CN1329524C - 评估抗体治疗反应的方法和组合物 - Google Patents
评估抗体治疗反应的方法和组合物 Download PDFInfo
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- CN1329524C CN1329524C CNB02820672XA CN02820672A CN1329524C CN 1329524 C CN1329524 C CN 1329524C CN B02820672X A CNB02820672X A CN B02820672XA CN 02820672 A CN02820672 A CN 02820672A CN 1329524 C CN1329524 C CN 1329524C
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Abstract
本发明涉及用于评估或估计个体对特定治疗方法的反应的方法和组合物。具体来说,本发明提供了方法,用于确定个体的反应,或使治疗方案适合于用治疗抗体疗法的个体。本发明是基于确定个体的FCGR3A基因型。本发明可用于患有恶性肿瘤,特别是淋巴瘤的病人,并且适合于选择最好的反应者和/或为低反应者调整治疗条件或方案。
Description
本发明涉及用于评估或估计个体对特定治疗方法的反应的方法和组合物。具体来说,本发明提供了方法,用于确定个体的反应、或使治疗方案适合于用治疗抗体疗法的个体。本发明可用于患有恶性肿瘤、特别是淋巴瘤的病人,适合于选择最好的反应者和/或为低反应者调整治疗条件或方案。
简介
人类中的多种治疗策略是基于使用治疗抗体。其中包括例如使用被开发用于减少靶细胞、特别是患病的细胞如病毒感染的细胞、肿瘤细胞或其它病原细胞包括异源的免疫活性细胞的治疗抗体。这样的抗体一般是IgG种类的单克隆抗体,典型的是IgG1和IgG3。这些抗体可以是重组抗体和人源化抗体,含有不同物种或来源或特异性的功能结构域。这样的治疗抗体的一个具体的例子是美罗华(rituximab)(Mabthera、Rituxan),它是一个嵌合的抗CD20 IgG1单克隆抗体,由人类的γ1和κ恒定区与鼠类的可变结构域连接而制成1。几年来,美罗华已显著地改变了针对B淋巴组织增生的恶性肿瘤、特别是非-Hodgkin′s淋巴瘤(NHL)的治疗策略。其它完整的人源化IgG1抗体的例子包括alemtuzumab(Campath-1H),它用于治疗B细胞恶性肿瘤,或trastuzumab(Herceptin),它用于治疗乳腺癌。其它的正在开发的治疗抗体的例子在本领域内也有公开。
这些抗体代表了人类治疗、特别是肿瘤治疗的一种新的有效的方法,但是它们并不总是能够表现出强的效力,它们的使用可以通过评估个体对它们的反应来加以改进。例如,对于美罗华来说,单独使用或与化学治疗结合使用已显示出在治疗低中度2-8和高度NHL6,9中是有效的,30%到50%患有低度NHL的病人对美罗华没有临床反应4,5。已经建议CD20在淋巴细胞中的表达水平2,在治疗时存在高肿瘤负荷6或者低的血清美罗华浓度2可以解释美罗华在某些病人中缺乏效力。然而,治疗失败的真实原因大部分仍然不了解。
允许评估病人对抗体治疗的反应的方法的可用性将极大地增强这些产品的治疗效力。但是,这样的治疗抗体在体内的精确的作用方式尚未清楚地被证明。事实上,尽管体外研究建议了美罗华的多种可能的作用方式(抗体依赖性细胞介导的细胞毒性(ADCC)10,11、补体依赖性细胞毒性10,12,13、导致凋亡的直接信号14,15等),这些减少靶细胞的抗体在人体内的清楚的作用还没有被证明。此外,尽管ADCC在消灭细胞内病原和肿瘤细胞中是一种重要的效应机制,但ADCC的作用仍然是有争议的12,13。
本发明现在提出的新方法和组合物可以评估个体对治疗抗体的治疗反应。本发明还提出了筛选对治疗抗体疗法具有最好的反应图的病人的方法。本发明还涉及了用治疗抗体治疗病人的方法,包含了一个优先评估病人的反应的步骤。本发明还涉及了适合于执行本发明的组合物和试剂盒。本发明也可以用于临床试验或实验设置,以评估或监测个体的反应,或者证实抗体的作用方式。
本发明部分地基于证实了个体的基因型及其对治疗抗体疗法反应的能力之间的相关性。具体来说,本发明显示了FcγRIIIa受体的基因型与个体对治疗抗体疗法的反应直接相关。
在人类中,三类FcγR(FcγRI、FcγRII和FcγRIII)及其亚型由8个基因编码,都位于染色体1的长臂上。这些基因中有些表现出功能性等位多态性,产生了具有不同的受体性质的同种异型。这些多态性已经被鉴定为能够增加对自身免疫病或传染疾病易感性的遗传因子19-21。这些遗传因子之一是FCGR3A中的基因二态性,它编码的FcγRIIIa在第158位氨基酸是一个苯丙氨酸(F)或缬氨酸(V)22,23。这个残基与IgG1的下铰链区直接相互作用,正如近来由IgG1-FcγRIII共嵴化(co-cristallization)所显示24。已经清楚地证明人类的IgG1与纯合的FcγRIIIa-158V自然杀伤细胞(NK)的结合比与纯合的FcγRIIIa-158F或杂合的NK细胞的结合更强22,23。
我们着手评估了体内FCGR3A基因型和病人对治疗抗体疗法的反应之间的可能的相关性。我们的发明部分起源于出乎意料的发现,即在该基因型与该反应图之间存在非常强的相关性,在第158位存在一个缬氨酸残基是高反应速率的一个指示。更具体来说,对以前没有治疗过的滤泡性NHL病人仅使用美罗华,这是一种反应速率非常高的特别情况5,然后进行FCGR3A基因型分析。FCGR2A-131H/R也被测定以作为对照,因为这个基因与FCGR3A共同位于染色体lq22上,并编码巨噬细胞FcγRIIa受体。
在47个以前没有治疗过而接受美罗华治疗的滤泡性非-Hodgkin′s淋巴瘤病人中确定了FCGR2A-158V/F的基因型。在两个月(M2)和1年(M12)时评估了临床和分子反应。阳性的分子反应被定义为在外周血和骨髓中BCL2-JH基因重排的消失。FCGR2A-158V纯合的病人是21%,而FCGR2A-158F纯合的和杂合的病人(FCGR2A-158F携带者)分别为34%和45%。在M2和M12时的目标反应速率在FCGR2A-158V纯合的病人中是100%和90%,相比而言在FCGR2A-158F携带者中是65%(p=0.02)和51%(p=0.03)。在M12时,在六分之五的FCGR3A-158V纯合的病人中观察到了阳性的分子反应,相比而言在FCGR3A-158F携带者中只有十六分之五有阳性反应(p=0.04)。此外,纯合的FCGR3A-158V基因型在多变量分析中被证实是与临床和分子反应相关的单一参数,也与较低速度的疾病发展相关(p=0.05)。
因此,本发明第一次在FCGR3A基因型和对治疗抗体的临床和分子反应之间建立了联系。本发明还提供了第一个独特的标记物,可用于监测、评估或筛选病人的反应。因此,本发明在患有恶性肿瘤、病毒感染或其它与个体中存在病原细胞相关的疾病、特别是非-Hodgkin氏淋巴瘤的病人的管理方面引入了一种新的药物遗传学方法。
本发明的一个目的在于一种评估个体对治疗抗体疗法的反应的方法,包括在体外确定FCGR3A基因型和/或在该个体FcγRIIIa受体中多态性的存在。具体来说,该方法包括在体外确定该个体的FCGR3A158基因型。
本发明的另一个目的是一种为治疗抗体疗法筛选病人的方法,该方法包括在体外确定FCGR3A基因型和/或在该个体FcγRIIIa受体中多态性的存在。具体来说,该方法包括在体外确定该个体的FCGR3A158基因型。
本发明的另一个目的是一种在个体中改善治疗抗体疗法的效力或治疗条件或方案的方法,该方法包括在体外确定FCGR3A基因型和/或在该个体FcγRIIIa受体中多态性的存在。具体来说,该方法包括在体外确定该个体的FCGR3A158基因型。
具体来说,在体外确定个体的FCGR3A158基因型包含了确定FcγRIIIa受体的第158位的氨基酸残基(或在FCGR3A基因的相应密码子),第158位是缬氨酸表明对该疗法有较好的反应,而第158位是苯丙氨酸表明对该疗法有较低的反应。
在本发明的上下文中,术语“治疗抗体”更具体来说是指任何能够减少病人中的靶细胞的抗体。这样的靶细胞的具体的例子包括参与了过敏、自身免疫疾病、异源反应等的肿瘤细胞、病毒感染的细胞、异源的细胞、病原性免疫活性细胞(例如B淋巴细胞、T淋巴细胞、抗原呈递细胞等)、或者甚至是健康的细胞(例如在一个抗血管增生(anti-angiogenic)治疗方案中的内皮细胞)。在本文的上下文中最优选的细胞是肿瘤细胞和病毒感染的细胞。治疗抗体可以例如介导一种细胞毒性效应或细胞裂解,特别是通过抗体依赖性细胞介导的细胞毒性(ADCC)。ADCC需要白细胞受体来结合IgG(FcγR)的Fc部分,其功能是将IgG致敏的抗原与带有FcγR的细胞毒性细胞连接起来并启动细胞活化机制。尽管这种作用的机理在人体内还没有被证实,它可以解释这样的减少靶细胞的治疗抗体的效用。治疗抗体可以是多克隆的,最好是单克隆的。它们可以通过杂交瘤或通过改建能够表达所需的可变和恒定结构域的重组细胞来生产。抗体可以是单链抗体或其它的保留了抗原特异性和下铰链区的抗体衍生物或其变体。它们可以是多功能抗体、重组抗体、ScFV、人源化抗体或其变体。治疗抗体对表面抗原例如膜抗原是特异性的。最优选的治疗抗体对肿瘤抗原(例如由肿瘤细胞特异性表达的分子)是特异性的,例如CD20、CD52、ErbB2(或HER2/Neu)、CD33、CD22、CD25、MUC-1、CEA、KDR、αVβ3等,特别是淋巴瘤抗原(例如CD20)。治疗抗体优选为IgG1或IgG3,更优选为IgG1。
本发明的治疗抗体的典型例子是美罗华、alemtuzumab和trastuzumab。这样的抗体可以按照经审定的在人类个体中使用的临床规程来使用。其它的治疗抗体的具体的例子包括例如epratuzumab、basiliximab、daclizumab、cetuximab、labetuzumab、sevirumab、tuvirumab、palivizumab、infliximab、omalizumab、efalizumab、natalizumab、clenoliximab等,如同在下面的表中所列:
抗体特异性 | DCI | 商品名 | 典型的指示 |
抗CD20 | 美罗华 | MabThera,Rituxan | LNH B |
抗CD52 | alemtuzumab | CAMPATH-1H | LLC,同种异体移植 |
抗CD33 | ZamylTM | 急性骨髓性白血病 | |
抗-HLA-DR | RemitogenTM | LNH B | |
抗CD22 | epratuzumab | LymphoCideTM | LNH B |
抗-erbB2 | trastuzumab | Herceptin | 转移性乳腺癌 |
(HER-2/neu) | |||
抗-EGFR(HER-1、erbB1) | cetuximab | ORL和结肠直肠癌 | |
抗-MUC-1 | Therex | 乳腺和上皮癌 | |
抗CEA | labetuzumab | CEA-CideTM | |
抗-αVβ3 | Vitaxin | 癌症(抗-angiogenic) | |
抗-KDR(VEGFR2) | 癌症(抗-angiogenic) | ||
抗VRS融合蛋白 | palivizumab | Synagis | 病毒疾病 |
同上 | NumaxTM | 同上 | |
CMV | sevirumab | Protovir | CMV感染 |
HBs | tuvirumab | OstavirTM | 乙型肝炎 |
抗-CD25 | basiliximab | Simulect | 同种异体移植排斥的预防/治疗 |
抗-CD25 | daclizumab | Zenapax | 同种异体移植排斥的预防/治疗 |
抗-TNF-a | infliximab | RemicadeTM | Crohn病,polyarthriterhumatoid |
抗IgE | omalizumab | XolairTM | 哮喘 |
抗整合蛋白αL(CDlla,LFA-1) | efalizumab | XanelimTM | 牛皮癣 |
抗CD4 | keliximab | ||
抗CD2 | siplizumab | ||
抗CD64 | 贫血 | ||
抗CD147 | GvH | ||
抗整合蛋白α4(α4β1-α4β7) | natalizumab | Antegren | 硬化症,Crohn |
抗整合蛋白β7 | Crohn,RCH | ||
抗CD4* | clenoliximab |
在本发明的上下文中,个体或病人包括了任何哺乳动物个体或病人,更优选为人类个体或病人。
按照本发明,术语FCGR3A基因是指任何在个体中编码FcγRIIIa多肽的核酸分子。该术语具体来说包括了基因组DNA、cDNA、RNA(rRNA前体、信使RNA等)等或任何含有其序列的全部或部分的合成核酸。合成核酸包括从RNAs制备的cDNA,其含有FCGR3A基因组DNA的序列的至少一部分,例如作为一个或多个内含子或含有一个或多个突变的一部分。最优选的情况下,术语FCGR3A基因是指基因组DNA、cDNA或mRNA,典型为基因组DNA或mRNA。FCGR3A基因优选是人类的FCGRIIIa基因或核酸,即含有一个核酸序列,它编码了一个具有人类的FcγRIIIa多肽序列的FcγRIIIa多肽的全部或部分。这样的核酸可以根据现有的技术分离或制备。例如,它们可以通过杂交技术从基因文库或库中分离。它们也可以被遗传地或化学地合成。图2描述了一个人类FCGRIIIa基因的遗传结构。图3代表了人类FcγRIIIa的氨基酸序列。第158位的氨基酸是从成熟蛋白的第一个残基开始计数的。它相当于具有信号肽的蛋白前体的第176位残基。图4代表了野生型FCGR3A基因的序列(也可参见Genbank登记号AL590385,或者部分序列可参见NM_000569)。
在本发明的上下文中,一部分或部分意味着至少3个核苷酸(例如一个密码子),优选为至少9个核苷酸,更优选为至少15个核苷酸,可以包含多达1000个核苷酸。这样的一部分可以通过本技术领域内任何广为人知的技术来获得,例如酶法和/或化学切割、化学合成或这些方法的组合。为了清楚起见,编码第158位氨基酸的FCGR3A基因的一部分序列表述如下:
cDNA 540 550 560 570 580
基因组DNA 4970 4980 4990 5000.
158F等位基因 tcctacttctgcagggggctttttgggagtaaaaatgtgtcttca
S Y F C R G L
F G S K N V S S
158V等位基因 tcctacttctgcagggggcttgttgggagtaaaaatgtgtcttca
S Y F C R G L
V G S K N V S S
正如上面指出的,本发明包含了在体外确定该个体的FCGR3A158基因型的方法。更具体地说包含了确定在FcγRIIIa多肽的第158位存在(或被编码)的氨基酸残基的性质的方法。
在该个体中的FCGR3A基因或相应的多肽的基因分型可以通过多种技术来完成,包括对编码的核酸分子或被编码的多肽进行分析。分析可以包括测序、迁移、电泳、免疫技术、扩增、特异性消化或杂交等。
在一个特定的实施方案中,FcγRIIIa受体第158位氨基酸残基的确定包括一个对FCGR3A受体基因或RNA或其中含有编码第158位氨基酸残基的一部分进行测序的步骤。
在另一个特定的实施方案中,FcγRIIIa受体第158位氨基酸残基的确定包括一个对FCGR3A受体基因或RNA或其中含有编码第158位氨基酸残基的一部分进行扩增的步骤。扩增可以通过聚合酶链反应(PCR)进行,例如简单的PCR、RT-PCR或嵌套PCR,可以使用例如常规的方法和引物。
在这点上,用于本发明的扩增引物更优选为含有少于大约50个核苷酸,更优选为少于30个核苷酸,一般来说少于大约25个或20个核苷酸。另外,优选的引物通常含有至少5个、优选为至少8个核苷酸,以确保特异性。引物的序列可以基于FCGR3A基因的序列制备,以优选允许完全的互补性。探针可以使用任何现有的技术标记,例如放射性、荧光、酶法、化学法标记等。标记可以使用例如磷32、生物素(16-dUTP)、地高辛(11-dUTP)。应该理解本发明并不被特定的检测或标记技术所束缚或限制。如下面公开的,引物还可以包含限制性内切酶位点,以便在扩增的核酸中引入等位基因特异性的限制性内切酶位点。
这样的扩增引物的具体的例子例如SEQ ID NO:1-4。
应该理解专业技术人员也可以设计其它的引物,例如任何FCGR3A基因的片段,以用于扩增步骤,特别是一对含有正向序列和反向序列的引物,其中该引物对中的引物与FCGR3A基因的一个区域杂交,以允许扩增FCGR3A基因至少含有第158位密码子的一部分。在一个优选实施方案中,每对引物含有至少一个与第158位密码子互补并重叠的引物,以允许区分158V(gtt)和158F(ttt)。扩增的条件也可以由专业技术人员根据通用的知识和说明书中包含的指南进行调整。
因此,在一个特定的实施方案中,本发明的方法包含了用特异性的寡核苷酸引物对细胞中或生物样品中的FCGR3a的mRNA或gDNA的一部分进行PCR扩增,该部分中包含了第158位密码子,并且通过例如电泳,特别是变性凝胶梯度电泳(DGGE)对PCR产物进行直接或间接的分析。
在另一个特定的实施方案中,FcγRIIIa受体第158位氨基酸残基的确定包括了一个等位基因特异性的限制性内切酶消化的步骤。这可以通过使用能够切割特定的等位基因的编码序列(例如158V等位基因)而不能切割其它的等位基因(例如158F等位基因)的限制性酶来进行,反之亦然。当这样的等位基因特异性的限制性酶位点在序列中不是天然存在时,它们可以通过使用在其序列中含有这样的位点的等位基因特异性扩增引物来扩增核酸而被人工地引入。扩增后,可以通过例如电泳来分析消化的产物以确定一个等位基因的存在。这种技术也可以鉴别对于选定的等位基因是纯合的或杂合的目标。
等位基因特异性扩增引物的例子包括例如SEQ ID NO:3。SEQ IDNO:3引入了NlaIII位点(5’-CATG-3’)的前3个核苷酸。切割发生在G后。该引物含有11个不能与FCGR3A杂交的碱基,使引物延伸以便于对扩增的产物进行电泳分析,以及含有21个能够与FCGR3A杂交的碱基,只有第31位的核苷酸(A)例外,它产生了限制性酶位点。
在另一个特定的实施方案中,FcγRIIIa受体第158位氨基酸残基的确定包括了一个将FCGR3A受体基因或RNA或其含有编码第158位氨基酸残基的一部分与一个特异性针对缬氨酸或苯丙氨酸基因型的核酸探针进行杂交,以确定杂交的存在或不存在的步骤。
应该理解上面的方法可以单独使用也可以不同的组合使用。此外,其它专业技术人员熟知的技术也可用于确定FCGR3A158基因型,例如任何使用扩增(例如PCR)、特异性引物、特异性探针、迁移等的方法,一般使用定量RT-PCR、LCR(连接酶链反应)、TMA(转录介导的扩增)、PCE(一种酶放大的免疫分析)和bDNA(分支DNA信号扩增)分析。
在本发明的一个优选实施方案中,FcγRIIIa受体第158位氨基酸残基的确定包括:
●从生物样品中获得基因组DNA,
●扩增FcγRIIIa受体基因或其含有编码第158位氨基酸残基的一部分,以及
●确定该FcγRIIIa受体基因第158位的氨基酸残基。
扩增可以使用任何特异性的技术来完成,例如PCR、包括嵌套PCR,使用上述的特异性引物。在一个最优选的实施方案中,第158位氨基酸残基的确定通过等位基因特异性限制性酶消化来进行。在这种情况下,方法包括:
●从生物样品中获得基因组DNA,
●扩增FcγRIIIa受体基因或其含有编码第158位氨基酸残基的一部分,
●引入一个等位基因特异性限制性酶位点,
●用特异性针对该限制性位点的酶消化核酸,以及
●通过电泳分析消化产物,消化产物的存在表明了等位基因的存在。
在另一个特定的实施方案中,确定基因型的方法包括:从细胞或生物样品或体外或离体的生物体液中提取总(或信使)RNA,任选进行cDNA合成,然后用FCGR3A特异性的寡核苷酸引物进行(PCR)扩增,以及分析PCR产物。
本发明的方法也可以包括直接对FcγRIIIa受体多肽或其含有第158位氨基酸残基的一部分进行测序、或通过使用特异性针对FcγRIIIa多肽的每个等位基因的试剂,来确定FcγRIIIa受体第158位的氨基酸残基。这可以通过任何适合的专业技术人员熟知的技术来确定,包括通过免疫分析(ELISA、EIA、RIA等)。这可以使用任何特异性针对FcγRIIIa158多肽的亲和试剂来进行,更优选为任何抗体或其片段或衍生物。在一个特定的实施方案中,FcγRIIIa158多肽用一个能够分辨FcγRIIIa158V和FcγRIIIa158F的抗FcγRIIIa158抗体(或其一个片段)来检测,更优选为一个单克隆抗体。抗体(或亲和试剂)可以通过任何适当的方法(放射性、荧光、酶法、化学法等)来标记。此外,FcγRIIIa158抗体免疫复合物可以使用第二种试剂(例如抗体)来确定(和/或定量),例如能够与抗FcγRIIIa158抗体结合的标记。
上述的方法是基于在个体的生物样品中的FCGR3A158的基因分型。生物样品可以是任何含有FCGR3A基因或相应的多肽的样品,特别是血液、骨髓、淋巴结或含有FCGR3A基因或多肽的体液,尤其是血液和尿液。此外,因为FCGR3A158基因一般存在于上述的细胞、组织或体液内,本发明的方法经常使用一种处理样品,以使基因或多肽可用于检测或分析。处理可以包括任何常规的固定技术、细胞裂解(机械的或化学的或物理的)、或任何其它常规的用于例如免疫组织化学或生物学的方法。
本方法特别适合于确定个体对抗肿瘤治疗抗体疗法的反应。关于这一点,在一个特定的实施方案中,个体患有肿瘤,治疗抗体疗法的目的在于减少肿瘤的负荷,特别是减少肿瘤细胞。在更优选的情况下,肿瘤是淋巴瘤,例如更优选为B淋巴瘤,特别是NHL。正如前面指出的,抗体优选为IgG1或IgG3,特别是抗CD20的IgG1或IgG3,更优选为人源化抗体,例如美罗华。
本发明还涉及了一种双特异性抗体,其中该双特异性抗体特异性地结合CD16和一种肿瘤抗原,例如CD20抗原。本发明还包括了含有这样的双特异性抗体和可药用的赋形剂或佐剂的药物组合物。
本发明的其它方面和优点将在下面的实施例中公开,这些实施例应该被当作是说明性的,并没有对本申请的范围进行限制。
附图说明
图1:根据FcγR3a-158V/F基因型在美罗华治疗后肿瘤没有发展的存活率的调整的KAPLAN-MEIER估计(p=0.05)。
图2:人类FCGR3A基因的遗传结构
图3:人类FcγRIIIa158F的氨基酸序列(SEQ ID NO:7)
图4:人类FCGR3A158F的核酸序列(SEQ ID NO:8)
材料和方法
病人与治疗
临床试验设计、合格性判据和终点评估以前已经报道5。简单地说,包含在本研究中的病人,如果根据REAL分类他们患有以前未治疗过的滤泡性CD20阳性NHL,那么他们就是合格的病人。病人需要表现出根据Ann-Arbor分类为II期到IV期的病症,并具有至少一个可测量的病灶。所有的病人需要具有按照GELF标准为低度的肿瘤负载27。总共4剂375mg/m2的美罗华(Roche,Neuilly,France)通过静脉内灌注给药(在第1、8、15、22天)。灌注及不利事件的管理已经被报道过5。研究规程被一个伦理委员会批准,所有的病人被告知同意。
监测和终点
基线评估包括临床检查、X-射线胸透、胸部、腹部和骨盆计算机断层分析(CT)、以及单侧骨髓活组织检查。反应由一组独立的放射学家评估,他们总结了所有被包括的病人的CT扫描图。最初的效力终点是目标反应率,即获得了按照一个国际专家委员会最近提出的标准28为完全的缓解(CR)、未经确认的CR(CRu)或部分的反应(PR)的病人的比例。临床反应在第50天和第78天进行评估。只考虑了最大的反应,该评估的时间点被命名为M2。在1年时(M12)评估所有的病人的进展情况。在M2时骨髓浸润消失并且在M12时淋巴细胞重新在骨髓中出现的CR或CRu病人被当作“有进展的”;在M2时骨髓活组织检查为阴性并且在M12时活组织检查为阳性的PR病人被当作PR。
BCL2-JH基因重排的分子分析如前所述5通过PCR在M2和M12诊断时获得的淋巴结上和既在外周血和又在骨髓上进行。
FCGR3A-158V/F基因分型
在包含在临床试验中的50个病人中,一个病人在组织学检查后被排除,另外有两个病人的DNA没有获得。因此有47个病人可以进行FCGR3A基因型分析。所有的样品在同一个实验室中进行分析,使用标准的程序提取DNA,并小心以避免交叉污染。DNA从外周血(n=43)、骨髓(n=3)或淋巴结(n=1)中分离。FCGR3A-158V/F多态性的基因分型按照Koene等22描述的方法进行,使用嵌套PCR然后进行等位基因特异性限制性酶消化。简单地说,两个FCGR3A特异性引物(5’-ATATTTACAGAATGGCACAGG-3’,SEQ ID NO:1;5’-GACTTGGTACCCAGGTTGAA-3’,SEQ ID NO:2)(Eurobio,Les Ulis,France)被用于扩增一个含有多态性位点的1.2kb的片段。PCR分析按照生产商的推荐使用了1.25μg基因组DNA、200ng每种引物、200μmol/L每种dNTP(MBI Fermentas,Vilnius,Lithuania)和1U TaqDNA聚合酶(Promega,Charbonniere,France)。第一次PCR为95℃ 10分钟,然后进行35个循环(每个循环包括3个步骤:95℃ 1分钟、57℃1.5分钟、72℃ 1.5分钟),以及72℃ 8分钟进行完全的延伸。第二次PCR使用引物(5’-ATCAGATTCGATCCTACTTCTGCAGGGGGCAT-3’SEQ ID NO:3;5’-ACGTGCTGAGCTTGAGTGATGGTGATGTTCAC-3’SEQ ID NO:4)(Eurobio)扩增了一个94bp的片段,只在FCGR3A-158V等位基因中产生了一个NlaIII限制性位点。该嵌套的PCR使用了1μL扩增的DNA、150ng每种引物、200μmol/L每种dNTP和1U Taq DNA聚合酶。第一个循环为在95℃ 5分钟,然后进行35个循环(每个循环包括3个步骤:95℃ 1分钟、64℃ 1分钟、72℃ 1.5分钟),以及72℃ 9.5分钟进行完全的延伸。然后将扩增的DNA(10μL)用10U的NlaIII(NewEngland Biolabs,Hitchin,England)在37℃消化12小时,然后在8%的聚丙烯酰胺凝胶上通过电泳分离。用溴化乙锭染色后,DNA带在紫外光下观察。对于纯合的FCGR3A-158F病人,只能够看见一条不被消化的带(94bp)。在杂合的病人中可以看见3条带(94bp,61bp和33bp),而对于纯合的FCGR3A-158V病人,只能获得两条被消化的带(61bp和33bp)。
FCGR2A-131H/R基因分型
FCGR2A-131H/R的基因分型按照Liang等28的方法使用PCR、然后通过等位基因特异性的限制性酶消化来进行。有义引物(5’-GGAAAATCCCAGAAATTCTCGC-3’SEQ ID NO:5)(Eurobio)已经被修饰成在R等位基因的情况下产生一个BstUI限制性位点,而反义引物(5’-CAACAGCCTGACTACCTATTACGCGGG-3’SEQ IDNO:6)(Eurobio)已经被修饰成带有第二个BstUI限制性位点作为内部对照。PCR扩增在50μL反应体系中进行,使用1.25μg基因组DNA、170ng每种引物、200μmol/L每种dNTP、0.5U Taq DNA聚合酶以及生产商的缓冲液。第一个循环为94℃ 3分钟,然后进行35个循环(每个循环包括3个步骤:94℃ 15秒、55℃ 30秒、72℃ 40秒),以及72℃ 7分钟进行完全的延伸。然后将扩增的DNA(7μL)用20U的BstUI(NewEngland Biolabs)在60℃消化12小时。进一步的分析参照在FCGR3A基因分型中的描述。FCGR2A-131H和-131R等位基因分别可被显示为337bp和316bp的DNA片段。
统计分析
使用平方分布检验法或在合适时使用费歇耳恰当检验法对不同基因型组的病人的临床和生物学特征及临床和分子反应进行比较。对数回归分析包括:性别、年龄(>或≤60岁)、包括的额外结点数(≥或<2)、骨髓的介入、诊断时BCL2-JH的重排状态和FCGR3A基因型,被用于鉴定影响临床和分子反应的独立的预后变量。无进展的幸存率按照Kaplan和Meier29的方法计算,测量从治疗开始直到进展/恶化或死亡。FCGR3A基因型的无进展幸存率使用对数等级检验法进行比较。P<0.05被认为是统计学显著的。
结果
临床反应
在49个进行FCGR3A-158V/F多态性测试的病人中,10个(20%)和17个(35%)分别是FCGR3A-158V和FCGR3A-158F纯合的,而22个(45%)是杂合的。3个组在诊断时的性别、疾病阶段、骨髓的介入、包括的额外结点数或外周血和骨髓中BCL2-JH重排的存在这些方面没有区别(表1)。当将纯合的FCGR3A-158V病人与FCGR3A-158F携带者(FCGR3A-158F纯合的和杂合的病人)进行比较时,或将纯合的FCGR3A-158F病人与FCGR3A-158V携带者(FCGR3A-158V纯合的和杂合的病人)进行比较时,没有发现区别。在M2时的目标反应率在FCGR3A-158V纯合、FCGR3A-158F纯合和杂合的病人中分别是100%(CR+CRu=40%)、70%(CR+CRu=29%)和64%(CR+CRu=18%)(P=0.09)。在FCGR3A-158V纯合病人和FCGR3A-158F携带者之间观察到了目标反应率的显著差别,后者的目标反应率为67%(CR+CRu=23%)(相对风险=1.5;95%CI,1.2-1.9;P=0.03)(表2)。在FCGR3A-158F纯合病人和FCGR3A-158V携带者之间没有观察到差别。在M12时,目标反应率在FCGR3A-158V纯合、FCGR3A-158F纯合和杂合的病人中分别是90%(CR+CRu=70%)、59%(CR+CRu=35%)和45%(CR+CRu=32%)(P=0.06)。在治疗1年后,在FCGR3A-158V纯合组和FCGR3A-158F携带者之间目标反应率的不同仍然存在,后者的目标反应率为51%(CR+CRu=33%)(相对风险=1.7;95%CI,1.2-2.5;P=0.03)。对数回归分析显示纯合的FCGR3A-158V基因型在M2(P=0.02)和M12(P=0.01)时是唯一的可以预测临床反应的因素。在3年时的无进展幸存率(中值随访35个月;31-41)(图1)在FCGR3A-158V纯合病人中是56%,而在FCGR3A-158F携带者中是35%。在进行了FCGR2A-131H/R多态性分析的45个病人中,FCGR2A-131R纯合和FCGR2A-131H纯合的分别为9个(20%)和13个(29%),而杂合的为23个(51%)。对于这3个组,或对于纯合的FCGR2A-131H病人和FCGR2A-131R携带者,或对于纯合的FCGR2A-131R病人和FCGR2A-131H携带者,在包含的特征和对美罗华治疗的临床反应方面没有区别(数据未显示)。
分子反应
在诊断时,在30个(64%)能够继续随访的病人的外周血和骨髓中均检测到了BCL2-JH重排。在M2和M12时分别对25个病人(6个FCGR3A-158V纯合病人和19个FCGR3A-158F携带者)和23个病人(6个FCGR3A-158V纯合病人和17个FCGR3A-158F携带者)在外周血和骨髓中均分析了BCL2-JH重排(表3)。在M2时,在3/6的FCGR3A-158V纯合病人和5/19的FCGR3A-158F携带者中观察到BCL2-JH重排的清除。相反,在M12时,在FCGR3A-158V纯合病人中的BCL2-JH重排清除率(5/6)高于在FCGR3A-158F携带者中的清除率(5/17)(相对风险=2.8;95%CI,1.2-6.4;P=0.03)。对数回归分析显示FCGR3A-158V纯合基因型是唯一与在M12时具有较高几率表现出BCL2-JH重排清除相关的因素(P=0.04)。有一个FCGR3A-158V纯合病人在M12时仍然在外周血和骨髓中均存在BCL2-JH重排,是位于美罗华治疗后23个月的CR组中。相反,在M2和M12时的分子反应不受FCGR2A-131H/R多态性的影响(数据未显示)。
讨论
因为美罗华在B细胞淋巴增殖性恶性肿瘤中的应用越来越多,因此需要对治疗失败和美罗华的作用方式有更深入的了解。为此,我们对具有明确的临床和实验室特征并且只用美罗华治疗过的滤泡性NHL病人进行了FCGR3A基因分型5。具体来说,所有包含在这个研究中的病人患有低肿瘤负荷的NHL,并在诊断时和随访中进行了BCL2-JH分子分析。在该人群中FCGR3A等位基因频率与普通的高加索人群中的频率相同23,24。我们的结果显示了在FCGR3A基因型和对美罗华的反应之间有联系。事实上,占总人群五分之一的纯合的FCGR3A-158V病人,具有较高的经历临床反应的可能性,在M2和M12时的目标反应率分别为100%和90%。此外,被分析了BCL2-JH重排的6个FCGR3A-158V纯合病人中有5个在M12时显示了分子反应,与此相比在17个FCGR3A-158F携带者中有5个显示了反应。FCGR3A-158V的纯合性是与临床和分子反应相关的唯一因素。但是,这些较高的临床和分子反应仍然不足以在纯合的FCGR3A-158V病人中提高无进展的存活率。
这是第一个用于对美罗华的临床和分子反应可容易评估的遗传预测因素的报告。但是,遗传联系并不表明美罗华参与FcγRIIIa的作用方式。在FCGR3A基因型和对美罗华的反应之间所观察到的联系可能是由于另一个在连接不均衡性中的遗传多态性。那些多态性可能位于FCGR3A本身中类似三等位基因的FCGR3A-48L/H/R多态性31,或位于其它的FcγR编码基因中,因为FCGR3A位于染色体1的长臂中,该长臂包含了3个FCGR2基因和FCGR3B32。已经报道了在FCGR2A和FCGR3B之间具有连接不均衡性33。但是,FCGR2A-131H/R多态性与对美罗华的反应较好无关这一事实,强烈地支持了一个非常靠近FCGR3A的基因或FCGR3A自身的直接参与。
几个体外研究的证据支持FCGR3A-158V/F多态性的直接参与。首先,Koene等23已经表明,以前报道的IgG与3种FcγRIIIa-48L/H/R同种型之间结合的不同31是连锁的FcγRIIIa-158V/F多态性的结果,有几个组已经证实来自FCGR3A-158V等位基因同种异型纯合的个体的自然杀伤细胞对人的复合IgG1有较高的亲和性,并对IgG1致敏的靶有更高的细胞毒性23,24,34。我们现在的结果表明FCGR3A-158V纯合的病人对美罗华有较好的反应,这可能是由于嵌合的人IgG1在体内与FcγRIIIa结合得更好。其次,自然杀伤细胞和巨嗜细胞介导的ADCC在体外8,11,12和鼠模型体内17,19是由抗CD20抗体引发的一种机制,而美罗华介导的凋亡被FcγR表达细胞放大了15,16。在所有的FcγR中,FcγRIIIa是唯一为自然杀伤细胞和巨嗜细胞所共有的受体。因此,我们推测FCGR3A-158V病人表现出对美罗华较好的反应是因为它们有较好的针对淋巴细胞的ADCC活性。超过50%的FCGR3A-158F携带者仍然表现出对美罗华的临床反应这一事实,可以被虽然较低但是仍然足够的ADCC活性来解释,或者更可能的是通过其它在体内操纵的机制例如补体依赖性细胞毒性、补体依赖性细胞介导的细胞毒性11,13,14和/或细胞凋亡15,16来解释。然后可以将ADCC看作是对美罗华反应的另一个机制,它在FCGR3A-158V纯合的病人中特别有效。
体外的研究表明了一种“基因-剂量”效应,IgG1与来自FCGR3A杂合供体的自然杀伤细胞的结合水平介于用来自FCGR3A-158V和FCGR3A-158F纯合子的自然杀伤细胞所观察到的结合水平之间23。但是,杂合的病人的临床反应显示出与FCGR3A-158F纯合的病人相似。为了在体内得出与“基因-剂量”效应相反的结论需要在较大组病人中进行进一步的研究。
因为FcγRIIIa与对美罗华的较好反应有很强的相关性,在开发新的针对CD20抗原的药物时需要把它考虑在内。例如,有可能可以使用改造的美罗华来治疗患有B细胞淋巴瘤的FCGR3A-158F携带病人。事实上,通过对IgG1下铰链区的不同残基进行修饰,Shields等最近获得了能够比天然的IgG1更强烈地与FcγRIIIa-158F结合的IgG1突变体34。
合在一起,这些结果允许在首先确定病人FCGR3A基因型的基础上建立新的针对B淋巴增殖性紊乱的治疗策略。因为该多态性在不同种族的人群,包括黑人和日本人中,具有相同的分布,这样的策略可以在世界范围内应用23,35,36。此外,这样的药物遗传学方法也可以适用于其它用于治疗B细胞恶性肿瘤,例如Campath-1H的完整的人源化IgG1抗体,或者那些用于治疗其它恶性肿瘤的人源化IgG1抗体,例如trastuzumab(Herceptin)。在更普遍的意义上,本方法可以适用于其它被开发用来减少靶细胞的完整的(人源化)治疗(IgG1)抗体。
参考文献
1.Maloney DG,Liles TM,Czerwinski DK等:Phase I clinical trialusing escalating single-dose infusion of chimeric anti-CD20 monoclonalantibody(IDEC-C2B8)in patients with recurrent B-cell lymphoma(在B细胞淋巴瘤复发的病人中逐步增加地灌注单一剂量的嵌合的抗CD20单克隆抗体(IDEC-C2B8)的1期临床试验).Blood.1994;84:2457-2466.
2.McLaughlin P,Grillo-Lopez AJ,Link BK等:Rituximabchimeric anti-CD20 monoclonal antibody therapy for relapsed indolentlymphoma:half of patients respond to a four-dose treatment program(复发的无痛性淋巴瘤的美罗华嵌合抗CD20单克隆抗体治疗:一半的病人对四剂的治疗程序有反应).J Clin Oncol.1998;16:2825-2833.
3.Maloney DG,Grillo-Lopez AJ,White CA等:IDEC-C2B8(Rituximab)anti-CD20 monoclonal antibody therapy in patients withrelapsed low-grade non-Hodgkin′s lymphoma(在复发的低度非Hodgkin氏淋巴瘤病人中IDEC-C2B8(美罗华)单克隆抗体治疗).Bolld.1997;90:2188-2195.
4.Hainsworth JD,Burris HA 3rd,Morrissey LH等:Rituximabmonoclonal antibody as initial systemic therapy for patients with low-grade non-Hodgkin lymphoma(美罗华单克隆抗体作为患有低度非Hodgkin淋巴瘤的病人的起始的全身疗法).Blood.2000;95:3052-3056.
5.Colombat P,Salles G,Brousse N等:Rituximab(anti-CD20monoclonal antibody)as first-line therapy of follicular lymphoma patientswith a low tumor burden:clinical and molecular evaluation(美罗华(抗CD20单克隆抗体)作为随时可使用的治疗方法用于患有低肿瘤负载的滤泡性淋巴瘤的病人:临床和分子评估).Blood.200;97:101-106.
6.Coiffier B,Haioun C,Ketterer N等:Rituximab(anti-CD20monoclonal antibody)for the treatment of patients with relapsing orrefractory aggressive lymphoma:a multicenter phase II study(美罗华(抗CD20单克隆抗体)用于治疗复发的或顽固性攻击性淋巴瘤病人:多通道II期研究).Blood.1998;92:1927-1932.
7.Foran JM,Rohatiner AZ,Cunningham D等:European phase IIstudy of rituximab(chimeric anti-CD20 monoclonal antibody)for patientswith newly diagnosed mantle-cell lymphoma and previously treatedmantle-cell lymphoma,immunocytoma,and small B-cell lymphocyticlymphoma(美罗华(嵌合的抗CD20单克隆抗体)用于新诊断的套细胞淋巴瘤和以前治疗过的套细胞淋巴瘤、免疫细胞瘤和小B细胞淋巴细胞淋巴瘤病人的欧洲II期研究).J Clin Oncol.2000;18:317-324.
8.Anderson DR,Grillo-Lopez A,Varns C,Chamber KS,Hanna N:Targeted anti-cancer therapy using rituximab,a chimaeric anti-CD20antibody(IDEC-C2B8)in the treatment of non-Hodgkin′s B-celllymphoma(使用美罗华、一种嵌合的抗CD20抗体(IDEC-C2B8)的靶向抗癌疗法用于治疗非Hodgkin氏B细胞淋巴瘤).Biochem Soc Trans.1997;25:705-708.
9.Vose J,Link B,Grossbard M等:Phase II study of rituximab incombination with CHOP chemotherapy in patients with preiously untreatedintermediate or high-grade non-Hodgkin’s lymphoma(NHL)(美罗华与CHOP化学疗法相结合用于以前未治疗过的中度或高度非Hodgkin氏淋巴瘤(NHL)病人的II期研究).Ann Oncol.1999;10:58.
10.Berinstein NL,Grillo-Lopez AJ,White CA等:Association ofserum Rituximab(IDEC-C2B8)concentration and anti-tumor response inthe treatment of recurrent low-grade or follicular non-Hodgkin′slymphoma(在治疗复发性低度或滤泡性非Hodgkin氏淋巴瘤中血清的美罗华(IDEC-C2B8)浓度与抗肿瘤反应的关系).Ann Oncol.1998;9:995-1001.
11.Harjunpaa A,Junnikkala S,Meri S:Rituximab(anti-CD20)therapy of B-cell lymphomas:direct complement killing is superior tocellular effector mechanisms(B细胞淋巴瘤的美罗华(抗CD20)疗法:直接的补体杀死超过了细胞效应机制).Scand J Immunol.2000;51:634-641.
12.Reff ME,Carner K,Chambers KS等:Depletion of B cells invivo by a chimeric mouse human monoclonal antibody to CD20(在体内通过一个针对CD20的嵌合的小鼠-人单克隆抗体减少B细胞).Blood.1994;83:435-445.
13.Idusogie EE,Presta LG,Gazzano-Santoro H等:Mapping of theClq binding site on rituxan,a chimeric antibody with a human IgG1Fc(Rituxan,一个带有人IgG1 Fc的嵌合抗体上的Clq结合位点的作图).J Immunol.2000;164:4178-4184.
14.Golay J,Zaffaroni L,Vaccari T等:Biologic response of Blymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro:CD55 and CD59 regulate complement-mediated cell lysis(B淋巴细胞在体外对抗CD-20单克隆抗体美罗华的生物学反应:CD55和CD59调节补体介导的细胞裂解).Blood.2000;95:3900-3908.
15.Shan D,Ledbetter JA,Press OW:Apoptosis of malignant humanB cells by ligation of CD20 with monoclonal antibodies(通过用单克隆抗体连接CD20引起的人恶性B细胞的凋亡).Blood.1998;91:1644-1652.
16.Shan D,Ledbetter JA,Press OW:Signaling events involved inanti-CD20-induced apoptosis of malignant human B cells(参与抗CD20诱导的人恶性B细胞凋亡的信号事件).Cancer Immunol Immunother.2000;48:673-683.
17.Hooijberg E,Sein JJ,van den Berk PC等:Eradication of largehuman B cell tumors in nude mice with unconjugated CD20 monoclonalantibodies and interleukin 2(在裸鼠中用非结合的CD20单克隆抗体和白细胞介素2根除大的人B细胞肿瘤).Cancer Res.1995;55:2627-2634.
18.Funakoshi S,Longo DL,Murphy WJ:Differential in vitro and invivo antitumor effects mediated by anti-CD40 and anti-CD20 monoclonalantibodies against human B-cell lymphomas(由针对人B细胞淋巴瘤的抗CD-40和抗CD-20单克隆抗体介导的体外和体内抗肿瘤效应的区别).JImmunother Emphasis Tumor Immunol.1996;19:93-101.
19.Clynes RA,Towers TL,Presta LG,Ravetch JV:Inhibitory Fcreceptors modulate in vivo cytoxicity against tumor targets(抑制性Fc受体在体内调节了针对肿瘤靶的细胞毒性).Nat Med.2000;6:443-446.
20.Fijen CA,Bredius RG,Kuijper EJ等:The role of FcD receptorpolymorphisms and C3 in the immune defence against Neisseriameningitidis in complement-deficient individuals(在补体缺乏的个体中Fcγ受体多态性和C3在针对脑膜炎奈瑟氏菌的免疫防御中的作用).ClinExp Immunol.2000;120:338-345.
21.Dijstelbloem HM,Scheepers RH,Oost WW等:Fcγ receptorpolymorphisms in Wegener′s granulomatosis:risk factors for diseaserelapse(Wegener氏肉芽肿病中的Fcγ受体多态性:疾病复发的风险因素).Arthritis Rheum.1999;42:1823-1827.
22.Myhr KM,Raknes G,Nyland H,Vedeler C:Immunoglobulin GFc-receptor(FcγR)IIA and IIIB polymorphisms related to disability inMS(免疫球蛋白G Fc受体(FcγR)IIA和IIIB多态性与MS中的能力丧失相关).Neurology.1999;52:1771-1776.
23.Koene HR,Kleijer M,Algra J Roos D,von clem Borne AE,deHaas M:FcγRIIIa-158V/F polymorphism influences the binding of IgG bynatural killer cell FcγRIIIa,independently of the FcγRIIIa-48L/R/Hphenotype(FcγRIIIa-158V/F多态性影响IgG与自然杀伤细胞FcγRIIIa的结合,与FcγRIIIa-48L/R/H基因型无关).Blood.1997;90:1109-1114.
24.Wu J,Edberg JC,Redecha PB等:A novel polymorphism ofFcγRIIIa(CD16)alters receptor function and predisposes to autoimmunedisease(FcγRIIIa(CD16)的一个新的多态性改变了受体的功能并导致易感染自身免疫疾病).J Clin Invest.1997;100:1059-1070.
25.Sondermann P,Huber R,Oosthuizen V,Jacob U:The 3.2-Acrystal structure of the human IgG1 Fc fragment-FcγRIII complex(人类IgG1 Fc片段-FcγRIII复合物的3.2-A晶体结构).Nature.2000;406:267-273.
26.Harris NL,Jaffe ES,Stein H等:A revised European-Americanclassification of lymphoid neoplasms:a proposal from the InternationalLymphoma Study Group(修订的欧洲-美国淋巴赘生物分类:国际淋巴瘤研究组的提议).Blood.1994;84:1361-1392.
27.Brice P,Bastion Y,Lepage E等:Comparison in low-tumor-burden follicular lymphomas between an initial no-treatment policy,prednimustine,or interferon alfa:a randomized study from the Grouped′Etude des Lymphomes Folliculaires.Groupe d′Etude des Lymphomes del′Adulte(最初未治疗的、松龙苯芥治疗的或α干扰素治疗的组之间低肿瘤负荷的滤泡性淋巴瘤的比较:随机化的研究).J Clin Oncol.1997;15:1110-1117.
28.Cheson BD,Horning SJ,Coiffier B等:Report of an internationalworkshop to standardize response criteria for non-Hodgkin′s lymphomas.NCI Sponsored International Working(NCI赞助的国际工作组关于对非Hodgkin氏淋巴瘤的反应标准进行标准化的国际研讨会报告).J ClinOncol.1999;17:1244.
29.Jiang XM,Arepally G,Poncz M,McKenzie SE:Rapid detectionof the FcγRIIA-H/R 131 ligand-binding polymorphism using an allele-specific restriction enzyme digestion(ASRED)(使用等位基因特异性限制性酶消化(ASRED)快速检测FcγRIIA-H/R131配体结合的多态性).JImmunol Methods.1996;199:55-59.
30.Kaplan E,Meier P:Nonparametric estimation from incompleteobservations(根据不完全观察进行的非参数性估算).J Am Stat Assoc.1958;53:457-481.
31.de Haas M,Koene HR,Kleijer M等:A triallelic Fcγ receptortype IIIA polymorphism influences the binding of human IgG by NK cellFcγ RIIIa(一个三等位基因的Fcγ受体型IIIA多态性影响了人IgG与自然杀伤细胞Fcγ RIIIa的结合).J Immunol.1996;156:3948-3955.
32.Peltz GA,Grundy HO,Lebo RV,Yssel H,Barsh GS,Moore KW:Human Fcγ RIII:cloning,expression,and identification of thechromosomal locus of two Fc receptors for IgG(人的Fcγ RIII:两个IgG的Fc受体的克隆、表达及染色体上的定位).Proc Natl Acad Sci U S A.1989;86:1013-1017.
33.Schnackenberg L,Flesch BK,Neppert J:Linkage disequilibriabetween Duffy blood groups,Fcγ IIa and Fcγ IIIb allotypes(Duffy血液组FcγIIa和FcγIIIb等种型之间的连接不均衡性).Exp Clin Immunogenet.1997;14:235-242.
34.Shields RL,Namenuk AK,Hong K等:High resolution mappingof the binding site on human IgG1 for FcγRI,FcγRII,FcγRIII,and FcRnand design of IgG1 variants with improved binding to the Fcγ R(人类IgG1上的FcγRI、FcγRII、FcγRIII和FcRn结合位点的高分辨率作图以及与FcγR具有改进结合的IgG1变异体的设计).J Biol Chem.;276:6591-6604.
35.Leppers-van de Straat FG,van der Pol WL,Jansen MD等:Anovel PCR-based method for direct Fcγ receptor IIIa(CD16)allotyping(使用一种基于PCR的新方法对Fcγ受体IIIa(CD16)进行直接的同种异型分型).J Immunol Methods.2000;242:127-132.
36.Lehrnbecher T,Foster CB,Zhu S等:Variant genotypes of thelow-affunity Fcγ receptors in two control populations and a review of low-affinity Fcγ receptor polymorphisms in control and disease populations(低亲和性Fcγ受体在两个对照人群中的变异基因型以及对照和疾病人群中低亲和性Fcγ受体多态性的综述).Blood.1999;94:4220-4232.
表1、按照FCGR3A-158V/F多态性分类的病人的特征
FCGR3A-158VV | FCGR3A-158VF | FCGR3A-158FF | P* | |
n(%) | 10(20%) | 22(45%) | 17(35%) | |
性别男女 | 37 | 1210 | 107 | ns |
疾病阶段II-III期IV期 | 37 | 616 | 611 | ns |
是否包括骨髓是否 | 73 | 166 | 98 | ns |
包括的额外结点数<2≥2 | 82 | 202 | 134 | ns |
在外周血中的BCL-JH重排 | 8 | 12 | 11 | ns |
在骨髓中的BCL-JH重排 | 7 | 12 | 11 | ns |
*纯合的FCGR3A-158V病人和FCGR3A-158F携带者和纯合的FCGR3A-158F病人三个组对FCGR3A-158V携带者的统计学比较。
表2、通过FCGR3A-158V/F多态性对美罗华的临床反应
FCGR3A-158VV | FCGR3A-158F携带者 | P* | |
M2时的临床反应 | |||
目标反应 | 10(100%) | 26(67%) | 0.03 |
完全的症状缓解 | 3 | 7 | |
未证实的完全症状缓解 | 1 | 2 | |
部分反应 | 6 | 17 | |
无反应 | 0(0%) | 13(33%) | |
无变化 | 0 | 10 | |
疾病发展 | 0 | 3 | |
M12时的临床反应 | |||
目标反应 | 9(90%) | 20(51%) | 0.03 |
完全的症状缓解 | 6 | 11 | |
未证实的完全症状缓解 | 1 | 2 | |
部分反应 | 2 | 7 | |
无反应 | 1(10%) | 19(49%) | |
无变化 | 0 | 2 | |
疾病发展 | 1 | 17 |
*纯合的FCGR3A-158V病人对FCGR3A-158F携带者的统计学比较。与三个基因型亚类相关的数据在文中给出。
表3、通过FCGR3A-158V/F多态性在M2和M12时对美罗华的分子反应
FCGR3A-158VV | FCGR3A-158F携带者 | P* | |
M2时的分子反应 | ns | ||
BCL2-JH重排的清除 | 3 | 5 | |
BCL2-JH重排的保持 | 3 | 14 | |
M12时的分子反应 | 0.03 | ||
BCL2-JH重排的清除 | 5 | 5 | |
BCL2-JH重排的保持 | 1 | 12 |
序列表
<110>图尔地区及大学医疗中心(CHRU de Tours)
英那特发玛公司(INNATE PHARMA)
<120>评估抗体治疗反应的方法和组合物
(Methods and compositions to evaluate antibody treatment response)
<130>SCT041074-47
<140>
<141>
<160>8
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<223>人工序列描述:FCGR3A特异引物
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atatttacag aatggcacag g 21
<210>2
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<212>DNA
<213>人工序列
<220>
<223>人工序列描述:FCGR3A特异引物
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gacttggtac ccaggttgaa 20
<210>3
<211>32
<212>DNA
<213>人工序列
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<223>人工序列描述:扩增引物
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atcagattcg atcctacttc tgcagggggc at 32
<210>4
<211>32
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<213>人工序列
<220>
<223>人工序列描述:扩增引物
<400>4
acgtgctgag cttgagtgat ggtgatgttc ac 32
<210>5
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<212>DNA
<213>人工序列
<220>
<223>人工序列描述:扩增引物
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ggaaaatccc agaaattctc gc 22
<210>6
<211>27
<212>DNA
<213>人工序列
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<223>人工序列描述:扩增反义引物
<400>6
caacagcctg actacctatt acgcggg 27
<210>7
<211>254
<212>PRT
<213>人工序列
<220>
<223>人工序列描述:人FCGR3A158F的氨基酸序列
<400>7
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
165 170 175
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
210 215 220
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
245 250
<210>8
<211>22685
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:人FCGR3A158F的核酸引物
<400>8
cagcctggct gacacagtga gacctcatct ctaaaaaaaa aagcaagcag aatattttct 60
taaaaggcaa ttatattcct tcttggccag gcccagtggc tcacacctgt aatcccagca 120
ctttgggagg ccgagatggg tggatcacct gaggtcagga gttcgagacc agcctggcca 180
acatggcgaa aacccgtgtc tactaaaaat acaaaaatta gctgggcatg ggggcatatg 240
cctgtaatcc cagctacttg ggaggctgag acaggagaat cgcttgtacc cgggaggcag 300
agattgcagt gagccgagat catgccactg cactccagcc ttggcgacag agtgaggctt 360
tgtctaaaaa aaaaaaggta ttttttgcct ctctgttggt accaattgtt aaattctttg 420
tggaccactg atgcttacca aaaaaaaaaa aaaaaaaagt gggggcatca tatttcctct 480
agttgacatt aagacacagt aatttagcca gaggagatct tagcaaacat acagtccaca 540
ctccactttc tcatttcatg attgtagaga ctgagatcta gacaatttaa tcggtggtca 600
ccctgggtga catagctagg tctagagctc ctggtctcca ggtcagcatt tctttcttct 660
tcattaaatg tcaagtttcc tcccctgttc attattagct ccttccagaa agagagtttc 720
ttatcttttt agtaggtact cagtaaatac caaggtattc actaggatgc ctttggatga 780
aggtaacaag ccctgactta aattggctta aacagcaggg aaatttactt gaaattgtaa 840
gaaatctggg ttggttgcgt tagaagctca gtgatgtcac caagaccata ttctatccct 900
ccactctgtc ctccttggct atttggcatt gacctcagac tggctgcctt caaaatctta 960
ggttttgcca gcagaaccta ggacaaaatg agcccttgtt catgtacagt gggagagaga 1020
gatcatctct cccaaacatg gcactcccct ctaccagatt ggccctattt aggacaaagt 1080
tccgtcttct cccacttaac caataaaagc caggggaatg ctacaccctg agtggcttag 1140
atcagtcaag atccacctct gcatatgagg gtgattcctg aatagaatca aggttatatt 1200
agaagggagg gagagggatg gatatcaggc tagtacatca tattctattt gttgagttaa 1260
ctgagtcata gcaattgttg agttggaaaa aactcagaac ctactgtgga ttcaagttca 1320
agaaatcatt ctttcctaca tacaacagca ttgctctgta gccctgagct aagagagcat 1380
cacgaaatac agtcttcttg ctgtttataa tcgtaagcaa actcttggac ctgggagggg 1440
atgaatggat aatgtctgtc tgacttgctt ctttctagtt agtaccaact acctcccttc 1500
ttcctgtgat tgttcttaga ataggataaa aaatcttccc ttccctagat cttacagtct 1560
ccccttcccc caggcctttc tatttttcag gattttactc taatcacacc accgaagaat 1620
caagaaatct ttaaagtgta ttagagtagc tagttgtggc agcactaaaa cacggctgca 1680
aattctttga cactctctcc atcaagaaat gaggcctaca tcctctaccc ttgaatctgg 1740
gtgggcttat aacttctggt gattagacta cagcagaaag agaagctgta tagcttccta 1800
aatgttataa agtcttaagg atagctgcca gcaagacctt aggacaaaat gagcccttgt 1860
tcacgtccag tgggagagag agagagagac cctctctccc aaatatggca atcctctcta 1920
cctataacat ttacataaat gttataggtt aaacgttcca caacaaacta agtactattt 1980
aacatcaaga ggaaaaagag acaggagaaa gggttaataa gcctgttgat gaggatctaa 2040
gaagaacaaa ggaggcctgg tttgggcctg gctacccgtt ggtcttgcaa agaagagtct 2100
gaggtggcag agccttcagt ggcagatgcc aaattatcat catgagtgac tgcaagacag 2160
tgtcagctaa gatagccatt tcaagctgct gaaggccttc tcttttagtc gtggagtcct 2220
gtgataagaa ctgaaagttg gaagagtgtg cttgtctgtg gccttatttg gtcggatgca 2280
gtctttatca tttttaattt gtttcttaga acattttatc ttgttggcca aatgccctac 2340
gaaatataaa atggagtctt tttctaagat ggagttagtt atgtcaaggg tcctttatac 2400
agtcttcatc ctttttcctg gcatacaact cctaaaatcc ttagaatctc caaagtgatg 2460
tcttttggtg tgctaatgag gtaactgatg gctggcagct cttaggtagc ttcataacag 2520
gggctgggca caagaaagat catggcaagg tcagaggatt ggggctttca gctccaccct 2580
ccaaactccc tctgggaagt ggagaggggc tgaaggttga attgatcacc aatagccaat 2640
gacttaatta atcattccta agtaataaag ctcccataaa aacccaaaag gacagggttt 2700
ggagatcctc cagagagccg aacacagaga ggttcttgga gggtagtgca ccagagggca 2760
tggaagctcc aagccccttc ccacaggtct tgccctatgt actctttact tgtgtccttt 2820
gtaatattct ttatcacaaa ctgataaatg taaatgtttc cctgagtact gagagccact 2880
ctagcaaatt aattgaaccc aagatgcagg tggtgggaac ccccatttat aactggttgg 2940
tcaaaagcac aggtaaaaca acctggggct tcatcctgga gtatcagaag tgtcttgtga 3000
gactgagccc ttcacttgtg tcacctgatg ctatttccag ttagatagtg ttggaattca 3060
attgaatttg agcagaagtc ccaatcccca gacctgtagt tgtcagtgac ctcttaggaa 3120
ctgggctgca cagcaggaag tgaggggcag gtggggagca aagctttatc tgtatttaca 3180
gtagatcccc atggctcaca tcaccgcctg agctcctcct cctatcagat cagctgtggc 3240
attaaattat cacaggagca tgaaccctat tgtgaagtac gcatgcaagg gatctaggtt 3300
gcattctcct tatgagaatc taatgcctga tgacctggca ttgtctccca tcaccctaga 3360
tgggactgtc tagttgcaag aaaacaagct cggggctccc cctgattcta cattatggta 3420
agttgtataa ttacttcatt aaatattaca ctgtaataat aatagaaatg aagtgcacaa 3480
taaatgtaat gcacttgaat catcctgaaa ccatccccca cccccctacc cctgtccgag 3540
gaaaaattgt cttccatgaa gccaatccct ggtgccaaaa attttggaga ccactggatt 3600
agaagacacc cagttggtgc ccactgctga attgcttgct tgcttgcttg cttgcttgcc 3660
agtggagaga aatccccaca tatctgttgt cagaaatgtg ttgtgagagc atagtgggag 3720
gaactgagtt tgttttttct acagttacag caataggtaa ctggaattca actgctggac 3780
tataccaaag actgccaggc cagcctacct ttctcacagc cttcttgact acctgtcttg 3840
gatgagctca ctgaaagccc acataccttc attctagcat ttcctcagtc tggttgagct 3900
gctttggagg taatacaggt tgtaggactt ccctccactc ctgctcagga ccgttttcag 3960
caggctaatc agacagcagt tggcactgag tacaactgga gaaatgttat cagcactgaa 4020
gactgctcca gctacaaatg cacagcgacc cacctcagct ggacctttgg cattgcttgg 4080
caatccttac aactgtttgg aattcttggt aattcttgta catacaaaca gtcggctcct 4140
tctcccatgc accacaatgg ccattctcaa ctctggtgtt ctcaaacccc caccacaact 4200
gttacccact ctttctcgac aagtgtcttt gactcctcct taaccaagaa aatcaggacc 4260
agcagatgtg gatggtcact caacatctga aaatggattt gcatatgtac cccctcagct 4320
cctgccttca gctcagagcg agaggtaatc cgtatccagt tcacagccaa ctccctgtcc 4380
atgtcccatt cccatctcct caggacccac acttgcttct ctaggtgttc cgtccctgtt 4440
aggcatccaa cttctcccac cctgctggct tcttcccaaa gacctataac caagctcgta 4500
tctttcactt aaaagaaaac aaaaactccc attctcttgt aactaccttt gcagaattgt 4560
gtctgaaaca gtgagagaga tctaacttaa ttgactccat cttgcttcta acctccaagc 4620
tgtctttcct cattcttggg cataggctga actaactttg ggagaaactt agtttatagt 4680
ttgtggttta aagcaaagat gataacagcc ctttcccagg gcagacctcc tttttttctg 4740
aagactagat tgtctttgta ggactaacat tagccacaag attggaaatt atggtttagg 4800
aatcatgcag gtggaggcta caagattctg acccttccta agcactgatc ctaagatcgg 4860
tgcttgagat attttgcaga ccctgcactt gatggatcac ctggcaacac ccagatcaat 4920
aaactggctc atctgatctt gtggtgccca cccaggaact gactcagaac aagaagacag 4980
cttcaacttc ctgtgatttc atccctgacc aatcaacact cctggctcac tggcttcccc 5040
tccaccaacc aagttgtcct taaaaactct gctccccgaa tgctctggaa gactgatttg 5100
agtaataata aaactccagt ctctggctca gtcagctctg catgaattac tctttctcta 5160
ttgcaattcc cctgtcttga tgaatcagct ctgtctagtt accatcctcc acttctcctt 5220
tcttattgtg tcacttaggg ctctgggtta taaacaactt tatcagaatc cagatctttt 5280
aagtagagga aaaagattta ttggatggaa actagaggag gtgagcccat cccactgctg 5340
tgatgactgg gacccaacca tctccctctc ctagaagtga atctccctta tgagtaaaca 5400
agtgtcactt attcaggatt catctcagaa gagactctaa tgggccaaac ctcagttata 5460
tgcctgtcct ctgtctgcct gtatcagtta gctagcactt ttataacaaa gtaccacaga 5520
ctgggtggct taaacaacaa aaatgtattt tcttacagtt cttgaggctg caagtccaag 5580
atcaaggtgt tgacagggtt ggtttctttt aatttttttt ttaaaaattt tattttagat 5640
ttaggggtat gcgtgcaggt ttgctacata ggtaaactcc tatcacgggg gtttgttgca 5700
cacattattt catcacccat gtactaagcc tagttactca atagttattt tttccgatcc 5760
tctccctctt cctaccctcc accctcaagt aggccccagt gtgtctgttg ttcttccttt 5820
gagtccatag ggttggtttc ttctgaggcc tctctccttg gcttgtaggt gtccatcttc 5880
tccctgtatg ggtctgtgtc ccaactaaca aggacatcag tcatattgga ctagagccca 5940
cctaatgatt ttattttaac ttaattacct ctttaaaggc tctatatcca catatagtca 6000
catcctgagg tatagggggt tccacatgtg gacttcaaca tatgaactgg gagagacaaa 6060
atttagtcct taatagtgcc ccaaagtggg gaaaaggaag atctggaccc tcgggtttcc 6120
atagtagaaa gcaatcactg ctttctatta agtactcaca gtggggcttc tccagaaaga 6180
atgatatgct aataagaagg ggaggaggaa gtgatcctgg acagccagat gatatgtgca 6240
ctattccttc ataatggaga ttctgaagag gagaagcact tgactaaaca ctttttcatt 6300
cctactccgt cttcaaccaa aagctgtcaa acttctgttt ctcagcccca gcccctgaaa 6360
ttgctcagga aaaggtcatt aatagttcct tgattgccat atttcaatca aactcttgtt 6420
tgaattcttt tctacaacat taatactgtt actgttgact actccttcct tgaagatctg 6480
ttcccactaa acttccttgt tccctcctct tcagcccctc ctatacaaac tcctttgtca 6540
gctatttttc ctgtgcacgc ttcaaaaatg tttgcatgtc aagtttctgt cattgactct 6600
ctcctcttct cctctccctc tcaatccctc cttcctttcc ctcactgttt ccctttaatt 6660
ctctctcaat actcttacag tttcagagat cttatcctta ctttatctta acctaggatc 6720
tctggatgga ttcaaataga gcttcttaaa ttaaaggaaa cataatgtgt atatttgcat 6780
cctttcttgg gagaaggccc aaaggttttt atcagaggtt tgaaacctca accgtgttgg 6840
tgcctcctaa attgtgtctt ttgtcaagac ctgtcttctg agttccaggg ccatgtgtct 6900
cactgcctac tggaaatctt cacctgaaac cttcacagct acctcaaact caataacatc 6960
aaaagctgaa atcattgtct ctccctccca aagcctgctc atcttcccat ttttcttttg 7020
tccatgaaag ctactgccat cctccttatc acccaaatta gaaatccgag catcacccag 7080
acctctcccc cttcatcacc cctcagccaa tcactcacca agtcttgtcc atccttcctt 7140
cctaacttct ctcctggatg cttccattgc atatccactt tttaaacaga gtggctcttg 7200
tctcaactag actgttgaaa taatcttcca acttttccct ccaccttcca tctctctccc 7260
ctctaactca ttccttggac tgctgtcaga gtacttttca taaaatataa aacagatctt 7320
gtgattcccc agtctaaagc ctttttatta gttcccatta ccttttagaa taaaatatgt 7380
actgttcatc ctgacacaca aaactcttcg tgataaatac taattgagtg cctagtatgt 7440
gcctgccctt gtgctaaatg ttgagggtac aggggtaaac aaggtgaaca gcttccctgc 7500
tctccaagac ctttcagtcc acaaatgcaa tgagtttaca gaggagaagc acaagctcct 7560
aaaggagttg gggtggggtt gggggtcaga acctaattta gaaaattgag gaaggtctca 7620
acctcccatc ttgcatttac aatagtaatc agcaggtgtg gtaccaaata tggaaccaac 7680
aattttatct gcattatctc atttaagcca tgagtgccat tattgttagc ctcactttac 7740
agataaggaa actgagggct agaaggttaa ataagtggca gagttgggat ttcctccaga 7800
ttcctgtgag acccagacat cttaatcctt ttggaacctg tgcttctcct ttgtagtact 7860
cactacactt gtggaactac atccaactac acttgtggaa ctacagccag ctctgcaaac 7920
atgacagtct acttcactcc aagtctttgc tcatgctgct cctcttgcct ggaatgccta 7980
tttctctcaa aaatcttcct gctgaatatt ttgcgatcta attaaagtgt tctctcttcc 8040
atgtacactc ctccctcaga tagaattagc cactgtcttc tttgtgcata cacagcattt 8100
cataaatact gtcacagtcc ctctagcact tcaaatactt atctgatgtt ctccccctaa 8160
gaaactgtaa gtcctagagg atgacaatca actgaattcc atagtcagaa acttctgctg 8220
tgcctggcct tccaatgaga aaaggagaga agaggagggg aaggaagaaa aagggaagga 8280
gaagaaagaa aagcaaacat gaagataaac acttcaatat atgatatccc aagaccatct 8340
acccttttgt aaaaattttg cttttttttt ttccccccca agagtcaggg tctcactctg 8400
tcgcccaggc tagagtgcag tgccatgaac ataactcact gtagcctcta actccggggc 8460
tcaagcaatc ctcctgcctc agcctcctgg gtagctggga ctacaggcat gcaccaccac 8520
atctggctat tattattatt actatattag tagagatggg gtctttctat gttgcctagg 8580
ctggtctcaa attcctggcc tcaagcaatt cttccacctc acattggcct tccaaagtgc 8640
tgggattaca ataagccacc ataggccaaa attttgcatt ttatccatta ctgtaaaatt 8700
aacccttaga aatccaacaa cactcaattt gagaattgtt caacaaccac ttaatgaaaa 8760
ccccctgaaa gcttcccatc ctgttgcagt ccctttctct cctcctgtgc tctctcctct 8820
tcttcctatc tagcccaccc ttttggcagc taagaattcc tccctccatt ggagagccac 8880
agaccaaaga ggagtcaaat aagaaaataa gacctcaaag aaggaaaaca aagtgaaggc 8940
cttgcatcag aagtcacgtg gcagaaagcc acctggatat ctgaaaagaa gaaagaattg 9000
agggatatcc gctttttgcc tcagagacca tccttagccc tgaaggcttt gtttctgctt 9060
taggtttccc agataagcat ccgaagtgct acagcaagga actttaagtt tccagatact 9120
tgtctggatt ttgcaaggcg tagatgagtc acttgagaag gagaactgga atggctgcct 9180
gggttcattt ccattgtcca atccaagggc ctgtggagaa ggggctgctg caagactctg 9240
tgtgtggcgg ggggaggggt gggtacgtgg atggcaatgg gaggatcaat taactccacc 9300
caggagccaa atgaaacaca caaataaaaa acaaaacctg agtagtggtt tttaggtcat 9360
tctggagtag aaagagcatt catttatagc aaaggttggc gggcacctgt gtcagcccct 9420
gcctccactc cacccctaac aagtatcagg tgcccacacg ggcctgctgc tcgcctcctg 9480
ggcttttcta agccaggtga gacctgtccc agatgtccac gaatccactg ggggagtggc 9540
actatcaagc agagtcatct gattttctgc ctgggacctg gaccattgtg agagtaacca 9600
acgtggggtt acgggggaga atctggagag aagagaagag gttaacaacc ctcccacttc 9660
ctggccaccc ccctccacct tttctggtaa ggagccctgg agccccggct cctaggctga 9720
cagaccagcc cagatccagt ggcccggagg ggcctgagct aaatccgcag gacctgggta 9780
acacgaggaa ggtaaagagt tcctgtcctc gcccctcccc acccccacct tttctgtgat 9840
cttttcagcc tttcgctggt gacttgttct tccagggccc atttctctac cctacctggg 9900
tttcttctaa cctggaaatc taatgatcaa atcacactaa aaagtcagta gctcctgtgg 9960
attacatatc ccaggagcat atagattttg aattttgaat tttgaaagaa attctgcgtg 10020
gagataatat tgaggcagag acactgctag tggtctgaag atttgaaagg accactttct 10080
gtgtgcaggc agggcctcag ctggagatag atgggtctgg gcgaggcagg agagtgacaa 10140
gttctgaggt gaaatgaagg aagccctcag agaatgctcc tcccaccttg aatctcatcc 10200
ccagggtctc actgtcccat tcttggtgct gggtggatcc aaatccagga gatggggcaa 10260
gcatcctggg atggctgagg gcacactctg gcagattctg tgtgtgtcct cagatgctca 10320
gccacagacc tttgagggag taaagggggc agacccaccc accttgcctc caggctcttt 10380
ccttcctggt cctgttctat ggtggggctc ccttgccaga cttcagactg agaagtcaga 10440
tgaagtttca agaaaaggaa attggtgggt gacagagatg ggtggagggg ctggggaaag 10500
gctgtttact tcctcctgtc tagtcggttt ggtcccttta gggctccgga tatctttggt 10560
gacttgtcca ctccagtgtg gcatcagggg ctggggaaag gctgtttact tcctcctgtc 10620
tagtcggttt ggtcccttta gggctccgga tatctttggt gacttgtcca ctccagtgtg 10680
gcatcatgtg gcagctgctc ctcccaactg ctctgctact tctaggtaag tcagggtctc 10740
cctggttgag ggagaagttt gagatgcctt gggttcagca gagacccctt ttcaggctac 10800
gaatgagact cccacgaagg gatgggaccc ctcaccacat ctatagctgt ggattgagct 10860
cctaggacaa gccaagatgg ggctagaaat gaggagaatg ctggttccaa ttggggcata 10920
ctcatgagtg aggccagtca cttcacccct ctgggtccca gaatcactct gtggaaccaa 10980
agagcttcga ctagatggtc cctagggtct gtctctttca gtttgacatt ccagggttct 11040
cctctatgat tttcaatttc taccctttct tgtggggata tgggttgagg ctctttctgt 11100
agcttggttc agggaaattc aacctgtacc cttaatttgt gagtttgcac agggagcaag 11160
gggtaaggga gcagtgttga aaatagggat ttgtgttgac agtggcgcaa gaggcatgaa 11220
cagtggagac cagagagcag gtagcaaggt ttccaccaga aacatcctga ttcttgggaa 11280
aattgggctc ctggggcaga ggagggcagg ggagttttaa actcactcta tgttctaatc 11340
actctgatct ctgcccctac tcaatatttg atttatcttt tttcttgcag tttcagctgg 11400
catgcggact ggtgagtcag cttcatggtc ttggattgac ccagtggggc acatatgggg 11460
acaaaggcca taagatattg ggaaatgctt gttgaatggg aaaatgctga tgtggggtta 11520
gcagggatag ttcctccaac acagcagaac ttggccctgt gcttctctgg ccagctttcc 11580
ttaagatact gaacaggcca aaaatggggc caagatgctc taagactgag ccaccaagca 11640
tgggtttgca atgagctcat tctggctttg aggctccctg ggaatggcag tgtagagcct 11700
gctcctctcc ctgtcctcac cccacattat cttggctcct cagaagatct cccaaaggct 11760
gtggtgttcc tggagcctca atggtacagg gtgctcgaga aggacagtgt gactctgaag 11820
tgccagggag cctactcccc tgaggacaat tccacacagt ggtttcacaa tgagagcctc 11880
atctcaagcc aggcctcgag ctacttcatt gacgctgcca cagtcgacga cagtggagag 11940
tacaggtgcc agacaaacct ctccaccctc agtgacccgg tgcagctaga agtccatatc 12000
ggtgagttga tgaaggggaa gaggaaaatc accaataaag ggtgaaacaa agggtcctga 12060
aatacttggt aagagccaga gatgatattc ttagagataa aagctaagat gagatgatgt 12120
gtggtcccac tgaatggtat cagagttgta gtcctagctc taagtaggtc ttgggcaaaa 12180
tgtcaaagcc tgtcagacag tagatatagg actgctgcat tgcacaattc caagaatccc 12240
catatggagt gcatacaatg tgaatgtgtc atgtgaaggt taggccatgg catagatgct 12300
caataatagt tatttatata tttattttca ttttttttaa ttttattttt tgagacagag 12360
tatcactctg tcacccaggc tggagtgcaa tgcggcaatc tcagctcact gcaacttctg 12420
cccccttggg ttgtagtgat tctcctgcct cagcctcccg agtagctgag attacaggca 12480
cccgccacca cgcccagcta atttttgtat ttttagtaga gacagggttt caccatgttg 12540
gtcagtctgg tctcaaactc ctgacctcag gtgattcacc agccttggct tcccaaagtg 12600
ctgggactac aggcgtgagc caccacacct ggccaataat atttattgaa taaattaatg 12660
aatttggtgt taggacctca atctccttct cgctctcaga catgtaatgc cctaagccac 12720
ctcccaaagc aatcctagtg gcctagcatc atatctttct gtctcctcat caatgctata 12780
ctcaaaccta taattaagca taaatttggt aatgtgatag ctcttccaat agaggcagat 12840
acatgttcag cctgcacatt aatcatgaca tgaaagttct tgtgtactat taacagaata 12900
tagacgtcag acacaggtag gagaaatatt ttgaaggcag aggtctttcc tggtgtccct 12960
acaatcttac cacataggct ggtccctgca gtgtcgccct gcaaacctaa ctctacttcc 13020
acggctgttc cattcataca atgtttatgg gtggaacaag ctttggggga agaagggcat 13080
aaggaggtgg atctgcaaga gagctccatg gaattgggcc tctgaaactg atttttgtgg 13140
ctctttggcc tctgacagta ccactcaact gacatggtct tcactctcca gagctacaag 13200
aagatatgtc catttctagc taggtaagag atgtccacct acaaccaaat aaaatggggg 13260
aattaccaag agaaagcaat agaaaaatca agtctaagag ttactagttt gccttgaact 13320
tggctctaga aactggcttt agaagtctag ccaatcaagg ctatattaaa ctgtgaccat 13380
gagaattagc ttcaccaggt aaacttctga gcatccttta atcctttagg acccatttca 13440
cttatgtcct cctctgagaa gcatttttta cttctttttt tgtttgtttg tttgtgtttg 13500
tttttgtttt tgtttttgag acagagtctc tctctgtcac ccaagctgga gtgcagtggc 13560
gcaatcttgg ctcactgcaa cctccacctc ccgggttcaa gcaattctcc tgcctcagcc 13620
tcccaagtag ctgggactac aggtgcatgc caccacgccc ggctaatttt ttgtattttt 13680
agtagagaca gggtttcgca gcgttagcca ggatggtctt gatctcctga cttcatgatc 13740
tgcccacctc ggcctcccaa agtactagga ttacagatgt gagccaccgc gcccagcctg 13800
cattttttac ttctttcagg cagaatttct ttattccaat ctagtcagcc ccgcagtcct 13860
ttattcttag cctgttgtag cacttgtcat attgtattgt gattatttct gaatatttat 13920
gtttctatgt ctagactgta gattctttga ggctgagaac tatatgtccc atcatctggg 13980
tatctccagt ccacagtgtg tcatacatag tgagtgcttg atgaaatatc acttgaagga 14040
atatacatat ggacattcac tgggtccatg acaggataga ttcgaacaag aatgttcctc 14100
caaaggccac cagactatat actaaccatg actttatgct aataatgatt catctctctg 14160
ctgaaaaagt aagtggatag ataggcacat ggcttctttt gataaatgat atctcttaat 14220
aggtaatgaa gattactttc tgtttggcaa atctttgtgg tagagaatca tgaccaacac 14280
acgtcctacc aattttgttt agcatcaggt agtagatttt ttaaattata gtaattcaag 14340
ctgagaatgt agatttaaaa aataaaatta ttgtaaattt tgttttgttc ttattacaaa 14400
agtcatttgg ggtcaatttc aaaaatatat aaaagtaaac aggagaaatt taaaatgtcc 14460
ttcagtccca ctccttcaga gaaaacccct gttaatatgt aagtgcatat ccttcttttt 14520
tctgtgcata atacttttta aaatatttga agtattatgc ttttttaact taaaattgtc 14580
tcatgaatat tttcttatgc cattataata cttacctata acatcattat tttttaatta 14640
ttcaggccct ttcccgacca tgacctcatg ttctctcttt gtgaagtctg attacttggt 14700
gacatgatcg tgagaataag ctctggcgat ataagaattt cctctcttga aggccatgct 14760
cagtaaatta cttggtgaca tgatcgtgag aataagctct ggcgatacaa gaatttcctc 14820
tcttgaaggc catgctcagt aataaagttg gtctcaccga ggccctgtga caccttagaa 14880
accacgaatt gccaggctga gcaataccag tcccgccctt cccctccctg gtgtttacat 14940
tgagttctcc ttcacaattt ctgcagccac tccgtggcca ccgtcacctt attcctgact 15000
gccacaagag tctttcaata ttcctttgat tgcctattcc ttctgaaatc taccttttcc 15060
tctaataggg caattcatca ttttcaaatg caatttttac tctgatctag aacttactgt 15120
gaatccttgt cacctgccac agcaaatcta agtctagcac ttaaggatcc tgcagatatg 15180
ctcatcgttg cttctcactt acctcattgc ttagtccctc tgctctaacc ctgtgtgttg 15240
atcacatgtg tgtgtgtccc tcttccccat tagacaaagg tcttggtatg acttcagttc 15300
tcttgcaggg ccccatcagc tcttccccaa agggagctat gcagggttga ctcccaatct 15360
ggctttccct tatgtctcag gatctgggtg gtacgtggcc ccttcacaaa gctctgcact 15420
gagagctgag gcctcccggg cctggggtgt ctgtgtcttt caggctggct gttgctccag 15480
gcccctcggt gggtgttcaa ggaggaagac cctattcacc tgaggtgtca cagctggaag 15540
aacactgctc tgcataaggt cacatattta cagaatggca aaggcaggaa gtattttcat 15600
cataattctg acttctacat tccaaaagcc acactcaaag acagcggctc ctacttctgc 15660
agggggcttt ttgggagtaa aaatgtgtct tcagagactg tgaacatcac catcactcaa 15720
ggtgagacat gtgccaccct ggaatgccca gggacgcctg tgtgtggaac ctgcaatcac 15780
actgggaagt tgagttggga ggagattcct gattcttaca cgcacttctt catatgtggt 15840
tccctcctgg tgatcaccag gaggtcccca aaagtccctg attgcagggt aggtttgcag 15900
ctctgtttca gtccattctt ttggggtagc taggaggtgt cattcactct gcagcatgat 15960
ggcaggagca gaagccacat ctcctcccca ataaatacct ctgtctttcc ttacgctaat 16020
cacacccacg gtgtcatatg ttcctatcgt gctggcctcc ttcttatcca agccttttag 16080
ccacgatcca aactggcagg agcccctcat cccctcacag aaagagccca gaacctgggt 16140
tctggccctg cagctaatta accatctgac cagaggtgag ccacttagtc tctctgaacc 16200
ccaatttctt cttccgtaac aaaaataagc tgacatttat tgggcacctt tcagtgtgct 16260
agactctgtg ctaaacaatt ctttacatgc acctggtttg actatcacag tagaccttca 16320
caacatgaga taggtaatat tccattttac agatgaagta accgaggtgc aaaaataaat 16380
aaataagttt ccctaaggtc acatcaaaga cttcaaagcc tgtatattta accagtaagt 16440
aaaagatttg aacaagcact aatatcctat gatcccatta agtcatccac aaaacatctc 16500
taggttctgt agcaccagcc tccagaatca gagctctaga gtggtgtgcc tggactttcc 16560
agtttcacag aacttctatc tgtaactagc ccaagacata aattgtaaac aatttgcatg 16620
tagaaaggca gcaaaacacc ttttgagatt ttgacactac aatgccataa tttgtacaaa 16680
aataatttca tgacacttta aactgaaagt aaatactccc aagtggttag ggaaagagag 16740
caaataaagc aaatggggta acatgtaaac aatgagtgga tctgggtaaa ggatatacga 16800
gattaaacta ttctggtcat ttttttttta agtttggaaa tatatcaaaa tcaagagttt 16860
aaaaaattga aatgcaaaat caacaaattt gtcccagttt ctagaccata gcattgtctg 16920
acaatttctt aactgtcaca caaaacccag cttacaacct aacttgttaa cgctccctgt 16980
cacatctctg tcaaacaagc aggagccttt gctcagtgtt tggtgagctg tcctctgctc 17040
agatagcact aagatcagga accaatggga ggaagcaata ctttccccca gacttcccca 17100
ccattcctac cacttgcctg ttggctgttg tcaaagactt tctactggtg acctcactgt 17160
ttgttccaaa tatctgcctt agtgactgtc attttttttc atctctccac ttctcctaat 17220
aggtttggca gtgtcaacca tctcatcatt ctttccacct gggtaccaag tctctttctg 17280
cttggtgatg gtactccttt ttgcagtgga cacaggacta tatttctctg tgaagacaaa 17340
cattcgaagc tcaacaagag actggaagga ccataaattt aaatggagaa aggaccctca 17400
agacaaatga cccccatccc atgggggtaa taagagcagt agcagcagca tctctgaaca 17460
tttctctgga tttgcaaccc catcatcctc aggcctctct acaagcagca ggaaacatag 17520
aactcagagc cagatccctt atccaactct cgacttttcc ttggtctcca gtggaaggga 17580
aaagcccatg atcttcaagc agggaagccc cagtgagtag ctgcattcct agaaattgaa 17640
gtttcagagc tacacaaaca ctttttctgt cccaaccgtt ccctcacagc aaagcaacaa 17700
tacaggctag ggatggtaat cctttaaaca tacaaaaatt gctcgtgtta taaattaccc 17760
agtttagagg ggaaaaaaaa acaattattc ctaaataaat ggataagtag aattaatggt 17820
tgaggcagga ccatacagag tgtgggaact gctggggatc tagggaattc agtgggacca 17880
atgaaagcat ggctgagaaa tagcaggtag tccaggatag tctaagggag gtgttcccat 17940
ctgagcccag agataagggt gtcttcctag aacattagcc gtagtggaat taacaggaaa 18000
tcatgagggt gacgtagaat tgagtcttcc aggggactct atcagaactg gaccatctcc 18060
aagtatataa cgatgagtcc tcttaatgct aggagtagaa aatggtccta ggaaggggac 18120
tgaggattgc ggtggggggt ggggtggaaa agaaagtaca gaacaaaccc tgtgtcactg 18180
tcccaagttg ctaagtgaac agaactatct cagcatcaga atgagaaagc ctgagaagaa 18240
agaaccaacc acaagcacac aggaaggaaa gcgcaggagg tgaaaatgct ttcttggcca 18300
gggtagtaag aattagaggt taatgcaggg actgtaaaac caccttttct gcttcaatat 18360
ctaattcctg tgtagctttg ttcattgcat ttattaaaca aatgttgtat aaccaatact 18420
aaatgtacta ctgagcttcg ctgagttaag ttatgaaact ttcaaatcct tcatcatgtc 18480
agttccaatg aggtggggat ggagaagaca attgttgctt atgaaagaaa gctttagctg 18540
tctctgtttt gtaagcttta agcgcaacat ttcttggttc caataaagca ttttacaaga 18600
tcttgcatgc tactcttaga tagaagatgg gaaaaccatg gtaataaaat atgaatgata 18660
aaattctttc ttcttccctt tgtccaacat tgtaacagag attggtttgg attggtaaga 18720
aacaccccct cctcccagca accatctcac cacaactcat ataaattagc cagcttgctt 18780
tccaaatctt gctgagacaa ttgggctaag gaggattctt atgggaagta tgggatagga 18840
gggtgaataa gcattagaga tcgttttaga gcattggggc agataggaga aggcacagct 18900
acacaggagg tagaggcctg ggcagaggta gagggtcagc ctgattgtat gaattatgag 18960
ctatatacca agacgattca agctagattg catacataaa tattacataa gattccgaca 19020
cgacacaggt gcatttggaa accttggaca ttcaactcac atttatttac tacctacaat 19080
gtgcaagctt gagttcaggt gctgaagata ccagatgaac aacacagggt cattccctgg 19140
agaagcttta tttctagtga gaaaaacagt taaataggaa gagaatgaag aaagggctgc 19200
agaaaagagg cttgatttgg ggggtgtggt catgaaggat gagtaggagt tcgccaggca 19260
aagaagagaa gaaaagccca aggttcatag gcaaagattc aaaaaccaga gtgtgagttc 19320
aagaaagcag tttggttctg tgtcggtgag ggagaggaaa gagtttcagg gccagatcat 19380
gaagggcatt accttccaaa ctaaggagat cgtatcagac cctgcaatac attgagagag 19440
tttaagcaga ccaggtttgt accgtatagt attttagaag gattctctcg caactacttg 19500
atggatggac gggacaggag agttgaagac cagaagccaa atagggcagc aaggcaggat 19560
gcagtaaccc aaagggagca atgaggaagt aactggcggt gaggctggag aggaaggtgc 19620
ttaatcaaca aggtatttag gaggccgact ctccaagaat tggcagccag cagtacacgg 19680
cgtgactaag gaccaggttc cacacatagt gcccgttttc tgagttagga aatagaaagg 19740
caaggcaggt acaggtttgg tggaaagaca aacaattcgt tttggtatta ttagtactta 19800
cttcctttgg tcagtaaatt ttcttaaagt gtcagtttcc ataacgtaat tgccgtggtt 19860
aagcagctaa gagttatcac tacaacccta gtcggaaaaa ccaaatacct caaaattacc 19920
cgtacagcac taaggcagaa gaggacattg ggaaccacac aacgcggagg tctgctacca 19980
gagctccctg cggttagcac cgcggctggt tttgagcgcc aaggccccag cgctcccagc 20040
ggatagcatc gcacgcagtt ttttcagtca aagtttcaaa aacccagggt tcacaaaatg 20100
cgacttccgt ccctgggtgg gatcgaacca ccaacctttc ggttaacagc cgaacgcgct 20160
aaccgattgc gccacagaga cgggcgttgg cgattttggc tgccaagtca cttcactgaa 20220
gaaaaaatgc tcagcactca cgtctccaaa aaaattgagg ttgatttgaa accagtgaca 20280
caattagctt tccgtgcttc agggcgcggc tcatagccct gagcgaggca ggtctttttt 20340
ctgcgctagc acttgcctag atctggagca ggactcagct tccagcagaa gaggttgaga 20400
aaaggagagc agaagagaat gcaggaacga agggtcttcg gggaatccaa aatggatgct 20460
ctctgtgggt tcgggggttc cgttgatttt ggtcagagaa gtacgacgat aagctttttt 20520
tgctgatgta gacaacttat gtatgcatgt gcacacgttt agtgctgact cataataagc 20580
ttattatcgt gagcattaaa aatattttct ttcaggtcca atcacgtcca gcaaaatgtg 20640
atgtctaagt aagtgagttt tgtgttacaa aattagtctt caacccacgc tgttttgaaa 20700
ggtttctacc ggcatattag acatgcagac agaacacgga gcttaaaaag cctgtaacat 20760
tccaattaat ggtattcagc ttggaaataa aaaatatttt ttaaaaaatg cgtgcaactt 20820
aaggactttc atgctgacat atccagatcc aaatatctga ggacagagac ccctaattcc 20880
accaccatcg acctagggaa cctcgtcagt gctgggtcta aaaaggcttt ttttttttct 20940
ttaattcata tgtatatata ctttattcat atatatatat actttaagtt ctaggctaca 21000
tgtgcacaag gtgcaggttc gttacatata catgtgccat gttggtgtgc tgcacccatt 21060
aactcgtcat ctacattagg tgtttctcct aatgttatcc ctccctcctt cccccaccca 21120
cgacaggtcc cggtgtgtga tgttccctac catgcacacg tatgtttatt gtggaactat 21180
tcacaatagc aaagacttgg aaccaaccca aatgtccatc aatgatagac tggattaaga 21240
aaatgtggca catatacact atggaatact atgcagccat aaaaaaggat gagttcatgt 21300
cctttgtagg gacatagatg aagctggaaa ccatcagtct gaacaaacta tcacaaggac 21360
agaaaatcag acaccgcatg ttctcactca taggtgggaa ttgaacaata agaacacttg 21420
gacgaaagcc attttctata ttgcccaaaa accagggtct ctccatagcc tccacacaga 21480
atctcctttc tttctgccct gccatcctct gtcatcagtg ggctccagtt taggagcagg 21540
tggaagtttt caatgatgtt cagtgaaatg agaagacatg caaacataga tatgtatatg 21600
cagaaattat atatgcatat atgtttatat gtacacagta tcatatgtat aataaataag 21660
taaataaata aataaatttg ccaaatgatc tttaaactag agtcatttat tttttttatt 21720
aatttttttt tttgagatgg agtcttgctc tgtcgcccag gctggagtgc agtggcgcaa 21780
tcttggctca ctgcaacctc cacctcccac attcaagcaa ttctcctgtc tcagcctcct 21840
gagtagctga gattacagtc atgggccacc atgcccggct aatttttgta tttttttttt 21900
tttttttttg agacagagtc tcgctgtcgc ccaggttgga gtgcagtggc gcgatctcgg 21960
ctcactgcag gctccgcccc ccgggattca cgccattctc ctgcctcagc ctcctgagta 22020
gctgggacta caggcacctg ccacctcgcc cggctaattt ttttgtattt ttggtagaga 22080
tgggctttcg ctatgttggc caggctggtc tcaaactcct gacctcaggt gatcctcctg 22140
cctcagcctc ccaaactgct gggattacag gtgagagcca ctgtaccagg cctagagtca 22200
tttcttttat actttaaatt tttgtctctg ttcttttgct cagacctgtg gagctggcaa 22260
tatgggcaag tgtcatggac tgtctactgc caggaagctc cattgtcacc gacaggatca 22320
gaagtggcat ggtaaatggt acaagaaagc ccattcgggc acagtcctga agaccagcct 22380
ttttggaggt gcttctcatg caaagggaat tgggctggaa aaagtaggga ttggagccaa 22440
atagcccagc tctgccactg agaagtgtgc cagggccaag ctgatcatcc agcataagct 22500
agatgctgtg gtctccactg gcacagctga tcctcttgtt acaggatgga ggctgtgagg 22560
cagatgagag aacagcaaga aaatcacagc ctttgtacct gatgatgatt gcttgaattt 22620
tattgaaaaa aatgatgaag ttctgtatca ggggaaccag cacccaatat ttcaatgtag 22680
gttct 22685
Claims (21)
1.适于体外确定FcγRIIIa受体第158位上的氨基酸残基的特异性针对FcγRIIIa受体基因的核酸引物或探针在制备用于评估个体对减少患者靶细胞的IgG治疗抗体疗法的反应的诊断试剂盒中的应用,其中第158位上的苯丙氨酸是对所述治疗低反应性的指示,而第158位上的缬氨酸是对所述治疗高反应性的指示。
2.权利要求1的应用,其中核酸引物适于对FcγRIIIa受体基因或其mRNA或其含有编码第158位氨基酸残基的一部分核苷酸序列进行测序。
3.权利要求1的应用,其中核酸引物适于对FcγRIIIa受体基因或其mRNA或其含有编码第158位氨基酸残基的一部分核苷酸序列进行扩增。
4.权利要求2的应用,其中核酸引物为包括正向序列和反向序列的引物对,所述引物对中的引物与FcγRIIIa受体基因区杂交,并扩增含有与第158位氨基酸残基相对应的密码子的至少部分FcγRIIIa受体基因。
5.权利要求4的应用,其中各对引物包含至少一个与所述密码子互补和重叠以区分第158位缬氨酸(gtt)和第158位苯丙氨酸(ttt)的引物。
6.权利要求3的应用,其中扩增通过聚合酶链式反应进行,所述聚合酶链式反应选自PCR、RT-PCR或嵌套PCR。
7.权利要求1的应用,其中核酸引物的序列选自SEQ ID NOs:1-4。
8.权利要求1的应用,其中特异性针对FcγRIIIa受体基因的核酸引物与限制性酶组合应用,所述酶切割第158位处特定等位基因的编码序列,而不切割第158位处的其他等位基因,从而使得等位基因特异的限制性酶消化。
9.权利要求8的应用,其中引物含有等位基因特异的限制性酶位点。
10.权利要求9的应用,其中引物包括SEQ ID NO:3。
11.权利要求1的应用,其中核酸探针特异性针对第158位基因型缬氨酸或苯丙氨酸。
12.权利要求11的应用,其中探针被标记。
13.权利要求1的应用,其中个体是人。
14.权利要求13的应用,其中个体患有肿瘤、病毒感染或与异源或病理性免疫活性细胞有关的病症。
15.权利要求14的应用,其中个体患有肿瘤,以及治疗抗体疗法的目的在于减小肿瘤的负荷。
16.权利要求15的应用,其中肿瘤是淋巴瘤。
17.权利要求16的应用,其中淋巴瘤是NHL。
18.权利要求1的应用,其中抗体是IgG1或IgG3。
19.权利要求18的应用,其中抗体是抗CD20抗体。
20.权利要求19的应用,其中抗体是美罗华。
21.权利要求18的应用,其中抗体减少B淋巴细胞。
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Families Citing this family (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003035904A2 (en) | 2001-10-19 | 2003-05-01 | Centre Hospitalier Regional Et Universitaire De Tours | Methods and compositions to evaluate antibody treatment response |
EP2385137A1 (en) * | 2002-07-31 | 2011-11-09 | University of Southern California | Polymorphisms for predicting disease and treatment outcome |
KR20060038461A (ko) * | 2003-07-24 | 2006-05-03 | 이나뜨 파르마 | Nk 세포 강화 화합물을 사용하여 치료용 항체의 효율을높이는 방법 및 조성물 |
GB0324778D0 (en) * | 2003-10-23 | 2003-11-26 | Forinnova As | Method |
MY162179A (en) * | 2004-04-01 | 2017-05-31 | Elan Pharm Inc | Steroid sparing agents and methods of using same |
RU2006138865A (ru) * | 2004-04-07 | 2008-05-20 | Чирон Корпорейшн (Us) | Анализы на основе нуклеиновой кислоты для идентификации полиморфизмов fc-рецептора |
CA2569196A1 (en) * | 2004-06-01 | 2005-12-15 | Centre Hospitalier Regional Et Universitaire De Tours | Fcgr3a gebotype and methods for evaluating treatment response to non-depleting antibodies |
US20070190657A1 (en) * | 2004-06-15 | 2007-08-16 | Universite Francois Rabelais | Methods of assessing susceptibility to drug-induced thrombocytopenia |
US9109255B2 (en) * | 2004-06-18 | 2015-08-18 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for determining responsiveness to antibody therapy |
AU2005267148A1 (en) * | 2004-07-01 | 2006-02-02 | University Of Southern California | Genetic markers for predicting disease and treatment outcome |
GB0510790D0 (en) * | 2005-05-26 | 2005-06-29 | Syngenta Crop Protection Ag | Anti-CD16 binding molecules |
KR20080073725A (ko) * | 2005-11-01 | 2008-08-11 | 노파르티스 아게 | 항-cd40 항체의 용도 |
WO2007053661A2 (en) * | 2005-11-01 | 2007-05-10 | Novartis Ag | Uses of anti-cd40 antibodies |
CA2631630A1 (en) * | 2005-11-30 | 2007-06-07 | University Of Southern California | Fc.gamma. polymorphisms for predicting disease and treatment outcome |
AU2007244683A1 (en) * | 2006-04-27 | 2007-11-08 | Pikamab, Inc. | Methods and compositions for antibody therapy |
CA2675352A1 (en) * | 2007-01-18 | 2008-07-24 | University Of Southern California | Genetic markers for predicting responsiveness to combination therapy |
US20090142763A1 (en) * | 2007-08-22 | 2009-06-04 | Wyeth | Nested pcr-based method for specific genotyping of the fc gamma receptor iiia gene |
US20120070432A1 (en) * | 2009-05-28 | 2012-03-22 | Amgen Inc. | Treatment of pancreatic cancer using a dr5 agonist in combination with gemcitabine |
EP2383572A1 (en) * | 2010-04-28 | 2011-11-02 | Assistance Publique Hôpitaux de Marseille | Cellular humoral activation test (CHAT) |
KR101938699B1 (ko) | 2012-07-23 | 2019-01-16 | 삼성전자주식회사 | Lrig1의 항 c―met 항체 적용 대상 환자 선별을 위한 용도 |
KR101938698B1 (ko) | 2012-07-23 | 2019-01-16 | 삼성전자주식회사 | Cbl의 항 c-met 항체 적용 대상 환자 선별을 위한 바이오마커로서의 용도 |
FR3037340A1 (fr) | 2015-06-09 | 2016-12-16 | Univ Montpellier | Methode de pronostic d'hemopathies lymphoides |
EP3356561B1 (en) * | 2015-10-02 | 2021-10-27 | Momenta Pharmaceuticals, Inc. | Therapeutic and diagnostic methods for autoimmune diseases and/or inflammation |
US10226088B1 (en) * | 2017-03-30 | 2019-03-12 | Robert Russell | Pet waste disposable glove |
CN108330180A (zh) * | 2017-10-25 | 2018-07-27 | 广州和康医疗技术有限公司 | 一种对fcgr3a位点进行基因型检测的方法及试剂盒 |
CN108676875B (zh) * | 2018-04-10 | 2022-01-18 | 哈尔滨医科大学 | Cd20、fcgriia及fcgriiia基因在弥漫大b细胞淋巴瘤预后中的用途 |
CA3125503A1 (en) * | 2019-01-18 | 2020-07-23 | Acepodia Biotechnologies Ltd. | A novel cd16+ natural killer cell and a method of culturing cd16+ natural killer cell |
US20230036481A1 (en) * | 2020-01-16 | 2023-02-02 | Acepodia Biotechnologies Ltd. | A novel cd16+ natural killer cell and a method of culturing cd16+ natural killer cell |
CN114277149B (zh) * | 2021-12-30 | 2023-09-22 | 苏州方科生物科技有限公司 | 一种检测cd16a基因多态性的试剂盒及其应用和使用方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1094965A (zh) * | 1992-11-13 | 1994-11-16 | 艾德药品公司 | 抗人类b淋巴细胞限制分化抗原的嵌合及放射标记抗体 |
WO2001012848A2 (en) * | 1999-08-14 | 2001-02-22 | University Of Leeds | Genetic marker for rheumatoid arthritis |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4446314A (en) * | 1980-09-30 | 1984-05-01 | Cutter Laboratories, Inc. | Fractionation of heparin |
ES2262163T3 (es) * | 1988-05-27 | 2006-11-16 | Applied Research Systems Ars Holding N.V. | Receptor iii de fc gamma humano. |
EP0633268B1 (en) * | 1993-05-14 | 2004-07-28 | Cancer Institute | MDC proteins and DNAs encoding the same |
US5830652A (en) * | 1994-08-30 | 1998-11-03 | New York Society For The Ruptured And Crippled Maintaining The Hospital For Special Surgery | Method for determining predisposition to severe forms of autoimmune disease by determining Fcy receptor allelic patterns |
US5866337A (en) * | 1995-03-24 | 1999-02-02 | The Trustees Of Columbia University In The City Of New York | Method to detect mutations in a nucleic acid using a hybridization-ligation procedure |
US6444789B1 (en) * | 1995-05-03 | 2002-09-03 | Applied Research Systems Ars Holding N.V. | CD16-II variants |
WO1997046715A1 (en) * | 1996-06-03 | 1997-12-11 | New York Society For The Ruptured And Crippled Maintaining The Hospital For Special Surgery | Fc receptor polymorphism |
ATE211740T1 (de) * | 1996-06-27 | 2002-01-15 | Pfizer | Substituierte indazolderivate |
US6676927B1 (en) * | 1999-01-20 | 2004-01-13 | The Rockefeller University | Animal model and methods for its use in the selection of cytotoxic antibodies |
EP1130122A3 (en) * | 2000-02-17 | 2001-10-17 | AstraZeneca AB | Methods for the diagnosis of polymorphisms in the human EP1-R gene |
WO2003035904A2 (en) * | 2001-10-19 | 2003-05-01 | Centre Hospitalier Regional Et Universitaire De Tours | Methods and compositions to evaluate antibody treatment response |
US20070231813A1 (en) * | 2004-06-01 | 2007-10-04 | Centre Hospitalier Regional Et Universitaire De Tours | Fcgr3a Gebotype and Methods for Evaluating Treatment Response to Non-Depleting Antibodies |
US20070190657A1 (en) * | 2004-06-15 | 2007-08-16 | Universite Francois Rabelais | Methods of assessing susceptibility to drug-induced thrombocytopenia |
-
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Patent Citations (2)
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---|---|---|---|---|
CN1094965A (zh) * | 1992-11-13 | 1994-11-16 | 艾德药品公司 | 抗人类b淋巴细胞限制分化抗原的嵌合及放射标记抗体 |
WO2001012848A2 (en) * | 1999-08-14 | 2001-02-22 | University Of Leeds | Genetic marker for rheumatoid arthritis |
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CA2463746C (en) | 2014-09-02 |
CN1571850A (zh) | 2005-01-26 |
DE60228108D1 (de) | 2008-09-18 |
ATE403750T1 (de) | 2008-08-15 |
US20150017633A1 (en) | 2015-01-15 |
EP1436427A2 (en) | 2004-07-14 |
EP1436427B1 (en) | 2008-08-06 |
WO2003035904A2 (en) | 2003-05-01 |
US20110123996A1 (en) | 2011-05-26 |
AU2002346999B2 (en) | 2007-11-08 |
CA2463746A1 (en) | 2003-05-01 |
JP4216720B2 (ja) | 2009-01-28 |
US7858300B2 (en) | 2010-12-28 |
ES2311634T3 (es) | 2009-02-16 |
US20050064417A1 (en) | 2005-03-24 |
JP2005506093A (ja) | 2005-03-03 |
DE02782887T1 (de) | 2005-01-13 |
WO2003035904A3 (en) | 2003-10-16 |
DK1436427T3 (da) | 2008-12-08 |
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