CN1152924A - 得自哺乳动物肝脏的蛋白质及其在肿瘤学中的应用 - Google Patents
得自哺乳动物肝脏的蛋白质及其在肿瘤学中的应用 Download PDFInfo
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Abstract
本发明涉及可以用高氯酸从哺乳动物肝脏、特别是山羊肝脏中提取的蛋白质及其在肿瘤学中的应用。
Description
本发明涉及得自动物组织特别是哺乳动物肝脏的蛋白质及其在肿瘤学中的应用。
WO 92/10197公开了哺乳动物器官特别是山羊肝脏的提取物,提取物由至少三种不同的蛋白质组成,其特征在于不寻常的药物学和免疫学性质。对其实际作用和各蛋白成分的序列的信息未予报道。
在Eur.J.Biochem.272,665,1993中公开了用5%高氯酸从大鼠肝脏和肾脏中提取的23-KDa二聚体蛋白质。相应的cDNA序列保藏于EMBL数据库,入藏号为X70825。
据认为,这种蛋白质(据报导,其与高迁移率族(HMG)蛋白质一同提取出来)在蛋白质折叠中起作用,所以可认为它是所谓“侣伴蛋白”类中的一员。
WO 93/18146公开了一种从兔肝脏提取的、分子量为59Kd、能与侣伴蛋白复合的蛋白质和一种90 Kd的热激蛋白质。
现已发现,一种从WO 92/10197所公开的提取物中纯化的新蛋白质具有序列Id n.1中所描述的部分氨基酸序列。
鉴于下列性质,所述蛋白质在肿瘤学中非常有用:
-用该蛋白质免疫过的动物的血清对人肿瘤细胞培养物显示出细胞毒性;
-对患有鼠结肠腺癌的Balb/c小鼠和患有胸膜内Yoshida腹水肿瘤的大鼠,该蛋白质在0.015μg/kg的剂量下有显著抗肿瘤活性;
-当给动物(包括人)施用时,它能引发能识别人癌细胞的抗体。
所述性质阐明了所进行的临床实验中观察到的活性。该临床实验中,把WO92/10197的提取物施用给受晚期肺癌、乳腺癌、胃癌、结肠癌和肝癌损害的患者。
本发明的蛋白质与Eur.J.Biochem.272,665,1993中公开的从大鼠肝脏提取的蛋白质具有高度同源性。
在不同动物品种特别是牛和马的肝脏中还发现了与序列Id.n.1的蛋白质具有高度同源性的蛋白质。本发明还涉及所述同源序列,得自大鼠肝脏的已知序列除外。
因此,已经发现了新的蛋白质家族:这个先前未知的家族的成员的特征在于各哺乳动物品种间的高度保守性和同源性以及分子量范围在约10KDa~约14KDa。
术语“高度同源性”是指氨基酸序列的同源性约为80%或更高,优选90%或更高。
本发明进一步涉及上述可用高氯酸从哺乳动物肝脏提取的蛋白质在肿瘤学中作为治疗和/或诊断工具的应用。
因此,本发明提供了含具有部分氨基酸序列n.1的蛋白质或与序列Id n.1具有至少80%同源性、优选至少90%同源性的蛋白质的药物组合物。
本发明的药物组合物将通过胃肠外途径施用,优选皮下或肌肉施用;通常每单位剂量含0.1~50mg总蛋白。可以把常规方法纯化的蛋白质活性成分在适宜的无毒载体上冻干并分装于小瓶或瓶子中。
合适的溶剂包括无菌水或盐水溶液。
根据本发明的进一步的实施方案,例如用化学合成法生产的本发明的蛋白质或其片断可以用于生产多克隆抗体或单克隆抗体。特别引人关注的抗体能识别肿癌抗原,因而能用于诊断、治疗或研究目的。这些抗体中的两种已于1993年7月27日保藏于the European collection ofAnimal Cell Cultures(EGACC),Porton Down,Salisbury,UK,入藏号为930806103和930806104。
在对人癌的几个活检样品进行的免疫细胞化学实验中,这些抗体被用于识别。
当本发明的蛋白质施用给患有致癌性疾病的患者时,除了象抑制或消除肿块、减轻痛疼和改善普通感觉(cenestesis)这样的有益效果外,还能引发对培养的肿瘤细胞具有显著细胞毒性作用的抗体。没有除去补体级联的全血清是所述细胞毒性作用所需要的。
当用于治疗目的或作为诱导对致癌性转化的免疫性的疫苗时,本发明的蛋白质可以通过皮下、肌肉或静脉途径注射,剂量范围为0.1~30mg/天/患者。这种治疗将重复进行,甚至长期进行,直到引发的抗体的浓度达到适宜水平为止。
可以用常规方法测定引发的抗体的浓度,例如采用免疫酶学技术。为此目的,本发明提供了诊断用试剂盒,它含有适当标记的试剂,例如,用作抗原的本发明的蛋白质或其片断,它选择性地固定于适宜支撑物上,抗-Ig抗体以及能够检测例如通过比色反应检测抗原-抗体复合物的适宜试剂。
本发明的蛋白质可以有利地与充当佐剂的适宜载体一起施用。例如,适宜的佐剂可以从无毒蛋白质中选取,优选从异种蛋白中选取,例如从相同品种(免疫原性蛋白就是从中提取的)得到的蛋白。
本发明的蛋白质是通过使粗提取物如下文实施例中所述在HPLC和疏水交换层析(FPLC)中经各个纯化步骤而制备的。所述粗提取物是这样得到的:用高氯酸提取器官,再用高渗盐水溶液(例如3M KCl)提取,然后透析。
得自山羊肝脏的蛋白质在N-末端封闭,从而在用CNBr切割后已进行部分测序,分别得到分子量(用MALDI-TOF法测定)分别为10263和4063p的两个主要片断,而切割前分子量为14.290道尔顿,与SDS-PAGE电泳测定的值一致。
下列实施例进一步阐述本发明。
实施例1
把按WO92/10197所述制备、下文称之为UK101的山羊肝脏提取物在Amicon PM 10膜上浓缩,然后用NaH2PO4/Na2HPO4(0.01M,pH6.5)透析。产物于在所述缓冲液中平衡过的TSKDEAE 5 PW上通过HPLC而纯化;收集初始缓冲液并用1M NaCl洗脱吸附于树脂上的蛋白质。然后,在TSK SW3000柱上通过HPLC纯化初始缓冲液中洗脱的峰。
用该层析法得到两个主要峰:丢弃第一个峰,因为它主要由糖原组成;然后,在Protein-Pac HIC Phenyl 5 PW柱上通过FPLC对尤其富含低分子量蛋白质的第二个峰进行纯化。
在下列条件下在这种疏水交换柱上进行纯化:首先洗脱初始缓冲液,Tris HCl(20mM,pH7,含1M(NH4)2SO4);然后进行线性梯度洗脱,以不合硫酸铵的20mM Tris-HCl结束。弃去初始缓冲液,而收集在(NH4)2SO4摩尔浓度范围为0.6~0.8M的梯度中洗脱的区域,并用H2O透析。
得到一种下文称之为UK114的样品,该样品在SDS-PAGE中显示出约14Kda的蛋白质区带,纯度约为90%。
实施例2
在免疫细胞化学实验中,使用兔体内引发的多克隆抗体,所述兔是每周与弗氏完全佐剂一起皮下施用PBS中的山羊肝脏提取物(WO92/10197)、共进行2个月而免疫过的。
从每周皮下注射含不完全弗氏佐剂的100μg UK101一个月后的Balb/c小鼠获得单克隆抗体。用常规方法进行与得自针对UK101进行过免疫的动物的淋巴细胞的骨髓瘤细胞的融合。所得杂交瘤中的两种于1993年7月27日保藏于the EuropeanCollection of Animal Cell Cultures(ECACC)Porton Down,Salisbury,UK,入藏号为930806103和930806104。
由上述杂交瘤分泌的抗体可识别本发明的蛋白质。
在对分离自不同器官的恶性肿瘤的30个活检样品进行的实验中,对单克隆抗体和多克隆抗体进行了分析。所述器官例如有乳腺、肺、膀胱、胃、结肠/直肠、子宫、软组织、前列腺。将这些组织固定于10%缓冲福尔马林中,用Mistostain Kit SP,ZymedLab.Inc.对石蜡中的制剂进行染色。
将切片于4℃与抗体(0.5μg/ml的Ig,含1%BSA/PBS)一起温育过夜。洗涤后,将切片与抗-兔猪生物素化Ig一起温育60分钟,然后与稀释度为1∶100的过氧化的链霉亲和素-生物素复合物一起再温育60分钟。用3,3-二氨基联苯胺/H2O2反应检测过氧化物酶结合。只有那些对胞质中的抗体显示特异反应的组织被认为是阳性的。免疫反应性对正常组织来说,被认为是阴性、略呈阳性、阳性(++)和高度阳性(+++)。结果报告于表中。在大多数恶性肿瘤中,可检测到与抗山羊、牛和马肝脏提取物的不同多克隆抗体的免疫细胞化学反应性(对抗马肝脏提取物的抗体而言是82.7%,对抗牛肝脏提取物的抗体而言是100%)。由杂交瘤n.930806103所分泌的单克隆抗体对93.7%受试肿瘤显示阳性结果。
表
恶性肿瘤的免疫细胞化学反应性(+/-)
UK101部位 山羊 牛 马 Mab n.930806103乳腺 4/0 1/0 1/0 1/0胃 4/3 3/0 3/0 3/0结肠/直 7/0 5/0 5/0 5/0肺 1/1 n.a. n.a. 1/0膀胱 2/0 1/0 1/0 1/0前列腺 3/0 1/0 1/0 1/1子宫 1/0 1/0 1/0 1/0肾上腺 1/0 1/0 1/0 1/0NOS 2/1 1/0 0/1 1/0总计 25/5 14/0 13/1 15/1
序列表(1)一般信息:
(i)申请人:
(A)名称:ZETESIS
(B)街道:Galleria del Corso 2
(C)城市:米兰
(E)国家:意大利
(F)邮政编码(ZIP):20122
(ii)本发明名称:得自哺乳动物肝脏的蛋白质及其在肿瘤学中的应用
(iii)序列数目:1
(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:Patentin Release#1.0,Version#1.25(EPO)(2)SEQ ID NO:1的信息:
(i)序列特征:
(A)长度:53个氨基酸
(B)类型:氨基酸
(C)拓朴学:线性
(0ii)分子类型:蛋白质
(v)片断类型:N-末端的
(vi)来源:
(A)生物体:Capra hircus
(F)组织类型:肝脏
(xi)序列描述:SEQ ID NO:1:Met Asp Pro Ala Ser Gly Gln Leu Val Pro Gly Gly Val Val1 5 10Glu Glu Ala Lys Gln Ala Leu Thr Asn Ile Gly Glu Ile Leu15 20 25Lys Ala Ala Gly Xaa Asp Phe Thr Asn Val Val Lys Ala Thr
30 35 40Val Leu Leu Ala Asp Ile Asn Asp Phe Xaa Ala
45 50
INTERNATIONAL FORMRECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentlfied at the bottom of this page1 where Rule 6.4(d) applies,such date is the date on which the status of international depositary
authority was acquired.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORMVIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page1 Indicate the date of the original deposit or,where a new deposit or a transfer has been
made,the most recent relevant date(date of the new deposit or date of the transfer).2 In the cases referred to in Rule 10.2(a)(ii) and (iii),refer to the most recent viability
tcct.3 Mark with a cross the applicable box.
Form BP/9(first page)4 Fill in if the information has been requested and ifthe results of the test were negative.Porm BP/9(second and last Page)
Claims (10)
1.可以从哺乳动物肝脏但不能从大鼠肝脏中提取的蛋白质,具有序列Id n.1的部分氨基酸序列或者与所述序列Id n.1的同源性至少为80%的序列。
2.根据权利要求1的蛋白质,与所述序列Id n.1的同源性至少为90%。
3.根据权利要求1或2的可以从山羊、马或牛肝脏中提取的蛋白质。
4.根据权利要求3的可以从山羊肝脏中提取的蛋白质。
5.根据前述任一权利要求的蛋白质,分子量从约10到约14Kda。
6.根据权利要求5的蛋白质,分子量约为14Kda。
7.根据前述任一权利要求的蛋白质,可以被由保藏于ECACC、入藏号为930806103和930806104的杂交瘤分泌的抗体所识别。
8.可以从哺乳动物肝脏中提取的蛋白质在抗肿瘤治疗中的应用,所述蛋白质具有序列Id n.1的部分氨基酸序列或者与所述序列Id n.1的同源性至少为80%的序列。
9.含有与合适载体混合的作为活性成分的权利要求1或8的蛋白质的药物组合物。
10.可以从哺乳动物肝脏中提取的蛋白质在诊断中的应用,所述蛋白质具有序列Id n.1的部分氨基酸序列或者与所述序列Id n.1的同源性至少为80%的序列。
Applications Claiming Priority (2)
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ITMI94A001469 | 1994-07-14 | ||
ITMI941469A IT1270618B (it) | 1994-07-14 | 1994-07-14 | Proteine ad attivita' antitumorale |
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CN1152924A true CN1152924A (zh) | 1997-06-25 |
CN1146576C CN1146576C (zh) | 2004-04-21 |
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CNB951941186A Expired - Fee Related CN1146576C (zh) | 1994-07-14 | 1995-07-12 | 得自哺乳动物肝脏的蛋白质及其在肿瘤学中的应用 |
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US (1) | US5792744A (zh) |
EP (1) | EP0770093A1 (zh) |
JP (1) | JPH10502814A (zh) |
KR (1) | KR100425627B1 (zh) |
CN (1) | CN1146576C (zh) |
AU (1) | AU702294B2 (zh) |
BR (1) | BR9508382A (zh) |
CA (1) | CA2194861A1 (zh) |
CZ (1) | CZ289380B6 (zh) |
FI (1) | FI970097A (zh) |
HU (1) | HU218285B (zh) |
IL (1) | IL114561A (zh) |
IT (1) | IT1270618B (zh) |
MX (1) | MX9602557A (zh) |
NO (1) | NO970114L (zh) |
RU (1) | RU2163243C2 (zh) |
TR (1) | TR199500854A2 (zh) |
TW (1) | TW434257B (zh) |
WO (1) | WO1996002567A1 (zh) |
ZA (1) | ZA955837B (zh) |
Cited By (1)
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CN104893316A (zh) * | 2015-01-16 | 2015-09-09 | 青岛新诺科铸造材料科技有限公司 | 一种填充型精密铸造中温调制蜡及其制备方法 |
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IT1276707B1 (it) * | 1995-06-13 | 1997-11-03 | Zetesis Spa | Composizioni farmaceutiche ad attivita' analgesica |
IT1282608B1 (it) | 1996-02-13 | 1998-03-31 | Zetesis Spa | Sequenza oligonocleotidica da fegato di capra |
IT1284524B1 (it) * | 1996-09-13 | 1998-05-21 | Zetesis Spa | Uso di proteine come agenti anti-retrovirali |
DK0928197T3 (da) | 1996-09-18 | 2003-06-23 | Zetesis Spa | Anvendelse af proteiner som midler mod autoimmune sygdomme |
IT1290828B1 (it) | 1997-03-25 | 1998-12-11 | Zetesis Spa | Uso di proteine estraibili da organi animali per la preparazione di medicamenti per il trattamento di condizioni patologiche |
IT1298442B1 (it) * | 1998-02-24 | 2000-01-10 | Zetesis Spa | Composizioni orali a basso dosaggio di proteine citotossiche |
US20010014471A1 (en) * | 1999-04-15 | 2001-08-16 | Vytautas Naktinis | Recombinant protein and its use in therapy and diagnostics |
AU7700900A (en) * | 1999-09-03 | 2001-04-10 | Vigen Laboratories, Inc. | Enriched fraction from a porcine liver extract for treating human diseases |
ITMI20010762A1 (it) * | 2001-04-10 | 2002-10-10 | Zetesis Spa | Uso della proteina uk114 o di suoi frammenti per il trattamento e la prevenzione dello shock endotossico |
ITMI20022307A1 (it) * | 2002-10-30 | 2004-04-30 | Zetesis Spa | Associazioni anti-tumorali comprendenti proteine e chemioterapici. |
IT201600127428A1 (it) | 2016-12-16 | 2018-06-16 | Cusani Alberto Bartorelli | Nuova proteina ricombinante uk 114 in forma stabile polimerica per uso nella terapia, nella diagnostica e nella prevenzione di neoplasie maligne |
IT201900022203A1 (it) | 2019-11-26 | 2021-05-26 | Cusani Alberto Bartorelli | Proteine uk 114 da salmone per uso nella terapia, nella diagnostica e nella prevenzione di neoplasie maligne |
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IT1244879B (it) * | 1990-12-11 | 1994-09-12 | Alberto Bartorelli | Estratti da tessuti animali, utili in terapia e in diagnostica. |
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- 1995-07-12 AU AU30779/95A patent/AU702294B2/en not_active Ceased
- 1995-07-12 CA CA002194861A patent/CA2194861A1/en not_active Abandoned
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- 1995-07-12 MX MX9602557A patent/MX9602557A/es not_active IP Right Cessation
- 1995-07-12 CZ CZ199769A patent/CZ289380B6/cs not_active IP Right Cessation
- 1995-07-12 US US08/765,957 patent/US5792744A/en not_active Expired - Fee Related
- 1995-07-12 CN CNB951941186A patent/CN1146576C/zh not_active Expired - Fee Related
- 1995-07-13 ZA ZA955837A patent/ZA955837B/xx unknown
- 1995-07-14 TR TR95/00854A patent/TR199500854A2/xx unknown
- 1995-07-17 TW TW084107362A patent/TW434257B/zh not_active IP Right Cessation
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1997
- 1997-01-10 NO NO970114A patent/NO970114L/no not_active Application Discontinuation
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104893316A (zh) * | 2015-01-16 | 2015-09-09 | 青岛新诺科铸造材料科技有限公司 | 一种填充型精密铸造中温调制蜡及其制备方法 |
Also Published As
Publication number | Publication date |
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CZ289380B6 (cs) | 2002-01-16 |
TR199500854A2 (tr) | 1996-06-21 |
RU2163243C2 (ru) | 2001-02-20 |
NO970114D0 (no) | 1997-01-10 |
TW434257B (en) | 2001-05-16 |
BR9508382A (pt) | 1997-12-23 |
FI970097A0 (fi) | 1997-01-10 |
JPH10502814A (ja) | 1998-03-17 |
AU702294B2 (en) | 1999-02-18 |
WO1996002567A1 (en) | 1996-02-01 |
HU218285B (en) | 2000-07-28 |
CA2194861A1 (en) | 1996-02-01 |
FI970097A (fi) | 1997-03-06 |
CZ6997A3 (en) | 1997-08-13 |
US5792744A (en) | 1998-08-11 |
IL114561A (en) | 2000-11-21 |
ITMI941469A0 (it) | 1994-07-14 |
EP0770093A1 (en) | 1997-05-02 |
HU9700057D0 (en) | 1997-02-28 |
AU3077995A (en) | 1996-02-16 |
ZA955837B (en) | 1996-02-21 |
IT1270618B (it) | 1997-05-07 |
KR100425627B1 (ko) | 2004-06-18 |
NO970114L (no) | 1997-03-05 |
IL114561A0 (en) | 1995-11-27 |
CN1146576C (zh) | 2004-04-21 |
HUT76328A (en) | 1997-08-28 |
MX9602557A (es) | 1997-05-31 |
ITMI941469A1 (it) | 1996-01-14 |
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