CN1315966A - 高亲和力抗体 - Google Patents
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Abstract
高亲和力单克隆抗体,其亲和力的特征为：(ⅰ)在抗原包被的微量滴定板的孔内温育第一种和第二种抗体样品,其浓度选择在标准曲线的线性部分之中,pH7.2,37℃,1小时;(ⅱ)去除两种样品中未结合的抗体;(ⅲ)将第一种样品同PBS在pH7.2条件下37℃温育1小时,并将第二种样品的pH值降至pH3或更低在37℃温育1小时;(ⅳ)去除两种样品中未结合的抗体;(ⅴ)将两种样品同抗－抗体磷性酸酶偶联物在37℃温育1小时;(ⅵ)去除两种样品中未结合的偶联物;以及(ⅶ)样品中加入PNPP底物,测定样品在405nm的光吸收值,以确定与抗原结合的抗体量,其中第二种样品的结合量大于50%的第一种样品的结合量。
Description
发明领域
本发明涉及抗体及其治疗用途。
发明背景
抗体曾经长期被认为是治疗癌症和其它疾病潜在的有力工具。然而，尽管存在一些明显的例外，这种潜力尚未获得普遍的认识。
这种成功的相对缺乏可能至少在某种程度上是由于使用了来源于啮齿类的单克隆抗体，该抗体很少具有高于10-9M的亲和力。具有这种亲和力水平的抗体治疗效用有限，因为已证明很难将足够的抗体运送至靶标以实现有用的生物学活性。抗体与抗原结合是可以逆转的，并且在体内实际使用的抗体浓度下，更倾向于解离而不是结合。原则上，有可能通过增加抗体浓度来抵消抗原的解离。然而，这可能会引起无法接受的临床副作用并且也将增加治疗相关的费用。
发明概述
本发明基于如下认识：可生产“酸耐受性”抗体或其片段，并且这种酸耐受性与抗体和抗原的高亲和力结合有关。
根据本发明，一种高亲和力抗体具有如下特征的亲和力：
(ⅰ)在抗原包被的微量滴定板的孔内温育第一种和第二种抗体样品，其浓度选择在标准曲线的线性反应部分之中，pH7.2,37℃,1小时；
(ⅱ)从两种样品中去除未结合的抗体；
(ⅲ)将第一种样品与PBS在pH7.2,37℃条件下温育1小时，并将第二种样品的pH值降至pH3或更低后37℃温育1小时；
(ⅳ)去除两种样品中未结合的抗体；
(ⅴ)将两种样品与抗抗体碱性磷酸酶偶联物在37℃温育1小时；
(ⅵ)去除两种样品中未结合的偶联物；以及
(ⅶ)向样品中加入PNPP底物，测定样品在405nm的光吸收值，以确定与抗原结合的抗体量，其中第二种样品的结合量大于50％的第一种样品的结合量。
优选地，步骤(ⅲ)中最大pH值为2.5，更优选为2.0。
预计具有酸耐受性的抗体或抗体片段更有利于结合而非解离，因而它们在靶位点具有更长的定居时间，导致更高的定居于靶位点的抗体浓度。
特别地，本发明涉及一种高亲和力单链Fv抗体片段的生产。这种ScFv具有特别的优点，它可允许其在体内更好地靶定位点。
附图描述
图1表明了在多种pH值下具有对癌胚抗原亲和力的绵羊和小鼠单克隆抗体以及单链Fv的酸耐受性结果。
发明描述
可以使用多种技术获得根据本发明的酸耐受性单克隆抗体。例如，可以使用经典的杂交瘤技术，包括分泌高亲和力抗体的免疫动物的B淋巴细胞与另一种适宜的融合伴侣的融合。另外一种可选择的方法为纯化所挑选的淋巴细胞的mRNA，并使用PCR技术扩增所需要的抗体基因。噬茵体展示技术和其它用于抗体片段展示的技术也可以用来在适当的筛选过程之后从原始文库或免疫文库中获得抗体基因。
抗体基因可以同毒素、放射性同位素、酶或其它任何合乎需要的分子共表达或化学交联，以提供具有强结合特点的融合蛋白。进一步，抗体可通过US-A-5770429所述的方法由转基因动物进行生产。
抗体可以是包括重链和轻链以及恒定区和可变区的完整抗体。另外可选择地，抗体可以是抗体片段，例如F(ab′)2、Fab、Fv或单链Fv片段，只要包含了赋予“酸耐受性”的可变区的至少一部分。抗体也可以是动物抗体、嵌合抗体或人源化抗体。专利WO-A-92/15699中公开了一种生产人源化抗体的适当方法。
在本发明一个优选的实施方案中，抗体为单链Fv片段。单链Fv片段包含由一适宜的肽连接的重链和轻链的可变区。
本发明的抗体可以由其酸耐受性定义，其可由如上所述的酸洗的酶联免疫吸附实验(EIA)表征。一般，与pH7.2条件下样品由EIA获得的A405值相比较，pH3或更低pH值条件下样品的A405值将代表＞50％的抗体结合。优选地，样品在pH2条件下的A405值将代表＞60％，更优选地为70％的pH7.2条件下获得的抗体结合。
经受免疫接种的动物不是啮齿类，而是选择可产生更高亲和力抗体的动物。可以使用任何大型哺乳类动物，且适宜的动物包括兔子、山羊、母牛和绵羊。
本发明的抗体可用于治疗，并且可以与生理可接受的赋形剂、稀释剂或载体一起配制入任何适当的组合物。
以下实施例阐明了本发明。
实施例1
用完全福氏佐剂中的癌胚抗原(CEA)免疫绵羊，然后用不完全福氏佐剂中的抗原加强免疫三次。在末次加强免疫之后处死动物并取出淋巴结。
淋巴结细胞经冲洗后与绵羊异骨髓瘤融合伴侣SFP3.2进行融合。融合细胞在含HAT(生命技术公司)的培养基中以大约106细胞/m的总浓度进行铺板。然后用常规EIA和酸洗EIA两种方法筛选样品中分泌针对特异抗原的高亲和力抗体的杂交瘤。
标准的EIA筛选实验如下进行：
用CEA(0.4μg/ml CEA于磷酸缓冲盐中，pH7.2)包被Maxisorb实验板(NUNC)，每孔100μl,4℃过夜。然后用pH7.2含0.01％Tween20去污剂的磷酸缓冲盐洗板3次。然后每孔加入200μl含0.2％脱雕牛奶蛋白的pH7.2的PBS溶液37℃半小时，以封闭所有板上遗留的反应位点。如上述方法用PBS洗板3次，然后板内每孔加入45μl抗体样品。样品在37℃温育1小时后，如前所述方法洗板。结合的抗体用磷性磷酸酶偶联的驴抗-绵羊抗体(Sigma A5187，经含1％BSA的PBS(pH7.2)1/5000稀释)进行检测。然后洗板，每孔加100μl PNPP(SigmaN2770)溶液。使用分光光度计测定样品在405nm的光吸收值，并以磷酸缓冲盐作为对照。
酸洗EIA筛选实验如下进行：
如以上标准EIA所述方法进行抗体样品的包被和结合。然而不同的是，抗体样品温育后洗板，并在每孔加入200μlpH2的HCl(10mM贮存液)37℃1小时。洗3次后，按上述方法用碱性磷酸酶偶联的驴抗-绵羊抗体和PNPP检测残留的与抗原结合的抗体。为确保对不同浓度下抗体的适当比较，选取每种样品使之在常规EIA(即EIA曲线中的线性反应部分)中的A405值约为1.0。
与在非酸洗的EIA中的抗体结合相比较，3个分泌抗体的杂交瘤(1D2,6G11和6H9)在酸洗的EIA中保留了大于50％的抗体结合。
实施例2
来源于上述杂交瘤6H9的单链Fv片段按以下方法生产：
用oligo-dT纤维素从培养的杂交瘤细胞中纯化mRNA。与mRNA互补的单链DNA(cDNA)通过反转录合成。由绵羊抗体基因的重链和轻链恒定区设计的通用引物用于在独立的反转录反应中，以合成抗体可变区的cDNA。
然后通过聚合酶链式反应扩增cDNA，并使用由重链和轻链可变区框架序列设计的引物生成双链DNA。扩增重链和轻链区分别采用独立的聚合酶链式反应。然后通过琼脂糖凝胶电泳分析产物，并从凝胶中切下轻链和重链基因对应的DNA条带进行纯化。
等摩尔量的重链和轻链可变区DNA与一条寡核苷酸连接头DNA混合在一起。连接头DNA编码氨基酸序列(Gly4Ser)3，并含有与重链可变区3′末端和轻链可变区5′末端互补的附加核苷酸。3条DNA分子在两阶段PCR反应中的第一阶段进行变性、退火和延伸(无引物)，使片段连接并由此排列成单链Fv。
单链FvDNA在第二阶段的PCR扩增所使用的引物对来源于重链和轻链可变区末端，并添加了限制酶识别位点Alw44i和NotⅠ。单链Fv基因产物经过琼脂糖凝胶电泳分析并进行纯化。然后用限制酶Alw44i和NotⅠ消化单链Fv并克隆至表达载体中。然后此载体用于转化大肠杆菌HB2151，从而进行蛋白质表达。载体经设计以在SFv的羧基COOH末端包括6-组氨酸(6-His)尾。单链Fv用镍螯合物亲和层析进行纯化，并通过SDS-PAGE进行分析。重链可变区和轻链可变区的氨基酸序列分别公开于SEQ ID No.2和SEQ ID No.4。酸洗EIA也用来测定单链Fv的酸耐受性。
酰洗EIA如下进行：
癌胚抗原(CEA)包被的微量滴定板按前述方法制备。单链Fv样品(6H9)由含1％牛血清白蛋白(BSA)的pH7.2 PBS稀释至1ng/ml～100ng/ml的浓度范围。微量滴定板每孔加入100μl样品37℃温育1小时。洗板后每孔加入200μl柠檬酸盐，37℃温育1小时。此时酸的制备是采用100mM柠檬酸盐贮存液在反应混合物中稀释至pH值分另为4.0、3.5、3.0、2.5和2.0。pH7.2的PBS用作参比对照。洗板后每孔加入100μl小鼠抗-四组氨酸抗体(Qiagen)(由含1％BSA的pH7.2PBS稀释至100ng/mB,37℃温育1小时。洗板后样品与每孔100μl山羊抗-小鼠碱性磷酸酶偶联物(Sigma A3688，由含1％BSA的pH7.2PBS 1/1000稀释)在37℃温育1小时。然后洗板，如前所述用PNPP处理，并用分光光度计测定405nm时的光吸收值。
作为酸耐受性的对照，SFv样品与PBS在pH7.2条件下共温育以产生SFv样品的EIA反应曲线。在线性区，SFv样品浓度为10-20ng/ml，对应给出的光吸收值A405为1.0-1.5，因而该样品浓度用于确定在酸洗样品中抗体结合量占对照样品中抗体结合量的百分率。
6H9完整抗体和6H9单链Fv的酸耐受性用于同小鼠来源的抗一癌胚抗原的完整抗体、ASB7和单链Fv MFE的酸耐受性进行比较。结果如图1所示，与抗原结合的小鼠来源的抗体在pH3.5显著减小，在pH2.5低于5％。相反，6H9抗体在pH3.5仍保持＞70％的抗原结合，在pH2.5＞60％，在pH2.0＞50％。
序列表<110>KS Biomedix Ltd<120>抗体<130>rep05827wo<140><141><160>4<170>PatentIn Ver.2.1<210>1<211>363<212>DNA<213>人工序列<220><223>人工序列的描述：抗体片段<220><221>CDS<222>(1)..(363)<400>1cag gtg cag ctg cag gag tcg gga ccc agc ctg gtg aag ccc tca cag 48Gln Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln1 5 10 15acc ctc tcc ctc acc tgc acg gtc tct gga ttc tca tta acc aag tat 96Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr
20 25 30ggt gtt agt tgg gtc cgc cag gct cca gga aag gcg ctt gag tgg cta 144Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45ggt ggt gtg tcc agt ggt gca cta aca gcc tat aac aca gcc cta cag 192Gly Gly Val Ser Ser Gly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln
50 55 60tcc cga ctc agc gtc acc agg gac acc tcc aag agc caa ttc tcc ctg 240Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu65 70 75 80tca ctg agc agc gtg act act gag gac acg gcc att tac tac tgt gcg 288Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95aaa tct gtc aat ggt gac agt gtt cct tat ggt ttg gac tac tgg agc 336Lys ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser
100 105 110cca gga crc cra crc acc gtc tcc rca 363Pro Gly Leu Leu Leu Thr Val Ser Ser
115 120<210>2<211>121<212>PRT<213>人工序列<223>人工序列的描述：抗体片段<400>2Gln Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr
20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45Gly Gly Val Ser Ser Sly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln
50 55 60Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu65 70 75 80Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser
100 105 110Pro Gly Leu Leu Leu Thr Val Ser Ser
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20 25 30gct tat gtg ggc Tgg tac caa cag gtc cca gga rca gcc ccc aga ctc 144Ala Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu
35 40 45ctc atc agt gct aca acc gat cga gcc tcg ggg atc ccc gac cga ttc 192Leu Ile Ser Ala Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe
50 55 60tcc ggc tcc agg tct ggg aac aca gcc acc ctg acc atc agc tcg ctc 240Ser Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu65 70 75 80cag gct gag gac gag gcc gat tat tac tgt gca tcg tat caa agt act 288Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr
85 90 95tac agt ggt gtt ttc ggc agc ggg acc agg ctg acc gtc ctg ggt 333Tyr Ser Gly Val Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly
100 105 110<210>4<211>111<212>PRT<213>人工序列<223>人工序列的描述：抗体片段<400>4Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln1 5 10 15Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn
20 25 30Ala Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu
35 40 45Leu Ile Ser Ala Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe
50 55 60Ser Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr
85 90 95Tyr Ser Gly Val Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly
100 105 110
Claims (10)
1．一种高亲和力单克隆抗体，其亲和力的特征为：
(ⅰ)在抗原包被的微量滴定板的孔内温育第一种和第二种抗体样品，其浓度选择在标准曲线的线性反应部分之中，pH7.2,37℃,1小时；
(ⅱ)从两种样品中去除未结合的抗体；
(ⅲ)将第一种样品与PBS在pH7.2条件下37℃温育1小时，并将第二种样品的pH值降至pH3或更低后37℃温育1小时；
(ⅳ)去除两种样品中未结合的抗体；
(ⅴ)将两种样品分别同抗抗体碱性磷酸酶偶联物在37℃温育1小时；
(ⅵ)去除两种样品中未结合的偶联物；以及
(ⅶ)样品中加入PNPP底物，测定样品在405nm的光吸收值，以确定与抗原结合的抗体量，其中第二种样品的结合量大于50％的第一种样品的结合量。
2．根据权利要求1的抗体，其中第二种样品的抗体结合量大于60％的第一种样品的抗体结合量。
3．根据权利要求1或权利要求2的抗体，其中步骤(ⅲ)的pH值降至pH2.5-pH2.0。
4．根据前面任一权利要求的非啮齿类的抗体。
5．根据前面任一权利要求的抗体，其中有针对肿瘤相关抗原的亲和力。
6．根据权利要求5的抗体，其中抗原为癌胚抗原。
7．根据前面任一权利要求的抗体，其为单链Fv、F(ab′)2、Fv或Fab。
8．根据权利要求7的抗体，该抗体具有一个包含SEQ ID No.2中定义的氨基酸序列的重链可变区和一个包含SEQ ID No.4中定义的氨基酸序列的轻链可变区，或其变体。
9．一种编码根据权利要求8的抗体的多聚核苷酸分子，其中多聚核苷酸包含SEQ ID No.1和3中定义的核苷酸序列，或其变体。
10．一种包含根据权利要求9的多聚核苷酸分子的克隆载体。
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| CN112724255A (zh) * | 2021-01-28 | 2021-04-30 | 成都金昆生物科技有限公司 | 靶向癌胚抗原的小分子抗体 |
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| US20030232401A1 (en) * | 2002-06-12 | 2003-12-18 | Pugia Michael J. | Bacterial test method by glycated label binding |
| JP4982183B2 (ja) * | 2003-12-12 | 2012-07-25 | ジェネンコー・インターナショナル・インク | Ｃａｂ分子 |
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| CN111848790B (zh) * | 2020-08-07 | 2022-02-22 | 上海交通大学 | 一种牛源抗金黄色葡萄球菌的单链抗体及其制备和应用 |
| CN112724255A (zh) * | 2021-01-28 | 2021-04-30 | 成都金昆生物科技有限公司 | 靶向癌胚抗原的小分子抗体 |
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| GB9818915D0 (en) | 1998-10-21 |
| HUP0103233A3 (en) | 2003-10-28 |
| AU5435099A (en) | 2000-03-21 |
| KR20010089174A (ko) | 2001-09-29 |
| WO2000012556A1 (en) | 2000-03-09 |
| JP2002525082A (ja) | 2002-08-13 |
| NO20011006D0 (no) | 2001-02-27 |
| ZA200101573B (en) | 2002-02-26 |
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