JPH07507570A - Anti-zona pellucida antibodies for delivery of therapeutic agents to the ovary - Google Patents
Anti-zona pellucida antibodies for delivery of therapeutic agents to the ovaryInfo
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- JPH07507570A JPH07507570A JP6517261A JP51726194A JPH07507570A JP H07507570 A JPH07507570 A JP H07507570A JP 6517261 A JP6517261 A JP 6517261A JP 51726194 A JP51726194 A JP 51726194A JP H07507570 A JPH07507570 A JP H07507570A
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- therapeutic agent
- antibody
- zona pellucida
- pharmaceutical composition
- ovary
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
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- 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
- A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
Abstract
(57)【要約】本公報は電子出願前の出願データであるため要約のデータは記録されません。 (57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】 治療剤の卵巣への送達のための抗透明帯抗体Ｉユ立ユＩ この発明は、透明帯に特異的に結合する抗体及びそれらの抗体から作られる免疫 化学薬剤、並びにかがる免疫化学薬剤を用いる治療方法に関するものである。[Detailed description of the invention] Anti-zona pellucida antibody I for delivery of therapeutic agents to the ovary This invention provides antibodies that specifically bind to the zona pellucida and antibodies produced from these antibodies. The present invention relates to chemical agents and therapeutic methods using immunochemical agents.
ｌ１立１１ 卵巣の病気特に卵巣癌は、好ましくは、特異的に卵巣を標的とする薬剤により治 療する。更に、受精率制御のための薬剤は、かかる薬剤が特異的に卵巣に向けら れて分配され、他の卵巣でない組織で希釈されないときに一層効果的であり得る 。l1 standing 11 Ovarian diseases, particularly ovarian cancer, are preferably treated with drugs that specifically target the ovaries. Treat. Furthermore, drugs for fertility control may be used to control fertility if such drugs are specifically directed to the ovaries. May be more effective when distributed as .
嗜乳動物の透明帯は、卵巣中にある卵母細胞を囲んでいる。その独特の構造は、 新規な糖蛋白質ネットワークを含む。透明帯蛋白質の独特の性質の故に、これら の蛋白質に対して高められた抗体は、一般に、？ｉ（巣に対して特異的である。In mammals, the zona pellucida surrounds the oocyte in the ovary. Its unique structure is Contains a novel glycoprotein network. Because of the unique properties of zona pellucida proteins, these Antibodies raised against proteins in ? i (specific for the nest).
従って、抗透明帯抗体は卵巣を標的とする治療剤のキャリアーとして理想的に適 している。Therefore, anti-zona pellucida antibodies are ideally suited as carriers for therapeutic agents targeting the ovary. are doing.
腫瘍組織を標的とした細胞毒性剤の送達の目的のために、細胞毒性剤が卵巣腫瘍 細胞及び特異的な腫瘍細胞の抗原に結合された（例えば、Ｂｊｏｒｎ等の米国特 許第４゜９５６．４５３号、Ｐａ５ｔａｎ等の第４．８０６．４９４号及びＢｙ ｅｒｓ等の４．９２５，９２２号を参照されたい）。この卵巣への薬剤の送達は 、卵巣組織が抗体により認識される特異的な腫瘍抗原を含むことを必要とする。For the purpose of cytotoxic agent delivery targeted to tumor tissue, cytotoxic agents can be used to target ovarian tumors. cells and specific tumor cell antigens (e.g., Bjorn et al. No. 4.956.453, Pa5tan et al. No. 4.806.494 and By 4.925,922). This delivery of drugs to the ovaries is , requires that ovarian tissue contain specific tumor antigens that are recognized by antibodies.
かかる抗体認識は、抗体が患者自身の腫瘍細胞に対して高められたときに期待さ れるが、腫瘍抗原は、−人の患者と他の者とで変わり、単一抗体がすべての患者 の卵巣に細胞毒性剤を運ぶことは期待出来ない。更に、卵巣の非発癌性条件は、 治療剤の標的への送達から恩恵を得るであろう。治療剤の標的への特異的な送達 は、一層少量の治療剤及び非特異的薬剤の特異的な卵巣作用を可能にすることが 望まれる。治療剤の標的への特異的な送達は、治療剤特に正常組織に有害であろ うものを一層少量で使用することを可能にし及び非特異的薬剤の特異的な卵巣作 用を可能にすることが望まれる。治療剤を特異的に卵巣を標的とすることにおい て用いるための抗体であって患者間で変わる抗原の認識に依存しない抗体を提供 することは非常に有用なことである。Such antibody recognition is expected when antibodies are raised against the patient's own tumor cells. However, tumor antigens vary from one patient to another, and a single antibody cannot be expected to deliver cytotoxic agents to the ovaries of patients. Furthermore, non-carcinogenic conditions of the ovary are would benefit from targeted delivery of therapeutic agents. Targeted specific delivery of therapeutic agents may enable specific ovarian effects of smaller amounts of therapeutic agents and non-specific agents. desired. Specific delivery of a therapeutic agent to a target is important because the therapeutic agent may be particularly harmful to normal tissues. allows the use of smaller amounts of ovarian fluid and eliminates the specific ovarian action of non-specific drugs. It is desirable to enable the use of In targeting therapeutic agents specifically to the ovary Provides an antibody for use in patients that does not depend on antigen recognition that varies between patients. It is a very useful thing to do.
ｌユニｌ上 透明帯抗原に対して高められたモノクローナル及びポリクローナル抗体は、所望 の治療剤を卵巣以外の組織への有意の損失を伴わずに特異的に卵巣に運ぶという ことが見出された。かかる抗体は、卵巣間で有意の認識の変動を伴わずに治療剤 を特異的に卵巣に向けるために用いられる。好適具体例において、抗透明帯抗体 は、特定の受精及び避妊効果のために薬剤を卵巣を標的として送達する。l uni l top Raised monoclonal and polyclonal antibodies against zona pellucida antigens The drug is delivered specifically to the ovary without significant loss to tissues other than the ovary. It was discovered that Such antibodies can be used as therapeutic agents without significant recognition variation between ovaries. used to specifically target the ovaries. In a preferred embodiment, an anti-zona pellucida antibody Targets delivery of drugs to the ovaries for specific fertilization and contraceptive effects.
日の６細なｆ日 抗透明帯抗体は、哨乳動物の全透明帯若しくは細分した透明帯に対して高められ た抗体及び特定の透明帯蛋白質に対して高められた抗体（天然若しくは組換え体 又はそれらの断片）を含む。これらの抗体は、ゲラ歯頚以外が好適であるが、任 意の哨乳動物種の透明帯に対して高めることが出来る。ゲラ菌類以外の透明帯抗 体はゲラ歯頚の透明帯と交差反応性でないが、非ゲッ巾動物種間では交差反応性 であることが報告されている。6 small f days of the day Anti-zona pellucida antibodies are raised against the entire zona pellucida or subdivided zona pellucida in sentinel mammals. and antibodies raised against specific zona pellucida proteins (natural or recombinant). or fragments thereof). These antibodies are suitable for antibodies other than the galley tooth neck, but can be applied to any target. It can be enhanced against the zona pellucida in mammalian species. Zona pellucida other than Gera fungi The body is not cross-reactive with the zona pellucida of the gelatinous neck, but it is cross-reactive among non-germinous species. It has been reported that
抗体（モノクローナル又はポリクローナルのもの）は、一般にこの分野で公知の 方法によって調製することが出来る。一般に、ポリクローナル抗体は、宿主動物 において透明帯抗原を宿主動物に注射することによって高められる。宿主動物の 血清を取り出して分画して抗血清を得る。抗血清を、透明帯及び透明帯抗原との 示された反応性により抗透明帯抗体の存在についてスクリーニングする（例えば 、公知のイムノアッセイ技術による）。Antibodies (monoclonal or polyclonal) are generally of the type known in the art. It can be prepared by a method. Generally, polyclonal antibodies are by injecting the host animal with zona pellucida antigen. host animal Serum is extracted and fractionated to obtain antiserum. Antiserum was tested against zona pellucida and zona pellucida antigen. Screen for the presence of anti-zona pellucida antibodies by demonstrated reactivity (e.g. , by known immunoassay techniques).
これらの抗体は又、この分野で公知の方法例えば免疫細胞化学又はウェスタンプ ロット分析によって、卵巣組織に対する特異性についてもスクリーニングする。These antibodies can also be prepared using methods known in the art, such as immunocytochemistry or Western PCR. Lot analysis will also screen for specificity to ovarian tissue.
モノクローナル抗体は、この分野で公知の方法によってハイブリドーマ細胞がら 生成される。一般に、宿主動物に透明帯抗原を腹腔内注射する。免疫化動物の肺 臓を採取し、これを、Ｋｏｈｌｅｒ及びＭｉｌｓｔｅｉｎ、 Ｎａｔｕｒｅ、 １９７５゜２５６：４９５−４９７の一般的な細胞雑種形成技術を用いて、マウ ス腫瘍パートナ−と共に用いてバイブリドーマを調製する。この技術は、腫瘍細 胞と肺臓細胞とをポリエチレングリコール等の融合剤を用いて融合させることを 含んでいる。融合の後に、雑種細胞を融合培地から分離し、選択成長培地（例え ば、ＨＡＴ培地）にて成長させて雑種形成してない親細胞を除く。所望であれば 、ハイブリドーマを増殖させて条件培養培地を、抗透明帯活性について従来のイ ムノアッセイ手順（ラジオイムノアッセイ、酵素イムノアッセイ又は蛍光イムノ アッセイを含む）をによってアッセイする。Monoclonal antibodies are isolated from hybridoma cells by methods known in the art. generated. Generally, the host animal is injected intraperitoneally with the zona pellucida antigen. Lungs of immunized animals The viscera was collected and used as described by Kohler and Milstein, Nature, 1975°256:495-497 using general cell hybridization techniques. A hybridoma is prepared using a tumor partner. This technique Fusing cells and lung cells using a fusion agent such as polyethylene glycol Contains. After fusion, the hybrid cells are separated from the fusion medium and placed in a selective growth medium (e.g. (e.g., HAT medium) to remove non-hybridized parental cells. if desired , hybridomas were grown and conditioned culture media were tested for anti-zona pellucida activity using conventional methods. immunoassay procedures (radioimmunoassay, enzyme immunoassay or fluorescent immunoassay) assay).
モノクローナル抗体を生成するハイブリドーマは、公知の手順を用いて、イン・ ビトロ又はイン・ビボで成長させることが出来る。好ましくは、これらのバイプ リドーマをマウスの腹水として維持する。これらのモノクローナル抗体を培養培 地又は体液から硫安沈殿、ゲル電気泳動、透析、クロマトグラフィー及び所望で あれば限外濾過等の従来の免疫グロブリン生成手順によって単離することが出来 る。Hybridomas producing monoclonal antibodies are produced in vitro using known procedures. It can be grown in vitro or in vivo. Preferably these vipes Maintain the ridoma as ascites in mice. These monoclonal antibodies were added to the culture medium. ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and optionally If available, they can be isolated by conventional immunoglobulin production procedures such as ultrafiltration. Ru.
本発明のモノクローナル及びポリクローナル抗透明帯抗体の重要な性質は、それ らの透明帯抗原との反応性及び卵巣組織に対する特異性を含む。本発明の抗体は 、治療される種と同じ種の透明帯抗原に対して高めることが出来（自己抗原）、 又は第２の種の透明帯に対して高めることが出来る（外来抗原）（種は、抗原性 交差反応性を示す）。例久ば、第２の宿主棟内で第１の種の透明帯に対して生成 されたポリクローナル抗血清は、ウェスタンプロット、ＥＬ Ｉ ＳＡ又は免疫 組織化学的技術により試験した際に、第１、第２及び更なる種の透明帯を認識す ることが出来る。抗体は、第１、第２又はその他の種の卵巣へのかかる薬剤の特 異的送達における利用のために、標識し又は治療剤と結合することが出来る。同 様に、組換λの透明帯蛋白質（外来又は自己抗原）及び合成により生成した透明 帯ペプチドを抗原として用いてこの方法の発明において有用な抗体を生成するこ とが出来る。An important property of the monoclonal and polyclonal anti-zona pellucida antibodies of the invention is that they including their reactivity with zona pellucida antigens and specificity for ovarian tissue. The antibody of the present invention , can be raised against zona pellucida antigens of the same species as the species being treated (autoantigens); or can be raised against the zona pellucida of a second species (foreign antigen) (species has antigenic (indicating cross-reactivity). For example, it is generated against the zona pellucida of the first species in the second host building. The polyclonal antiserum was analyzed by Western blot, EL ISA or immunoassay. The first, second and further species of zona pellucida are recognized when examined by histochemical techniques. Rukoto can. The antibodies may be specific to the ovaries of the first, second or other species. It can be labeled or conjugated with a therapeutic agent for use in heterogeneous delivery. same Similarly, recombinant λ zona pellucida protein (foreign or autoantigen) and synthetically produced zona pellucida The band peptide can be used as an antigen to generate antibodies useful in this method invention. I can do that.
所望の免疫化学剤を形成するために、抗透明帯抗体を公知の方法によって化学治 療剤に結合する。この結合は、種々の二官能性蛋白質カップリング剤を用いて行 なうことが出来る。かかる試薬の例は、Ｎ−スクシンイミジル−３−（２−ピリ ジルジチオ）プロピオネート、イミノチオラン、イミドエステルの二官能性誘導 体、アルデヒド例＾ばグルタルアルデヒド、ビス−アジド化合物例えばビス（Ｐ −ジアゾニウムベンゾイル）−エチレンジアミン、ジイソシアネート例λばトリ レン２．６−ジイソシアネート、及びビスー活性フルオレン化合物例えば１．５ −ジフルオロ−２，４−ジニトロベンゼン等である。Anti-zona pellucida antibodies can be chemically treated by known methods to form the desired immunochemical agent. binds to therapeutic agents. This coupling can be performed using a variety of bifunctional protein coupling agents. I can become. An example of such a reagent is N-succinimidyl-3-(2-pyri Bifunctional induction of dildithio)propionate, iminothiolane, and imidoester aldehydes such as glutaraldehyde, bis-azide compounds such as bis(P -diazoniumbenzoyl)-ethylenediamine, diisocyanate e.g. 2.6-diisocyanate, and bis-active fluorene compounds e.g. 1.5 -difluoro-2,4-dinitrobenzene and the like.
抗透明帯抗体に結合するための所望の治療剤の例は、放射性同位体、化学療法剤 、細胞毒素等を含む。放射性同位体は、好ましくは、高い線エネルギー放射性の 同位体例えば、Ｙ、Ｐｒ等である。この細胞毒素は、細胞毒性薬剤又は細菌、カ ビ又は植物起源の酵素的に活性な毒素であってよい。かかる酵素的に活性な毒素 の例は、ジフテリア毒素Ａ、外毒素Ａ、リシンＡ、アブリンＡ、モデシンＡ、ア ルファサルシン イシンである。好ましい細胞毒素は、リポソーム阻害蛋白質例えばりシンのＡ鎖 又はアブリンのＡ鎖である。リシンのＡ鎖は、米国特許第４，５９０，０７１号 で開示されているように細胞毒性結合体に用いられている。Examples of desired therapeutic agents for binding to anti-zona pellucida antibodies include radioisotopes, chemotherapeutic agents , cytotoxins, etc. The radioactive isotope is preferably a high linear energy radioactive Isotopes such as Y, Pr, etc. This cytotoxin is a cytotoxic drug or a bacterial or bacterial The toxin may be an enzymatically active toxin of bacterial or plant origin. such enzymatically active toxins Examples are diphtheria toxin A, exotoxin A, ricin A, abrin A, modecin A, Rufasarcin It's Ishin. A preferred cytotoxin is the A chain of liposome inhibitory protein such as lysin. Or the A chain of abrin. The A chain of ricin is disclosed in U.S. Patent No. 4,590,071. have been used in cytotoxic conjugates as disclosed in .
本発明の治療用免疫化学剤は、卵巣を特異的に標的とする治療剤の送達が望まし い種々の治療応用に用いることが出来る。かかる治療応用の例は、卵巣癌、受精 率制御等を含む。好適具体例において、リシン結合した抗透明帯抗体を用いて， 標的の卵巣中の原始濾胞の補充を加速する。加速した補充は，例えば、後期ステ ージ例えば第三次濾胞に有効な避妊方法において有用である．この抗透明帯抗体 は、細胞毒性削りシンを特異的に卵巣へ送達する．リシン処理は、卵巣からの大 型濾胞（例えば、第三次濾胞）の消失を生じ且つ成長プールへの濾胞（例えば、 原始濾胞）の補充をも生じる（該プールにおいてそれらは排卵誘発剤により作用 を受け得る）。The therapeutic immunochemical agent of the present invention desirably delivers a therapeutic agent that specifically targets the ovary. It can be used for a variety of therapeutic applications. Examples of such therapeutic applications are ovarian cancer, fertilization. Including rate control etc. In a preferred embodiment, a lysine-conjugated anti-zona pellucida antibody is used to Accelerates the replenishment of primordial follicles in the target ovary. Accelerated replenishment may occur during late stages, e.g. For example, it is useful in contraceptive methods that are effective against tertiary follicles. This anti-zona pellucida antibody delivers cytotoxic shavingsin specifically to the ovary. Ricin treatment removes large amounts from the ovary. resulting in loss of type follicles (e.g. tertiary follicles) and follicles (e.g. This also results in the replenishment of primordial follicles (in which pool they are acted upon by ovulation-inducing agents). ).
本発明の免疫化学剤は、公知の従来の方法例えば注射、吸入又は卵巣への送達用 に改変した方法によって生者に投与することが出来る。好ましくは、この薬剤を 、非経口で、例えば、静脈経由、腹腔経由等によって投与する。この薬剤を、製 薬上許容し得るキャリアー中、例文ば、水性媒質例えば水、緩衝液、塩溶液、グ リシン、又は油ベースのキャリアー中で、特異的抗体−薬剤カップルに適するよ うに調製する。治療上有効な量即ち患者のＩｌｌ ｆｌの成長を除き、減じ若し くは遅らせるか又は所望の受精率効果を誘導する量を投与する。治療上有効な投 与量及び投与量養生法は、患者の特定の特性、治療適応症及び治療剤の性質によ って変化する。この投与量及び養生法は、治療する内科医が、この分野で公知の 許容し得る手順によって計算することが出来る。The immunochemical agents of the invention can be delivered by known conventional methods such as injection, inhalation or delivery to the ovary. It can be administered to living subjects by a method modified from . Preferably, this drug , administered parenterally, for example, intravenously, intraperitoneally, etc. This drug is manufactured in a pharmaceutically acceptable carrier, eg, an aqueous medium such as water, buffers, saline solutions, gels, etc. in a lysine- or oil-based carrier as appropriate for specific antibody-drug couples. Prepare sea urchin. A therapeutically effective amount, i.e., removes, reduces or reduces the growth of the patient's Illfl. administer an amount that delays or induces the desired fertility effect. Therapeutically effective Dosage and dosage regimen will depend on the specific characteristics of the patient, the therapeutic indication and the nature of the therapeutic agent. It changes. This dosage and regimen will be determined by the treating physician as known in the art. It can be calculated by an acceptable procedure.
一般に、治療上有効な投与量は、愚者の体重に対して計算した治療剤の計算され たＬＤ、。より少ない。薬剤の量は、典型的に、約０．Ｏｌ 〜１００ｍｇ／ｋ ｇ体重（好ましくは、０．０１〜１０ｍｇ／ｋ ｇ）の範囲内にある。Generally, the therapeutically effective dose is the calculated dose of the therapeutic agent relative to the patient's body weight. LD,. Fewer. The amount of drug is typically about 0. Ol ~100mg/k g body weight (preferably 0.01 to 10 mg/kg).
この発明は、下記の実施例をづ照することにより、−暦良く理解され得る。This invention can be better understood by referring to the following examples.
夫１０１± 一゛０刊″Ｉこの１−。Husband 101± 1゛0th edition''I this 1-.
熱可溶化したイヌの透明帯（Ｈ５ＤＺ）にヌ・ｊする抗透明帯抗体を、一般に、 Ｄｕｎｂａｒ等（Ｂｉｏｃｈｅｍｉｓｔｒ 、 １９：３５６−３６５．１９８ ０）により記載された手順に従って調製した（イリし、揃えたカミソリの刃を用 いて卵巣をミンスした）、Ｈ３ＤＺ （２５０ＩＬｇ）とＭＤＰ （２５０ｕｇ ）との混合物でウサギを免疫化した。約３週間の間隔で更なる２回の増強を追加 した。その結果生じたウサギ血清を、次いで、ＩｇＧ分画のために用いた。Anti-zona pellucida antibodies that bind to heat-solubilized canine zona pellucida (H5DZ) are generally Dunbar et al. (Biochemistr, 19:356-365.198 Prepared according to the procedure described by minced ovaries), H3DZ (250ILg) and MDP (250ug ) was used to immunize rabbits. Added 2 more boosts approximately 3 weeks apart did. The resulting rabbit serum was then used for IgG fractionation.
ザイムド（Ｚｙｍｅｄ ）プロティンＡカラムと共に与えられた指示に従って、 ＩｇＧ画分を採取した。集めた抗血清を１時間プロティンＡカラム上でインキュ ベートした。次いで、カラムをＰＢＳで４回洗い、ＩｇＧを０．１Ｍ酸性酸（ｐ Ｈ２，８）を用いて溶出し、直ちに１Ｍトリス（ｐＨ９，５）で緩衝した。集め た試料を、次いで、ＰＢＳ （ｐＨ７，２）に対して長（透析した。Following the instructions given with the Zymed Protein A column, The IgG fraction was collected. The collected antiserum was incubated on a protein A column for 1 hour. I bet. The column was then washed four times with PBS and the IgG was washed with 0.1M acidic acid (p Elution was performed with H2.8) and immediately buffered with 1M Tris (pH 9.5). gather The sample was then dialyzed against PBS (pH 7.2).
このＩｇＧ画分のＥＬＩＳＡ力価は、ＨＳ Ｄ Ｚに対して６４により大きかっ た。The ELISA titer of this IgG fraction was greater than 64 against HSDZ. Ta.
見立±１ ｚｐ 木−リシンＡ士ム 実施例１について記載したように生成した抗体を脱グリコジル化したりシンＡ鎖 に結合した（ Ｒ，Ｆｕｌｔｏｎ等、Ｃａｎｃｅｒ Ｒｅ５ｅａｒｃｈ、 ４８ ：２６１８−２６２５．１９８９に記載された手順に従い、テキサス州、Ｄａｌ ｌａｓ、 Ｉｎ１ａｎｄ Ｌａｂｏｒａｔｏｒｉｅｓによる）。結合した抗体を 、続いて、動物研究において用いた。Mitate ±1 zp Tree - Rishin A Shimu Antibodies produced as described for Example 1 can be deglycosylated or synthesized with syn A chain. (R, Fulton et al., Cancer Research, 48 :2618-2625.1989, Dal, Texas. las, In1 and Laboratories). The bound antibody , subsequently used in animal studies.
上記のように調製したりシン結合した抗体（１ｍｇ）を０．５ｍｌ ＰＢＳ中に 置いた。この溶液を、次いで、７月齢の雌イヌに静脈注射した。最初の４８時間 にわたって、注射されたイヌは、高体温、嗜眠及び幾らかの嘔吐を示したが、そ の後、正常な健康に戻った。注射の７日後、このイヌの片方の卵巣を摘出した。Antibody (1 mg) prepared or syn-conjugated as above was added to 0.5 ml of PBS. placed. This solution was then injected intravenously into a 7 month old female dog. first 48 hours Over time, the injected dogs showed hyperthermia, lethargy and some vomiting; After that, he returned to normal health. Seven days after injection, one ovary of the dog was removed.
摘出した卵巣を組織学的分析のために切片化して染色した。この卵巣の染色した 切片の分析は、対照の未処理の卵巣と比較して、処理した卵巣内で第三次濾胞の 促進された閉鎖を示し、た。驚くべきことに、原始濾胞及び第二次濾胞の補充が 、対照に比較して、処理した卵巣内で促進された。灯明の及び処理した組織学的 切片を、図１〜９に示す。図１〜４は、それぞれ、７５．１０．３０及び１００ 倍の倍率の対照の未処理の卵巣の切片を示している。図５〜８は、処理した卵巣 の切片を、それぞれ、７．５．１０．３０及び１００倍の倍率で示している。The removed ovaries were sectioned and stained for histological analysis. This ovary was stained Analysis of sections showed that the number of tertiary follicles in treated ovaries compared to control untreated ovaries was showed accelerated closure. Surprisingly, recruitment of primordial and secondary follicles , was promoted in treated ovaries compared to controls. illuminated and processed histology Sections are shown in Figures 1-9. Figures 1-4 are 75.10.30 and 100, respectively. Control untreated ovary sections are shown at 2x magnification. Figures 5-8 show treated ovaries Sections are shown at 7.5.10.30 and 100x magnification, respectively.
五五玉ユ 目５■−゛ ０帯 実施例１について記載した手順に従って、ネコのハ可溶化透明帯（Ｈ５ＣＺ）に 対して抗体を生成した。アフィニティー精製したウサギ抗Ｈ３ＣＺ■ｇＧをネブ ラス力大学のＲ，Ｋｉｔｔｏｋｅ博士によって、ヨードケン法を用いてヨウ素化 した６２＝１の ＋ｚ５Ｉ、蛋白質のモル比を用いた。反応生成物をＢｌｏ−Ｇ ｅ１ Ｐ−６０カラム上で分離し、ヨウ素化蛋白質をボイドボリュームに溶出し た。約１２４百万ＤＰＭの放射能が、１５μｇの回収した蛋白質に結合した。55 balls Eye 5■-゛ 0 band Following the procedure described for Example 1, the feline Ha-solubilized zona pellucida (H5CZ) was Antibodies were generated against it. Affinity-purified rabbit anti-H3CZ gG was Iodination using the iodoken method by Dr. R. Kittoke of Las Power University +z5I and protein molar ratio of 62=1 were used. The reaction product is Blo-G e1 Separate on P-60 column and elute iodinated protein into void volume Ta. Approximately 124 million DPM of radioactivity was bound to 15 μg of recovered protein.
Ｏ１％ゼラチンを含む０．５９ｍ１．ＰＢＳ中の放射能櫻識した蛋白質（２，９ ５μｇ、１０９９マイクロキユリー）を、雌ネコに静脈注射した。この動物に、 ５０ｍ１ＰＢＳ中の５０μｇのナトリウムイオディクル（Ｉｏｄｉｃｌｅ ）を も注射した。この量のナトリウムイオディクルは、モルベースで計算した場合、 放射能標識した蛋白質中の １２ ｊ ■の量の１０，０００倍であった。0.59ml containing O1% gelatin. Radioactively recognized proteins (2,9 5 μg, 1099 microcuries) was injected intravenously into a female cat. to this animal, 50 μg of sodium iodicle in 50 ml PBS was also injected. This amount of sodium iodicle, when calculated on a molar basis, is It was 10,000 times the amount of 12 j ■ in the radioactively labeled protein.
注射の２４時間後、この動物を安楽死させて、種々の組織を取り出した。組織を ホモジェナイズして、マイクロメゾイック（Ｍｉｃｒｏ−Ｍｅｄｉａ ）ガンマ −カウンターを用いて放射能を分析した。表１に示すように、２４時間以内にお いて、卵巣内の放射能ｆｆｉ（ＣＰＭ／ｇ）は、他の再生性組織のそれの４倍で あった。Twenty-four hours after injection, the animals were euthanized and various tissues were removed. organization Homogenize and micro-media gamma - Radioactivity was analyzed using a counter. As shown in Table 1, within 24 hours The radioactivity ffi (CPM/g) in the ovary is four times that of other regenerative tissues. there were.
表１ ■ 二し鉱ユ 心臓 ２５３４ 腎臓 ５０３４ 肝臓 ５５３７ 脳 ４１５ 筋肉 ８８２ 肺臓 ３２０２ 子宮 ７９８１ 卵巣 ３１３４８ フロントページの続き （５１） Ｉｎｔ、 Ｃ１，’ 識別記号 庁内整理番号Ａ６１Ｋ ５１１００ ＣＯ７Ｋ １６／１８ ８３１８−４Ｈ（７２）発明者 ボウマン、ディピッド アメリカ合衆国 ７７３８１ テキサス、ザウッドランズ、ヒースストーン ブ レイスＩTable 1 ■ Nishiko Yu Heart 2534 Kidney 5034 Liver 5537 Brain 415 Muscle 882 Lungs 3202 Uterus 7981 Ovary 31348 Continuation of front page (51) Int, C1,' Identification symbol Internal office reference number A61K 51100 CO7K 16/18 8318-4H (72) Inventor Bowman, Dipid United States 77381 Texas, The Woodlands, Heathstone Bu Wraith I
Claims (16)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US768993A | 1993-01-22 | 1993-01-22 | |
| US007,689 | 1993-01-22 | ||
| PCT/US1994/000858 WO1994016735A1 (en) | 1993-01-22 | 1994-01-21 | Anti-zona pellucida antibodies for delivery of therapeutic agents to the ovary |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07507570A true JPH07507570A (en) | 1995-08-24 |
Family
ID=21727611
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6517261A Pending JPH07507570A (en) | 1993-01-22 | 1994-01-21 | Anti-zona pellucida antibodies for delivery of therapeutic agents to the ovary |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0632729A1 (en) |
| JP (1) | JPH07507570A (en) |
| AU (1) | AU6127694A (en) |
| CA (1) | CA2132632A1 (en) |
| WO (1) | WO1994016735A1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4795634A (en) * | 1986-03-14 | 1989-01-03 | Aphton Corporation | Method for contraception by immunization against the zona pellucida |
-
1994
- 1994-01-21 CA CA002132632A patent/CA2132632A1/en not_active Abandoned
- 1994-01-21 EP EP94907879A patent/EP0632729A1/en not_active Withdrawn
- 1994-01-21 WO PCT/US1994/000858 patent/WO1994016735A1/en not_active Application Discontinuation
- 1994-01-21 AU AU61276/94A patent/AU6127694A/en not_active Abandoned
- 1994-01-21 JP JP6517261A patent/JPH07507570A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994016735A1 (en) | 1994-08-04 |
| AU6127694A (en) | 1994-08-15 |
| CA2132632A1 (en) | 1994-08-04 |
| EP0632729A1 (en) | 1995-01-11 |
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