CN1207686A - 定向治疗变应性应答的Fcε－PE嵌合蛋白及制法和含有它的药物组合物 - Google Patents
定向治疗变应性应答的Fcε－PE嵌合蛋白及制法和含有它的药物组合物 Download PDFInfo
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Abstract
本发明大体涉及治疗变应性应答的新方法,它基于通过嵌合细胞毒素Fc2’－3－PE40定向消除表达FcεRI受体的细胞。编码小鼠IgE分子Fc区的氨基酸301—437的序列被遗传融合至PE40－缺乏细胞结合域的PE的截短了的形式。产生于大肠杆菌的该嵌合蛋白,特异性地且有效地杀伤表达FcεRI受体的小鼠肥大细胞系,以及得自骨髓的初级肥大细胞。本发明提供了定向消除FcεRI表达细胞的嵌合蛋白,它特别适用于治疗变应性应答。所述嵌合蛋白包括FcεRI表达细胞的细胞导向部分和细胞杀伤部分。优选的杀伤部分是细菌毒素假单胞菌属外毒素(PE)。该假单胞菌属外毒素是铜绿假单胞菌的产物。本发明还涉及制备所述蛋白质的方法。该嵌合蛋白是通过将小鼠IgE分子的Fc区与PE40(缺乏细胞结合域的PE的截短了的形式)遗传融合而制备的。本发明还提供了用于治疗变应性疾病以及治疗增生症和恶性肿瘤的药物组合物,它包括上述嵌合蛋白作为活性成分和常规的佐剂产品。
Description
本发明大体涉及治疗变应性应答的新方法。更具体地说，本发明涉及定向消除FcεRI表达细胞的Fcε-PE嵌合蛋白，它的生产方法，以及含有它的药物组合物。该嵌合蛋白包括与细胞杀伤部分连接的IgE分子一部分的细胞导向部分，所述杀伤部分可识别和消灭超量表达特异性受体的细胞。用于本发明的嵌合蛋白中的杀伤部分是细菌毒素假单胞菌属(Pseudomonas)外毒素(PE)(铜绿假单胞菌(Pseudomonasaeruginosa)的产物)。
世界人口的约百分之二十患有各种变应性疾病如哮喘、变应性鼻炎、食物过敏、特应性皮炎和过敏症。过去十年中变应性疾病流行的惊人的增多已导致明确需要更有效的疗法。
IgE和肥大细胞或嗜碱性粒细胞间的相互作用是变应性应答中的主要效应途径。IgE结合高亲和性受体(FcεRI)的恒定区，几乎全部见于这些细胞的表面。虽然解离速度低，但结合作用本身不会刺激细胞。然而，细胞表面结合的IgE被多价抗原交联会引起受体凝聚，触发爆发性细胞脱粒由此变态反应介体如细胞脱粒由此释放变态反应介体如组胺和5-羟色胺(seretonin)。
FcεRI受体的分布限于参加变应性应答的细胞这一事实使它成为利用嵌合细胞毒素的定向免疫疗法的有吸引力候选物。嵌合细胞毒素是一类通过基因融合技术构建的新型定向分子。这些分子包括细胞导向部分和细胞杀伤部分，使它们能识别和消灭超量表达特异性受体的细胞。
用于嵌合蛋白构建物的细菌毒素假单胞菌属外毒素(PE)，是铜绿假单胞菌的产物。进入胞质后，PE通过它的ADP-核糖基化活性抑制蛋白质合成，于是引起细胞死亡(Middlebrook，J.I.，和Dorland，R.B.1984.Bacterial toxins：cellular mechanisms of action.Microbiol.Rev.48，199.)。有效的嵌合细胞毒素已通过将编码各种生长因子或单链抗体的cDNAs与缺乏内在细胞结合能力的PE衍生物融合而构建。这些嵌合蛋白之一称为IL2-PE40，被构建以导向和选择性地消除超量表达IL2受体的活化T细胞，已证实能在自身免疫病、移植物排异反应和癌的各种模型中提供有效的和选择性的免疫抑制作用(Lorberboum-Galski，H.1994.Interleukin 2-Pseudomonas exotoxin A(IL2-PE40)chimeric proteinfor targeted immunotherapy and the study of immune responses.J.Toxicol.-Toxin Rewiews，13(1)，105.)。
在细菌中表达的IgE的整个重组恒定区(Fcε)对FcεRI受体的亲和性，比得上天然IgE的，以及具有敏化嗜碱性粒细胞关于抗IgE诱导的组胺释放的能力。当测试在细菌中表达的人Fcε的重组片段的受体结合性时，发现相应于该恒定区的域2和域3接头处残基301-376的肽足以高亲和性地结合该受体。还有人报道了受体结合不需ε链二聚化(Helm，B.，Marsc，P.，Vercelli，D.，Padlan，E.，Gould，H.，和Geha，R.1988.The mast cell binding site on human immunoglobulin E.Nature331，180.)。
本发明大体涉及治疗变应性应答的新方法。目前，用于治疗哮喘和变应性疾病的主要已知药物有如下几类：
1.β2兴奋剂-通过刺激β2肾上腺受体引起气道扩张。
2.甲基黄嘌呤(Methylxantines)-平滑肌松弛药，引起支气管扩张。
3.糖皮质素-减轻炎症。
4.色甘酸钠-防止肥大细胞脱粒。
5.抗组胺药-防止组胺作用于它的靶细胞。
虽然已广为应用，但这些药物具有下列显著的缺点：
1.特异性：所有这些药物(色甘酸钠除外)的作用都不是肥大细胞特异性的。因此，它们不能防止变态反应介体的释放，而是颠倒或阻止由它们的作用引起的效果。这些药物是根据症状而治疗的，它只在变应性反应开始后才开始作用，所以不能用于预防方面。
2.毒性：由于不是特异性的，所以这些药物作用于各种组织和器官会产生严重的副作用。β2兴奋剂的主要副作用是震颤，但它们还可引起心脏的心律失常；甲基黄嘌呤刺激中枢神经系统，引起神经质、恶心、呕吐、厌食、头痛和心肌引起的心搏过速。在高血浆含量时存在癫痫发作和心律失常的危险。抗组胺药影响中枢神经系统起镇静作用。类固醇最有害，可抑制垂体的-肾上腺的作用，引起液体和电解质失调，引起高血压、血糖过多，增大对感染的敏感性，引起骨质疏松症以及阻止儿童的生长。
3.效果的持久性：β-肾上腺素能兴奋剂、氨基黄嘌呤(aminoxantines)和抗组胺药大多数是短期作用药物，所以必须经常施用。类固醇是长期作用药物，也具有长的诱导时间，所以对于急症价值很小。
仅有的肥大细胞特异性药物是色甘酸钠。该药物可用作预防药，基本上没有副作用。然而，它的半寿命很短，诱导时间很长，它只能局部应用并且只有部分病人能响应它。所有这些使色甘酸钠的应用很有限。
近年来已报道了一些关于干扰IgE和它的高亲和性受体之间相互作用的尝试，作为抗变态反应疗法的基础。业已研究了作为IgE-FcεRI相互作用的竞争性抑制剂的重组肽，它们包括来自IgE(Helm，B.，Kebo，D.，Vercelli，D.，Glovsky，M.M.，Gould，H.，Ishizaka，K.，Geha，R.，和Ishizaka，T.1989.Blocking the passive sensatization of human mastcells and basophil granolocytes with IgE antibodies by a recombinanthumanε-chain fragment of 76 amino acids.Proc.Natl.Acad.Sci.USA86，9465.)或FcεRI(Ra，C.，Kuromitsu，s.，Hirose，T.，Yasuda，S.，Furuichi，K.，和Okumura，K.1993.Soluble human high affinityreceptor for IgE abrogates the IgE-mediated allergic reaction.Int.Immunol.5，47.；Haak-Frendscho，M.，Ridgway，J.，Shields，R.，Robbins，K.，Gorman，C.，和Jardieu，P.1993.Human IgE receptor a-chain IgGchimera blocks passive cutaneous anaphylaxis reaction in vivo.J.Immunol.151，351.)的结构元件。还试验了生成的单克隆抗体，它们抗IgE(Baniyash，M.，和Eshhar，Z.1984.Inhibition of IgE binding to mastcells and basophils by monoclonal antibodies to murine IgE.Eur.J.Immunol.14，799)或FcεRI(Kitani，S.，Kraft，D.，Fischler，C.，Mergenhagen，S.E.，和Siraganian，R.P.1988.Inhibition of allergicreactions with monoclonal antibody to the high affinity IgE receptor.J.Immunol.140，2585.)，能阻止IgE与受体的结合而不会引起肥大细胞脱粒。然而，IgE对FcεRI的亲和性很高(KM＝10-10M)，所以一旦与它的受体结合，IgE分子就保持与细胞膜连接达数周。此外，肥大细胞能在低受体占据下活化：少到5％的受体的交联度就足以引起肥大细胞脱粒。该系统的这两项性能妨碍竞争剂的抑制作用，因此限制它们的临床价值。我们的抗变态反应分子在远为更小的程度上依赖于同IgE竞争的能力。一旦通过非占据的IgE受体进入靶细胞，该嵌合蛋白就影响靶细胞。此外，受体在肥大细胞成熟过程中的早期表达，会使得在未成熟的靶细胞能释放介体之前而消除它们。由于受体不是在干细胞上表达的，所以大体上未损坏骨髓。
IgE系统相当复杂且性质多种多样。IgE及其结合结构之间的相互作用除变应性应答之外还有很多功能，其中的一些刚开始出现。抗IL-4的单克隆抗体、IL-4受体或低亲和性IgE受体消除IgE在小鼠内的表达但具有更普遍的免疫抑制效果。因此本发明(其中高亲和性IgE受体而不是整个IgE系统被定向)的优点是显而易见的。
本发明大体涉及治疗变应性应答的新方法，它基于通过嵌合细胞毒素Fc2’-3-PE40而定向消除表达FcεRI受体的细胞。编码小鼠IgE分子Fc区的氨基酸301-437的序列被遗传融合至PE40-缺乏细胞结合域的PE截短了的形式。产生于大肠杆菌(E.coli.)的该嵌合蛋白，特异性地且有效地杀伤表达FcεRI受体的小鼠肥大细胞系，以及得自骨髓的初级肥大细胞。
本发明提供了定向消除FcεRI表达细胞的嵌合蛋白，它特别适用于治疗变应性应答。所述嵌合蛋白包括FcεRI表达细胞的细胞导向部分和细胞杀伤部分。优选的杀伤部分是细菌毒素假单胞菌属外毒素(PE)。该假单胞菌属外毒素是铜绿假单胞菌的产物。
本发明还涉及制备所述蛋白质的方法。该嵌合蛋白是通过将小鼠IgE分子的Fc区与PE40(缺乏细胞结合域的PE截短了的形式)遗传融合而制备的。
本发明还提供了用于治疗变应性疾病以及治疗增生症和恶性肿瘤的药物组合物，它包括上述嵌合蛋白作为活性成分和常规的佐剂产品。
本发明进一步涉及制备这些药物组合物的方法，它包括将小鼠IgE分子的Fc区与PE40遗传融合以及，如果需要的话，添加常规的佐剂产品。本发明的药物组合物可呈用于注射、局部施药或口服的任意合适的形式。
与现有已知药物相比，本发明的Fc-PE嵌合蛋白具有如下一些优点：
1.特异性：Fc-PE是高度特异性的，影响负责释放变应性介体的细胞(肥大细胞和嗜碱性粒细胞)。由于它可防止变应性侵袭，所以它作为预防性疗法有很大的价值。
2.毒性：由于它作用于效应细胞(affector cells)而不是作用于它的靶器官，所以如果有的话，Fc-PE也只有很小的副作用。此外，由于该受体不是在干细胞上表达的，所以可预测它不会损坏骨髓和没有免疫抑制作用。预计在治疗结束后的数周以内会重新出现正常的生理状态。
3.效果的持久性：因为肥大细胞需经历数周才能成熟，所以可预计Fc-PE的效果是长期的，不需经常施用。此外，由于体外研究指出，在不到4小时内观察到细胞蛋白质合成减少80％，所以预计Fc-PE的诱导时间比较短，使它可应用于急性期变应性反应中。
Fcε-PE还可适用于治疗肥大细胞和嗜碱性粒细胞的增生症和恶性肿瘤，如全身性肥大细胞增生病(呈良性和恶性形式)和嗜碱细胞性白血病。化疗不适于良性肥大细胞增生病患者，因为它有严重的副作用。另一方面，没有良好的治疗恶性病的临床方案。Fcε-PE嵌合蛋白具有高度的潜能和选择性，能用于涉及表达FcεRI受体的细胞的良性病和恶性病。
下述实验结果指出，本发明的Fc2’-3-PE40嵌合蛋白是有效的和选择性的变态反应疗法中有希望的候选物。
本发明提供了用于定向消除FcεRI表达细胞的Fcε-PE嵌合细胞毒素蛋白，特别适用于治疗变应性应答如哮喘、变应性鼻炎、食物过敏、特应性皮炎和过敏症。
本发明将通过下列实验而得以进一步详细描述。这些实验不是想限定本发明的范围而只是演示和阐释它。
1.Fcε-PE40嵌合蛋白的构建。
小鼠IgE恒定区的片段(Fcε)被用作嵌合蛋白的导向部分，因为它结合人高亲和性IgE受体和小鼠高亲和性IgE受体(Conrad，D.H.，Wingard，J.R.，和Ishizaka，T.1983 The interaction of human androdent IgE with the human basophil IgE receptor.J.Immunol.130，327.)。
我们应用相应于a.a.301-437的一条序列，它包含区域2的COOH端和整个区域3(C2’-C3)。我们还应用相应于a.a.225-552的一条序列，它包含全部C2-C4区域。这些片段的cDNA是应用从小鼠B细胞分离的RNA和一特定的引物组通过RT-PCR而得到的，所述小鼠B细胞被同位素转换以分泌IgE。应用抗-Thy1.2和兔补体通过负选择分离得自6周龄BALB/C小鼠的脾的B细胞。在脂多糖(LPS，10μg/ml)和IL4(500u/ml)存在下以2×106个细胞/ml的密度将细胞培养5天以诱导同种型转换而生产IgE。5天后，分离全部细胞RNA(由BIOTECK Laboratories，Houston，USA生产的RNAzol TM B分离试剂盒)。然后应用逆转录系统(Promega，USA)在生产厂商建议的条件下将全部RNA(2.5μg)逆转录成第一链cDNA。用TE缓冲液(10mMTris-HCl，pH7.6，1mM EDTA)将cDNA稀释成总体积为1ml后在4℃下贮存备用。
Fcε片段是应用cDNA和一对合成的寡核苷酸引物通过PCR产生的，这对引物是用于Fcε2’-3序列的5’-GCG GAT CCC ATA TGG AGCAAT GGA TGT CGT-3’(有义，起始于核苷酸406，根据基因库序列J00476)和5’-GCG GAT CCC ATA TGT GGG GTC TTG GTG ATGGAA C-3’(反义，起始于核苷酸813)以及用于Fcε2-4序列的5’-GCGGAT CCC ATA TGC GAC CTG TCA ACA TCA CTG-3’(有义，起始于核苷酸175)和5’-GCG GAT CCC ATA TGG GAG GGA CGG AGGGAG G-3’(反义，起始于核苷酸1167)。
合成的寡核苷酸是在Applied Biosystems DNA合成仪上合成并在寡核苷酸纯化柱体上纯化的。将Vent聚合酶(Biolabs)用于扩增。将反应混合物在DNA热循环仪(MJ Research，Inc，USA)中保温33次循环。每次循环包括：在95℃下1min.，在退火温度下1min.和在72℃2min。用于每个引物对的MgSO4浓度和退火温度为：对于Fc2’-3’是2.5mM和61℃，对于Fc2-4是2mM和57℃。
编码IL2-PE40的pHL 906质粒以前已被描述过(Fishman，A.，Bar-Kana，Y.，Steinberger，I.，和Lorberboum-Galski，H.1994.Increased cytotoxicity of IL2-PE chimeric proteins containing targetingsignal for lysosomal membranes，Biochem.33，6235.)。用Ndel切割pHL906质粒，得更大的3596bp片段。将上述Fcε片段插入pHL906的Ndel位点。所得质粒pAF 2302和pAF 2415分别编码C2’-C3片段和C2-C4片段，各自融合PE40的5’，这两种质粒通过限制酶切分析和序列分析而表征(未示出结果)。应用大肠杆菌菌株HB101来转化和制备这些质粒。
2.嵌合蛋白的表达和部分纯化
由质粒pAF 2302编码的新设计的嵌合蛋白Fcε-PE40是在大肠杆菌菌株BL21(λ-DE3)内表达的，该大肠杆菌菌株携带了呈溶源性和可诱导形式的T7 RNA聚合酶基因。在异丙基硫代-β-D-半乳糖苷(IPTG，最终浓度为1mM)存在下在O.D.6000.5时诱导180min。将一片表达细胞悬浮于含0.2mg/ml溶菌酶(lysosyme)的TE缓冲液(50mM Tris pH8.0，1mM EDTA)，声波处理(三个30s脉冲)再在30,000×g下离心30min。分出上清液(可溶性部分)并保存供分析用。将离心片置于提取缓冲液(6M盐酸胍，0.1M Tris pH8.6，1mM EDTA，0.05M NaCl和10mM DTT)中变性并在4℃下搅拌30min。在30,000×g下离心该悬浮液达15min使它变清亮，弃去离心片。然后将上清液对0.1MTris(pH8.0)、1mM EDTA、0.25mM NaCl和0.25mM L-精氨酸透析16h。在15,000×g下离心该透析液达15min，将所得上清液(不溶性部分，盐酸胍处理过)用作嵌合蛋白来源。应用凝胶电泳法鉴定蛋白质(图2)。整个细胞提取物的蛋白质分布图显示了该嵌合蛋白的高表达水平。
通过Western印迹分析应用抗PE的抗体(图3A)和抗IgE的抗体(Serotec，England)(图3B)进一步鉴定该蛋白质。将电泳后的样品转到硝酸纤维素上并按所述方法(Lorberboum-Galski，H.，Fitzgerald，D.J.，Chaudhary V.，Ashya，S.，和Pastan，I.1988.Cytotoxicactivity of an interleukin 2-Pseudomonas exotoxin chimeric proteinproduced in Escherichia coli.Proc.Natl.Acad.Sci.USA 85，1992.)进行免疫印迹分析。按生产厂商的说明使用Vectastain ABC试剂盒(VectorLaboratories，USA)。该嵌合体与两种抗体反应，于是证实了框内的全长嵌合蛋白的克隆和产生。
表达细胞的亚细胞分级揭示了不溶性部分(包含体)尤其富含嵌合蛋白(图2)。因此将该部分用作嵌合蛋白的来源。
如前所述，应用富合作底物的延伸因子2的小麦胚提取物测定试验样品的ADP-核糖基化活性，表明该新型嵌合蛋白是酶促活泼的(未示出结果)。
3.Fc2’-3-PE40嵌合蛋白对小鼠肥大细胞系的效应。
测试了该嵌合蛋白对各种已知能表达FcεRI受体的小鼠肥大细胞系的细胞毒性效应。通过蛋白质合成的抑制作用估测该嵌合蛋白的细胞毒性活性，用[3H]亮氨酸掺入测定。用含0.25％牛血清白蛋白的磷酸盐缓冲盐水稀释后，不同浓度的该嵌合蛋白被加入接种于96孔板中达20h的2×104个细胞/0.2ml，接着用2μCi[3H]-亮氨酸脉冲处理8h。结果以对比实验的百分数表示，对比实验中的细胞未接触该嵌合蛋白。所有分析都在三个独立的实验中重复进行三次。
应用了表达FcεRI受体的三个靶细胞系：MC-9，一种来源于胎小鼠肝且依靠IL3生长的肥大细胞系；C57，一种来源于小鼠骨髓不依赖于IL3的肥大细胞系；以及来源于小鼠中期受孕(midgestation)初期胎盘的、Abelson病毒转化的肥大细胞系。
发现Fcε-PE40对试验的所有细胞系以剂量依赖性方式呈细胞毒性(图4)。MC-9系和C57系对嵌合毒素特别敏感，它们的ID50分别为50-75ng/ml和100-125ng/ml。Abelson细胞系的敏感性要小得多(ID50为1200-1500ng/ml)。
4.Fcε-PE40应答的特异性
为了检验Fc2’-3-PE40活性的特异性，产生两种对比蛋白质PE40和Fc2’-3-PE40M，估测它们对靶细胞和非靶细胞的效应。为构建Fc2’-3-PE40M，用EcoRI和BamHI切割编码PE的C端122个氨基酸的区，并用在氨基酸553处有缺失的相应片段置换。
正如所料，没有内在导向能力的PE40对靶细胞系无影响(图4)。Fc2’-3-PE40M(它的Fc2’-3部分与突变的、酶促惰性形式PE40连接)也对靶细胞无细胞毒性(图4)。
此外，有可能通过整个小鼠IgE(40μg/ml，图5A)或通过αPE多克隆抗体(10μg/ml，图5B)阻止Fc2’-3-PE40对靶细胞的细胞毒性作用。
还测试了Fc2’-3-PE40对小鼠各种非靶细胞系的影响(表1)。造血源的所有细胞系都未受嵌合蛋白的影响。意外地发现，虽然成纤维细胞系和血肿细胞系的ID50值比MC-9细胞的值高20倍，但它们对嵌合毒素表现出一定的敏感性(表1)。
上述信息表明，Fc2’-3-PE40对肥大细胞系的毒性效果是由于Fc2’-3部分介导的特异性响应的缘故，所述Fc2’-3部分将嵌合体的细胞毒性部分(PE40)导向细胞。
5.嵌合蛋白对初级肥大细胞的作用。
看来得自建立的各细胞系的新鲜鼠肥大细胞反应各不相同，我们还测试了得自正常小鼠的初级肥大细胞对Fc2’-3-PE40的敏感性。在IL3存在下培养二周后，小鼠骨髓分化成几乎纯的细胞群，它们具有未成熟肥大细胞的形态，含有颗粒并表达FcεRI受体。
杀死4～6周大小的BALB/C小鼠，使它们的骨髓在无菌下从股骨涌入冷的0.9％NaCl。用0.9％NaCl将细胞悬浮液洗两次，在300×g下离心10min，最后重悬浮于RPMI 1640培养基，该培养基中含10％热灭活的胎牛血清、4mM L-谷氨酰胺、1mM丙酮酸钠(sodiumpiruvate)、0.1mM非必需氨基酸、5×10-5M β-巯基乙醇、100u/ml青霉素、100μg/ml链霉素和20u/ml重组小鼠IL3。以106个细胞/ml的密度将细胞置于37℃的组织培养瓶中在5％CO2加湿的气氛中生长2-3周。每7天更换一次培养基。从第7天开始在培养物中加入重组IL4(10u/ml)。
为观察成熟程度，将细胞固定在载玻片上，用酸性甲苯胺蓝(pH1.0)染色后在油浸显微镜下观测。
在培养的第16天测试了嵌合蛋白对得自骨髓的肥大细胞(BMMC)的影响。如图6所示，Fc2’-3-PE40对BMMC以剂量依赖性方式表现出细胞毒性，ID50为125ng/ml。在高嵌合蛋白剂量时，几乎100％抑制蛋白质合成。对比蛋白质Fc2’-3-PE40M或PE40都未对BMMC表现细胞毒性(图6)。因此，初级肥大细胞对嵌合蛋白的应答类似于建立的肥大细胞系(图4和图6)。
6.Fc2’-3-PE40的受体特异性
除了高亲和性FcεRI受体外，还报道了其它三种低亲和性结合IgE的膜表面结构-低亲和性FcεRII受体、εBP半乳糖苷结合蛋白(也称为MAC-2或CBP35)和FcγRII/III受体。这些结构出现在各类细胞上，主要是造血源细胞，但也出现在成纤维细胞(εBP)上。除了FcεRI之外，FcγRII/III和εBP也出现在肥大细胞膜上。由于我们的目标是只定向肥大细胞，所以重要的是要证实嵌合蛋白不识别这些结构因此不能通过它们被内化。理论上，我们的嵌合蛋白不符合低亲和性IgE结合结构FcεRII、εBP和FcγRII/III的结合要求。FcεRII只结合二硫键连接的ε链二聚物，而我们的蛋白质缺乏对二聚化极为重要的区域4。εBP只结合糖基化的IgE；由于Fc2’-3-PE40是在细菌内产生的，所以它不被糖基化。FcγRII/III结合IgE-免疫复合体而不结合游离的IgE。然而受体结合的问题通过实验解决。
对已知能表达除FcεRI外的这些受体的C57肥大细胞进行了关于εBP和FcγRII/III的实验。为了测试该嵌合蛋白是否能通过FcγRII/III受体进入细胞，在添加该嵌合蛋白之前将细胞与2.4G2抗体(Pharmigen)(50μg/m)预培养。能与FcγRII受体和FcγRIII受体的胞外域结合的该单克隆抗体被证实为IgE结合的竞争性抑制剂。从图7A中可见，对比细胞以及与抗体预培养的细胞对Fc2’-3-PE40的细胞应答没有差别。
我们随后检验了εBP是否与Fc2’-3-PE40的细胞毒性有关。由于εBP与膜糖决定子连接，所以在培养基中加入乳糖会导致它从细胞表面解离。我们发现，当存在或不存在乳糖(25mM，图7B)时细胞对Fc2’-3-PE40的应答没有差别。
在2.4G2抗体和乳糖存在下另外对成纤维细胞系进行了实验，发现该细胞系对嵌合蛋白是部分应答的(表1)。再次发现，Fc2’-3-PE40细胞毒性对处理的细胞和对比细胞没有差别(未示出结果)。
为了检测Fc2’-3-PE40是否影响携带FcεRII的细胞，我们应用了0.12A3细胞系，即表达FcεRII受体的小鼠B细胞杂交瘤。甚至在高剂量(＞5000ng/ml，图8A)时，0.12A3细胞也对Fc2’-3-PE40完全没有应答。由于该细胞系在长期培养时失去受体，所以该检测之后用抗该受体的B3B4抗体(Pharmigin)进行FACS分析。结果表明，该受体在54％的细胞上表达了(结果未示出)。
另外对新鲜的小鼠B脾细胞进行了实验，所述B细胞与LPS(50μg/ml)一起预培养16小时以刺激FcεRII的表达。由FACS分析测得，虽然有69％的细胞表达了该受体，但Fc2’-3-PE40对这些B脾细胞无影响(图8B)。
总之，这些结果表明，Fc2’-3-PE40不结合低亲和性IgE结合的结构，即FcεRII、FcγRII/III和εBP。
7.Fc2’-3-PE40对细胞脱粒的影响
因为Fc2’-3-PE40可能的临床应用性，所以重要的是要测试用Fc2’-3-PE40处理肥大细胞是否会导致FcεRI被该嵌合蛋白结合时触发的变应性介体释放。
将C57细胞用[3H]-羟色胺(10μci/ml)预标记一夜，洗涤，以2×105个细胞/孔的密度于96孔组织培养板中置于含10％FCS的DMEM中，再在37℃下与Fc2’-3-PE40(10μg/ml)一起培养。在不同的时间点上，分离上清液并测定释放入上清液中的5-羟色胺(seretonin)。将未标记的细胞也与Fc2’-3-PE40一起培养，在相同的时间间隔用[3H]亮氨酸进行脉冲处理1hr以测定嵌合毒素对蛋白质合成的抑制。添加嵌合蛋白后1/2、4或8hr，在Fc2’-3-PE40处理过的细胞和未处理的细胞之间，上清液中[3H]5-羟色胺含量没有差别(图9A)。在4h时蛋白质合成的抑制达80％，且8h时为90％(图9B)。这些结果表明，在受体结合期间或在抑制蛋白质合成时Fc2’-3-PE40不会导致释放应变性介体。
8.Fcε-PE40的电泳特征
在非还原条件下对电泳处理过的样品进行Western印迹分析(从样品缓冲液中除去2-巯基乙醇)表明Fc2’-3-PE40嵌合蛋白主要作为单体存在(图10b)。至于非变性PAGE，从样品缓冲液中除去2-巯基乙醇，且未对样品加热。此外，用凝胶中等体积的水、样品缓冲液和电极操作缓冲液置换SDS。在非变性条件下，该嵌合蛋白以宽带形式(图10c)泳动。单独的非变性系统不能区别分子量、电荷和构象对蛋白质电泳迁移率的影响。然而，泳道中分子的近似性指示它们在这些参数方面不可能差别很大。
9.内化分析
该嵌合蛋白的体外活性只是靠它的内化实现。为了测试该嵌合蛋白是否被内化，在37℃下将5×105个细胞/3ml与20μg该嵌合蛋白一起培养1小时。用冷PBS洗涤3次后，将该片状物用0.5ml酸溶液(0.15MNaCl，0.15M乙酸(pH3))在冰上处理3min以除去膜结合的嵌合蛋白。然后加入50％FCS中和该pH，接着用RPMI/10％FCS洗涤3次。用0.3ml RIPA裂解缓冲液(150mM NaCl，1mM EDTA，20mM tris-HClpH7.4，1mM苯甲基磺酰氟，15％SDS，1％脱氧胆酸(deoxycholycacid)，1％Nonidet P-40)裂解该细胞片状物。各样品经过电泳后应用α-PE和ECL检测系统(Amersham)进行免疫印迹分析。Western印迹分析揭示，Fc2’-3-PE40嵌合蛋白无疑地被内化入靶细胞(图11)。
10.Fc2’-3-PE40对细胞脱粒的影响
在37℃下将C57细胞与[3H]-羟色胺(10μci/ml)一起培养一夜。洗涤细胞3次以除去游离的[3H]-羟色胺，在Tyrod’s缓冲液(10mMHepes pH7.4，130mM NaCl，5mM KCl，5.6mM葡萄糖，0.5％BSA)中以2.5×105个细胞/0.5ml置于24孔组织培养板中，再在4℃下与IgE(10μg/ml)培养1小时。然后添加MgCl2和CaCl2至最终浓度分别为1mM和1.6mM，接着在37℃下与二硝基苯基-人血清白蛋白(DNP-HSA，50ng/ml)一起培养30分钟或与不同浓度的嵌合蛋白一起培养不同的时间。通过离心收集无细胞的上清液并测定释放的[3H]-羟色胺的量。对测试的任意浓度的嵌合蛋白未观测到脱粒作用(图12a)。作为对比，在相同条件下将同IgE一起预培养过的细胞与DNP接触。DNP触发脱粒的效应清晰可见(图12a)。Fc2’-3-PE40也在其与靶细胞相互作用的后期未引起任何脱粒(图12b)，但它抑制蛋白质合成达80％以上(图12c)。我们的结果表明，Fc2’-3-PE40在其与细胞的相互作用期间的任何阶段都不触发脱粒。
Claims (11)
1.通过定向消除FcεRI表达细胞而治疗变应性应答的嵌合蛋白，其中所述嵌合蛋白包括FcεRI表达细胞的细胞导向部分和细胞杀伤部分。
2.权利要求1的嵌合蛋白，其中的杀伤部分是细菌毒素假单胞菌属外毒素(PE)。
3.权利要求1的嵌合蛋白，其中的细胞导向部分是小鼠IgE分子的Fc区。
4.权利要求1的嵌合蛋白，其中的细胞导向部分和细胞杀伤部分被遗传融合。
5.权利要求1的嵌合蛋白，其中编码小鼠IgE分子Fc区的氨基酸225-552的序列被遗传融合至PE40、即缺乏细胞结合域的PE截短了的形式。
6.权利要求1的嵌合蛋白，其中编码小鼠IgE分子Fc区的氨基酸301-437的序列被遗传融合至PE40、即缺乏细胞结合域的PE截短了的形式。
7.用于选自下列的变应性疾病的权利要求1的嵌合蛋白：哮喘、变应性鼻炎、食物过敏、特应性皮炎和过敏症。
8.用于治疗变应性疾病以及治疗增生症和涉及表达FcεRI受体的细胞的恶性肿瘤的药物组合物，它包括权利要求1-6中定义的嵌合蛋白作为活性成分和常规的佐剂产品。
9.权利要求8的用于治疗变应性疾病以及治疗增生症和恶性肿瘤的药物组合物，其中所述组合物呈适当的形式供注射(静脉内、关节内、皮下、肌内、腹膜内)，鼻内，鞘内，皮内，经皮，吸入，局部施用，口服，持续释放，或通过包括经肠途径的其它任意途径。
10.制备权利要求9定义的药物组合物的方法，它包括将小鼠IgE分子的Fc区与PE进行遗传融合以及，如果需要的话，添加常规的佐剂产品。
11.一种质粒，它包括一种启动子，该启动子与编码权利要求1-5中定义的肽的DNA分子操作性地连接。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL116436A IL116436A (en) | 1995-12-18 | 1995-12-18 | Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND |
| IL116436 | 1995-12-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1207686A true CN1207686A (zh) | 1999-02-10 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96199668A Pending CN1207686A (zh) | 1995-12-18 | 1996-12-18 | 定向治疗变应性应答的Fcε－PE嵌合蛋白及制法和含有它的药物组合物 |
Country Status (11)
| Country | Link |
|---|---|
| US (3) | US6919079B1 (zh) |
| EP (1) | EP0868198B1 (zh) |
| JP (2) | JP2000516570A (zh) |
| KR (1) | KR100464699B1 (zh) |
| CN (1) | CN1207686A (zh) |
| AT (1) | ATE261315T1 (zh) |
| AU (1) | AU718110B2 (zh) |
| DE (1) | DE69631837T2 (zh) |
| ES (1) | ES2217331T3 (zh) |
| IL (1) | IL116436A (zh) |
| WO (1) | WO1997022364A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324735A (zh) * | 2017-01-20 | 2018-07-27 | 李莉 | 用于疾病治疗的胞外体制剂及其应用 |
| CN108473551A (zh) * | 2016-01-13 | 2018-08-31 | 阿费里斯股份公司 | 高亲和力IgE受体的α链(FceRIa) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL116436A (en) | 1995-12-18 | 2006-12-31 | Yissum Res Dev Co | Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND |
| AU725408B2 (en) * | 1996-05-08 | 2000-10-12 | F. Hoffmann-La Roche Ag | Treatment of asthma with TNFR-Ig |
| IL118570A (en) * | 1996-06-04 | 2007-06-17 | Shai Yarkoni | Use of a chimeric protein that includes Met – GnRH in the preparation of a pharmaceutical preparation for the treatment of adenocarcinoma or patocarcinoma |
| US6645490B2 (en) | 1998-03-02 | 2003-11-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Chimeric proteins with cell-targeting specificity and apoptosis-inducing activities |
| CA2331274C (en) * | 1998-05-13 | 2010-04-06 | Biotecon Gesellschaft Fur Biotechnologische Entwicklung Und Consulting Mbh | Hybrid protein for inhibiting the degranulation of mastocytes and the use thereof |
| US6787524B2 (en) | 2000-09-22 | 2004-09-07 | Tanox, Inc. | CpG oligonucleotides and related compounds for enhancing ADCC induced by anti-IgE antibodies |
| ZA200305980B (en) | 2001-02-12 | 2007-01-31 | Res Dev Foundation | Modified proteins, designer toxins, and methods of making thereof |
| EP1414471B1 (en) | 2001-07-17 | 2012-06-13 | Research Development Foundation | Therapeutic agents comprising pro-apoptotic proteins |
| PT1863849E (pt) | 2005-03-11 | 2011-05-10 | Syngenta Ltd | Controlo de pragas de roedores |
| US9388686B2 (en) | 2010-01-13 | 2016-07-12 | Halliburton Energy Services, Inc. | Maximizing hydrocarbon production while controlling phase behavior or precipitation of reservoir impairing liquids or solids |
| US8505625B2 (en) | 2010-06-16 | 2013-08-13 | Halliburton Energy Services, Inc. | Controlling well operations based on monitored parameters of cement health |
| WO2012169735A2 (ko) * | 2011-06-07 | 2012-12-13 | (주)네오팜 | FCεRI의 수용성 단편을 포함하는 복합체 및 이를 포함하는 IGE 매개 알레르기성 질환 치료용 조성물 |
| US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
Family Cites Families (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4215051A (en) | 1979-08-29 | 1980-07-29 | Standard Oil Company (Indiana) | Formation, purification and recovery of phthalic anhydride |
| GB8616166D0 (en) * | 1986-07-02 | 1986-08-06 | Research Corp Ltd | Polypeptide competitor |
| US4902495A (en) * | 1986-07-22 | 1990-02-20 | The United States Of America As Represented By The Department Of Health And Human Services | IgE Fc directed delivery system |
| US5082927A (en) | 1986-09-24 | 1992-01-21 | The United States Of America As Represented By The Department Of Health And Human Services | Selectively cytotoxic IL-4-PE40 fusion protein |
| EP0378666A4 (en) | 1988-07-05 | 1992-01-22 | Amgen Inc. | Interleukin ii analogs |
| WO1990009799A1 (en) | 1989-02-23 | 1990-09-07 | Colorado State University Research Foundation | GnRH ANALOGS FOR DESTROYING GONADOTROPHS |
| DE69027210T2 (de) * | 1989-04-21 | 1997-01-23 | Protein Design Labs, Inc., Palo Alto, Calif. | Rekombinantes antikörper-toxin-fusion-protein |
| US5458878A (en) * | 1990-01-02 | 1995-10-17 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | P. exotoxin fusio proteins have COOHG220101al alterations which increase cytotoxicity |
| AU645783B2 (en) | 1990-01-23 | 1994-01-27 | Tanox Biosystems, Inc. | Extracellular segments of human epsilon immunoglobulin anchoring peptides and antibodies specific therefor |
| ATE182175T1 (de) | 1990-05-11 | 1999-07-15 | Us Health | Verbesserte exotoxine aus pseudomonas mit geringer toxität bei tieren und hoher zelltötender aktivität |
| US5723129A (en) | 1991-10-16 | 1998-03-03 | University Of Saskatchewan | GnRH-leukotoxin chimeras |
| US5837268A (en) | 1991-10-16 | 1998-11-17 | University Of Saskatchewan | GnRH-leukotoxin chimeras |
| WO1993015751A1 (en) | 1992-02-14 | 1993-08-19 | Merck & Co., Inc. | CHIMERIC TOXINS BINDING TO THE GnRH RECEPTOR |
| ATE314475T1 (de) | 1992-06-18 | 2006-01-15 | Us Gov Health & Human Serv | Rekombinantes pseudomonas exotoxin mit gesteigerter aktivität |
| WO1994004689A1 (en) | 1992-08-14 | 1994-03-03 | The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | Recombinant toxin with increased half-life |
| US5672686A (en) | 1994-08-09 | 1997-09-30 | Immunogen, Inc. | Bcl-Y - specific antibodies |
| US5587459A (en) | 1994-08-19 | 1996-12-24 | Regents Of The University Of Minnesota | Immunoconjugates comprising tyrosine kinase inhibitors |
| AU5965296A (en) | 1995-05-31 | 1996-12-18 | Regents Of The University Of Minnesota | Recombinant polypeptide cytotoxins for cancer treatment |
| US5759782A (en) | 1995-06-07 | 1998-06-02 | The United States Of America | Cellular apoptosis susceptibility protein (CSP) and antisense CSP |
| WO1997012632A1 (en) | 1995-10-05 | 1997-04-10 | Tkb Associates Limited Partnership | Methods for treatment of diseases associated with a deficiency of fas ligand activity |
| IL116559A (en) | 1995-11-17 | 2005-11-20 | Yissum Res Dev Co | Chimeric protein consisting of a bacterial toxin and a myelin basic protein sequence |
| IL116436A (en) * | 1995-12-18 | 2006-12-31 | Yissum Res Dev Co | Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND |
| WO1997034620A1 (en) | 1996-03-18 | 1997-09-25 | The Regents Of The University Of California | Peptide inhibitors of neurotransmitter secretion by neuronal cells |
| US6008042A (en) | 1996-05-16 | 1999-12-28 | Smithkline Beecham Corporation | Interleukin-1 beta converting enzyme like apoptotic protease-7 |
| US5834234A (en) | 1996-05-29 | 1998-11-10 | Immunogen, Inc. | Apoptosis associated protein Bbk |
| IL118570A (en) | 1996-06-04 | 2007-06-17 | Shai Yarkoni | Use of a chimeric protein that includes Met – GnRH in the preparation of a pharmaceutical preparation for the treatment of adenocarcinoma or patocarcinoma |
| US6218363B1 (en) | 1996-08-28 | 2001-04-17 | Thomas Jefferson University | MHC peptides and methods of use |
| US6172213B1 (en) | 1997-07-02 | 2001-01-09 | Genentech, Inc. | Anti-IgE antibodies and method of improving polypeptides |
| US6645490B2 (en) | 1998-03-02 | 2003-11-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Chimeric proteins with cell-targeting specificity and apoptosis-inducing activities |
| US6140066A (en) | 1998-03-24 | 2000-10-31 | Lorberboum-Galski; Haya | Methods of cancer diagnosis using a chimeric toxin |
-
1995
- 1995-12-18 IL IL116436A patent/IL116436A/en not_active IP Right Cessation
-
1996
- 1996-12-18 US US09/091,645 patent/US6919079B1/en not_active Expired - Fee Related
- 1996-12-18 ES ES96940708T patent/ES2217331T3/es not_active Expired - Lifetime
- 1996-12-18 KR KR10-1998-0704636A patent/KR100464699B1/ko not_active IP Right Cessation
- 1996-12-18 DE DE69631837T patent/DE69631837T2/de not_active Expired - Lifetime
- 1996-12-18 AT AT96940708T patent/ATE261315T1/de not_active IP Right Cessation
- 1996-12-18 JP JP09522641A patent/JP2000516570A/ja active Pending
- 1996-12-18 CN CN96199668A patent/CN1207686A/zh active Pending
- 1996-12-18 EP EP96940708A patent/EP0868198B1/en not_active Expired - Lifetime
- 1996-12-18 AU AU10707/97A patent/AU718110B2/en not_active Ceased
- 1996-12-18 WO PCT/IL1996/000181 patent/WO1997022364A1/en not_active Application Discontinuation
-
2002
- 2002-03-14 US US10/096,840 patent/US7740853B2/en not_active Expired - Fee Related
-
2007
- 2007-07-31 US US11/888,167 patent/US7704506B2/en not_active Expired - Fee Related
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- 2008-11-05 JP JP2008284755A patent/JP2009106286A/ja active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108473551A (zh) * | 2016-01-13 | 2018-08-31 | 阿费里斯股份公司 | 高亲和力IgE受体的α链(FceRIa) |
| CN108324735A (zh) * | 2017-01-20 | 2018-07-27 | 李莉 | 用于疾病治疗的胞外体制剂及其应用 |
| CN108324735B (zh) * | 2017-01-20 | 2024-02-09 | 李莉 | 用于疾病治疗的胞外体制剂及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2217331T3 (es) | 2004-11-01 |
| WO1997022364A1 (en) | 1997-06-26 |
| JP2009106286A (ja) | 2009-05-21 |
| US20030158390A1 (en) | 2003-08-21 |
| DE69631837T2 (de) | 2005-03-17 |
| DE69631837D1 (de) | 2004-04-15 |
| US7740853B2 (en) | 2010-06-22 |
| IL116436A (en) | 2006-12-31 |
| AU1070797A (en) | 1997-07-14 |
| IL116436A0 (en) | 1996-03-31 |
| US20090082549A1 (en) | 2009-03-26 |
| KR100464699B1 (ko) | 2005-07-28 |
| EP0868198A1 (en) | 1998-10-07 |
| EP0868198B1 (en) | 2004-03-10 |
| KR20000064465A (ko) | 2000-11-06 |
| US7704506B2 (en) | 2010-04-27 |
| US6919079B1 (en) | 2005-07-19 |
| AU718110B2 (en) | 2000-04-06 |
| ATE261315T1 (de) | 2004-03-15 |
| JP2000516570A (ja) | 2000-12-12 |
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