a monoclonal anti-idiotype antibody against a membrane surface antigen of cells derived from nasopharyngeal carcinoma as an active ingredient.
本発明の上咽頭癌治療剤の有効成分である上咽頭癌由
来細胞の膜表面抗原に対するモノクローナル抗イデイオ
タイプ抗体は、例えば矢野間らによつて報告された５−
９株をクローニングして得たハイブリドーマ76AC6−B9
株（第42回日本癌学会総会）を無血清培地中で培養して
得られる抗NPC−204モノクローナル抗体から次の如くし
て製造される。A monoclonal anti-idiotype antibody against a membrane surface antigen of cells derived from nasopharyngeal cancer, which is an active ingredient of the therapeutic agent for nasopharyngeal cancer of the present invention, was reported by Yanoma et al.
Hybridoma 76AC6-B9 obtained by cloning 9 strains
It is produced as follows from an anti-NPC-204 monoclonal antibody obtained by culturing a strain (42nd Annual Meeting of the Japanese Cancer Society) in a serum-free medium.
抗HPC−204モノクローナル抗体によりBALB/Cマウスを
免疫し、抗体価が充分高まつたところで免疫マウスの脾
臓を摘出して得た脾細胞とマウスの骨髄腫細胞NS−１又
はP3u−１とを融合させてハイブリドーマを作製する。
このハイブリドーマを無血清培地中で培養しNPC−204株
の膜表面抗原に対する抗イデイオタイプ抗体、即ちモノ
クローナル抗NPC−204イデイオタイプ抗体を産生するハ
イブリドーマをアルカリフオスフアターゼ標識したモノ
クローナル抗NPC−204抗体を用いる酸素免疫測定法（EI
A法）により選択する。効率良く高い力価のモノクロー
ナル抗イデイオタイプ抗体を産生するハイブリドーマを
限界希釈法により無血清培地を用いてクローニングし、
モノクローナル抗NPC−204イデイオタイプ抗体を産生す
る安定株IVN−63、IVP−41、IVN−４を得た。BALB / C mice were immunized with anti-HPC-204 monoclonal antibody, and spleen cells obtained by removing the spleen of immunized mice when the antibody titer was sufficiently high and mouse myeloma cells NS-1 or P3u-1 were prepared. Hybridize to produce hybridomas.
This hybridoma is cultivated in a serum-free medium and an anti-idiotype antibody against the membrane surface antigen of the NPC-204 strain, that is, a hybridoma producing a monoclonal anti-NPC-204 idiotype antibody is used as a monoclonal anti-NPC-204 antibody labeled with alkaline phosphatase. Oxygen immunoassay (EI
Select according to method A). A hybridoma that efficiently produces a high titer monoclonal anti-idiotype antibody was cloned using a serum-free medium by the limiting dilution method,
Stable strains IVN-63, IVP-41 and IVN-4 producing monoclonal anti-NPC-204 idiotype antibodies were obtained.
これ等の安定株を無血清培地中に培養するか、動物に
投与して腹水化し、培養物又は腹水を採取すればクラス
IgG型か又はIgM型のモノクローナル抗NPC−204イデイオ
タイプ抗体が得られる。Culture these stable strains in serum-free medium or administer to animals to ascites and collect the culture or ascites.
IgG type or IgM type monoclonal anti-NPC-204 idiotype antibodies are obtained.
斯くして得られるモナクローナル抗NPC−204イデイオ
タイプ抗体は次の如き性状及び生物学的性質を有する。The monaclonal anti-NPC-204 idiotype antibody thus obtained has the following properties and biological properties.
（１）物理化学的性状 （２）安定性 本発明の上咽頭癌治療剤を調製するには、上記の如く
して得られたモノクローナル抗イデイオタイプ抗体を常
法により、生理食塩液、精製水、リン酸塩等に溶解し
て、皮下注射用、筋肉内注射用、静脈内注射用又は腹腔
内注射用の製剤ににすればよい。またこれらの製剤の形
態としては、溶液の他に、凍結乾燥品等の用時溶解して
使用する形態のものが挙げられる。(1) Physicochemical properties (2) Stability To prepare the therapeutic agent for nasopharyngeal cancer of the present invention, the monoclonal anti-idiotype antibody obtained as described above is dissolved in physiological saline, purified water, phosphate or the like by a conventional method, and then subcutaneously injected. The preparation may be for intramuscular injection, intravenous injection or intraperitoneal injection. Examples of the form of these preparations include solutions, as well as solutions in which freeze-dried products and the like are dissolved before use.
本発明の癌治療剤の投与量は、症状により異なるが、
モノクローナル抗イデイオタイプ抗体として50μg/kg〜
50mg/kg/日が好ましい。Although the dose of the therapeutic agent for cancer of the present invention varies depending on symptoms,
50 μg / kg ~ as monoclonal anti-idiotype antibody
50 mg / kg / day is preferred.
本発明の上咽頭癌治療剤は、これを担癌患者に投与す
ることにより、脾リンパ球を賦活化し、免疫系を介して
上咽頭癌細胞の増殖を抑制し、優れた治療効果を発揮す
る。The therapeutic agent for nasopharyngeal cancer of the present invention, when administered to a cancer-bearing patient, activates splenic lymphocytes, suppresses proliferation of nasopharyngeal cancer cells via the immune system, and exerts an excellent therapeutic effect. .
次に参考例および実施例を挙げて本発明を説明する。 Next, the present invention will be described with reference to Reference Examples and Examples.
参考例１ （１）モノクローナル抗NPC−204イデイオタイプ抗体産
生細胞株と作製： NPC−204株の膜表面抗原に対するモノクローナル抗NP
C−204抗体を産生するハイブリドーマ76AC6−B9株を無
血清培地SFM−101（日水製薬株式会社製）中で培養し、
培養上清中より得た抗NPC−204抗体を56℃、30分温浴中
で反応させたのち、コンプリート・フロイント・アジバ
ント中に混和してBALB/C♀マウス６〜８週令に100μｇ
皮下注射した。初回注射の２週間後に抗NPC−204抗体を
インコンプリート・フロイント・アジバント中に混和し
て100μｇ皮下注射した。更に２週間経過後、抗NPC−20
4抗体をPBS中に溶解して100μｇマウスの腹腔内に注射
し、３日後に脾臓を摘出して単核脾細胞とした後、８−
アザグアニン耐性BALB/cマウス骨髄細胞株NS−１又はP3
u−１と50％PEG（シグマ社製、分子量4,000）により融
合させた。Reference Example 1 (1) Monoclonal anti-NPC-204 idiotype antibody-producing cell line and preparation: Monoclonal anti-NP against membrane surface antigen of NPC-204 strain
The hybridoma 76AC6-B9 strain producing the C-204 antibody was cultured in a serum-free medium SFM-101 (manufactured by Nissui Pharmaceutical Co., Ltd.),
Anti-NPC-204 antibody obtained from the culture supernatant was reacted in a warm bath at 56 ° C for 30 minutes, and then mixed in complete Freund's adsorbent to 100 µg of BALB / C♀ mouse 6 to 8 weeks old.
Injected subcutaneously. Two weeks after the first injection, 100 μg of anti-NPC-204 antibody was mixed in Incomplete Freund's adjuvant and subcutaneously injected. After a further 2 weeks, anti-NPC-20
4 antibody was dissolved in PBS and intraperitoneally injected into 100 μg mouse, and 3 days later, the spleen was excised to obtain mononuclear splenocytes.
Azaguanine resistant BALB / c mouse bone marrow cell line NS-1 or P3
U-1 was fused with 50% PEG (Sigma, molecular weight 4,000).
融合細胞の選択は無血清培地SFM−101により行なつ
た。融合細胞の培養上清についてEIA法により抗NPC−20
4抗体との結合性を調べ、反応性の高いものについて限
界希釈法によりクローニングを実施し、モノクローナル
抗NPC−204イデイオタイプ抗体産生株IVN−63、IVP−4
1、IVN−４を得た。The selection of fused cells was carried out by the serum-free medium SFM-101. The culture supernatant of the fused cells was treated with anti-NPC-20 by EIA method.
4 The binding with the antibody was examined, and those with high reactivity were cloned by the limiting dilution method to obtain monoclonal anti-NPC-204 idiotype antibody-producing strains IVN-63 and IVP-4.
1, IVN-4 was obtained.
（２）抗NPC−204イデイオタイプ抗体の製造： （１）で得られたモノクローナル抗NPC−204イデイオ
タイプ抗体産生株を無血清培地中で培養し、培養液を1,
500rpmで15分間遠心分離したのち、上清を更に10,000rp
mで20分間遠心分離し上清を得た。この上清を限外ろ過
膜Amicon Diaflo YMIOを用いて1/10〜1/20容量まで濃縮
したのち、等容量の飽和硫安を加えて蛋白分画を沈澱さ
せた。冷所に１夜置いたのち10,000rpm、15分間遠心分
離し、得られた沈澱物をPBSに溶解した後、0.9％NaCl液
で48時間透析して、それぞれ３種のモノクローナル抗NP
C−204イデイオタイプ抗体を得た。(2) Production of anti-NPC-204 idiotype antibody: The monoclonal anti-NPC-204 idiotype antibody-producing strain obtained in (1) was cultured in a serum-free medium,
After centrifuging at 500 rpm for 15 minutes, the supernatant is added to 10,000 rp
The mixture was centrifuged at m for 20 minutes to obtain a supernatant. This supernatant was concentrated to 1/10 to 1/20 volume using an ultrafiltration membrane Amicon Diaflo YMIO, and then an equal volume of saturated ammonium sulfate was added to precipitate the protein fraction. After allowing to stand overnight in a cold place, centrifugation was performed at 10,000 rpm for 15 minutes, the resulting precipitate was dissolved in PBS and dialyzed against 0.9% NaCl solution for 48 hours to obtain three monoclonal anti-NPs.
A C-204 idiotype antibody was obtained.
実施例１ 癌細胞増殖抑制作用： 試験方法 ６週令、雌のBALB/cマウスにモノクローナル抗NPC−2
04イデイオタイプ抗体（IVN−63抗イデイオタイプ抗
体）100μｇを皮下注射して免疫し、２週間後に脾臓を
摘出し、RPMI−1640倍地（10％FCS含有）中で単核脾細
胞とした。ポリスチレン製の96穴マイクロプレートに脾
細胞数:NPC−204細胞数の比率が200:1になる様に調製し
たそれぞれの浮遊液を、１ウエル当りNPC−204細胞数が
５×103個となる様に播いたのち、37℃のCO2インキユベ
ーター中にて培養し、経時的に細胞数を観察した。Example 1 Cancer Cell Growth Inhibitory Effect: Test Method Monoclonal anti-NPC-2 in 6-week-old female BALB / c mice
04 Idiotype antibody (IVN-63 anti-idiotype antibody) (100 μg) was subcutaneously injected to immunize, and two weeks later, the spleen was removed and used as mononuclear splenocytes in RPMI-1640 medium (containing 10% FCS). Each suspension was prepared in a polystyrene 96-well microplate so that the ratio of spleen cell number: NPC-204 cell number was 200: 1, and NPC-204 cell number was 5 × 10 3 per well. After sowing, the cells were cultured in a CO 2 incubator at 37 ° C, and the number of cells was observed with time.
対照群にはモノクローナル抗NPC−204イデイオタイプ
抗体による免疫のかわりにPBSを注射したマウスの脾細
胞を用いた。その他に脾細胞を加えないNPC−204細胞群
を無処置群とし比較観察した。As a control group, spleen cells of a mouse injected with PBS instead of immunization with a monoclonal anti-NPC-204 idiotype antibody were used. In addition, the NPC-204 cell group to which splenocytes were not added was used as a non-treatment group for comparative observation.
試験結果 結果を図１および図２に示す。その結果、モノクロー
ナル抗NPC−204イデイオタイプ抗体免疫群では、播種７
日目より対照群よりもNPC−204細胞の生存率が著しく低
下した。従つて担癌患者に該抗イデイオタイプ抗体を投
与すれば、脾リンパ球賦活化によつて上咽頭癌細胞の増
殖を抑制することができる。Test results The results are shown in FIGS. 1 and 2. As a result, in the monoclonal anti-NPC-204 idiotype antibody immunized group, seeding 7
From day one, the survival rate of NPC-204 cells was significantly lower than that of the control group. Therefore, if the anti-idiotype antibody is administered to a cancer-bearing patient, the proliferation of nasopharyngeal cancer cells can be suppressed by activating splenic lymphocytes.
実施例２ 急性毒性： 体重20〜25gのddY系雌雄マウスを１群５匹として用
い、本発明のモノクローナル抗イデイオタイプ抗体を腹
腔内又は皮下注射することにより急性毒性試験を行なつ
た。観察は７日間行なつた。その結果、本発明モノクロ
ーナル抗イデイオタイプ抗体は500mg/kgの投与で死亡例
はなく、何ら中毒症状を発現しなかつた。Example 2 Acute Toxicity: An acute toxicity test was conducted by intraperitoneally or subcutaneously injecting the monoclonal anti-idiotype antibody of the present invention using 5 ddY male and female mice each having a body weight of 20 to 25 g as one group. Observation was carried out for 7 days. As a result, the monoclonal anti-idiotype antibody of the present invention was found to have no deaths even after administration of 500 mg / kg, and did not develop any toxic symptoms.
図１および図２は実施例１の試験における培養日数と生
存癌細胞数との関係および培養日数と癌細胞生存率との
関係をそれぞれ示す図面である。1 and 2 are drawings showing the relationship between the number of days of culture and the number of surviving cancer cells and the relationship between the number of days of culture and the survival rate of cancer cells in the test of Example 1, respectively.
Claims (1)
ノクローナル抗イデイオタイプ抗体を有効成分とする上
咽頭癌治療剤。1. A therapeutic agent for nasopharyngeal cancer comprising a monoclonal anti-idiotype antibody against a membrane surface antigen of cells derived from nasopharyngeal carcinoma as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62164780A JP2544146B2 (en) | 1987-07-01 | 1987-07-01 | Nasopharyngeal cancer treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62164780A JP2544146B2 (en) | 1987-07-01 | 1987-07-01 | Nasopharyngeal cancer treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS649939A JPS649939A (en) | 1989-01-13 |
| JP2544146B2 true JP2544146B2 (en) | 1996-10-16 |
Family
ID=15799805
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62164780A Expired - Lifetime JP2544146B2 (en) | 1987-07-01 | 1987-07-01 | Nasopharyngeal cancer treatment |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2544146B2 (en) |
-
1987
- 1987-07-01 JP JP62164780A patent/JP2544146B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS649939A (en) | 1989-01-13 |
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