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CN104204230A - Methods related to treatment of inflammatory diseases and disorders - Google Patents

Methods related to treatment of inflammatory diseases and disorders Download PDF

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CN104204230A
CN104204230A CN201380019432.0A CN201380019432A CN104204230A CN 104204230 A CN104204230 A CN 104204230A CN 201380019432 A CN201380019432 A CN 201380019432A CN 104204230 A CN104204230 A CN 104204230A
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K.S.弗雷德里克森
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Abstract

本发明涉及基因标记,所述标记与在患有炎性疾病或病症的患者中预测针对抗炎治疗的临床反应的方法相关。The present invention relates to genetic markers that are relevant to methods of predicting clinical response to anti-inflammatory therapy in patients suffering from an inflammatory disease or disorder.

Description

治疗炎性疾病和病症的相关方法Related methods of treating inflammatory diseases and conditions

技术背景 technical background

本发明涉及炎性疾病和病症的诊断、预后和治疗优化领域中的方法,目的在于通过提供预测对治疗药的反应性的方法而改进对患者的治疗选项和治疗方案。 The present invention relates to methods in the field of diagnosis, prognosis and treatment optimization of inflammatory diseases and disorders with the aim of improving treatment options and regimens for patients by providing methods for predicting responsiveness to therapeutic agents.

背景 background

对于大量患者来说,炎性疾病和病症和尤其是自身免疫疾病严重影响患者健康并且治疗选项是令人不满意的。 For a large number of patients, inflammatory diseases and disorders and especially autoimmune diseases seriously affect patient health and treatment options are unsatisfactory.

类风湿性关节炎(RA)是临床上重要的,慢性系统性自身免疫RA是未知病因的自身免疫病。大多数RA患者具有慢性疾病过程,甚至用当前可用的治疗,仍可导致渐进的关节破坏、变形、失能和甚至过早死亡。RA的诊断通常依赖于对患者体征和症状的临床和实验室评价。通常,对疑似患有RA的患者的实验室评价可包括测定血清中的被认为是类风湿因子(RF)的某些抗体和环状瓜氨酸化肽抗体(抗CCP)的水平。尽管这些抗体在RA患者血清中通常存在,但并非所有RA患者都有。还可使用被称为红细胞沉降速率(ESR)的额外血液检验。升高的ESR表明炎性过程普遍存在,尽管不一定是RA。进一步血液检验可用于评价其它因子的水平,所述因子例如已知与RA相关的C-反应蛋白(CRP)。另外,对受累关节可进行放射照相分析。总之,这样的当前可用的诊断RA的实验室检验是不精确和不完善的。 Rheumatoid arthritis (RA) is a clinically important, chronic systemic autoimmune RA is an autoimmune disease of unknown etiology. Most RA patients have a chronic disease course that, even with currently available treatments, can lead to progressive joint destruction, deformation, disability and even premature death. The diagnosis of RA usually relies on clinical and laboratory evaluation of the patient's signs and symptoms. Typically, laboratory evaluation of patients suspected of having RA may include measuring serum levels of certain antibodies known to be rheumatoid factor (RF) and cyclic citrullinated peptide antibodies (anti-CCP). Although these antibodies are commonly present in the sera of RA patients, not all RA patients have them. An additional blood test called erythrocyte sedimentation rate (ESR) may also be used. An elevated ESR indicates a prevalent inflammatory process, although not necessarily RA. Further blood tests can be used to assess levels of other factors such as C-reactive protein (CRP), which is known to be associated with RA. Alternatively, radiographic analysis of the affected joint may be performed. In conclusion, such currently available laboratory tests for diagnosing RA are imprecise and incomplete.

美国风湿病学会(The American College of Rheumatology,ACR)标准常用于诊断和判定严重程度(http://www.rheumatology.org)。 The American College of Rheumatology (ACR) criteria are commonly used for diagnosis and severity (http://www.rheumatology.org).

已经进行了尝试以便根据生物标记而改善诊断和预后(参见例如Rioja等人, Arthritis and Rheum. 58(8):2257-2267 (2008);Pyrpasopoulou等人, Mol. Diagn. Ther. 14(l):43-48 (2010);WO 2004/0009479;WO 2007/0105133;WO 2007/038501;WO 2007/135568;WO 2008/104608;WO 2008/056198;WO 2008/132176;和WO 2008/154423)。目前,已经给出了将RA患者分为亚组和鉴定患者组的方法,其基于特定分子谱表明对抗CD20治疗的更高反应性(WO2011028945)。然而,并没有鉴定出能够让临床医生或其他人准确定义类风湿性关节炎的病理生理方面、临床活性、对治疗的反应、预后或发展所述疾病的风险的经临床确证的诊断或预后标记。 Attempts have been made to improve diagnosis and prognosis based on biomarkers (see e.g. Rioja et al., Arthritis and Rheum. 58(8):2257-2267 (2008); Pyrpasopoulou et al., Mol. Diagn. Ther. 14(l) :43-48 (2010); WO 2004/0009479; WO 2007/0105133; WO 2007/038501; WO 2007/135568; WO 2008/104608; WO 2008/056198; WO 2008/132176; and WO 2008/154). Currently, methods have been given to classify RA patients into subgroups and to identify groups of patients that, based on specific molecular profiles, showed higher responsiveness to anti-CD20 therapy (WO2011028945). However, no clinically validated diagnostic or prognostic markers have been identified that would allow clinicians or others to accurately define the pathophysiological aspects of rheumatoid arthritis, clinical activity, response to treatment, prognosis, or risk of developing the disease .

因此,随着RA患者寻求治疗,有重要的试验和误差涉及对特定患者有效的治疗药的探索。为了找到最有效的治疗,这样的试验和误差通常包括相当大的风险并使患者不适。因此,需要更有效手段用于确定哪些患者将对哪些治疗有反应和用于将这样的判定加入到对RA患者的更有效的治疗方案中。 Thus, as RA patients seek treatment, there is significant trial and error involved in the search for therapeutic agents that are effective in a particular patient. Such trial and error often involves considerable risk and discomfort to the patient in order to find the most effective treatment. Therefore, there is a need for more efficient means for determining which patients will respond to which treatments and for incorporating such determinations into more effective treatment regimens for RA patients.

因此高度有利的是具有额外的方法,用于在患者中客观地鉴定疾病的存在,限定类风湿性关节炎的病理生理方面、临床活性、对治疗的反应、包括对用不同RA治疗药的治疗的反应、预后和/或发展类风湿性关节炎的风险。 It would therefore be highly advantageous to have additional methods for objectively identifying the presence of disease in patients, defining the pathophysiological aspects of rheumatoid arthritis, clinical activity, response to treatment, including treatment with different RA therapeutics response, prognosis and/or risk of developing rheumatoid arthritis.

因此,一直都需要鉴定与类风湿性关节炎以及其它自身免疫病相关的新的分子诊断或预后标记。 Therefore, there is a continuing need to identify new molecular diagnostic or prognostic markers associated with rheumatoid arthritis as well as other autoimmune diseases.

概述 overview

如本文所述,本发明人提供用于改善炎性疾病或病症、自身免疫疾病和尤其是RA的治疗的多种方法。 As described herein, the inventors provide various methods for improving the treatment of inflammatory diseases or disorders, autoimmune diseases, and RA in particular.

本发明一方面涉及预测患者对抗炎药的反应的方法,包括:在来自所述患者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,预测所述患者对所述抗炎药的反应。 One aspect of the present invention relates to a method for predicting a patient's response to an anti-inflammatory drug, comprising: obtaining information on the expression level of one or more genes of FIG. 1 in a biological sample from the patient, wherein the reference level of the gene is Compared to the altered expression of one or more of the genes, the response of the patient to the anti-inflammatory agent is predicted.

本发明另一方面涉及预测患者对抗炎药的反应的方法,包括: Another aspect of the invention relates to a method of predicting a patient's response to an anti-inflammatory drug, comprising:

a) 在来自所述患者的生物样品中检测图1的一个或多个基因的表达水平,和 a) detecting the expression level of one or more genes of Figure 1 in a biological sample from said patient, and

b) 将所述水平与所述基因的参考水平比较, b) comparing said level to a reference level for said gene,

其中与所述参考水平相比的一个或多个所述基因的表达改变,预测所述患者对所述抗炎药的反应。 wherein the change in expression of one or more of said genes compared to said reference level predicts said patient's response to said anti-inflammatory drug.

本发明进一步描述了鉴定对抗炎药的反应具有增加的可能性的受试者的方法,包括:在来自所述受试者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,表明已经鉴定了对抗炎药的反应具有增加的可能性的受试者。 The present invention further describes a method of identifying a subject with an increased likelihood of responding to an anti-inflammatory drug, comprising: obtaining an expression level of one or more genes of FIG. 1 in a biological sample from said subject. Information wherein expression of one or more of said genes is altered compared to a reference level for said genes indicates that a subject having an increased likelihood of responding to an anti-inflammatory drug has been identified.

一方面本发明涉及鉴定对抗炎药的反应具有增加的可能性的患者的方法,包括: In one aspect the invention relates to a method of identifying a patient with an increased likelihood of responding to an anti-inflammatory drug comprising:

a. 在来自所述患者的生物样品中检测图1的一个或多个基因的表达水平 a. Detecting the expression level of one or more genes of Figure 1 in a biological sample from said patient

b. 将所述水平与所述基因的参考水平比较, b. comparing said level to a reference level for said gene,

其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,表明已经鉴定了对抗炎药的反应具有增加的可能性的患者。 Wherein expression of one or more of said genes is altered compared to a reference level of said genes, a patient having an increased likelihood of responding to an anti-inflammatory drug has been identified.

本发明的方法可涉及这样的情况:其中表达改变是与参考水平相比图1A的基因的表达增加;和/或其中表达改变是与参考水平相比图1B的基因的表达减少。 The methods of the invention may relate to situations where the altered expression is increased expression of the gene of Figure 1A compared to a reference level; and/or wherein the altered expression is decreased expression of the gene of Figure IB compared to the reference level.

所述方法进一步描述了可以使用qRT-PCR或使用微阵列芯片,基于mRNA,在血液样品中检测所述表达水平。在本发明的具体的实施方案中,测定了补体因子D (CFD)的表达水平并发现所述水平高于参考水平,所述参考水平可根据用于检测所述转录物的方法而有不同定义。 The method further describes that said expression level can be detected in a blood sample based on mRNA using qRT-PCR or using a microarray chip. In a specific embodiment of the invention, the expression level of complement factor D (CFD) was determined and found to be above a reference level, which may be defined differently depending on the method used to detect the transcript .

本发明一方面涉及用于治疗自身免疫疾病或病症的抗炎药,其中与图1的一个或多个基因的参考水平相比,患者具有图1的一个或多个基因的表达改变。 One aspect of the invention pertains to anti-inflammatory agents for use in the treatment of an autoimmune disease or condition, wherein the patient has altered expression of one or more genes of Figure 1 compared to a reference level of one or more genes of Figure 1 .

本发明的进一步的方面涉及治疗患有炎性疾病或其中与参考水平相比图1的一个或多个基因的表达水平被改变的受试者的方法,包括将治疗量的抗炎药给予所述受试者。 A further aspect of the invention relates to a method of treating a subject having an inflammatory disease or wherein the expression level of one or more genes of Figure 1 is altered compared to a reference level, comprising administering a therapeutic amount of an anti-inflammatory drug to the subject the subject.

所述方法可包括进一步的步骤,包括:考虑与参考水平相比,在所述患者中图1的一个或多个基因的表达水平被改变。 The method may comprise a further step comprising: taking into account that the expression level of one or more genes of Figure 1 is altered in said patient compared to a reference level.

本发明一方面涉及在患者中治疗炎性疾病或病症的方法,包括: One aspect of the invention relates to methods of treating an inflammatory disease or condition in a patient, comprising:

a. 在来自所述患者的生物样品中测定图1的一个或多个基因的表达水平 a. Determining the expression level of one or more genes of Figure 1 in a biological sample from said patient

b. 将所述水平与所述基因的参考水平比较, b. comparing said level to a reference level for said gene,

c. 测定与所述参考水平相比,图1的一个或多个基因的表达水平是否被改变 c. Determining whether the expression level of one or more genes of Figure 1 is altered compared to said reference level

d. 将治疗量的抗炎药给予所述患者。 d. Administering a therapeutic amount of an anti-inflammatory drug to the patient.

在某些情况下,可使用基因表达的信息以确定患者是否实际上给予抗炎药和上述方法可以因此包括对表达数据的评价,例如结论是,与所述参考水平相比,所述生物样品中图1的一个或多个基因的表达水平被改变。考虑到适当考虑与参考水平相比,表1A的一个或多个基因的表达水平是否增加和/或与参考水平相比,表1B的一个或多个基因的表达水平是否降低。 In some cases, information on gene expression can be used to determine whether a patient is actually being administered an anti-inflammatory drug and the methods described above can thus include evaluation of expression data, e.g. concluding that the biological sample The expression level of one or more genes in Figure 1 is altered. Due consideration is given to whether the expression level of one or more genes of Table IA is increased compared to the reference level and/or whether the expression level of one or more genes of Table IB is decreased compared to the reference level.

在另一方面,本发明涉及制品,包括包装在一起的药物组合物和标签,所述药物组合物包含抗炎药和药学上可接受的载体,所述标签说明所述药物组合物可用于治疗患有自身免疫疾病或病症并具有图1的一个或多个基因的表达改变的患者。 In another aspect, the invention relates to an article of manufacture comprising a packaged pharmaceutical composition comprising an anti-inflammatory agent and a pharmaceutically acceptable carrier and a label stating that the pharmaceutical composition is used for the treatment of A patient suffering from an autoimmune disease or disorder having altered expression of one or more genes of Figure 1 .

本发明一方面涉及试剂盒,包括: One aspect of the present invention relates to a kit comprising:

a) 包括至少一种检测试剂的一种或多种组合物,所述检测试剂用于测定表1A和/或表1B的一个或多个基因的表达水平,和 a) one or more compositions comprising at least one detection reagent for determining the expression level of one or more genes of Table 1A and/or Table 1B, and

b) 所述试剂盒的使用说明书,包括如何将表达水平与受试者的反应可能性关联。 b) Instructions for use of the kit, including how to relate expression levels to the subject's likelihood of response.

基于现有数据,可向患者建议改善的治疗并可使试验的影响和误差最小化。技术人员显而易见的是,除了本文的具体实施例之外,还可以对本发明进行某些改动。 Based on the available data, improved treatments can be suggested to patients and the effects and errors of trials can be minimized. It will be apparent to a skilled artisan that certain modifications to the invention may be made in addition to the specific examples herein.

序列表 sequence listing

本申请包括序列表,包括以下序列: This application includes a Sequence Listing, including the following sequences:

SEQ ID NO 1: CFD mRNA探针: CCTGCTGCTACAGCTGTCGGAGAAG SEQ ID NO 1: CFD mRNA probe: CCTGCTGCTACAGCTGTCGGAGAAG

SEQ ID NO 2: 18S rRNA对照探针: TGGAGGGCAAGTCTGGTGCCAGCAG SEQ ID NO 2: 18S rRNA control probe: TGGAGGGCAAGTCTGGTGCCAGCAG

SEQ ID NO 3: rs1683565:  SEQ ID NO 3: rs1683565:

AGAGCCCAAAGCTCATGGAAAAGAGXATATAAAGGAGTCCCTGCAGTAGA AGAGCCCAAAGCTCATGGAAAAGAG X ATATAAAGGAGTCCCTGCAGTAGA

其中位置26的X是A或G where X at position 26 is A or G

SEQ ID NO 4:  rs1683591: SEQ ID NO 4: rs1683591:

TCTGTCCACAGGCGGGGGTGGAGGGXATGGCCGGCCTCACACCATCTGCCA TCTGTCCACAGGCGGGGGTGGAGGG X ATGGCCGGCCTCACACCATCTGCCA

其中位置26的X是A或G where X at position 26 is A or G

SEQ ID NO 5: rs1683590: SEQ ID NO 5: rs1683590:

AATATCTGAAATTTTCCCAGTTTACXAGCCTCTGACGTAACCGTCCTCTCT AATATCTGAAATTTTCCCAGTTTAC X AGCCTCTGACGTAACCGTCCTCTCT

其中位置26的X是A或G where X at position 26 is A or G

SEQ ID NO 6: ACTB探针:  SEQ ID NO 6: ACTB probe:

CCTTTGCCGATCCGCCGCCCGTCCA CCTTTGCCGATCCGCCGCCCGTCCA

附图简述 Brief description of the drawings

图 1显示在抗IL20 RA试验中,与28个关节的疾病活性评分 – C-反应蛋白(DAS28-CRP)变化(在第8周)正相关的转录物列表(表1a)和负相关的转录物列表(表1b)。在给药的患者(不包括安慰剂对照)中显示明显相关性(假发现率(FDR)=5%)的转录物包括在列表中(等级次序为每表的顶端具有最显著的相关性)。表1A中列出的基因已被鉴定为对在本发明的方法中使用是相关的,其中相对高的表达水平具有影响。 Figure 1 shows the list of transcripts positively correlated (Table 1a) and negatively correlated with changes in Disease Activity Score – C-reactive protein (DAS28-CRP) (at week 8) in 28 joints in the anti-IL20 RA trial List of objects (Table 1b). Transcripts showing a significant association (false discovery rate (FDR) = 5%) in dosed patients (excluding placebo controls) are included in the list (rank order with the top of each table having the most significant association) . The genes listed in Table 1A have been identified as being relevant for use in the methods of the invention where relatively high expression levels have an effect.

表1B中列出的基因已被鉴定为对在本发明的方法中使用是相关的,其中相对低的表达水平具有影响。 The genes listed in Table IB have been identified as being relevant for use in the methods of the invention where relatively low expression levels have an effect.

图2显示表2包括从表1A和1B中选择基因,所述基因被认为对在本发明的方法和尤其应用多变量分析的方法中使用是相关的。对于基于多变量的DAS28-CRP预测而言,所选的转录物/基因是相关的。 Figure 2 shows that Table 2 includes a selection of genes from Tables 1A and 1B that are considered relevant for use in the methods of the invention and in particular methods employing multivariate analysis. Selected transcripts/genes are relevant for multivariate based DAS28-CRP prediction.

图3显示在抗IL20试验中,在来自RA-患者的PaxGene全血样品中的CFD (补体因子D)转录物的转录水平的分布。强健的多芯片平均(Robust Multichip Average,RMA)标准化值示于Y轴上(log2标度(scale))。来自单个患者的样品以交替颜色(黑色或白色)呈现和单个患者被任意编号为1-82。 Figure 3 shows the distribution of transcript levels of CFD (complement factor D) transcripts in PaxGene whole blood samples from RA-patients in the anti-IL20 assay. Robust Multichip Average (RMA) normalized values are shown on the Y-axis (log2 scale). Samples from individual patients are presented in alternating colors (black or white) and individual patients are arbitrarily numbered 1-82.

图4显示在2a期抗IL20试验中的CFD mRNA的接受者操作特征(ROC)曲线和美国风湿病学会50%综合标准(ACR50)反应。ROC曲线上的X表明阈值10.32 (RMA标准化的表达值)。 Figure 4 shows the Receiver Operating Characteristic (ROC) curve and American College of Rheumatology 50% Composite Criteria (ACR50) response for CFD mRNA in the Phase 2a anti-IL20 trial. The X on the ROC curve indicates a threshold of 10.32 (RMA normalized expression value).

图5显示在2a期抗IL20试验中的CFD mRNA的ROC曲线和美国风湿病学会70 %综合标准(ACR70)反应。 Figure 5 shows the ROC curve and American College of Rheumatology Composite Criteria 70% (ACR70) response for CFD mRNA in the Phase 2a anti-IL20 trial.

图6显示在给药前(第1天)样品中,CFD mRNA的定量RT-PCR (qRT-PCR)检测与来自在更多时间点的基于微阵列检测的数据的关联。将线性标度上的微阵列信号(反转化的RMA数据(Y轴))与来自qRT-PCR分析的18S标准化的CFD水平比较。 Figure 6 shows the correlation of quantitative RT-PCR (qRT-PCR) detection of CFD mRNA in pre-dose (Day 1 ) samples with data from microarray-based detection at additional time points. The microarray signal (back-transformed RMA data (Y-axis)) on a linear scale was compared to 18S normalized CFD levels from qRT-PCR analysis.

图7显示在具有基于CFD的层化的两个备选阈值(alternative treshold)的抗IL20的2a期试验(临床试验政府识别号NCT01282255)中的ACR20、ACR50和ACR70反应率。如果使用基于来自图4的ROC曲线的CFD mRNA水平的患者层化(例如使用的阈值>10.32 (RMA标准化的表达值)),得到高反应的患者群体(图表A的底部)。如果仅包括CFD水平低于阈值的个体,则图表A的上部展示反应。当使用CFD的备选阈值(基于CFD和β肌动蛋白(ACTB)的绝对定量测定)时,得到有反应患者的类似富集(图表B的下面部分)。 Figure 7 shows ACR20, ACR50 and ACR70 response rates in a Phase 2a trial of anti-IL20 (Clinical Trial Government Identification Number NCT01282255) with two alternative thresholds for CFD-based stratification. If patient stratification based on CFD mRNA levels from the ROC curve of Figure 4 is used (e.g. using a threshold >10.32 (RMA normalized expression value)), a high responding patient population is obtained (bottom of graph A). The upper part of graph A shows the response if only individuals with CFD levels below the threshold are included. A similar enrichment of responding patients was obtained when using an alternative threshold for CFD (based on absolute quantification of CFD and β-actin (ACTB)) (lower part of panel B).

图8显示在RA患者滑液中的Bb水平,显示在PaxGene样品中高或低的CFD表达水平,如通过qPCR所评价。 Figure 8 shows Bb levels in synovial fluid of RA patients showing high or low CFD expression levels in PaxGene samples, as assessed by qPCR.

描述 describe

本发明涉及预测抗炎化合物的治疗效果的方法。正如背景部分所述,当前可用的治疗至少在某种程度上具有低成功率,并且治疗通常包括某种程度的试验和误差。本发明提供鉴定患者亚组的方法,其具有高的治疗成功率,从而使大量患者可以避免与寻找有效治疗的困难相关的不适的风险。 The present invention relates to methods of predicting the therapeutic effect of anti-inflammatory compounds. As noted in the Background section, currently available treatments have low success rates, at least in part, and treatments often involve some degree of trial and error. The present invention provides methods for identifying subgroups of patients with a high rate of treatment success, thereby allowing a large number of patients to avoid the risk of discomfort associated with the difficulty of finding effective treatments.

定义 definition

为了解释本申请的目的,在本文中较少的另外说明以使用以下定义。 For the purpose of explaining the present application, the following definitions are used less otherwise herein.

如本文可互换使用的,术语“多核苷酸”或“核酸”指任何长度的核苷酸聚合物,并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或其类似物、或可以由DNA或RNA聚合酶掺入聚合物内的任何底物。多核苷酸可以包含修饰的核苷酸,例如甲基化核苷酸及其类似物。如果存在的话,那么对核苷酸结构的修饰可以在聚合物装配之前或之后赋予。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以,例如通过与标记组分缀合或本领域已知的其它类型修饰,在聚合后进一步修饰。 As used interchangeably herein, the term "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, can be imparted before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, eg, by conjugation with labeling components or other types of modifications known in the art.

如本文使用的,“寡核苷酸”指短的单链多核苷酸,其长度为至少约7个核苷酸和小于约250个核苷酸。寡核苷酸可以是合成的。术语“寡核苷酸”和“多核苷酸”不是相互排斥的。关于多核苷酸的上文描述同等地和完全地适用于寡核苷酸。 As used herein, "oligonucleotide" refers to short, single-stranded polynucleotides that are at least about 7 nucleotides and less than about 250 nucleotides in length. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. What has been said above with respect to polynucleotides applies equally and fully to oligonucleotides.

术语“引物”指能够与核酸杂交并且一般通过提供游离3'-OH基团而允许互补核酸聚合的单链多核苷酸。 The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing the polymerization of a complementary nucleic acid, typically by providing a free 3'-OH group.

术语“阵列”或“微阵列”指可在基质上有序排列的可杂交阵列元件,优选多核苷酸探针(例如寡核苷酸)。基质可以是固体基质例如玻璃载玻片或半固体基质例如硝酸纤维素膜。 The term "array" or "microarray" refers to a hybridizable array of elements, preferably polynucleotide probes (eg, oligonucleotides), arranged in an orderly manner on a substrate. The substrate may be a solid substrate such as a glass slide or a semi-solid substrate such as a nitrocellulose membrane.

术语“扩增”指产生参考核酸序列或其互补体的一个或多个拷贝的过程。扩增可以是线性或指数的(例如PCR)。“拷贝”不一定意味相对于模板序列的完全序列互补性或同一性。例如,拷贝可以包括核苷酸类似物例如脱氧肌苷、故意的序列改变(例如通过引物引入的序列改变,所述引物包括与模板可杂交但并非完全互补的序列)、和/或在扩增过程中出现的序列错误。 The term "amplification" refers to the process of producing one or more copies of a reference nucleic acid sequence or its complement. Amplification can be linear or exponential (eg PCR). "Copy" does not necessarily imply complete sequence complementarity or identity with respect to the template sequence. For example, copies may include nucleotide analogs such as deoxyinosine, deliberate sequence changes (such as those introduced by primers that include sequences that are hybridizable but not fully complementary to the template), and/or A sequence error occurred during the process.

术语“多重PCR”指使用超过一个引物组在得自单个样品(例如一个患者)的核酸上进行的单个PCR反应,用于在单个反应中扩增2个或更多个DNA序列的目的。 The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acid from a single sample (eg, a patient) using more than one primer set for the purpose of amplifying 2 or more DNA sequences in a single reaction.

杂交反应的“严格性”可容易地由本领域普通技术人员确定,并且一般是取决于探针长度、洗涤温度和盐浓度的经验计算。一般而言,更长的探针需要更高的温度用于恰当的退火,而更短的探针需要更低的温度。当互补链存在于低于其解链温度的环境中时,杂交一般取决于变性DNA再退火的能力。在探针和可杂交序列之间的所需同源性程度越高,可以使用的相对温度越高。由此,较高的相对温度将趋于使得反应条件更严格,而较低的温度趋于使得反应条件较不严格。关于杂交反应的严格性的另外细节和说明,参见Ausubel等人,Current Protocols in Molecular Biology, Wiley Interscience Publishers,(1995)。 "Stringency" of a hybridization reaction can be readily determined by one of ordinary skill in the art and is generally an empirical calculation dependent on probe length, washing temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures will tend to make the reaction conditions less stringent. For additional details and clarification on the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

如本文定义的,“严格条件”或“高严格条件”可以通过下述进行定义:(1)采用低离子强度和高温用于洗涤,例如0.015 M氯化钠/0.0015 M柠檬酸钠/0.1%十二烷基硫酸钠,在50℃;(2)在杂交过程中采用变性剂,例如甲酰胺,例如,50% (v/v)甲酰胺与0.1%牛血清白蛋白/0.1% Ficoll/0.1%聚乙烯吡咯烷酮/50mM磷酸钠缓冲液,在pH6.5与750mM氯化钠、75mM柠檬酸钠,在42℃;或(3)在42℃在采用50%甲酰胺、5 x SSC(0.75M NaCl, 0.075M柠檬酸钠)、50mM磷酸钠(pH6.8)、0.1%焦磷酸钠、5 x Denhardt溶液、超声处理的鲑精DNA (50微克/ml)、0.1% SDS和10%硫酸葡聚糖的溶液中过夜杂交,在42℃在0.2 x SSC (氯化钠/柠檬酸钠)中10分钟洗涤,随后在55℃由含有EDTA的0.1 x SSC组成的10分钟高严格洗涤。 As defined herein, "stringent conditions" or "highly stringent conditions" can be defined by: (1) using low ionic strength and high temperature for washing, e.g. 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% Sodium dodecyl sulfate at 50°C; (2) Use denaturants such as formamide during hybridization, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750mM sodium chloride, 75mM sodium citrate at 42°C; or (3) at 42°C in 50% formamide, 5 x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10% glucosulphate Glycans were hybridized overnight in solution, washed in 0.2 x SSC (sodium chloride/sodium citrate) for 10 min at 42°C, followed by a high stringency wash consisting of 0.1 x SSC with EDTA for 10 min at 55°C.

“中等严格条件”可以如Sambrook等人,Molecular Cloning : A Laboratory Manual, New York : Cold Spring Harbor Press,1989所述进行定义,包括使用比上述较不严格的洗涤溶液和杂交条件(例如温度、离子强度和%SDS)。中等严格条件的实例是在37℃在包含下述的溶液中过夜温育:20%甲酰胺、5 x SSC (150mM NaCl、15mM柠檬酸三钠)、50mM磷酸钠(pH7.6)、5 x Denhardt溶液、10%硫酸葡聚糖和20mg/ml变性剪切鲑精DNA,随后在约37-50℃在1 x SSC中洗涤滤膜。技术人员知道如何根据需要调节温度、离子强度等,以适应例如探针长度等因素。 "Moderately stringent conditions" can be defined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, including the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ion Strength and %SDS). An example of moderately stringent conditions is an overnight incubation at 37°C in a solution containing: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filter in 1 x SSC at approximately 37-50°C. The skilled artisan knows how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

术语“检测”包括任何检测手段,包括直接和间接检测。 The term "detection" includes any means of detection, including direct and indirect detection.

术语“表达的水平”或“表达水平”一般而言可互换使用,并且指生物样品中多核苷酸或氨基酸产物或蛋白质的量。“表达”一般指基因编码的信息被转换成细胞中存在和运作的结构的过程。因此,如本文使用的,基因的“表达”可以指转录成多核苷酸、翻译成蛋白质、或甚至蛋白质的翻译后修饰。转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段也应视为表达的,无论它们是源于通过可变剪接生成的转录物或降解的转录物,还是源于蛋白质的翻译后加工例如蛋白质水解。“表达的基因”包括转录为作为mRNA的多核苷酸并且随后翻译成蛋白质的那些,以及转录成RNA但不翻译成蛋白质的那些(例如转移RNA和核糖体RNA)。 The terms "level of expression" or "expression level" are generally used interchangeably and refer to the amount of a polynucleotide or amino acid product or protein in a biological sample. "Expression" generally refers to the process by which the information encoded by a gene is converted into the structures that exist and function in the cell. Thus, as used herein, "expression" of a gene can refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins shall also be considered expressed, whether they arise from transcripts generated by alternative splicing or from degraded transcripts, or from post-translational Processing such as proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and subsequently translated into protein, as well as those that are transcribed into RNA but not translated into protein (eg, transfer RNA and ribosomal RNA).

术语“表达谱”可用于定义一组基因的表达水平,提供样品中的转录活性的更复杂的图像。 The term "expression profile" can be used to define the expression levels of a set of genes, providing a more complex picture of transcriptional activity in a sample.

如本文使用的,术语“生物标记”指患者状态的指示物,因为这样的生物标记可用于评价患者的疾病状态(包括在个体中诊断和评价治疗的反应)。生物标记是分子实体,可在来自患者的生物样品中对其进行检测。生物标记包括但不限于DNA、RNA、蛋白质、碳水化合物和其它生物化学实体或部分,包括其组合,例如基于糖脂或糖蛋白的分子标记。“诊断标记”和“预后标记”是“生物标记”详细说明,其表明分子实体的存在或不存在或水平可提供对诊断和/或预后的信息,包括例如对一种或多种治疗类型的反应。某些生物标记可能适于诊断,某些适于后续疾病发展和治疗反应,而其它适于预测对治疗的临床反应。 As used herein, the term "biomarker" refers to an indicator of a patient's state, as such biomarkers can be used to assess a patient's disease state (including diagnosing and assessing response to treatment in an individual). Biomarkers are molecular entities that can be detected in biological samples from patients. Biomarkers include, but are not limited to, DNA, RNA, proteins, carbohydrates, and other biochemical entities or moieties, including combinations thereof, such as glycolipid or glycoprotein based molecular markers. "Diagnostic markers" and "prognostic markers" are specifications for "biomarkers" that indicate the presence or absence or levels of a molecular entity that are diagnostic and/or prognostic, including, for example, for one or more types of treatment reaction. Certain biomarkers may be suitable for diagnosis, some for subsequent disease development and treatment response, and others for prediction of clinical response to treatment.

与患者的临床益处增加相关的“预后标记”的“量”或“水平”在来自所述患者的生物样品中是可检测水平。可通过本领域技术人员已知的并且也在本文中公开的方法检测表达水平。所评价的生物标记的表达水平或量可用于判定或预测治疗的反应。 The "amount" or "level" of a "prognostic marker" that correlates with increased clinical benefit to a patient is a detectable level in a biological sample from said patient. Expression levels can be detected by methods known to those skilled in the art and also disclosed herein. The expression level or amount of the assessed biomarker can be used to determine or predict response to treatment.

术语“表达改变”是指通常在mRNA或蛋白水平上检测的增加的或减少的基因表达水平。认为表达水平相对于参考水平而改变,例如表达水平“高于”或“低于”相关的预定水平。基因表达水平改变可以代表其表达“高于”或“低于”其它基因和/或其它个体的表达水平的基因。 The term "altered expression" refers to an increased or decreased level of gene expression, usually detected at the mRNA or protein level. An expression level is considered to be altered relative to a reference level, for example an expression level is "above" or "below" a relevant predetermined level. Altered gene expression levels may represent genes whose expression is "higher" or "lower" than the expression levels of other genes and/or other individuals.

术语“增加的表达”或“增加的水平”是指通常在mRNA或蛋白水平上检测的升高的或增加的基因表达水平。认为表达水平相对于参考水平而增加,例如表达水平“高于”相关的预定水平。增加的基因表达水平可以代表与其它基因和/或其它个体的表达水平相比,在个体中以“高”水平表达的基因。 The term "increased expression" or "increased level" refers to an elevated or increased level of gene expression, usually detected at the mRNA or protein level. An expression level is considered to be increased relative to a reference level, eg, an expression level is "above" a relevant predetermined level. An increased level of gene expression may represent a gene that is expressed at a "high" level in an individual compared to the expression levels of other genes and/or other individuals.

术语“减少的表达”或“减少的水平”是指通常在mRNA或蛋白水平上检测的降低的或减少的基因表达水平。认为表达水平相对于参考水平而减少,例如表达水平“低于”相关的预定水平。减少的基因表达水平可以代表与其它基因和/或其它个体的表达水平相比,在个体中以“低”水平表达的基因。 The term "reduced expression" or "reduced level" refers to a reduced or reduced level of gene expression, usually detected at the mRNA or protein level. An expression level is considered to be reduced relative to a reference level, eg, an expression level is "below" a relevant predetermined level. A reduced level of gene expression may represent a gene that is expressed at a "low" level in an individual compared to the expression levels of other genes and/or other individuals.

术语“类风湿因子”或“RF”指,在患者血清中检测到的并且针对人和动物IgG上存在的抗原决定簇的单独或任何组合的IgM、IgG或IgA同种型抗体。 The term "rheumatoid factor" or "RF" refers to antibodies of the IgM, IgG or IgA isotypes, alone or in any combination, detected in the serum of a patient and directed against epitopes present on human and animal IgG.

术语“RF阳性”指用于RF的测定例如ELISA测定的结果,其中所述结果超过用于被认为可重现地含有可检测水平的RF的样品的测定的阈值或截止值。 The term "RF positive" refers to the result of an assay for RF, such as an ELISA assay, wherein the result exceeds the threshold or cut-off value of the assay for samples considered to reproducibly contain detectable levels of RF.

术语“RF阴性”指用于RF的测定例如ELISA测定的结果,其中所述结果等于或低于用于被认为可重现地含有不可检测水平的RF的样品的测定的阈值或截止值。 The term "RF negative" refers to the result of an assay for RF, such as an ELISA assay, wherein the result is at or below the threshold or cutoff value of the assay for samples considered reproducibly containing undetectable levels of RF.

如本文所用,术语“样品”或“生物样品”指得自或源自目标受试者的组合物,其包含待检测、测定或鉴定的一种或多种分子实体。例如,短语“患者样品”、“受试者样品”及其变体指得自目标患者或受试者的任何样品,其被预期含有或已知含有待表征的细胞和/或分子实体,包括但不限于组织样品、细胞样品或血液样品。 As used herein, the term "sample" or "biological sample" refers to a composition obtained or derived from a subject of interest comprising one or more molecular entities to be detected, assayed or identified. For example, the phrase "patient sample", "subject sample" and variations thereof refer to any sample obtained from a patient or subject of interest that is expected to contain or is known to contain the cellular and/or molecular entities to be characterized, including But not limited to tissue samples, cell samples or blood samples.

术语“组织样品”或“细胞样品”或“血液样品”意指包含得自受试者的一种或多种细胞的样品。组织或细胞样品的来源可以是实体组织如来自新鲜、冷冻和/或保存的器官或组织样品或活检组织或抽吸物;体液例如脑脊髓液、羊膜液、腹膜液、间质液或血液或任何血液组分。组织样品或细胞样品还可以是原代或培养的细胞或细胞系。任选地,组织或细胞样品得自疾病组织/器官(例如表现出病理特征)。组织样品可以含有在自然界中不与组织天然地混合的化合物,例如防腐剂、抗凝剂、缓冲剂、固定剂、营养素、抗生素等。 The term "tissue sample" or "cell sample" or "blood sample" means a sample comprising one or more cells obtained from a subject. The source of the tissue or cell sample can be solid tissue such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; bodily fluid such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid or blood or any blood component. The tissue sample or cell sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ (eg exhibiting pathological features). A tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

术语“血清样品”是指得自个体的任何血清样品。从哺乳动物中获得血清的方法是本领域众所周知的。 The term "serum sample" refers to any serum sample obtained from an individual. Methods of obtaining serum from mammals are well known in the art.

如本文使用的,“对照样品”、“对照细胞”或“对照组织”,是指得自已知或被认为未患有待用本发明的方法或组合物鉴定的疾病或病症的来源的生物样品、细胞或组织。在一个实施方案中,对照样品、对照细胞或对照组织得自待使用本发明的组合物或方法鉴定疾病或病症的同一受试者或患者的身体的表面上未受影响的部分。在一个实施方案中,对照样品、对照细胞或对照组织得自如下个体的身体的部分,所述个体并非待使用本发明的组合物或方法鉴定疾病或病症的受试者或患者。 As used herein, "control sample", "control cell" or "control tissue" refers to a biological sample obtained from a source known or believed not to suffer from the disease or condition to be identified using the methods or compositions of the invention, cells or tissues. In one embodiment, the control sample, control cell or control tissue is obtained from an apparently unaffected part of the body of the same subject or patient whose disease or disorder is to be identified using a composition or method of the invention. In one embodiment, the control sample, control cell or control tissue is obtained from a part of the body of an individual who is not a subject or patient for whom a disease or disorder is to be identified using a composition or method of the invention.

术语“诊断”在本文中用于指分子或病理学状态、疾病或病症的鉴定或分类。例如,“诊断”可以指特定类型的炎性疾病或病症或特异性自身免疫疾病例如RA的鉴定。 The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or disorder. For example, "diagnosing" can refer to the identification of a particular type of inflammatory disease or disorder or a specific autoimmune disease such as RA.

术语“预测”或其变体用于指患者对药物或药物组将具有有利或不利反应的可能性。在一个实施方案中,预测涉及所述反应的程度。在一个实施方案中,预测涉及这样的可能性:患者将改善后续治疗,例如用特定治疗药的治疗,和在一定的时间段中无疾病复发。本发明的预测法可以在临床上使用,通过选择对于任何特定患者合适的治疗模式而作出治疗决策。对于判定患者是否可能有利地响应治疗方案例如给定治疗方案,包括例如给予给定的药物或治疗药或其组合,本发明的预测方法是有价值的工具。 The term "prediction" or variations thereof is used to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or group of drugs. In one embodiment, the extent to which the prediction is related to said response is predicted. In one embodiment, the prediction relates to the likelihood that the patient will improve on subsequent treatment, eg, treatment with a particular therapeutic agent, and be disease-free for a certain period of time. The predictive methods of the present invention can be used clinically to make treatment decisions by selecting the appropriate treatment modality for any particular patient. The predictive methods of the present invention are valuable tools for determining whether a patient is likely to respond favorably to a treatment regimen, eg, a given treatment regimen, including eg administration of a given drug or therapeutic agent or combination thereof.

术语“指示(indication)”、“指示的(indicative)”或其变体用于指所得的指导;因为基于如本文所述的基因表达水平改变的“指示”提供以下信息:受试者或患者对抗炎治疗可能有反应。基于这样的指导,本发明的方法可以在临床上使用,通过选择对于任何特定患者合适的治疗模式而作出治疗决策。 The terms "indication", "indicative" or variants thereof are used to refer to resulting guidance; as an "indication" based on a change in gene expression level as described herein provides information that a subject or patient May respond to anti-inflammatory treatment. Based on such guidance, the methods of the present invention can be used clinically to make treatment decisions by selecting the appropriate treatment modality for any particular patient.

如本文使用的,“治疗”指试图改变接受治疗的个体的天然过程的临床干预,可以在临床病理学过程之前或期间执行。期望的治疗效果包括阻止疾病或其病况或症状的出现或复发,减轻疾病的病况或症状,减少疾病的任何直接或间接的病理学后果,降低疾病进展速率,改善或缓和疾病状态,和/或实现缓解或改善的预后。在某些实施方案中,本发明的方法和组合物在延迟疾病或病症发展的尝试中是有用的。 As used herein, "treatment" refers to clinical intervention that seeks to alter the natural course of the individual being treated, and may be performed prior to or during the course of clinical pathology. Desired therapeutic effects include arresting the onset or recurrence of the disease or its condition or symptoms, alleviation of the condition or symptoms of the disease, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, amelioration or palliation of the disease state, and/or Achieving remission or improved prognosis. In certain embodiments, the methods and compositions of the invention are useful in attempts to delay the development of a disease or condition.

“有效量”指以所需剂量和在所需的时间段内有效达到期望的治疗或预防结果的量。治疗药的“治疗有效量”可以根据以下因素而改变:例如疾病状态,个体的年龄、性别和重量,和抗体在个体中引发所需应答的能力。治疗有效量还是其中治疗药的治疗有利效应胜过其任何毒性或有害效应的量。“预防有效量”指以所需剂量和在所需的时间段内有效达到期望的预防结果的量。典型地但不是必须的,因为预防剂量在疾病的早期阶段前或在疾病的早期阶段时在受试者中使用,所以预防有效量将小于治疗有效量。 An "effective amount" refers to an amount effective, at dosages and for periods of time required, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent can vary depending on factors such as the disease state, the age, sex and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also that amount in which any toxic or detrimental effects of the therapeutic agent outweigh any toxic or detrimental effects thereof. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time required, to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered to the subject before or at an early stage of the disease.

如本文所用的术语“个体”、“受试者”或“患者”,可以互换使用,通常是指脊椎动物。在某些实施方案中,所述脊椎动物是哺乳动物。哺乳动物包括但不限于灵长类动物(包括人和非人灵长类动物)和啮齿类动物(例如小鼠和大鼠)。在某些实施方案中,所述哺乳动物是人。术语“患者”进一步表示并非健康受试者的受试者。在一个实施方案中,“患者”是经诊断或患有与炎性疾病或病症相关的体征或症状的个体。在一个实施方案中,“患者”患有自身免疫疾病或病症,例如RA。 As used herein, the terms "individual", "subject" or "patient", used interchangeably, generally refer to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (eg, mice and rats). In certain embodiments, the mammal is a human. The term "patient" further means a subject who is not a healthy subject. In one embodiment, a "patient" is an individual diagnosed with or suffering from signs or symptoms associated with an inflammatory disease or disorder. In one embodiment, a "patient" has an autoimmune disease or disorder, such as RA.

“对照受试者”指未曾被诊断和/或未患有与炎性疾病或病症相关的任何体征或症状的表面上健康的受试者。 A "control subject" refers to an apparently healthy subject who has not been diagnosed and/or is not suffering from any signs or symptoms associated with an inflammatory disease or disorder.

“关联(correlate或correlating)”意指以任何方式将第一分析或方案的性能和/或结果与第二分析或方案的性能和/或结果进行对比。例如,可以将第一分析或方案的结果用于执行第二方案,和/或可以使用第一分析或方案的结果以确定是否应执行第二分析或方案。就基因表达分析或方案的实施方案而言,可以使用基因表达分析或方案的结果以确定是否应执行特定的治疗方案。 "Correlating or correlating" means comparing the performance and/or results of a first assay or protocol with the performance and/or results of a second assay or protocol in any way. For example, the results of a first analysis or protocol can be used to perform a second protocol, and/or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. With regard to the gene expression analysis or protocol embodiments, the results of the gene expression analysis or protocol can be used to determine whether a particular treatment protocol should be performed.

术语“患者反应”或“反应”可以使用指示患者受益的任何终点进行评估,包括但不限于,a)对疾病进展的抑制;b)疾病发作次数和/或症状的减少;c)损伤尺寸的缩小;d)对疾病细胞浸润到邻近周围器官和/或组织内的抑制;e)对疾病传播的抑制;f)自身免疫应答的降低,这可以但不一定导致疾病损伤的消退或消除;g)与所述病症相关的一种或多种症状在一定程度上的缓解;h)在治疗后无疾病表现的时间增加;和/或i)在治疗后在给定时间点上死亡率减少。为了患者反应的目的,抑制是指掩蔽、缩小、延迟或完全终止相关症状。 The term "patient response" or "response" may be assessed using any endpoint indicative of patient benefit, including, but not limited to, a) inhibition of disease progression; b) reduction in number of disease episodes and/or symptoms; c) improvement in lesion size shrinkage; d) inhibition of disease cell infiltration into adjacent surrounding organs and/or tissues; e) inhibition of disease spread; f) reduction of autoimmune response, which may, but does not necessarily, lead to regression or elimination of disease damage; g ) some relief of one or more symptoms associated with the condition; h) an increase in the time free from disease manifestations after treatment; and/or i) a decrease in mortality at a given time point after treatment. For purposes of patient response, suppression refers to masking, diminishing, delaying, or complete cessation of associated symptoms.

当涉及受试者或患者对于先前已给予他们的一种或多种药物的反应时,表述“对……无反应”描述所述受试者或患者,在所述药物给予后,未曾显示出对于所治疗的病症而言任何的或足够的治疗迹象,或他们显示出了对于所述药物在临床上无法接受的高度毒性,或他们未维持在第一次施用所述药物后的治疗迹象,其中在该背景中使用的词语治疗如本文所定义。短语“无反应的”包括描述对于先前给予的一种或多种药物具有抵抗性和/或难治性的那些受试者,并且包括下述情况:其中受试者或患者在接受他或她正被给予的药物时已发展,和其中受试者或患者在完成方案后12个月内(例如6个月内)已发展,其中所述方案包括他或她不再对其有反应的药物。对于一种或多种药物的无反应性因此包括在先前或当前的对其治疗后继续具有活动性疾病的受试者。例如,在用患者对其无反应的药物治疗约1-3个月后,患者可以具有活动性疾病活性。这样的反应性可以由在治疗所述病症中熟练的临床医生进行评估。为了对药物的非反应性的目的,自用一种或多种药物进行的先前或目前治疗中经历“临床上不可接受的高水平的毒性”的受试者经历与之相关的一种或多种不良副作用或不利事件,所述副作用或不利事件被有经验的临床医生视为是重要的,例如严重感染、充血性心力衰竭、脱髓鞘(导致多发性硬化)、显著超敏反应、神经病理学事件、高度自身免疫、癌症例如子宫内膜癌、非霍奇金淋巴瘤、乳腺癌、前列腺癌、肺癌、卵巢癌或黑素瘤、肺结核(TB)等。 The expression "unresponsive" when referring to a subject's or patient's response to a drug or drugs that have been previously administered to them describes the subject or patient who, after administration of the drug, has not shown Any or sufficient evidence of treatment for the condition being treated, or they exhibit a clinically unacceptable high degree of toxicity for the drug, or they do not maintain evidence of treatment after the first administration of the drug, Wherein the word therapy as used in this context is as defined herein. The phrase "unresponsive" includes descriptions of those subjects who are resistant and/or refractory to one or more previously administered drugs, and includes situations in which a subject or patient is receiving his or her Has developed while being administered the drug, and wherein the subject or patient has developed within 12 months (eg, within 6 months) of completing a regimen that includes the drug to which he or she no longer responds . Non-responsiveness to one or more drugs thus includes subjects who continue to have active disease following previous or current treatment therefor. For example, a patient may have active disease activity after about 1-3 months of treatment with a drug to which the patient has not responded. Such responsiveness can be assessed by a clinician skilled in the treatment of the disorder. For the purposes of non-responsiveness to a drug, a subject who has experienced "clinically unacceptably high levels of toxicity" from prior or current treatment with one or more drugs has experienced one or more Adverse side effects or adverse events deemed significant by experienced clinicians, such as severe infection, congestive heart failure, demyelination (leading to multiple sclerosis), marked hypersensitivity, neuropathology Events, high levels of autoimmunity, cancers such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer or melanoma, tuberculosis (TB), etc.

术语“不充分的反应”或“不充分反应者”用于描述经历不满意的给定治疗效果的患者。这可以表征为低治疗效果和/或大量副作用。认为所述标准相当于无反应性。术语“不充分的反应”用于涉及其中给定反应被预期或旨在基于先前的试验的治疗。如果一段时间之后未获得“充分的反应”,则治疗通常被中断,认为所述患者是“不充分反应者”。也可以是,所述患者继续治疗,但与其它治疗组合以改善治疗反应。 The term "inadequate responder" or "inadequate responder" is used to describe a patient who experiences an unsatisfactory effect of a given treatment. This can be characterized by low therapeutic efficacy and/or high number of side effects. The criteria were considered equivalent to anergy. The term "adequate response" is used to refer to treatments where a given response is expected or intended based on previous trials. If an "adequate response" is not achieved after a period of time, treatment is usually discontinued and the patient is considered an "under-responder". It is also possible that the patient continues treatment, but in combination with other treatments to improve treatment response.

术语“充分的反应”用于描述当治疗功效的预期实现时患者中的治疗效果。 The term "adequate response" is used to describe the effect of a treatment in a patient when the intended efficacy of the treatment is achieved.

“药物”是治疗疾病、病症和/或病况的活性药物。在一个实施方案中,疾病、病症和/或病况是RA或其症状或副作用。 A "drug" is an active drug that treats a disease, disorder and/or condition. In one embodiment, the disease, disorder and/or condition is RA or a symptom or side effect thereof.

“抗炎药”是这样的化合物、药物或药剂:其可以或有希望减少炎性疾病或病症的炎性反应或症状。 An "anti-inflammatory drug" is a compound, drug or agent that can or is expected to reduce the inflammatory response or symptoms of an inflammatory disease or disorder.

如本文使用的,“RA治疗药”或“有效治疗RA的治疗药”及其语法变体指这样的药剂:当以有效量提供时,其被已知、临床上证实或被临床医生预期在RA受试者中提供治疗益处。 As used herein, "RA therapeutic agent" or "a therapeutic agent effective in the treatment of RA" and grammatical variants thereof refer to an agent which, when provided in an effective amount, is known, clinically proven, or expected by a clinician to be effective in the treatment of RA. Provides therapeutic benefit in RA subjects.

“拮抗剂”指能够中和、阻断、抑制、废除、减少或干扰特定或指定蛋白质的活性的分子,所述活性包括在配体的情况下其与一种或多种受体的结合或在受体的情况下其与一种或多种配体的结合。拮抗剂包括抗体及其抗原结合片段、蛋白质、肽、糖蛋白、糖肽、糖脂、多糖、寡糖、核酸、生物有机分子、拟肽、药理学活性剂及其代谢产物、转录和翻译控制序列等。拮抗剂还包括蛋白质的小分子抑制剂、和融合蛋白、与蛋白质特异性结合从而隔离其与其靶标的结合的受体分子和衍生物、蛋白质的拮抗剂变体、针对蛋白质的反义分子、RNA适体、和针对蛋白质的核酶。 "Antagonist" refers to a molecule capable of neutralizing, blocking, inhibiting, abolishing, reducing or interfering with the activity of a specific or designated protein, including in the case of a ligand, its binding to one or more receptors or In the case of a receptor its binding to one or more ligands. Antagonists include antibodies and their antigen-binding fragments, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacologically active agents and their metabolites, transcriptional and translational controls sequence etc. Antagonists also include small molecule inhibitors of proteins, and fusion proteins, receptor molecules and derivatives that specifically bind to proteins thereby sequestering their binding to their targets, antagonist variants of proteins, antisense molecules directed against proteins, RNA Aptamers, and ribozymes against proteins.

发明详述 Detailed description of the invention

本发明的发明人已经发现,根据某些基因表达谱的检查,可以鉴定具有成功治疗的高可能性的患者。本发明是基于按照实施例中所述而获得的数据。本发明的患者典型地患有炎性疾病或病症和尤其是自身免疫疾病或病症。基于所得的数据,可在多种方法中使用所述信息,因为可以选择对治疗的反应具有增加的可能性的患者。 The inventors of the present invention have discovered that, based on examination of certain gene expression profiles, patients with a high likelihood of successful treatment can be identified. The invention is based on data obtained as described in the Examples. Patients of the invention typically suffer from inflammatory diseases or disorders and especially autoimmune diseases or disorders. Based on the data obtained, the information can be used in a variety of ways, as patients with an increased likelihood of responding to treatment can be selected.

本发明一方面涉及预测受试者对抗炎药的反应的方法,包括:在来自所述受试者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,预测所述受试者对所述抗炎药的反应。 One aspect of the present invention relates to a method for predicting a subject's response to an anti-inflammatory drug, comprising: obtaining information on the expression level of one or more genes of FIG. 1 in a biological sample from the subject, wherein the A change in the expression of one or more of the genes compared to a reference level of the gene predicts the subject's response to the anti-inflammatory agent.

可使用不同的措辞来解释评价图1的一个或多个基因的基因表达的情况。这同样可用于获得表达水平的信息,评价水平或表达或考虑表达水平。所有这些方法不必包括获得血液样品,因为这可能在先前已发生,因此所述方法详述,为在指定患者中预测临床反应或临床反应的可能性的目的,使用来自生物样品的基因表达的信息。这进一步表明先前也已获得的基因表达水平的信息可以用于本发明的方法。 Different wording can be used to explain the evaluation of gene expression of one or more genes of FIG. 1 . The same can be used to obtain information on expression levels, evaluate levels or expression or take expression levels into account. All of these methods do not necessarily include obtaining a blood sample, as this may have occurred previously, so the method details the use of information on gene expression from a biological sample for the purpose of predicting a clinical response or the likelihood of a clinical response in a given patient . This further demonstrates that information on gene expression levels that has also been obtained previously can be used in the methods of the present invention.

本发明一方面涉及鉴定对抗炎药的反应具有增加的可能性的受试者的方法,包括在来自所述受试者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述样品中的所述基因的参考水平相比的一个或多个所述基因的表达改变,鉴定了对抗炎药的反应具有增加的可能性的受试者。 One aspect of the invention relates to a method of identifying a subject with an increased likelihood of responding to an anti-inflammatory drug comprising obtaining information on the expression level of one or more genes of Figure 1 in a biological sample from said subject , wherein altered expression of one or more of said genes compared to a reference level of said genes in said sample identifies subjects with an increased likelihood of responding to an anti-inflammatory drug.

正如以上的替代措辞,例如按照本发明的方法可以使用评价表达水平或考虑表达水平,和此外,也如上所述,所述方法不一定包括获得血液样品和在所述血液样品中评价表达水平的步骤。 Just like the above alternative wording, for example, the method according to the invention can use the evaluation of the expression level or take into account the expression level, and moreover, also as mentioned above, the method does not necessarily comprise obtaining a blood sample and evaluating the expression level in said blood sample step.

在本发明的其它方面,所述方法确实包括在来自所述患者的生物样品中测定图1的一个或多个基因的表达水平和将所述水平与所述基因的参考水平比较的步骤。 In other aspects of the invention, the method does include the steps of determining the expression level of one or more genes of Figure 1 in a biological sample from said patient and comparing said level to a reference level of said gene.

因此本发明一方面涵盖预测受试者对抗炎药的反应的方法,包括: Thus one aspect of the invention encompasses methods of predicting a subject's response to an anti-inflammatory drug comprising:

a) 在来自所述受试者的生物样品中检测图1的一个或多个基因的表达水平,和 a) detecting the expression level of one or more genes of Figure 1 in a biological sample from said subject, and

b) 将所述水平与所述基因的参考水平比较, b) comparing said level to a reference level for said gene,

其中与所述参考水平相比的一个或多个所述基因的表达改变,预测了所述受试者对所述抗炎药的反应。 Wherein the change in expression of one or more of said genes compared to said reference level predicts said subject's response to said anti-inflammatory drug.

进一步的方面涉及鉴定对抗炎药的反应具有增加的可能性的受试者的方法,包括: Further aspects relate to methods of identifying subjects with an increased likelihood of responding to anti-inflammatory drugs, comprising:

a) 在来自所述受试者的生物样品中检测图1的一个或多个基因的表达水平, a) detecting the expression level of one or more genes of Figure 1 in a biological sample from said subject,

b) 将所述水平与所述基因的参考水平比较, b) comparing said level to a reference level for said gene,

其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,表明已经鉴定出对抗炎药的反应具有增加的可能性的受试者。 Wherein expression of one or more of said genes is altered compared to a reference level of said genes, a subject having an increased likelihood of responding to an anti-inflammatory drug has been identified.

基因表达 gene expression

许多炎性疾病或病症(包括自身免疫疾病或病症)的分子背景并未完全了解,因此诊断是复杂的并且倾向于不准确的。目前可用的治疗对某些患者有用,但对其它患者无用,这其中的原因尚未阐明。为了增加治疗的成功性,进行了尝试,以便根据各种参数将患者分为亚组。 The molecular background of many inflammatory diseases or disorders, including autoimmune diseases or disorders, is not fully understood and thus diagnosis is complex and prone to inaccuracy. Currently available treatments are helpful for some patients but not others for reasons that have not been elucidated. In order to increase the success of the treatment, attempts were made to divide the patients into subgroups according to various parameters.

一个选项是表征患者的基因表达谱并据此而将患者分组。通常,在RNA水平或在蛋白水平上测定基因表达,因为测定了给定mRNA或翻译产物的水平。或者,可以检测或间接确定基因表达,例如通过与其它基因或标记的关联,也包括多态性例如SNP的标记。SNP通常是双等位基因的并且容易被检验。因此可以在不同水平上通过本领域技术人员已知的多种方法来检测基因表达。 One option is to characterize the gene expression profile of patients and group patients accordingly. Typically, gene expression is measured at the RNA level or at the protein level, as the level of a given mRNA or translation product is measured. Alternatively, gene expression can be detected or determined indirectly, for example by association with other genes or markers, also including markers of polymorphisms such as SNPs. SNPs are usually biallelic and easily tested. Gene expression can thus be detected at various levels by a variety of methods known to those skilled in the art.

用于通过mRNA水平来检测基因表达的试验可以基于PCR技术,例如多重-PCR,其中使用两个以上引物组,用于在分离自生物样品(例如患者的血液样品)的核酸上进行的单个反应中扩增2个或更多个DNA序列的目的。所述方法可以是两步法,其还包括扩增前的cDNA合成的步骤。微阵列芯片也可用于分析大群体的基因,这样的微阵列芯片可以经特别设计,以包括相关探针,或者可以收集来自关于目标基因的标准芯片的信息以评价被认为相关的基因的基因表达谱。 Assays for detection of gene expression by mRNA levels may be based on PCR techniques, such as multiplex-PCR, in which more than two primer sets are used for a single reaction on nucleic acid isolated from a biological sample, such as a patient's blood sample The purpose of amplifying 2 or more DNA sequences. The method may be a two-step method that also includes a step of cDNA synthesis prior to amplification. Microarrays can also be used to analyze large populations of genes, such microarrays can be specially designed to include relevant probes, or information can be collected from standard arrays on genes of interest to assess gene expression of genes thought to be relevant Spectrum.

PCR和阵列技术的特异性取决于引物和探针与所分析样品中的mRNA分子的杂交,可以通过本领域技术人员已知的参数调整严格性。 The specificity of PCR and array techniques depends on the hybridization of primers and probes to mRNA molecules in the sample being analyzed, the stringency of which can be adjusted by parameters known to those skilled in the art.

当有证据表明其它基因或标记的检测与目标基因表达水平关联时,通过关联到所述其它基因或所述标记,可以进行基因表达的检测。如本文的实施例4所述,技术人员可以进行目标基因的表达数量特征基因座(eQTL)分析和鉴定SNP与该基因的表达水平的关联或关系。在进一步的替代方案中,通过与其它基因或标记关联可以由此测定表达水平。 When there is evidence that the detection of other genes or markers correlates with the expression level of the gene of interest, detection of gene expression can be performed by correlating to said other genes or said markers. As described in Example 4 herein, skilled artisans can perform expression quantitative trait locus (eQTL) analysis of a target gene and identify the association or relationship between the SNP and the expression level of the gene. In a further alternative, expression levels can thus be determined by correlation with other genes or markers.

还考虑了在蛋白水平上测量基因表达,只要翻译产物是在得自患者的样品中可以检测的蛋白质。 Measuring gene expression at the protein level is also contemplated, as long as the translation product is a detectable protein in a sample from the patient.

可使用合适的技术检测蛋白质,其通常是基于抗体的,因为根据本领域已知技术,可以产生和使用对指定蛋白具有特异性的抗体。当使用来自少数基因的基因表达数据时,基于抗体的技术是最可用的。使用蛋白质组分析,可在蛋白质水平上进行更复杂的基因表达分析。 Proteins can be detected using suitable techniques, which are usually antibody-based, since antibodies specific for a given protein can be generated and used according to techniques known in the art. Antibody-based techniques are most usable when using gene expression data from a small number of genes. Using proteome analysis, more complex analysis of gene expression can be performed at the protein level.

对于某些基因产物,功能测定可同样好地用于确定表达水平的目的。功能测定可以是测试生物活性或酶活性的测定,这取决于蛋白质的功能和本领域有关这类蛋白质活性的知识。 For certain gene products, functional assays may serve equally well for the purpose of determining expression levels. Functional assays can be assays that test biological activity or enzymatic activity, depending on the function of the protein and the knowledge in the art about the activity of such proteins.

如上所述,本发明的一个实施方案涉及鉴定对抗炎药的反应具有增加的可能性的患者的方法,包括: As noted above, one embodiment of the invention is directed to a method of identifying patients with an increased likelihood of responding to an anti-inflammatory drug comprising:

a) 在来自所述患者的生物样品中测定图1的一个或多个基因的表达水平 a) Determining the expression level of one or more genes of Figure 1 in a biological sample from said patient

b) 将所述水平与所述基因的参考水平比较, b) comparing said level to a reference level for said gene,

其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,预测了所述患者对所述抗炎药的反应。 wherein the altered expression of one or more of said genes compared to a reference level of said genes predicts said patient's response to said anti-inflammatory agent.

同样的标准可用于鉴定或选择使用所述抗炎药进行治疗的患者,这基于鉴定对使用所述抗炎药的治疗的反应具有增加的可能性的患者的期望。 The same criteria can be used to identify or select patients for treatment with the anti-inflammatory drug based on the desire to identify patients with an increased likelihood of responding to treatment with the anti-inflammatory drug.

如本文公开的实施例中可见,图1的一个或多个基因的表达水平改变指示对抗炎药的临床反应。此外吸引力集中在与参考水平相比增加的一个或多个基因。或者,重点可在于与参考水平相比减少的一个或多个基因。这些特征的任一个与患者中的改善的反应率关联的基因,分别列于图1A和图1B中。在另一个实施方案中,本文所述的方法涉及这样的情况:其中与参考水平相比,图1A的基因的改变的表达是增加的。在另一个实施方案中,本文所述的方法涉及这样的情况:其中与参考水平相比,图1B的基因的改变的表达是降低的。在其它实施方案中,可包括更多基因,例如表达水平高于参考水平的图1A的一个或多个基因与表达水平低于参考水平的图1B的一个或多个基因的组合,从而使用多个基因的表达信息。在一个实施方案中,将至少两个基因的表达水平与个体参考水平比较。进一步可能的是将以下信息组合:与参考水平相比图1A的基因的改变的表达是增加的信息,以及与参考水平相比图1B的基因的改变的表达是降低的信息。 As seen in the Examples disclosed herein, changes in the expression levels of one or more genes of Figure 1 are indicative of clinical response to anti-inflammatory drugs. Further attractiveness focuses on one or more genes that are increased compared to reference levels. Alternatively, the focus can be on one or more genes that are reduced compared to a reference level. Genes for which any of these features were associated with improved response rates in patients are listed in Figures 1A and 1B, respectively. In another embodiment, the methods described herein relate to the situation wherein the altered expression of the gene of Figure 1A is increased compared to a reference level. In another embodiment, the methods described herein relate to the situation wherein the altered expression of the gene of Figure IB is reduced compared to a reference level. In other embodiments, more genes may be included, such as a combination of one or more genes of FIG. 1A whose expression level is higher than a reference level and one or more genes of FIG. 1B whose expression level is lower than a reference level, thereby using multiple expression information of a gene. In one embodiment, the expression levels of at least two genes are compared to an individual reference level. It is further possible to combine the information that the altered expression of the genes of Figure 1A is increased compared to the reference level and the altered expression of the genes of Figure IB is decreased compared to the reference level.

用于本发明的基于多变量的方法的基因组合举例于表2中,在这样的方法中,可以使用1个或多个例如至少2个、至少3个、至少4个基因的表达水平的信息。 Gene combinations for multivariate-based methods of the present invention are exemplified in Table 2. In such methods, information on the expression levels of one or more, e.g., at least 2, at least 3, at least 4 genes can be used .

如下文所进一步描述,参考水平是基因的特性,取决于所述方法的特定目的。 As described further below, the reference level is a property of the gene, depending on the particular purpose of the method.

参考水平 reference level

参考水平可以是在未受影响的或健康受试者中的表达水平,其最可能是这样的基因情况:其中基因的表达改变是炎性疾病或病症的特征,和所述基因可用作诊断标记。在一个实施方案中,参考水平可以是在未受影响的或健康个体中的表达水平。 The reference level may be the expression level in an unaffected or healthy subject, which is most likely the case of a gene in which altered expression of a gene is characteristic of an inflammatory disease or disorder, and which is useful for diagnosis mark. In one embodiment, the reference level may be the expression level in an unaffected or healthy individual.

根据本文的数据,显而易见的是,与患者相比,在未受影响的或健康个体中其它生物标记不一定表现出不同的表达。在一个实施方案中,参考水平可以是在健康个体或患者或其混合物的群体中测定的表达水平的平均值。在其它情况下,某些基因的基因表达水平可提供使用抗炎药的患者的能治疗性的预测信息,尽管表达谱与疾病状态或诊断标准可能无关联。这可能是因为以下事实:诊断的疾病不准确,因为诊断工具不反映疾病的变异性。这样的生物标记可能依然具有很大价值,因为它们可根据本文的方法使用,以将治疗指向更可能对指定治疗具有反应的个体患者。 From the data herein it is evident that other biomarkers do not necessarily exhibit different expression in unaffected or healthy individuals compared to patients. In one embodiment, the reference level may be the mean of the expression levels determined in a population of healthy individuals or patients or a mixture thereof. In other cases, the gene expression levels of certain genes may provide predictive information of the treatability of patients on anti-inflammatory drugs, although expression profiles may not correlate with disease state or diagnostic criteria. This may be due to the fact that the diagnosed disease is inaccurate because the diagnostic tool does not reflect the variability of the disease. Such biomarkers may still be of great value, as they can be used according to the methods herein to direct treatment to individual patients who are more likely to respond to a given treatment.

基于这样的信息,参考水平可以是预定水平、任意表达水平,其用于筛选对抗炎药具有反应的患者。在本发明的实施方案中,参考水平是预定水平。 Based on such information, the reference level can be a predetermined level, any expression level, which is used to screen patients for response to anti-inflammatory drugs. In an embodiment of the invention, the reference level is a predetermined level.

本文的实例证明根据本发明的方法可使用单个基因的表达水平。该预定水平因此可以是指示给定的反应的表达水平,所述给定的反应例如通过DAS28-CRP、ACR20、ACR50和/或ACR70反应测定的反应。参考水平或预定水平可以认为是阈值,并且可以选择所述阈值,旨在患者组中的某一反应水平。然后,所选阈值将指示在一部分患者中达到ACR20、ACR50和/或ACR70反应的可能性。同样,可以基于DAS28-CRP评分选择阈值,旨在患者组中的某一评分。因此,可设定水平,以便对于每一标准,取消选择无反应者,或增加达到阳性反应的一个或多个标准的患者部分。因此,生物标记预测治疗反应,并且如果仅旨在高反应者的话,甚至可用于预测反应程度。 The examples herein demonstrate that the expression levels of individual genes can be used in accordance with the methods of the invention. The predetermined level may thus be an expression level indicative of a given response, eg a response as determined by a DAS28-CRP, ACR20, ACR50 and/or ACR70 response. A reference level or predetermined level may be considered a threshold and said threshold may be chosen aiming at a certain level of response in a patient group. The selected threshold will then indicate the likelihood of achieving an ACR20, ACR50 and/or ACR70 response in a subset of patients. Likewise, thresholds can be chosen based on DAS28-CRP scores, targeting a certain score in a patient group. Thus, levels can be set such that for each criterion, non-responders are deselected, or the fraction of patients meeting one or more criteria for a positive response is increased. Thus, biomarkers predict treatment response, and can even be used to predict the extent of response if only aimed at high responders.

在一个实施方案中,预定参考水平可以基于ROC曲线,其经设定以选择在用抗炎药治疗的至少40%的患者、例如45%、例如50%、例如55%、例如60%、例如65%或至少70%的患者中达到ACR50反应的表达水平。 In one embodiment, the predetermined reference level may be based on a ROC curve set to select at least 40% of patients, such as 45%, such as 50%, such as 55%, such as 60%, such as ACR50-responsive expression levels were achieved in 65% or at least 70% of patients.

在一个实施方案中,预定参考水平可以基于ROC曲线,其经设定以选择在用抗炎药治疗的至少25%的患者、例如30%、例如35%、例如40%、例如45%、例如50%的患者中达到ACR70反应的表达水平。 In one embodiment, the predetermined reference level may be based on a ROC curve set to select at least 25% of patients, such as 30%, such as 35%, such as 40%, such as 45%, such as ACR70-responsive expression levels were achieved in 50% of patients.

在本发明的一个实施方案中,补体因子D (CFD,也称为adiposin)的表达水平,用于选择、鉴定和/或判定患者对抗炎药的反应是否具有更高的可能性。所述基因列于图1A (和图2)中,并举例说明具有增加的表达水平的基因如何可用于本发明的方法。在本文的实施例中,使用微阵列技术和qRT-PCR测定表达水平,但根据本发明其亦合适考虑用于测定CFD的mRNA水平的替代方法和用于测定CFD的蛋白水平或活性的通用方法。 In one embodiment of the present invention, the expression level of complement factor D (CFD, also known as adiposin) is used to select, identify and/or determine whether a patient has a higher probability of responding to an anti-inflammatory drug. The genes are listed in Figure 1A (and Figure 2) and illustrate how genes with increased expression levels can be used in the methods of the invention. In the examples herein, expression levels were determined using microarray technology and qRT-PCR, but alternative methods for determining mRNA levels of CFD and general methods for determining protein levels or activity of CFD are also suitable according to the invention .

因此,本发明的方法可包括,当通过微阵列技术测定时,补体因子D (CFD)的表达水平在标准化表达值的log2-标度(scale)上超过9.5。基于本文给出的ROC数据,将阈值增加至例如9.8、10.0、10.2、10.3、10.4或甚至10.5提供了具有更高特异性但同时更低灵敏性的方法。 Accordingly, the methods of the invention may comprise expression levels of complement factor D (CFD) exceeding 9.5 on the log2-scale of normalized expression values when determined by microarray techniques. Based on the ROC data presented here, increasing the threshold to eg 9.8, 10.0, 10.2, 10.3, 10.4 or even 10.5 provides a method with higher specificity but at the same time lower sensitivity.

基于PCR测定例如RT-PCR,也可测定表达水平,无论是用内部对照进行的qRT-PCR,或是使用可同时测定超过一个转录物的多重PCR,其也通常包括内部对照。 Expression levels can also be determined based on PCR assays such as RT-PCR, either qRT-PCR with internal controls, or using multiplex PCR that can simultaneously measure more than one transcript, which also typically includes internal controls.

因此,本发明的方法可包括以下方法:其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中测定转录物的循环阈值(Ct)为30 (使用Assay ID: Hs00157263_m1(Life technologies 的Applied Biosystems)。在进一步的这类实施方案中,在PCR循环28或甚至26内可检测CFD转录物。进一步优选的是在相同时间,在相同的cDNA样品上检测18S RNA具有循环阈值(Ct) 12.5,证实了PCR分析的质量。 Accordingly, the methods of the present invention may include methods wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the cycle threshold (Ct) of transcripts is determined to be 30 (using Assay ID: Hs00157263_m1 (Applied Biosystems). In further such embodiments, CFD transcripts are detectable within PCR cycles 28 or even 26. It is further preferred that 18S RNA is detected on the same cDNA sample at the same time with a cycle threshold (Ct) of 12.5 , confirming the quality of the PCR analysis.

也可检测扩增反应的效率,其应当高于95%,其中100%表明每一循环的扩增子的理论倍增。或者,PCR扩增的效率应当为至少1.9或优选地至少1.93,其中2.0代表每一循环的扩增子的理论倍增。 The efficiency of the amplification reaction can also be tested, which should be higher than 95%, where 100% indicates a theoretical doubling of the amplicon per cycle. Alternatively, the efficiency of PCR amplification should be at least 1.9 or preferably at least 1.93, where 2.0 represents a theoretical doubling of the amplicon per cycle.

在进一步的实施方案中,本发明的方法包括其中间接通过SNP检测而测量补体因子D (CFD)的表达水平的方法。在这样的方法中,可评价一个或多个SNP。与基因表达相反,SNP检测提供是/是、是/非(=非/是)或非/非反应,而不是高或低表达的相对尺度(relative scale),因此所述评价必须集中在对应于目标基因的表达改变的基因型。 In further embodiments, the methods of the invention include methods wherein the expression level of complement factor D (CFD) is measured indirectly by SNP detection. In such methods, one or more SNPs can be evaluated. In contrast to gene expression, SNP detection provides a yes/yes, yes/no (=no/yes) or no/no response, not a relative scale of high or low expression, so the evaluation must focus on the Genotypes with altered expression of the target gene.

根据所选的SNP,参考水平可以是“非/非”和与至少一个等位基因应当提供“是”的表达改变相关联。在替代的实施方案中,所述参考水平可以是“是/是”或甚至“是/非”。 Depending on the SNP selected, the reference level can be "no/no" and associated with an expression change that at least one allele should provide a "yes". In alternative embodiments, the reference level may be "yes/yes" or even "yes/no".

在CFD的实例中,增加的CFD表达与每个SNP的特异性参考水平相关。因此,这类方法将包括其中间接通过检测以下的一种或多种而测量补体因子D (CFD)的表达水平的方法:rs1683565、rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898。 In the case of CFD, increased CFD expression correlated with specific reference levels for each SNP. Accordingly, such methods would include those in which the level of expression of complement factor D (CFD) is measured indirectly by detecting one or more of: rs1651891, rs1651890 and rs2930898.

因为这些SNP都涉及其是或A或G的单核苷酸,因此所述参考水平将是AA、AG或GG。 Since these SNPs all involve single nucleotides which are either A or G, the reference level will be AA, AG or GG.

对于实施例中描述的SNP rs1683591,所述参考水平将是AA基因型(低CFD表达),并且AG和GG基因型将表明CFD的表达改变(高CFD表达)。 For SNP rs1683591 described in the Examples, the reference level would be the AA genotype (low CFD expression), and the AG and GG genotypes would indicate altered expression of CFD (high CFD expression).

本发明的方法因此包括其中间接通过SNP rs1683591的AG基因型或GG基因型的存在而测量CFD水平的方法。 The methods of the invention thus include methods wherein the level of CFD is measured indirectly by the presence of the AG genotype or the GG genotype of SNP rs1683591.

正如使用微阵列数据和RT-PCR数据的方法所示,通过降低阈值可增加所述方法的特异性,尽管因此损失某些灵敏性。 As shown for methods using microarray data and RT-PCR data, the specificity of the method can be increased by lowering the threshold, although some sensitivity is thus lost.

如果基因的表达增加与本发明的方法相关,例如对于CFD,则增加阈值将提供具有更高特异性但同时更低灵敏性的方法。 If increased expression of a gene is associated with the method of the invention, eg for CFD, increasing the threshold will provide a method with higher specificity but at the same time lower sensitivity.

反之亦然,如果基因的表达减少与本发明的方法相关,则减少表达水平阈值将提供具有更高特异性但同时更低灵敏性的方法。也显而易见的是,为了满足该情况下的阈值标准,表达水平低于阈值。 Vice versa, if reduced expression of a gene is relevant to the method of the invention, reducing the expression level threshold will provide a method with higher specificity but at the same time lower sensitivity. It is also evident that, in order to meet the threshold criteria in this case, the expression level is below the threshold.

根据以上,显而易见的是,如本文所述鉴定的其它基因,可以同样单独或组合用于在患者中预测临床反应可能性的方法,并且藉此根据所选患者对所述治疗具有反应的高可能性而选择患者进行给定的治疗。 From the above, it is evident that other genes identified as described herein can likewise be used alone or in combination in a method for predicting the likelihood of clinical response in a patient, and thereby based on the high likelihood that the selected patient will respond to said treatment Patients are selected for a given treatment based on their nature.

生物样品 Biological samples

在最初诊断后,对个体患者进行任何分亚组或表征的起点,是生物样品或基于先前已得到的生物样品的信息。根据本发明,所述生物样品可以是在使用本发明之前或期间得自患者的任何样品。所述样品优选地是可通过常规方法容易获取的血液样品,但其它类型的样品也可使用。所述样品也可以是血清样品。在某些情况下,可以使用组织样品例如滑液样品。本领域技术人员将会理解在测定给定基因组的表达水平之前如何处理给定的样品。可采集全血样品作为PaxGene样品。 After the initial diagnosis, the starting point for any subgrouping or characterization of individual patients is the biological sample or information based on previously obtained biological samples. According to the invention, said biological sample may be any sample obtained from a patient before or during use of the invention. The sample is preferably a blood sample readily obtainable by conventional methods, but other types of samples may also be used. The sample can also be a serum sample. In some cases, tissue samples such as synovial fluid samples may be used. Those skilled in the art will understand how to treat a given sample prior to determining the expression level of a given genome. Whole blood samples can be collected as PaxGene samples.

如果目标基因在特定细胞类型中表达,这可反映出样品用于表达研究。因此,生物样品可以是来自血液样品的外周血单核细胞样品,也称为PBMC部分,或甚至是其仅包括单核细胞的亚部分,例如CD14+、CD4+和/或CD8+阳性细胞中的一种或多种。 If the gene of interest is expressed in a particular cell type, this may reflect the sample being used for expression studies. Thus, the biological sample may be a sample of peripheral blood mononuclear cells from a blood sample, also called a PBMC fraction, or even a subfraction thereof comprising only monocytes, such as one of CD14+, CD4+ and/or CD8+ positive cells or more.

在基因编码可溶性蛋白标记的情况下,表达研究可在血清样品上进行,和可评价蛋白质而非转录物的存在。可使用特异性抗体检测血清中的可溶性蛋白,例如通过本领域已知的ELISA。如果评价更多蛋白质,可考虑使用蛋白质组分析进行蛋白质水平上的更复杂的基因表达分析。 In the case of genetically encoded soluble protein markers, expression studies can be performed on serum samples, and the presence of proteins rather than transcripts can be assessed. Soluble proteins in serum can be detected using specific antibodies, for example by ELISA as known in the art. If more proteins are evaluated, consider using proteome analysis for more complex gene expression analysis at the protein level.

除了表达水平及其变化之外,生物样品也可用于测定患者的类风湿因子(RF)状态。 In addition to expression levels and changes thereof, biological samples can also be used to determine a patient's rheumatoid factor (RF) status.

患者和患者状态 Patient and Patient Status

在一个实施方案中,受试者是患者,例如经诊断或患有与本文所述的炎性疾病或病症相关的体征或症状的个体。在一个实施方案中,所述患者患有自身免疫疾病或病症。在具体的实施方案中,所述患者是RA患者或患有RA症状。 In one embodiment, the subject is a patient, eg, an individual diagnosed with or suffering from signs or symptoms associated with an inflammatory disease or disorder described herein. In one embodiment, the patient has an autoimmune disease or disorder. In specific embodiments, said patient is a patient with RA or suffers from symptoms of RA.

样品可得自从未进行炎性疾病或病症治疗的患者,意思是先前从未将针对炎性疾病或病症的治疗给予所述患者。对于患有可能罕见发生的自身炎性疾病的患者,在考虑本文所述的抗炎药(尤其是对于生物药物类)之前通常考虑多种治疗。在某些情况下,基因表达信息可得自先前获得的样品,因此所述样品可被认为是从未接受治疗的,而患者不再是从未接受给定治疗的。所述样品在某些情况下可以得自正在接受炎性疾病或病症治疗的患者。所述患者可以是处于基本使用任何药物的治疗之中,所述药物不限于本文提出的抗炎药。 A sample may be obtained from a patient who has not been treated for an inflammatory disease or disorder, meaning that no treatment for an inflammatory disease or disorder has been previously administered to said patient. For patients with autoinflammatory diseases that may occur infrequently, multiple therapies are often considered before considering the anti-inflammatory agents described herein, especially for biologic drugs. In some cases, gene expression information can be obtained from a previously obtained sample, so the sample can be considered treatment naive and the patient is no longer naive to a given treatment. The sample may in some cases be obtained from a patient being treated for an inflammatory disease or disorder. The patient may be under treatment with essentially any drug, not limited to the anti-inflammatory drugs set forth herein.

通常将用作治疗炎性疾病或病症的一线药物的药物给予患者,然后评价本发明的治疗是否具有高的成功可能性。 Drugs used as first-line drugs for the treatment of inflammatory diseases or conditions are typically administered to patients and then evaluated to see if the treatment of the present invention has a high probability of success.

用于正在治疗或先前已治疗患者的药物可包括以下的一种或多种:非甾体抗炎药(NSAID)例如Asprin?、Ibuprofen?等、皮质类固醇、疾病-修饰性抗风湿药(DMARD)例如Plaquenil?、Azulfidine?、Methotrexate?等、Copaxone? (glatirimer acetate)、Gilneya? (芬戈莫德)、抗生素例如Flagyl?、Cipro?、局部(皮肤施用)药物包括局部皮质类固醇、维生素D类似物乳膏(Dovonex?)、局部类视色素(Tazorac?)、保湿剂、局部免疫调节剂(他克莫司和吡美莫司)、煤焦油、地蒽酚和其它、Raptiva?、Ustekimumab?、光治疗例如PUVA、UVB和CellCept? (麦考酚酸吗乙酯)。还包括生物抗炎药,包括但不限于IFN-β、Orencia? (CTLA4-Ig)、Humira? (抗TNF)、Cimzia? (抗TNF、PEG Fab)、Tysabri? (a4-整联蛋白 mAb)、Simponi?、Rituxan/MabThera?、Actemra/RoActemra?和Kineret?。 Medications for current or previously treated patients may include one or more of the following: nonsteroidal anti-inflammatory drugs (NSAIDs) such as Asprin®, Ibuprofen®, etc., corticosteroids, disease-modifying antirheumatic drugs (DMARDs) ) such as Plaquenil®, Azulfidine®, Methotrexate®, etc., Copaxone® (glatirimer acetate), Gilneya® (fingolimod), antibiotics such as Flagyl®, Cipro®, topical (skin applied) medications including topical corticosteroids, vitamin D similar creams (Dovonex®), topical retinoids (Tazorac®), moisturizers, topical immunomodulators (tacrolimus and pimecrolimus), coal tar, dithranol and others, Raptiva®, Ustekimumab® , light therapy such as PUVA, UVB and CellCept? (mycophenolate mofetil). Also included are biologic anti-inflammatory agents, including but not limited to IFN-β, Orencia® (CTLA4-Ig), Humira® (anti-TNF), Cimzia® (anti-TNF, PEG Fab), Tysabri® (a4-integrin mAb) , Simponi™, Rituxan/MabThera™, Actemra/RoActemra™, and Kineret™.

如果不使用治疗之前获得的样品,可使用一种或多种宽范围的抗炎药来治疗患者,所述抗炎药包括如上所述的NSAID、DMARD和TNF-α抑制剂。通常,将用甲氨蝶呤(MTX)治疗患者。 If samples obtained prior to treatment are not used, the patient may be treated with one or more of a wide range of anti-inflammatory drugs including NSAIDs, DMARDs and TNF-alpha inhibitors as described above. Typically, the patient will be treated with methotrexate (MTX).

另外,先前采用的治疗在患者中不提供充分的反应,和因此所述患者不再是从未接受所述治疗,但被认为是不充分反应者。对于诊断和临床反应,用于判定给定的患者是否是不充分反应者的标准将取决于如下所述的待治疗的疾病或病症。 In addition, a previously employed therapy did not provide an adequate response in the patient, and thus the patient is no longer never received the therapy, but is considered an inadequate responder. For diagnosis and clinical response, the criteria used to determine whether a given patient is an inadequate responder will depend on the disease or condition being treated as described below.

因此,患者对于MTX治疗和/或对于TNF-α抑制剂治疗可以是不充分反应者,其中TNF-α抑制剂治疗是指被认为TNF-α抑制剂的药物中的一种或多种,包括抗体药物和可溶性受体药物两者。 Thus, patients may be inadequate responders to MTX therapy and/or to TNF-alpha inhibitor therapy, where TNF-alpha inhibitor therapy refers to one or more of the drugs that are considered TNF-alpha inhibitors, including Both antibody drugs and soluble receptor drugs.

优选的是,在获得所述生物样品之前或同时,选择用于本文所述的鉴定方法的基因的表达不受先前或同时的用任何抗炎药治疗的影响。 Preferably, the expression of the genes selected for the identification methods described herein is not affected by previous or concurrent treatment with any anti-inflammatory drug before or while said biological sample is obtained.

如上所述,还可涉及到考虑患者的类风湿因子(RF)状态和抗环状瓜氨酸化蛋白抗体(抗CCP)状态。确定RF和抗CCP状态的测定是本领域已知的,技术人员可以按照来自制造商的说明书,毫无困难地使用任何这样的测定。RF阳性患者的类风湿因子水平超过指示样品中存在RF的某一阈值。如果阴性,类风湿因子水平低于所述阈值,指示所述样品中不存在RF。 As noted above, it may also involve consideration of the patient's rheumatoid factor (RF) status and anti-cyclic citrullinated protein antibody (anti-CCP) status. Assays to determine RF and anti-CCP status are known in the art and the skilled artisan will have no difficulty using any such assay following instructions from the manufacturer. Rheumatoid factor levels in RF positive patients exceed a certain threshold indicating the presence of RF in the sample. If negative, the rheumatoid factor level is below the threshold, indicating that RF is not present in the sample.

在一个实施方案中,患者RF状态是阳性或阴性。在进一步的具体的实施方案中,患者RF状态是要么阳性要么阴性。 In one embodiment, the patient's RF status is positive or negative. In further specific embodiments, the patient's RF status is either positive or negative.

在一个实施方案中,所述患者抗CCP状态是阳性或阴性。在进一步的具体的实施方案中,所述患者抗CCP状态是要么阳性要么阴性。 In one embodiment, the patient's anti-CCP status is positive or negative. In a further specific embodiment, said patient's anti-CCP status is either positive or negative.

适应症 Indications

如上所述,本发明涉及各种疾病、尤其包括自身免疫和炎性疾病或病症的治疗。 As noted above, the present invention relates to the treatment of various diseases, including autoimmune and inflammatory diseases or conditions, among others.

用抗炎药治疗的所述病况或病症是类风湿性关节炎、青少年类风湿性关节炎、银屑病、银屑病性关节炎、强直性脊椎炎、斯耶格伦综合征(Sjogren's syndrome)、多发性硬化、炎性肠病例如溃疡性结肠炎和克罗恩病、系统性红斑狼疮或狼疮性肾炎、其任何组合、以及与这些疾病相关的伴随疾病,其中心血管疾病是所述伴随疾病的非限制性实例。又一方面,其它示例性的病况包括但不限于青少年慢性关节炎、骨关节炎、除强直性脊椎炎之外的其它脊椎关节病、系统性硬化(硬皮病)、原发性炎性肌病(皮肌炎、多肌炎)、脉管炎、系统性脉管炎、颞动脉炎、动脉粥样硬化、结节病、重症肌无力、自身免疫性溶血性贫血(免疫性全血细胞减少症、阵发性睡眠性血红蛋白尿症)、恶性贫血、自身免疫性血小板减少症(原发性血小板减少性紫癜、免疫-介导的血小板减少症)、甲状腺炎(格雷夫斯氏病、桥本甲状腺炎、幼年型淋巴细胞性甲状腺炎、萎缩性甲状腺炎)、糖尿病、II型糖尿病、免疫-介导的肾病(肾小球肾炎、肾小管间质性肾炎、自身免疫性卵巢炎)、胰腺炎、自身免疫性睾丸炎、自身免疫性葡萄膜炎、抗磷脂综合征,除了多发性硬化、原发性脱髓鞘性多神经病或格-巴二氏综合征和慢性炎症性脱髓鞘性多神经病以外的中枢和外周神经系统的脱髓鞘疾病,肝胆疾病诸如传染性肝炎(甲、乙、丙、丁、戊型肝炎和其它非亲肝(hepatotropic)病毒)、自身免疫性慢性活动性肝炎、病毒性肝炎、原发性胆汁性肝硬变、肉芽肿性肝炎、韦格纳氏肉芽肿病、贝赫切特病和硬化性胆管炎,炎性肠疾病诸如乳糜泻、谷蛋白敏感性肠病和惠伯尔病,自身免疫或免疫-介导的皮肤病包括大疱性皮肤病、多形红斑和接触性皮炎、特应性皮炎、疱疹样皮炎、寻常天疱疮、白癜风(白斑病),变态反应性疾病诸如哮喘、变应性鼻炎、特应性皮炎、食物超敏反应和荨麻疹,脓毒病,内毒素血症,肺的免疫性疾病诸如嗜酸细胞性肺炎、原发性肺纤维化和过敏性肺炎、慢性阻塞肺疾病,和器官或骨髓移植有关的疾病包括移植物排斥和移植物抗宿主病。 Said condition or disorder to be treated with anti-inflammatory drug is rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Sjogren's syndrome ), multiple sclerosis, inflammatory bowel disease such as ulcerative colitis and Crohn's disease, systemic lupus erythematosus or lupus nephritis, any combination thereof, and concomitant diseases associated with these diseases, wherein cardiovascular disease is the Non-limiting examples of concomitant diseases. In yet another aspect, other exemplary conditions include, but are not limited to, juvenile chronic arthritis, osteoarthritis, spondyloarthropathy other than ankylosing spondylitis, systemic sclerosis (scleroderma), primary inflammatory muscle disease (dermatomyositis, polymyositis), vasculitis, systemic vasculitis, temporal arteritis, atherosclerosis, sarcoidosis, myasthenia gravis, autoimmune hemolytic anemia (immune pancytopenia syndrome, paroxysmal nocturnal hemoglobinuria), pernicious anemia, autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves' disease, bridge primary thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, type II diabetes mellitus, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis, autoimmune oophoritis), Pancreatitis, autoimmune orchitis, autoimmune uveitis, antiphospholipid syndrome, except multiple sclerosis, primary demyelinating polyneuropathy or Guillain-Barr syndrome, and chronic inflammatory demyelination Demyelinating diseases of the central and peripheral nervous system other than polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic activity hepatitis, viral hepatitis, primary biliary cirrhosis, granulomatous hepatitis, Wegener's granulomatosis, Behçet's disease, and sclerosing cholangitis, inflammatory bowel disease such as celiac disease, gluten Sensitive enteropathy and Whipper's disease, autoimmune or immune-mediated skin diseases including bullous dermatosis, erythema multiforme and contact dermatitis, atopic dermatitis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo (vitiligo), allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, sepsis, endotoxemia, immune diseases of the lungs such as eosinophilic pneumonia , primary pulmonary fibrosis and hypersensitivity pneumonitis, chronic obstructive pulmonary disease, and diseases associated with organ or bone marrow transplantation including graft rejection and graft-versus-host disease.

炎性疾病的原因是多方面的,涉及多个途径和组分。炎症是包括多组分的事件级联,包括管脉系统(例如内皮细胞、周皮细胞、平滑肌细胞)、免疫系统细胞(例如T和B淋巴细胞;多形核白细胞或粒细胞,例如单核细胞和嗜中性粒细胞;树突细胞、巨噬细胞和NK细胞)、细胞衍生的可溶性介质(细胞因子、趋化因子)以及在靶组织中居留的细胞(例如上皮细胞、滑膜成纤维细胞、神经元细胞)。这些元件的每一个(包括其调节物)在疾病发展中都可能具有作用,并且随后也可能作为上述疾病和病症的治疗靶标。炎性疾病因此也可通过受影响的途径来表征,例如作为B或T-细胞介导的疾病或病症,作为细胞因子介导的病症或受体介导的病症等等。 The causes of inflammatory diseases are multifaceted, involving multiple pathways and components. Inflammation is a cascade of events involving multiple components, including the vasculature (eg, endothelial cells, pericytes, smooth muscle cells), immune system cells (eg, T and B lymphocytes; polymorphonuclear leukocytes, or granulocytes, eg, monocytes cells and neutrophils; dendritic cells, macrophages, and NK cells), cell-derived soluble mediators (cytokines, chemokines), and cells resident in target tissues (e.g., epithelial cells, synovial fibroblasts cells, neurons). Each of these elements, including their regulators, may have a role in disease development and may subsequently also serve as a therapeutic target for the diseases and disorders described above. Inflammatory diseases can thus also be characterized by the pathways affected, for example as a B or T-cell mediated disease or disorder, as a cytokine-mediated disorder or a receptor-mediated disorder, and the like.

对于本发明,所述适应症因此可以是经抗炎药治疗而得以改善的任何病症,例如由IL-10家族例如如下所述的受体和配体的信号转导/活性的下调所介导的病症。 For the purposes of the present invention, the indication may thus be any condition that is ameliorated by anti-inflammatory drug treatment, e.g. mediated by downregulation of signaling/activity of the IL-10 family such as receptors and ligands as described below disease.

可使用IL-10家族的细胞因子和受体的调节剂治疗的适应症包括自身免疫疾病和病症,例如类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、多发性硬化(MS)、炎性肠病(IBD)、银屑病或银屑病性关节炎(PSA)。 Indications treatable with modulators of cytokines and receptors of the IL-10 family include autoimmune diseases and conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) , inflammatory bowel disease (IBD), psoriasis, or psoriatic arthritis (PSA).

抗炎药 anti-inflammatory drugs

如上所述,多个途径参与炎症,可在多个水平上靶向每个途径。通过阻断受体,通过提供可溶性受体片段或通过阻止配体结合受体或经受体转导信号,可以获得对受体信号转导的抑制,其实例是用于治疗某些自身免疫疾病和/或癌症的靶向生物治疗药。例如,癌症患者可以用针对CD20的抗体(抗CD20)来治疗;类风湿性关节炎患者可以用抗CD20 (一种TNF拮抗剂) (可溶性TNFR或抗TNF-α)来治疗;银屑病患者可以用抗CD11a来治疗;多发性硬化患者可以用INF-β来治疗;溃疡性结肠炎患者可以用抗TNF-α来治疗和克罗恩病患者可以用抗TNF-α或抗α4整联蛋白来治疗。不幸的是,这些治疗并不完全有效。 As noted above, multiple pathways are involved in inflammation and each pathway can be targeted at multiple levels. Inhibition of receptor signaling can be obtained by blocking the receptor, by providing soluble receptor fragments, or by preventing ligand binding to the receptor or transduction of the signal via the receptor, examples of which are used in the treatment of certain autoimmune diseases and/or targeted biotherapeutics for cancer. For example, cancer patients can be treated with antibodies against CD20 (anti-CD20); rheumatoid arthritis patients can be treated with anti-CD20 (a TNF antagonist) (soluble TNFR or anti-TNF-alpha); psoriasis patients Can be treated with anti-CD11a; patients with multiple sclerosis can be treated with INF-β; patients with ulcerative colitis can be treated with anti-TNF-α and patients with Crohn's disease can be treated with anti-TNF-α or anti-α4 integrin Come for treatment. Unfortunately, these treatments are not completely effective.

先前已经描述过,IL-10家族成员是治疗炎性疾病或病症的有用靶标(WO 2001/46261)。 It has been described previously that members of the IL-10 family are useful targets for the treatment of inflammatory diseases or disorders (WO 2001/46261).

IL-10家族包括IL-10、IL19、IL-20、IL-22、IL-24和IL-26,其与以下受体异源二聚体结合: The IL-10 family includes IL-10, IL19, IL-20, IL-22, IL-24, and IL-26, which bind to the following receptor heterodimers:

IL-10: 与IL-10R1 / IL-10R2结合  IL-10: binds to IL-10R1 / IL-10R2

IL-19: 与IL-20R1 / IL-20R2结合 IL-19: Binds to IL-20R1 / IL-20R2

IL-20: 与IL-20R1 / IL-20R2和IL-22R / IL-20R2结合 IL-20: Binds to IL-20R1/IL-20R2 and IL-22R/IL-20R2

IL-22: 与IL-22R / IL-10R2结合 IL-22: Binds to IL-22R / IL-10R2

IL-24: 与IL-20R1 / IL-20R2和IL-22R / IL-20R2结合 IL-24: Binds to IL-20R1/IL-20R2 and IL-22R/IL-20R2

IL-26: 未知受体  IL-26: unknown receptor

该受体重叠表明,尽管某些功能对每个家族成员是特异性的,但也有某些共享的效应。每种配体和受体在炎性疾病中的准确作用尚未确定,但若干已经关联到疾病。实例包括:IL-20,其可通过抗体或受体片段来靶向,用于治疗某些炎性疾病(WO 2001/45261);IL-22和IL-19、IL-17 (WO10025369、WO2010102251),其是IL-10家族细胞因子的所有成员。 This receptor overlap suggests that although certain functions are specific to each family member, there are also certain shared effects. The precise role of each ligand and receptor in inflammatory diseases has yet to be determined, but several have been linked to disease. Examples include: IL-20, which can be targeted by antibodies or receptor fragments for the treatment of certain inflammatory diseases (WO 2001/45261); IL-22 and IL-19, IL-17 (WO10025369, WO2010102251) , which are all members of the IL-10 family of cytokines.

白介素-19 (IL-19)、IL-20和白介素-24 (IL-24)是白介素-10 (IL-10) 细胞因子家族成员。正如以上所见,这3种白介素与IL-20R1/IL-20R2 异源二聚体受体结合并通过所述受体而转导信号。IL-20和IL-24 (而不是IL-19)也是受体复合物的配体,所述受体复合物由IL-20R2和IL-22R1组成(Parrish-Novak等人, J Biol Chem 2002;277: 47517- 47523;Dumoutier 等人, J Immunol 2001;167:3545-3549)。已经提出,IL-19和IL-20,以及其它IL-10家族成员,构成螺旋状细胞因子的独特亚家族,其中至少IL-19和IL-20具有类似的三维结构(Chang等人, J Biol Chem 2003;278: 3308-13)。 Interleukin-19 (IL-19), IL-20, and interleukin-24 (IL-24) are members of the interleukin-10 (IL-10) cytokine family. As seen above, these 3 interleukins bind to and transduce signals through the IL-20R1/IL-20R2 heterodimeric receptor. IL-20 and IL-24 (but not IL-19) are also ligands for a receptor complex consisting of IL-20R2 and IL-22R1 (Parrish-Novak et al., J Biol Chem 2002; 277: 47517-47523; Dumoutier et al., J Immunol 2001; 167:3545-3549). It has been suggested that IL-19 and IL-20, along with other IL-10 family members, constitute a distinct subfamily of helical cytokines, of which at least IL-19 and IL-20 have similar three-dimensional structures (Chang et al., J Biol Chem 2003; 278: 3308-13).

因此已经描述了使用受体片段或单克隆抗体来拮抗IL-20活性,作为治疗多种炎性病况的有希望的方法。也已描述了人IL-20 (hlL-20)的抗原性表位以及结合hulL-20的大鼠或鼠单克隆抗体(例如WO2005052000、US20060142550和WO2007081465)。在一个或多个物种(包括人)中可以降低IL-20-介导的IL-20R1/IL-20R2和IL-22R1/IL-20R2受体复合物的活化的抗lL-20单克隆抗体已经描述于WO 2010/000721。 Antagonism of IL-20 activity using receptor fragments or monoclonal antibodies has thus been described as a promising approach for the treatment of a variety of inflammatory conditions. Antigenic epitopes of human IL-20 (hlL-20) and rat or murine monoclonal antibodies that bind hulL-20 have also been described (eg WO2005052000, US20060142550 and WO2007081465). Anti-IL-20 monoclonal antibodies that reduce IL-20-mediated activation of IL-20R1/IL-20R2 and IL-22R1/IL-20R2 receptor complexes in one or more species, including humans, have been Described in WO 2010/000721.

因此抗炎药可以是能够降低IL-20介导的IL-20R1 / IL-20R2以及IL-22R / IL-20受体这两者的活化的IL-20拮抗剂。所述抗炎药可以通过不经IL-19或IL-24受体降低受体活化而具有特异性。 Anti-inflammatory drugs may thus be IL-20 antagonists capable of reducing IL-20-mediated activation of both IL-20R1/IL-20R2 and IL-22R/IL-20 receptors. The anti-inflammatory drug may be specific by reducing receptor activation without the IL-19 or IL-24 receptors.

基于至少共享的作用方式,靶向每一配体和受体可提供类似的生物学效应。本发明的抗炎药因此可以是IL-10家族成员及其受体的拮抗剂,例如通过结合配体或受体而调节上述受体的信号转导的化合物,因此降低了所述配体或受体的生物活性。测定IL-10家族成员的拮抗活性的测定法是本领域已知的,也描述于WO 2010/000721。 Targeting each ligand and receptor may provide similar biological effects based on at least a shared mode of action. Anti-inflammatory drugs of the present invention may thus be antagonists of IL-10 family members and their receptors, for example compounds that modulate signal transduction of said receptors by binding to ligands or receptors, thus reducing the Receptor biological activity. Assays for determining the antagonistic activity of IL-10 family members are known in the art and are also described in WO 2010/000721.

本发明的抗炎药可以在药物组合物中,例如包含抗炎药和药学上可接受的载体和标签的药物组合物。所述抗炎药或药物组合物可以是适于口服、i.v.和/或s.c.给药。所述抗炎药或药物组合物可以重复给予,例如每月一次或每周一次。 The anti-inflammatory drug of the present invention can be in a pharmaceutical composition, for example, a pharmaceutical composition comprising an anti-inflammatory drug and a pharmaceutically acceptable carrier and label. The anti-inflammatory drug or pharmaceutical composition may be suitable for oral, i.v. and/or s.c. administration. The anti-inflammatory drug or pharmaceutical composition may be administered repeatedly, for example monthly or weekly.

本文的方法也考虑到给药途径或方案,因为所述反应可能取决于所用的治疗方案。在一个实施方案中,所述反应的预测或指示是基于每周一次给予抗IL-20抗体。在一个实施方案中,所述抗体经皮下给予。 The methods herein also take into account the route or regimen of administration, as the response may depend on the therapeutic regimen used. In one embodiment, the prediction or indication of response is based on weekly administration of an anti-IL-20 antibody. In one embodiment, the antibody is administered subcutaneously.

临床反应 clinical response

根据适应症,可以通过各种方法确定诊断和临床反应。在给予给定的抗炎药后,不表现出所治疗的病症的任何或足够的治疗体征的患者被认为是无反应的。相反,在给予给定的抗炎药后,通过表现出所治疗的病症的足够的治疗体征而有反应的患者就被认为是有反应的。足够的治疗体征因疾病的不同和患者的不同而异,并不暗示患者经历“完全”治疗,而仅仅是观察到一种或多种临床参数的改善。可在给予抗炎药之后的不同时间点来考虑反应性,并且患者可在一次或多次给予后短时间内或长时间内具有反应,但只要得到阳性结果,就认为该患者是有反应的。 Depending on the indication, the diagnosis and clinical response can be determined by various methods. Patients who do not exhibit any or sufficient signs of treatment of the condition being treated after administration of a given anti-inflammatory drug are considered non-responsive. Conversely, a patient is considered to respond after administration of a given anti-inflammatory drug by showing adequate signs of treatment of the condition being treated. Signs of adequate treatment vary by disease and by patient and do not imply that a patient undergoes "full" treatment, but merely that an improvement in one or more clinical parameters is observed. Responsiveness can be considered at various time points after administration of the anti-inflammatory drug, and a patient can be responsive for a short or prolonged period after one or more administrations, but as long as a positive result is obtained, the patient is considered responsive .

根据本发明,可以增加成功率(例如将抗炎药给予将有反应的患者的频率),而且使用本发明在被给予的患者中可获得达到不仅高成功率而且强反应的频率。 According to the present invention, the success rate (eg the frequency with which anti-inflammatory drugs are administered to patients who will respond) can be increased, and using the present invention can achieve not only high success rates but also the frequency of strong responses in administered patients.

可通过本领域已知方法测定临床反应。优选使用由政府主管部门批准的官方疾病评分。据说这样的疾病评分随时间而发展,所以获得临床评分的未来方法被认为与本发明相关。 Clinical response can be measured by methods known in the art. Preference is given to using official disease scores approved by government authorities. Such disease scores are said to develop over time, so future methods of obtaining clinical scores are considered relevant to the present invention.

考虑的是本领域技术人员能够鉴定给定的疾病或病症的相关的临床参数,因此本文包括的仅是少量关键的临床参数。根据不同标准诊断自身免疫疾病。 Only a small number of key clinical parameters are included herein, given that one skilled in the art is able to identify relevant clinical parameters for a given disease or condition. Autoimmune diseases are diagnosed according to different criteria.

本文的方法涉及患者对抗炎药的反应的指示和预测,这取决于适应症和症状,可在不同时间点预测预期反应。在个别的实施方案中,所述指示和预测涉及在12个月内、10个月内、8个月内、6个月内、5个月内、4个月内、3个月内或2个月内获得的反应。 The method herein involves the indication and prediction of the patient's response to anti-inflammatory drugs, depending on the indication and symptoms, predicting the expected response at different time points. In individual embodiments, the indication and prediction relate to within 12 months, within 10 months, within 8 months, within 6 months, within 5 months, within 4 months, within 3 months or within 2 months Responses obtained within a month.

类风湿性关节炎(RA) Rheumatoid Arthritis (RA)

类风湿性关节炎可根据美国风湿病学会(ACR)等所定义的标准来诊断。当使用所述标准时,对治疗的反应可以基于degrease评分。放射照相损伤的预防或延迟也是RA治疗的目标。美国风湿病学会(ACR) 20%综合改善标准描述了如果在触痛(tender)和肿胀关节计数中存在20%改善和在5个另外的量度(疼痛、身体功能、患者的综合健康评估、医生的综合健康评估和急性期反应物水平)的至少3个中存在20%改善,那么将患者归为“改善的”。类似地,ACR50和ACR70代表甚至更高程度的患者改善。 Rheumatoid arthritis can be diagnosed according to criteria defined by the American College of Rheumatology (ACR) and others. When using the criteria, response to treatment can be based on a degree score. Prevention or delay of radiographic injury is also a goal of RA treatment. The American College of Rheumatology (ACR) 20% Composite Improvement Criteria describe if there is a 20% improvement in tender and swollen joint counts and in 5 additional measures (pain, physical function, patient's general health assessment, physician Patients were classified as "improved" if there was a 20% improvement in at least 3 of the overall health assessment and acute phase reactant levels). Similarly, ACR50 and ACR70 represent an even higher degree of patient improvement.

根据获得ACR20、ACR50和/或ACR70的患者数量或患者部分,抗炎药作为RA治疗药的有效性可以因此而量化。 The effectiveness of an anti-inflammatory drug as a treatment for RA can thus be quantified in terms of the number or fraction of patients who achieve ACR20, ACR50 and/or ACR70.

除了ACR评分之外,也可使用28关节的疾病活性评分(DAS28)追踪类风湿性关节炎的发展。它是1980年代在Nijmegen开发的综合指数,已广泛用作RA疾病活性和对治疗的反应的指示物,以及基于DAS的欧洲抗风湿病联盟(European League Against Rheumatism,EULAR)反应标准。DAS28中包括的关节是(双侧):近端指间关节(10关节)、掌指关节(10)、腕(2)、肘(2)、肩(2)和膝(2)。当考虑这些关节时,同时统计触痛(TJC28)和肿胀(SJC28)的关节数量。可以包括C-反应蛋白(CRP)水平(以mg/l计)的测定,并且患者还在前面7天期间在0-100的数值范围上对疾病活性进行主观评价(SA),其中0是“无活性”,100是“最高活性可能”。据此计算DAS28。 In addition to the ACR score, the disease activity score of 28 joints (DAS28) can also be used to track the development of rheumatoid arthritis. It is a composite index developed in Nijmegen in the 1980s and has been widely used as an indicator of RA disease activity and response to treatment, as well as the DAS-based European League Against Rheumatism (EULAR) response criteria. The joints included in DAS28 are (bilateral): proximal interphalangeal joint (10 joints), metacarpophalangeal joint (10), wrist (2), elbow (2), shoulder (2), and knee (2). When considering these joints, count both tender (TJC28) and swollen (SJC28) joints. Measurement of C-reactive protein (CRP) levels (in mg/l) may be included, and patients also have a subjective assessment (SA) of disease activity on a scale of 0-100 during the previous 7 days, where 0 is " No activity", 100 is the "highest activity possible". Calculate DAS28 accordingly.

使用所述DAS,对于高疾病活性、低疾病活性或甚至缓解,已经开发了若干阈值。所述评分也可用作反应标准,当在两个时间点(例如治疗开始前和治疗后)测定患者DAS,可以评价患者的临床反应。 Using the DAS, several thresholds have been developed for high disease activity, low disease activity or even remission. The score can also be used as a response criterion, when the patient's DAS is measured at two time points (eg, before treatment initiation and after treatment), the patient's clinical response can be assessed.

本发明涉及改善RA治疗的有效性。尽管若干化合物已获批准并用于RA治疗,但治疗结果对于所有患者而言很少是优化的,并包括一些方面的试验和误差(arrow),因为没有使用预测RA治疗的有效性的方法。 The present invention relates to improving the effectiveness of RA treatment. Although several compounds have been approved and used for RA treatment, treatment outcomes are rarely optimal for all patients and involve some aspect of trial and error (arrow), as no method for predicting the effectiveness of RA treatment is used.

最近,已经描述了使用针对CD20的抗体疗法(利妥昔单抗)增加RA治疗有效性的方法(WO2011/028945, Owczarczyk等人 2011, Science translational medicine)。在WO2011/028945中,基于患者的表达谱定义了不同亚组的RA患者,并且还通过鉴定对抗CD20疗法不可能发生反应的RA患者亚组而包括了一些与临床反应的关联。FcRH5和CXCL13中的一种或多种的高(超过阈值) mRNA水平增加了RA患者的ARC50率。根据RF状态的进一步的亚组分类允许进一步细分,由此对于大约40%患者获得ARC50标准,其可以反映依赖于B细胞途径的亚组,所述B细胞途径是淋巴样亚组的标志和利妥昔单抗的靶标。 Recently, an approach to increase the effectiveness of RA treatment using an antibody therapy against CD20 (rituximab) has been described (WO2011/028945, Owczarczyk et al. 2011, Science translational medicine). In WO2011/028945 different subgroups of RA patients were defined based on the expression profiles of the patients and some correlations with clinical response were also included by identifying subgroups of RA patients unlikely to respond to anti-CD20 therapy. High (above threshold) mRNA levels of one or more of FcRH5 and CXCL13 increased ARC50 rates in RA patients. Further subgroup classification according to RF status allows for further subdivision whereby ARC50 criteria are obtained for approximately 40% of patients, which can reflect subgroups dependent on the B-cell pathway that is a marker of the lymphoid subgroup and target of rituximab.

本发明表明图1和2的基因的表达水平改变,指示高于RA患者的平均临床反应的临床反应。 The present invention demonstrates that the expression levels of the genes of Figures 1 and 2 are altered, indicating a clinical response above the average clinical response of RA patients.

系统性红斑狼疮(SLE) Systemic Lupus Erythematosus (SLE)

对于RA,SLE治疗效果可以是基于美国风湿病学会(ACR)分类标准的基础。建立这些标准,主要是用于科学研究和临床试验,而不是为了诊断目的,所以并非所有SLE患者都通过全部标准。 For RA, SLE treatment effects can be based on the American College of Rheumatology (ACR) classification criteria. These criteria were established mainly for scientific research and clinical trials, not for diagnostic purposes, so not all SLE patients pass all the criteria.

多发性硬化(MS) Multiple Sclerosis (MS)

存在着所述疾病的若干亚类并观察到不同的预后和进展。 Several subtypes of the disease exist and different prognosis and progression are observed.

美国国立多发性硬化学会(The United States National Multiple Sclerosis Society)于1996年标准化4个亚类定义:1) 复发-缓解型,2) 继发进展型,3) 原发进展型,和4) 进展复发型。对于诊断和评价,使用不同标准,使得对MS治疗潜在有效的药物的测试极大地复杂化。基于MS是自身免疫疾病,免疫调节剂包括抗炎药可用于治疗或控制MS The United States National Multiple Sclerosis Society standardized definitions of 4 subcategories in 1996: 1) relapsing-remitting, 2) secondary progressive, 3) primary progressive, and 4) progressive recurrent type. For diagnosis and evaluation, different criteria are used, greatly complicating the testing of drugs potentially effective in MS treatment. Based on the fact that MS is an autoimmune disease, immunomodulators including anti-inflammatory drugs can be used to treat or manage MS .

银屑病性关节炎(PSA) Psoriatic Arthritis (PSA)

银屑病性关节炎可通过美国风湿病学会(ACR)等所定义的标准来诊断。当使用所述标准时,对治疗的反应可以基于degrease评分。放射照相损伤的预防或延迟也是PSA治疗的目标。美国风湿病学会(ACR)20%综合改善标准描述了如果在触痛(tender)和肿胀关节计数中存在20%改善和在5个另外的量度(疼痛、身体功能、患者的综合健康评估、医生的综合健康评估和急性期反应物水平)的至少3个中存在20%改善,那么将患者归为“改善的”。类似地,ACR50和ACR70代表甚至更高程度的患者改善。 Psoriatic arthritis can be diagnosed by criteria defined by the American College of Rheumatology (ACR) and others. When using the criteria, response to treatment can be based on a degree score. Prevention or delay of radiographic injury is also a goal of PSA therapy. The American College of Rheumatology (ACR) 20% Composite Improvement Criteria describe if there is a 20% improvement in tender and swollen joint counts and in 5 additional measures (pain, physical function, patient's comprehensive health assessment, physician Patients were classified as "improved" if there was a 20% improvement in at least 3 of the overall health assessment and acute phase reactant levels). Similarly, ACR50 and ACR70 represent an even higher degree of patient improvement.

根据获得ACR20、ACR50和/或ACR70的患者数量或患者部分,抗炎药作为PSA治疗药的有效性可以因此而量化。 The effectiveness of an anti-inflammatory drug as a PSA treatment can thus be quantified in terms of the number or fraction of patients who achieve ACR20, ACR50 and/or ACR70.

除了ACR评分之外,还可使用28关节的疾病活性评分(DAS28)追踪银屑病性关节炎的发展。它是80年代在Nijmegen开发的综合指数,已广泛用作PSA疾病活性和对治疗的反应的指示物以及EULAR反应标准。DAS28中包括的关节是(双侧): 近端指间关节(10关节)、掌指关节(10)、腕(2)、肘(2)、肩(2)和膝(2)。当考虑这些关节时,同时统计触痛(TJC28)和肿胀(SJC28)的关节数量。 In addition to the ACR score, the progression of psoriatic arthritis can be tracked using the Disease Activity Score of 28 joints (DAS28). It is a composite index developed in Nijmegen in the 80s and has been widely used as an indicator of PSA disease activity and response to treatment as well as an EULAR response criterion. The joints included in DAS28 are (bilateral): proximal interphalangeal joint (10 joints), metacarpophalangeal joint (10), wrist (2), elbow (2), shoulder (2), and knee (2). When considering these joints, count both tender (TJC28) and swollen (SJC28) joints.

可以包括C-反应蛋白(CRP)水平(以mg/l计)的测定,并且患者还在前面7天期间在0-100的数值范围上对疾病活性进行主观评价(SA),其中0是“无活性”,100是“最高活性可能”。据此计算DAS28。 Measurement of C-reactive protein (CRP) levels (in mg/l) may be included, and patients also have a subjective assessment (SA) of disease activity on a scale of 0-100 during the previous 7 days, where 0 is " No activity", 100 is the "highest activity possible". Calculate DAS28 accordingly.

使用所述DAS,对于高疾病活性、低疾病活性或甚至缓解,已经开发了若干阈值。所述评分还可用作反应标准,当在两个时间点(例如治疗开始前和治疗后)测定患者的DAS时,可以评价患者的临床反应。 Using the DAS, several thresholds have been developed for high disease activity, low disease activity or even remission. The score can also be used as a response criterion, allowing the patient's clinical response to be assessed when the patient's DAS is measured at two time points (eg, before treatment initiation and after treatment).

皮肤银屑病是PsA的主要方面,尽管皮肤中的活性程度不一定与关节活性相关。已经开发出用于评价皮肤银屑病的多种手段。一种广泛使用的手段是银屑病面积和严重程度指数(PASI)。PASI评价单个银屑病性损伤的红斑、厚度/硬结和范围,然后使用公式来计算所涉及皮肤的体表面积的总范围,其评分范围为0-72。 Cutaneous psoriasis is a major aspect of PsA, although the degree of activity in the skin does not necessarily correlate with joint activity. Various means have been developed for the evaluation of cutaneous psoriasis. One widely used measure is the Psoriasis Area and Severity Index (PASI). PASI evaluates individual psoriatic lesions for erythema, thickness/induration, and extent, and then uses a formula to calculate the total extent of body surface area of the skin involved on a scale of 0-72.

银屑病性关节炎反应标准(PsARC)经专门开发用于PSA临床试验。PsARC由4项测量组成:1)患者疾病活性综合评价(反应需要在5点李克特量表(Likert scale)上改善为1),2)医生疾病活性综合评价(反应需要在5点李克特量表上改善为1),3)关节疼痛(反应需要在总评分上减少30%或以上,评价或68或78个关节,使用4点量表),和4)关节肿胀(反应需要在总评分上减少30%或以上,评价或66或76个关节,使用4点评分量表)。为了成为“PsARC反应者”,患者在4项测量中必须达到2项改善,其中之一必须是关节疼痛或肿胀,其在任何测量中无恶化。 The Psoriatic Arthritis Response Criteria (PsARC) were developed specifically for use in PSA clinical trials. PsARC consists of 4 measures: 1) Patient Comprehensive Assessment of Disease Activity (response needs to improve to 1 on a 5-point Likert scale), 2) Physician Comprehensive Assessment of Disease Activity (response requires improvement to 1 on a 5-point Likert scale). Improvements on specific scales were 1), 3) joint pain (response required a reduction of 30% or more on the total score, assessed or 68 or 78 joints, using a 4-point scale), and 4) joint swelling (response required at A reduction of 30% or more in the total score, evaluating either 66 or 76 joints, using a 4-point scale). In order to be a "PsARC responder," a patient must achieve improvement in 2 of 4 measures, one of which must be joint pain or swelling, without worsening in any measure.

治疗 treat

本发明一方面涉及基于来自本文的实例的信息的治疗方法。预测抗炎药的临床成功的方法随后提供用所述方法鉴定的患者的治疗方法。因为可以容易地进行鉴定满足某一预定标准的患者的方法,其与患者的实际治疗分开(所用的方法不一定包括确定患者满足某些预定标准的步骤,尽管它是本发明的优选的实施方案)。当使用本发明的治疗方法时,预期患者将以高确定度反应,这不是没有预先了解患者满足某一预定标准的事实的情况。 One aspect of the invention relates to methods of treatment based on information from the examples herein. A method of predicting the clinical success of an anti-inflammatory drug then provides a method of treatment for the patients identified by the method. Because the method of identifying patients meeting certain predetermined criteria can be readily performed, separate from the actual treatment of the patient (the method used does not necessarily include the step of determining that the patient meets certain predetermined criteria, although it is a preferred embodiment of the present invention ). When using the treatment methods of the present invention, it is expected that the patient will respond with a high degree of certainty, which is not the case without prior knowledge of the fact that the patient meets some predetermined criteria.

本发明的一个实施方案涉及在患者中治疗炎性疾病或病症的方法,其中与参考水平相比图1的一个或多个基因的表达水平被改变,所述方法包括将治疗量的抗炎药给予所述患者。 One embodiment of the invention relates to a method of treating an inflammatory disease or condition in a patient, wherein the expression level of one or more genes of Figure 1 is altered compared to a reference level, the method comprising administering a therapeutic amount of an anti-inflammatory drug administered to the patient.

再一个实施方案涉及在患者中治疗炎性疾病或病症的方法: Yet another embodiment relates to a method of treating an inflammatory disease or condition in a patient:

a. 与参考水平相比,在所述患者中考虑图1的一个或多个基因的表达水平是否被改变, a. Consider whether the expression level of one or more genes of Figure 1 is altered in said patient compared to a reference level,

b. 包括将治疗量的抗炎药给予所述患者。 b. comprising administering a therapeutic amount of an anti-inflammatory drug to said patient.

此外,在一个实施方案中,本发明涉及在患者中治疗炎性疾病或病症的方法 Furthermore, in one embodiment, the present invention relates to a method of treating an inflammatory disease or disorder in a patient

a. 在来自所述患者的生物样品中测定与参考水平相比,图1的一个或多个基因的表达水平是否被改变, a. determining in a biological sample from said patient whether the expression level of one or more genes of Figure 1 is altered compared to a reference level,

b. 包括将治疗量的抗炎药给予所述患者。 b. comprising administering a therapeutic amount of an anti-inflammatory drug to said patient.

甚至进一步,在一个实施方案中,本发明涉及在患者中治疗炎性疾病或病症的方法,包括: Even further, in one embodiment, the invention relates to a method of treating an inflammatory disease or condition in a patient comprising:

a. 在来自所述患者的生物样品中测定图1的一个或多个基因的表达水平 a. Determining the expression level of one or more genes of Figure 1 in a biological sample from said patient

b. 将所述水平与所述基因的参考水平比较, b. comparing said level to a reference level for said gene,

c. 判定与所述参考水平相比,图1的一个或多个基因的表达水平是否被改变 c. Determine whether the expression level of one or more genes of Figure 1 is altered compared to the reference level

d. 将治疗量的抗炎药给予所述患者。 d. Administering a therapeutic amount of an anti-inflammatory drug to the patient.

在上述方法的替代方案中,所述参考水平可以是预定水平。 In an alternative to the above method, the reference level may be a predetermined level.

在另外的实施方案中,所述方法的每一种可包括与所述参考水平相比,在所述生物样品中图1的一个或多个基因的表达水平被改变。 In additional embodiments, each of said methods may comprise the expression level of one or more genes of Figure 1 being altered in said biological sample compared to said reference level.

参考本文以上的描述,包括预测方法的更详细的信息,其涉及本发明的治疗方法。 Reference is made to the description herein above, including more detailed information on methods of prediction, which relate to the methods of treatment of the present invention.

本发明一方面涉及在受试者中治疗炎性疾病或病症的抗炎药,其中所述受试者表现出与图1的一个或多个基因的参考水平相比,图1的一个或多个基因的表达改变。 One aspect of the invention pertains to anti-inflammatory agents for the treatment of an inflammatory disease or condition in a subject, wherein the subject exhibits one or more of the genes of Figure 1 compared to a reference level of one or more of the genes of Figure 1 . Altered gene expression.

对于治疗方法,参考本文以上的描述,包括预测或鉴定方法的更详细信息,其明显地同样涉及定义抗炎药的特征及其药学用途。 For methods of treatment, reference is made to the description herein above, including more detailed information on methods of prediction or identification, which obviously also relate to defining the characteristics of anti-inflammatory drugs and their pharmaceutical use.

制品 products

本发明在另一方面涉及制品,包括包装在一起的药物组合物和标签,所述药物组合物包含抗炎药和药学上可接受的载体,所述标签说明所述药物组合物用于治疗患有自身免疫疾病或病症并具有图1的一个或多个基因的表达改变的患者。 In another aspect the present invention relates to an article of manufacture comprising a packaged pharmaceutical composition comprising an anti-inflammatory agent and a pharmaceutically acceptable carrier and a label stating that the pharmaceutical composition is used to treat a patient A patient with an autoimmune disease or disorder having altered expression of one or more genes of Figure 1 .

参考本文以上的描述,包括预测方法的更详细的信息,其涉及本发明的制品。 Reference is made to the description herein above, including more detailed information on methods of prediction, which relate to the articles of manufacture of the present invention.

检测试剂和试剂盒 Assay Reagents and Kits

本发明进一步涉及组合物,包括至少一种检测试剂,用于测定图1的一个或多个基因、尤其是图2的一个或多个基因和尤其是CFD的表达水平。所述检测试剂可以是对每个基因包括mRNA及其所编码的蛋白具有特异性的抗体、探针或引物。 The invention further relates to a composition comprising at least one detection reagent for determining the expression level of one or more genes of figure 1, especially of one or more genes of figure 2 and especially of CFD. The detection reagents may be antibodies, probes or primers specific to each gene including mRNA and its encoded protein.

本发明另一方面涉及试剂盒,其包括如上所述的检测试剂或包含所述检测试剂的组合物和使用说明书。在使用内部对照的情况下,试剂盒可以进一步包含参考基因组合物。所述试剂盒还可包含用于表达标准化的检测试剂,所述检测试剂用于检测球蛋白基因。本发明的进一步的部分包括关于如何将表达水平与对本文所述的抗炎药的反应可能性关联的描述。尤其是考虑到试剂盒,其用于检测补体因子D (CFD)的表达水平及其评价。 Another aspect of the present invention relates to a kit, which includes the above-mentioned detection reagent or a composition comprising the detection reagent and instructions for use. Where internal controls are used, the kit may further comprise a reference genetic composition. The kit may further comprise detection reagents for expression normalization, the detection reagents being used for the detection of globin genes. A further part of the invention includes a description of how to correlate expression levels with likelihood of response to the anti-inflammatory drugs described herein. Especially considering the kit for detecting the expression level of complement factor D (CFD) and its evaluation.

治疗靶标 therapeutic target

抗炎药是炎性疾病或病症的表型所需途径的调节剂。根据本文的数据,显而易见的是,所选基因可被单独考虑为迄今为止用于治疗炎性疾病或病症的新的治疗靶标,因为对于自身免疫疾病和尤其是RA,先前未描述过它们。 Anti-inflammatory drugs are modulators of pathways required for the phenotype of an inflammatory disease or disorder. From the data herein, it is evident that the selected genes may individually be considered as novel therapeutic targets for the treatment of inflammatory diseases or disorders to date, since they have not been described previously for autoimmune diseases and especially RA.

实施方案 implementation plan

如本文所述的本发明概述于、但不限于以下实施方案。 The invention as described herein is summarized in, but not limited to, the following embodiments.

1. 预测受试者对抗炎药的反应的方法,包括:在来自所述受试者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述基因的参考水平相比的一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 1. A method for predicting a subject's response to an anti-inflammatory drug, comprising: obtaining information on the expression level of one or more genes of Figure 1 in a biological sample from said subject, wherein the reference to said gene The change in the expression level of one or more of the genes compared to the level predicted the subject's response to the anti-inflammatory drug.

2. 预测患者对抗炎药的反应的方法,包括: 2. Methods for predicting patient response to anti-inflammatory drugs, including:

a. 在来自所述患者的生物样品中检测图1的一个或多个基因的表达水平,和 a. detecting the expression level of one or more genes of Figure 1 in a biological sample from said patient, and

b. 将所述水平与所述基因的参考水平比较 b. Comparing said level to a reference level for said gene

其中与所述参考水平相比的一个或多个所述基因的表达改变,预测了所述患者对所述抗炎药的反应。 wherein the altered expression of one or more of said genes compared to said reference level predicts said patient's response to said anti-inflammatory drug.

3. 鉴定对抗炎药的反应具有增加的可能性的受试者的方法,包括:在来自所述受试者的生物样品中获得图1的一个或多个基因的表达水平的信息,其中与所述基因的参考水平相比的一个或多个所述基因的表达水平改变,表明已经鉴定了对抗炎药的反应具有增加的可能性的受试者。 3. A method of identifying a subject with an increased likelihood of responding to an anti-inflammatory drug, comprising: obtaining information on the expression level of one or more genes of Figure 1 in a biological sample from said subject, wherein An altered expression level of one or more of the genes compared to a reference level of the gene indicates that a subject with an increased likelihood of responding to an anti-inflammatory drug has been identified.

4. 鉴定对抗炎药的反应具有增加的可能性的患者的方法,包括: 4. A method of identifying patients with an increased likelihood of responding to anti-inflammatory drugs, comprising:

a. 在来自所述患者的生物样品中检测图1的一个或多个基因的表达水平 a. Detecting the expression level of one or more genes of Figure 1 in a biological sample from said patient

b. 将所述表达水平与所述基因的参考水平比较, b. comparing said expression level to a reference level for said gene,

其中与所述基因的参考水平相比的一个或多个所述基因的表达改变,表明已经鉴定了对抗炎药的反应具有增加的可能性的患者。 Wherein expression of one or more of said genes is altered compared to a reference level of said genes, a patient having an increased likelihood of responding to an anti-inflammatory drug has been identified.

5. 先前实施方案中任一项的方法,其中在生物样品中测定补体因子D (CFD)的表达。 5. The method of any one of the previous embodiments, wherein the expression of complement factor D (CFD) is determined in the biological sample.

6. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B, 成员9 (SERPINB9)的表达。 6. The method of any one of embodiments 1-4, wherein expression of complement factor D (CFD) and serpin peptidase inhibitor, clade B, member 9 (SERPINB9) is determined in the biological sample.

7. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B, 成员9 (SERPINB9)和/或锌指, CCHC域含有24 (ZCCHC24)的表达。 7. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or serpin peptidase inhibitor, clade B, member 9 (SERPINB9) and/or are assayed in the biological sample or zinc finger, expression of CCHC domain containing 24 (ZCCHC24).

8. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或果糖胺3激酶相关蛋白(FN3KRP)和/或间质同源框1 (MEOX1)的表达。 8. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or fructosamine 3-kinase-related protein (FN3KRP) and/or mesenchymal homeobox 1 (MEOX1) are determined in the biological sample )expression.

9. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B, 成员9 (SERPINB9)和/或锌指, CCHC域含有24 (ZCCHC24)和/或果糖胺3激酶相关蛋白(FN3KRP)的表达。 9. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or serpin peptidase inhibitor, clade B, member 9 (SERPINB9) and/or are assayed in the biological sample or zinc finger, CCHC domain containing 24 (ZCCHC24) and/or expression of fructosamine 3-kinase-related protein (FN3KRP).

10. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B, 成员9 (SERPINB9)和/或锌指, CCHC域含有24 (ZCCHC24)和/或果糖胺3激酶相关蛋白(FN3KRP)和/或间质同源框1 (MEOX1)的表达。 10. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or serpin peptidase inhibitor, clade B, member 9 (SERPINB9) and/or are assayed in the biological sample or zinc finger, CCHC domain containing 24 (ZCCHC24) and/or expression of fructosamine 3-kinase-related protein (FN3KRP) and/or mesenchymal homeobox 1 (MEOX1).

11. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或成纤维细胞生长因子13 (FGF13)和/或微管蛋白, β2A (TUBB2A)和/或溶质载体家族39 (金属离子转运蛋白, 成员11) (SLC39A11)和/或跨膜通道-样4 (TMC4)的表达。 11. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or fibroblast growth factor 13 (FGF13) and/or tubulin, beta 2A (TUBB2A) are determined in the biological sample and/or expression of solute carrier family 39 (metal ion transporters, member 11) (SLC39A11) and/or transmembrane channel-like 4 (TMC4).

12. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或核孔复合物相互作用蛋白-样2 (NPIPL2)和/或锌指蛋白880 (ZNF880)和/或醛脱氢酶5家族, 成员A1 (ALDH5A1)的表达。 12. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or nuclear pore complex interacting protein-like 2 (NPIPL2) and/or zinc finger protein 880 are assayed in the biological sample (ZNF880) and/or expression of aldehyde dehydrogenase family 5, member A1 (ALDH5A1).

13. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或成纤维细胞生长因子13 (FGF13)和/或微管蛋白, β2A (TUBB2A)和/或溶质载体家族39 (金属离子转运蛋白, 成员11) (SLC39A11)和/或跨膜通道-样4 (TMC4)和/或核孔复合物相互作用蛋白-样2 (NPIPL2)和/或锌指蛋白880 (ZNF880)和/或醛脱氢酶5家族, 成员A1 (ALDH5A1)的表达。 13. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or fibroblast growth factor 13 (FGF13) and/or tubulin, β2A (TUBB2A) are determined in the biological sample and/or solute carrier family 39 (metal ion transporter, member 11) (SLC39A11) and/or transmembrane channel-like 4 (TMC4) and/or nuclear pore complex interacting protein-like 2 (NPIPL2) and/or Expression of zinc finger protein 880 (ZNF880) and/or aldehyde dehydrogenase family 5, member A1 (ALDH5A1).

14. 实施方案1-4中任一项的方法,其中在生物样品中测定了补体因子D (CFD)和/或丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B, 成员9 (SERPINB9)和/或锌指, CCHC域含有24 (ZCCHC24)和/或果糖胺3激酶相关蛋白(FN3KRP)和/或间质同源框1 (MEOX1)和/或成纤维细胞生长因子13 (FGF13)和/或微管蛋白, β2A (TUBB2A)和/或溶质载体家族39 (金属离子转运蛋白, 成员11) (SLC39A11)和/或跨膜通道-样4 (TMC4)和/或核孔复合物相互作用蛋白-样2 (NPIPL2)和/或锌指蛋白880 (ZNF880)和/或醛脱氢酶5家族, 成员A1 (ALDH5A1)的表达。 14. The method of any one of embodiments 1-4, wherein complement factor D (CFD) and/or serpin peptidase inhibitor, clade B, member 9 (SERPINB9) and/or are assayed in the biological sample or zinc finger, CCHC domain containing 24 (ZCCHC24) and/or fructosamine 3-kinase-related protein (FN3KRP) and/or mesenchymal homeobox 1 (MEOX1) and/or fibroblast growth factor 13 (FGF13) and/or Tubulin, β2A (TUBB2A) and/or solute carrier family 39 (metal ion transporter, member 11) (SLC39A11) and/or transmembrane channel-like 4 (TMC4) and/or nuclear pore complex interacting protein- Expression of 2 (NPIPL2) and/or zinc finger protein 880 (ZNF880) and/or aldehyde dehydrogenase family 5, member A1 (ALDH5A1).

15. 前述实施方案中任一项的方法,其中与参考水平相比,图1A的基因的表达改变是增加。 15. The method of any one of the preceding embodiments, wherein the change in expression of the gene of Figure 1A is an increase compared to a reference level.

16. 前述实施方案中任一项的方法,其中与参考水平相比,图1B的基因的表达改变是降低。 16. The method of any one of the preceding embodiments, wherein the altered expression of the gene of Figure 1B is a decrease compared to a reference level.

17. 前述实施方案中任一项的方法,其中将至少两个基因的表达水平与所述至少两个基因的个体参考水平比较。 17. The method of any one of the preceding embodiments, wherein the expression levels of at least two genes are compared to individual reference levels of said at least two genes.

18. 前述实施方案中任一项的方法,其中将至少两个基因的表达水平与所述至少两个基因的个体参考水平比较,并且其中与参考水平相比图1A的基因的表达改变是增加,并且其中与参考水平相比图1B的基因的表达改变是降低。 18. The method of any one of the preceding embodiments, wherein the expression level of at least two genes is compared with an individual reference level of said at least two genes, and wherein the change in expression of the gene of Figure 1A is an increase compared to the reference level , and wherein the change in expression of the gene of FIG. 1B is a decrease compared to the reference level.

19. 前述实施方案中任一项的方法,其中所述参考水平是预定水平。 19. The method of any one of the preceding embodiments, wherein the reference level is a predetermined level.

20. 前述实施方案中任一项的方法,其中所述预定水平是指示使用DAS28-CRP、ACR20、ACR50和/或ACR70测量的反应的阈值。 20. The method of any one of the preceding embodiments, wherein the predetermined level is a threshold indicative of a response measured using DAS28-CRP, ACR20, ACR50 and/or ACR70.

21. 前述实施方案中任一项的方法,其中所述生物样品是血液样品或血清样品。 21. The method of any one of the preceding embodiments, wherein the biological sample is a blood sample or a serum sample.

22. 前述实施方案中任一项的方法,其中所述生物样品是Paxgene全血样品。 22. The method of any one of the preceding embodiments, wherein the biological sample is a Paxgene whole blood sample.

23. 前述实施方案中任一项的方法,其中所述生物样品是来自血液样品的PBMC部分。 23. The method of any one of the preceding embodiments, wherein the biological sample is a PBMC fraction from a blood sample.

24. 前述实施方案中任一项的方法,其中所述生物样品是来自血液样品的细胞亚组。 24. The method of any one of the preceding embodiments, wherein the biological sample is a subset of cells from a blood sample.

25. 前述实施方案中任一项的方法,其中所述生物样品是来自血液样品的细胞亚组,其中在所述亚组中可以是CD14+、CD4+和CD8+阳性细胞中的一种或多种。 25. The method of any one of the preceding embodiments, wherein said biological sample is a subset of cells from a blood sample, wherein in said subset may be one or more of CD14+, CD4+ and CD8+ positive cells.

26. 前述实施方案中任一项的方法,其中基于mRNA而测定所述表达水平。 26. The method of any one of the preceding embodiments, wherein said expression level is determined based on mRNA.

27. 前述实施方案中任一项的方法,其中使用PCR来测定所述表达水平。 27. The method of any one of the preceding embodiments, wherein PCR is used to determine said expression level.

28. 前述实施方案中任一项的方法,其中所述PCR选自多重PCR和qRT-PCR。 28. The method of any one of the preceding embodiments, wherein the PCR is selected from multiplex PCR and qRT-PCR.

29. 前述实施方案中任一项的方法,其中使用微阵列芯片测定所述表达水平。 29. The method of any one of the preceding embodiments, wherein the expression level is determined using a microarray chip.

30. 前述实施方案中任一项的方法,其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中使用Assay ID: Hs00157263_m1 (Applied Biosystems)测定转录物的循环阈值(Ct)为30。 30. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the cycle threshold (Ct) of transcripts determined using Assay ID: Hs00157263_m1 (Applied Biosystems) is 30.

31. 前述实施方案中任一项的方法,其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中使用CFD的Assay ID: Hs00157263_m1 (Applied Biosystems/ Invitrogen)和ACTB的Assay ID: Hs99999903_m1 (Applied Biosystems/ Invitrogen)检测转录物的绝对数为至少0.03个CFD拷贝/β-肌动蛋白mRNA (基因符号ACTB)拷贝。 31. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the Assay ID of CFD: Hs00157263_m1 (Applied Biosystems/Invitrogen) and ACTB: Hs99999903_m1 ( Applied Biosystems/Invitrogen) detected an absolute number of transcripts of at least 0.03 CFD copies/β-actin mRNA (gene symbol ACTB) copies.

32. 前述实施方案中任一项的方法,其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中使用CFD的Assay ID: Hs00157263_m1 (Applied Biosystems/ Invitrogen)和ACTB的Assay ID: Hs99999903_m1 (Applied Biosystems/ Invitrogen)检测转录物的绝对数为至少0.04个CFD拷贝/β-肌动蛋白mRNA (基因符号ACTB)拷贝。 32. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the Assay ID of CFD: Hs00157263_m1 (Applied Biosystems/Invitrogen) and ACTB: Hs99999903_m1 ( Applied Biosystems/Invitrogen) detected the absolute number of transcripts to be at least 0.04 CFD copy/β-actin mRNA (gene symbol ACTB) copy.

33. 前述实施方案中任一项的方法,其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中使用CFD的Assay ID: Hs00157263_m1 (Applied Biosystems/ Invitrogen)和ACTB的Assay ID: Hs99999903_m1 (Applied Biosystems/ Invitrogen)检测转录物的绝对数为至少0.05个CFD拷贝/β-肌动蛋白mRNA (基因符号ACTB)拷贝。 33. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the Assay ID of CFD: Hs00157263_m1 (Applied Biosystems/Invitrogen) and ACTB: Hs99999903_m1 ( Applied Biosystems/Invitrogen) detected the absolute number of transcripts to be at least 0.05 CFD copy/β-actin mRNA (gene symbol ACTB) copy.

34. 前述实施方案中任一项的方法,其中通过qRT-PCR测定补体因子D (CFD)的表达水平和其中使用CFD的Assay ID: Hs00157263_m1 (Applied Biosystems/ Invitrogen)和ACTB的Assay ID: Hs99999903_m1 (Applied Biosystems/ Invitrogen)检测转录物的绝对数为至少0.06个CFD拷贝/β-肌动蛋白mRNA (基因符号ACTB)拷贝。 34. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined by qRT-PCR and wherein the Assay ID of CFD: Hs00157263_m1 (Applied Biosystems/Invitrogen) and ACTB: Hs99999903_m1 ( Applied Biosystems/Invitrogen) detected the absolute number of transcripts to be at least 0.06 CFD copy/β-actin mRNA (gene symbol ACTB) copy.

35. 前述实施方案中任一项的方法,其中当使用微阵列芯片测定时,补体因子D (CFD)的表达水平在RMA或GC-RMA标准化表达值的log2标度上超过9.5。 35. The method of any one of the preceding embodiments, wherein when using a microarray chip assay, the expression level of complement factor D (CFD) exceeds 9.5 on the log2 scale of RMA or GC-RMA normalized expression values.

36. 前述实施方案中任一项的方法,其中根据一个或多个SNP间接测定所述表达水平。 36. The method of any one of the preceding embodiments, wherein said expression level is determined indirectly from one or more SNPs.

37. 前述实施方案中任一项的方法,其中根据一个或多个CFD表达相关的SNP间接测定补体因子D (CFD)的表达水平。 37. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs associated with CFD expression.

38. 前述实施方案中任一项的方法,其中根据在CFD单倍体块(haploblock)中的一个或多个SNP间接测定补体因子D (CFD)的表达水平。 38. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs in the CFD haploblock.

39. 前述实施方案中任一项的方法,其中根据选自以下的一个或多个SNP间接测定补体因子D (CFD)的表达水平:rs1683565、rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898。 39. The method of any one of the preceding embodiments, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs selected from the group consisting of: rs1683565, rs1683591, rs1683590, rs1683569, rs1683574, rs1651888, rs2930894, rs2930891 , rs4417648, rs1651891, rs1651890 and rs2930898.

40. 前述实施方案中任一项的方法,其中通过SNP rs1683591的AG或GG基因型的存在而间接测定CFD的表达水平。 40. The method of any one of the preceding embodiments, wherein the expression level of CFD is determined indirectly by the presence of the AG or GG genotype of SNP rs1683591.

41. 前述实施方案1-25中任一项的方法,其中在蛋白质水平上测定所述表达水平。 41. The method of any one of the preceding embodiments 1-25, wherein said expression level is determined at the protein level.

42. 实施方案41的方法,其中使用抗体测定所述表达水平。 42. The method of embodiment 41, wherein said expression level is determined using an antibody.

43. 实施方案41的方法,其中使用蛋白质组分析测定所述表达水平。 43. The method of embodiment 41, wherein said expression level is determined using proteomic analysis.

44. 前述实施方案中任一项的方法,其中所述受试者或患者是患有炎性疾病或病症的患者。 44. The method of any one of the preceding embodiments, wherein the subject or patient is a patient suffering from an inflammatory disease or disorder.

45. 前述实施方案中任一项的方法,其中所述患者患有自身免疫疾病或病症。 45. The method of any one of the preceding embodiments, wherein the patient suffers from an autoimmune disease or disorder.

46. 实施方案44或45的方法,其中所述患者患有类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、多发性硬化(MS)、炎性肠病(IBD)、银屑病性关节炎(PSA)或银屑病性关节炎。 46. The method of embodiment 44 or 45, wherein the patient suffers from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), psoriasis Psoriatic arthritis (PSA) or psoriatic arthritis.

47. 实施方案46的方法,其中所述患者患有RA。 47. The method of embodiment 46, wherein the patient has RA.

48. 实施方案44-47中任一项的方法,其中所述患者正在接受或已经接受MTX治疗。 48. The method according to any one of embodiments 44-47, wherein said patient is receiving or has received MTX therapy.

49. 实施方案44-48中任一项的方法,其中所述患者是对MTX治疗的不充分反应者。 49. The method of any one of embodiments 44-48, wherein the patient is an inadequate responder to MTX treatment.

50. 前述实施方案44-49中任一项的方法,其中所述患者正在接受TNF-α抑制剂治疗。 50. The method according to any one of the preceding embodiments 44-49, wherein said patient is being treated with a TNF-alpha inhibitor.

51. 实施方案44-50中任一项的方法,其中所述患者从未接受TNF-α抑制剂治疗。 51. The method according to any one of embodiments 44-50, wherein said patient has never received TNF-alpha inhibitor treatment.

52. 实施方案44-51中任一项的方法,其中所述患者是对TNF-α抑制剂治疗的不充分反应者。 52. The method according to any one of embodiments 44-51, wherein said patient is an inadequate responder to TNF-alpha inhibitor therapy.

53. 实施方案44-52中任一项的方法,其中所述患者是对用于所述炎性疾病或病症的一种或多种治疗的不充分反应者。 53. The method according to any one of embodiments 44-52, wherein said patient is an inadequate responder to one or more treatments for said inflammatory disease or disorder.

54. 实施方案44-53中任一项的方法,其中所述患者是对MTX和TNF-α抑制剂治疗的不充分反应者。 54. The method according to any one of embodiments 44-53, wherein the patient is an inadequate responder to treatment with MTX and TNF-alpha inhibitors.

55. 实施方案44-54中任一项的方法,其中所述患者是RF阳性。 55. The method of any one of embodiments 44-54, wherein the patient is RF positive.

56. 实施方案44-55中任一项的方法,其中所述患者是RF阴性。 56. The method according to any one of embodiments 44-55, wherein said patient is RF negative.

57. 前述实施方案中任一项的方法,其中所述抗炎药是抗体。 57. The method of any one of the preceding embodiments, wherein the anti-inflammatory drug is an antibody.

58. 前述实施方案中任一项的方法,其中所述抗炎药是受体拮抗剂。 58. The method according to any one of the preceding embodiments, wherein the anti-inflammatory agent is a receptor antagonist.

59. 前述实施方案中任一项的方法,其中所述抗炎药是IL-10家族的一个或多个成员的拮抗剂。 59. The method according to any one of the preceding embodiments, wherein the anti-inflammatory agent is an antagonist of one or more members of the IL-10 family.

60. 前述实施方案中任一项的方法,其中所述抗炎药是IL-10、IL19、IL-20、IL-22、IL-24和IL-26中的一种或多种的拮抗剂。 60. The method of any one of the preceding embodiments, wherein the anti-inflammatory agent is an antagonist of one or more of IL-10, IL19, IL-20, IL-22, IL-24 and IL-26 .

61. 前述实施方案中任一项的方法,其中所述抗炎药是IL-19、IL-20和IL-24中的一种或多种的拮抗剂。 61. The method of any one of the preceding embodiments, wherein the anti-inflammatory drug is an antagonist of one or more of IL-19, IL-20 and IL-24.

62. 前述实施方案中任一项的方法,其中所述抗炎药是IL-20的拮抗剂。 62. The method according to any one of the preceding embodiments, wherein the anti-inflammatory drug is an antagonist of IL-20.

63. 前述实施方案中任一项的方法,其中所述抗炎药是IL-20的拮抗剂,其降低IL-20介导的(medicated) IL-20R1/IL-20R2和IL-22R/IL-20R2受体两者的活化。 63. The method of any one of the preceding embodiments, wherein the anti-inflammatory agent is an antagonist of IL-20, which reduces IL-20-mediated (mediated) IL-20R1/IL-20R2 and IL-22R/IL - Activation of both 20R2 receptors.

64. 前述实施方案中任一项的方法,其中所述抗炎药是IL-20的拮抗剂,其降低IL-20介导的IL-20R1/IL-20R2和IL-22R/IL-20R2受体两者的活化,而不是IL19或IL24介导的受体活化。 64. The method of any one of the preceding embodiments, wherein the anti-inflammatory agent is an antagonist of IL-20, which reduces IL-20-mediated IL-20R1/IL-20R2 and IL-22R/IL-20R2 receptors. activation of both, but not IL19- or IL24-mediated receptor activation.

65. 前述实施方案中任一项的方法,其中所述抗炎药是抗人IL-20抗体。 65. The method of any one of the preceding embodiments, wherein the anti-inflammatory agent is an anti-human IL-20 antibody.

66. 在受试者中治疗炎性疾病或病症的方法,在所述受试者中与参考水平相比图1的一个或多个基因的表达水平被改变,所述方法包括将治疗量的抗炎药给予所述受试者。 66. A method of treating an inflammatory disease or condition in a subject in which the expression level of one or more genes of Figure 1 is altered compared to a reference level, the method comprising administering a therapeutic amount of Anti-inflammatory drugs are administered to the subject.

67. 在患者中治疗炎性疾病或病症的方法,包括: 67. A method of treating an inflammatory disease or condition in a patient, comprising:

a. 在所述患者中考虑与参考水平相比,图1的一个或多个基因的表达水平是否被改变, a. Consider whether the expression level of one or more genes of Figure 1 is altered compared to the reference level in said patient,

b. 将治疗量的抗炎药给予所述患者。 b. Administering a therapeutic amount of an anti-inflammatory drug to the patient.

68. 在患者中治疗炎性疾病或病症的方法,包括: 68. A method of treating an inflammatory disease or condition in a patient, comprising:

a. 在来自所述患者的生物样品中测定与参考水平相比,图1的一个或多个基因的表达水平是否被改变, a. determining in a biological sample from said patient whether the expression level of one or more genes of Figure 1 is altered compared to a reference level,

b. 将治疗量的抗炎药给予所述患者。 b. Administering a therapeutic amount of an anti-inflammatory drug to the patient.

69. 在患者中治疗炎性疾病或病症的方法,包括: 69. A method of treating an inflammatory disease or condition in a patient, comprising:

a. 在来自所述患者的生物样品中测定图1的一个或多个基因的表达水平, a. determining the expression level of one or more genes of Figure 1 in a biological sample from said patient,

b. 将所述水平与所述基因的参考水平比较, b. comparing said level to a reference level for said gene,

c. 测定与所述参考水平相比,图1的一个或多个基因的表达水平是否被改变,和 c. determining whether the expression level of one or more genes of Figure 1 is altered compared to said reference level, and

d. 将治疗量的抗炎药给予所述患者。 d. Administering a therapeutic amount of an anti-inflammatory drug to the patient.

70. 前述实施方案68-69中任一项的方法,其中与所述参考水平相比,所述生物样品中图1的一个或多个基因的表达水平被改变。 70. The method of any one of the preceding embodiments 68-69, wherein the expression level of one or more genes of Figure 1 in the biological sample is altered compared to the reference level.

71. 前述实施方案68-70中任一项的方法,其中所述方法的特征在于前述实施方案15-65的特征中的任何一种或多种。 71. The method of any one of the preceding embodiments 68-70, wherein the method is characterized by any one or more of the features of the preceding embodiments 15-65.

72. 制品,包括包装在一起的药物组合物和标签,所述药物组合物包含抗炎药和药学上可接受的载体,所述标签说明所述药物组合物用于治疗患有自身免疫疾病或病症并具有图1的一个或多个基因的表达改变的患者。 72. An article of manufacture comprising a packaged pharmaceutical composition comprising an anti-inflammatory agent and a pharmaceutically acceptable carrier and a label stating that the pharmaceutical composition is used for the treatment of patients with an autoimmune disease or A patient with a disorder and having altered expression of one or more of the genes of Figure 1.

73. 实施方案72的制品,其中所述制品的特征在于前述实施方案5-46的特征中的任何一种或多种。 73. The article of embodiment 72, wherein the article is characterized by any one or more of the features of the preceding embodiments 5-46.

74. 在受试者中治疗炎性疾病或病症的抗炎药,其中所述受试者表现出与所述基因的参考水平相比,图1的一个或多个基因的表达改变。 74. An anti-inflammatory agent for treating an inflammatory disease or condition in a subject, wherein said subject exhibits altered expression of one or more genes of Figure 1 as compared to a reference level of said gene.

75. 实施方案74的抗炎药,特征在于前述实施方案15-65的特征中的任何一种或多种。 75. The anti-inflammatory agent of embodiment 74, characterized by any one or more of the features of the preceding embodiments 15-65.

76. 组合物,包括至少一种检测试剂,其用于测定表1的一个或多个基因的表达水平。 76. A composition comprising at least one detection reagent for determining the expression level of one or more genes of Table 1.

77. 实施方案76的组合物,其中所述检测试剂用于测定补体因子D (CFD)的表达。 77. The composition of embodiment 76, wherein the detection reagent is used to determine the expression of complement factor D (CFD).

78. 实施方案76的组合物,其中所述检测试剂是CFD探针。 78. The composition of embodiment 76, wherein the detection reagent is a CFD probe.

79. 实施方案76的组合物,其中所述检测试剂是CFD引物。 79. The composition of embodiment 76, wherein the detection reagent is a CFD primer.

80. 实施方案76的组合物,其中所述检测试剂是用于检测CFD表达相关的SNP的引物。 80. The composition of embodiment 76, wherein the detection reagent is a primer for detecting a SNP associated with CFD expression.

81. 实施方案76的组合物,其中所述检测试剂是用于检测选自以下的CFD表达相关的SNP的一种或多种引物:rs1683565、rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898。 81. The composition of embodiment 76, wherein the detection reagent is one or more primers for detecting a SNP associated with CFD expression selected from the group consisting of: rs1683565, rs1683591, rs1683590, rs1683569, rs1683574, rs1651888, rs2930894, rs2930891 , rs4417648, rs1651891, rs1651890 and rs2930898.

82. 试剂盒,包括:实施方案76-81中任一项的组合物和使用说明书。 82. A kit comprising: the composition of any one of embodiments 76-81 and instructions for use.

83. 实施方案82的试剂盒,进一步包括参考样品。 83. The kit of embodiment 82, further comprising a reference sample.

84. 实施方案82-83的试剂盒,进一步包括用于标准化的检测试剂。 84. The kit of embodiments 82-83, further comprising detection reagents for normalization.

85. 实施方案82-84的试剂盒,其中所述使用说明书包括关于如何将表达水平与反应可能性关联的描述。 85. The kit of embodiments 82-84, wherein the instructions for use include a description of how to correlate expression levels with likelihood of response.

86. 实施方案82-85的试剂盒,其中所述试剂盒用于测定患者对抗炎药将具有反应的可能性。 86. The kit of embodiments 82-85, wherein the kit is used to determine the likelihood that a patient will respond to an anti-inflammatory drug.

87. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 87. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administration of said anti-inflammatory drug, at least one test has shown , the expression level of one or more genes of Figure 1 is altered in a biological sample from said patient.

88. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 88. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administration of said anti-inflammatory drug, at least one test has shown , the expression level of one or more genes of FIG. 1 is altered in a biological sample from the patient; and wherein the expression level of one or more of the genes is altered compared to a reference level of the gene, predicting the The experimenter's response to the anti-inflammatory drug.

89. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 89. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering said anti-inflammatory drug, it has been determined that the The expression level of one or more genes of Figure 1 is altered in a biological sample from the patient.

90. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 90. A method of treating an inflammatory disease or condition in a patient, comprising administering to the patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering the anti-inflammatory drug, it has been determined that the The expression level of one or more genes of Figure 1 is altered in the biological sample of the patient; and wherein the expression level of one or more of the genes is altered compared to a reference level of the gene, predicting that the subject Response to the anti-inflammatory drugs.

91. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,实施方案1-43中任一项的至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 91. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering said anti-inflammatory drug, at least one of any of embodiments 1-43 Such tests have shown that the expression level of one or more genes of Figure 1 is altered in a biological sample from said patient compared to a reference level.

92.  92.

93. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,实施方案1-43中任一项的至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 93. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering said anti-inflammatory drug, at least one of any one of embodiments 1-43 The test has shown that the expression level of one or more genes of Figure 1 is altered in a biological sample from the patient compared to a reference level; and wherein one or more of the genes is compared to a reference level of the gene The change in the expression level of the gene predicts the response of the subject to the anti-inflammatory drug.

94. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,实施方案1-43中任一项已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 94. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein any one of embodiments 1-43 has determined The expression level of one or more genes of Figure 1 is altered in a biological sample from said patient compared to a reference level.

95. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前实施方案1-43中任一项已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 95. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein any one of embodiments 1-43 has been determined to be associated with In the biological sample from said patient, the expression level of one or more genes of Figure 1 is altered compared to a reference level; and wherein the expression level of one or more of said genes is altered compared to the reference level of said gene , predicting the subject's response to the anti-inflammatory drug.

96. 实施方案87-94中任一项的方法,其中所述受试者或患者是患有炎性疾病或病症的患者。 96. The method of any one of embodiments 87-94, wherein the subject or patient is a patient suffering from an inflammatory disease or disorder.

97. 实施方案87-94中任一项的方法,其中所述患者患有自身免疫疾病或病症。 97. The method of any one of embodiments 87-94, wherein the patient suffers from an autoimmune disease or disorder.

98. 实施方案87-96中任一项的方法,其中所述患者患有类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、多发性硬化(MS)、炎性肠病(IBD)、银屑病性关节炎(PSA)或银屑病性关节炎。 98. The method of any one of embodiments 87-96, wherein the patient suffers from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD ), psoriatic arthritis (PSA), or psoriatic arthritis.

99. 实施方案97的方法,其中所述患者患有RA。 99. The method of embodiment 97, wherein the patient has RA.

100. 实施方案87-98的方法,其中所述患者正在接受或已经接受MTX治疗。 100. The method of embodiments 87-98, wherein the patient is receiving or has received MTX therapy.

101. 实施方案87-99的方法,其中所述患者是对MTX治疗的不充分反应者。 101. The method of embodiments 87-99, wherein the patient is an inadequate responder to MTX treatment.

102. 前述实施方案87-100中任一项的方法,其中所述患者正在接受TNF-α抑制剂治疗。 102. The method according to any one of the preceding embodiments 87-100, wherein said patient is being treated with a TNF-alpha inhibitor.

103. 实施方案87-101的方法,其中所述患者从未接受TNF-α抑制剂治疗。 103. The method of embodiment 87-101, wherein the patient has never received TNF-alpha inhibitor treatment.

104. 实施方案87-103的方法,其中所述患者是对TNF-α抑制剂治疗的不充分反应者。 104. The method of embodiments 87-103, wherein the patient is an inadequate responder to TNF-alpha inhibitor treatment.

105. 实施方案87-103中任一项的方法,其中所述患者是对用于所述炎性疾病或病症的一种或多种治疗的不充分反应者。 105. The method according to any one of embodiments 87-103, wherein said patient is an inadequate responder to one or more treatments for said inflammatory disease or disorder.

106. 实施方案87-104的方法,其中所述患者是对MTX和TNF-α抑制剂治疗的不充分反应者。 106. The method of embodiments 87-104, wherein the patient is an inadequate responder to MTX and TNF-alpha inhibitor therapy.

107. 实施方案87-105的方法,其中所述患者是RF阳性。 107. The method of embodiments 87-105, wherein the patient is RF positive.

108. 实施方案87-106的方法,其中所述患者是RF阴性。 108. The method of embodiments 87-106, wherein said patient is RF negative.

109. 实施方案87-107的方法,其中所述抗炎药是抗体。 109. The method of embodiments 87-107, wherein the anti-inflammatory agent is an antibody.

110. 实施方案87-108的方法,其中所述抗炎药是受体拮抗剂。 110. The method of embodiments 87-108, wherein the anti-inflammatory agent is a receptor antagonist.

111. 实施方案87-109的方法,其中所述抗炎药是IL-10家族的一个或多个成员的拮抗剂。 111. The method of embodiments 87-109, wherein the anti-inflammatory drug is an antagonist of one or more members of the IL-10 family.

112. 实施方案87-110的方法,其中所述抗炎药是IL-10、IL19、IL-20、IL-22、IL-24和IL-26中的一种或多种的拮抗剂。 112. The method of embodiments 87-110, wherein the anti-inflammatory agent is an antagonist of one or more of IL-10, IL19, IL-20, IL-22, IL-24, and IL-26.

113. 实施方案87-111的方法,其中所述抗炎药是IL-19、IL-20和IL-24中的一种或多种的拮抗剂。 113. The method of embodiments 87-111, wherein the anti-inflammatory agent is an antagonist of one or more of IL-19, IL-20, and IL-24.

114. 实施方案87-112的方法,其中所述抗炎药是IL-20的拮抗剂。 114. The method of embodiments 87-112, wherein the anti-inflammatory drug is an antagonist of IL-20.

115. 实施方案87-113的方法,其中所述抗炎药是IL-20的拮抗剂,其降低IL-20介导的IL-20R1/IL-20R2和IL-22R/IL-20R2受体两者的活化。 115. The method of embodiments 87-113, wherein the anti-inflammatory agent is an antagonist of IL-20, which reduces IL-20-mediated IL-20R1/IL-20R2 and IL-22R/IL-20R2 receptor duality. the activation of the

116. 实施方案87-114的方法,其中所述抗炎药是IL-20的拮抗剂,其降低IL-20介导的IL-20R1/IL-20R2和IL-22R/IL-20R2受体两者的活化,而不是IL19或IL24介导的受体活化。 116. The method of embodiments 87-114, wherein the anti-inflammatory agent is an antagonist of IL-20, which reduces IL-20-mediated IL-20R1/IL-20R2 and IL-22R/IL-20R2 receptor duality. receptor activation, but not IL19- or IL24-mediated receptor activation.

117. 实施方案87-115的方法,其中所述抗炎药是抗人IL-20抗体。 117. The method of embodiments 87-115, wherein the anti-inflammatory agent is an anti-human IL-20 antibody.

118. 治疗炎性疾病的方法,包括将药学有效量的抗炎药给予炎性疾病患者,所述患者具有这样的表达谱:其中相对于在对所述抗炎药无反应的人中的第一生物标记的表达而言,第一生物标记的表达是增加的,其中所述第一生物标记是补体因子D (CFD)。 118. A method for treating an inflammatory disease, comprising administering a pharmaceutically effective amount of an anti-inflammatory drug to a patient with an inflammatory disease, said patient having an expression profile wherein relative to the first in a non-responsive person to said anti-inflammatory drug With respect to the expression of a biomarker, the expression of a first biomarker is increased, wherein the first biomarker is complement factor D (CFD).

119. 实施方案118的方法,其中所述炎性疾病患者中的CFD的表达比在对所述抗炎药无反应的人中的CFD的表达高至少一个标准偏差。 119. The method of embodiment 118, wherein the expression of CFD in said inflammatory disease patient is at least one standard deviation higher than the expression of CFD in a person unresponsive to said anti-inflammatory drug.

120. 实施方案118的方法,其中所述炎性疾病是自身免疫疾病或病症。 120. The method of embodiment 118, wherein the inflammatory disease is an autoimmune disease or disorder.

121. 实施方案120的方法,其中所述自身免疫疾病或病症选自类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、多发性硬化(MS)、炎性肠病(IBD)和银屑病性关节炎(PSA)。 121. The method of embodiment 120, wherein the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD) and Psoriatic arthritis (PSA).

122. 实施方案121的方法,其中所述自身免疫疾病是RA。 122. The method of embodiment 121, wherein the autoimmune disease is RA.

123. 实施方案118的方法,其中所述炎性疾病患者具有这样的表达谱:其中相对于在对所述抗炎药无反应的人中的第二生物标记的表达而言,第二生物标记的表达是增加的,其中所述第二生物标记是SERPINB9 (丝氨酸蛋白酶抑制蛋白肽酶抑制剂和进化枝B (卵清蛋白), 成员9)。 123. The method according to embodiment 118, wherein said inflammatory disease patient has an expression profile wherein the second biomarker is Expression of is increased where the second biomarker is SERPINB9 (serine protease inhibitory protein peptidase inhibitor and clade B (ovalbumin), member 9).

124. 实施方案118的方法,其中所述炎性疾病患者具有这样的表达谱:其中相对于在对所述抗炎药无反应的人中的第二生物标记的表达而言,第二生物标记的表达是增加的,其中所述第二生物标记选自以下的一种或多种:SERPINB9 (丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B (卵清蛋白)成员9)和ZCCHC24 (锌指, CCHC域含有24 )。 124. The method according to embodiment 118, wherein said inflammatory disease patient has an expression profile wherein the second biomarker is The expression of is increased, wherein the second biomarker is selected from one or more of the following: SERPINB9 (serine protease inhibitory protein peptidase inhibitor, clade B (ovalbumin) member 9) and ZCCHC24 (zinc finger , CCHC domain contains 24 ).

125. 实施方案118的方法,其中所述炎性疾病患者具有这样的表达谱:其中相对于在对所述抗炎药无反应的人中的第二生物标记的表达而言,第二生物标记的表达是增加的,其中所述第二生物标记选自以下的一种或多种:SERPINB9 (丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B (卵清蛋白)成员9)、ZCCHC24 (锌指, CCHC域含有24 )、FN3KRP (果糖胺3激酶相关蛋白)、FN3KRP (果糖胺3激酶相关蛋白)、MEOX1 (间质同源框1)、FGF13 (成纤维细胞生长因子13)、TUBB2A (微管蛋白, β2A)、SLC39A11 (溶质载体家族39 (金属离子转运蛋白), 成员11)、TMC4 (跨膜通道-样4)、NPIPL2 (核孔复合物相互作用蛋白-样2)、ZNF880 (锌指蛋白880)和ALDH5A1 (醛脱氢酶5家族, 成员A1)。 125. The method according to embodiment 118, wherein said inflammatory disease patient has an expression profile wherein the second biomarker is The expression of is increased, wherein the second biomarker is selected from one or more of the following: SERPINB9 (serine protease inhibitory protein peptidase inhibitor, clade B (ovalbumin) member 9), ZCCHC24 (zinc finger , CCHC domain contains 24 ), FN3KRP (fructosamine 3-kinase-related protein), FN3KRP (fructosamine 3-kinase-related protein), MEOX1 (interstitial homeobox 1), FGF13 (fibroblast growth factor 13), TUBB2A (micro Tubulin, β2A), SLC39A11 (solute carrier family 39 (metal ion transporter), member 11), TMC4 (transmembrane channel-like 4), NPIPL2 (nuclear pore complex interacting protein-like 2), ZNF880 (zinc Refers to protein 880) and ALDH5A1 (aldehyde dehydrogenase 5 family, member A1).

126. 实施方案118-125的方法,其中所述抗炎药是IL-10、IL-19、IL-20、IL-22、IL-24和IL-26中的一种或多种的拮抗剂。 126. The method of embodiments 118-125, wherein the anti-inflammatory agent is an antagonist of one or more of IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 .

127. 实施方案118-126的方法,其中所述抗炎药是IL-19、IL-20和IL-24中的一种或多种的拮抗剂。 127. The method of embodiments 118-126, wherein the anti-inflammatory agent is an antagonist of one or more of IL-19, IL-20, and IL-24.

128. 实施方案118-127的方法,其中所述抗炎药是IL-20的拮抗剂。 128. The method of embodiments 118-127, wherein the anti-inflammatory drug is an antagonist of IL-20.

129. 治疗自身免疫疾病的方法,包括: 129. A method of treating an autoimmune disease, comprising:

鉴定自身免疫疾病患者; Identify patients with autoimmune diseases;

判定所述患者表达第一生物标记,其中第一生物标记是补体因子D (CFD); determining that the patient expresses a first biomarker, wherein the first biomarker is complement factor D (CFD);

基于以下认识,选择抗炎药作为患者的治疗:所述抗炎药在自身免疫疾病患者中有效,相对于在对所述抗炎药无反应的受试者中的第一生物标记的表达而言,在所述患者中第一生物标记的表达谱是增加的;和 Anti-inflammatory drugs were selected as treatment for patients based on the recognition that the anti-inflammatory drugs are effective in patients with autoimmune diseases relative to the expression of the first biomarker in subjects unresponsive to the anti-inflammatory drugs In other words, the expression profile of the first biomarker is increased in the patient; and

将所述抗炎药给予所述患者。 The anti-inflammatory drug is administered to the patient.

130. 实施方案129的方法,其中所述自身免疫疾病或病症选自类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、多发性硬化(MS)、炎性肠病(IBD)和银屑病性关节炎(PSA)。 130. The method of embodiment 129, wherein the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD) and Psoriatic arthritis (PSA).

131. 实施方案130的方法,其中所述自身免疫疾病是RA。 131. The method of embodiment 130, wherein the autoimmune disease is RA.

132. 实施方案129的方法,其中选择抗炎药作为患者的治疗进一步包括以下的认识:所述抗炎药在自身免疫疾病患者中有效,相对于在对所述抗炎药无反应的受试者中的第二生物标记的表达而言,在所述患者中第二生物标记的表达谱是增加的;和其中第二生物标记选自以下的一种或多种:SERPINB9 (丝氨酸蛋白酶抑制蛋白肽酶抑制剂, 进化枝B (卵清蛋白) 成员9)、ZCCHC24 (锌指, CCHC域含有24 )、FN3KRP (果糖胺3激酶相关蛋白)、FN3KRP (果糖胺3 激酶相关蛋白)、MEOX1 (间质同源框1)、FGF13 (成纤维细胞生长因子13)、TUBB2A (微管蛋白, β2A)、SLC39A11 (溶质载体家族39 (金属离子转运蛋白), 成员11)、TMC4 (跨膜通道-样4)、NPIPL2 (核孔复合物相互作用蛋白-样2)、ZNF880 (锌指蛋白880)和ALDH5A1 (醛脱氢酶5家族, 成员A1)。 132. The method of embodiment 129, wherein selecting an anti-inflammatory drug as treatment for the patient further comprises the recognition that the anti-inflammatory drug is effective in patients with an autoimmune disease, as opposed to being effective in subjects who are unresponsive to the anti-inflammatory drug. In the patient, the expression profile of the second biomarker is increased in said patient; and wherein the second biomarker is selected from one or more of the following: SERPINB9 (serine protease inhibitory protein Peptidase inhibitors, clade B (ovalbumin) member 9), ZCCHC24 (zinc finger, CCHC domain contains 24), FN3KRP (fructosamine 3-kinase-related protein), FN3KRP (fructosamine 3-kinase-related protein), MEOX1 ( Interstitial homeobox 1), FGF13 (fibroblast growth factor 13), TUBB2A (tubulin, β2A), SLC39A11 (solute carrier family 39 (metal ion transporter), member 11), TMC4 (transmembrane channel- NPIPL2 (nuclear pore complex interacting protein-like 2), ZNF880 (zinc finger protein 880) and ALDH5A1 (aldehyde dehydrogenase 5 family, member A1).

133. 实施方案129-132中任一项的方法,其中所述抗炎药是IL-10、IL-19、IL-20、IL-22、IL-24和IL-26中的一种或多种的拮抗剂。 133. The method of any one of embodiments 129-132, wherein the anti-inflammatory agent is one or more of IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 species of antagonists.

134. 实施方案129-133中任一项的方法,其中所述抗炎药是IL-19、IL-20和IL-24中的一种或多种的拮抗剂。 134. The method according to any one of embodiments 129-133, wherein the anti-inflammatory drug is an antagonist of one or more of IL-19, IL-20 and IL-24.

135. 实施方案129-134中任一项的方法,其中所述抗炎药是IL-20的拮抗剂。 135. The method of any one of embodiments 129-134, wherein the anti-inflammatory drug is an antagonist of IL-20.

136. 实施方案129的方法,其中对所述患者表达第一生物标记的判定包括mRNA的检测。 136. The method of embodiment 129, wherein determining that the patient expresses the first biomarker comprises detection of mRNA.

137. 实施方案136的方法,其中对mRNA的检测包括多重PCR和qRT-PCR。 137. The method of embodiment 136, wherein the detection of mRNA comprises multiplex PCR and qRT-PCR.

138. 实施方案129的方法,其中根据一个或多个CFD表达相关的SNP间接测定补体因子D (CFD)的表达水平。 138. The method of embodiment 129, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs associated with CFD expression.

139. 实施方案129的方法,其中根据在CFD单倍体块(haploblock)中的一个或多个SNP间接测定补体因子D (CFD)的表达水平。 139. The method of embodiment 129, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs in the CFD haploblock.

140. 实施方案138-139的方法,其中根据选自以下的一个或多个SNP间接测定补体因子D (CFD)的表达水平:rs1683565、rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898。 140. The method of embodiments 138-139, wherein the expression level of complement factor D (CFD) is determined indirectly based on one or more SNPs selected from the group consisting of rs1683565, rs1683591, rs1683590, rs1683569, rs1683574, rs1651888, rs2930894, rs2930891, rs4417648 , rs1651891, rs1651890 and rs2930898.

141. 实施方案140的方法,其中通过SNP rs1683591的AG或GG基因型的存在而间接测定CFD的表达水平。 141. The method of embodiment 140, wherein the expression level of CFD is determined indirectly by the presence of the AG or GG genotype of SNP rs1683591.

142. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 142. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administration of said anti-inflammatory drug, at least one test has shown , the expression level of one or more genes of Figure 1 is altered in a biological sample from said patient.

143. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,至少一种测试已经显示了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 143. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administration of said anti-inflammatory drug, at least one test has shown , the expression level of one or more genes of FIG. 1 is altered in a biological sample from the patient; and wherein the expression level of one or more of the genes is altered compared to a reference level of the gene, predicting the The experimenter's response to the anti-inflammatory drug.

144. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变。 144. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering said anti-inflammatory drug, it has been determined that the The expression level of one or more genes of Figure 1 is altered in a biological sample from the patient.

145. 在患者中治疗炎性疾病或病症的方法,包括将治疗量的抗炎药给予所述患者,其中,在给予所述抗炎药之前,已经测定了与参考水平相比,在来自所述患者的生物样品中图1的一个或多个基因的表达水平被改变;和其中与所述基因的参考水平相比一个或多个所述基因的表达水平改变,预测了所述受试者对所述抗炎药的反应。 145. A method of treating an inflammatory disease or condition in a patient, comprising administering to said patient a therapeutic amount of an anti-inflammatory drug, wherein, prior to administering said anti-inflammatory drug, it has been determined that the The expression level of one or more genes of Figure 1 is altered in the biological sample of the patient; and wherein the expression level of one or more of the genes is altered compared to a reference level of the gene, predicting that the subject Response to the anti-inflammatory drugs.

通用方法 general method

总RNA纯化Total RNA purification

可通过本领域技术人员已知的多种方法自任何类型的生物样品得到总RNA。 Total RNA can be obtained from any type of biological sample by a variety of methods known to those skilled in the art.

本文的实施例是基于使用PaxGene blood RNA KIT IVD (QIAGEN)而获得的数据,所述试剂盒特别适合延长时间采集并随后分析的样品。按照制造商(Qiagen)说明书处理PaxGene血液样品并按照PaxGene PAXgene Blood RNA Kit (QIAGEN)的方案分离总RNA。 The examples herein are based on data obtained using the PaxGene blood RNA KIT IVD (QIAGEN), which is particularly suited for samples collected over extended periods of time and subsequently analyzed. PaxGene blood samples were processed according to the manufacturer's (Qiagen) instructions and total RNA was isolated according to the protocol of the PaxGene PAXgene Blood RNA Kit (QIAGEN).

球蛋白mRNA减少Decreased globin mRNA

按照制造商的说明书,使用GLOBINClear试剂盒(Applied Biosystems, Foster City, CA, USA),可获得总RNA样品中的球蛋白mRNA的减少。 Reduction of globin mRNA in total RNA samples was obtained using the GLOBINClear kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions.

RNA完整性证实RNA integrity confirmed

在进行进一步分析之前,证实RNA样品的完整性是合适的。 It is appropriate to verify the integrity of the RNA sample before proceeding to further analysis.

可以按照制造商说明书,使用Agilent 2100 Bioanalyzer和总RNA Nano芯片(Agilent Technologies, Santa Clara, CA, USA)。通常RNA完整性计数(RIN-评分)高于7的样品就被认为可以接受用于进一步分析。 Agilent 2100 Bioanalyzer and Total RNA Nanochip (Agilent Technologies, Santa Clara, CA, USA) can be used according to the manufacturer's instructions. Typically samples with an RNA Integrity Count (RIN-score) above 7 were considered acceptable for further analysis.

AffyMetrix GeneChip杂交、扫描和分析AffyMetrix GeneChip hybridization, scanning and analysis

通过3' IVT Express试剂盒(Affymetrix, Santa Clara, Ca, USA),按照制造商说明书,使用总RNA样品,自50毫微克总RNA制备标记的cRNA (靶)。按照制造商所述,制备杂交合剂并在杂交炉640 (Affymetrix)中在45℃杂交到人类基因组U133 Plus 2.0 GeneChips? (Affymetrix)达17h (60 RPM)。杂交后,使用fluidics方案“EukGE-WS2v5_450” (Affymetrix),将基因芯片在GeneChip?fluidics station 450中洗涤和染色。将GeneChips?在GeneChip?扫描仪3000 (Affymetrix)中扫描。通过使用R环境和可在以下URL: cran.r-project.org & bioconductor.org中找到的Bioconductor包“Affy”,将输出“*.cel文件”用于基因芯片数据的RMA (Robust Multiarray Average)标准化。 Labeled cRNA (target) was prepared from 50 nanograms of total RNA using the total RNA sample by the 3' IVT Express Kit (Affymetrix, Santa Clara, Ca, USA) following the manufacturer's instructions. Hybridization mixes were prepared and hybridized to Human Genome U133 Plus 2.0 GeneChips® (Affymetrix) in a Hybridization Oven 640 (Affymetrix) at 45°C for 17h (60 RPM) as described by the manufacturer. After hybridization, the gene chip was washed and stained in GeneChip® fluidics station 450 using the fluidics protocol "EukGE-WS2v5_450" (Affymetrix). GeneChips® were scanned in a GeneChip® Scanner 3000 (Affymetrix). By using the R environment and the Bioconductor package "Affy" which can be found at the following URLs: cran.r-project.org & bioconductor.org, the output "*.cel files" will be used for RMA (Robust Multiarray Average) of microarray data standardization.

用可得自统计学编程环境R的开源工具(可得自URL: cran.r-project.org)以及QluCore Omics explorer 2.2 (QluCore AB, Sweden)进行微阵列数据的统计学分析。微阵列通过RMA (Robust Multiarray Average)而标准化,使用Affy包(可得自URL: cran.r-project.org)和定制芯片定义文件(custom Chip Definition File) (HGU133Plus2_Hs_ENSG),其可得自URL: brainarray.mbni.med.umich.edu)。 Statistical analysis of microarray data was performed with open source tools available from the statistical programming environment R (available at URL: cran.r-project.org) and QluCore Omics explorer 2.2 (QluCore AB, Sweden). Microarrays were standardized by RMA (Robust Multiarray Average) using the Affy package (available at URL: cran.r-project.org) and a custom Chip Definition File (HGU133Plus2_Hs_ENSG), available at URL: brainarray.mbni.med.umich.edu).

使用Simca-P +11软件(Umetrics, Ume?, Sweden)和偏最小二乘(PLS)工具进行多变量预测。 Multivariate predictions were performed using Simca-P+11 software (Umetrics, Ume?, Sweden) and the partial least squares (PLS) tool.

使用GraphPad Prism 5 (GraphPad软件, CA, USA)制备ROC (接受者操作特征)曲线。 ROC (Receiver Operating Characteristic) curves were prepared using GraphPad Prism 5 (GraphPad Software, CA, USA).

定量RT-PCRquantitative RT-PCR

按照制造商的说明书,使用随机引物和TaqMan逆转录试剂(Applied Biosystems, Foster City, CA, USA),通过自200 ng总RNA制备25微升cDNA,进行定量RT-PCR分析。 Quantitative RT-PCR analysis was performed by preparing 25 microliters of cDNA from 200 ng of total RNA using random primers and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions.

以总体积25微升进行qPCR分析,每个样品一式两份(6,95微升的10倍稀释的cDNA),使用TaqMan PCR core试剂(Applied Biosystems)和ABI PRISM? 7900HT序列检测系统(Applied Biosystems)。 qPCR analysis was performed in a total volume of 25 μl in duplicate per sample (6,95 μl of 10-fold diluted cDNA), using TaqMan PCR core reagents (Applied Biosystems) and ABI PRISM™ 7900HT sequence detection system (Applied Biosystems ).

使用CFD mRNA和18S rRNA的引物和FAM标记的探针,检测CFD mRNA、ACTB mRNA和18S rRNA的表达水平。引物和探针以按需检测(Assays-on-Demand) (Applied Biosystems)的方式订购。这些测定的探针序列如下:CFD (CCTGCTGCTACAGCTGTCGGAGAAG (Assay ID: Hs00157263_m1)), ACTB (CCTTTGCCGATCCGCCGCCCGTCCA (Assay ID: Hs Hs99999903_m1)和18S rRNA (TGGAGGGCAAGTCTGGTGCCAGCAG;assay Hs99999901_s1)。使用ABI Prism SDS 2.2软件(Applied Biosystems)分析数据,并将表达水平标准化至18S rRNA或ACTB mRNA。 The expression levels of CFD mRNA, ACTB mRNA and 18S rRNA were detected using primers for CFD mRNA and 18S rRNA and FAM-labeled probes. Primers and probes were ordered as Assays-on-Demand (Applied Biosystems).这些测定的探针序列如下:CFD (CCTGCTGCTACAGCTGTCGGAGAAG (Assay ID: Hs00157263_m1)), ACTB (CCTTTGCCGATCCGCCGCCCGTCCA (Assay ID: Hs Hs99999903_m1)和18S rRNA (TGGAGGGCAAGTCTGGTGCCAGCAG;assay Hs99999901_s1)。使用ABI Prism SDS 2.2软件(Applied Biosystems)分析data and normalize expression levels to 18S rRNA or ACTB mRNA.

PCR产物的可靠检测应在CFD测定(ID: Hs00157263_m1)中在循环(Ct-值) 26内可检测到,和18S rRNA的检测(assay ID Hs99999901_s1) 应在Ct=12,5时获得。还可通过以下进行标准化:通过计算Δ-Ct值,或通过使用稀释的质粒编码的CFD、ACTB和18S的标准曲线,和将定量测定的CFD拷贝数与定量测定的ACTB或18S拷贝数相关联。 Reliable detection of PCR products should be detectable within cycle (Ct-value) 26 in CFD assay (ID: Hs00157263_m1), and detection of 18S rRNA (assay ID Hs99999901_s1) should be obtained at Ct=12,5. Normalization can also be performed by calculating delta-Ct values, or by using a standard curve of diluted plasmid-encoded CFD, ACTB, and 18S, and correlating the quantified CFD copy number with the quantified ACTB or 18S copy number .

在上文,正式基因符号标识用于分析的转录物。 Above, formal gene symbols identify transcripts used for analysis.

实施例 Example

实施例1-预测性转录物的鉴定 Example 1 - Identification of predictive transcripts

在以下时间点自患者采集来自1b期和2a期试验的血液样品,所述试验检查抗IL-20在类风湿性关节炎(RA)患者中的安全性、耐受性和功效(临床试验政府识别号:NCT01038674和NCT01282255):在1b期试验中给药前(第1天)和给药后第8、15、29、43和99天,以及在2a期试验中给药前(第1天)和给药后第15、36天。分别在6周期间每周一次给予患者(总共7剂)和在11周期间每周一次给予患者(总共12剂)。 Blood samples from Phase 1b and Phase 2a trials examining the safety, tolerability and efficacy of anti-IL-20 in patients with rheumatoid arthritis (RA) were collected from patients at time points (Clinical Trials Government Identification numbers: NCT01038674 and NCT01282255): pre-dose (Day 1) and post-dose days 8, 15, 29, 43, and 99 in the Phase 1b trial, and pre-dose (Day 1 ) and the 15th and 36th day after administration. The patients were dosed weekly for 6 weeks (total of 7 doses) and weekly for 11 weeks (total of 12 doses), respectively.

如上所述地获取总RNA。在球蛋白mRNA减少和RNA完整性证实后,按照上述程序通过AffyMetrix GeneChip杂交,分析RNA样品。 Total RNA was obtained as described above. After globin mRNA reduction and RNA integrity were confirmed, RNA samples were analyzed by AffyMetrix GeneChip hybridization following the procedure described above.

为了在全血PaxGene样品中鉴定转录物(其与在DAS28-CRP和其它疾病评分测定例如ACR20、ACR50和ACR70中的变化相关),我们对来自单个患者的表达谱进行了回归分析,所述患者纳入1b和2a期试验,所述试验检查抗IL20抗体在RA患者中的功效。根据主要在给药前(基线)和在第8、15、29、36和43天得到的样品,选择随时间表现出相对稳定性并因此合适作为给药前层化标记的转录物。 To identify transcripts in whole blood PaxGene samples that correlate with changes in DAS28-CRP and other disease score measures such as ACR20, ACR50, and ACR70, we performed regression analysis on expression profiles from individual patients who Phase 1b and 2a trials examining the efficacy of anti-IL20 antibodies in RA patients were included. Based on samples taken primarily pre-dose (baseline) and at days 8, 15, 29, 36 and 43, transcripts were selected that exhibited relative stability over time and were therefore suitable as pre-dose stratification markers.

还进行了将所获微阵列数据点与临床功效关联的多变量方法。通过使用偏最小二乘(PLS)推测潜在结构,鉴定用于在接受抗IL20的单个患者中预测临床效果的转录物的最佳组合。经鉴定的PLS模型通过排列检验(将观察数据与预测数据间相关性的R2-系数从0.8降至0.1)而得以交叉验证。这表明PLS模型是有效的而非不适合的(over-fitted)。使用任何多变量分析软件工具(Like Simca-P +11 (Umetrics)或Unscrambler (CAMO软件AS, Oslo, Norway)可以进行基于PLS的预测。首先使用来自AffyMetrix微阵列的18954数据点,进行多变量预测。一组14种预测性转录物的实例示于图2。 A multivariate approach linking the obtained microarray data points to clinical efficacy was also performed. The optimal combination of transcripts for predicting clinical effect in individual patients receiving anti-IL20 was identified by inferring the underlying structure using partial least squares (PLS). The identified PLS models were cross-validated by permutation testing (reducing the R2 -coefficient of the correlation between observed and predicted data from 0.8 to 0.1). This indicates that the PLS model is efficient rather than over-fitted. PLS-based predictions can be performed using any multivariate analysis software tool (Like Simca-P+11 (Umetrics) or Unscrambler (CAMO Software AS, Oslo, Norway). Multivariate predictions were first performed using 18954 data points from the AffyMetrix microarray An example of a set of 14 predicted transcripts is shown in Figure 2.

基于多变量的预测模型中的目标基因是补体因子D(CFD),也称为Adipsin。RA患者中的CFD转录物水平见图3。单个患者中的mRNA表达水平是随时间而稳定的,但患者之间具有明显差异。具有最低水平的CFD mRNA的患者与具有最高水平的患者之间的差异为大约8倍。 The target gene in the multivariate-based prediction model was complement factor D (CFD), also known as Adipsin. CFD transcript levels in RA patients are shown in Figure 3. The level of mRNA expression in a single patient is stable over time, but there are significant differences between patients. The difference between patients with the lowest levels of CFD mRNA and those with the highest levels was approximately 8-fold.

如图1所示,在单变量回归分析中,CFD也在阳性相关的转录物中。同时,这些发现促使对CFD作为高反应预测物在给予抗IL20抗体的RA-患者中的有用性的检查。这通过制备接受者操作特征(ROC)曲线来完成,见图4 (ACR50反应)和图5 (ACR70反应)。发现ACR50反应的曲线下面积(AUC)为0,81 (p=0,00068),指示基于CFD mRNA水平的ACR50反应的良好预测。 As shown in Figure 1, CFD was also among the positively associated transcripts in univariate regression analysis. At the same time, these findings prompted examination of the usefulness of CFD as a predictor of high response in RA-patients administered anti-IL20 antibodies. This is done by preparing receiver operating characteristic (ROC) curves, see Figure 4 (ACR50 response) and Figure 5 (ACR70 response). The area under the curve (AUC) for the ACR50 response was found to be 0,81 (p=0,00068), indicating a good prediction of the ACR50 response based on CFD mRNA levels.

为了测试CFD mRNA在抗IL20 RA-试验中是否是高反应的预测物,在ACR50反应者和无反应者、以及对ACR70反应者和无反应者定义了患者的最佳分类的阈值。使用得自在第1天(给药前)和在给药后第15和36天采集的4个样品的平均CFD-mRNA水平进行这样的定义。如前所述,在这些访问中发现CFD mRNA水平是随时间而稳定的。对于ACR50分类,发现CFD mRNA阈值为10.32 (RMA标准化AffyMetrix 微阵列水平的log2标度) (在图4中通过X表示)。 To test whether CFD mRNA is a predictor of high response in the anti-IL20 RA-test, thresholds for optimal classification of patients were defined in ACR50 responders and non-responders, and for ACR70 responders and non-responders. Such definition was performed using mean CFD-mRNA levels from 4 samples taken at day 1 (pre-dose) and at days 15 and 36 post-dose. As previously described, CFD mRNA levels were found to be stable over time at these visits. For ACR50 classification, the CFD mRNA threshold was found to be 10.32 (log2 scale of RMA normalized AffyMetrix microarray levels) (indicated by X in Figure 4).

当在2a期试验数据中在给予抗IL20抗体的RA患者中使用该阈值(10.32或以上)时,发现ACR50反应率从37%明显增加至65% (图7)。当使用10.32或以上的入选阈值时,将包括大约半数的纳入2a期试验中的患者。同样对于ACR70反应,发现在给药的RA患者中的阈值10.32将反应率从25%增加至45%。当使用阈值10.32时的反应率的改进(enrichment)示于图7。 When this threshold (10.32 or above) was used in RA patients administered anti-IL20 antibody in the phase 2a trial data, a significant increase in ACR50 response rate was found from 37% to 65% (Figure 7). When an entry threshold of 10.32 or above was used, approximately half of the patients included in the phase 2a trial would be included. Also for ACR70 response, a threshold of 10.32 was found to increase the response rate from 25% to 45% in dosed RA patients. The enrichment in response rate when using a threshold of 10.32 is shown in FIG. 7 .

在2a期试验的给予安慰剂的个体中,CFD mRNA水平为10.32或以上的患者的ACR50反应率是17%,相比之下,CFD mRNA水平低于10.32的患者的反应率为11%。对于ACR70,CFD mRNA水平为10.32或以上的给予安慰剂的患者的反应率为8 %,相比之下,CFD mRNA水平低于10.32的患者的反应率为0%。 Among individuals given placebo in the phase 2a trial, the ACR50 response rate was 17% for those with CFD mRNA levels of 10.32 or above, compared with 11% for those with CFD mRNA levels below 10.32. For ACR70, the response rate for placebo-treated patients with CFD mRNA levels of 10.32 or above was 8 percent, compared with 0 percent for patients with CFD mRNA levels below 10.32.

基于以上分析,结论是CFD mRNA水平是在RA患者中针对抗IL-20的高反应的良好预测物,和如果仅包括CFD mRNA水平为10.32或以上的RA患者时,可获得尤其ACR50和ACR70反应的明显增加。 Based on the above analysis, it is concluded that CFD mRNA level is a good predictor of high response to anti-IL-20 in RA patients, and especially ACR50 and ACR70 responses can be obtained if only RA patients with CFD mRNA level of 10.32 or above are included obvious increase.

实施例2 - qRT-PCR与阵列数据的关联 Example 2 - Correlation of qRT-PCR to array data

为了检测单用CFD mRNA基线(给药前)测定是否与来自在微阵列分析中评价的不同时间点的CFD mRNA水平关联,在可用的给药前样品上对CFD mRNA进行qRT-PCR,并且将这些与来自微阵列的记录的CFD水平关联。如上所述地进行定量RT-PCR分析和自每个cDNA样品的重复分析获得数据。将来自qRT-PCR平台的CFD mRNA水平标准化到也通过qRT-PCR检测的18S rRNA水平。为了将微阵列水平与qRT-PCR水平关联,将RNA标准化的微阵列水平转化到线性尺度。如图6所示,在基线的qRT-PCR测定和来自若干访问的微阵列测定之间发现高度关联(R2 =0,86)。这表明在基线(给药前)的基于CFD的RA-患者层化是可行的,因为图1和图2中所述的其它预测性转录物例如CFD经鉴定不仅与基线样品关联,而且与2a期试验中的若干时间点关联,这些也被选择以展示随时间的稳定性(正如在图6中通过CFD的相关性所示例)。本文所述的转录物因此特别适合于高度反应的患者的给药前预测性层化。 To test whether baseline (pre-dose) determination of CFD mRNA alone correlates with CFD mRNA levels from different time points evaluated in the microarray analysis, qRT-PCR was performed on CFD mRNA on available pre-dose samples, and the These correlate with recorded CFD levels from the microarray. Quantitative RT-PCR analysis was performed as described above and data were obtained from duplicate analyzes of each cDNA sample. CFD mRNA levels from the qRT-PCR platform were normalized to 18S rRNA levels also detected by qRT-PCR. To correlate microarray levels with qRT-PCR levels, RNA-normalized microarray levels were transformed to a linear scale. As shown in Figure 6, a high correlation (R 2 =0,86) was found between the qRT-PCR assay at baseline and the microarray assay from several visits. This suggests that CFD-based stratification of RA-patients at baseline (pre-dose) is feasible, as other predictive transcripts such as CFD described in Figures 1 and 2 were identified not only associated with baseline samples, but also with 2a Several time point correlations in the long-term trial were also chosen to demonstrate stability over time (as exemplified by the CFD correlations in Figure 6). The transcripts described herein are therefore particularly suitable for pre-dose predictive stratification of highly responsive patients.

为了获得基于qRT-PCR数据的层化(类似使用微阵列数据用阈值10.32而得到的层化),在CFD测定(ID: Hs00157263_m1))中PCR产物应在循环(Ct-值) 26内检测到,和18S rRNA的检测(assay Hs99999901_s1)应在Ct=12,5时获得。这些值相当于根据来自单个患者的样品而估计的在微阵列值的RMA标度上的大约10.25,其中两次阵列检测接近10.32,平均为10.24。使用qRT-PCR测定,在该患者中的CFD的检测用Ct-值26得到,和18S对照的Ct值为12.5。 To obtain stratification based on qRT-PCR data (similar to that obtained using microarray data with a threshold of 10.32), the PCR product should be detected within cycle (Ct-value) 26 in the CFD assay (ID: Hs00157263_m1) , and the detection of 18S rRNA (assay Hs99999901_s1) should be obtained at Ct=12,5. These values correspond to approximately 10.25 on the RMA scale of microarray values estimated from samples from a single patient, with two array assays approaching 10.32 and an average of 10.24. Detection of CFD in this patient was obtained with a Ct-value of 26 using qRT-PCR assay, and a Ct-value of 12.5 for the 18S control.

还可能通过qRT-PCR测定补体因子D (CFD)的表达水平,其中使用CFD的Assay ID: Hs00157263_m1 (Applied Biosystems/ Invitrogen)和ACTB的Assay ID: Hs99999903_m1 (Applied Biosystems/ Invitrogen),对于改善的反应的阈值是至少0.04个CFD拷贝/β-肌动蛋白拷贝的绝对数。 It is also possible to determine the expression level of complement factor D (CFD) by qRT-PCR using Assay ID: Hs00157263_m1 (Applied Biosystems/Invitrogen) for CFD and Assay ID: Hs99999903_m1 (Applied Biosystems/Invitrogen) for ACTB, for improved response The threshold was an absolute number of at least 0.04 CFD copies/β-actin copies.

实施例3 – 在PaxGene样品中CFD mRNA水平的疾病关联性Example 3 - Disease Correlation of CFD mRNA Levels in PaxGene Samples

为了评价RA患者的外周血中的CFD水平是否可与局部关节中的相关活性标记相关联,采集成对的PaxGene (全血)和滑液样品。补体因子D在替代补体途径活化中是起始的丝氨酸蛋白酶,并切割与因子B络合的C3b为C3bBb和Ba。C3bBb是C3转化酶,其将切割其它C3分子为C3a和C3b。因为Bb分子对于替代补体活化是独特的,Bb蛋白水平用作该途径的活性标记。有力证据支持经典和替代补体活化两者参与类风湿性关节炎的病理生理学。 To evaluate whether CFD levels in peripheral blood of RA patients could be correlated with relevant activity markers in local joints, paired PaxGene (whole blood) and synovial fluid samples were collected. Complement factor D is the initial serine protease in the activation of the alternative complement pathway and cleaves C3b complexed with factor B to C3bBb and Ba. C3bBb is a C3 convertase that will cleave other C3 molecules into C3a and C3b. Because the Bb molecule is unique to alternative complement activation, Bb protein levels serve as an activity marker for this pathway. Strong evidence supports the involvement of both classical and alternative complement activation in the pathophysiology of rheumatoid arthritis.

按照试剂盒方案准备Bb plus EIA (MicroVue cat# A027)测定(测定人血浆或血清中补体片段Bb的量的一种测定法)。 Follow the kit protocol to prepare the Bb plus EIA (MicroVue cat# A027) assay (an assay for determining the amount of complement fragment Bb in human plasma or serum).

将洗涤缓冲液(x20)在去离子水中稀释。将1% HBR1 (一种嗜异染封闭试剂(HBR1) 18,42mg/ml,来自Scantibodies Lab. Part 3KC533)加入到保湿试剂,和将1% HBR加入到补体标本稀释剂(80μl HBR1 + 7,92ml)。将重配的标准和对照在1ml保湿试剂/HBR1中稀释并静置15min。将样品在补体标本稀释剂+1% HBR1中稀释10倍(45μl+405μl)和20倍(22μl+418μl)。 Wash buffer (x20) was diluted in deionized water. Add 1% HBR1 (a Heterophilic Blocking Reagent (HBR1) 18,42mg/ml from Scantibodies Lab. Part 3KC533) to the Moisture Reagent, and 1% HBR to the Complement Specimen Diluent (80μl HBR1 + 7, 92ml). The reconstituted standards and controls were diluted in 1ml Humidity Reagent/HBR1 and allowed to stand for 15min. Samples were diluted 10-fold (45 μl + 405 μl) and 20-fold (22 μl + 418 μl) in complement specimen diluent + 1% HBR1.

将样品预洗涤3次并与100μl标准孵育30 min,洗涤5次,与50μl 缀合物孵育30 min,洗涤5次,与100μl底物孵育15 min,最后用100μl终止液终止。 Samples were prewashed 3 times and incubated with 100 μl standard for 30 min, washed 5 times, incubated with 50 μl conjugate for 30 min, washed 5 times, incubated with 100 μl substrate for 15 min, and finally terminated with 100 μl stop solution.

通过ELISA阅读器测定吸光度,将阅读器设置在450 nm (ref在600-690nm),用线性曲线拟合。 Absorbance was determined by an ELISA reader set at 450 nm (ref at 600-690 nm) with a linear curve fit.

通过使用MicroVue Bb Plus EIA测定和HBR1,我们能够在RA患者滑液中测定Bb水平。当将这些水平针对来自同一患者的成对PaxGene样品中的CFD mRNA水平作图时,发现显著相关,如图8所示。 By using the MicroVue Bb Plus EIA assay and HBR1, we were able to measure Bb levels in the synovial fluid of RA patients. When these levels were plotted against CFD mRNA levels in paired PaxGene samples from the same patient, a significant correlation was found, as shown in Figure 8.

该发现证明,来自RA患者的全血中的CFD mRNA水平指示在局部关节中的替代补体途径的活化状态。 This finding demonstrates that CFD mRNA levels in whole blood from RA patients indicate the activation status of the alternative complement pathway in local joints.

因为来自RA患者的全血中的CFD mRNA水平与局部关节中的替代补体途径的活化状态相关(PaxGene样品中的CFD mRNA与成对滑液水平中的Bb水平之间相关),吸引人的是推测来自RA患者的PaxGene样品中的CFD mRNA水平还可能是靶向补体活化的治疗的预测物。 Because CFD mRNA levels in whole blood from RA patients correlate with the activation status of the alternative complement pathway in local joints (correlation between CFD mRNA in PaxGene samples and Bb levels in paired synovial fluid levels), it is intriguing that It was speculated that CFD mRNA levels in PaxGene samples from RA patients might also be predictive of treatments targeting complement activation.

实施例4 – 使用单核苷酸多态性(SNP)的分析 Example 4 - Analysis Using Single Nucleotide Polymorphisms (SNPs)

作为检测PaxGene样品中的CFD mRNA水平的替代方法,对CFD单倍体块中的某些单核苷酸多态性(SNP)的分析可提供预测反应的便利方法。在PaxGene和其它样品中的CFD mRNA的双峰分布清楚表明遗传多态性可能是CFD mRNA表达模式的基础解释。本领域技术人员可以对CFD进行表达数量特征基因座(eQTL)分析,并鉴定SNP与CFD表达水平的相关性或关联。显示与CFD及其邻近基因的单倍体块中的CFD mRNA表达水平强烈相关的SNP的实例具有身份rs1683565。可以通过多种不同方法进行该SNP或显示与rs1683565强烈连锁不平衡的其它SNP的测定,所述方法包括但不限于,基于杂交的方法(例如SNP微阵列)和基于酶的方法(例如PCR和限制性片断长度多态性方法)。显示与rs1683565强烈连锁不平衡的其它SNP包括但不限于具有以下身份的SNP:rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898。 As an alternative to measuring CFD mRNA levels in PaxGene samples, analysis of certain single nucleotide polymorphisms (SNPs) in CFD haplotype blocks provides a convenient way to predict response. The bimodal distribution of CFD mRNA in PaxGene and other samples clearly suggests that genetic polymorphisms may be the underlying explanation for CFD mRNA expression patterns. Those skilled in the art can perform expression quantitative trait locus (eQTL) analysis on CFD, and identify the correlation or association between SNP and CFD expression level. An example of a SNP showing a strong association with CFD mRNA expression levels in the haplotype block of CFD and its neighboring genes has the identity rs1683565. Determination of this SNP, or other SNPs exhibiting strong linkage disequilibrium with rs1683565, can be performed by a number of different methods including, but not limited to, hybridization-based methods such as SNP microarrays and enzyme-based methods such as PCR and restriction fragment length polymorphism method). Other SNPs showing strong linkage disequilibrium with rs1683565 include, but are not limited to, SNPs with the following identities: rs1683591, rs1683590, rs1683569, rs1683574, rs1651888, rs2930894, rs2930891, rs4417648, rs1651891, rs1651890, and rs293089

作为实例,SNP rs1683591提供AA、AG或GG基因型。基于与CFD表达的关联,AA基因型对应于低CFD表达(具有相对低的反应率),而AG和GG基因型对应于更高水平的CFD表达和因此对抗炎药的反应具有高的可能性。 As an example, SNP rs1683591 provides AA, AG or GG genotypes. Based on the association with CFD expression, the AA genotype corresponds to low CFD expression (with a relatively low response rate), whereas the AG and GG genotypes correspond to higher levels of CFD expression and thus a high likelihood of response to anti-inflammatory drugs sex.

可通过许多充分描述的方法,检测上述SNP、它们的组合或显示与所述SNP (rs1683565、rs1683591、rs1683590、rs1683569、rs1683574、rs1651888、rs2930894、rs2930891、rs4417648、rs1651891、rs1651890和rs2930898)强烈连锁不平衡的其它SNP,所述方法包括但不限于基于杂交的方法(例如SNP微阵列)和基于酶的方法(例如PCR和限制性片断长度多态性方法)。 The above SNPs, their combinations or showing strong linkage disequilibrium with said SNPs (rs1683565, rs1683591, rs1683590, rs1683569, rs1683574, rs1651888, rs2930894, rs2930891, rs4417648, rs1651891, rs1651890 and rs29) can be detected by a number of well-described methods Other SNPs, including but not limited to hybridization-based methods (such as SNP microarrays) and enzyme-based methods (such as PCR and restriction fragment length polymorphism methods).

在一个实例中,从患者中采集血液样品,和按照制造商说明书,通过试剂DNAzol? (Becton Dickinson)分离基因组DNA。简而言之,通过涡旋或手工混合,将1 ml DNAzol?与0.5 ml全血混合。通过将0.4 ml异丙醇加入到DNAzol? BD-血液裂解物中,从样品中沉淀DNA。将所得混合物涡旋并将其在室温下存放5 min。通过在6,000 × g离心6 min将沉淀的DNA离心下来。去除上清液和将0.5 ml DNAzol加入到DNA沉淀物中。涡旋或振摇DNA沉淀物,直到完全溶解。将所得混合物在6,000 × g离心5 min。然后,去除上清液和通过与1 ml 75%乙醇混合而洗涤DNA沉淀物,然后在6,000 × g离心5 min。去除乙醇,无需干燥,向DNA沉淀物中加入200 μl 8 mM NaOH并通过在室温下温育3-5 min,然后涡旋震荡,使DNA溶解。将碱性DNA溶液用0.1 M HEPES中和。从分离的DNA中,制备具有特异性测定所需SNP的定量聚合酶链式反应(qPCR)。qPCR设置可以根据经设计用于特异性检测所需SNP的TaqMan探针。作为实例,将通过任何探针和/或引物组合来检测具有身份rs1683565的SNP,所述组合可以区分以下序列:AGAGCCCAAAGCTCATGGAAAAGAG[A/G]ATATGAAAGGAGTCCCTGCAGTAGA。这可通过来自Invitrogen的市售的测定 (目录号: 4351379 ID: C___9612061_10)而进行。在另一实例中,将通过任何探针和/或引物组合来检测具有身份rs1683591的SNP,所述组合可以区分以下序列: TCTGTCCACAGGCGGGGGTGGAGGG[A/G]ATGGCCGGCCTCACACCATCTGCCA。这可通过来自Invitrogen的市售的测定 (目录号: 4351379 ID: C___9612100_10)而进行。在第三个实例中,将通过任何探针和/或引物组合来检测具有身份rs1683590的SNP,所述组合可以区分以下序列:AATATCTGAAATTTTCCCAGTTTAC[A/G]AGCCTCTGACGTAACCGTCCTCTCT。这可通过来自Invitrogen的市售的测定 (目录号4351379 ID: C___3153459_10)而进行。对于TaqMan?基因分型测定,你必须在每个反应孔中添加相当于1-10 ng的DNA模板。为了定量测定基因组DNA,使用可靠方法例如A260测定。如制造商所述,通过旋转瓶子而彻底混合TaqMan? GTXpress? Master Mix (Invitrogen (目录号4403311))并与TaqMan?基因分型测定和基因组DNA模板混合。在可兼容的PCR仪器(例如ABI PRISM? 7900HT序列检测系统,具有FAST组件)中进行PCR达40循环(首先95℃ 20秒,然后40循环与引物退火并在60℃延伸20秒,然后在95℃变性3秒。PCR扩增后,你在实时PCR上进行终点读板。使用来自Invitrogen的SDS软件,其使用读板期间进行的来自各孔的荧光测定,然后对信号值作图。所述软件确定哪个等位基因在各孔样品中,用于随后的等位基因区别分析。 In one example, a blood sample is collected from a patient, and genomic DNA is isolated by reagent DNAzol™ (Becton Dickinson) according to the manufacturer's instructions. Briefly, mix 1 ml DNAzol™ with 0.5 ml whole blood by vortexing or mixing by hand. Precipitate DNA from samples by adding 0.4 ml of isopropanol to DNAzol™ BD-blood lysate. The resulting mixture was vortexed and stored at room temperature for 5 min. The precipitated DNA was centrifuged down by centrifugation at 6,000 × g for 6 min. Remove the supernatant and add 0.5 ml DNAzol to the DNA pellet. Vortex or shake the DNA pellet until completely dissolved. The resulting mixture was centrifuged at 6,000 × g for 5 min. Then, the supernatant was removed and the DNA pellet was washed by mixing with 1 ml of 75% ethanol, followed by centrifugation at 6,000 × g for 5 min. Remove the ethanol without drying, add 200 μl of 8 mM NaOH to the DNA pellet and dissolve the DNA by incubating at room temperature for 3-5 min followed by vortexing. Neutralize the alkaline DNA solution with 0.1 M HEPES. From the isolated DNA, prepare a quantitative polymerase chain reaction (qPCR) with specific determination of the desired SNP. The qPCR setup can be based on TaqMan probes designed to specifically detect the desired SNP. As an example, the SNP with identity rs1683565 will be detected by any probe and/or primer combination that can distinguish the following sequence: AGAGCCCAAAGCTCATGGAAAAGAG[A/G]ATATGAAAGGAGTCCCTGCAGTAGA. This can be done by a commercially available assay from Invitrogen (Catalogue Number: 4351379 ID: C___9612061_10). In another example, the SNP with identity rs1683591 will be detected by any probe and/or primer combination that can distinguish the following sequence: TCTGTCCACAGGCGGGGGTGGAGGG[A/G]ATGGCCGGCCTCACACCATCTGCCA. This can be done by a commercially available assay from Invitrogen (Catalogue Number: 4351379 ID: C___9612100_10). In a third example, the SNP with identity rs1683590 will be detected by any combination of probes and/or primers that can distinguish the following sequence: AATATCTGAAATTTTCCCAGTTTAC[A/G]AGCCTCTGACGTAACCGTCCTCTCT. This can be done by a commercially available assay from Invitrogen (Cat. No. 4351379 ID: C___3153459_10). For TaqMan™ genotyping assays, you must add the equivalent of 1-10 ng of DNA template per reaction well. For the quantitative determination of genomic DNA, reliable methods such as the A260 assay are used. TaqMan™ GTXpress™ Master Mix (Invitrogen (Cat# 4403311)) was mixed thoroughly by swirling the bottle and mixed with TaqMan™ genotyping assay and genomic DNA template as described by the manufacturer. Perform PCR in a compatible PCR instrument (e.g. ABI PRISM® 7900HT Sequence Detection System with FAST module) for 40 cycles (first at 95°C for 20 seconds, then 40 cycles with primer annealing and extension at 60°C for 20 seconds, then at 95°C °C denaturation for 3 seconds. After PCR amplification, you do an endpoint read of the plate on a real-time PCR. Use the SDS software from Invitrogen, which uses the fluorescence measurements from the wells made during the plate read, and then plots the signal values. The The software determines which allele is in each well sample for subsequent allelic discrimination analysis.

Claims (16)

1. the method for the reaction of prediction patient to antiphlogiston, comprise: the information that obtains the expression level of one or more genes of Fig. 1 in the biological sample from described patient, wherein the expression of one or more described genes changes compared with the reference level of described gene, predicts the reaction of described patient to described antiphlogiston.
2. the method for the reaction of prediction patient to antiphlogiston, comprising:
A. in the biological sample from described patient, detect the expression level of one or more genes of Fig. 1, and
B. by the reference level comparison of described level and described gene,
Wherein the expression of one or more described genes changes compared with described reference level, predicts the reaction of described patient to described antiphlogiston.
3. qualification has the experimenter's of the possibility of increase method to the reaction of antiphlogiston, comprise: the information that obtains the expression level of one or more genes of Fig. 1 in the biological sample from described experimenter, wherein the expression of one or more described genes changes compared with the reference level of described gene, shows to have identified the experimenter reaction of antiphlogiston to the possibility of increase.
4. qualification has the patient's of the possibility of increase method to the reaction of antiphlogiston, comprising:
A. in the biological sample from described patient, detect the expression level of one or more genes of Fig. 1
B. by the reference level comparison of described level and described gene,
Wherein the expression of one or more described genes changes compared with the reference level of described gene, shows to have identified the patient reaction of antiphlogiston to the possibility of increase.
5. the method for any one in aforementioned claim,
A. wherein compared with reference level, the expression of the change of the gene of Figure 1A increases, and/or
B. wherein compared with reference level, the expression of the change of the gene of Figure 1B reduces.
6. the method for any one in aforementioned claim, is wherein used PCR, and for example multiplex PCR or qRT-PCR, or micro-array chip, based on mRNA, detect described expression level in blood sample.
7. the method for any one in aforementioned claim, wherein the expression level of Complement Factor D (CFD) is higher than reference level.
8. the method for any one in aforementioned claim,
A. wherein measure the expression level of Complement Factor D (CFD) by qRT-PCR and wherein use Assay ID:Hs00157263_m1 (Applied Biosystems) detection transcript to there is cycle threshold (Ct) 30, or
B. the expression level and the wherein said expression level that wherein use micro-array chip to measure Complement Factor D (CFD) exceed 9.5 in the log2 scale of RMA or GC-RMA normalized expression value, or
C. wherein measure the expression level of Complement Factor D (CFD) by qRT-PCR and wherein use the Assay ID:Hs00157263_m1 (Applied Biosystems/ Invitrogen) for CFD, the absolute number that detects transcript is at least 0.04 CFD copy/beta-actin mRNA copy, or
D. wherein express the expression level of relevant SNP indirect measurement Complement Factor D (CFD) according to one or more CFD.
9. in patient, treat the method for inflammatory diseases or illness, comprise and give described patient by the antiphlogiston of therapeutic dose, wherein, before giving described antiphlogiston, in claim 1-8, at least one test of any one has shown in the biological sample from described patient, compared with reference level, the expression level of one or more genes of Fig. 1 is changed; Wherein the expression level of one or more described genes changes compared with the reference level of described gene, predicts the reaction of described patient to described antiphlogiston.
10. the method for any one in aforementioned claim, wherein said experimenter or patient suffer from autoimmune disease or illness, for example rheumatoid arthritis (RA), systemic lupus erythematous (SLE), multiple sclerosis (MS), inflammatory bowel (IBD), psoriasis arthropathica (PSA) or psoriatic.
The method of any one in 11. aforementioned claims, wherein said antiphlogiston is one or more the antagonist in IL-19, IL-20 and IL-24.
The method of any one in 12. aforementioned claims, wherein said antiphlogiston is anti-human IL-20 antibody.
The antiphlogiston of 13. treatment autoimmune diseases or illnesss, wherein, compared with the reference level of one or more genes of Fig. 1, the expression that patient has one or more genes of Fig. 1 changes.
14. goods, comprise pharmaceutical composition packaging together and label, described pharmaceutical composition comprises antiphlogiston and pharmaceutically acceptable carrier, and described label illustrates that described pharmaceutical composition can be used for the patient that treatment suffers from autoimmune disease or illness and has the expression change of one or more genes of Fig. 1.
15. test kits, comprising:
A. comprise one or more compositions of at least one detection reagent, described detection reagent is used for the expression level of one or more genes of measuring table 1A and/or table 1B, and
B. how that expression level is associated with experimenter's reaction possibility the working instructions of described test kit, comprise.
The test kit of 16. claims 15, wherein said detection reagent is used for measuring the expression of Complement Factor D (CFD).
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