CN1930300A - In vitro test system for predicting patient tolerability of therapeutic agents - Google Patents

In vitro test system for predicting patient tolerability of therapeutic agents Download PDF

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CN1930300A
CN1930300A CN 200580007070 CN200580007070A CN1930300A CN 1930300 A CN1930300 A CN 1930300A CN 200580007070 CN200580007070 CN 200580007070 CN 200580007070 A CN200580007070 A CN 200580007070A CN 1930300 A CN1930300 A CN 1930300A
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曹颖
K·德尼斯-米泽
S·E·威尔森
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希龙公司
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Abstract

描述了预测患者对治疗药物如细胞因子、淋巴因子和免疫毒素的耐受性的方法。 We describe a prediction as a method of treating patients with drug resistant cytokines, lymphokines, and immunotoxin. 该方法利用血管渗透综合征(VLS)体外模型来评价药物对大分子蛋白质通过内皮细胞(EC)融合单层的作用。 The method to evaluate the effects of drugs on a confluent monolayer of protein molecules by endothelial cells (EC) by using vascular permeability syndrome (VLS) in vitro model.

Description

预测患者治疗药物耐受性的体外试验系统 Patients predicted drug resistance in vitro test system

技术领域 FIELD

本发明一般涉及体外分析方法。 The present invention relates generally to in vitro analytical methods. 具体地说,本发明涉及预测患者对特定治疗药物,包括免疫治疗药物如IL-2突变蛋白的耐受能力的方法。 More specifically, the present invention relates to a method for specific therapeutic agents, including tolerance immunotherapeutic drugs, such as IL-2 mutein of predicting patient.

背景技术 Background technique

白介素-2(IL-2)是天然杀伤细胞(NK)和T-细胞增殖和功能的强效刺激物(Morgan等,(1976),Science 193:1007-1011)。 Interleukin -2 (IL-2) is a natural killer (NK) cells and T- cell proliferation and function potent stimulator (Morgan et, (1976), Science 193: 1007-1011). 这种天然存在的淋巴因子单独或与淋巴因子激活的杀伤(LAK)细胞或肿瘤浸润淋巴细胞(TIL)联合(使用)显示对各种恶性肿瘤具有抗肿瘤活性(参见,例如Rosenberg等,(1987),N.Engl.J.Med.,316:889-897;Rosenberg,(1988),Ann.Surg.,208:121-135;Topalian等,(1988),J.Clin.Oncol.6:839-853;Rosenberg等,(1988),N.Engl.J.Med.,319:1676-1680;和Weber等,(1992),J.Clin.Oncol.,10:33-40)。 This naturally occurring lymphokine alone or with lymphokine activated killer (LAK) cells or tumor-infiltrating lymphocytes (of TIL) combined (used) exhibit an antitumor activity (see for various malignancies, e.g. Rosenberg et al., (1987 ), N.Engl.J.Med, 316: 889-897; Rosenberg, (1988), Ann.Surg, 208:.. 121-135; Topalian et, (1988), J.Clin.Oncol.6: 839 -853; Rosenberg et, (1988), N.Engl.J.Med, 319:. 1676-1680; and Weber et al, (1992), J.Clin.Oncol, 10: 33-40).. 在转移性黑色素瘤和肾细胞癌患者中,当使用Proleukin(一种从Chiron Corporation,Emeryville,CA商品化可购得的IL-2制剂)时,IL-2的抗肿瘤活性得到最充分的描述。 In patients with metastatic melanoma and renal cell carcinoma, when Proleukin (one kind of Chiron Corporation, Emeryville, IL-2 formulation available from commercial CA), the anti-tumor activity of IL-2 to give the fullest description. 其它疾病,包括淋巴瘤也显示对IL-2治疗有反应(Gisselbrecht等,(1994),Blood,83(8):2020-2022)。 Other diseases, including lymphoma, also showed a therapeutic response to IL-2 (Gisselbrecht et, (1994), Blood, 83 (8): 2020-2022).

也可使用具有抗肿瘤活性的许多其它治疗药物来治疗癌症。 It may also be used to treat many other cancer therapeutic agents having anti-tumor activity. 这些药物包括白介素如IL-3、IL-4、干扰素(IFN)-α、GM-CSF、抗神经节苷抗体、环孢素A、环磷酰胺、丝裂霉素C、FK973、野百合碱吡咯和阿糖胞苷;以及许多免疫毒素,例如由蓖麻毒蛋白A链(RTA)、封端的蓖麻毒蛋白(blR)、皂草素(SAP)、美洲商陆抗病毒蛋白(PAP)和假单胞菌外毒素(PE)构建的免疫毒素。 Such agents include interleukins, such as IL-3, IL-4, interferon (IFN) -α, GM-CSF, an anti-ganglioside antibody, cyclosporin A, cyclophosphamide, mitomycin C, FK973, lily alkali pyrrole and cytarabine; and many immunotoxins, for example ricin a chain (the RTA), blocked ricin (BLR), saporin (the SAP), pokeweed antiviral protein (PAP ) and Pseudomonas exotoxin (PE) immunotoxin construct.

然而,许多这些治疗剂的治疗应用受到其差的耐受性和毒性的限制。 However, many of these therapeutic agents in therapeutic use is limited by its poor tolerability and toxicity. 例如,高剂量IL-2免疫治疗常伴随着严重的毒副作用,最显著的有血管渗漏综合征(VLS)、严重的流感样症状(发热、寒颤、呕吐)、低血压和神经变化(参见,例如Duggan等,(1992),J.Immunotherapy,12:115-122;Gisselbrecht等,(1994),Blood,83:2081-2085;和Sznol与Parkinson,(1994),Blood,83:2020-2022)。 For example, high-dose IL-2 immunotherapy is often associated with serious side effects, most notably vascular leak syndrome (VLS), severe flu-like symptoms (fever, chills, vomiting), low blood pressure and neurological changes (see , Duggan et e.g., (1992), J.Immunotherapy, 12: 115-122; Gisselbrecht et, (1994), Blood, 83: 2081-2085; and Sznol and Parkinson, (1994), Blood, 83: 2020-2022 ). 采用如上所述的其它化学治疗药物时也可观察到VLS。 VLS may be observed when using other chemotherapeutic drugs as described above. VLS的机制可能是由于活化的PBMC与内皮细胞相互作用所介导的内皮损伤。 VLS mechanism may be due to activated PBMC and endothelial cell interaction mediated by endothelial injury. 细胞因子、炎症介质或药物固有的结构基序的产生(Baluna和Vitetta(1997)Immunopharmacology 37:117-132;Baluna等,(1999)Proc.Natl.Acad.Sci.USA 96:3957-3962)。 Cytokines, inherent structural motifs or drug to produce inflammatory mediators (Baluna and Vitetta (1997) Immunopharmacology 37: 117-132; Baluna et, (1999) Proc.Natl.Acad.Sci.USA 96: 3957-3962). 虽然尚不清楚毒性和VLS的确切机制,但累积的数据提示,由于激活单核细胞/巨噬细胞的致炎细胞因子如IFN-γ、TNF-α、TNF-β、IL-1β和IL-6的过度产生,IL-2-诱导的天然杀伤(NK)细胞触发了剂量限制性毒性(DLT),诱导一氧化氮(NO)产生,导致后续内皮细胞的损伤(Dubinett等,1994;Samlowski等,1995)。 Although the exact mechanism is unclear toxicity and VLS, but the cumulative data suggest that, due to activation of monocytes / pro-inflammatory cytokines such as macrophages IFN-γ, TNF-α, TNF-β, IL-1β and IL- overproduction 6, IL-2- induced natural killer (NK) cells triggers the dose-limiting toxicity (the DLT), inducible nitric oxide (NO) is generated, resulting in subsequent endothelial cell injury (Dubinett et, 1994; Samlowski et , 1995).

已开发了几种IL-2突变蛋白用来克服天然分子所显示的毒性。 Several have been developed for IL-2 muteins against the toxicity of the native molecule shown. 这些突变蛋白的例子参见2004年3月5日提交的共同拥有、共同待批的美国临时专利申请序列号60/550,868。 Examples of these mutant proteins see co-owned 2004 March 5 filed, co-pending US Provisional Patent Application Serial No. 60 / 550,868.

已开发了体外模型,用来检测VLS的机制。 In vitro models have been developed for detecting the VLS mechanism. 例如,Damle和Doyle在J.Bacteriol.(1989)142:2660-2669中描述了一种体外试验系统,用来检测IL-2-活化的杀伤(IAK)淋巴细胞和许多细胞因子对穿过内皮细胞单层的经内皮大分子通量的作用。 For example, in Doyle and Damle J.Bacteriol (1989) 142:. 2660-2669 describes an in vitro assay system to detect IL-2- activated killer (the IAK) lymphocytes and a number of cytokines on endothelial through macromolecule transendothelial cell monolayers flux effect. 该系统测定穿过人脐带内皮细胞的FITC-白蛋白通量。 The assay system FITC- albumin flux through human umbilical endothelial cells. Kotasek等,Cancer Res.(1988)48:5528-5532描述了一种体外试验,用来测定淋巴因子活化的细胞介导的细胞毒机制,也采用内皮细胞单层。 Kotasek etc., Cancer Res (1988) 48:. 5528-5532 describes an in vitro assay for measuring cytotoxic mechanism mediated lymphokine activated cells, endothelial monolayer also be employed. Lindstrom等,Blood(1997)90:2323-2334,描述了一种用于毒素介导的VLS的体外模型,采用生长在微孔支持物上,并在存在或不存在蓖麻毒蛋白毒素A链的情况下,低压下培养的人内皮细胞。 Lindstrom et, Blood (1997) 90: 2323-2334, describes an in vitro model for VLS toxin-mediated, using grown on a microporous support, and the presence or ricin toxin A chain is present in the case of cultured human endothelial cells under low pressure.

虽然采用了这些试验系统来研究VLS的机制,但迄今尚未采用这些系统来预测患者对各种治疗,例如采用修饰的淋巴因子和化学治疗免疫毒素的免疫治疗的耐受性。 Although the use of these experimental system to study the VLS mechanism, but so far has not been used to predict patients various treatments, such as the use of modified lymphokines and chemical treatment was well tolerated immunotherapy immunotoxin adoption of these systems.

发明概述本发明提供一种用于预测患者对特定治疗药物如免疫治疗药物的耐受能力的简单有效的体外分析方法,从而预测这种分子的治疗应用。 SUMMARY The present invention provides a simple and effective method for in vitro analysis of a particular therapeutic drugs such as drugs immune tolerance of a patient for the prediction to predict the therapeutic use of this molecule. 该方法利用检测蛋白质通过内皮细胞单层的渗漏的分析系统,作为受试治疗耐受性的预测因子。 The method using the detection of protein leakage through the endothelial cell monolayer assay system, as a predictor of treatment of a subject tolerance. 这种分析尤其适合在人体中预测各种免疫治疗的耐受性,因为人的DLT(发热/寒颤,VLS和低血压)都与致炎细胞因子和一氧化氮(NO)的产生具有衍生相关性。 This analysis is particularly suitable for the treatment of various immune tolerance prediction in humans, because the person is the DLT (fever / chills, hypotension, and the VLS) and have derived the production of nitric oxide (NO), induced by inflammatory cytokines associated sex.

因此,在一个实施方式中,本发明涉及一种预测患者对选定治疗药物耐受或不耐受的体外方法。 Thus, in one embodiment, the present invention relates to an in vitro method of predicting drug resistance or intolerance to treatment of selected patients. 该方法包括:(a)提供贴附于粘附基质的内皮细胞的融合单层;(b)使该单层与以下成分接触:(i)选定的治疗药物或淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用治疗药物或LAK细胞的上清液激活外周血单核细胞产生LAK细胞;和(ii)可检测的标记大分子,其中,当融合单层保持完整时,所述融合单层可基本上保留该可检测的标记大分子;(c)在该单层的完整性受损时,可检测的标记大分子能通过该融合单层和粘附基质的条件下,孵育步骤(b)的单层一段时间;和(d)检测穿过融合单层和粘附基质的大分子,作为患者对特定治疗药物耐受或不耐受的指标。 The method comprising: (a) providing adherent stromal attached to confluent monolayers of endothelial cells; (b) The following components in contact with the monolayer: (i) a selected therapeutic or lymphokine activated killer (LAK ) cell preparation, wherein generating LAK cells by activating peripheral blood mononuclear cells or supernatant of LAK cell therapy; macromolecular markers and (ii) a detectable, wherein, when confluent monolayer intact, the confluent monolayer of molecules that can substantially retain detectable marker; (c) when the integrity of the monolayer is compromised, the labeled macromolecules can be detected under the conditions of a confluent monolayer and adherent matrix, incubation step (b) a single period of time; and (d) detecting through the confluent monolayer and adherent matrix of macromolecules as indicators of a patient to a particular therapeutic drug tolerance or intolerance.

在该方法的某些实施方式中,治疗药物是免疫治疗剂如IL-2突变蛋白,或免疫毒素,或小分子化学治疗剂。 In certain embodiments of the method, the therapeutic agent is an immunotherapeutic agents such as IL-2 muteins, or immunotoxins, or a small molecule chemotherapeutic agent.

在其它实施方式中,本方法中使用的粘附基质包括胶原基质。 In other embodiments, the adhesive used in the method of the present matrix comprises a collagen matrix.

在又一些实施方式中,本方法中使用的内皮细胞是人脐静脉内皮细胞(HUVEC)。 In still other embodiments, endothelial cells used in this method is a human umbilical vein endothelial cells (HUVEC).

在其它实施方式中,本方法中使用的可检测的标记大分子是可检测的标记白蛋白,例如标记的牛血清白蛋白(BSA)。 In other embodiments, the labeled macromolecules can be detected in the present method is used in a detectable marker albumin, e.g. labeled bovine serum albumin (BSA). BSA可被荧光标记,例如用FITC标记。 BSA may be fluorescently labeled, for example, labeled with FITC.

在又一些其它的实施方式中,本发明涉及预测患者对IL-2突变蛋白耐受或不耐受的体外方法。 In still other embodiments, the present invention relates to in vitro methods or predicted protein tolerant patients intolerant of mutant IL-2. 该方法包括:(a)提供贴附于粘附基质的内皮细胞融合单层;(b)使该单层与以下成分接触:(i)淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用IL-2突变蛋白激活外周血单核细胞产生LAK细胞,和(ii)可检测的标记大分子,其中,融合单层保持完整时,所述融合单层可基本上保留该可检测的标记大分子;(c)在该单层的完整性受损时,可检测的标记大分子能通过该融合单层和粘附基质的条件下,孵育步骤(b)的单层一段时间;和(d)检测穿过融合单层和粘附基质的大分子,作为患者对IL-2突变蛋白耐受或不耐受的指标。 The method comprising: (a) providing adherent stromal attached to confluent monolayers of endothelial cells; (b) The single component in contact with the following: (i) a lymphokine activated killer (LAK) cell preparation, wherein, by activated peripheral blood mononuclear cells to produce LAK cells, labeled macromolecules, and (ii) can be detected by the IL-2 mutein, wherein, when confluent monolayer intact, the confluent monolayer may substantially retain the detectable label macromolecules; (c) when the integrity of the monolayer is compromised, the labeled macromolecules can be detected under the conditions of a confluent monolayer and adherent matrix, incubation in step (b) a single period of time; and ( d) detecting through the confluent monolayer and adherent matrix of macromolecules as IL-2 patients resistant or intolerant muteins indicators.

在某些实施方式中,本方法中使用的粘附基质包括胶原基质。 In certain embodiments, the present method includes the use of adherent stromal collagen matrix.

在另一些实施方式中,本方法中使用的内皮细胞是人脐静脉内皮细胞(HUVEC)。 In other embodiments, endothelial cells used in this method is a human umbilical vein endothelial cells (HUVEC).

在其它实施方式中,本方法中使用的可检测的标记大分子是可检测的标记白蛋白,例如标记的BSA。 In other embodiments, the labeled macromolecules can be detected in the present method is used in the detection of labeled albumin, such as a labeled BSA. BSA可被荧光标记,例如用FITC标记。 BSA may be fluorescently labeled, for example, labeled with FITC.

在另一个实施方式中,本发明涉及预测患者对IL-2突变蛋白耐受或不耐受的体外方法。 In another embodiment, the present invention relates to a method of in vitro protein prediction tolerant patients intolerant or IL-2 mutant. 该方法包括:(a)提供贴附于粘附基质如胶原基质的人脐静脉内皮细胞(HUVEC)的融合单层;(b)使该单层与以下成分接触:(i)淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用IL-2突变蛋白激活外周血单核细胞产生LAK细胞,和(ii)荧光标记的白蛋白;(c)在该单层的完整性受损时,标记荧光白蛋白能通过该融合单层和粘附基质的条件下,孵育步骤(b)的单层一段时间;和(d)检测穿过融合单层的荧光标记的白蛋白,作为患者对IL-2突变蛋白耐受或不耐受的指标。 The method comprising: (a) providing adherent matrix is ​​attached to the collagen matrix, such as human umbilical vein endothelial cells (HUVEC) confluent monolayers; (b) The following components in contact with the monolayer: (i) lymphokine activated killer (LAK) cell preparation, wherein the generating LAK cells, and (ii) a fluorescent labeled albumin by activating peripheral blood mononuclear cells with IL-2 mutein; (c) damaged when the integrity of the monolayer, albumin by labeling fluorescent under which a confluent monolayer and adherent matrix, single incubation period of step (b) a; and (d) detecting the fluorescence labeled albumin fusion single pass through a patient IL -2 mutein tolerance or intolerance index.

在其它实施方式中,荧光标记的白蛋白是BSA。 In other embodiments, the fluorescent-labeled albumin is BSA. BSA可被荧光标记,例如用FITC标记。 BSA may be fluorescently labeled, for example, labeled with FITC.

参考本说明书,本领域技术人员容易明白本发明的这些和其它实施方式。 Reference to this specification, those skilled in the art will readily understand that these and other embodiments of the present invention.

附图简要说明图1A-1C描述了在分别来自三位不同对象的刺激培养物上清液的存在下,采用25nM IL-2突变蛋白-刺激的LAK细胞进行实验的结果。 BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C is described in the presence of culture supernatant of each stimulus from three different objects using 25nM IL-2 muteins - LAK cells stimulated results of tests performed. 使用荧光强度作为与LAK细胞和上清液孵育22小时后,FITC-BSA穿过HUVEC单层的迁移测定指标。 Using a fluorescent intensity as 22 hours after incubation with LAK cells and supernatant, FITC-BSA through the HUVEC monolayer migration assay indicators. *:与培养液对照相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);$:与Proleukin相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);&amp;:与F42E相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);@:与rIL-2相比,P值<0.1,t检验:配对比较两样品的平均值(n=5)。 *: Compared with control culture medium, P value <0.1, t test: Comparison of the average of two paired samples (n = 5); $: as compared with Proleukin, P value <0.1, t test: paired two sample comparison the average (n = 5); & amp ;: comparison with F42E, P value <0.1, t test: comparison of the average of two paired samples (n = 5); @: compared with rIL-2, P-value < 0.1, t test: paired samples comparing the average of two (n = 5).

图2A-2C描述了采用分别来自三位不同对象的25nM IL-2突变蛋白-刺激的LAK细胞进行实验的结果。 Figures 2A-2C describe the use of 25nM IL-2 muteins were from three different objects - LAK cells stimulated results for experiments. 使用荧光强度作为与不含上清液的LAK细胞孵育22小时后,FITC-BSA穿过HUVEC单层的迁移测定指标。 After using the fluorescent intensity of the LAK cell-free supernatants were incubated for 22 hours, FITC-BSA through HUVEC monolayer migration assay indicators. *:与培养液对照相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);$:与Proleukin相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);&amp;:与F42E相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);@:与rIL-2相比,P值<0.1,t检验:配对比较两样品的平均值(n=5)。 *: Compared with control culture medium, P value <0.1, t test: Comparison of the average of two paired samples (n = 5); $: as compared with Proleukin, P value <0.1, t test: paired two sample comparison the average (n = 5); & amp ;: comparison with F42E, P value <0.1, t test: comparison of the average of two paired samples (n = 5); @: compared with rIL-2, P-value < 0.1, t test: paired samples comparing the average of two (n = 5).

图3A-3C显示了采用分别来自三位不同对象的25nM IL-2突变蛋白-刺激的LAK细胞的上清液进行实验的结果。 Figures 3A-3C show the use of 25nM IL-2 muteins were from three different objects - LAK cells stimulated supernate of the experiment. 使用荧光强度作为与上清液孵育22小时后,FITC-BSA穿过HUVEC单层的迁移测定指标。 Using the fluorescent intensity after 22 hours incubation the supernatant, FITC-BSA through the HUVEC monolayer migration assay indicators. *:与培养液对照相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);$:与Proleukin相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);&amp;:与F42E相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);@:与rIL-2相比,P值<0.1,t检验:配对比较两样品的平均值(n=5)。 *: Compared with control culture medium, P value <0.1, t test: Comparison of the average of two paired samples (n = 5); $: as compared with Proleukin, P value <0.1, t test: paired two sample comparison the average (n = 5); & amp ;: comparison with F42E, P value <0.1, t test: comparison of the average of two paired samples (n = 5); @: compared with rIL-2, P-value < 0.1, t test: paired samples comparing the average of two (n = 5).

图4A-4C显示了采用25nM IL-2突变蛋白进行三次独立实验的结果。 Figures 4A-4C show the results of using 25nM IL-2 muteins three separate experiments. 使用荧光强度作为与IL-2孵育22小时后,FITC-BSA穿过HUVEC单层的迁移测定指标。 Using the fluorescent intensity after 22 hours incubation IL-2, FITC-BSA through the HUVEC monolayer migration assay indicators. *:与培养液对照相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);$:与Proleukin相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);&amp;:与F42E相比,P值<0.1,t检验:配对比较两样品的平均值(n=5);@:与rIL-2相比,P值<0.1,t检验:配对比较两样品的平均值(n=5)。 *: Compared with control culture medium, P value <0.1, t test: Comparison of the average of two paired samples (n = 5); $: as compared with Proleukin, P value <0.1, t test: paired two sample comparison the average (n = 5); & amp ;: comparison with F42E, P value <0.1, t test: comparison of the average of two paired samples (n = 5); @: compared with rIL-2, P-value < 0.1, t test: paired samples comparing the average of two (n = 5).

发明详述除非另有说明,本发明的实施采用本领域内药理学、化学、生物化学、重组DNA技术、免疫学的常规技术。 DETAILED DESCRIPTION Unless otherwise indicated, the embodiment of the present invention using conventional techniques in the art of pharmacology, chemistry, biochemistry, recombinant DNA technology, immunology. 文献中详细解释了这些技术。 Document Such techniques are explained in detail. 例如,参见《实验免疫学手册》(Handbook of Experimental Immunology),第I-IV卷(DMWeir和CCBlackwell编,Blackwell Scientific Publications);ALLehninger,Biochemistry(Worth Publishers,Inc.,目前的附刊);Sambrook等,Molecular Cloning:A LaboratoryManual(第2版,1989);Methods In Enzymology(S.Colowick和N.Kaplan编,Academic Press,Inc.)。 For example, see "Experimental Immunology Handbook" (Handbook of Experimental Immunology), the first I-IV volume (DM Weir and CCBlackwell eds, Blackwell Scientific Publications); ALLehninger, Biochemistry (. Worth Publishers, Inc, currently attached published); Sambrook et , Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N.Kaplan eds, Academic Press, Inc.).

I.定义为了描述本发明,将采用以下术语,其定义如下所述。 I. Definitions In order to describe the present invention, the following terms will be employed, which is defined as follows.

应当注意,如说明书和权利要求书所述,除非内容中清楚地另行指出,单数形式“一”、“一个”和“这个”包括复数形式。 It should be noted that, as the book specification and claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms. 因此,例如,“一细胞”包括两个或多个细胞,等等。 Thus, for example, "a cell" includes two or more cells, and the like.

术语“包含”涵盖了“由……组成”和“含有”,例如,组合物“包含”X可以是只由X构成,或是包含一些其它成分,例如X+Y。 The term "comprising" encompasses "consisting ...... consisting of" and "comprising", e.g., a composition "comprising" X may be composed of only X, or contain other ingredients, such as X + Y.

术语“基本上”不排除“完全”,例如,组合物“基本上不含”Y可以是完全不含Y。 The term "substantially" does not exclude "completely" e.g., the composition "substantially free of" Y may be completely free from Y. 必要时,本发明定义中可省略“基本上”。 If necessary, definition of the invention may be omitted "substantially."

术语“来源于”在这里指分子的来源,而不限于分子制备的方法(例如,化学合成或重组方法)。 The term "derived" herein refers to the source of molecules, without being limited to the method (e.g., chemical synthesis or recombinant methods) preparing a molecule.

术语“免疫治疗剂”在这里指可用于治疗癌症的免疫促进剂或免疫抑制剂。 The term "immunotherapeutic agent" used herein refers to the treatment of cancer or immune enhancers immunosuppressant. 这些药剂包括但不限于各种细胞因子和淋巴因子,例如各种白介素,如IL-1、IL-2、IL-3、IL-4、IL-5、IL-12和这些分子的突变蛋白;干扰素,包括但不限于IIFN-α、IFN-β、IFN-γ及其突变蛋白;集落刺激因子如GM-CSF和GM-CSF的突变蛋白;肿瘤坏死因子,例如TNF-α和TNF-β及这些分子的突变蛋白。 These agents include but are not limited to, various cytokines and lymphokines, such as various interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-12 and these molecules mutein; interferons, including but not limited to IIFN-α, IFN-β, IFN-γ and its muteins; colony stimulating factors such as GM-CSF and muteins of GM-CSF; tumor necrosis factor such as TNF-α and TNF-β and muteins of these molecules. 术语“免疫治疗剂”还包括免疫毒素。 The term "immunotherapeutic agent" also includes immunotoxins. “免疫毒素”指抗体-毒素偶合物,用于破坏具有与该抗体同源的抗原的特定靶细胞(例如,肿瘤细胞)。 "Immunotoxin" refers to an antibody - toxin conjugate, for destroying specific target cells (e.g., tumor cells) with homology with the antigens. 与这种抗体偶合毒素的例子包括但不限于:蓖麻毒蛋白A链(RTA)、封端的蓖麻毒蛋白(blR)、皂草素(SAP)、美洲商陆抗病毒蛋白(PAP)和假单胞菌外毒素(PE),以及其它毒性化合物,例如放射性同位素和其它化学治疗药物。 Examples of coupling toxins with such antibodies include, but are not limited to: ricin A chain (RTA), blocked ricin (blR), saporin (SAP), pokeweed antiviral protein (PAP), and Pseudomonas exotoxin (PE), and other toxic compounds, such as other radioisotopes and chemotherapeutic drugs.

术语“IL-2”在这里指来源于淋巴因子的蛋白质,淋巴因子由正常外周血淋巴细胞产生,体内低浓度存在。 The term "IL-2" herein refers to a protein derived from a lymphokine, lymphokines produced by normal peripheral blood lymphocytes, the presence of low concentrations in vivo. IL-2首先由Morgan等,(1976),Science,193:1007-1008描述,由于它能诱导激活的T淋巴细胞增殖而最初称为T细胞生长因子。 IL-2 first described by Morgan et al., (1976), Science, 193: 1007-1008 described, since the proliferation of T lymphocytes can induce activation and originally called T cell growth factor. 这是一种报道分子量为13,000-17,000的蛋白质(Gillis和Watson,(1980),J.Exp.Med.,159:1709),其等电点为6-8.5。 This is a report of 13,000-17,000 molecular weight protein (Gillis and Watson, (1980), J.Exp.Med, 159:. 1709), its isoelectric point is 6-8.5. 该定义包括全长度IL-2蛋白及其生物学活性片段,该术语还包括IL-2的表达后修饰,例如,糖基化、乙酰化、磷酸化等。 This definition includes full-length IL-2 proteins and biologically active fragments, the term also includes post-expression modifications of IL-2, for example, glycosylation, acetylation, phosphorylation and the like.

术语“突变蛋白”在这里指包括修饰的蛋白质,例如,对原有序列的删除、去头、添加和取代。 The term "mutein" as used herein refers to a modified protein comprising, e.g., to delete the original sequence, to the head, additions and substitutions. 一般,该蛋白质保持生物学活性,即抗肿瘤活性。 Typically, the protein to retain biological activity, i.e., anti-tumor activity. 这些修饰可以是精心设计的,例如通过定向诱变,或是意外的,例如通过产生该蛋白质的宿主的突变或由于PCR扩增的误差。 These modifications may be designed, for example, by site-directed mutagenesis, or accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. 术语“突变蛋白”可与术语“变体”和“类似物”互换使用。 The term "muteins" may be used interchangeably with the term "variant" and "analog" are used interchangeably. 这些突变蛋白的氨基酸序列与参比序列具有高度序列同源性,例如,当两个序列比对时,氨基酸序列同源性大于50%,通常大于60-70%,甚至大于80-85%或以上,例如至少90-95%或以上。 These muteins of the amino acid sequence of the reference sequence having a high degree of sequence homology, e.g., when the two sequences, the amino acid sequence homology of more than 50%, usually greater than 60-70%, 80-85%, or even greater than above, for example at least 90-95% or more. 常常,类似物包括相同数量的氨基酸但包括取代物,如本文所述。 Often, the same number of analogs include amino acid substitutions include, but, as described herein. 制备多肽突变蛋白的方法是本领域已知的,如下所述。 The method of preparing a polypeptide muteins are known in the art, as described below.

突变蛋白包括性质上保守性或非保守性的取代。 Muteins include conservative substitutions or non-conservative in nature. 保守性取代指发生在与其侧链相关的氨基酸家族中的取代。 Refers to conservative substitutions occur at amino acid side chain family associated therewith substitutions. 具体地说,氨基酸通常分为四个家族:(1)酸性--天冬氨酸和谷氨酸;(2)碱性--赖氨酸、精氨酸、组氨酸;(3)非极性--丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸;和(4)不带电荷的极性--甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。 Specifically, amino acids are generally divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non- polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; polarity and (4) uncharged - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. 苯丙氨酸、色氨酸和酪氨酸有时也可归类为芳香族氨基酸。 Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. 例如,可合理地预测,用异亮氨酸或缬氨酸独立地取代亮氨酸,用谷氨酸独立地取代天冬氨酸,用丝氨酸独立地取代苏氨酸,或用结构上相关的氨基酸类似地保守性取代一个氨基酸,将不会对生物学活性有重大影响。 For example, can be reasonably predicted, substituted with isoleucine or valine, leucine, independently, is independently substituted with glutamic acid, aspartic acid, serine independently substituted with threonine, or with a structurally related Similarly conservative amino acid substitution of one amino acid, will not have a significant impact on the biological activity. 例如,感兴趣的蛋白质可包括多达约5-10个保守性或非保守性氨基酸取代,或甚至多达约15-25个、50个或75个保守性或非保守性氨基酸取代,或5-75之间的任何整数,只要不影响该分子的所需功能。 For example, the protein of interest may include up to about 5-10 conservative substituted or non-conservative amino acids, or even up to about 15-25, 50 or 75 substituted with conservative or non-conservative amino acids, or 5 any integer between -75, it does not affect the desired function of the molecule. 本领域技术人员容易确定感兴趣的蛋白质耐受改变的区域。 Those skilled in the art can readily determine regions of the protein of interest is resistant to change.

术语“突变蛋白”也指天然分子的衍生物。 The term "mutein" also refers to derivatives of the natural molecule. “衍生物”指感兴趣天然多肽、天然多肽片段或它们各自的类似物的任何合适的修饰,例如糖基化、磷酸化、聚合物偶联(如与聚乙二醇偶联)或加入其它外来成分的类似物,只要保留天然多肽所需的生物学活性。 "Derivative" refers to any suitable modification of the native polypeptide of interest, or their respective native polypeptide fragment analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol conjugated) was added, or other analogs extraneous components, as long as the native polypeptide retains a desired biological activity. 制备多肽片段、类似物和衍生物的方法是本领域公知的。 Preparation of polypeptide fragments, analogs and derivatives thereof are known in the art.

“片段”指仅由完整全长序列和结构的一部分所形成的分子。 A "fragment" refers to a molecule consisting of an intact full length sequence and a portion of the formed structure only. 片段包括天然多肽的C-端删除、N-端删除和/或中间删除。 Polypeptide comprising a fragment of the native C- terminal deletions, N- terminal deletion and / or intermediate deleted. 特定蛋白质的活性片段通常包括至少约5-10个全长分子的连续氨基酸残基,优选至少约15-25个全长分子的连续氨基酸残基,最优选至少约20-50或更多个全长分子的连续氨基酸残基,或5个氨基酸与全长序列之间的任何整数,只要感兴趣片段保留生物学活性,例如抗肿瘤活性,如本文所述。 Active fragments of a particular protein generally comprises at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more full contiguous amino acid residues long molecule, or any integer between 5 amino acids and the full-length sequence, as long as the fragment retains the biological activity of interest, such as an anti-tumor activity, as described herein.

当指多肽时,“分离的”表示所指的分子与完整生物体(该分子在自然界中在该生物体中被发现)离析并分开,或该分子以基本上不存在相同类型的其它生物大分子的状态存在。 When referring to polypeptides, "isolated" indicates whole organism referred molecule (the molecule is found in that organism in nature) isolated and separated, or other biological molecule which is substantially absent of the same type of large presence status molecule. 指多核苷酸时,术语“分离的”指全部或部分地缺少天然状态下与其正常相关的序列的核酸分子;或自然存在,但具有与其相关的异源序列的序列;或从染色体解离的分子。 Refers to a polynucleotide, the term "isolated" refers to a whole or in part the lack of nucleic acid molecules normally associated therewith in its natural state sequence; or naturally occurring, but having heterologous sequences associated with the sequence; or dissociate from chromosomes molecule.

术语“细胞培养”和“组织培养”可互换使用,指在液体介质的悬浮培养液中,或在具有液体介质的表面如玻璃、塑料、琼脂或其它合适的基质上,体外维持细胞。 The term "cell culture" and "tissue culture" are used interchangeably, refer to a liquid suspension culture medium, or on the surface with a liquid medium such as glass, plastic, or other suitable matrix agar, cells are maintained in vitro. 一般来说,“细胞培养”需要缓冲以维持恒定适当pH的介质。 Typically, "cell culture" medium buffered to maintain a constant need to appropriate pH. 通常配制细胞培养中使用的介质,以包含足够的必需营养物质,且渗透压调节到适合需维持的特定细胞,温度和气体也控制在合适的范围内。 Typically used in cell culture media formulated to contain enough of the essential nutrients, and osmotic pressure regulator into specific cells need to maintain a suitable temperature and the gas is also controlled within an appropriate range. 细胞培养技术是本领域已知的。 Cell culture techniques are known in the art. 例如,参见Morgan等,Animal Cell Culture,BIOS Scientific Publishers,Oxford,UK(1993),和Adams,RLPCell Culture for Biochemists,第2版,Elsevier(I990)。 For example, see Morgan et al., Animal Cell Culture, BIOS Scientific Publishers, Oxford, UK (1993), and Adams, RLPCell Culture for Biochemists, 2nd ed., Elsevier (I990).

术语“内皮细胞”在这里指来源于心脏空腔和血管和淋巴管细胞最内层的分化的扁平细胞。 The term "endothelial cells" herein refers to the cavity from the heart and blood vessels and lymphatic cells flat innermost differentiated cells. 内皮细胞来源于中胚层胚胎细胞层。 Endothelial cells derived from embryonic mesoderm cell layer.

内皮细胞的例子如下所述。 Examples of endothelial cells as described below.

“外周血单核细胞”或“PBMC”指采用例如密度离心,从哺乳动物如人的外周血分离的细胞群。 "Peripheral blood mononuclear cells" or "PBMCs" refers to, for example, using density centrifugation, from a mammal such as a human peripheral blood cell populations isolated. 通常,PBMC群包括大多数淋巴细胞和单核细胞,不包括血红细胞和大多数多形核白细胞和粒细胞。 Typically, most of PBMCs group comprising lymphocytes and monocytes, red blood cells and do not include most of polymorphonuclear leukocytes and granulocytes.

“代”指传代培养细胞群的行为。 "Generation" refers to the behavior of cultured cells were passaged population. “传代培养”指用上一次培养的样品接种新鲜无菌培养液建立的细胞培养。 "Subculture" means a culture with the last sample of fresh sterile culture was inoculated cell culture establishment. 每次重复的传代培养计为一次传代。 Each iteration count a subculture passages.

本发明分析中使用的合适的“大分子”是足够大的大分子,除非单层破裂,融合单层可基本上保留该大分子,从而提高大分子通过融合单层的渗透性。 Suitable "macromolecule" analysis in the present invention is sufficiently large macromolecules, unless broken monolayer, confluent monolayers can substantially retain the macromolecule, thereby increasing the permeability of macromolecules confluent monolayers. “提高”的渗透性指与不存在破裂试剂(即用导致血管渗漏综合征的免疫治疗剂刺激LSK细胞)条件下转运通过单层相比,转运通过单层的大分子的量更大,速率更快。 "Improving" refers to fracture permeability agent is absent (i.e. with vascular leakage syndrome lead to immune stimulation therapeutic cells LSK) transport compared by mono-, transported by a larger amount under the condition of single macromolecules, faster rate. 尤其有用的大分子包括血清白蛋白如牛血清白蛋白(BSA)、卵清蛋白、钥孔血蓝蛋白、免疫球蛋白分子、甲状球蛋白及本领域技术人员已知的其它蛋白质。 Particularly useful macromolecules include serum albumin such as bovine serum albumin (BSA), ovalbumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin and other proteins known to those skilled in the art.

如本文所用,术语“标记”和“可检测的标记”指能够检测的分子,包括但不限于,放射性同位素、荧光剂、半导体纳米结晶、化学发光物质、发色团、酶、酶底物、酶辅因子、酶抑制剂、染料、金属离子、金属溶胶、配基(例如,生物素、抗生蛋白链菌素或半抗原)等。 As used herein, the terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescent substances, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens) and the like. 术语“荧光剂”指在检测范围内能够显示荧光的物质或其一部分。 The term "fluorescer" refers to a substance or a portion thereof capable of displaying the fluorescence in the detection range. 本发明中可使用的标记的具体例子包括但不限于:辣根过氧化物酶(HRP),荧光素化合物如称为“FITC”的荧光素5(6)-异氰酸酯或荧光素异氰酸酯异构体I,罗丹明、丹酰、羟基香豆素、二甲基吖啶鎓酯(DMAE)、德克萨斯红、鲁米诺、NADPH和α-β-半乳糖苷酶。 Specific examples of the marker of the invention may be used include, but are not limited to: horseradish peroxidase (HRP), fluorescein compound referred to as "FITC" fluorescein 5 (6) - fluorescein isocyanate or isocyanate isomers I, rhodamine, dansyl, hydroxycoumarin, dimethyl acridinium ester (the DMAE), Texas red, luminol, of NADPH and α-β- galactosidase.

II.实施本发明的方式在详细描述本发明之前,应理解本发明并不限于这些具体制剂或方法参数,这些制剂或方法参数可变化。 II. In the embodiment of the present invention Before describing the invention in detail, it should be understood that the present invention is not limited to particular formulations or process parameters, these formulations or process parameters may vary. 还应理解,本文所用术语仅仅是为了描述本发明的具体实施方式,而不是限制性的。 It should also be understood that the terminology used herein is merely for describing particular embodiments of the present invention, and not restrictive.

虽然可采用许多与本发明所述类似或等价的方法和材料来实施本发明,本文描述了优选的材料和方法。 Although many methods and materials may be employed with the present invention is similar to or equivalent to the present embodiment of the invention, described herein, the preferred materials and methods.

如上所述,采用免疫疗法如IL-2疗法来治疗各种癌症,例如转移性黑色素瘤、肾细胞癌和淋巴瘤。 As described above, the use of immunotherapy such as IL-2 therapy to treat various cancers, for example, metastatic melanoma, renal cell carcinoma and lymphoma. 但是,与免疫疗法相关的严重毒性限制了这些治疗方法,例如血管渗漏综合征(VLS)、严重的流感样症状(发热、寒颤、呕吐)、低血压、神经变化、一氧化氮(NO)产生,导致后续内皮细胞的损伤。 However, severe toxicity associated with immunotherapy limits of these treatment methods, such as vascular leak syndrome (VLS), severe flu-like symptoms (fever, chills, vomiting), low blood pressure, neurological changes, nitric oxide (NO) generation, resulting in subsequent endothelial cell damage. 因此,制备了许多免疫治疗剂的突变蛋白,如淋巴因子突变蛋白,希望能够克服上述副作用。 Thus, a number of muteins of preparing an immunotherapeutic agent, a lymphokine such as muteins, can be desirable to overcome the aforementioned side effects. 本发明提供了试验这些突变蛋白,和其它治疗剂的体外方法,来预测毒性。 The present invention provides muteins of these tests, and other therapeutic agents in vitro method to predict toxicity.

具体地说,本发明基于以下发现:体外方法可用于精确和有效地预测患者对采用特定IL-2突变蛋白的耐受能力,而不发生当患者经历IL-2免疫疗法时所产生的常见副作用。 More specifically, the present invention is based on the discovery: in vitro methods may be used when the common side effects and effectively predict the precise tolerance of the patient using a specific IL-2 mutein, occurs when the patient experiences without IL-2 immunotherapy generated . 该分析方法利用体外内皮渗透性模型来监测大分子通过融合内皮细胞单层的渗漏。 The analysis method using an in vitro model to monitor the permeability of the endothelium leakage through the fusion molecules of the endothelial cell monolayers. 该分析中使用的大分子一般被融合单层保留,或仅少量泄漏。 Analysis of the macromonomer used is generally confluent monolayers retained, or only a small leak. 然而,当细胞单层的完整性受到损伤时,例如,暴露于可引起血管渗漏综合征的治疗药物,或暴露于由引起血管渗漏综合征等的治疗药物刺激产生的LAK细胞(或其上清液),大分子的内皮渗透性增加。 However, when the integrity of the cell monolayer damage, e.g., exposure to therapeutic drugs can cause vascular leakage syndrome, or LAK cells exposed to the therapeutic agent irritation caused by vascular leakage syndrome (or the supernatant), increased permeability of the endothelium macromolecules.

本分析方法尤其适用于预测人对各种基于淋巴因子的免疫疗法如IL-2免疫疗法的耐受性,因为人的DLT(发热/寒颤,VLS和低血压)都与致炎细胞因子和NO具有衍生相关性。 This analysis method is particularly suitable for the prediction-based immunotherapy of various people such as lymphokines IL-2 tolerability immunotherapy, because the DLT person (fever / chills, hypotension, and the VLS) are associated with pro-inflammatory cytokines and NO having derived correlation.

本领域已知许多渗透性试验,可用于检测治疗药物如IL-2突变蛋白。 Many permeability test known in the art, may be used to detect drugs such as IL-2 muteins. 例如,参见Damle和Doyle,J.Bacteriol.(1989)142:2660-2669;Kotasek等,Cancer Res.(1988)48:5528-5532;Stone-Wolff等,J Exp.Med.(1984)159:828;Lindstrom等,Blood(1997)90:2323-2334。 See, eg, Damle and Doyle, J.Bacteriol (1989) 142:. 2660-2669; Kotasek the like, Cancer Res (1988) 48: 5528-5532; Stone-Wolff et, J Exp.Med (1984) 159.:. 828; Lindstrom et, Blood (1997) 90: 2323-2334.

一般来说,这些试验采用具有融合单层的粘附基质,它通常允许可溶性营养物、代谢物和激素因子通过,但基本上阻止细胞迁移通过,除非融合单层的完整性受损。 In general, these experiments using a fusion adherent stromal monolayer, which generally allow soluble nutrients, metabolites and hormones by factors, but basically by blocking cell migration, unless the integrity of the confluent monolayers impaired. 本发明试验中采用的融合单层通常来自内皮细胞,例如血管和淋巴内皮细胞。 Confluent monolayer of test employed in the present invention are typically derived from endothelial cells, such as vascular and lymphatic endothelial cells. 这些细胞的例子包括但不限于:人脐静脉内皮细胞(HUVEC),可根据例如Jaffe等,J.Clin.Invest.(1973)52:2745;Stroncek等,Arteriosclerosis(1986)137:1735-1742所述的方法获得,购自各供应商如美国标准培养菌种库(ATCC),Manassas,VA(例如商品目录CRL-1730)和Cascade Biologics,Poland,OR;WB572细胞(自发转化的人隐静脉平滑肌细胞);SV-E6细胞(稳定转染E6病毒癌基因的人隐静脉细胞);A7R5细胞(自发转化的大鼠胸主动脉平滑肌细胞;ATCC);GH3B6细胞(大鼠脑垂体细胞;ATCC,Bethesda,Md.);PVEC细胞(大鼠肺静脉内皮细胞;J.Tissue CultureRes.(1986)10:9);CPA47(牛内皮细胞;ATCC CRL 1733);CPAE细胞(牛内皮细胞;ATCC CCL 209);EJG细胞(牛内皮细胞;ATCC CRL 8659);FBHE(牛内皮细胞;ATCC CRL 1395);HW-EC-C细胞(人内皮细胞;ATCC CRL 1730);和T/GHA-VSMC细胞(人血管平滑肌细胞;ATCC CRL 1999)。 Examples of such cells include, but are not limited to: human umbilical vein endothelial cells (HUVEC), for example according to Jaffe et al, J.Clin.Invest (1973) 52:. 2745; Stroncek et, Arteriosclerosis (1986) 137: 1735-1742 that the method described is obtained, commercially available from suppliers such as the American Type culture culture Collection (ATCC), Manassas, VA (e.g. catalog CRL-1730) and Cascade Biologics, Poland, OR; human WB572 cells (spontaneously transformed saphenous vein smooth muscle cells ); human SV-E6 cells (stably transfected with viral oncogenes E6 cells saphenous vein); A7r5 cells (spontaneously transformed rat thoracic aortic smooth muscle cells; ATCC); GH3B6 cells (rat pituitary cells; ATCC, Bethesda , Md); PVEC cells (rat pulmonary vein endothelial cells; J.Tissue CultureRes (1986.) 10:. 9); CPA47 (bovine endothelial cells; ATCC CRL 1733); CPAE cells (bovine endothelial cells; ATCC CCL 209); EJG cells (bovine endothelial cells; ATCC CRL 8659); FBHE (bovine endothelial cells; ATCC CRL 1395); HW-EC-C cells (human endothelial cells; ATCC CRL 1730); and T / GHA-VSMC cells (human vascular smooth muscle cells; ATCC CRL 1999).

在使用本发明试验之前,在合适的培养液中常规传代细胞并培养,如本领域技术人员所公知。 Before using the assays of the invention in a suitable culture medium and cultured routinely passaged cells, as those skilled in the art. 例如,可在市售培养液,例如实施例所述的RPMI-10AB培养液;如Damel和Doyle,J.Bacteriol.(1989)142:2660-2669所述的RPMI1640补给性培养液;如Lindstrom等,Blood(1997)90:2323-2334所述的M199补给性培养液;得自Cambrex,Baltimore,MD,含2%胎牛血清的内皮生长培养液以及本领域技术人员已知的其它组织培养介质中,培养内皮细胞。 For example, commercially available medium, for example RPMI-10AB embodiment of the culture broth; as Damel and Doyle, J.Bacteriol (1989) 142: RPMI1640 culture solution supply of said 2660-2669; as Lindstrom et. , Blood (1997) 90: M199 replenishment of the culture broth 2323-2334; available from Cambrex, Baltimore, MD, containing 2% fetal bovine serum endothelial growth medium, and other tissues known to those skilled in the culture medium cultured endothelial cells. 可使用其它因子,如内皮细胞生长因子(ECGF)、肝素等。 Other factors may be used, such as endothelial cell growth factor (ECGF), heparin. 例如,参见Thornton等,Science(1983)222:623。 For example, see Thornton et al., Science (1983) 222: 623. 使用前合适的代数可变化,取决于所用细胞系的期望寿命。 Suitable algebraic before use can vary, depending on the desired lifetime of the cell lines used. 通常,本发明中使用的HUVEC的代数为2-20代,优选3-10代,甚至更优选,少于5代,例如2、3、4代。 Typically, the algebraic of HUVEC in the present invention is 2 to 20 generations, preferably 3 to 10 generations, even more preferably, less than 5 generations, e.g. 2,3,4 generations.

经过所需次数的传代之后,将大约1×103-1×107,有效1×104-1×107,例如1×105-1×106的细胞加入到合适的粘附基质中。 After the desired number of passages, approximately 1 × 103-1 × 107, effectively 1 × 104-1 × 107, for example, cell 1 × 105-1 × 106 was added to a suitable adhesive matrix. 根据细胞类型,培养适当时间,得到融合单层。 Depending on cell type, culture appropriate time to give a confluent monolayer. 例如,HUVAC一般培养约1-7天,通常2-5天,例如1、2、3、4、5、6或7天,直到形成融合单层。 For example, HUVAC typically cultured for about 1-7 days, usually 2-5 days, 5, 6, or 7 days until a confluent monolayer. 可采用本领域已知的技术评价融合度,例如用结晶紫检测。 Known in the art can be evaluated degree of integration techniques, such as detection with crystal violet.

本发明试验中试验的合适的基质包括膜支持物,例如用于组织培养的渗透性微孔薄膜,通常允许物质如可溶性营养物、代谢物和激素因子自由通过该膜,同时阻止细胞迁移通过该膜。 Experimental testing of the present invention is suitable matrix comprises a membrane support, such as for tissue culture permeable microporous film, generally allow soluble materials such as nutrients, metabolites and hormonal factors freely through the membrane, while preventing migration of cells through the membrane. 例如,粘附支持物包括葡聚糖聚合物、聚氯乙烯、聚乙醇酸、聚乳酸、聚乳酸乙醇酸共聚物(polylactic coglycolic acids)和/或硅。 For example, the adhesion support comprises a dextran polymer, polyvinyl chloride, polyglycolic acid, polylactic acid, polylactic-glycolic acid (polylactic coglycolic acids) and / or silicon. 通常,基质还可包括胶原、纤维蛋白、纤连蛋白、层粘连蛋白和/或透明质酸。 Typically, the matrix may further comprise collagen, fibrin, fibronectin, laminin, and / or hyaluronic acid. 这些支持物是本领域所公知的,例如,可购自Costar(Cambridge,MA)。 These supports are known in the art, e.g., commercially available from Costar (Cambridge, MA). 一个例子是TrranswellTM支持物(例如,TRANSWELL-COL PTFE膜,膜厚度25-50μm,孔径0.4-3.0μm,用来自牛胎盘的I型和II型胶原处理)。 One example is TrranswellTM support (e.g., TRANSWELL-COL PTFE film, the film thickness of 25-50 microns, a pore size 0.4-3.0μm, from bovine placental type I and type II collagen treatment). 这些支持物允许物质从上室通过单层进入下室,在下室中收集和测定物质。 These supports allow the material from the chamber into the lower chamber by a single layer, the lower collection chamber and the test substance. 然而,本发明方法可使用任何合适的渗透膜支持物,只要可监测通过单层的迁移。 However, the method of the present invention may use any suitable permeable membrane support, as long as the migration can be monitored by a single layer.

融合单层一旦建立,在不存在试验物质的情况下,将可检测的标记大分子加入到单层(例如转移孔支持物的上室),以提供背景测定。 Once confluent monolayer, in the case where the test material is not present, the detectable label molecules added to the monolayer (for example, a transfer chamber of the support hole), to provide a background measurement. 可检测的标记大分子的尺寸大到足以被融合单层保留,除非由于可使单层破裂的分子的存在而导致单层渗漏。 Labeled macromolecules can be detected size large enough to be retained confluent monolayers, unless there was a monolayer can rupture due to the molecular monolayer resulting leakage. 如上所述,本发明方法中一般使用的大分子包括血清白蛋白如牛血清白蛋白(BSA)、卵清蛋白、钥孔血蓝蛋白、免疫球蛋白分子、甲状球蛋白及本领域技术人员已知的其它蛋白质。 As described above, the method of the present invention is generally used in macromolecules include serum albumin such as bovine serum albumin (BSA), ovalbumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin skilled in the art and have been other known proteins. 加入标记大分子之后,试验进行15分钟到几个小时,例如,30分钟到48小时,优选1小时到24小时,更优选2小时到12小时,例如1...2...3...4...5...6...7...8...12...20...24...40...48或更多个小时。 After adding a labeled macromolecule, the test for 15 minutes to several hours, e.g., 30 minutes to 48 hours, preferably 1 hour to 24 hours, more preferably 2 to 12 hours, e.g. 1 ... 2 ... 3 .. .4 ... 5 ... 6 ... 7 ... 8 ... 12 ... 20 ... 24 ... 40 ... 48 or more hours. 例如,采用分光光度计,可检测通过单层进入(例如)底室的标记分子,以提供基线测定。 For example, using a spectrophotometer, it may be detected by a single entry (e.g.) labeled molecule bottom chamber to provide a baseline measurement. 如果采用荧光标记,可使用荧光光度计测定荧光,表示为相对荧光单位。 If a fluorescent label, fluorescence can be measured using a fluorescent photometer, expressed as relative fluorescence units. 测定可以是定性或定量的。 Assay may be qualitative or quantitative. 例如,可采用以下公式计算白蛋白清除:白蛋白清除(μl)=VL×A/L,其中,VL是取样时的底室体积,A是取样时每微升底室中的荧光单位,L是试验开始时,每微升顶室中的荧光单位。 For example, the following formula may be employed Clear albumin: Albumin clearance (μl) = VL × A / L, where, VL is the volume at the bottom of the sampling chamber, A per microliter bottom chamber fluorescence units sampling, L It is the start of the test, per microliter of the top chamber fluorescence units. 通过线性回归分析计算白蛋白的清除率(μl/分钟)。 Calculated by linear regression analysis of albumin clearance (μl / min). 例如,可测定清除(μl±SEM/分钟)。 For example, the measurement may be cleared (μl ± SEM / min).

进行适当测定之后,除去任何残留的标记大分子,将试验化合物以及含标记大分子的培养液加入到顶孔中。 After appropriate assay, remove any remaining labeled macromolecules, and the medium containing the test compound labeled macromolecules were added to the top wells. 例如,加入感兴趣的治疗药物,或已用治疗药物刺激活化的LAK细胞(或其上清液),以及对照,如下所述。 For example, addition of a therapeutic agent of interest, or treatment with LAK cells activated by stimulating drugs (or supernatant), and a control, as described below. 采用选定的治疗药物以激活PBMC,通过活化外周血单核细胞(PBMC),产生LAK细胞。 Using the selected therapeutic agents to activate PBMC, peripheral blood mononuclear cells by activation (PBMC), generating LAK cells. 可采用本领域公知的技术,例如采用Ficoll-Hypaque密度梯度离心,从全血分离用于激活的PBMC。 Known in the art may be employed in the art, for example by density gradient centrifugation Ficoll-Hypaque, for activating PBMC were isolated from whole blood. 离心后,通过在37℃下,粘附塑料的相继循环持续45分钟,粘附的单核细胞可与(但不必需)非粘附单核细胞(NAMNC)分离。 After centrifugation, the at 37 ℃, successive circulating adhesion plastic 45 minutes, adherent mononuclear cells may be separated (but not necessarily) a non-adherent monocytes (NAMNC). 为了制备活化细胞,联合(使用)治疗药物和PBMC。 To prepare the activated cells, combined (using) a therapeutic agent and PBMC. 加入的药物量取决于特定的受试物质。 The amount of drug added depends on the particular test substance. 因此,例如,当分析淋巴因子如IL-2突变蛋白时,突变蛋白的加入浓度为10-500nM,通常浓度为25-250nM,甚至更优选浓度为35-100nM。 Thus, for example, when the analysis lymphokines such as IL-2, when mutated protein, the mutant protein is added at a concentration 10-500 nm, typically at a concentration of 25-250nM, and even more preferably at a concentration of 35-100nM. 本领域技术人员可容易地确定合适的使用浓度。 Those skilled in the art can readily determine the appropriate concentration. 例如,参见Damle和Doyle,J.Bacteriol.(1989)142:2660-2669;Damle等,J.Immunol.(1987)138:1779;Damle和Doyle,Int.J.Cancer(1987)40:519;Damle等,J.Immunol.(1986)137:2814。 See, eg, Damle and Doyle, J.Bacteriol (1989) 142: 2660-2669; Damle et, J.Immunol (1987) 138:. 1779; Damle and Doyle, Int.J.Cancer (1987) 40:. 519; Damle et, J.Immunol (1986) 137:. 2814.

参考如上所述基线测定,进行试验。 As described above with reference to a baseline measurement, test. 可在多个时间点从底室取样,测定存在的标记大分子。 Samples may be taken at various time points from the bottom chamber, detecting the presence of labeled macromolecules. 可进行各种对照,如下所述。 Various controls can be, as described below. 具体地说,可使用具有改善的耐受性的治疗药物作为阴性对照,以建立不存在引起VLS的治疗药物时发生的基线渗漏。 In particular, resistance may be used with improved therapeutic agents as a negative control to establish baseline leak occurs when the therapeutic agent due to the absence of VLS. 例如,IL-2突变蛋白F42E和Y107R是耐受性提高的取代突变蛋白。 For example, IL-2 muteins are Y107R F42E and increased tolerance substitution mutein. 参见,2005年3月5日提交的共同拥有、共同待批的美国临时专利申请序列号60/550,868。 See, co-owned 2005 March 5 filed, co-pending US Provisional Patent Application Serial No. 60 / 550,868. 体内外模型中,就NK和T细胞增殖,以及NK/LAK/ADCC活性方面而言,这些突变蛋白也维持效应子作用。 Outer vivo models, on the proliferation of NK and T cells, and in terms of NK / LAK ADCC activity of /, these muteins maintained effector role. 此外,可采用阳性对照,例如去污剂和已知可破坏细胞单层的物质等,例如皂苷。 In addition, a positive control may be employed, such as detergents and the cell monolayer destruction of known substances, such as saponin. 还可使用培养液对照。 It may also be used broth control.

如上所述,使用本发明方法测定治疗剂如IL-2突变蛋白的患者耐受性。 As described above, the therapeutic agent was measured using the method of the present invention, such as IL-2 mutant proteins patient tolerance. 已知许多IL-2突变蛋白,如下所述。 Many known that IL-2 muteins, as described below. 然而,本发明方法也可应用于本文未具体描述的其它IL-2突变蛋白。 However, the method of the present invention is also applicable to other IL-2 muteins not specifically described herein. 这些IL-2突变蛋白可来源于任何种类的IL-2。 The IL-2 muteins may be derived from any kind of IL-2. 这些变体应保留天然多肽所需的生物学活性,使得给予患者时,含变体多肽的药物组合物具有与含天然多肽的药物组合物相同的治疗效果。 Such variants should retain the desired biological activity of the native polypeptide such that when administered to a patient, including variant polypeptides and pharmaceutical compositions having pharmaceutical composition comprising the native polypeptide same therapeutic effect. 即,变体多肽可以类似于天然多肽的方式,用作药物组合物中的治疗活性成分。 That is, the variant polypeptide may be similar to the way native polypeptide, as therapeutically active ingredient in the pharmaceutical composition. 本领域已知测定变体多肽是否保留所需的生物学活性的方法,因而可用作药物组合物中的治疗活性成分。 Those skilled in process variant polypeptide retains the desired biological activity if the known assays therefore useful as therapeutically active ingredient in the pharmaceutical composition. 可采用特别用于测定天然多肽或蛋白质活性的试验来测定生物学活性。 It can be employed in particular for assays to determine the biological activity of the native polypeptide or protein activity. 天然或天然来源的IL-2的合适的生物学活性突变蛋白可以是该多肽的片段、类似物以及衍生物,如上所述。 Suitable natural or natural origin of the biological activity of IL-2 muteins may be a fragment of the polypeptide, analogs and derivatives, as described above.

例如,可通过在编码感兴趣天然多肽的克隆DNA序列中产生突变来制备多肽的氨基酸序列变体。 For example, amino acid sequence variants may be prepared in a mutation of a polypeptide of interest by cloning a DNA sequence encoding the native polypeptide. 诱变和改变核苷酸序列的方法是本领域所所熟知的。 And changing the nucleotide sequence of the mutagenesis methods are well known in the art. 例如,参见以下文献:Walker和Gaastra编,(1983),《分子生物学技术》(Techniques in Molecular Biology),(MacMillan Publishing Company,New York);Kunkel,(1985),Proc.Natl.Acad.Sci.,USA 82:488-492;Kunkel等,(1987),Methods Enzymol.,154:367-382;Sambrook等,(1989),《分子克隆:实验室手册》(Molecular Cloning:A Laboratory Manual),(冷泉港实验室出版社,Plainview,New York);美国专利号4,873,192;和本文引用的参考文献。 For example, see the following documents: Walker and Gaastra, eds., (1983), "Molecular Biology Techniques" (Techniques in Molecular Biology), (MacMillan Publishing Company, New York); Kunkel, (1985), Proc.Natl.Acad.Sci ., USA 82: 488-492; Kunkel et, (1987), Methods Enzymol, 154:. 367-382; Sambrook et al., (1989), "molecular cloning: A laboratory Manual" (molecular cloning: A laboratory Manual), (Cold Spring Harbor laboratory Press, Plainview, New York); U.S. Pat. No. 4,873,192; and references cited herein. 不影响感兴趣的多肽的生物学活性的合适氨基酸取代的指南可参见Dayhoff等,(1978),在《蛋白质序列和结构图》(Atlas of Protein Sequence andStructure)(Natl.Biomed.Res.Found.,Washington,DC)中所述的模型。 Suitable amino acid substitutions do not affect the guidance of the biological activity of the polypeptide of interest may be found in Dayhoff et al., (1978), in "Protein Sequence and Structure FIG." (Atlas of Protein Sequence andStructure) (Natl.Biomed.Res.Found., washington, DC) in the model. 优选保守性取代,例如用具有相似性质的另一种氨基酸替换一种氨基酸。 Conservative substitutions, such as replacing an amino acid with another amino acid having similar properties. 保守性取代的例子包括但不限于:GlyAla、ValIleLeu、AspGlu、LysArg、AsnGln和PheTrpTyr。 Examples of conservative substitution include, but are not limited to: GlyAla, ValIleLeu, AspGlu, LysArg, AsnGln and PheTrpTyr.

可在专业文献中找到残基取代、删除或插入来改变IL-2蛋白质诸区域的指导。 It can be found in the specialist literature residue substitutions, deletions or insertions to alter the protein with various regions of IL-2. 例如,参见以下文献所述的结构/功能关系和/或结合研究:Bazan,(1992),Science,257:410-412;McKay,(1992),Science,257:412;Theze等,(1996),Immunol.Today,17:481-486;Buchli和Ciardelli,(1993),Arch.Biochem.Biophys.,307:411-415;Collins等,(1988),Proc.Natl.Acad.Sci.,USA 85:7709-7713;Kuziel等,(1993),J.Immunol.,150:5731;Eckenberg等,(1997),Cytokine,9:488-498。 For example, see the following documents the structure / function relationships and / or binding studies: Bazan, (1992), Science, 257: 410-412; McKay, (1992), Science, 257: 412; Theze et al., (1996) , Immunol.Today, 17: 481-486; Buchli and Ciardelli, (1993), Arch.Biochem.Biophys, 307:. 411-415; Collins et, (1988), Proc.Natl.Acad.Sci, USA 85. : 7709-7713; Kuziel et, (1993), J.Immunol, 150:. 5731; Eckenberg et, (1997), Cytokine, 9: 488-498.

在构建感兴趣IL-2多肽的变体的过程中,进行的修饰应使变体继续具有所需活性。 In constructing the polypeptide of interest IL-2 variants, modifications should be made variants continue to possess the desired activity. 显然,在编码变体多肽的DNA中产生的任何突变不能将该序列置于读码框外,并且优选不会产生可能产生二级mRNA结构的互补区域。 Obviously, any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame outside, and preferably no complementary regions may produce secondary mRNA structure. 参见,欧洲专利申请号75,444。 See, European Patent Application No. 75,444.

IL-2的生物学活性变体一般与用作比较基准的参比IL-2多肽分子,例如天然的人IL-2具有至少约70%、优选至少约80%、更优选至少约90%-95%或更高、最优选至少约98%、99%或更高的氨基酸序列相同性。 A biologically active variant of IL-2 is generally used as the baseline reference IL-2 polypeptide molecule, such as native human IL-2 having at least about 70%, preferably at least about 80%, more preferably at least about 90% - 95% or higher, most preferably at least about 98%, 99% or greater identity of the amino acid sequence. 序列相同性百分比可用Smith-Waterman同源性检索算法,以亲族缺口(affine gap)检索测定,所用参数为缺口开放罚分12、缺口延伸罚分2和BLOSUM矩阵62。 Percentage of sequence identity are available Smith-Waterman homology search algorithm to relatives notch (affine gap) measured retrieved, the parameters used are gap open penalty of 12, gap extension penalty of 2, and BLOSUM 62 matrix. Smith-Waterman同源性检索算法的说明见Smith和Waterman,(1981),Adv.Appl.Math.2:482-489。 Description Smith-Waterman homology search algorithm is taught in Smith and Waterman, (1981), Adv.Appl.Math.2: 482-489. 例如,变体可有少至1-15个氨基酸残基、少至1-10个氨基酸残基,如6-10,少至5个、少至4个、3个、2个、甚至1个氨基酸残基不同。 For example, the variant may have as few as 1-15 amino acid residues, as few as 1-10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 different amino acid residues.

就两条氨基酸序列的最佳比对而言,与参比氨基酸序列相比,变体氨基酸序列的连续区段可具有相同的氨基酸数、加入的氨基酸残基或删除的氨基酸残基。 Of two amino acid sequences for optimal alignment, compared with the reference amino acid sequences, the contiguous segment of the variant amino acid sequence may have the same number of amino acids, amino acid residues is added or deleted amino acid residues. 用于和参比氨基酸序列比较的连续区段包含至少20个连续的氨基酸残基,可以是30、40、50或更多个氨基酸残基。 For comparison to the reference amino acid sequence comprising a contiguous segment of at least 20 contiguous amino acid residues, and may be 30,40, 50 or more amino acid residues. 可对与保守性残基取代或缺口相关的序列相同性进行校正(参见Smith-Waterman同源性检索算法)。 It may be on the conservative residue substitutions or gaps for sequence identity associated correction (see Smith-Waterman homology search algorithm). 感兴趣的天然IL-2多肽的生物学活性变体与天然多肽相比有少至1-15个氨基酸残基、少至1-10个氨基酸残基,如6-10,少至5个、少至4个、3个、2个、甚至1个氨基酸残基的不同。 Interest native IL-2 polypeptide is biologically active variant compared to the native polypeptide has few as 1-15 amino acid residues, as few as 1-10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even a different amino acid residues.

具有IL-2活性的多肽的精确化学结构取决于许多因素。 Exact chemical structure of a polypeptide having IL-2 activity depends on many factors. 由于分子中存在可电离的氨基和羧基,获得的特定多肽可以是酸性盐或碱性盐,或中性形式。 As ionizable amino and carboxyl groups in the molecule, a particular polypeptide may be obtained in acidic or basic salt, or in neutral form. 在合适的环境中可保留它们的生物学活性的所有这些制剂包括在本文所用具有IL-2活性的多肽的定义中。 In a suitable environment to retain their biological activity include all such formulations defined herein as having IL-2 activity of the polypeptide. 此外,可通过利用糖分子衍生化(糖基化)或其它补充分子,例如脂质、磷酸、乙酰基团等增加该多肽的一级氨基酸序列。 Further, by derivatizing sugar molecules (glycosylation) or by using other supplementary molecules such as lipids, phosphate, acetyl groups and the like increase in the primary amino acid sequence of the polypeptide. 也可通过和糖偶联来增加该多肽的一级氨基酸序列。 It may also be an increase in the amino acid sequence of the polypeptide by coupling sugar. 这种增加的某些方面可通过生产宿主的翻译后加工系统来实现;其它这种修饰可在体外引入。 Certain aspects of this increase may be achieved by post-translational processing systems of the producing host; other such modifications may be introduced in vitro. 在任何情况中,只要多肽的IL-2活性未遭破坏,这种修饰就包括在本文所用的IL-2多肽的定义中。 In any case, as long as the IL-2 activity of the polypeptide is not destroyed, such modifications are included in the definition as used herein, IL-2 polypeptide. 在各种试验中,期望这种修饰可通过提高或降低该多肽的活性来定量或定性地影响其活性。 In various tests, it is desirable that such modifications may quantitatively or qualitatively by increasing or decreasing the activity of the polypeptide to affect its activity. 此外,可通过氧化、还原或其它衍生化方式来修饰链中的单个氨基酸残基,可切割该多肽而得到保留活性的片段。 Further, by oxidation, reduction, or other derivatization modified embodiment individual amino acid residues in the chain, which may be cut obtained polypeptide fragments that retain activity. 这种不破坏活性的改变不将这种多肽序列排除在本文所用感兴趣的IL-2多肽的定义之外。 This change does not destroy the activity of such a polypeptide sequence not excluded from the definition of interest as used herein, IL-2 polypeptide.

本领域为多肽变体的制备和使用提供了基本指导。 The present art provides the basic guidelines for the preparation and use of polypeptide variants thereof. 在制备IL-2变异蛋白过程中,本领域的技术人员不难确定对天然蛋白质的核苷酸或氨基酸序列的哪种修饰将导致产生适合用作本发明方法药物组合物中治疗活性组分的变体。 In the preparation process of IL-2 variant protein, those skilled in the art can readily determine what nucleotide or amino acid sequence of the native protein will result in a modification of the method of the present invention is suitable for use as a pharmaceutical composition therapeutically active component Variants.

用于本发明方法中的IL-2或其变异蛋白可以是任何来源,但优选重组产生。 For IL-2 protein or a variant method of the present invention may be of any origin, but is preferably recombinantly produced. “重组IL-2”或“重组IL-2变异蛋白”指具有与天然序列IL-2相当生物学活性并通过例如Taniguchi等,(1983),Nature,302:305-310和Devos,(1983),NucleicAcids Research,11:4307-4323所述重组DNA技术制备的白介素-2或其变体;或者如Wang等,(1984),Science,224:1431-1433所述经突变而改变的IL-2。 "Recombinant IL-2" or "recombinant IL-2 variant protein" refers to the biological activity of IL-2 has a relatively native sequence, for example, by Taniguchi et al., (1983), Nature, 302: 305-310 and Devos, (1983) , NucleicAcids Research, 11: 4307-4323 the preparation of recombinant DNA techniques interleukin-2 or a variant thereof; or, as Wang et al, (1984), Science, 224: 1431-1433 mutated and the altered IL-2 . 大体上,克隆编码IL-2的基因,然后在转化的生物,优选微生物中表达。 Gene general, encoding the IL-2 is cloned and then expressed in the organism, preferably a transformed microorganism. 宿主生物能在表达条件下表达外来基因而产生IL-2。 IL-2 generated a host organism capable of expressing a foreign gene under expression conditions. 生长、收集、破裂(disrupt)或自细胞者中提取IL-2的方法的基本描述见,例如全文纳入本文作为参考的美国专利号4,604,377;4,738,927;4,656,132;4,569,790;4,748,234;4,530,787;4,572,798;4,748,234。 Growth was collected, cracking (Disrupt) or substantially the method described IL-2 extracted from the cells were in the see, for example, incorporated herein by reference U.S. Patent Nos. 4,604,377; 4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798; 4,748,234.

例如IL-2突变蛋白,参见欧洲专利(EP)公布号EP 136,489(公开了天然存在的IL-2的氨基酸序列中的一种或多种以下改变:Asn26-Gln26;Trp121-Phe121;Cys58-Ser58或Ala58;Cys105-Ser105或Ala105;Cys125-Ser125或Ala125;删除Arg 120之后的所有残基;和它们的Met-1形式);1983年10月13日提交的欧洲专利申请号83306221.9所述的重组IL-2突变蛋白(1984年5月30日以公布号EP 109,748公布),该专利是比利时专利号893,016和共有的美国专利4,518,584的等同专利(这两份专利公开了重组的人IL-2突变蛋白,其中天然人IL-2编号的125位半胱氨酸被删除或被中性氨基酸所取代;丙氨酰-ser125-IL-2;和脱-丙氨酰-ser125-IL-2)。 IL-2 muteins e.g., see European Patent (EP) Publication No. EP 136,489 (discloses the amino acid sequence of a naturally occurring IL-2 in one or more of the following changes: Asn26-Gln26; Trp121-Phe121; Cys58-Ser58 or Ala58; Cys105-Ser105 or Ala105; Cys125-Ser125 or Ala125; remove all residues following Arg 120; and the Met-1 forms thereof); recombinant according to European Patent application No. 83306221.9 October 13, 1983, filed IL-2 mutant proteins (May 30, 1984 published as publication No. EP 109,748), which is patent Belgian patent No. 893,016 and a total of US patent 4,518,584 patent equivalent of (these two patents disclose recombinant human IL-2 mutation protein, wherein the native human IL-2 cysteine ​​125 number is deleted or replaced by a neutral amino acid; alanyl -ser125-IL-2; and de - alanyl -ser125-IL-2). 也参见美国专利号4,752,585(该专利公开了以下IL-2突变蛋白:ala104 ser125 IL-2、ala104 IL-2、ala104 ala125 IL-2、val104 ser125IL-2、val104 IL-2、val104 ala125 IL-2、脱-ala1 ala104 ser125 IL-2、脱-ala1 ala104IL-2、脱-ala1 ala104 ala125 IL-2、脱-ala1 val104 ser125 IL-2、脱-ala1 val104 IL-2、脱-ala1 val104 ala125 IL-2、脱-ala1脱-pro2 ala104 ser0125 IL-2、脱-ala1脱-pro2ala104 IL-2、脱-ala1脱-pro2 ala104 ala125IL-2、脱-ala1脱-pro2 val104 ser125IL-2、脱-ala1脱-pro2 val104 IL-2、脱-ala1脱-pro2 val104 ala125 IL-2、脱-ala1脱-pro2脱-thr3 ala104 ser125 IL-2、脱-ala1脱-pro2脱-thr3 ala104 IL-2、脱-ala1脱-pro2脱-thr3 ala104 ala125 IL-2、脱-ala1脱-pro2脱-thr3 val104 ser125 IL-2、脱-ala1脱-pro2脱-thr3 val104 IL-2、脱-ala1脱-pro2脱-thr3 val104 ala125IL-2、脱-ala1脱-pro2脱-thr3脱-ser4 ala104 ser125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4 ala104 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4 ala104 ala125 IL-2、脱-ala1脱-pro2脱 See also U.S. Patent No. 4,752,585 (which discloses the IL-2 mutant proteins: ala104 ser125 IL-2, ala104 IL-2, ala104 ala125 IL-2, val104 ser125IL-2, val104 IL-2, val104 ala125 IL-2 , de -ala1 ala104 ser125 IL-2, off -ala1 ala104IL-2, off -ala1 ala104 ala125 IL-2, off -ala1 val104 ser125 IL-2, off -ala1 val104 IL-2, off -ala1 val104 ala125 IL- 2, de de -ala1 -pro2 ala104 ser0125 IL-2, de de -ala1 -pro2ala104 IL-2, de de -ala1 -pro2 ala104 ala125IL-2, de de -ala1 -pro2 val104 ser125IL-2, de de -ala1 -pro2 val104 IL-2, de de -ala1 -pro2 val104 ala125 IL-2, -pro2 de de de -ala1 -thr3 ala104 ser125 IL-2, -pro2 de de de -ala1 -thr3 ala104 IL-2, off - ala1 de de -pro2 -thr3 ala104 ala125 IL-2, -pro2 de de de -ala1 -thr3 val104 ser125 IL-2, -pro2 de de de -ala1 -thr3 val104 IL-2, -pro2 de de de -ala1 - thr3 val104 ala125IL-2, -pro2 de de de -ala1 -thr3 off -ser4 ala104 ser125 IL-2, -pro2 de de de -ala1 -thr3 off -ser4 ala104 IL-2, -ala1 de de de -thr3 -pro2 de -ser4 ala104 ala125 IL-2, -pro2 de de de -ala1 -thr3脱-ser4 val104 ser125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4 val104 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4 val104 ala125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 ala104 ser125IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 ala104 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 ala104ala125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 val104 ser125IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 val104 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 val104 ala125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6ala104 ala125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 ala104IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 ala104 ser125 IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 val104 ser125IL-2、脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 val104 IL-2和脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 val104 ala125IL-2)和美国专利号4,931、543(该专利公开了用于本文实施例的IL-2突变蛋白脱-丙氨酰-1,丝 -thr3 off -ser4 val104 ser125 IL-2, -pro2 de de de -ala1 -thr3 off -ser4 val104 IL-2, -pro2 de de de -ala1 -thr3 off -ser4 val104 ala125 IL-2, de de -ala1 -pro2 -thr3 de de de -ser4 -ser5 ala104 ser125IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 ala104 IL-2, -ala1 de de de -thr3 -pro2 -ser4 off off - ser5 ala104ala125 IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 val104 ser125IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 val104 IL-2, de de -ala1 -pro2 -thr3 de de de -ser4 -ser5 val104 ala125 IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 off -ser6ala104 ala125 IL-2, -ala1 de de de -thr3 off -pro2 -ser4 de de -ser5 -ser6 ala104IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 off -ser6 ala104 ser125 IL-2, -ala1 de de de -thr3 -pro2 -ser4 off off - ser5 off -ser6 val104 ser125IL-2, -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 off -ser6 val104 IL-2 and deprotection -ala1 -thr3 de de de -pro2 -ser4 de de -ser6 -ser5 val104 ala125IL-2) and U.S. Patent No. 4,931,543 (which discloses the IL-2 mutein used in the examples herein, the removal - alanyl-1, wire 氨酸-125人IL-2以及其它IL-2突变蛋白)。 -125 acid human IL-2 as well as other IL-2 muteins).

也参见欧洲专利公布号EP 200,280(1986年12月10日公布),该专利公开了重组IL-2突变蛋白,其中104位的甲硫氨酸被保守性氨基酸取代。 See also European Patent Publication No. EP 200,280 (published December 10, 1986), which discloses recombinant IL-2 muteins wherein the methionine at position 104 is substituted with a conservative amino acid. 例子包括以下突变蛋白:ser4脱-ser5 ala104 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 ala104 ala125 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 glu104 ser125IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 glu104 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5 glu104 ala125IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 ala104 ala125 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 ala104 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 ala104ser125 IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 glu104 ser125IL-2;脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 glu104 IL-2和脱-ala1脱-pro2脱-thr3脱-ser4脱-ser5脱-ser6 glu104 ala125 IL-2。 Examples include the following muteins: ser4 off -ser5 ala104 IL-2; -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 ala104 ala125 IL-2; -ala1 de de de -thr3 -pro2 -ser4 off off - ser5 glu104 ser125IL-2; -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 glu104 IL-2; -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 glu104 ala125IL-2; de de -ala1 -pro2 -ser4 de de de -thr3 -ser5 off -ser6 ala104 ala125 IL-2; -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 off -ser6 ala104 IL-2; -pro2 de de de -ala1 -thr3 -ser4 de de de -ser5 -ser6 ala104ser125 IL-2; -ala1 de de de -thr3 -pro2 de de -ser4 -ser5 off -ser6 glu104 ser125IL-2; -pro2 de de de -ala1 -thr3 off - ser4 de de -ser5 -ser6 glu104 IL-2 and deprotection -ala1 -pro2 de de de -ser4 -thr3 de de -ser5 -ser6 glu104 ala125 IL-2. 也参见欧洲专利申请号EP 118,617和美国专利号5,700,913,该两份专利公开了以丙氨酸取代天然IL-2的甲硫氨酸作为N-末端氨基酸的未糖基化人IL-2变体;删除了起始甲硫氨酸从而使脯氨酸是N-末端氨基酸的未糖基化人IL-2;和在N-末端甲硫氨酸和脯氨酸之间插入丙氨酸的未糖基化人IL-2。 See also European Patent Application No. EP 118,617 and U.S. Patent No. 5,700,913, which patent discloses a two alanine substituted IL-2 as the native N- terminal methionine amino acid unglycosylated human IL-2 variants ; initial methionine deleted such that proline is non-glycosylated human N- terminal amino acid of IL-2; and alanine inserted between the N- terminal methionine and proline did not glycosylated human IL-2.

其它IL-2突变蛋白包括公开于WO 99/60128的(用组氨酸或异亮氨酸取代20位的天冬氨酸,用精氨酸、甘氨酸或异亮氨酸取代88位的天冬酰胺,或者用亮氨酸或谷氨酸取代126位的谷氨酰胺),据报道这些突变蛋白对T细胞受体表达细胞所表达的高亲和力IL-2受体具有优于NK细胞所表达受体的选择性活性并且IL-2毒性降低;美国专利号5,229,109所公开的突变蛋白(用丙氨酸取代38位的精氨酸,或者用赖氨酸取代42位的苯丙氨酸),与天然IL-2相比这些突变蛋白显示与高亲和力IL-2受体结合(能力)降低但仍能激活LAK细胞;国际公布号WO 00/58456所公开的突变蛋白(改变或删除天然IL-2中天然存在的(x)D(y)序列,其中D是天冬氨酸,(x)是亮氨酸、异亮氨酸、甘氨酸或缬氨酸,(y)是缬氨酸、亮氨酸或丝氨酸);公开于国际公布号WO 00/04048中的IL-2 p1-30肽(对应于IL-2的前30个氨基酸,该肽含有IL-2的 Other IL-2 muteins include aspartame is disclosed in WO 99/60128 (with histidine or isoleucine at position 20 substituted with aspartic acid, arginine, glycine or isoleucine substitution at position 88 amides or substituted with leucine or glutamic acid glutamine 126), which is reported to muteins of T-cell receptors expressed by cells in the high affinity IL-2 receptor expressed by NK cells than the expression by selective activity of the IL-2 toxicity and reduced; U.S. Patent No. 5,229,109 disclosed mutein (position 38 substituted with alanine, arginine, lysine or phenylalanine substitution at position 42), and these compared to native IL-2 muteins show high affinity binding to IL-2 receptor (capacity) while still reducing the activation of LAK cells; international Publication No. WO 00/58456 disclosed mutein Publication (changed or deleted native IL-2 naturally occurring (x) D (y) sequence, wherein D is aspartic acid, (X) is leucine, isoleucine, glycine, or valine, (y) is valine, leucine acid or serine); disclosed in international Publication IL-2 peptide P1-30 No. WO 00/04048 (corresponding to the first 30 amino acids of IL-2, containing the peptide of IL-2 个α-螺旋A并能与IL-2受体的b链相互作用);和公开于WO 00/04048的IL-2 p1-30肽的突变形式(用赖氨酸取代20位的天冬氨酸)。 And an α- helix A and IL-2 receptor capable of interacting b chain); and a mutant form disclosed in the IL-2 peptide P1-30 WO 00/04048 (the lysine at position 20 substituted aspartyl acid).

具有预计的毒性降低的IL-2突变蛋白的其它例子公开于2004年3月5日提交的美国临时专利申请序列号60/550,868中。 Other examples of U.S. Provisional Patent IL-2 mutein having a predicted reduced toxicity are disclosed in March 5, 2004, filed Application Serial No. 60 / 550,868. 这些突变蛋白包括在成熟人IL-2序列的125位用丝氨酸取代半胱氨酸的成熟人IL-2氨基酸序列,并且在该成熟人IL-2序列中具有至少一处额外的氨基酸取代从而使该突变蛋白具有以下功能特征:在可比试验条件下与类似量的脱-丙氨酰-1、C125S人IL-2或C125S人IL-2相比,1)能维持或提高天然杀伤(NK)细胞的增殖,和2)诱导NK细胞产生的致炎细胞因子水平降低。 These muteins comprise the amino acid sequence of IL-2 substituted with cysteine ​​in mature human mature human IL 2-sequence 125 with serine, and having at least one additional amino acid in the mature human IL-2 sequence such that the substituted the mutein has the following functional characteristics: similar amount off under comparable test conditions -, C125S human IL-2 or C125S human IL-2 as compared alanyl-1, 1) can be maintained or improved natural killer (NK) cell proliferation, and 2) reduced proinflammatory cytokine levels induced by NK cells. 在一些实施方式中,此额外取代选自:T7A、T7D、T7R、K8L、K9A、K9D、K9R、K9S、K9V、K9W、T10K、T10N、Q11A、Q11R、Q11T、E15A、H16D、H16E、L19D、L19E、D20E、124L,K32A、K32W、N33E、P34E、P34R、P34S、P34T、P34V、K35D、K35I、K35L、K35M、K35N、K35P、K35Q、K35T、L36A、L36D、L36E、L36F、L36G、L36H、L36I、L36K、L36M、L36N、L36P、L36R、L36S、L36W、L36Y、R38D、R38G、R38N、R38P、R38S、L40D、L40G、L40N、L40S,T41E、T41G、F42A、F42E、F42R、F42T、F42V、K43H、F44K、M46I、E61K、E61M、E61R、E62T、E62Y、K64D、K64E、K64G、K64L、K64Q、K64R、P65D、P65E、P65F、P65G、P65H、P65I、P65K、P65L、P65N、P65Q、P65R、P65S、P65T、P65V、P65W、P65Y、L66A、L66F、E67A、L72G、L72N、L72T、F78S、F78W、H79F、H79M、H79N、H79P、H79Q、H79S、H79V、L80E、L80F、L80G、L80K、L80N、L80R、L80T、L80V、L80W、L80Y、R81E、R81K、R81L、R81M、R81N、R81P、R81T、D84R、S87T、N88D、N88H、N88T、V91A、 In some embodiments, this additional substituents selected from: T7A, T7D, T7R, K8L, K9A, K9D, K9R, K9S, K9V, K9W, T10K, T10N, Q11A, Q11R, Q11T, E15A, H16D, H16E, L19D, L19E, D20E, 124L, K32A, K32W, N33E, P34E, P34R, P34S, P34T, P34V, K35D, K35I, K35L, K35M, K35N, K35P, K35Q, K35T, L36A, L36D, L36E, L36F, L36G, L36H, L36I, L36K, L36M, L36N, L36P, L36R, L36S, L36W, L36Y, R38D, R38G, R38N, R38P, R38S, L40D, L40G, L40N, L40S, T41E, T41G, F42A, F42E, F42R, F42T, F42V, K43H, F44K, M46I, E61K, E61M, E61R, E62T, E62Y, K64D, K64E, K64G, K64L, K64Q, K64R, P65D, P65E, P65F, P65G, P65H, P65I, P65K, P65L, P65N, P65Q, P65R, P65S, P65T, P65V, P65W, P65Y, L66A, L66F, E67A, L72G, L72N, L72T, F78S, F78W, H79F, H79M, H79N, H79P, H79Q, H79S, H79V, L80E, L80F, L80G, L80K, L80N, L80R, L80T, L80V, L80W, L80Y, R81E, R81K, R81L, R81M, R81N, R81P, R81T, D84R, S87T, N88D, N88H, N88T, V91A, V91D、V91E、V91F、V91G、V91N、V91Q、V91W、L94A、L94I、L94T、L94V、L94Y、E95D、E95G、E95M、T102S、T102V、M104G、E106K、Y107H、Y107K、Y107L、Y107Q、Y107R、Y107T、E116G、N119Q、T123S、T123C、Q126I和Q126V;其中氨基酸残基位置相对于成熟人IL-2氨基酸序列编号。 V91D, V91E, V91F, V91G, V91N, V91Q, V91W, L94A, L94I, L94T, L94V, L94Y, E95D, E95G, E95M, T102S, T102V, M104G, E106K, Y107H, Y107K, Y107L, Y107Q, Y107R, Y107T, E116G, N119Q, T123S, T123C, Q126I, and Q126V; wherein amino acid residue positions relative to the mature human IL-2 amino acid SEQ ID NO. 在其它实施方案中,这些突变蛋白包括在成熟人IL-2序列的125位用丙氨酸取代半胱氨酸的成熟人IL-2氨基酸序列,并且在该成熟人IL-2序列中具有至少一处额外的氨基酸取代从而使该突变蛋白具有相同的功能特征。 In other embodiments, these muteins comprise the amino acid sequence of IL-2 substituted with cysteine ​​in mature human sequence of mature human IL 2-125 with alanine, and at least the mature human IL-2 sequence an additional amino acid substitution such that the mutein has the same functional characteristics. 在一些实施方案中,此额外取代选自:T7A、T7D、T7R、K8L、K9A、K9D、K9R、K9S、K9V、K9W、T10K、T10N、Q11A、Q11R、Q11T、E15A、H16D、H16E、L19D、L19E、D20E、124L、K32A、K32W、N33E、P34E、P34R、P34S、P34T、P34V、K35D、K35I、K35L、K35M、K35N、K35P、K35Q、K35T、L36A、L36D、L36E、L36F、L36G、L36H、L36I、L36K、L36M、L36N、L36P、L36R、L36S、L36W、L36Y、R38D、R38G、R38N、R38P、R38S、MOD、L40G、L40N、L40S、T41E、T41G、F42A、F42E、F42R、F42T、F42V、K43H、F44K、M46I、E61K、E61M、E61R、E62T、E62Y、K64D、K64E、K64G、K64L、K64Q、K64R、P65D、P65E、P65F、P65G、P65H、P65I、P65K、P65L、P65N、P65Q、P65R、P65S、P65T、P65V、P65W、P65Y、L66A、L66F、E67A、L72G、L72N、L72T、F78S、F78W、H79F、H79M、H79N、H79P、H79Q、H79S、H79V、L80E、L80F、L80G、L80K、L80N、L80R、L80T、L80V、L80W、L80Y、R81E、R81K、R81L、R81M、R81N、R81P、R81T、D84R、S87T、N88D、N88H、N88T、V91A、V In some embodiments, this additional substituents selected from: T7A, T7D, T7R, K8L, K9A, K9D, K9R, K9S, K9V, K9W, T10K, T10N, Q11A, Q11R, Q11T, E15A, H16D, H16E, L19D, L19E, D20E, 124L, K32A, K32W, N33E, P34E, P34R, P34S, P34T, P34V, K35D, K35I, K35L, K35M, K35N, K35P, K35Q, K35T, L36A, L36D, L36E, L36F, L36G, L36H, L36I, L36K, L36M, L36N, L36P, L36R, L36S, L36W, L36Y, R38D, R38G, R38N, R38P, R38S, MOD, L40G, L40N, L40S, T41E, T41G, F42A, F42E, F42R, F42T, F42V, K43H, F44K, M46I, E61K, E61M, E61R, E62T, E62Y, K64D, K64E, K64G, K64L, K64Q, K64R, P65D, P65E, P65F, P65G, P65H, P65I, P65K, P65L, P65N, P65Q, P65R, P65S, P65T, P65V, P65W, P65Y, L66A, L66F, E67A, L72G, L72N, L72T, F78S, F78W, H79F, H79M, H79N, H79P, H79Q, H79S, H79V, L80E, L80F, L80G, L80K, L80N, L80R, L80T, L80V, L80W, L80Y, R81E, R81K, R81L, R81M, R81N, R81P, R81T, D84R, S87T, N88D, N88H, N88T, V91A, V 91D、V91E、V91F、V91G、V91N、V91Q、V91W、L94A、L94I、L94T、L94V、L94Y、E95D、E95G、E95M、T102S、T102V、M104G、E106K、Y107H、Y107K、Y107L、Y107Q、Y107R、Y107T、E116G、N119Q、T123S、T123C、Q126I和Q126V;其中氨基酸残基位置相对于成熟人IL-2氨基酸序列编号。 91D, V91E, V91F, V91G, V91N, V91Q, V91W, L94A, L94I, L94T, L94V, L94Y, E95D, E95G, E95M, T102S, T102V, M104G, E106K, Y107H, Y107K, Y107L, Y107Q, Y107R, Y107T, E116G, N119Q, T123S, T123C, Q126I, and Q126V; wherein amino acid residue positions relative to the mature human IL-2 amino acid SEQ ID NO. 在其它实施方案中,这些突变蛋白包括在成熟人IL-2序列内至少一处额外的氨基酸取代的成熟人IL-2氨基酸序列,从而使该突变蛋白具有相同的功能特征。 In other embodiments, these muteins comprise the amino acid sequence of at least one additional amino acid substitution in the mature human 2 IL-within the mature human IL-2 sequence such that the mutein has the same functional characteristics. 在一些实施方案中,此额外取代选自:T7A、T7D、T7R、K8L、K9A、K9D、K9R、K9S、K9V、K9W、T10K、T10N、Q11A、Q11R、Q11T、E15A、H16D、H16E、L19D、L19E、D20E、124L、K32A、K32W、N33E、P34E、P34R、P34S、P34T、P34V、K35D、K35I、K35L、K35M、K35N、K35P、K35Q、K35T、L36A、L36D、L36E、L36F、L36G、L36H、L36I、L36K、L36M、L36N、L36P、L36R、L36S、L36W、L36Y、R38D、R38G、R38N、R38P、R38S、L40D、L40G、L40N、L40S、T41E、T41G、F42A、F42E、F42R、F42T、F42V、K43H、F44K、M46I、E61K、E61M、E61R、E62T、E62Y、K64D、K64E、K64G、K64L、K64Q、K64R、P65D、P65E、P65F、P65G、P65H、P65I、P65K、P65L、P65N、P65Q、P65R、P65S、P65T、P65V、P65W、P65Y、L66A、L66F、E67A、L72G、L72N、L72T、F78S、F78W、H79F、H79M、H79N、H79P、H79Q、H79S、H79V、L80E、L80F、L80G、L80K、L80N、L80R、L80T、L80V、L80W、L80Y、R81E、R81K、R81L、R81M、R81N、R81P、R81T、D84R、S87T、N88D、N88H、N88T、V91A、 In some embodiments, this additional substituents selected from: T7A, T7D, T7R, K8L, K9A, K9D, K9R, K9S, K9V, K9W, T10K, T10N, Q11A, Q11R, Q11T, E15A, H16D, H16E, L19D, L19E, D20E, 124L, K32A, K32W, N33E, P34E, P34R, P34S, P34T, P34V, K35D, K35I, K35L, K35M, K35N, K35P, K35Q, K35T, L36A, L36D, L36E, L36F, L36G, L36H, L36I, L36K, L36M, L36N, L36P, L36R, L36S, L36W, L36Y, R38D, R38G, R38N, R38P, R38S, L40D, L40G, L40N, L40S, T41E, T41G, F42A, F42E, F42R, F42T, F42V, K43H, F44K, M46I, E61K, E61M, E61R, E62T, E62Y, K64D, K64E, K64G, K64L, K64Q, K64R, P65D, P65E, P65F, P65G, P65H, P65I, P65K, P65L, P65N, P65Q, P65R, P65S, P65T, P65V, P65W, P65Y, L66A, L66F, E67A, L72G, L72N, L72T, F78S, F78W, H79F, H79M, H79N, H79P, H79Q, H79S, H79V, L80E, L80F, L80G, L80K, L80N, L80R, L80T, L80V, L80W, L80Y, R81E, R81K, R81L, R81M, R81N, R81P, R81T, D84R, S87T, N88D, N88H, N88T, V91A, V91D、V91E、V91F、V91G、V91N、V91Q、V91W、L94A、L94I、L94T、L94V、L94Y、E95D、E95G、E95M、T102S、T102V、M104G、E106K、Y107H、Y107K、Y107L、Y107Q、Y107R、Y107T、E116G、N119Q、T123S、T123C、Q126I和Q126V;其中氨基酸残基位置相对于成熟人IL-2氨基酸序列编号。 V91D, V91E, V91F, V91G, V91N, V91Q, V91W, L94A, L94I, L94T, L94V, L94Y, E95D, E95G, E95M, T102S, T102V, M104G, E106K, Y107H, Y107K, Y107L, Y107Q, Y107R, Y107T, E116G, N119Q, T123S, T123C, Q126I, and Q126V; wherein amino acid residue positions relative to the mature human IL-2 amino acid SEQ ID NO. 除了在成熟人IL-2序列1位删除了起始丙氨酸残基之外,美国临时专利申请序列号60/550,868中描述的其它突变蛋白包括上述突变蛋白。 In addition to the initial alanine residue deleted in the sequence of a mature human IL-2 addition, U.S. Provisional Patent Application Serial No. other muteins described in 60 / 550,868 comprises the above-described muteins.

IL-2突变蛋白也可以是IL-2融合物或偶联物,所述融合物或偶联物包括与第二蛋白融合或与聚脯氨酸或水溶性聚合物共价偶联的IL-2,以降低给药频率或提高IL-2耐受性。 IL-2 muteins may be IL-2 fusions or conjugates, the conjugate comprises a fusion or fusion with a second protein or conjugated to polyproline or a water-soluble polymer covalently IL- 2, to reduce the frequency of administration, or to improve IL-2 tolerability. 例如,可采用本领域已知的方法(参见,WO 01/79258),使IL-2突变蛋白与人白蛋白或白蛋白片段融合。 For example, a method may be employed (see, WO 01/79258) known in the art, so that IL-2 muteins and fused to human albumin or an albumin fragment. 或者,可采用本领域已知的方法(参见,例如美国专利号4,766,106;5,206,344和4,894,226),使IL-2突变蛋白与聚脯氨酸或聚乙二醇均聚物和聚氧乙基化多元醇共价偶联,其中所述均聚物未取代或一端被烷基取代,所述多元醇未取代。 Alternatively, a method known in the art (see, e.g. U.S. Patent Nos. 4,766,106; 5,206,344 and 4,894,226), so that IL-2 muteins to polyproline or polyethylene glycol homopolymers and polyoxyethylated polyols covalently coupling an alcohol, wherein said homopolymer is unsubstituted or substituted alkyl group at one end, said polyol is unsubstituted.

本发明分析方法也可由于检测其它治疗药物,例如其它免疫治疗剂、细胞因子和淋巴因子突变蛋白,以及免疫毒素和小分子化学治疗剂。 Analysis method of the present invention may also be due to the detection of other therapeutic agents, for example, other immunotherapeutic agents, cytokines and lymphokines muteins, immunotoxins and chemotherapeutic agent and a small molecule. 这些药物包括但不限于:白介素,例如IL-1、IL-2、IL-3、IL-4,IL-5、IL-12和这些分子的突变蛋白;干扰素,例如但不限于IFN-α、IFN-β、IFN-γ及其突变蛋白;GM-CSF和GM-CSF的突变蛋白;肿瘤坏死因子,例如TNF-α和TNF-β及这些分子的突变蛋白;抗神经节苷抗体、环孢素A、环磷酰胺、丝裂霉素C、FK973、野百合碱吡咯(monocrotalinepyrrole)和阿糖胞苷及这些分子的突变蛋白;以及各种免疫毒素。 These drugs include but are not limited to: interleukins, such as IL-1, IL-2, IL-3, IL-4, mutein IL-5, IL-12 and these molecules; interferons such as, but not limited to IFN-α , IFN-β, IFN-γ and its muteins; GM-CSF, and GM-CSF muteins; tumor necrosis factor, e.g. muteins TNF-α and TNF-β, and these molecules; anti-ganglioside antibodies, ring mutein cyclosporin a, cyclophosphamide, mitomycin C, FK973, monocrotaline pyrrole (monocrotalinepyrrole) and these molecules and cytarabine; and various immunotoxins.

III.实验下面是进行本发明的具体实施方式的例子。 III. Experimental Below are examples of the present invention for specific embodiments. 提供这些实施例仅仅是为了说明目的,而不是为了以任何方式限制本发明的范围。 These examples are merely for illustrative purposes and are not intended to limit the scope of the invention in any way.

为确保使用的各种数值的准确性(例如,量、温度等),进行了研究,但应当允许一定的实验误差和偏差。 To ensure the accuracy of various values ​​used (e.g., amounts, temperature, etc.), it has been studied, but should allow some experimental errors and deviations.

材料和方法A.培养液和试剂:-PBS(不含Ca/Mg),冰冷;-培养液200(Cascade Biologics,Portland,OR);-培养液200PRF(Cascade Biologics,Portland,OR);-LSGS:低血清生长添加剂(50×)。 Materials and Methods A. Reagents and culture medium: -PBS (excluding Ca / Mg), ice; - medium 200 (Cascade Biologics, Portland, OR); - broth 200PRF (Cascade Biologics, Portland, OR); - LSGS : low serum growth supplement (50 ×). (Cascade Biologics,Portland,OR)。 (Cascade Biologics, Portland, OR). 补给性培养液中这些组分的最终浓度为:2%v/v胎牛血清;1μg/ml氢化可的松;10ng/ml人表皮生长因子;10ng/ml人表皮生长因子;3ng/ml基本的成纤维细胞生长因子和10g/ml肝素;-PSA溶液(Cascade Biologics,Portland,OR):完全培养液中的最终浓度含有100U/ml青霉素,100μg/ml硫酸链霉素和0.25μg/ml两性霉素B;-胰蛋白酶(0.25%)/EDTA(0.1%)(Cellgro,Hemdon,VA);-FITC-白蛋白(50mg)(Sigma,St.Louis,MO);-FITC-BSA储备液(20×)。 The final concentration of the culture solution supply of these components is: 2% v / v fetal calf serum; 1μg / ml hydrocortisone; 10ng / ml human epidermal growth factor; 10ng / ml human epidermal growth factor; 3ng / ml basic fibroblast growth factor and 10g / ml heparin; solution-PSA (Cascade Biologics, Portland, OR): final concentration in complete medium containing 100U / ml penicillin, 100μg / ml streptomycin sulfate and 0.25μg / ml amphoteric amphotericin B; - trypsin (0.25%) / EDTA (0.1%) (Cellgro, Hemdon, VA); - FITC- albumin (50mg) (Sigma, St.Louis, MO); - FITC-BSA stock solution ( 20 ×). FITC-BSA悬浮在2.5ml完全培养液200中。 FITC-BSA were suspended in 2.5ml complete culture solution 200.

-人AB血清(SeraCare Life Sciences,Oceanside,CA)-人AB培养液(500mL)RPMI(不含酚红)............................................426.5ml人AB-加热灭活*............................................50ml - human AB serum (SeraCare Life Sciences, Oceanside, CA) - human AB broth (500mL) RPMI (without phenol red) ...................... ...................... 426.5ml heat-inactivated human AB- * ................... ......................... 50ml

青霉素/链霉素(终浓度100μg/ml).................................5mlHEPES(1M储备液,终浓度25mM)....................................12.5mlL-谷氨酰胺(100×储备液,终浓度2mM).............................5ml两性霉素B(250μg/ml储备液,终浓度0.5μg/ml)....................1ml4℃下,储存培养液至多4周。 Penicillin / streptomycin (final concentration 100μg / ml) ................................. 5mlHEPES (1M stock solution final concentration 25mM) .................................... 12.5mlL- glutamine (100 × stock solution, final concentration 2mM) ............................. 5ml amphotericin B (250μg / ml stock solution, final concentration 0.5μg / ml) .................... at 1ml4 ℃, up to four weeks storage medium.

*4℃下过夜解冻血清,56℃加热灭活45分钟-RPMI:冰冷,不含血清-PBS/EDTA:5.7ml 0.5M EDTA加入到500ml PBS(不含Ca/Mg)中,最终pH7.2;-台盼蓝染料(Invitrogen Life Technologies,Carlsbad,CA,或等价的0.4%溶液);-10%皂苷储备液:2.5g皂苷在25ml PBS中,4℃下储存;-完全培养液200:培养液200/培养液200PRF..........................................500mlLSGS............................................................10mlPSA溶液.........................................................1mlB.细胞培养:HUVEC(人脐静脉内皮细胞)得自Cascade Biologics,Portland,OR。 * Thawed at 4 ℃ overnight serum, 56 ℃ heat inactivated 45 minutes -RPMI: cold, serum-free -PBS / EDTA: 5.7ml 0.5M EDTA was added to 500ml PBS (containing no Ca / Mg), the final pH7.2 ; - trypan blue dye (Invitrogen Life Technologies, Carlsbad, CA, or equivalent to 0.4% solution); - 10% saponin stock solution: 2.5 g of saponin in 25ml PBS and stored at 4 ℃; - 200 complete medium: 200 broth / culture medium 200PRF .......................................... 500mlLSGS .................................................. .......... 10mlPSA solution ...................................... . ................... 1mlB cell culture: HUVEC (human umbilical vein endothelial cells) obtained from Cascade Biologics, Portland, OR. 每小管含有≥5×105个细胞。 Each vial contains ≥5 × 105 cells. 通过浸入37℃的水,解冻小管。 37 [deg.] C by immersion in water, thawed vials. 用完全培养液200将细胞稀释至10ml,采用台盼蓝计数确定每毫升中的活细胞数。 200 cells were diluted to 10ml with complete medium, counted using trypan blue to determine the number of viable cells per ml. 然后,再用完全培养液200将小管内容物稀释至1.25×104活细胞/毫升。 Then, complete medium and then diluted with 200 to vial contents were 1.25 × 104 viable cells / mL. 将5ml或15ml细胞悬浮液分别加入到25cm2或75cm2培养瓶中,在培养瓶中涡旋培养液以分散细胞。 To 5ml or 15ml of the cell suspension were added 25cm2 to 75cm2 flasks or in flask culture was vortexed to disperse the cells. 在湿润的5%CO2孵育箱中,37℃下孵育培养物。 In a humidified 5% CO2 incubator, the cultures were incubated at 37 ℃. 24-36小时后,将培养液换成新鲜补给性培养液200,以后每天换液,直到培养物达到约80%融合。 After 24-36 hours, the medium was changed to fresh supply of the culture medium 200, after the medium was changed every day until the cultures reached approximately 80% confluence. 这大概需要5-6天。 It takes about 5-6 days.

然后如下所述传代培养HUVEC。 Then follows subcultured HUVEC. 除去培养液,将胰蛋白酶/EDTA溶液加入到培养瓶中,通过轻拍和吸取并加入完全培养液200取出细胞,将细胞转移至15ml无菌锥形试管中。 Culture medium was removed, the trypsin / EDTA solution was added to the flask, 200 complete medium and the cells are removed by suction, and added tapping, the cells were transferred to a 15ml sterile conical tube. 细胞在1000rpm下离心10分钟,计数,并以2.5×103活细胞/平方厘米接种。 Cells were centrifuged at 1000rpm for 10 minutes, counted and 2.5 × 103 viable cells / cm inoculation.

2或3代期间的细胞在冻存培养液(90%FBS,10%DMSO)中冷冻保存,储存在液氮中备用。 2 or 3 generations cells during the freezing medium (90% FBS, 10% DMSO) cryopreserved, stored in liquid nitrogen backup.

实施例1筛选IL-2突变蛋白的体外试验为了试验预测IL-2突变蛋白患者耐受性的能力,进行了以下试验。 In vitro tests of Example 1 Screening of mutein IL-2 To test the prediction embodiment ability of an IL-2 protein in patients with mutations, we performed the following tests. 试验中使用两种耐受性提高的IL-2突变蛋白,作为原理证据。 Tolerance test using two improved IL-2 muteins, as proof of principle. 这两种突变蛋白是F42E和Y107R取代突变蛋白。 Both proteins are F42E mutation Y107R and substitution mutein. 参见2005年3月5日提交的共同拥有、共同待批的美国临时专利申请序列号60/550,868。 See also co-owned 2005 March 5 filed, co-pending US Provisional Patent Application Serial No. 60 / 550,868. 体内外模型中,在NK和T细胞增殖,以及NK/LAK/ADCC活性方面,这些突变蛋白也保持了效应子功能。 Outer vivo models, in NK and T cell proliferation, and NK / LAK / ADCC activity of these muteins also maintained effector functions. 此外,使用由Emeryville,CA的Chiron Corporation生产的Proleukin作为引起VLS的代表性IL-2分子。 Further, by the use of Emeryville, CA produced by the Chiron Corporation Proleukin as a representative VLS induced IL-2 molecule. 该制剂中的IL-2是重组产生的,未糖基化的人IL-2突变蛋白,称为aldesleukin,其删除了起始丙氨酸残基并用丝氨酸残基取代125位半胱氨酸残基(称为脱-丙氨酰-1,丝氨酸-125人白介素-2)而与天然人IL-2氨基酸序列不同。 The formulation of IL-2 is recombinantly produced, unglycosylated human IL-2 mutein, called aldesleukin, which deleted the initial alanine residue 125 and a cysteine ​​residue substituted with serine residue group (referred to as de - alanyl-1, serine -125 human interleukin-2) with the amino acid sequence of native human 2 IL-different. 该IL-2突变蛋白在大肠杆菌中表达,然后如美国专利号4,931543所述经渗滤和离子交换层析纯化。 The IL-2 muteins expressed in E. coli, and as described in U.S. Patent No. 4,931543 was purified by diafiltration and ion exchange. IL-2制剂以Proleukin商品名提供,为无菌、白色至灰白色无防腐剂的冻干粉,每小管含有1.3mg蛋白质(22MIU)。 IL-2 formulation Proleukin tradename provided as a sterile, white to off-white preservative-free lyophilized powder, containing 1.3mg protein per vial (22MIU).

如下制备本试验中使用的淋巴因子-活化的杀伤(LAK)细胞。 Prepared lymphokines used in this test - activated killer (LAK) cells. 从正常供体收集全血,置于Vacutainer CPT管(ACDA,Becton Dickinson,Franklin Lakes,NJ)中。 Whole blood was collected from normal donors, was placed Vacutainer CPT tubes (ACDA, Becton Dickinson, Franklin Lakes, NJ) in. 根据CPT生产商的说明书分离PBMC。 PBMC were isolated according to the manufacturer's instructions CPT. 简言之,倒置试管以混合,1500-1800×g下离心20分钟,除去血沉棕黄层,置于锥形管中,用PBS 2%FBS洗(最多300×g,15分钟)。 Briefly, tubes inverted to mix, 1500-1800 × g rpm for 20 minutes to remove the buffy coat, disposed conical tube, washed with PBS 2% FBS (up to 300 × g, 15 minutes). 除去上清液,洗涤细胞2次。 The supernatant was removed, cells were washed twice. 将PBMC悬浮在RPMI-10AB培养液中,采用血细胞计数器进行台盼蓝排斥试验计数。 The PBMC were suspended in RPMI-10AB broth using a hemacytometer for counting trypan blue exclusion test. 将PBMC以1.5×106细胞/毫升悬浮在RPMI-10AB中(不含酚红)。 PBMC were 1.5 × 106 cells / ml were suspended in RPMI-10AB (without phenol red). 24孔组织培养板的每孔中加入1ml PBMC悬浮液,并加入含50nm所需IL-2突变蛋白的1ml/孔37℃RPMI-10AB培养液。 Per well of 24-well tissue culture plate was added 1ml PBMC suspension was added 37 ℃ RPMI-10AB broth IL-2 1ml / 50nm bore containing the desired mutein. 盖上培养板,在湿润的37℃、5%CO2培养箱中孵育3天,然后将培养板置于冰上约30分钟,除去上清液,4℃,300×g下离心5分钟,收集在冰上的50ml锥形试管中。 Cover plates, in a humidified 37 ℃, 5% CO2 incubator for 3 days and then the plates were placed on ice for about 30 minutes, the supernatant was removed, 4 ℃, 300 × centrifuged g 5 minutes to collect in 50ml conical tube on ice. 每孔中加入1ml冰冷的PBS/EDTA,孵育20分钟。 Each well was added 1ml ice-cold PBS / EDTA, incubated for 20 min. 取出粘附细胞,加入到冰上的试管中。 Adherent cells were removed and added to a test tube on ice. 每孔中加入1ml冷的PBS。 Each well was added 1ml of cold PBS. 4℃,将50ml锥形试管在300×g下离心5分钟。 4 ℃, a 50ml conical tube centrifuged at 300 × g for 5 minutes. 同时,洗涤各孔,将洗涤液收集在冰上50ml锥形试管中。 At the same time, each well was washed, the washing liquid collected in a 50ml conical tube on ice. 倒出上清液,细胞团悬浮在12ml冷RPMI中,加入到PBS洗涤液中。 The supernatant was decanted and the cell pellet resuspended in 12ml cold RPMI added to the wash solution in PBS. 4℃,300×g下再次离心5分钟。 4 ℃, at 300 × g rpm for 5 minutes again. 再次倒出上清液,悬浮细胞团,离心,如上所述。 The supernatant was decanted again, cell pellet was resuspended, centrifuged, as described above. 将洗涤的细胞悬浮在冷的完全RPMI中,台盼蓝排斥试验计数。 The washed cells were suspended in cold complete RPMI, counted by trypan blue exclusion test.

将600μl完全培养液200PRF加入到胶原涂覆转移孔(Transwell-COLTM,胶原涂覆PTFE膜,Costar(Corning,Inc.,Coming,NY)的下室中,从培养瓶中取出代系小于5的HUVEC,以1.0×106细胞/毫升悬浮在完全培养液200PRF中。将100μlHUVEC悬浮液加入到转移孔的上室中。设立含培养液但无细胞的对照转移孔,以测定FITC-BSA穿过胶原膜的最大转移。将转移孔在湿润的5%CO2培养箱中,37℃孵育3天,以建立融合单层。第4天,用结晶紫(Sigma,St.Louis,MO;2.3%w/v,草酸铵0.1%w/v,乙醇,SD3A 20%v/v)染色单层,检查融合。当单层达到90%融合时进行试验。 The 200PRF 600μl complete medium was added to the collagen-coated transfer holes (Transwell-COLTM, collagen-coated PTFE membrane, Costar (Corning, Inc., Coming, NY) under the chamber, removed from the flask on behalf of the Department of less than 5 of HUVEC to 1.0 × 106 cells / ml in complete culture medium were suspended in 200PRF. 100μlHUVEC the suspension was added to the upper chamber in the transfer holes. establishment but no cell control wells containing broth transferred to FITC-BSA measured through collagen the maximum transfer film transfer holes in a humidified 5% CO2 incubator, 37 [deg.] C incubation for 3 days to establish a confluent monolayer on day 4, with crystal violet (Sigma, St.Louis, MO;. 2.3% w / v, ammonium oxalate 0.1% w / v, ethanol, SD3A 20% v / v) single staining, examination fusion. when tested confluent monolayers reached 90%.

从转移孔的上室中全部取出完全培养液200,将100μl含FITC-BSA(1mg/ml)的完全培养液200加入到试验和对照转移孔中。 Remove complete medium transfer hole 200 from the upper chamber of all, the 100μl complete medium containing FITC-BSA (1mg / ml) was added to a 200 well test and control transfer. 用铝箔覆盖培养板,30分钟时,从转移孔的下室中取出10μl样品,置于具有透明底部的黑色96-孔板中。 Plates were covered with aluminum foil, 30 minutes, 10μl samples were taken from the transfer chamber hole, placed in a black clear bottom 96-well plates. 然后,补充10μl完全培养液至下室中。 Then, 10μl complete medium added to the lower chamber. 将40μl完全培养液200加入到黑色96-孔板中,混合。 The 40μl complete medium were added to black 96-well plate 200, and mixed. 利用IL-2突变蛋白-荧光素(485/575nm,1.0s)进行,样品读数。 IL-2 muteins using - fluorescein (485 / 575nm, 1.0s) for sample reading. 基于荧光强度读数,再次分配转移孔,对于90%融合或更高的融合单层,读数应低于6,000。 Based on the fluorescence intensity readings, dispensing wells were again transferred, for a 90% or more fused confluent monolayers, reading should be less than 6,000. 孵育3小时后,培养板再次取样,读数,如上所述。 After 3 hours of incubation, plates were again sampled readings, as described above. 3小时强度读数作为基线或时间0时的读数。 3 hours or a time intensity readings as a baseline reading of zero.

FITC-BSA从上室除去,将100μl含FITC-BSA(1mg/ml)的试验化合物加入到上室中:(1)IL-2突变蛋白F42E、Y107R或Proleukin(25nM);(2)3天25nM IL-2-突变蛋白-刺激的PBMC上清液;(3)3天25nM IL-2-突变蛋白-刺激的LAK细胞;和(4)3天25nM IL-2-突变蛋白-刺激的LAK细胞的上清液。 FITC-BSA was removed from the chamber, the 100μl containing FITC-BSA (1mg / ml) of the test compound was added to the upper chamber: (1) IL-2 muteins F42E, Y107R or Proleukin (25nM); (2) 3 day 25nM IL-2- mutein - stimulated PBMC supernatant; (3) 3 days 25nM IL-2- mutein - stimulated LAK cells; and (4) 3 days 25nM IL-2- mutein - stimulated LAK cell supernatants. 另外,采用0.05%皂苷作为阳性对照(皂苷能破坏单层),完全培养液200用作培养液对照。 Further, use as a positive control 0.05% saponin (saponin can damage monolayer), 200 complete medium was used as the control culture. 用铝箔覆盖培养板,37℃孵育。 Plate is covered with aluminum foil, 37 ℃ incubation. 与试验化合物孵育3小时后收集样品,荧光读数,如上所述。 After 3 hours of incubation were collected with the test compound sample, the fluorescence reading, as described above.

结果如图1-4所示。 The results shown in Figure 1-4. 所示数据例子为3次独立的实验,每种试验条件n=5或6,图中数据来自3位不同的正常人供体。 Data shown in Examples 3 independent experiments, each test condition n = 5 or 6, the data in FIG. 3 from a different normal human donors. 如图1A-1C所示,Proleukin-刺激的PBMC(LAK)和上清液导致内皮细胞单层明显损伤。 As shown in FIG. 1A-1C, Proleukin- stimulated PBMC (LAK) and a supernatant injury leading to endothelial cell monolayers significantly. 在3位受试供体的2位中,与Proleukin相比,F42E和Y107R突变蛋白的VLS显著降低。 In the three donors tested 2 as compared with Proleukin, F42E and Y107R muteins VLS significantly reduced. 如图2A-2C所示,3位受试供体的2位中,仅Proleukin-诱导的LAK细胞显示超过培养液对照样品的VLS显著增加,但培养液对照与F42E或Y107R之间没有显著性差异。 As shown in FIG. 2A-2C, 3 of two of the donor subject, only Proleukin- VLS induced LAK cells display broth over the control sample increased significantly, but no difference between the control and the culture medium or Y107R significant F42E difference. 如图3A-3C所示,仅来自IL-2-诱导的PBMC培养物上清液不足以在体外明显诱导VLS综合征。 As shown in FIG. 3A-3C, PBMC from only IL-2- induced culture supernatant insufficient to significantly induce VLS syndrome in vitro. 如图4A-4C所示,仅Proleukine或F42E和Y107R都不能直接引起EC单层损伤。 As shown in FIG 4A-4C, only Proleukine Y107R or F42E and can not cause direct damage EC monolayer. 如图1-4所示,单独用F42E和Y107R之间,F42E-和Y107R-刺激的上清液之间,单独用F42E-和Y107R-刺激的LAK细胞与培养液对照之间没有显著性差异。 No significant differences between F42E- Y107R- and LAK cells stimulated control culture solution shown in Figure 1-4, alone between F42E and Y107R, and between F42E- Y107R- stimulation supernatant alone .

因此,测定FITC-BSA穿过内皮细胞融合单层的渗透性的VLS的体外模型有效且一致。 Thus, FITC-BSA measured in vitro through cell fusion model VLS endothelial monolayer permeability effective and consistent.

因此,描述了用于预测IL-2突变蛋白患者耐受性的新型体外试验系统。 Accordingly, muteins described patient tolerance of new in vitro test systems for predicting IL-2. 虽然详细描述了本发明优选的实施方式,但应理解,不背离权利要求书所述的本发明精神和范围,可进行各种明显改变。 Although the detailed description of the presently preferred embodiments, it is to be understood that, without departing from the spirit and scope of the claims according to the present invention book, various obvious changes may be made.

Claims (19)

1.一种预测患者对选定的治疗药物耐受或不耐受的体外方法,所述方法包括:(a)提供贴附于粘附基质的内皮细胞融合单层;(b)使所述单层与以下成分接触:(i)所述选定的治疗药物或淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用所述治疗药物或所述LAK细胞的上清液激活外周血单核细胞产生所述LAK细胞,和(ii)可检测标记的大分子,其中,当所述单层保持完整时,所述融合单层可基本上保留所述可检测标记的大分子;(c)在所述单层的完整性受损时,在所述可检测标记的大分子能穿过所述融合单层和所述粘附基质的条件下,孵育步骤(b)的所述单层一段时间;和(d)检测穿过所述融合单层和所述粘附基质的大分子,作为患者对所述治疗药物耐受或不耐受的指标。 1. A method of predicting a patient selected in vitro method of treating drug resistance or intolerance, the method comprising: (a) providing adherent stromal attached to confluent monolayers of endothelial cells; (b) the a single layer in contact with the following components: (i) the selected killer (LAK) cells or a pharmaceutical preparation for treating lymphokine activated, wherein the activation of peripheral blood monocytes by treatment with the medicament or the LAK cell supernatant LAK cells produce the cell nucleus, and (ii) a detectable label macromolecules, wherein, when the single layer remains intact, the confluent monolayer may substantially retain the detectable label macromolecules; (c ) when the integrity of the monolayer is compromised, the detectably labeled macromolecules can pass through the single layer and under the condition of the confluent monolayer of adherent matrix, incubation in step (b) is period of time; and (d) detection of the macromolecules through the confluent monolayer and adherent matrix, as a patient the therapeutic index of the drug resistance or intolerance.
2.如权利要求1所述的方法,其特征在于,所述治疗药物是免疫治疗剂、免疫毒素或小分子化学治疗剂。 2. The method according to claim 1, wherein said therapeutic agent is an immunotherapeutic agents, immunotoxins, or a small molecule chemotherapeutic agent.
3.如权利要求2所述的方法,其特征在于,所述免疫治疗剂是白介素-2(IL-2)突变蛋白。 The method according to claim 2, wherein said immunotherapeutic agent is interleukin -2 (IL-2) mutein.
4.如权利要求1所述的方法,其特征在于,所述粘附基质包括胶原基质。 4. The method according to claim 1, wherein the adhesive matrix comprises a collagen matrix.
5.如权利要求1所述的方法,其特征在于,所述内皮细胞是人脐静脉内皮细胞(HUVEC)。 5. The method according to claim 1, wherein said endothelial cells are human umbilical vein endothelial cells (HUVEC).
6.如权利要求1所述的方法,其特征在于,所述可检测标记的大分子是可检测标记的白蛋白。 6. The method according to claim 1, wherein said detectable label is detectably labeled macromolecule albumin.
7.如权利要求6所述的方法,其特征在于,所述可检测标记的白蛋白是标记的牛血清白蛋白(BSA)。 7. The method according to claim 6, wherein said detectable label albumin is labeled bovine serum albumin (BSA).
8.如权利要求7所述的方法,其特征在于,所述BSA是荧光标记的。 8. The method according to claim 7, wherein the BSA is fluorescently labeled.
9.如权利要求8所述的方法,其特征在于,所述荧光标记是FITC。 9. The method according to claim 8, wherein said fluorescent label is FITC.
10.一种预测患者对白介素-2(IL-2)突变蛋白耐受或不耐受的体外方法,所述方法包括:(a)提供贴附于粘附基质的内皮细胞融合单层;(b)使所述单层与以下成分接触:(i)淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用所述IL-2突变蛋白激活外周血单核细胞产生所述LAK细胞,和(ii)可检测标记的大分子,其中,当所述单层保持完整时,所述融合单层可基本上保留所述可检测标记的大分子;(c)在所述单层的完整性受损时,在所述可检测标记的大分子能穿过所述融合单层和所述粘附基质的条件下,孵育步骤(b)的所述单层一段时间;和(d)检测穿过所述融合单层和所述粘附基质的大分子,作为患者对IL-2突变蛋白耐受或不耐受的指标。 10. A method of predicting a patient Interleukin -2 (IL-2) mutein tolerance or intolerance in vitro method, said method comprising: (a) providing affixed to adherent stromal confluent monolayer of endothelial cells; (b) contacting the monolayer and the following components: (i) a lymphokine activated killer (LAK) cell preparation, wherein said generating LAK cells by activating peripheral blood mononuclear cells with the IL-2 muteins, and (ii) a detectable label macromolecules, wherein, when the single layer remains intact, the confluent monolayer may substantially retain the detectable label macromolecules; (c) integrity of the monolayer when impaired, the detectably labeled molecules can pass through the confluent monolayer and under the adhesive matrix, incubation in step (b) the single period of time; and (d) detecting confluent monolayer of macromolecules through the matrix and the adhesive, as a patient indexes IL-2 muteins of tolerance or intolerance.
11.如权利要求10所述的方法,其特征在于,所述粘附基质包括胶原基质。 11. The method according to claim 10, wherein the adhesive matrix comprises a collagen matrix.
12.如权利要求10所述的方法,其特征在于,所述内皮细胞是人脐静脉内皮细胞(HUVEC)。 12. The method according to claim 10, wherein said endothelial cells are human umbilical vein endothelial cells (HUVEC).
13.如权利要求10所述的方法,其特征在于,所述可检测标记的大分子是可检测标记的白蛋白。 13. The method according to claim 10, wherein said detectable label is detectably labeled macromolecule albumin.
14.如权利要求13所述的方法,其特征在于,所述可检测标记的白蛋白是标记的牛血清白蛋白(BSA)。 14. The method according to claim 13, wherein said detectable label albumin is labeled bovine serum albumin (BSA).
15.如权利要求14所述的方法,其特征在于,所述BSA是荧光标记的。 15. The method according to claim 14, wherein the BSA is fluorescently labeled.
16.如权利要求15所述的方法,其特征在于,所述荧光标记是FITC。 16. The method according to claim 15, wherein said fluorescent label is FITC.
17.一种涉及预测患者对白介素-2(IL-2)突变蛋白耐受或不耐受的体外方法,所述方法包括:(a)提供贴附于粘附基质如胶原基质的人脐静脉内皮细胞(HUVEC)的融合单层;(b)使所述单层与以下成分接触:(i)淋巴因子活化的杀伤(LAK)细胞制剂,其中,通过用所述IL-2突变蛋白激活外周血单核细胞产生所述LAK细胞,和(ii)荧光标记的白蛋白;(c)在所述单层的完整性受损时,在所述荧光标记的白蛋白能穿过所述融合单层和所述粘附机制的条件下,孵育步骤(b)的所述单层一段时间;和(d)检测穿过所述融合单层的荧光标记的白蛋白,作为患者对所述IL-2突变蛋白耐受或不耐受的指标。 17. A method of in vitro involves predicting patient Interleukin -2 (IL-2) mutein tolerance or intolerance, said method comprising: (a) providing adherent matrix is ​​attached to the collagen matrix, such as human umbilical confluent monolayers vein endothelial cells (HUVEC) in; (b) the following components in contact with the monolayer: killer (LAK) cell preparation (i) lymphokine activated, wherein the activation by treatment with the IL-2 muteins the peripheral blood mononuclear cells produce LAK cells, and (ii) a fluorescent labeled albumin; (c) when the integrity of the monolayer is compromised, in the fluorescent-labeled albumin fusion can pass through the and the monolayer under conditions of adhesion mechanisms, incubation in step (b) the single period of time; and (d) detecting through said confluent monolayers fluorescently labeled albumin, a patient said IL -2 mutein tolerance or intolerance index.
18.如权利要求17所述的方法,其特征在于,所述荧光标记的白蛋白是标记的牛血清白蛋白(BSA)。 18. The method according to claim 17, wherein the fluorescent-labeled albumin was labeled bovine serum albumin (BSA).
19.如权利要求18所述的方法,其特征在于,所述荧光标记是FITC。 19. The method according to claim 18, wherein said fluorescent label is FITC.
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