WO1986005986A1 - Process for preparing carcinostatic substance complex - Google Patents

Process for preparing carcinostatic substance complex Download PDF

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Publication number
WO1986005986A1
WO1986005986A1 PCT/JP1985/000214 JP8500214W WO8605986A1 WO 1986005986 A1 WO1986005986 A1 WO 1986005986A1 JP 8500214 W JP8500214 W JP 8500214W WO 8605986 A1 WO8605986 A1 WO 8605986A1
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WO
WIPO (PCT)
Prior art keywords
complex
dextran
antibody
substance
anticancer
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Application number
PCT/JP1985/000214
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French (fr)
Japanese (ja)
Inventor
Hideo Nishimaki
Tomiyuki Matsunaga
Yoshio Kagitani
Masayuki Nishida
Tadakazu Suyama
Original Assignee
The Green Cross Corporation
Priority date (The priority date 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 date listed.)
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Publication date
Priority claimed from JP19813183A external-priority patent/JPS6089433A/en
Application filed by The Green Cross Corporation filed Critical The Green Cross Corporation
Priority to PCT/JP1985/000214 priority Critical patent/WO1986005986A1/en
Publication of WO1986005986A1 publication Critical patent/WO1986005986A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy

Definitions

  • Anticancer substance composite and method for producing the same
  • the present invention relates to a novel regulated active substance complex, a method for producing the same, and a method for stabilizing a poor anticancer compound complex.
  • the present inventors have been conducting research on a carcinostatic agent that is more stable and acts more specifically on cancer cells, ie, an antibody-dextran-carcinogen complex.
  • a carcinostatic agent that is more stable and acts more specifically on cancer cells
  • an antibody-dextran-carcinogen complex By binding 5 to 30 moles of dextran per 1 mole of the antibody, it is possible to obtain a complex that is excellent in storage stability, does not decrease the antibody titer, and does not decrease the anticancer effect.
  • By masking the end-reactive aldehyde group in the conventional conjugate to reduce the reaction with the antibody it is possible to stabilize the conjugate and to suppress the decrease in antibody titer.
  • the present inventors have found that the complex acts on cancer cells more specifically, and have completed the present invention.
  • the present invention relates to a complex of a carcinostatic agent comprising a dextran-inhibiting agent having a cleaved aldoseviroose ring and an antibody against a surface antigen, 5 to 30 mol per mol of the antibody. And a aldehyde group of a dextran which has a cleaved aldopyropyrose ring (hereinafter referred to as a cleaved dextran).
  • a method for producing a conjugate comprising an inhibitor and an antibody via cleavage dextran The present invention relates to a method for improving the stability, antibody titer, and anticancer action of a complex by masking an aldehyde group of a cleaved dextran in a body to thereby reduce reactivity with an antibody.
  • FIG. 1 shows the ⁇ -futoprotein-producing hepatoblastoma of the complex of Example 1 and the conventional complex (macromolecularized anti-fetobrotin-dima antibody-dextran-daunomycin complex).
  • FIG. 2 is a graph showing a comparison of the growth inhibitory effect on cells, and FIG. 2 is a diagram showing the separation of the complex of the present invention and an unreacted dextran-daricin-daunomycin complex.
  • the antibody used in the present invention is an antibody against a surface antigen of a cancer to be treated, and is a specific antibody derived from an animal obtained by immunizing an animal with the antigen, a monoclonal antibody obtained by genetic engineering or a cell fusion method. Any antibody such as a local antibody may be used.
  • the dextran is not particularly limited, and a dextran having a molecular weight in the range of 1,000 to 200,000 is preferably used.
  • the substance having an anticancer effect may be a substance having a group reactive with an aldehyde group (for example, an amino group or a hydroxyl group), and a substance having no such reactive group may be used.
  • an aldehyde group for example, an amino group or a hydroxyl group
  • Suitable inhibitory substances include, for example, dawn myosin, mitomycin C, and adriamycin.
  • masking the aldehyde group means inactivating the aldehyde group with respect to the antibody.
  • Masking includes, for example, reducing an aldehyde group to a hydroxymethyl group
  • reducing agents include, for example, metal borohydride and metal cyanide borohydride.
  • acetals by alcohols eg, methanol, ethanol, pranol, ethanol, ethylene glycol
  • amide compounds especially amino acids
  • Examples of the amino acids which include the generation of a Schiff base according to include ⁇ as shown below.
  • Aliphatic amino acids (glycine, alanine, norin, leucine, isoloisin, etc.), oxamino acids (serine, threonine, etc.), sulphate amino Acids (cystine, cystine, methionine, etc.), Aromatic amino acids (phenylalanine, tyrosine, triptophan, etc.)-'Acidic amino acids:
  • amino acids other than amino acid ⁇ —Aranine and others
  • the present invention provides, for example, cleavage dextran,
  • an aldehyde group and a carcinogen that is reactive with the aldehyde group are bound to the aldehyde group, and the aldehyde of the dextran It is produced through masking of an aldehyde group and binding of an antibody to the aldehyde group, but is preferably performed in the above order.
  • the cleavage dextran is known, and its production method is disclosed in Proc. Natl. Acad. Sci. USA, 78, 2128 (1976) and the like.
  • the composition ratio of each component in the complex obtained by the production method of the present invention is preferably 5 to 30 molecules of dextran and 20 to 70 molecules of the inhibitory substance per one molecule of the antibody.
  • the remaining aldehyde groups are molecules that are inactivated by masking as much as possible, usually at least 80% of the aldehyde groups are masked. Is preferred.
  • the outline of an example of the production method of the present invention is as follows.
  • Cleavage of the aldohyl viranose ring of dextran is usually carried out by oxidation.
  • the oxidizing agent it is preferable to use sodium periodate. For example, add about 100 to 70 oxidizing agents to dextran 500 and react for 5 to 24 hours. Respond. After completion of the reaction, for example, the reaction solution is bent through distilled water for 10 to 30 hours, and preferably lyophilized to obtain oxidized (cleaved) dextran.
  • the reaction between the cleaved dextran and the antibacterial substance it is preferable to couple 2 to 10 molecules of the anticancer substance to one molecule of the cleaved dextran.
  • the final concentration of cleaved dextran in an aqueous solvent is about 10-30 W / V%, Is preferably added to a final concentration of about 0.5 to 5 V% and reacted for about 5 to 15 hours.
  • a dextran-cancer substance is obtained.
  • Unreacted anticancer agents are removed by, for example, adsorption chromatography using Toyopar HW40 or other means known per se.
  • conditions sufficient to mask about 85 to 95% of the free aldehyde groups in the complex may be selected.
  • the reduction treatment is preferably performed with 0 to 10'c for 1 to 20 hours using 1 to 10 mol of a reducing agent.
  • the reaction time is usually 12 to 30 hours, and the pH is preferably about 5 to 8.
  • the antibody When binding the antibody to the complex, the antibody is added to a reaction solution containing 5 to 30 V% of the complex so that the final concentration becomes, for example, 0.1 to 3 WZV%, and the mixture is cooled. In 12 to 40 hours Let react.
  • the complex according to the present invention thus obtained can be purified by applying it to a gel filtration carrier ion exchange resin, antigen-Sepharose, or the like.
  • the complex thus obtained is, for example, sterilized and filtered, and then dispensed into a liquid frozen product or a lyophilized dry preparation.
  • the present invention solves the disadvantage that a large number of antibodies bind to the aldehyde group of a conventional cleavable dextran to form a macromolecule, thereby masking the aldehyde group to prevent cancer. That is what you do.
  • the molecular weight is 100,000 to 700,000, whereas in the complex of the present invention, 150,000 is used. It has a molecular weight of 2250,000, and is a combination of dextran of 5 to 30 molecules and a large number (20 to 70 molecules) of an anticancer agent per antibody molecule. This complex is more stable and more excellent in properties than the macromolecule as described below.
  • the conventional macromolecule complex shows sedimentation in the first month and a decrease in antibody activity even in cold storage.
  • insolubles are easily generated by freeze-drying.
  • no sedimentation was observed even after one month of liquid storage, and no decrease in antibody activity was observed in storage stability after freeze-drying.
  • the conventional macromolecularized complex shows that In contrast to the activity of only 0 to 60%, the complex obtained according to the present invention shows almost 100% of the antibody activity without impairing the antibody activity. This is probably because in the macromolecularized complex, since multiple antibodies are bound to dextran, the expression of sufficient activity is suppressed due to steric hindrance and the like.
  • the complex obtained by the present invention compared to the conventional macromolecularized complex, allows a large number of anticancer agents to bind to one antibody molecule via dextran, and is stable. It has excellent properties and antibody activity, and is close to the original properties of antibodies, and has advantages such as easy access to target cells and good membrane permeability. In addition, its stability as a lyophilized product has been confirmed.
  • the dosage and administration method of the conjugate obtained in the present invention for clinical use are as follows: 10 to 300 mg of this product is dissolved in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia, and the age, symptoms and progress Intravenous injection, intravenous drip infusion, and drip injection are used as appropriate according to the requirements.
  • Experimental example 1 10 to 300 mg of this product is dissolved in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia, and the age, symptoms and progress Intravenous injection, intravenous drip infusion, and drip injection are used as appropriate according to the requirements.
  • the medium used was RPMI 1640 (manufactured by Nissui Pharmaceutical Co., Ltd.).

Abstract

A carcinostatic substance complex comprising aldohexopyranose ring-cleaved dextran, a carcinostatic substance, and an antibody for cancer surface antigen, wherein 5 to 30 mol of the dextran is bound per mol of the antibody, is stable, shows a restrained reduction of antibody titer, and attacks cancer cells more specifically.

Description

明 細 書  Specification
制癌作用物質複合体及びその製造法  Anticancer substance composite and method for producing the same
技術分野  Technical field
本発明は、 新規制瘙作用物質複合体、 その製造方法及び制癌 作用物貧複合体の安定化方法に関する。  TECHNICAL FIELD The present invention relates to a novel regulated active substance complex, a method for producing the same, and a method for stabilizing a poor anticancer compound complex.
従来技術  Conventional technology
今日、 多数の制瘙作用物質が制癌剤として開発され、 臨床応 用されているが、 これらはいずれも癌細胞に対し非特異的であ つて、 正常細胞に対しても毒性を有するため、 制癌剤単独によ る治療効果はあまり得られていない。  Today, a large number of anticancer drugs have been developed and used clinically as anticancer drugs, but all of them are non-specific for cancer cells and toxic to normal cells. Has not obtained much therapeutic effect.
このこ とから、 制癌作用物質を癌細胞に親和性を有する物質 と結合させて複合体とし、 この複合体を特異的に瘙細胞に到達 させて制癌作用を発揮させよう とする治療法のアイデアが生ま れた この 1 つと して、 Ε· Hurwi tz ら (Europ. J. Cancer, Vol.14, 1213-1220. 1978 年） により瘙抗原に対する抗体にデキ ス ト ラ ンを介し、 制癌作用を有するダウノ マイ シ ン等を結合さ せた複合体が開発された。 この複合体の製法は、 アル ドへキソ ビラノ ース環の開裂したデキス ト ラ ンのアルデヒ ド基に抗体の ァ ミノ基と制癌作用物質のァ ミノ基とを結合させると言う もの である。 ところが、 酸化デキス ト ラ ンのアルデヒ ド基は非特異 的に結合容易な抗体のァ ミ ノ基と結合するため、 デキス ト ラ ン 1 モル当たり 6 〜 1 0 0 モルの抗体が結合した複合体が調製さ れる。 この複合体は分子量が 1 0 0万〜 7 0 0万の巨大分子で、 長期保存時における安定性の低下、 及び抗体価の低下さ らには 制瘙作用の低下などが認められ、 効果的な薬理作用が得られて いない。 For this reason, a therapeutic method in which an anticancer substance is combined with a substance having an affinity for cancer cells to form a complex, and the complex is specifically allowed to reach 瘙 cells to exert an anticancer effect. Hurwitz et al. (Europ. J. Cancer, Vol. 14, 1213-1220. 1978) reported that antibodies to the 瘙 antigen were mediated by dextran. A complex in which daunomycin and the like having a cancer action are bound has been developed. The method for producing this complex is to bond the amino group of the antibody and the amino group of the anticancer substance to the aldehyde group of the dextran in which the aldexolanose ring is cleaved. . However, since the aldehyde group of the oxidized dextran binds nonspecifically to the amino group of the easily bound antibody, a complex in which 6 to 100 moles of the antibody is bound per mole of dextran is used. Is prepared. This complex is a macromolecule with a molecular weight of 1,000,000 to 700,000.It is effective because it has reduced stability during long-term storage, reduced antibody titer, and reduced inhibitory action. A great pharmacological action Not in.
本発明の開示  Disclosure of the present invention
かかる実情下、 本発明者らは、 より安定で癌細胞に対してよ り特異的に作用する制癌剤、 即ち抗体-デキス ト ラ ン —制癌作 用物質複合体の研究を行って来たところ、 抗体 1 モル当り 5 〜 3 0 モルのデキス トランを結合させることによって、 保存安定 性に優れ、 抗体価の低下、 制癌作用の低下のない複合体が得ら れること及びかかる複合体は、 従来の複合体中の末反応アルデ ヒ ド基をマスキ ングして、 抗体との反応を低下せしめておけば 複合体の安定化が図られること、 抗体価の低下が抑制されるこ と、 当該複合体がより特異的に癌細胞に作用することを見出し て本発明を完成するに至った。  Under such circumstances, the present inventors have been conducting research on a carcinostatic agent that is more stable and acts more specifically on cancer cells, ie, an antibody-dextran-carcinogen complex. By binding 5 to 30 moles of dextran per 1 mole of the antibody, it is possible to obtain a complex that is excellent in storage stability, does not decrease the antibody titer, and does not decrease the anticancer effect. By masking the end-reactive aldehyde group in the conventional conjugate to reduce the reaction with the antibody, it is possible to stabilize the conjugate and to suppress the decrease in antibody titer. The present inventors have found that the complex acts on cancer cells more specifically, and have completed the present invention.
即ち、 本発明は、 アル ドへキソビラノ ー ス環の開裂したデス キ ト ラ ン制瘙作用物質及び瘙表面抗原に対する抗体よりなる制 癌作用物質複合体において、 抗体 1 モル当り 5 〜 3 0 モルの当 該デキス ト ラ ンが結合してなる制瘙作用物質複合体、 並びにァ ルドへキソピラノ ース環の開裂したデスキ ト ラ ン （以下、 開裂 デキス ト ラ ンという） のアルデヒ ド基に、  That is, the present invention relates to a complex of a carcinostatic agent comprising a dextran-inhibiting agent having a cleaved aldoseviroose ring and an antibody against a surface antigen, 5 to 30 mol per mol of the antibody. And a aldehyde group of a dextran which has a cleaved aldopyropyrose ring (hereinafter referred to as a cleaved dextran).
①アルデヒ ド基と反応性の制癌性物質を反応させる工程  ① Step of reacting aldehyde groups with reactive anticancer substances
(工程 A )  (Process A)
②当該アルデヒ ド基をマスキングする工程 （工程 B ) (2) Step of masking the aldehyde group (Step B)
③当該アルデヒ ド基に癌細胞表面に対する抗体 （以下、 単に 抗体という） を結合させる工程 （工程 C ) ③ Step of binding an antibody against the cancer cell surface (hereinafter simply referred to as “antibody”) to the aldehyde group (Step C)
をほどこしてなる制瘙作用物質複合体の製造方法更には開裂デ キス トラ ンを介して、 制瘙作用物質及び抗体を結合させた複合 体において、 開裂デキス ト ラ ンのアルデヒ ド基をマスキ ングす ることによつて抗体との反応性を低下せしめるる当該複合体の 安定性、 抗体価及び制癌作用の向上方法に関する。 A method for producing a conjugate comprising an inhibitor and an antibody via cleavage dextran The present invention relates to a method for improving the stability, antibody titer, and anticancer action of a complex by masking an aldehyde group of a cleaved dextran in a body to thereby reduce reactivity with an antibody.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 実施例 1 の複合体及び従来複合体 （巨大分子化し た抗 —フエ トブロティ ン · ゥマ抗体ーデキス ト ラ ン―ダウノ マイ シン複合体） の α —フユ トプロテイ ン産生肝芽腫細胞に対 する増殖抑制作用の比較を示すグラフであり、 第 2図は、 本発 明複合体と未反応デキス ト ラ ン—ダリ シン—ダウノマイ シ ン複 合体との分離を示す図である。  Fig. 1 shows the α-futoprotein-producing hepatoblastoma of the complex of Example 1 and the conventional complex (macromolecularized anti-fetobrotin-dima antibody-dextran-daunomycin complex). FIG. 2 is a graph showing a comparison of the growth inhibitory effect on cells, and FIG. 2 is a diagram showing the separation of the complex of the present invention and an unreacted dextran-daricin-daunomycin complex.
1 · · ♦ 本発明複合体 （実施例 1 )  1 · ♦ Complex of the present invention (Example 1)
2 ♦ ♦ · 従来の複合体  2 ♦ ♦ · Conventional complex
3 · · · 対照 ,  3 · · · contrast,
4 · · · 未反応のデキス ト ラ ン複合体  4 Unreacted dextran complex
発明の詳細な説明  Detailed description of the invention
本発明に用いる抗体は、 治療を意図する癌の表面抗原に対す る抗体であり、 抗原を動物に免疫して得られる動物由来特異抗 体、 遺伝子工学又は細胞融合法によつて得られるモノ ク ロ—ナ ル抗体などいずれのものでもよい。  The antibody used in the present invention is an antibody against a surface antigen of a cancer to be treated, and is a specific antibody derived from an animal obtained by immunizing an animal with the antigen, a monoclonal antibody obtained by genetic engineering or a cell fusion method. Any antibody such as a local antibody may be used.
デキス ト ラ ンは、 特に限定されるものでな く 、 好適には分子 量 1 0 0 0 〜 2 0 0万の範囲のものが用いられる。  The dextran is not particularly limited, and a dextran having a molecular weight in the range of 1,000 to 200,000 is preferably used.
制癌作用物質は、 アルデヒ ド基と反応性の基 （例えばァ ミ ノ 基、 水酸基） を有するものであればよ く 、 また、 かかる反応性 の基を有しないものについても、 当該制癌作用物質に適当な化 合物を反応させてアルデヒ ド基と反応性のものに導く こ とによ つて本発明に使用しう る。 好適な制瘙作用物質としてはたとえ ば、 ダウ ノ マイ シ ン、 マイ ト マイ シ ン C、 ア ド リ アマイ シ ンな どがあげられる。 The substance having an anticancer effect may be a substance having a group reactive with an aldehyde group (for example, an amino group or a hydroxyl group), and a substance having no such reactive group may be used. By reacting a substance with an appropriate compound to lead to one reactive with the aldehyde group This will be used in the present invention. Suitable inhibitory substances include, for example, dawn myosin, mitomycin C, and adriamycin.
本発明において、 アルデヒ ド基のマスキングとは、 アルデヒ ド基を抗体に対して不活化する こ とである。  In the present invention, masking the aldehyde group means inactivating the aldehyde group with respect to the antibody.
マスキ ングとしては、 例えばアルデヒ ド基を還元してヒ ドロ キシメ チル基に変ずる こと （還元剤としては、 例えば水素化ホ ゥ素金属塩、 水素化ホウ素シア ン金属塩などが挙げられる） 、 アルコ ール類 （例えば、 メ タノ ール、 エタノ ール、 プロ ノ、。ノ 一 ル、 エチ レ ング リ コ ール） による ァセタ ールの生成、 ア ミ ン化 合物 （特にア ミ ノ酸） による シ ッ フ塩基の生成等があげられる ァ ミ ノ酸としては、 たとえば次の如きも φが例示される。 中性ア ミ ノ酸 ： .  Masking includes, for example, reducing an aldehyde group to a hydroxymethyl group (reducing agents include, for example, metal borohydride and metal cyanide borohydride). Of acetals by alcohols (eg, methanol, ethanol, pranol, ethanol, ethylene glycol), and amide compounds (especially amino acids) Examples of the amino acids which include the generation of a Schiff base according to) include φ as shown below. Neutral amino acid:.
脂肪族ア ミ ノ酸 〔グリ シン、 ァラニン、 ノ リ ン、 ロイ シン、 イ ソ ロ イ シ ンなど〕 、 ォキ シア ミ ノ酸 〔セ リ ン、 ス レオニ ン など〕 、 舍硫ア ミ ノ酸 〔システィ ン、 シスチ ン、 メ チォニン など〕 、 芳香族ァ ミ ノ酸 〔フエ二ルァ ラ ニ ン、 チ ロ シ ン、 ト リ プ ト フ ァ .ンなど〕 - ' 酸性ァ ミ ノ酸 ：  Aliphatic amino acids (glycine, alanine, norin, leucine, isoloisin, etc.), oxamino acids (serine, threonine, etc.), sulphate amino Acids (cystine, cystine, methionine, etc.), Aromatic amino acids (phenylalanine, tyrosine, triptophan, etc.)-'Acidic amino acids:
ァスパラギン酸、 グルタ ミ ン酸など  Aspartic acid, glutamate, etc.
塩基性ア ミ ノ酸 ： Basic amino acids:
ヒスチジ ン、 リ ジ ン、 アルギニンなど  Histidine, lysine, arginine, etc.
ィ ミ ノ酸残基 ： Imino acid residue:
プロ リ ン、 ォキシプロ リ ンなど  Proline, oxyproline, etc.
—ァ ミ ノ酸以外のァ ミ ノ酸 ： ^ —ァラニンなど —Amino acids other than amino acid: ^ —Aranine and others
本発明は、 たとえば開裂デキス ト ラ ン、 即ち式  The present invention provides, for example, cleavage dextran,
Figure imgf000007_0001
Figure imgf000007_0001
(ただし、 分子量 1 0 0 0 〜 2 0 0万のものが好ま しい） のァ ルデヒ ド基に、 アルデヒ ド基と反応性の制癌作用物質を結合き せること、 当該デキス ト ラ ンのアルデヒ ド基をマスキングする こと、 及び当該アルデヒ ド基に抗体を結合させることを経て製 造されるが、 好適には上記の順序にて処理される。 (Although those having a molecular weight of 1,000 to 200,000 are preferred), an aldehyde group and a carcinogen that is reactive with the aldehyde group are bound to the aldehyde group, and the aldehyde of the dextran It is produced through masking of an aldehyde group and binding of an antibody to the aldehyde group, but is preferably performed in the above order.
上記開裂デキス ト ラ ンは公知であり、 その製法は、 Proc. Natl. Acad. Sci. USA, 78, 2128 (1976年） などに開示がある。 本発明の製造法によって得られる複合体における各成分の構 成の比は、 好まし く は抗体 1分子に対して、 デキス ト ラ ン 5 〜 3 0分子、 制瘙作用物質 2 0 〜 7 0分子であり、 残余のアルデ ヒ ド基は可及的多数がマスキ ングによって不活化されているこ とが好まし く 、 通常少な く とも 8 0 %のアルデヒ ド基がマスキ ングされている こ とが好適である。  The cleavage dextran is known, and its production method is disclosed in Proc. Natl. Acad. Sci. USA, 78, 2128 (1976) and the like. The composition ratio of each component in the complex obtained by the production method of the present invention is preferably 5 to 30 molecules of dextran and 20 to 70 molecules of the inhibitory substance per one molecule of the antibody. Preferably, the remaining aldehyde groups are molecules that are inactivated by masking as much as possible, usually at least 80% of the aldehyde groups are masked. Is preferred.
本発明の製造法の一例の概略は次の通りである。  The outline of an example of the production method of the present invention is as follows.
デキス ト ラ ンのアル ドへキソ ビラノ ース環の開裂は、 通常酸 化によって行われる。 酸化剤としては、 過ヨウ素酸ナ ト リ ウム を用いることが好ま しい。 たとえば、 デキス ト ラ ン 5 0 0 に 対して、 約 1 0 0 〜 7 0 の酸化剤を添加し 5 〜 2 4時間反 応を行う。 反応の終了後、 たとえば反応液を蒸溜水に対し 1 0 〜 3 0時間透折し、 好ま し く は凍結乾燥を行って酸化 （開裂） デキス ト ラ ンを得る。 Cleavage of the aldohyl viranose ring of dextran is usually carried out by oxidation. As the oxidizing agent, it is preferable to use sodium periodate. For example, add about 100 to 70 oxidizing agents to dextran 500 and react for 5 to 24 hours. Respond. After completion of the reaction, for example, the reaction solution is bent through distilled water for 10 to 30 hours, and preferably lyophilized to obtain oxidized (cleaved) dextran.
開裂デキス ト ラ ンと制瘙作用物質との反応に際して、 開裂デ キス ト ラ ン 1分子に対して制癌作用物質 2 〜 1 0分子を結合さ せることが好まし く 、 かかる比とするためには、 たとえば、 P H 6 〜 9 において水性溶媒 （好まし く は、 リ ン酸緩衝液） 中に、 開裂デキス ト ラ ンを最終濃度約 1 0 〜 3 0 W / V %、 制瘙作用 物質を最終濃度約 0. 5 〜 5 V %加えて、 5 〜 1 5時間程度 反応させることが好適である。 かく してデスキ ト ラ ン—制癌作 用物質が得られる。 未反応の制癌作用物質は、 たとえば ト ヨパ ール H W 4 0 などによる吸着ク ロマ トグラフィ ー、 その他自体 既知の手段にて除去される。  In the reaction between the cleaved dextran and the antibacterial substance, it is preferable to couple 2 to 10 molecules of the anticancer substance to one molecule of the cleaved dextran. For example, at pH 6-9, the final concentration of cleaved dextran in an aqueous solvent (preferably phosphate buffer) is about 10-30 W / V%, Is preferably added to a final concentration of about 0.5 to 5 V% and reacted for about 5 to 15 hours. Thus, a dextran-cancer substance is obtained. Unreacted anticancer agents are removed by, for example, adsorption chromatography using Toyopar HW40 or other means known per se.
かく して得られた複合体中のアルデヒ ド基をマスキングする に際しては、 当該複合体中の遊離のアルデヒ ド基の約 8 5 〜 9 5 %がマスキングされるに十分な条件を選択すればよい。  In masking the aldehyde groups in the complex thus obtained, conditions sufficient to mask about 85 to 95% of the free aldehyde groups in the complex may be selected. .
例えば、 還元処理は 1 〜 1 0 モルの還元剤によつて 0 〜 1 0 'cで 1 5 〜 2 0時間処理することが好ましい。 また、 α —ア ミ ノ酸を用いる場合は、 反応時間は通常 1 2 〜 3 0時間であり、 P Hは 5 〜 8程度であることが好ま しい。  For example, the reduction treatment is preferably performed with 0 to 10'c for 1 to 20 hours using 1 to 10 mol of a reducing agent. When α-amino acid is used, the reaction time is usually 12 to 30 hours, and the pH is preferably about 5 to 8.
かく して、 制癌作用物質-マスキ ングデキス ト ラ ン複合体が 得られる。 .  Thus, a carcinostatic substance-masking dextran complex is obtained. .
当該複合体に抗体を結合させるに際しては、 当該複合体 5 〜 3 0 V %を舍有する反応液に、 抗体を最終濃度が、 例えば 0. 1 〜 3 W Z V %になるように添加し、 冷所で 1 2 〜 4 0時間 反応させる。 When binding the antibody to the complex, the antibody is added to a reaction solution containing 5 to 30 V% of the complex so that the final concentration becomes, for example, 0.1 to 3 WZV%, and the mixture is cooled. In 12 to 40 hours Let react.
このようにして得られた本発明に関する複合体は、 ゲル濾過 担体ィォン交換樹脂、 抗原—セファ ロースなどにアプライ して 精製することができる。  The complex according to the present invention thus obtained can be purified by applying it to a gel filtration carrier ion exchange resin, antigen-Sepharose, or the like.
かく して得られた複合体は、 例えば除菌濾過を行つた後、 分 注し、 液状凍結品あるいは凍結乾燥した乾燥製剤とされる。  The complex thus obtained is, for example, sterilized and filtered, and then dispensed into a liquid frozen product or a lyophilized dry preparation.
本発明は、 従来の複合体が開裂'デキス ト ラ ンのアルデヒ ド基 に多数の抗体が結合して巨大分子化してしまう という欠点を、 当該アルデヒ ド基をマスキングすることにより、 防がんとする ものである。  The present invention solves the disadvantage that a large number of antibodies bind to the aldehyde group of a conventional cleavable dextran to form a macromolecule, thereby masking the aldehyde group to prevent cancer. That is what you do.
即ち、 従来の複合体では、 例えば分子量 10000 程度のデキス ト ラ ンを使用した場合、 分子量は 1 0 0万〜 7 0 0万であるの に対して、 本発明に関する複合体では、 1 5万〜 2 5万の分子 量をもち、 抗体 1分子に 5 〜 3 0分子のデキス ト ラ ンと多数 （ 2 0 〜 7 0分子） の制癌作用物質が結合したものである。 この 複合体は、 以下に述べるように巨大分子化したものに比べ、 安 定性、 性状さ らには癌細胞の増殖抑制作用において優れている。  That is, in the conventional complex, for example, when dextran having a molecular weight of about 10,000 is used, the molecular weight is 100,000 to 700,000, whereas in the complex of the present invention, 150,000 is used. It has a molecular weight of 2250,000, and is a combination of dextran of 5 to 30 molecules and a large number (20 to 70 molecules) of an anticancer agent per antibody molecule. This complex is more stable and more excellent in properties than the macromolecule as described below.
液状保存安定性に関して、 巨大分子化した従来の複合体では、 冷所保存においても、 1 ヶ月目で沈殺が認められ、 抗体活性の 低下が認められる。 また、 凍結乾燥処理により不溶物が生じや すい。 しかし、 本発明で得られた複合体では液状保存 1 ヶ月目 でも沈殺は認められず、 凍結乾燥後の保存安定性についても、 抗体活性の低下は認められない。  Regarding the liquid storage stability, the conventional macromolecule complex shows sedimentation in the first month and a decrease in antibody activity even in cold storage. In addition, insolubles are easily generated by freeze-drying. However, in the complex obtained in the present invention, no sedimentation was observed even after one month of liquid storage, and no decrease in antibody activity was observed in storage stability after freeze-drying.
抗体活性について、 本発明で得られた複合体と従来の複合体 とを比較すると、 従来の巨大分子化した複合体では、 抗体の 4 0 〜 6 0 %の活性を示すに過ぎないのに対して、 本発明で得ら れた複合体では、 ほぼ 1 0 0 %と抗体活性がそこなわれずに発 現している。 これは、 巨大分子化した複合体では、 デキス ト ラ ンに複数の抗体が結合しているために、 立体障害等により、 充 分な活性の発現が抑えられてしまう ことによると思われる。 When comparing the complex obtained according to the present invention with the conventional complex with respect to the antibody activity, the conventional macromolecularized complex shows that In contrast to the activity of only 0 to 60%, the complex obtained according to the present invention shows almost 100% of the antibody activity without impairing the antibody activity. This is probably because in the macromolecularized complex, since multiple antibodies are bound to dextran, the expression of sufficient activity is suppressed due to steric hindrance and the like.
電気泳動位置に関しては、 τ位にバン ドの認められる抗体に 対して、 巨大分子化した複合体では 位にバン ドが認められ、 また、 本発明で得られた複合体では、 その中間にバン ドが認め られる。 デキス ト ラン—制瘙作用物質の結合により、 荷電が変 化しているのがわかるが、 本発明で得られた複合体では、 より 元の抗体に近似した性質を持つことがわかる-。  Regarding the position of the electrophoresis, a band was recognized in the macromolecule complex, and a band was found in the middle of the complex obtained by the present invention, with respect to the antibody having a band at the τ position. Is allowed. It can be seen that the charge has been changed by the binding of the dextran-suppressing agent, but the complex obtained in the present invention has properties closer to those of the original antibody.
-フユ トプロティ ン産生肝芽腫培養株を用いた i n v i tro 実験では、 第 1図のように、 細胞数から求めた細胞増殖抑制効 果は、 従来の複合体より、 本発明で得られた複合体の方が、 よ り優れているという結果が得られている。  In an in vitro experiment using -futoprotein-producing hepatoblastoma cultures, as shown in Fig. 1, the cell growth inhibitory effect obtained from the cell number was higher than that of the conventional complex. The results show that the body is better.
以上のように、 従来の巨大分子化した複合体に比べて本発明 で得られた複合体は、 デキス ト ラ ンを介して抗体 1分子に多数 の制癌作用物質が結合可能であり、 安定性、 抗体活性に優れ、 抗体本来の性質に近いものであって、 このものの標的細胞への 到達しやすさや膜透過性の良さ等の有利な面があげられる。 ま た、 凍結乾燥製剤としての安定性も認められている。  As described above, the complex obtained by the present invention, compared to the conventional macromolecularized complex, allows a large number of anticancer agents to bind to one antibody molecule via dextran, and is stable. It has excellent properties and antibody activity, and is close to the original properties of antibodies, and has advantages such as easy access to target cells and good membrane permeability. In addition, its stability as a lyophilized product has been confirmed.
本発明で得られた複合体の臨床使用における投与量および投 与方法は、 10〜300 m gの本品を日本薬局方注射用蒸留水 0 . 5〜 5 m lに溶解し、 年齢、 症状および経過に応じて、 適宜加減して 静脈内注射、 点滴静注、 点滴注射して用いる。 実験例 1 The dosage and administration method of the conjugate obtained in the present invention for clinical use are as follows: 10 to 300 mg of this product is dissolved in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia, and the age, symptoms and progress Intravenous injection, intravenous drip infusion, and drip injection are used as appropriate according to the requirements. Experimental example 1
後記実施例.1 で得られた複合体及び比較として、 従来の抗 The complex obtained in Example 1.
— フ エ ト プロ ティ ン · ゥマ抗体ーデキス ト ラ ン―ダウ ノ マイ シ ン複合体 （分子量 1 0 0 X 1 0 4 ) を、 4 で にて保存したとこ ろ、 従来品は 1 ヶ月で沈澱が認められるのに対して、 本発明で 得られた複合体は、 1 ヶ月を経ても沈殺は認められなかった。 また、 両者の抗体活性を Ρ Η Α法にて測定したところ、 従来品 は 4 0〜 6 0 %の活性の低下が認められたのに対して、 実施例 1 の複合体は、 1 ヶ月後においても活性の低下は全く認められ なかった。 - off-et-door professional tee down-© Ma antibody Dekisu door run-- Dow Roh My Thin complex (molecular weight 1 0 0 X 1 0 4) , saved Toko filtration at 4, the conventional product in one month Precipitation was observed, whereas the complex obtained in the present invention did not show any slaughter even after one month. When the antibody activities of both antibodies were measured by the Ρ Η method, the activity of the conventional product was reduced by 40 to 60%. No decrease in activity was observed at all.
さ らに、 両複合体を凍結乾燥したところ、 従来品は不溶物が 生じたのに対して、 実施例 1 の複合体は不溶物が生ぜず、 また、 その活性の低下は認められなかった。  Furthermore, when both complexes were freeze-dried, insolubles were generated in the conventional product, whereas insolubles were not generated in the complex of Example 1 and no decrease in the activity was observed. .
実験例 2 Experimental example 2
実施例 1 で得られた複合体、 及び実験例 1 で用いた従来の複 合体の α —フユ トプロ ティ ン産生肝芽腫細胞 1 . O x 10 5 偭 m l に対する増殖抑制作用の比較実験を行っ た。 その結果は第 1図 に示す通りである。 A comparative experiment was conducted on the growth inhibitory effect of the complex obtained in Example 1 and the conventional complex used in Experimental Example 1 on α-futoprotein-producing hepatoblastoma cells 1.0 × 10 5 ml. Was. The results are as shown in Fig. 1.
なお、 培地は RPM I 1640 (日水製薬社製） を用いた。  The medium used was RPMI 1640 (manufactured by Nissui Pharmaceutical Co., Ltd.).
実施例 1 Example 1
デキス ト ラ ン T— 1 0、 1 0 0 |^を 0. 0 1 5 ^の過ョゥ素酸 ナ ト リ ウ ム 2 0 m 1 に溶解後、 遮光して室缉 8時間処理後、 4 てで 1晩、 蒸留水に対して充分に透折した。 その後、 凍結乾燥 して、 乾'燥酸化デキス ト ラ ン 1 0 0 mgを得た。  After dissolving dextran T—10, 10 0 | ^ in 0.015% sodium periodate 20 m 1, and after shielding from light in a room for 8 hours, After 4 nights, the membrane was sufficiently broken through distilled water. Thereafter, the resultant was freeze-dried to obtain 100 mg of dried and oxidized dextran.
こ の乾燥酸化デキス ト ラ ン 1 0 Q mgを食塩加リ ン酸缓衝液、 pH 9. 0 、 0. 5 m lに溶解後、 同量の同液で溶解したダウノ マイ シ ン 1 0 mgを混合して室温で 1時間反応させた。 続いて、 反応液 を トョパール樹脂をつめた力ラムに通して、 遊離のダウノ マイ シンを除去し、 通過液にグリ シ ン 5 0 mgを添加後、 4 でで 2 4 時間反応させた。 つぎに、 これに肝細胞性肝癌の表面抗原に対 する抗体の一種である抗ヒ ト ' アルフ ァ フヱ トブロティ ンゥ マ 抗体を 1 2. 5 〜 2 0 m g添加後、 4 でで 2 4時間反応させ、 反応 液をセフアデッ クス G— 2 0 0 のゲル濾過力 ラムにかけ、 本発 明の複合体の面分を得た （第 2図） 。 さらに、 これに安定剤を 添加して、. 凍結乾燥製剤を得た。 - 得られた複合体の性状は、 ダウノ マイ シ ンノ抗体モル比 4 0 〜 5 0 、 デキス ト ラ ンズ抗体モル比 1 2 、 残存抗体活性、 ほぼ 1 0 0 分子量 2 5万、 電気泳動位置 2 〜 r位という もの であった。 10 Q mg of the dried oxidized dextran was added to a phosphate-buffered saline solution, After dissolving in 0.5 ml of pH 9.0, 10 mg of daunomycin dissolved in the same amount of the same solution was mixed and reacted at room temperature for 1 hour. Subsequently, the reaction solution was passed through a force ram filled with TOPEARL resin to remove free daunomycin, 50 mg of glycine was added to the flow-through solution, and the mixture was reacted at 4 for 24 hours. Next, after adding 12.5 to 20 mg of anti-human alpha-brominated antibody, a kind of antibody against the surface antigen of hepatocellular liver cancer, 4 to 24 hours After the reaction, the reaction mixture was applied to a gel filtration column of Sephadex G-200 to obtain a surface area of the complex of the present invention (FIG. 2). Further, a stabilizer was added thereto to obtain a freeze-dried preparation. -The properties of the obtained complex were as follows: Daunomycin antibody molar ratio: 40-50, Dextrans antibody molar ratio: 12, Residual antibody activity: Almost 100, molecular weight: 250,000, Electrophoresis position: 2 ~ R rank.
実施例 2 . Example 2.
デキス ト ラ ン T — 4 0 、 1 gを 0. 0 3 Mの過ヨ ウ素酸ナ ト リ ゥ ム 1 0 0 m lに溶解後、 遮光して、 室温 8時間処理後、 4 'cで 1晩蒸溜水に対して透折した。 その後、 凍結乾燥して乾燥酸化 デキス ト ラ ンを得た。  After dissolving 1 g of dextran T — 40 g in 100 ml of 0.03 M sodium periodate, shield from light, treat at room temperature for 8 hours, and use 4'c Folded over distilled water overnight. Then, it was freeze-dried to obtain a dried oxidized dextran.
この乾燥酸化デキス ト ラ ン 5 0 m gを生理的食塩液 0. 5 m lに溶 解後、 同量の同液で溶解したァ ド リ アマイ シ ン 5 mgを混合して、 室温で 1時間反応させた。 反応液を トョパール樹脂を用いてバ ッチ法で処理し、 処理後液にリ ジ ン 2 0 を添加後、 4 °cで 2 4時間反応させた。 つぎに、 これにヒ トメ ラノ ーマに対するモ ノ ク ロ ーナル抗体 （マウ ス由来） を 2 m g添加後、 4 。C で 4 0時 間反応させ、 反応液をセフア ク リル S 3 0 0 のゲル濾過により、 本発明の複合体の画分をプールし、 安定剤を添加して凍結乾燥 製剤を得た。 After dissolving 50 mg of the dried oxidized dextran in 0.5 ml of physiological saline, mix with 5 mg of adoramicin dissolved in the same amount of the same solution, and allow the mixture to stand at room temperature for 1 hour. Reacted. The reaction solution was treated by a batch method using Toyopearl resin, and after the treatment, lysine 20 was added to the solution, followed by a reaction at 4 ° C for 24 hours. Next, 2 mg of a monoclonal antibody (derived from mouse) against human melanoma was added to the solution. 4 o'clock at C The reaction mixture was subjected to gel filtration of Sephacryl S300 to pool the fractions of the complex of the present invention, and a stabilizer was added to obtain a lyophilized preparation.

Claims

請求の範囲 The scope of the claims
(1) アルドへキソビラノ ース環の開裂したデスキ ト ラ ン、 制瘙 作用物質及び癌表面抗原に対する抗体よりなる制癌作用物質複 合体において、 抗体 1 モル当り 5 〜 3 0 モルの当該デキス ト ラ ンが結合してなる制瘙作用物質複合体。  (1) In a complex of a carcinostatic substance consisting of a dextran which has cleaved an aldohexobilanose ring, a carcinogenic substance and an antibody against a cancer surface antigen, 5 to 30 mol of the dextran per 1 mol of the antibody A complex of inhibitory substances formed by binding of lan.
(2) 抗体 1分子に対して 5 〜 3 0分子のデキス ト ラ ン、 2 0 〜 7 0分子の制瘙作用物質が結合してなる請求の範囲第 (1)項記載 の制癌作用物質複合体の製造法。  (2) The anticancer substance according to claim (1), wherein 5 to 30 molecules of dextran and 20 to 70 molecules of an anticancer substance are bound to one molecule of the antibody. A method for producing a composite.
(3) 制癌作用物質がダウノ マイ シンである請求の範囲第 (1)また は (2)項記載の複合体。  (3) The complex according to (1) or (2), wherein the anticancer substance is daunomycin.
(4) 制癌剤がァ ドリ ァマイ シンである請求の範囲第（1)または (2) 項記載の複合体。 (4) The complex according to (1) or ( 2 ), wherein the anticancer agent is adoriamycin.
(5) アル ドへキソ ビラノ ース環の開裂したデスキ ト ラ ンのアル デヒ ド基に、  (5) The aldehyde group of the dextran in which the aldo-hexa-vilanose ring is cleaved is
①アルデヒ ド基と反応性の制癌性物質を反応させる工程  ① Step of reacting aldehyde groups with reactive anticancer substances
(土程 A )  (Site A)
②当該アルデヒ ド基をマスキ ングする工程 （工程 B ) (2) Step of masking the aldehyde group (Step B)
③当該アルデヒ ド基に癌細胞表面抗原に対する抗体を結合さ せる工程 （工程 C ) · (3) Step of binding an antibody against the cancer cell surface antigen to the aldehyde group (Step C)
をほどこ してなる制瘙作用物質複合体の製造法。 A method for producing a complex with a control substance.
(6) 工程 A、 工程 B、 工程 Cの順序で処理するこ とを特徴とす る請求の範囲第 (1)項記載の制瘙作用物質複合体の製造法。  (6) The method for producing an inhibitory substance complex according to claim (1), wherein the treatment is performed in the order of step A, step B, and step C.
PCT/JP1985/000214 1983-10-21 1985-04-17 Process for preparing carcinostatic substance complex WO1986005986A1 (en)

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JP19813183A JPS6089433A (en) 1983-10-21 1983-10-21 Preparation of carcinostatic substance composite
PCT/JP1985/000214 WO1986005986A1 (en) 1983-10-21 1985-04-17 Process for preparing carcinostatic substance complex

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WO1986005986A1 true WO1986005986A1 (en) 1986-10-23

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Application Number Title Priority Date Filing Date
PCT/JP1985/000214 WO1986005986A1 (en) 1983-10-21 1985-04-17 Process for preparing carcinostatic substance complex

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 101, No. 21, 189406t. *
CHEMICAL ABSTRACTS, Vol. 99, No. 22, 181404x. *
International Journal of Cancer, Vol. 21, No. 6, (1978), E. HURWITZ et al., "The Effect in Vivo of Chemotherapeutic Drug-Antibody Conjungates in Two Murine Experimental Tumor Systems", p747-755. *
Journal of Medicinal Chemistry, Vol. 28, No. 1, (1985), E. HURWITZ et al., "The Covalent Linking of Two Nucleotide Analogues to Antibodies", p137-140. *

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