CN104650233A - ADC-drug-modified antibody IgG1-CH-K330C for establishing radioactive isotope coupling labelling - Google Patents

ADC-drug-modified antibody IgG1-CH-K330C for establishing radioactive isotope coupling labelling Download PDF

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CN104650233A
CN104650233A CN201510098479.1A CN201510098479A CN104650233A CN 104650233 A CN104650233 A CN 104650233A CN 201510098479 A CN201510098479 A CN 201510098479A CN 104650233 A CN104650233 A CN 104650233A
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heavy chain
amino acid
constant region
nucleotide sequence
acid sequence
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CN201510098479.1A
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Chinese (zh)
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梅岩
李涛
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南京任诺药业有限公司
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Abstract

The invention discloses an ADC-drug-modified antibody IgG1-CH-K330C for establishing radioactive isotope coupling labelling, which comprises (a) an amino acid sequence of a heavy chain constant region, an amino acid sequence of a heavy chain constant region CH2 and an amino acid sequence of a heavy chain constant region CH3 obtained by mutation modification; (b) an amino acid sequence of the heavy chain variable region disclosed as SEQ ID NO:2; (c) an amino acid sequence of the light chain variable region disclosed as SEQ ID NO:4; and (d) an amino acid sequence of the light chain constant region disclosed as SEQ ID NO:6. On the premise of not influencing the targeting properties and functions of the antibody, the antibody surface is subjected to site-directed mutagenesis, thereby controlling the number of the connected active micromolecules, and carrying a stable number of micromolecules, toxins or nuclides.

Description

用于构建放射性同位素偶联标记的ADC药物的改造抗体lgG1-CH-K330C ADC medicament for constructing engineered antibodies radioisotope labeled lgG1-CH-K330C

技术领域 FIELD

[0001] 本发明涉及生物医药领域,具体涉及一种用于构建放射性同位素偶联标记的ADC 药物的改造抗体IgGl-CH-K330C。 [0001] The present invention relates to the field of biomedicine, particularly relates to ADC engineered antibody drug for constructing a radioisotope-labeled IgGl-CH-K330C.

背景技术 Background technique

[0002] 现代生物学证明癌症有很强的多样性,并且选择性不好的药物会对正常细胞产生很大的负作用。 [0002] Modern biology of cancer has proved a strong diversity and selectivity bad drugs affect normal cells have a huge negative effect. 目前癌症新药研发局面缓慢,仍然有大量的癌症没有比较好的治疗办法。 The current situation slow cancer drug development, there are still a large number of cancer no better cure. 肝癌、肺癌、胰腺癌的五年存活率都在15%以下。 Liver cancer, lung cancer, pancreatic cancer five-year survival rates are 15% or less. 当前癌症新药研发的平台大致分为单克隆抗体大分子生物药和小分子化学药,小分子化学药选择性差,会对正常细胞产生难以预期的毒性。 Current cancer drug development platform roughly classified into biological macromolecules monoclonal antibodies and small molecule drugs chemical drugs, small chemical drug selectivity, normal cells will produce unpredictable toxicity. 单克隆抗体,简称单抗,对某一类细胞的选择性高,并可以在体内自然代谢,所以安全性相对较高,但单克隆抗体通常只能作用于细胞表面的靶点,因而经常只能起到抑制作用而并不能真正杀死癌细胞。 A monoclonal antibody, referred to as monoclonal antibody is selective for a certain type of cell is high, and may be metabolized naturally in the body, so the security is relatively high, but usually only the monoclonal antibody to act on the target cell surface, and it often only It can play an inhibitory effect and can not really kill cancer cells.

[0003] 理想的药物就是把优异临床疗效的靶向特异性抗体和小分子药物药代动力学结合起来,这就是抗体一药物偶联物ADC。 [0003] over the drug is to target specific antibodies excellent clinical efficacy and pharmacokinetics of the small molecule drug combination, which is an antibody drug conjugate ADC. 抗体-药物偶联物ADC是把具有细胞内吞作用的靶向特异性抗体和具有特定药理学特性,如细胞强毒作用的小分子化合物结合起来。 Antibody - drug conjugate the targeted ADC is specific antibody having endocytosis and have specific pharmacological properties, such as strong cytotoxic effects of small molecule compounds together. 即利用单克隆抗体把具有强烈毒副作用的毒素小分子化药靶向到肿瘤细胞,以期达到提高疗效, 减少毒副作用。 Namely the use of monoclonal antibodies to small molecule toxins Kayaku having a strong toxicity to targeted tumor cells, in order to improve efficacy, reduce side effects. 这项技术既克服了单抗药靶点可选性少,杀伤力差等缺点;同时又解决了小分子毒性高,代谢不稳定的问题。 This technology not only to overcome the monoclonal antibody drug targets for optional less lethal defects and poor; at the same time solve the high toxicity of small molecules, metabolic instability. 抗体-药物偶联物ADC因在血液中相对稳定,能有效地降低小分子细胞毒素化药本身对循环系统以及健康组织的毒性,是目前抗肿瘤领域的研宄热点之一〇 Antibody - drug conjugates because ADC is relatively stable in the blood, can effectively reduce the toxicity itself cytotoxic small molecule drugs for the circulatory system and of healthy tissue, it is one of the study based on hot square antitumor art

[0004] 抗体-药物偶联物ADC在抗癌领域拥有广泛的发展空间,成为近年来抗肿瘤药物领域中最受关注的靶向药物,成功应用于临床的例子有治疗乳癌的曲妥珠单抗,治疗非霍奇金淋巴瘤的利妥昔单抗等。 [0004] antibody - drug conjugates ADC has a broad space for development in the field of cancer, has become in recent years the field of anticancer drugs targeted drugs most attention, there are examples of successful used in clinical treatment of breast cancer trastuzumab alone anti, treatment of non-Hodgkin's lymphoma rituximab and so on. 抗体一药物偶联物ADC药物的抗体部分为单链抗体,效应分子通常由放射性核素、药物或毒素片段等组成,以抗体为载体的免疫偶联物将效应分子带到肿瘤区域的靶细胞,进而以效应分子对靶细胞的选择性杀伤作用来治疗癌症,因而抗体-药物偶联物ADC药物被称为治疗恶性肿瘤的"生物导弹"。 An antibody portion of the antibody drug conjugates ADC drug a single chain antibody, by the effector molecule is generally a radionuclide, drug or toxin fragment etc., antibody-carrier conjugate of the immune effector molecules to the tumor region of the target cells , and further to the effector molecule to selectively killing target cells for the treatment of cancer, and thus the antibody - drug conjugates ADC medicament for treatment of malignant tumors is called "biolistic." 抗体一药物偶联物ADC药物中单克隆抗体部分的靶向作用和抗体所连接的效应分子对靶细胞的杀伤作用两者都是至关重要的。 Antibody drug conjugates a monoclonal both ADC drug targeting and effector molecules linked antibody antibody portion of killing target cells is critical.

[0005] 在单抗新药的研发和改造中,分子影像学成为当今及其重要的研宄手段,正电子发射断层显像immunopositron emission tomography,以下简称ImmunoPET。 [0005] In the monoclonal antibody drug development and transformation, molecular imaging and become the important study based on means, positron emission tomography immunopositron emission tomography, hereinafter referred to as ImmunoPET. 为近年来发展的新兴分子影像学技术,借助于其PET的高灵敏性和抗体高特意性的结合,可用于寻找和量化单抗,从而提供了一种量化分子靶标的方法,成为在疾病诊断中广泛使用的发展设计技术。 Emerging molecular imaging technology developed in recent years, by means of a high sensitivity and high antibody binding specifically of PET which can be used to find and quantify the monoclonal antibodies, so as to provide a method for quantizing a target molecule, in the diagnosis of the disease become design technology is widely used.

[0006] 当前,抗体-药物偶联物ADC研发比较领先的公司主要有ImmunoGen和Seattle Genetics。 [0006] Currently, the antibody - drug conjugates ADC in leading R & D company mainly ImmunoGen and Seattle Genetics. ImmunoGen的主要技术是依靠抗体表面的Lysine将活性小分子和抗体连接起来制成ADC。 The main technique is to rely Lysine antibody ImmunoGen surface active small molecules and antibodies made to connect ADC. 这个技术的最大问题在于抗体表面有大量可以连接小分子的赖氨酸,这样就使得每次产生的ADC上一个抗体连接的活性小分子数量不能保持稳定。 The biggest problem is that the antibody technique a large number of surface-lysine can be attached to small molecules, so that the number of active small molecules of an antibody produced by each ADC connection can not be maintained stable. 有的抗体搭载多达8个小分子,有的却很少,甚至没有。 Some antibody molecules carry up to 8 hours, but some little or none. 过多承载小分子的抗体会失去抗体本身的特性,而搭载不足的抗体又不能在癌细胞内部产生足够的致死浓度。 Antibody carrying too small molecules will lose characteristics of the antibody itself, insufficient carrying antibodies can not produce enough lethal concentration inside the cancer cells. Seattle Genetics的主要技术是依靠还原抗体配对的SS键,然后依靠还原后的SH基团与连接物上的还原态的硫形成新的SS 键,从而将活性小分子和抗体连接起来制成ADC。 The main technique is to rely on Seattle Genetics reduced antibody paired SS bond, SS bond and the formation of new sulfur-reducing state relies on the reduced SH groups and the linker to the active small molecules and antibodies made to connect ADC. 他们的技术问题在于破坏了抗体原有的配对SS键,使得抗体的稳定性大大降低,而且每次还原的抗体配对的SS键数量不恒定,使得他们和ImmunoGen有同样的问题,每次产生的抗体-药物偶联物ADC上一个抗体连接的活性小分子数量不能保持一致。 Their technical problem that destroyed the original antibody SS key pair, such that stability of the antibody greatly reduced, and reduction of the number of SS bonds each antibody pair is not constant, so that they have the same problem and ImmunoGen, each resulting antibody - an antibody drug conjugate on the number of active small molecule ADC not consistent. ADC药物中单克隆抗体部分的靶向作用和抗体所连接的效应分子对靶细胞的杀伤作用两者都是至关重要的。 ADC drug targeting both the monoclonal antibody and the effector molecule linked antibody portion of killing target cells is critical.

[0007] 目前缺乏一种在不影响抗体靶向性和功能性的前提下,突变改造的突变点不影响抗体可变区与抗原结合的活性;维持可结晶片段区的构象和功能;点突变位于抗体分子表面;突变在温和条件下即可连接链接物的抗体。 [0007] A current lack of targeting and without affecting functional antibody, the mutated by point mutation does not affect the activity of the antibody to the antigen binding variable region; maintaining crystallizable fragment conformation and functional areas; point mutations located surface antibody molecules; mutant antibodies was to connect the link under mild conditions.

发明内容 SUMMARY

[0008] 本发明的目的是在不影响抗体靶向性和功能性的前提下,通过对抗体表面进行定点突变,从而控制连接活性小分子,突变在温和条件下即可连接连接物的用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C。 [0008] The object of the present invention without affecting the targeting and functional antibody, the antibody surface by site-directed mutagenesis, so as to control active small molecule, mutations can under mild conditions for connection thereof ADC drug radioisotope labeled constructs engineered antibody IgGl-CH-K330C.

[0009] 为了实现上述技术目的,本发明提供的技术方案为:一种用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C,包括 [0009] To achieve the above technical object, the technical solution of the present invention to provide: a method for constructing engineered antibody drug ADC radioisotope labeled IgGl-CH-K330C, comprising

[0010] (a)重链恒定区,包括重链恒定区CH1的氨基酸序列、重链恒定区CH2的氨基酸序列和通过突变改造过得到的重链恒定区CH3的氨基酸序列;所述重链恒定区CH1的氨基酸序列是SEQ ID NO:8所示的氨基酸序列,所述重链恒定区CH2的氨基酸序列是SEQ ID NO: 10 所示的氨基酸序列,所述重链恒定区CH3的氨基酸序列是SEQ ID NO: 12所示的氨基酸序列; [0010] (a) a heavy chain constant region comprising an amino acid sequence of the heavy chain constant region CH1, the amino acid sequence of the heavy chain constant region CH2 and transformed by mutation resulting heavy chain constant region CH3; and the heavy chain constant CH1 region amino acid sequence is SEQ ID NO: 8 amino acid sequence shown in the amino acid sequence of the heavy chain constant region CH2 is SEQ ID NO: 10 amino acid sequence shown in the amino acid sequence of the heavy chain constant region CH3 is SEQ ID NO: 12 amino acid sequence shown;

[0011] (b)重链可变区,所述重链可变区的氨基酸序列是SEQ ID NO:2所示的氨基酸序列; [0011] (b) a heavy chain variable region, the heavy chain variable region amino acid sequence is SEQ ID NO: 2 amino acid sequence;

[0012] (C)轻链可变区,所述轻链可变区的氨基酸序列是SEQ ID N0:4所示的氨基酸序列; The amino acid sequence [0012] (C) a light chain variable region, the light chain variable region is N0 SEQ ID: 4 amino acid sequences shown;

[0013] (d)轻链恒定区,所述轻链恒定区的氨基酸序列是SEQ ID N0:6所示的氨基酸序列。 [0013] (d) a light chain constant region, the amino acid sequence of the light chain constant region is SEQ ID N0: 6 shown in the amino acid sequence.

[0014] 进一步地,制备所述的用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C的方法。 [0014] Further, for preparing the engineered antibody drug ADC radioisotope labeled IgGl-CH-K330C constructed by the method.

[0015] 进一步地,用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C的搭载小分子、毒素或核素的方法。 [0015] Further, a method for ADC engineered antibody drug radioisotope labeled IgGl-CH-K330C mounted a small molecule, a toxin or radionuclide construct.

[0016] 更进一步地,本发明的一种分离的核酸,包括所述的重链恒定区的核苷酸序列、重链可变区的核苷酸序列、轻链恒定区的核苷酸序列和轻链可变区的核苷酸序列; [0016] Still further, the present invention is one kind of isolated nucleic acid comprising the nucleotide sequence of the heavy chain constant region, the nucleotide sequence of the heavy chain variable region, the nucleotide sequence of the light chain constant region and the light chain variable region nucleotide sequence;

[0017] 所述重链恒定区的核苷酸序列包括所述重链恒定区CH1的核苷酸序列、重链恒定区CH2的核苷酸序列和重链恒定区CH3的核苷酸序列; [0017] The nucleotide sequence of the heavy chain constant region comprises the nucleotide sequence of the heavy chain constant region CH1, and the nucleotide sequence of heavy chain CH2 constant region and the nucleotide sequence of the heavy chain constant region CH3;

[0018] 所述重链恒定区CH1的核苷酸序列是SEQ ID NO:7所示的核苷酸序列,所述重链恒定区CH2的核苷酸序列是SEQ ID N0:9所示的核苷酸序列,所述重链恒定区CH3的核苷酸序列是SEQ ID NO: 11所示的核苷酸序列; [0018] The nucleotide sequence of the heavy chain CH1 constant region is SEQ ID NO: 7 nucleotide sequence, the nucleotide sequence of the heavy chain CH2 constant region is SEQ ID N0: 9 shown in nucleotide sequence, the nucleotide sequence of the heavy chain constant region CH3 is SEQ ID NO: 11 nucleotide sequence;

[0019] 所述重链可变区的核苷酸序列是SEQ ID NO: 1所示的核苷酸序列,所述轻链恒定区的核苷酸序列是SEQ ID N0:5所示的核苷酸序列,所述轻链可变区的核苷酸序列是SEQ ID N0:3所示的核苷酸序列。 [0019] The nucleotide sequence of the heavy chain variable region is SEQ ID NO: 1 nucleotide sequence, the nucleotide sequence of the light chain constant region is SEQ ID N0: 5 as shown in nucleus nucleotide sequence, the nucleotide sequence of the light chain variable region is N0 SEQ ID: 3, the nucleotide sequence shown.

[0020] 进一步地,上述改造抗体IgGl-CH-K330C用于治疗和诊断工具以检测表达腱生蛋白的疾病的用途。 [0020] Further, the above-described engineered antibody IgGl-CH-K330C therapeutic and diagnostic tool to detect the expression of a disease tenascin.

[0021] 进一步地,所述疾病为恶性肿瘤或系统性自身免疫病或新生血管类疾病。 [0021] Further, the disease is cancer or autoimmune disease or systemic neovascular diseases.

[0022] 更进一步地,所述恶性肿瘤选自囊性脑肿瘤、神经胶质瘤、鼻咽癌、胰腺癌、肺癌、 食管癌、乳腺癌、胃癌、大肠癌、肝癌、前列腺癌、卵巢恶性肿瘤、宫颈癌、子宫内膜癌、恶性黑色素瘤、皮肤癌、淋巴瘤、白血病或甲状腺癌。 [0022] Furthermore, said cancer is selected from cystic brain tumors, gliomas, nasopharyngeal cancer, pancreatic cancer, lung cancer, esophageal cancer, breast cancer, stomach cancer, colorectal cancer, liver cancer, prostate cancer, ovarian malignant tumor, cervical cancer, endometrial cancer, malignant melanoma, skin cancer, lymphoma, leukemia or thyroid cancer.

[0023] 进一步地,所述系统性自身免疫病选自系统性红斑狼疮、类风湿性关节炎、系统性脉管炎、硬皮病、天疱疮、皮肌炎、混合结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病或溃疡性结肠炎。 [0023] Furthermore, the systemic autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, itself autoimmune hemolytic anemia, autoimmune thyroid disease or ulcerative colitis.

[0024] 更进一步地,所述新生血管类疾病选自黄斑水肿、脉络膜新生血管性疾病、新生血管青光眼、视网膜静脉阻塞或视网膜新生血管性疾病。 [0024] Furthermore, said neovascular diseases are selected from macular edema, choroidal neovascular diseases, neovascular glaucoma, retinal vein occlusion or retinal neovascularization.

[0025] 有益效果:本发明在单抗体表面进行定点突变。 [0025] Advantageous Effects: The present invention is directed mutagenesis on a single surface of the antibody. 选用IgGl的骨架,在可结晶片段Fc端进行半胱氨酸Cys突变,通过可结晶片段Fc端的突变点进行定向的化学偶联形成特定的抗体偶联物。 Selection IgGl backbone, for Cysteine ​​Cys mutation Fc fragment crystallizable side, by orienting point mutation Fc fragment crystallizable end chemically coupled to form specific antibody conjugate. 本发明具有如下优点: The present invention has the following advantages:

[0026] (1)本发明在不影响抗体靶向性和功能性的前提下,通过对抗体表面进行定点突变,从而解决控制连接活性小分子的数目这个问题。 [0026] (1) In the present invention, without affecting the antibody targeting and functionality, by site-directed mutagenesis of the antibody surface, to control the number of connections to solve this problem active small molecules. 通过控制突变基团的数目来控制连接活性小分子的数目,从而克服抗体-药物偶联物ADC上一个抗体连接的活性小分子数量不能保持一致的关键性技术障碍。 To control the number of connections by the number of active small molecules mutated control groups, thus overcoming the antibody - ADC on a number of active small molecules attached to the antibody is not consistent with the key technical barriers to drug conjugate.

[0027] (2)经过半胱氨酸Cys替换改造的抗体骨架,除去重链可变区和轻链可变区的IgGl人抗体部分,维持了正常的抗体空间构象和可结晶片段Fc功能,用基因工程的方法构建不同的抗体以达到多种靶向和结合多种抗原的功能。 [0027] (2) After replacing Cys cysteine ​​engineered antibody framework, human IgGl antibody was removed portion of the heavy chain variable region and light chain variable regions, to maintain normal antibody conformation and the function Fc fragment crystallizable, Construction of different antibodies by genetic engineering to achieve more targeting and binding function more antigens.

[0028] (3)经过突变的单抗体既保持了抗体的选择性和稳定性,又能搭载稳定数量的小分子、毒素或核素。 [0028] (3) through a single mutated antibody while maintaining the selectivity and stability of the antibody, but a stable number of small molecules is mounted, a toxin or radionuclide. 通过替换新增半胱氨酸Cys位点的巯基与恰当的连接物结合后,可以与小分子毒素、化疗药物、放射性同位素、亲和配体连接,连接复合物应用于多种类型的抗肿瘤治疗和体外体内病理诊断等各个方面。 Add Cys cysteine ​​site by replacing a mercapto group with an appropriate linker upon binding, it can be a small molecule toxins, chemotherapeutic agents, radioactive isotopes, affinity ligand, connecting the composite applied to many types of anti-tumor the treatment of various pathological diagnosis in vitro and in vivo.

[0029] 说明书附图 [0029] The accompanying drawings

[0030] 图1为Herceptin单克隆抗体的重链可变区DNA序列PCR产物琼脂糖凝胶电泳图; [0030] FIG. 1 is a heavy chain variable region of monoclonal antibody Herceptin DNA sequence of the PCR products by agarose gel electrophoresis;

[0031] 图2为Herceptin单克隆抗体的轻链可变区DNA序列PCR产物琼脂糖凝胶电泳图; [0031] FIG. 2 Herceptin is a monoclonal antibody light chain variable region DNA sequence of the PCR products by agarose gel electrophoresis;

[0032] 图3为重链可变区和轻链可变区克隆结果PCR鉴定图; [0032] FIG. 3 is a clone identified by PCR results in FIG heavy chain variable region and light chain variable region;

[0033] 图4为轻链可变区质粒抽提电泳图; [0033] FIG. 4 is a light chain variable domain plasmid extraction electrophoresis;

[0034] 图5为重链可变区质粒抽提电泳图; [0034] FIG. 5 is a heavy chain variable region of plasmid extraction electrophoresis;

[0035] 图6为Here印tin重链恒定区330位点突变后电泳图; [0035] Here FIG. 6 is a tin plate heavy chain constant region 330 After electrophoresis point mutation;

[0036] 图7为Here印tin重链恒定区330位点突变后质粒抽提电泳图; [0036] Here FIG. 7 is a tin plate heavy chain constant region 330 After plasmid extraction point mutation electrophoresis;

[0037] 图8为重链恒定区330位点突变后表达的蛋白SDS-PAGE图; [0037] FIG. 8 is a heavy chain constant region 330 protein expression SDS-PAGE FIG point mutation;

[0038] 图9为抗体结构简图; [0038] FIG. 9 is a structure diagram antibody;

[0039] 图10为本发明的突变抗体搭载核素的策略示意图; [0039] FIG 10 a schematic view of the present invention, the antibody mutant strategy mounted nuclides;

[0040] 图11为本发明的突变抗体搭载小分子的策略示意图; [0040] FIG 11 a schematic view of the present invention, the antibody mutant strategy mounted a small molecule;

[0041] 图12为本发明的突变抗体搭载毒素的策略示意图。 [0041] FIG 12 a schematic view of the present invention, the antibody mutant strategy mounted toxin.

具体实施方式 Detailed ways

[0042] 下面结合具体实施方式对本发明进行详细描述,所述的实施例有助于对本发明的理解和实施,并非构成对本发明的限制。 [0042] DETAILED DESCRIPTION The following embodiments of the present invention will be described in detail, for example, contribute to the understanding and practice of the invention, not to limit the invention to the embodiment. 实施本发明,除具体实施例中所涉及的物料和精馏操作条件外,本领域技术人员还可以根据不同的分离目的对其进行等同或等效变换。 Embodiment of the present invention, in addition to materials and operating conditions of Example rectification involved in specific embodiments, the skilled artisan can also be transformed according to the same or equivalent different separation purposes. 本发明的保护范围并不以具体实施方式为限,而是由权利要求加以限定。 The scope of the present invention is not limited to particular embodiments, but be defined by the claims.

[0043] 本发明的主要创新点在于:按照以下的标准进行突变设计(1)在不影响抗体结构的前提下对抗体氨基酸进行突变。 [0043] The main innovation of this invention is: designing a mutation (1) according to the following criteria in the antibody amino acid mutations without affecting the antibody structure. (2)突变位点的设计不会引入疏水基团。 (2) designed mutation site does not introduce a hydrophobic group. (3)位于溶剂裸露区。 (3) located in a solvent exposed region. ⑷空间位阻大,不易形成二聚体。 ⑷ sterically bulky, difficult to form a dimer. (5)对抗体的domain之间的相互作用无影响。 (5) no effect on the interaction between an antibody domain. (6)总体的Dstability降低。 (6) the overall Dstability reduced. (7)不影响Fc的功能。 (7) does not affect the Fc function.

[0044] IgGl重链恒定区的330位点的Lys很保守,在抗体表达中往往容易被切除,因此选择Lys为靶点进行突变不会影响Fc的功能,大量数据显示Lys对抗体的物理化学性质影响不大。 [0044] Lys 330 sites in IgGl heavy chain constant region is conserved, it is often easily cut antibody expression, the selection of target mutated Lys Fc does not affect the function of a large number of physical and chemical data Lys antibody nature has little influence. 在此位置进行偶联不会对抗体的功能造成影响。 The coupling in this position does not affect the function of antibodies.

[0045] 实施例1 [0045] Example 1

[0046] IgGl突变位点的设计 [0046] IgGl mutation site design

[0047] 为了得到抗HER2抗体人源化结构,我们把抗体蛋白序列(氨基酸序列在下面已列出)载入到分子操作平台(M0E)的同源建模软件中。 [0047] In order to obtain a humanized anti-HER2 antibody structure, we antibody protein sequence (amino acid sequence is listed below) to a loading platform molecule (M0E) homology modeling software. 在蛋白质结构数据库中选用1HZH的晶体结构作为抗J1ER2抗体的建模模板。 Selection of the crystal structure 1HZH J1ER2 as an anti-antibody protein structure modeling template in the database. 抗HER2抗体初始几何序列部分是从一个或多个1HZH 链的区域模板中复制的。 Anti-HER2 antibody is an initial geometric sequence portion copied from a template region of the one or more 1HZH chain. 在抗J1ER2抗体和1HZH序列中保守的残基部分,所有重原子被复制到抗J1ER2抗体,否则仅复制骨架部分。 Antibody and anti J1ER2 1HZH residues conserved sequence portion, is copied to all heavy atoms J1ER2 anti-antibody, or backbone moieties copy only. 模板链中可以形成二硫键的半胱氨酸与抗HER2 抗体序列的半胱氨酸的位置相对应时,将二硫键复制到模型中。 The position of the template strand of cysteine-cysteine ​​disulfide bond may be formed with an anti-HER2 antibody sequences relative seasonal copy disulfide to the model. 在确定了主干段和侧链构象的中间模型后,按照化合价的要求完成加氢原子,模型进行一系列的能量最小化设计以排除严重的空间位阻,最后对模型进行评分。 After determining the intermediate segment of the trunk model and the side-chain conformations, as required to complete the hydrogenation atomic valences, the model series of energy minimization designed to eliminate severe steric hindrance, scoring the model last. 然后将数据写入到输出数据库,并进行质量评估测量标记任何严重的几何问题。 Then writes the output data to the database, and evaluate the quality measurement mark any serious problems of geometry. 最终的模型为评分最佳的中间模型。 The final model for the best score among models. 同源建模阶段完成后,为了确认模型的立体化学与晶体结构典型值一致,使用Moe的蛋白质的几何立体化学质量检测工具来检验最终的抗HER2抗体模型。 After homology modeling phase is completed, in order to confirm the stereochemistry consistent model and crystal structure of typical values, geometric stereochemical quality detection means Moe protein to test the anti-HER2 antibody a final model. 将抗HER2抗体和1HZH的模型结构叠加,两者的均方偏根差(RMSD)不大于丨.5 A则被视为好的模型。 The anti-HER2 antibody and 1HZH superimposed model structures, the root mean square difference between the bias (the RMSD) of no greater than Shu .5 A is considered a good model. RMSD是两个结构之间匹配原子的距离的平方的均值的平方根。 RMSD is the matching distance between the two atoms of the root mean square of the structure. RMSD = SQRTRSUMWii)2}/^],dii是两个结构中相匹配原子的距离。 RMSD = SQRTRSUMWii) 2} / ^], dii configuration matches two atoms distance. N是两个结构的匹配的原子数。 N is the number of atoms of the two structures match. RMSD的值为0时则是相同的结构,值越大则二者的结构差异越大。 When RMSD value of 0 is the same structure, the greater the value the greater the difference between the two structures. RMSD值是评价二者结构变化的可靠指标。 RMSD value is a reliable indicator of the evaluation of both the structural change.

[0048] 下一步我们将设计抗HER2抗体模型结构表面的半胱氨酸的突变。 [0048] Next we will design an anti-HER2 cysteine ​​mutant antibody surface model. 改造恒定区以避免其干扰可变区与抗原的结合。 Transformation constant regions to avoid interference variable region binding to its antigen. 抗体表面半胱氨酸的改造考虑到不同的标准。 Cysteine ​​engineered antibodies of the surface taking into account the different criteria. 例如,从抗体域突变,突变点的裸露,氨基酸的取代等多方面考虑。 For example, from an antibody domain mutation, point mutation exposed amino acid substitutions and many other considerations. 开发高通量的筛选方法来确定适合半胱氨酸突变和偶联的位点。 Development of high throughput screening methods for determining the site of the cysteine ​​mutation and conjugated. 然而,还没有普遍的方法来预测抗体中半胱氨酸的突变能否稳定和高效的偶联,目前存在半胱氨酸突变点位于CL,CH1和CH3区域中。 However, there is no general way to predict whether the antibody mutant cysteine ​​stable and efficient coupling, cysteine ​​mutation site is located at present CL, CH1 and CH3 regions. 我们找到了一种新的半胱氨酸突变设计方法,通过这种方法开发了1个人源IgGl的半胱氨酸突变。 We found a new mutation of cysteine ​​design method, developed cysteine ​​mutants 1 individual source IgGl by this method. 为了避免专利冲突,我们仅专注于抗体的Fc区的改造而不是CH1。 In order to avoid patent conflicts, we only focus on the transformation of the Fc region of an antibody instead of CH1. 我们选择所有抗HER2抗体的Fc区裸露的残基作为半胱氨酸的突变目标。 Select all we anti-HER2 antibody Fc region residues as naked cysteine ​​mutant target. 设计算法的第一步,通过完全切断突变位点的连接a与0碳的键来划分系统。 The first step in the algorithm is designed, by connecting a carbon bond and 0 completely cut to divide the mutation site system. 分区后,形成一系列的与特定的残基不相交的原子集(侧链)。 After partition, the formation of a series of sets atom (side chains) does not intersect with a particular residue. 每个原子集连接一个或多个突变点和旋转异构体,这些旋转异构体从旋转异构体文库中读取。 Each atom connecting one or more mutations set points and rotational isomers, these rotamers read from the rotamer library. 旋转异构体和突变体中骨架原子叠加到突变残基的骨架原子中。 Rotamers and mutant backbone atoms superimposed mutated residue backbone atoms. 由于旋转异构体文库中不包含任何骨架原子,主干骨架中羰基氧的应变能加到侧链中。 Since the rotamer library does not contain any skeletal atom, trunk backbone carbonyl oxygen of the strain can be added to the side chain. 周边的残基被添加到空间构象的搜索中。 Surrounding residues are added to the spatial conformation search. 在生成半胱氨酸突变体后,有利于对突变的结构进行能量最小化。 After generating cysteine ​​mutant, beneficial mutations structure of energy minimization. 如果结构有严重的冲突,在使用保守的方法之前先使用一种特别的改良的方法来修饰。 If the structure has serious conflict, using conservative methods before the first use a special method to modify modified. 这种改良的方法允许原子在突变的残基自由移动,移动范围控制相邻原子在8A以内,重量lOkcal /A和偏差0.25A范围内,其他原子固定。 This improved method allows the atoms to move freely in the mutated residues, within the movement range control atoms. 8A, the weight lOkcal / A and the variation range of 0.25A, adjacent to other atoms fixed. 严重结构冲突的改良方法包括应用300kcal/A和偏差0.25A到所有原子,同时禁用静电。 The improved method comprises applying a serious conflict structure 300kcal / A to 0.25A and variations all of the atoms, while disabling static. 应用范德华缩放因子从〇. 1增加到1 进行多次能量最小化。 Application of van der Waals scaling factor. 1 to 1 billion from the multiple energy minimization. J1ER2的Fc区域半胱氨酸的突变的精确模型同时考虑突变体空间结构改变以及野生型到半胱氨酸突变型之间稳定性的改变。 An accurate model of the Fc region mutations in cysteine ​​J1ER2 change taking change of the spatial structure and stability of the mutants between cysteine ​​mutant to wild-type. 突变体稳定性的变化通过测定突变型(Mut)和野生型(WT)折叠自由能的不同来衡量。 Abrupt changes in the stability measured by the different assays the mutant (a Mut) and wild type (WT) in free energy of folding. 我们预测稳定性(s)的差异变化是通过HER2抗体结构在折叠(f)和展开(u)状态下的野生型和突变型之间进行的。 We predicted change stability difference (s) is in the folded configuration by HER2 antibody (f) and for deployment between wild-type and mutant at (u) state.

[0049]AA Gs = A GfMut- A GfWT = A GsWT - Mut- A GsWT - Mut [0050] 使用上述公式,我们在线性相互作用能(LIE)的基础上创建模型。 [0049] AA Gs = A GfMut- A GfWT = A GsWT - Mut- A GsWT - Mut [0050] Using the above formula, we create a model-based linear interaction energy (LIE) on. 根据LIE,从野生型到突变型残基的环境相互作用能量的改变通过AG表示,相互作用的能量是在蛋白的能量与不含相关残基的蛋白能量之间的改变。 The LIE, from wild-type to mutant residues environment interactions alter energy is represented by the AG, it is to change the interaction energy between the energy and protein free from protein to energy-related residues. 设计的应用程序将所有的突变写到数据库中,在表1中描述。 Applications designed mutations all written to the database, described in Table 1. 设计的应用程序自动对所有的野生型蛋白进行半胱氨酸突变同时计算稳定性的相对变化以及其它可预测性能。 Applications designed automatically to all wild type protein cysteine ​​mutants also calculated relative change prediction stability, and other properties. 突变设计输出数据如表1所示。 Mutant design output data shown in Table 1.

[0051]表1 [0051] TABLE 1

[0052] [0052]

Figure CN104650233AD00081

[0053] [0053]

Figure CN104650233AD00091

[0054] 在表1中我们重点注意三列,一列是突变列(mutation),一列是稳定性(stability)或稳定性变化(Dstability)列和最后一列是溶剂裸露区(solvent exposed surface area)。 [0054] In Table 1 we focus attention three, a mutated column (mutation), a is the stability (Stability) or stability changes (Dstability) column and the last column is solvent exposed region (solvent exposed surface area). 突变的定义如下面1:F306C,1代表链数,F代表野生型残基的单字母的名称,306代表残基的UID和插入代码,C代表突变成半胱氨酸单字母名称。 Mutation as defined in the following 1: F306C, 1 represents the number chain, letter F represents the name of a single wild-type residues, 306 residues represent UID and insert the code, C for cysteine ​​mutated into single letter. 稳定性列是突变体的绝对热稳定性。 Stability of the column is a mutant of absolute thermal stability. 当生成一个聚集体,稳定性是聚集体的稳定性的玻耳兹曼的平均值。 When generating an aggregate stability Boltzmann average stability of aggregates. 越负的值表示一个越稳定的突变。 The more negative value indicates a more stable mutation. 稳定性变化是从野生型蛋白到突变体的相对热稳定性。 Stability change relative to the wild-type protein thermostability mutants. 当生成一个聚集体,稳定性变化是聚集体的相对稳定性的玻耳兹曼的平均值。 When generating an aggregate stability Boltzmann average change of the relative stability of aggregates. 越负的值表示一个越稳定的突变。 The more negative value indicates a more stable mutation. 为了特定位点的偶联我们在CH3区域设计了1个IgGl的半胱氨酸的突变。 Conjugation to specific sites in the CH3 region we designed a mutant IgGl of cysteine. 我们的想法是将分布在抗体表面Fc区域里的氨基酸突变。 The idea is the distribution of amino acid mutations in the Fc region in the antibody surface. 我们倾向替代裸露在抗体表面的极性氨基酸(丝氨酸,苏氨酸,天冬氨酸)和带电氨基酸(赖氨酸,谷氨酸),替代极性氨基酸,半胱氨酸不会造成抗体表面电荷的巨大变化和蛋白结构的变化。 Alternatively we prefer antibody surface exposed to the polar amino acids (serine, threonine, aspartic acid), and charged amino acids (lysine, glutamic acid), polar amino acids Alternatively, the antibody does not cause surface cysteine great change in charge and changes the protein's structure. IgGl重链恒定区的330位点的Lys很保守,在抗体表达中往往容易被切除,因此选择Lys为祀点进行突变也不会影响Fc的功能,大量数据显示Lys对抗体的物理化学性质影响不大。 Lys 330. site IgGl heavy chain constant region is conserved, it is often easily cut antibody expression, mutated to Lys so selected point will not affect the Si Fc function, large amounts of data showing the effect of antibody Lys physicochemical properties little.

[0055] 实施例2 [0055] Example 2

[0056] 以抗HER2单克隆抗体Herceptin为例对该抗体进行重链恒定区CH3氨基酸位点突变 [0056] In the anti-HER2 monoclonal antibody Herceptin example amino acid point mutations CH3 heavy chain constant region of the antibody

[0057] (l)Herceptin单克隆抗体的轻、重链可变区的合成 [0057] Synthesis of (l) Herceptin monoclonal antibody light and heavy chain variable region

[0058] 采用PCR方法,分别以Herceptin单克隆抗体的轻链可变区、重链可变区DNA序列为模板合成Herceptin单克隆抗体的轻链可变区DNA和重链可变区DNA。 [0058] The PCR method, respectively, the light chain variable region of monoclonal antibody Herceptin, a heavy chain variable region DNA sequence of the light chain variable region and heavy chain variable region DNA DNA monoclonal antibody Herceptin for the template synthesis.

[0059] 合成轻、重链可变区的引物: [0059] Synthesis of the light and heavy chain variable region primer:

[0060] Herceptin 重链上游引物:accagggtgctgagcgaggtgcagctggtggagagcgg [0060] Herceptin heavy chain upstream primer: accagggtgctgagcgaggtgcagctggtggagagcgg

[0061] Herceptin 重链下游引物:gcccttggtgctagcgctgctcacggtcaccagggtg [0061] Herceptin heavy chain downstream primer: gcccttggtgctagcgctgctcacggtcaccagggtg

[0062] Herceptin 轻链上游引物:ataatgagtaggggagacatccagatgacccagag [0062] Herceptin light chain upstream primer: ataatgagtaggggagacatccagatgacccagag

[0063] Herceptin 轻链下游引物:gtgcagccaccgtacgcttgatctccaccttggtgc [0063] Herceptin light chain downstream primer: gtgcagccaccgtacgcttgatctccaccttggtgc

[0064] PCR反应体系 [0064] PCR reaction system

Figure CN104650233AD00101

[0065] [0065]

[0066] PCR反应条件 [0066] PCR reaction conditions

[0067] 预变性,95 °C,3分钟,1个循环 [0067] denaturation, 95 ° C, 3 min 1 cycle

[0068] 变性,95°C,1 分钟 [0068] denaturation, 95 ° C, 1 minutes

[0069] 退火,56°C,0• 5分钟30个循环 [0069] annealing, 56 ° C, 0 • 5 minutes and 30 cycles

[0070]延伸,72 °C 1.5 分钟 [0070] extension, 72 ° C 1.5 minutes

[0071] 最后延伸,72°C,10分钟 [0071] The final extension, 72 ° C, 10 minutes

[0072] PCR反应结束后进行琼脂糖凝胶电泳,如图1和图2,确认Herceptin单克隆抗体的重链可变区DNA序列PCR产物琼脂糖凝胶电泳图和Herceptin单克隆抗体的轻链可变区DNA序列PCR产物琼脂糖凝胶电泳图。 For [0072] After completion of PCR reactions by agarose gel electrophoresis, as shown in FIGS. 1 and 2, it was confirmed the heavy chain variable region of a monoclonal antibody Herceptin DNA sequence of the PCR product was agarose gel electrophoresis and the light chain of the monoclonal antibody Herceptin the variable region DNA sequence of the PCR product was agarose gel electrophoresis FIG.

[0073] (2)轻、重链可变区克隆至表达载体 [0073] (2) light and heavy chain variable region was cloned into an expression vector

[0074] 米用GcneArt®Seamless Cloning and Assembly (lifetechnologies Cat#A13288)无缝克隆上述Herceptin单克隆抗体的轻、重链可变区的PCR产物到表达载体,轻、重链可变区的反应体系一致。 [0074] m with GcneArt®Seamless Cloning and Assembly (lifetechnologies Cat # A13288) cloned seamless light above Herceptin monoclonal antibody, the PCR product of the heavy chain variable region into an expression vector, light, a reaction system of a heavy chain variable region consistent. 体系中的插入片段分别是Herceptin单克隆抗体的轻链可变区的PCR产物和Herceptin单克隆抗体的重链可变区的PCR产物。 System inserts were PCR product PCR product of the light chain variable region of the monoclonal antibody Herceptin and the heavy chain variable region of monoclonal antibody Herceptin.

[0075] [0075]

Figure CN104650233AD00111

[0076] 将上述反应体系混合,室温孵育30分钟,然后将混合物置于冰上立即进行下一步纯化操作。 [0076] The above reaction was mixed and incubated at room temperature for 30 minutes then the mixture was placed on ice immediately for the next purification operation. 如图3所示,重链可变区和轻链可变区克隆结果PCR鉴定图;通过PCR方法确定载体是否与轻、重链可变区正确连接,然后将相应的质粒转化至DH5 a感受态细胞中, 37°C,200rpm培养16小时,常规质粒柱抽提方法进行质粒抽提。 As shown, the heavy chain variable region and light chain variable regions were cloned PCR identification result of FIG. 3; and determining whether a carrier light and heavy chain variable regions are connected correctly, then the corresponding plasmid was transformed into competent DH5 a method by PCR competent cells, 37 ° C, culture 200rpm 16 hours post conventional plasmid extraction method for plasmid extraction. 如图4所示,为轻链可变区质粒抽提电泳图;如图5所示,为重链可变区质粒抽提电泳图。 4, the light chain variable region of plasmid extraction electrophoresis; shown in FIG. 5, as heavy chain variable region plasmid extraction electrophoresis.

[0077] (3)重链恒定区的位点突变 Mutation [0077] (3) a heavy chain constant region

[0078] 采用PCR方法对连接在表达载体的重链恒定区330位点的Lys突变为Cys。 [0078] The PCR method is connected at Lys 330 of expression vector of the heavy chain constant region is mutated to Cys.

[0079] 突变引物: [0079] The mutagenic primer:

[0080] 上游引物:tccctgtctccgggttgctaatctagag [0080] Forward primer: tccctgtctccgggttgctaatctagag

[0081]下游引物:gcaacccggagacagggagaggctcttc [0081] Reverse primer: gcaacccggagacagggagaggctcttc

[0082] PCR反应体系 [0082] PCR reaction system

[0083] [0083]

Figure CN104650233AD00112

[0084] PCR反应条件 [0084] PCR reaction conditions

[0085]预变性,95 °C,3分钟,1个循环 [0085] denaturation, 95 ° C, 3 min 1 cycle

[0086]变性,95°C,1分钟 [0086] denaturation, 95 ° C, 1 minutes

[0087]退火,62°C,0• 5分钟30个循环 [0087] annealing, 62 ° C, 0 • 5 minutes and 30 cycles

[0088]延伸,72 °C,1.5 分钟 [0088] extension, 72 ° C, 1.5 minutes

[0089] 最后延伸,72°C,10分钟 [0089] The final extension, 72 ° C, 10 minutes

[0090] Dpnl酶切PCR产物去除模板,10ul酶切产物加入到50ulDH5a感受态细胞中, 37°C过夜培养,每个平皿得到50个克隆。 [0090] Dpnl digested PCR product was the template is removed, the digestion product was added to 10 ul of competent cells 50ulDH5a, 37 ° C overnight incubation, each plate 50 obtained clones. 挑取单菌落扩大培养,抽提质粒。 Expansion for single colony was picked, plasmids were extracted. 如图6所示,为Herceptin重链恒定区330位点突变后电泳图;如图7所示,为Herceptin重链恒定区330 位点突变后质粒抽提电泳图; 6, the electrophoresis is a heavy chain constant region Herceptin 330 after the point mutation; 7, after heavy chain constant region Herceptin 330 plasmid extraction point mutations electrophoresis;

[0091] 实施例3 [0091] Example 3

[0092] 转染及抗体表达纯化 [0092] Transfection and Expression and Purification of Antibodies

[0093] 采用过柱法纯化Herceptin可变区轻、重链质粒和突变的重链恒定区质粒。 [0093] Herceptin was purified by column using the variable regions of the light and heavy chain plasmid and a mutant plasmid heavy chain constant region. 将合成并纯化好的Herceptin可变区轻、重链质粒和突变的重链恒定区质粒及轻链恒定区质粒转染至哺乳动物细胞中表达。 The synthesis and purification of Herceptin variable region good light and heavy chain plasmid and mutant heavy chain constant region and a light chain constant region of plasmid plasmid transfected into a mammalian cell expression.

[0094] 在8L灭菌的细胞培养瓶中接入3L的0.5 X 106cells/mL的HEK293细胞,37°C 150 转/分钟培养至活细胞浓度达到1. 5-2. 0 X 106cells/mL,加入4. 5L无血清的化学成分确定的培养基稀释细胞培养液。 [0094] The access 3L 0.5 X 106cells / mL HEK293 cells, 37 ° C 150 rev / min until the culture reached 1. 5-2 viable cell concentration in the cell culture flasks 8L sterilized. 0 X 106cells / mL, 4. 5L serum-free medium was added to the chemically defined cell culture medium dilution.

[0095]每106细胞中加入2ul Freestyle Max transfection reagent (Invitrogen),每l〇6细胞中加入lug的DNA。 [0095] per 106 cells was added 2ul Freestyle Max transfection reagent (Invitrogen), each lug l〇6 added to the cells in the DNA. 按照Invitrogen的说明书进行转染混合液的配制。 Transfection mixture is formulated according to Invitrogen's instructions. 转染24小时后以5 :100的比例加入Feed X (ACR0)至培养液中,在转染24小时后和96小时后加入葡萄糖和谷氨酰胺溶液。 24 hours after transfection, 5: 100 added Feed X (ACR0) to the culture medium 24 hours after transfection and after 96 hours the solution was added glucose and glutamine. 163小时后细胞培养液大约达到8. 4L。 After 163 hours the cell culture medium reached about 8. 4L.

[0096] 向培养液中加入ImM的PMSF灭活蛋白酶,在4°C离心机中3500rpm离心45分钟以分离细胞和杂质,通过0.6/0. 2micron ULTA HC Capsules (GE Healthcare Life Sciences)过滤后获得上清进行层析纯化。 [0096] After the culture obtained was added to ImM PMSF inactivate the protease, 3500rpm centrifuged for 45 minutes to separate cells and impurities,. 2micron ULTA HC Capsules (GE Healthcare Life Sciences) was filtered through a 0.6 / 0 at 4 ° C centrifuge The supernatant was purified by chromatography. HEK293细胞的上清载入MabSelect Sure层析柱(GE Healthcare Life Sciences),洗脱峰合并浓缩、米用Viva-spin 20ml,10k MWC0 超滤、0. 2um滤膜无菌过滤得到无菌的蛋白原液。 Loading supernatant of HEK293 cells MabSelect Sure column (GE Healthcare Life Sciences), elution peak were combined and concentrated, rice with Viva-spin 20ml, 10k MWC0 ultrafiltration, 0. 2um a sterile membrane sterile filtered protein dope. 如图8所示,为重链恒定区330位点突变后表达的蛋白SDS-PAGE图; For protein expression SDS-PAGE in FIG. 8, a point mutation heavy chain constant region shown in FIG 330;

[0097] 实施例4 [0097] Example 4

[0098] 通过ELISA方法在体外检测突变前后抗体结合抗原HER2的效应 [0098] Effect of antigen antibody binds HER2 in vitro before and after the detection of mutations by the ELISA method

[0099] 为确定突变的抗体与HER2结合的特异性,针对HER2和对照蛋白进行了ELISA。 [0099] To determine mutated specific antibody binds HER2, HER2 and for the control proteins were ELISA. 用包被缓冲液将J1ER2蛋白稀释至1~10 yg/ml,每孔加100ul,4°C过夜进行包被。 With coating buffer J1ER2 protein was diluted to 1 ~ 10 yg / ml, was added to each well 100ul, 4 ° C overnight for coating. 次日洗涤3次。 Washed 3 times the next day. 分别在相应的已包被的反应孔中加一定稀释的Herceptin样品及突变抗体0. lml,置37°C孵育1小时,洗涤。 Were added diluted in some wells have been coated with the respective samples and the Herceptin antibody mutant 0. lml, opposing 37 ° C for 1 hour, washed. (同时做空白、阴性及阳性孔对照)于反应孔中,加入新鲜稀释的酶标第二抗体(抗抗体)0. lml,37°C孵育30-60分钟,洗涤,最后一遍用DDW洗涤。 (At the same time a blank, negative control and positive hole) in the wells, was added freshly diluted enzyme-labeled secondary antibody (anti-antibody) 0. Lml, 37 ° C incubation for 30-60 minutes, washed, and finally washed again with DDW. 加底物液显色:于各反应孔中加入临时配制的TMB底物溶液0. lml,37°C 10~30分钟。 OPD color VIS: Extemporaneous added to each reaction wells TMB substrate solution 0. lml, 37 ° C 10 ~ 30 minutes. 终止反应:于各反应孔中加入2M硫酸0. 05ml。 The reaction was stopped: 0. 05ml 2M sulfuric acid was added to each reaction wells. 结果判定:可于白色背景上,直接用肉眼观察结果:反应孔内颜色越深,阳性程度越强,阴性反应为无色或极浅,依据所呈颜色的深浅,以" + 号表示。也可测0D450值:在ELISA检测仪上,于450nm处,以空白对照孔调零后测各孔0D450 值,若大于规定的阴性对照0D450值的2. 1倍,即为阳性。如表2所示,为ELISA检测重链恒定区330位点突变前后抗体活性变化; Results found: may be on a white background, with the naked eye observation: the darker the color reaction of the hole, the stronger the degree of positive, negative reaction is colorless or very light, was based on the color depth, a "+ sign also. 0D450 values ​​can be measured: in the ELISA reader at 450nm at each well to measure blank values ​​0D450 hole zeroing, if more than 2.1 times the negative control 0D450 predetermined value, is positive as shown in table 2. shown, before and after 330 point mutation by ELISA heavy chain constant region antibody activity change;

[0100] 表2 [0100] TABLE 2

[0101] [0101]

[0102] [0102]

Figure CN104650233AD00131

[0103] 由表2可知,抗体的重链恒定区330位点的氨基酸突变不会对抗体的活性造成影响。 [0103] As apparent from Table 2, the point 330 amino acid heavy chain constant region of the antibody mutant does not affect the activity of the antibody.

[0104] 实施例5 [0104] Example 5

[0105] 突变抗体与核素偶联方法 [0105] Method mutant antibodies conjugated radionuclide

[0106] 对核素的选择方面,主要通过对半衰期、精确度和代谢途径等方面的综合评估。 [0106] The choice of radionuclides, mainly through the half-life aspects of a comprehensive assessment of accuracy and metabolic pathways and the like. Zr89的半衰期约为3. 3天与抗体在体内的半衰期接近,I 124的半衰期约为4. 2天与Zr 89相近。 Zr89 half-life of about 3.3 days and the antibody in vivo half-life close, I 124 half-life of about 4.2 days similar to Zr 89. 但是l124(0+max. 1.5和2. IMeV)的正电子衰变灵敏度比较低。 However, l124 (0 + max. 1.5 and 2. IMeV) positron decay relatively low sensitivity. 而Zr89(0+max. 902keV) 的正电子衰变具有很高的灵敏度,可以与F18和C11相媲美。 And Zr89 (0 + max. 902keV) positron decay high sensitivity, F18 and C11 may be comparable. 1 124在代谢过程中通过蛋白水解作用,很快的经过酶催化反应去碘,被细胞代谢掉,PET技术不能准确的表现出抗体的作用。 1124 in the metabolic process by proteolysis, soon after the enzyme-catalyzed reaction to iodine, metabolized cells, PET technology can not accurately show the effect of the antibody. 而在Zr89-mAbs的代谢过程中,通过细胞内在化作用,Zr89-mAbs进入细胞内溶酶体中,进一步加强了抗体的PET成像作用。 And Zr89-mAbs in the metabolic process, the action of the cell by internalization, Zr89-mAbs into the cell lysosomes, to further enhance the effect of antibody PET imaging. 本发明中用射正电子的标记物Zr89标记抗体,通过电子发射断层照相技术(PET)对肿瘤进行诊断以及对个体化用药提供指导。 Shots with the present invention marker Zr89 labeled antibody electrons tumor diagnosed by electron emission tomography technique (PET) and provide guidance to individualized medicine.

[0107] 在抗体核素偶联物的linker设计方面,抗体表面有40个Lys和8个双链结合的Cys可以偶联,偶联的产物是不同位点以及不同linker和抗体比例的混合物。 [0107] In the linker design antibody radionuclide conjugate aspect, the antibody and the surface 40 Lys Cys 8 duplex binding can be coupled, the coupling product is a mixture of different and distinct sites and linker antibody ratio. 我们对抗体表面进行定点Cys突变,通过抗体表面自由的巯基链接核素可以产生位点单一,比例精准的偶联产物。 We Cys mutation site-directed surface of the antibody may be produced by a single site link mercapto surface free antibody species, the ratio of the coupling product precision. 抗体核素偶联物的linker的稳定性是至关重要的。 Stability linker antibody radionuclide conjugate is critical. 因此在linker方面选择非解离的硫醚链接,最大程度上保证linker的稳定性。 So choose non-sulfide link in the linker dissociated aspects, to ensure the stability of the linker maximum extent. 在确保linker稳定性的同时, linker的水溶性也将影响到最终抗体核素偶联物的作用。 At the same time ensuring the stability of the linker, linker-soluble will also affect the final effect of the antibody radionuclide conjugate. 避免过多的引入疏水基团,可以有效地提高抗体核素偶联物的水溶性,降低疏水基团聚集而引起的沉降的可能性。 The possibility of avoiding excessive settling incorporation of hydrophobic groups can improve the nuclide soluble antibody conjugate, hydrophobic groups to reduce aggregation caused.

[0108] 通过螯合剂络合的方式可以把Zr89标记到抗体表面。 [0108] can Zr89 labeled antibody to the surface by way of chelator. 螯合作用是具有两个或两个以上配位原子的多齿配体与同一个金属离子形成螯合环的化学反应。 Chelating polydentate ligand having two or more coordinating atoms of the chemical reaction to form a chelate ring with the same metal ion. 具有多齿配体的化合物称为螯合剂。 A compound having a multidentate ligand called a chelating agent. 螯合剂中配位原子的数目除了二齿、三齿外,还有四齿、五齿、六齿等。 The number of chelating agents coordinating atoms in addition to bidentate, tridentate, there tetradentate, pentadentate, hexadentate like. 金属离子和螯合配体生成的螯合物,比它和单齿配体生成的类似配合物有较高的稳定性。 Metal ions and chelating ligand chelates produced, than it generated and monodentate ligand complexes have similar high stability. 这是由于要同时断开螯合剂配位于金属上的两个键是困难的,如果已断开了一个键,则在第二个键未断开以前,它又可重新成键。 This is due to the chelating ligand and disconnect the two keys located on the metal is difficult, if a key has been disconnected, before the second key is not turned off, which in turn re-bonding.

[0109] 在螯合剂的选择方面,主要从螯合力、螯合剂稳定性以及水溶性等方面评估。 [0109] In the choice of the chelating agent, the chelating power from the main evaluation, stability and water-soluble chelating agents and so on. 多齿螯合剂可以为我们提供稳定的Zr 89螯合产物。 Polydentate chelating agent can provide a stable Zr 89 chelation product for us. 主要常用的螯合剂有二乙撑三胺五乙酸〇)TPA)、l,4,7, 10-四氮杂环十二烷_1,4,7, 10-四羧酸(DOTA)以及去铁敏B(desferrioxamine B,简称Df)等。 Commonly used chelating agents have the major diethylenetriamine pentaacetic acid square) TPA), l, 4,7, 10- tetraaza cyclododecane _1,4,7, 10-tetracarboxylic acid (DOTA), and to deferoxamine B (desferrioxamine B, referred Df of) the like. 我们采用与Zr络合比较强的去铁敏B(Df)。 We use Zr complex and relatively strong deferoxamine B (Df). 去铁敏B(Df)是双功能螯合剂,在螯合的同时还保持着反应活性位点,可以用于与抗体的偶联反应之中。 Desferrioxamine B (Df) is a bifunctional chelating agent, while also maintaining the chelated reactive sites, may be used in the coupling reaction with the antibody. 如图9至图12所示,本发明是从细胞株表达出来的抗体,其表面突变的Cys上的巯基被其他附着物所占据。 As shown in FIG. 9 to 12, the present invention is an antibody expressed from cell lines out of which the Cys sulfhydryl group on the surface is occupied by mutation other fixtures. 通过还原-氧化的方法可以剔除附着物,使巯基自由出来,为后面的偶联做准备,通过连接物与一种螯合剂去铁敏desferrioxamine,简称Df相连接,达到与放射性核素的螯合。 By reduction - oxidation method can remove attachments, the free sulfhydryl group out to prepare for the subsequent coupling with a chelating agent desferrioxamine desferrioxamine via a linker, referred Df is connected reaches a radionuclide chelating . 放射免疫显影是用放射性核素标记抗体、激素等生物制品作为亲肿瘤药物的阳性显像剂,借助于抗原与抗体,配基与受体的作用,达到定位诊断肿瘤的目的。 Radioimmunoassay developed with radiolabeled antibodies, hormones and other biological products as a positive pro-cancer drugs imaging agent by means of antigen and antibody, ligand receptor action, the purpose of diagnosis of tumor localization. 从而生产出来的抗体可以用于个体化医疗分子成像诊断。 Thus produced antibodies can be used for personalized medicine diagnostic molecular imaging. 通过与In 111,1^99111或I 131标记可用于平面扫描或单光子发射计算机断层照相技术SPECT。 By In 111,1 ^ 99111 I 131 labeled or may be used for planar scans or single photon emission computed tomography technology SPECT. 通过发射正电子的标记物,例如Zr 89 可用于正电子发射断层照相技术PET。 By positron-emitting labels such as Zr 89 it may be used for positron emission tomography techniques PET.

[0110] 以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。 [0110] The above are only preferred embodiments of the present invention, it should be noted: to those of ordinary skill in the art, in the present invention without departing from the principles of the premise, can make various improvements and modifications, such modifications and modifications should also be regarded as the protection scope of the present invention.

Claims (9)

1. 用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C,其特征在于:包括(a) 重链恒定区,包括重链恒定区CHl的氨基酸序列、重链恒定区CH2的氨基酸序列和通过突变改造过得到的重链恒定区CH3的氨基酸序列;所述重链恒定区CHl的氨基酸序列是SEQ ID N0:8所示的氨基酸序列,所述重链恒定区CH2的氨基酸序列是SEQ ID NO: 10所示的氨基酸序列,所述重链恒定区CH3的氨基酸序列是SEQ ID NO: 12所示的氨基酸序列; (b) 重链可变区,所述重链可变区的氨基酸序列是SEQ ID N0:2所示的氨基酸序列; (c) 轻链可变区,所述轻链可变区的氨基酸序列是SEQ ID N0:4所示的氨基酸序列; (d) 轻链恒定区,所述轻链恒定区的氨基酸序列是SEQ ID N0:6所示的氨基酸序列。 1. ADC engineered antibody drug used to construct radioisotope labeled IgGl-CH-K330C, characterized by: (a) a heavy chain constant region comprising the amino acid sequence of the heavy chain CHl constant region of the heavy chain constant region CH2 the amino acid sequence and transformed by mutating the amino acid sequence obtained CH3 heavy chain constant region; the amino acid sequence of the heavy chain CHl constant region is SEQ ID N0: 8 is the amino acid sequence shown in the amino acid heavy chain constant region of CH2 sequence is SEQ ID NO: 10 amino acid sequence shown in the amino acid sequence of the heavy chain constant region CH3 is SEQ ID NO: amino acid sequence shown in 12; (b) a heavy chain variable region, the heavy chain variable the amino acid sequence region is N0 SEQ ID: amino acid sequence represented 2; (c) a light chain variable region, the amino acid sequence of the light chain variable region is N0 SEQ ID: the amino acid sequence shown in 4; (d) light chain constant region, the amino acid sequence of the light chain constant region is SEQ ID N0: 6 shown in the amino acid sequence.
2. 制备权利要求1所述的用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C 的方法。 2. Preparation as claimed in claim ADC for the engineered antibody drug radioisotope labeled IgGl-CH-K330C method of claim 1 constructed.
3. 权利要求1的用于构建放射性同位素偶联标记的ADC药物的改造抗体IgGl-CH-K330C的搭载小分子、毒素或核素的方法。 The method of engineered antibody drug ADC radioisotope labeled IgGl-CH-K330C mounted small molecule toxins or radionuclides 3. Construction as claimed in claim 1.
4. 一种分离的核酸,包括编码权利要求1或2中所述的重链恒定区的核苷酸序列、重链可变区的核苷酸序列、轻链恒定区的核苷酸序列和轻链可变区的核苷酸序列; 所述重链恒定区的核苷酸序列包括所述重链恒定区CHl的核苷酸序列、重链恒定区CH2的核苷酸序列和重链恒定区CH3的核苷酸序列; 所述重链恒定区CHl的核苷酸序列是SEQ ID N0:7所示的核苷酸序列,所述重链恒定区CH2的核苷酸序列是SEQ ID N0:9所示的核苷酸序列,所述重链恒定区CH3的核苷酸序列是SEQ ID NO: 11所示的核苷酸序列; 所述重链可变区的核苷酸序列是SEQ ID NO: 1所示的核苷酸序列,所述轻链恒定区的核苷酸序列是SEQ ID N0:5所示的核苷酸序列,所述轻链可变区的核苷酸序列是SEQ ID N0:3所示的核苷酸序列。 4. An isolated nucleic acid, comprising a heavy chain constant region of claim 1 or claim 2 encoding nucleotide sequence, the nucleotide sequence of the heavy chain variable region nucleotide sequence and a light chain constant region the nucleotide sequence of the light chain variable region; the nucleotide sequence of the heavy chain constant region comprises the nucleotide sequence of the heavy chain constant region CHl, heavy chain constant region and the heavy chain nucleotide sequence of the constant CH2 CH3 region of the nucleotide sequence; a nucleotide sequence of the heavy chain constant region CHl is N0 SEQ ID: 7 nucleotide sequence, the nucleotide sequence of the heavy chain constant region CH2 is SEQ ID N0 : the nucleotide sequence shown in Figure 9, the nucleotide sequence of the heavy chain constant region CH3 is SEQ ID NO: 11 nucleotide sequence; a nucleotide sequence of the heavy chain variable region is SEQ ID NO: 1 nucleotide sequence, the nucleotide sequence of the light chain constant region is N0 SEQ ID: 5 is the nucleotide sequence shown, the light chain variable region nucleotide sequence SEQ ID N0: 3 nucleotide sequence.
5. 权利要求1用于治疗和诊断工具以检测表达腱生蛋白的疾病的用途。 Of claim 1 for therapeutic and diagnostic tools to detect the expression of a disease tenascin.
6. 权利要求5所述的用途,其中所述疾病为恶性肿瘤或系统性自身免疫病或新生血管类疾病。 The use of claim 5 wherein the disease is cancer or autoimmune disease systemic diseases or neovascular claim.
7. 权利要求6所述的用途,其中所述恶性肿瘤选自囊性脑肿瘤、神经胶质瘤、鼻咽癌、 胰腺癌、肺癌、食管癌、乳腺癌、胃癌、大肠癌、肝癌、前列腺癌、卵巢恶性肿瘤、宫颈癌、子宫内膜癌、恶性黑色素瘤、皮肤癌、淋巴瘤、白血病或甲状腺癌。 The use according to claim 6, wherein said malignant tumor is selected from cystic brain tumors, gliomas, nasopharyngeal cancer, pancreatic cancer, lung cancer, esophageal cancer, breast cancer, stomach cancer, colorectal cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, malignant melanoma, skin cancer, lymphoma, leukemia or thyroid cancer.
8. 权利要求6所述的用途,其中所述系统性自身免疫病选自系统性红斑狼疮、类风湿性关节炎、系统性脉管炎、硬皮病、天疱疮、皮肌炎、混合结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病或溃疡性结肠炎。 The use according to claim 6, wherein said systemic autoimmune diseases selected from systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune thyroid disease or ulcerative colitis.
9. 权利要求6所述的用途,其中所述新生血管类疾病选自黄斑水肿、脉络膜新生血管性疾病、新生血管青光眼、视网膜静脉阻塞或视网膜新生血管性疾病。 The use of claim 6 choroidal neovascular diseases, neovascular glaucoma, retinal vein occlusion or retinal neovascularization as claimed in claim 9, wherein said neovascular diseases are selected from macular edema.
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