WO2019096219A1

WO2019096219A1 - Humanized anti-il-13 antibody and preparation method and use thereof

Info

Publication number: WO2019096219A1
Application number: PCT/CN2018/115694
Authority: WO
Inventors: 杨沂蓁; 宫世勇; 吴建; 康立山; 梁绍勤; 段清; 刘礼乐
Original assignee: Shanghai Pharmaexplorer Co Ltd
Current assignee: Shanghai Pharmaexplorer Co Ltd
Priority date: 2017-11-15
Filing date: 2018-11-15
Publication date: 2019-05-23
Anticipated expiration: 2020-05-15
Also published as: CN109776677A; CN109776677B

Abstract

Disclosed are a humanized IL-13 antibody and a preparation method therefor. The IL-13 antibody has high affinity, inhibits the secretion of an IL-13-induced thymus activation regulated chemokine and a periostin and the expression of vascular cell adhesion molecule-1, inhibits IL-13-induced airway hyperresponsiveness in a mouse, and can be used in the preparation of a drug for preventing or treating diseases related to abnormal IL-13 expression or function.

Description

Humanized anti-IL-13 antibody, preparation method and application thereof

Technical field

本发明涉及抗体领域，具体涉及一种人源化抗IL-13抗体及其制备方法和应用。The invention relates to the field of antibodies, in particular to a humanized anti-IL-13 antibody, a preparation method and application thereof.

Background technique

支气管哮喘(简称哮喘)是一种常见的慢性气道性炎症疾病，通常伴随气道反应性增高，出现反复发作的喘息、气促、胸闷和/或咳嗽等症状。20世纪70年代以后，哮喘病广泛流行。到2011年，全球约有2.35亿至3亿人受到影响，大约有25万人因此失去生命。临床上，哮喘通常是以吸入类固醇等糖皮质激素并辅助长效β-肾上腺素能受体激动剂等方式来治疗。但是，约有10％的病人不能通过常规的气道吸入的方法加以缓解，这些病人往往需要口服副作用很大的类固醇类药物来控制病情，而且仍然伴随着较高的死亡率。患者不仅生活质量受到巨大影响，而产生的直接医疗费用和间接成本会造成个人和社会经济的巨大负担。Bronchial asthma (referred to as asthma) is a common chronic airway inflammatory disease, usually accompanied by increased airway responsiveness, recurrent wheezing, shortness of breath, chest tightness and/or cough. After the 1970s, asthma was widespread. By 2011, some 235 million to 300 million people worldwide were affected, and about 250,000 people lost their lives. Clinically, asthma is usually treated by inhaling glucocorticoids such as steroids and assisting long-acting beta-adrenergic receptor agonists. However, about 10% of patients cannot be relieved by routine airway inhalation. These patients often need oral steroids with high side effects to control the disease, and are still associated with higher mortality. Not only is the quality of life of patients greatly affected, but the direct medical costs and indirect costs that result are enormous burdens on the individual and the socio-economic.

近年来，随着对哮喘的深入研究，发现很大部分哮喘患者是由于Th2细胞过度释放可溶性细胞因子诱导IgE的产生，导致肥大细胞、嗜酸性粒细胞脱颗粒增多，从而引起多种炎症细胞参与的气道过敏反应和慢性气道炎症。其中白细胞介素13(以下简称IL-13)和白细胞介素4(以下简称IL-4)在这个过程中起着非常重要的作用。IL-13是一种主要由Th2细胞分泌的多效应细胞因子，分子量约为10KDa，其基因位于第5号染色体上，与IL-4基因紧密连接。IL-4和IL-13都由Th2细胞产生，他们共享同一条受体链，在功能上有很多相似之处。近年来，多项动物实验已经证实IL-4和IL-13均可导致气道反应性增高，嗜酸性粒细胞浸润和粘液分泌的增加。哮喘患者中普遍出现的血浆IgE水平的升高，也已经被证实是IL-13刺激B细胞增殖、分化的结果。近年来随着对IL-13基因单核苷酸的多态性的深入研究，还发现IL-13存在一个天然变体R130Q。人群中约25％的染色体上均出现这种变体的编码，但在哮喘患者中，这个比例可以提高到大约50％。文献报道指出R130Q变体与哮喘的发病直接相关，而且更容易出现过敏症状。深入的研究发现，表达IL-13变体R130Q的患者体内IL-13浓度更高，且效果更强，更易引发哮喘等症状(参见May and Fung 2015,Cytokine75:89)。In recent years, with the in-depth study of asthma, it is found that a large proportion of asthma patients are caused by excessive release of soluble cytokines by Th2 cells to induce IgE production, resulting in increased degranulation of mast cells and eosinophils, resulting in the involvement of various inflammatory cells. Airway allergic reactions and chronic airway inflammation. Among them, interleukin 13 (hereinafter referred to as IL-13) and interleukin 4 (hereinafter referred to as IL-4) play a very important role in this process. IL-13 is a multi-effect cytokine secreted mainly by Th2 cells with a molecular weight of about 10KDa. Its gene is located on chromosome 5 and is tightly linked to the IL-4 gene. Both IL-4 and IL-13 are produced by Th2 cells, which share the same receptor chain and have many similarities in function. In recent years, several animal experiments have confirmed that both IL-4 and IL-13 can cause an increase in airway responsiveness, an increase in eosinophil infiltration and mucus secretion. Elevated levels of plasma IgE, which are common in asthmatic patients, have also been shown to be the result of IL-13-stimulated B cell proliferation and differentiation. In recent years, with the in-depth study of the polymorphism of the single nucleotide of IL-13 gene, it was also found that there is a natural variant R130Q of IL-13. This variant is encoded on approximately 25% of the chromosomes in the population, but in asthma patients this ratio can be increased to approximately 50%. Reports in the literature indicate that R130Q variants are directly related to the onset of asthma and are more prone to allergic symptoms. In-depth studies have found that patients with IL-13 variant R130Q have higher levels of IL-13 and are more effective, leading to asthma and other symptoms (see May and Fung 2015, Cytokine 75:89).

因此，迫切需要可抑制IL-13的药物，如能够与IL-13相结合的特异性单克隆抗体。这些抗体能够结合人IL-13蛋白(野生型或R130Q变体)并能够在蛋白水平和细胞水平抑制IL-13与其受体IL-13Ra1/IL-4Ra异源二聚体或受体IL-13Ra2结合；能明显抑制IL-13诱导的胸腺活化调节趋化因子的分泌和骨膜蛋白的分泌以及血管细胞粘附分子-1的表达；且能够明显抑制IL-13诱导的小鼠气道高反应性。因此，IL-13抗体能够运用于预防、诊断和治疗支气管哮喘等疾病的药物的制备中。单克隆抗体由于具有靶向性、特异性、专一性、高亲和力等优势，正发展成为新型诊断和治疗药物。然而，早期的临床试验揭示，在人体中使用非人源单克隆抗体，常常因为人抗小鼠抗体(HAMA)和人抗大鼠抗体(HARA)应答，导致严重的免疫反应，抗体被快速清除。随后开发出免疫原性较小的抗体，包括嵌合抗体、人源化抗体和全人源抗体。根据人源化程度不同,治疗性单克隆抗体药物可分为4种：鼠源性抗体(无人源氨基酸序列)、嵌合抗体(60％～70％人源化氨基酸序列)、CDR移植抗体(90％～95％人源化氨基酸序列)以及全人源抗体(100％人源氨基酸序列)。随着人源化程度增加，非鼠源单克隆抗体可以减轻人体治疗过程中人抗鼠抗体反应(HAMA和HARA反应)，逐步消除异源性抗体的免疫原性问题，在保持对抗原高亲和力的同时，改善了抗体的药代动力学，临床上已大量使用这些抗体药物进行靶向治疗。Therefore, there is an urgent need for drugs that inhibit IL-13, such as specific monoclonal antibodies that bind to IL-13. These antibodies bind to human IL-13 protein (wild-type or R130Q variant) and are capable of inhibiting IL-13 and its receptor IL-13Ra1/IL-4Ra heterodimer or receptor IL-13Ra2 at the protein and cellular levels. Binding; can significantly inhibit IL-13-induced thymic activation, regulate chemokine secretion and periostin secretion, and expression of vascular cell adhesion molecule-1; and can significantly inhibit IL-13-induced airway hyperresponsiveness in mice. . Therefore, IL-13 antibodies can be used in the preparation of drugs for the prevention, diagnosis and treatment of diseases such as bronchial asthma. Monoclonal antibodies are developing into new diagnostic and therapeutic drugs due to their advantages of targeting, specificity, specificity and high affinity. However, early clinical trials revealed that the use of non-human monoclonal antibodies in humans often results in severe immune responses due to human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, and antibodies are rapidly cleared. . Subsequently, antibodies with less immunogenicity were developed, including chimeric antibodies, humanized antibodies, and fully human antibodies. According to the degree of humanization, therapeutic monoclonal antibody drugs can be divided into four types: murine antibody (unmanned amino acid sequence), chimeric antibody (60% to 70% humanized amino acid sequence), CDR graft antibody (90% to 95% humanized amino acid sequence) and fully human antibody (100% human amino acid sequence). As the degree of humanization increases, non-rat monoclonal antibodies can alleviate human anti-mouse antibody responses (HAMA and HARA responses) during human therapy, gradually eliminating the immunogenicity of heterologous antibodies, and maintaining high affinity for antigens. At the same time, the pharmacokinetics of the antibodies have been improved, and these antibody drugs have been used in a large amount in the clinic for targeted therapy.

目前，单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。At present, monoclonal antibodies can be developed by various methods and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

传统的杂交瘤制备技术由Kohler and Milstein在40年前建立(Kohler and Milstein 1975，Nature 256:495)，现在已广泛应用于科研、诊断、治疗等许多单克隆抗体相关的制备和生产之中。其基本方法虽然延用至今，但在许多方面都有所变化、改进和创新，包括不同品系动物如转基因动物的使用、电融合技术的引入、高效筛选技术设备的应用如ClonePix设备等。从而使杂交瘤技术的应用更多样化和高效化。常规动物如小鼠等制备的单抗，可以通过常规分子生物学方法克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体(参见美国专利文献U.S.Pat.No.4,816,567)，以大大降低人体使用时的免疫原性。此外，鼠源抗体可变区的CDR结构域可以嫁接到人源抗体架构上，从而使鼠源抗体成分降低到5％以下，大大增加了抗体在人体内使用的安全性。这一途径得到抗体称为人源化抗体，并且是目前抗体药物市场的主要产品(参见美国专利文献U.S.Pat.No.5,225,539等)。Traditional hybridoma preparation technology was established 40 years ago by Kohler and Milstein (Kohler and Milstein 1975, Nature 256:495), and is now widely used in the preparation and production of many monoclonal antibodies related to scientific research, diagnosis, and treatment. Although its basic methods have been used up to now, there have been changes, improvements and innovations in many aspects, including the use of different strains of animals such as genetically modified animals, the introduction of electrofusion technology, and the application of efficient screening technology equipment such as ClonePix equipment. Thereby the application of hybridoma technology is more diversified and efficient. The monoclonal antibody prepared by a conventional animal such as a mouse can clone the antibody heavy chain variable region and the light chain variable region gene by conventional molecular biological methods, and the variable region gene can be grafted to the human antibody constant region gene to form a human mouse. Chimeric antibodies (see U.S. Patent No. 4,816,567) to greatly reduce immunogenicity in human use. In addition, the CDR domain of the murine antibody variable region can be grafted onto the human antibody framework, thereby reducing the murine antibody component to less than 5%, greatly increasing the safety of the antibody for use in humans. The antibody obtained by this route is called a humanized antibody, and is a major product of the current antibody drug market (see U.S. Pat. No. 5,225,539, etc.).

因此，本领域迫切需要开发可抑制IL-13的药物，如IL-13抗体用于预防或治疗支气管哮喘。Therefore, there is an urgent need in the art to develop drugs that inhibit IL-13, such as IL-13 antibodies, for the prevention or treatment of bronchial asthma.

发明内容Summary of the invention

本发明要解决的技术问题是为了克服现有技术目前缺少IL-13抗体的不足，提供了一种亲和力高、特异性强的人源化抗IL-13抗体及其制备方法和应用。所述的人源化抗IL-13抗体与人IL-13蛋白(野生型或R130Q变体)具有高度亲和力，并能够在蛋白水平和细胞水平抑制IL-13与其受体IL-13Ra1/IL-4Ra异原二聚体或受体IL-13Ra2结合。在趋化因子释放实验和细胞外基质蛋白释放实验，细胞粘附分子表达实验中，这些抗体表现出良好的生物学活性。The technical problem to be solved by the present invention is to overcome the deficiency of the current lack of IL-13 antibody in the prior art, and to provide a humanized anti-IL-13 antibody with high affinity and high specificity, a preparation method and application thereof. The humanized anti-IL-13 antibody has high affinity to human IL-13 protein (wild type or R130Q variant) and is capable of inhibiting IL-13 and its receptor IL-13Ra1/IL- at the protein and cellular levels. 4Ra heterodimer or receptor IL-13Ra2 binds. These antibodies exhibited good biological activity in chemokine release assays and extracellular matrix protein release assays, cell adhesion molecule expression assays.

这些抗体的用途，包括但不仅限于抑制IL-13/IL-13受体介导的信号通路的负调节，抑制气道高反应性、嗜酸性粒细胞浸润及粘液过度分泌等哮喘症状，单独或与其他抗哮喘抗体联合应用用于哮喘的治疗，以及相关的哮喘患者的诊断等用途。随着这些抗体人源化程度增加，可减轻人体治疗过程中人抗鼠抗体反应(HAMA反应)，逐步消除异源性抗体的免疫原性问题，在保持对抗原高亲和力的同时，改善了抗体的药代动力学。The use of these antibodies includes, but is not limited to, inhibition of negative regulation of IL-13/IL-13 receptor-mediated signaling pathways, inhibition of asthmatic symptoms such as airway hyperresponsiveness, eosinophil infiltration, and excessive mucus secretion, either alone or Used in combination with other anti-asthmatic antibodies for the treatment of asthma, as well as the diagnosis of related asthma patients. As the degree of humanization of these antibodies increases, the human anti-mouse antibody response (HAMA reaction) can be alleviated during human treatment, the immunogenicity of the heterologous antibody is gradually eliminated, and the antibody is improved while maintaining high affinity for the antigen. Pharmacokinetics.

在本发明的第一方面，提供了一种人源化抗体，包括重链可变区和/或轻链可变区；In a first aspect of the invention, a humanized antibody comprising a heavy chain variable region and/or a light chain variable region is provided;

其中，所述人源化抗体的重链可变区包括以下三个互补决定区CDR：Wherein the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

SEQ ID NO:18、SEQ ID NO:2或SEQ ID NO:10所示的CDR1，CDR1 set forth in SEQ ID NO: 18, SEQ ID NO: 2 or SEQ ID NO: 10,

SEQ ID NO:19、SEQ ID NO:3或SEQ ID NO:11所示的CDR2，和CDR2 of SEQ ID NO: 19, SEQ ID NO: 3 or SEQ ID NO: 11, and

SEQ ID NO:20、SEQ ID NO:4或SEQ ID NO:12所示的CDR3；CDR3 of SEQ ID NO:20, SEQ ID NO:4 or SEQ ID NO:12;

所述人源化抗体的轻链可变区包括以下三个互补决定区CDR：The light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

SEQ ID NO:22、SEQ ID NO:6或SEQ ID NO:14所示的CDR1，CDR1 of SEQ ID NO:22, SEQ ID NO:6 or SEQ ID NO:14,

SEQ ID NO:23、SEQ ID NO:7或SEQ ID NO:15所示的CDR2，和CDR2 of SEQ ID NO: 23, SEQ ID NO: 7 or SEQ ID NO: 15, and

SEQ ID NO:24、SEQ ID NO:8或SEQ ID NO:16所示的CDR3；CDR3 of SEQ ID NO:24, SEQ ID NO:8 or SEQ ID NO:16;

其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留IL-13结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining IL-13 binding affinity.

在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2或3个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的 VH和VL所构成的衍生抗体能够保留与IL-13结合的亲和力。In another preferred embodiment, the amino acid sequence of any of the above CDRs comprises a derivative CDR sequence that has been added, deleted, modified and/or substituted 1, 2 or 3 amino acids, and which results in VH and VL containing the derived CDR sequences. The constructed derivative antibody is capable of retaining the affinity for binding to IL-13.

在另一优选例中，所述的衍生抗体与IL-13结合的亲和力F1与相应非衍生的抗体与IL-13结合的亲和力F0之比(F1/F0)为0.5-2，较佳地为0.7-1.5，和更佳地0.8-1.2。In another preferred embodiment, the ratio of the affinity F1 of the derivative antibody to IL-13 binding to the affinity F0 of the corresponding non-derivatized antibody to IL-13 (F1/F0) is 0.5-2, preferably 0.7-1.5, and more preferably 0.8-1.2.

在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。在另一优选例中，所述的人源化抗体在人中的免疫原性Z1与非人源化的抗体(如鼠源抗体)在人中的免疫原性Z0之比(Z1/Z0)为0-0.5，较佳地0-0.2，更佳地0-0.05(如0.001-0.05)。In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted is from 1 to 5 (e.g., from 1 to 3, preferably from 1 to 2, more preferably 1). In another preferred embodiment, the ratio of the immunogenic Z1 of the humanized antibody in human to the immunogenicity Z0 of a non-humanized antibody (such as a murine antibody) in humans (Z1/Z0) It is 0-0.5, preferably 0-0.2, more preferably 0-0.05 (e.g., 0.001-0.05).

在另一优选例中，所述重链可变区CDR1的氨基酸序列与如序列表中SEQ ID No.18、SEQ ID No.2、SEQ ID No.10所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性，In another preferred embodiment, the amino acid sequence of the heavy chain variable region CDR1 is at least 80% identical to the amino acid sequence set forth in SEQ ID No. 18, SEQ ID No. 2, and SEQ ID No. 10 of the Sequence Listing. Sequence homology of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,

所述重链可变区CDR2的氨基酸序列与如序列表中SEQ ID No.19、SEQ ID No.3、SEQ ID No.11所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性，和The amino acid sequence of the heavy chain variable region CDR2 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 19, SEQ ID No. 3, and SEQ ID No. 11 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology, and

所述重链可变区CDR3的氨基酸序列与如序列表中SEQ ID No.20、SEQ ID No.4、SEQ ID No.12所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性。The amino acid sequence of the heavy chain variable region CDR3 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 20, SEQ ID No. 4, and SEQ ID No. 12 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

在另一优选例中，所述轻链可变区CDR1的氨基酸序列与如序列表中SEQ ID No.22、SEQ ID No.6、SEQ ID No.14所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性，In another preferred embodiment, the amino acid sequence of the light chain variable region CDR1 is at least 80% identical to the amino acid sequence represented by SEQ ID No. 22, SEQ ID No. 6, and SEQ ID No. 14 in the sequence listing. Sequence homology of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,

所述轻链可变区CDR2的氨基酸序列与如序列表中SEQ ID No.23、SEQ ID No.7、SEQ ID No.15所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性，The amino acid sequence of the light chain variable region CDR2 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 23, SEQ ID No. 7, and SEQ ID No. 15 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology,

所述轻链可变区CDR3的氨基酸序列与如序列表中SEQ ID No.24、SEQ ID No.8、SEQ ID No.16所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性。The amino acid sequence of the light chain variable region CDR3 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 24, SEQ ID No. 8, and SEQ ID No. 16 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

在另一优选例中，所述人源化抗体的重链可变区包括以下三个互补决定区CDR：In another preferred embodiment, the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

SEQ ID NO:18所示的CDR1，CDR1 shown in SEQ ID NO: 18,

SEQ ID NO:19所示的CDR2，和CDR2 shown in SEQ ID NO: 19, and

SEQ ID NO:20所示的CDR3；CDR3 shown in SEQ ID NO:20;

或or

SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO: 2,

SEQ ID NO:3所示的CDR2，和CDR2 shown in SEQ ID NO: 3, and

SEQ ID NO:4所示的CDR3；CDR3 shown in SEQ ID NO: 4;

或or

SEQ ID NO:10所示的CDR1，CDR1 shown in SEQ ID NO: 10,

SEQ ID NO:11所示的CDR2，和CDR2 shown in SEQ ID NO: 11, and

SEQ ID NO:12所示的CDR3。CDR3 shown in SEQ ID NO:12.

在另一优选例中，所述人源化抗体的轻链可变区包括以下三个互补决定区CDR：In another preferred embodiment, the light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

SEQ ID NO:22所示的CDR1，CDR1 shown in SEQ ID NO: 22,

SEQ ID NO:23所示的CDR2，和CDR2 shown in SEQ ID NO: 23, and

SEQ ID NO:24所示的CDR3；CDR3 shown in SEQ ID NO:24;

或or

SEQ ID NO:6所示的CDR1，CDR1 shown in SEQ ID NO: 6,

SEQ ID NO:7所示的CDR2，和CDR2 shown in SEQ ID NO: 7, and

SEQ ID NO:8所示的CDR3；CDR3 shown in SEQ ID NO: 8;

或or

SEQ ID NO:14所示的CDR1，CDR1 shown in SEQ ID NO: 14,

SEQ ID NO:15所示的CDR2，和CDR2 shown in SEQ ID NO: 15, and

SEQ ID NO:16所示的CDR3。CDR3 shown in SEQ ID NO: 16.

在另一优选例中，所述人源化抗体的重链可变区序列选自下组：SEQ ID NO:17、1或9；和/或In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is selected from the group consisting of SEQ ID NO: 17, 1 or 9; and/or

所述人源化抗体的轻链可变区序列选自下组：SEQ ID NO:21、5或13。The light chain variable region sequence of the humanized antibody is selected from the group consisting of SEQ ID NO: 21, 5 or 13.

在另一优选例中，所述人源化抗体的重链可变区序列为SEQ ID NO:17，并且轻链可变区序列为SEQ ID NO:21。In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is SEQ ID NO: 17, and the light chain variable region sequence is SEQ ID NO:21.

在另一优选例中，所述人源化抗体的重链可变区序列为SEQ ID NO:1，并且轻链可变区序列为SEQ ID NO:5。In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is SEQ ID NO: 1, and the light chain variable region sequence is SEQ ID NO: 5.

在另一优选例中，所述人源化抗体的重链可变区序列为SEQ ID NO:9，并且轻链可变区序列为SEQ ID NO:13。In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is SEQ ID NO:9 and the light chain variable region sequence is SEQ ID NO:13.

SEQ ID NO:18所示的CDR1，CDR1 shown in SEQ ID NO: 18,

SEQ ID NO:19所示的CDR2，和CDR2 shown in SEQ ID NO: 19, and

SEQ ID NO:20所示的CDR3；CDR3 shown in SEQ ID NO:20;

和/或and / or

SEQ ID NO:22所示的CDR1，CDR1 shown in SEQ ID NO: 22,

SEQ ID NO:23所示的CDR2，和CDR2 shown in SEQ ID NO: 23, and

SEQ ID NO:24所示的CDR3。CDR3 shown in SEQ ID NO:24.

在另一优选例中，所述人源化抗体的重链可变区序列如SEQ ID NO:57、51、52、53、54、55或56所示；和/或所述人源化抗体的轻链可变区序列如SEQ ID NO:58、59或60所示。In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is set forth in SEQ ID NO: 57, 51, 52, 53, 54, 55 or 56; and/or the humanized antibody The light chain variable region sequence is set forth in SEQ ID NO: 58, 59 or 60.

在另一优选例中，所述人源化抗体的重链和轻链可变区序列如表A所示：In another preferred embodiment, the heavy and light chain variable region sequences of the humanized antibody are shown in Table A:

表ATable A

抗体编号Antibody number 重链可变区SEQ ID NO.Heavy chain variable region SEQ ID NO. 轻链可变区SEQ ID NO.Light chain variable region SEQ ID NO. Hu-29D9H8-1Hu-29D9H8-1 2626 3333 Hu-29D9H8-2Hu-29D9H8-2 2626 3434 Hu-29D9H8-3Hu-29D9H8-3 2626 3535 Hu-29D9H8-4Hu-29D9H8-4 2727 3232 Hu-29D9H8-5Hu-29D9H8-5 2727 3333 Hu-29D9H8-6Hu-29D9H8-6 2727 3434 Hu-29D9H8-7Hu-29D9H8-7 2727 3535 Hu-29D9H8-8Hu-29D9H8-8 2828 3232 Hu-29D9H8-9Hu-29D9H8-9 2828 3333 Hu-29D9H8-10Hu-29D9H8-10 2828 3434 Hu-29D9H8-11Hu-29D9H8-11 2828 3535 Hu-29D9H8-12Hu-29D9H8-12 2929 3232

Hu-29D9H8-13Hu-29D9H8-13 2929 3333 Hu-29D9H8-14Hu-29D9H8-14 2929 3434 Hu-29D9H8-15Hu-29D9H8-15 2929 3535 Hu-29D9H8-16Hu-29D9H8-16 3030 3232 Hu-29D9H8-17Hu-29D9H8-17 3030 3333 Hu-29D9H8-18Hu-29D9H8-18 3030 3434 Hu-29D9H8-19Hu-29D9H8-19 3030 3535 Hu-29D9H8-20Hu-29D9H8-20 3131 3232 Hu-29D9H8-21Hu-29D9H8-21 3131 3333 Hu-29D9H8-22Hu-29D9H8-22 3131 3434 Hu-29D9H8-23Hu-29D9H8-23 3131 3535 Hu-28A2E11-1Hu-28A2E11-1 3737 4343 Hu-28A2E11-2Hu-28A2E11-2 3737 4444 Hu-28A2E11-3Hu-28A2E11-3 3737 4545 Hu-28A2E11-4Hu-28A2E11-4 3737 4646 Hu-28A2E11-5Hu-28A2E11-5 3737 4747 Hu-28A2E11-6Hu-28A2E11-6 3737 4848 Hu-28A2E11-7Hu-28A2E11-7 3737 4949 Hu-28A2E11-8Hu-28A2E11-8 3838 4343 Hu-28A2E11-9Hu-28A2E11-9 3838 4444 Hu-28A2E11-10Hu-28A2E11-10 3838 4545 Hu-28A2E11-11Hu-28A2E11-11 3838 4646 Hu-28A2E11-12Hu-28A2E11-12 3838 4747 Hu-28A2E11-13Hu-28A2E11-13 3838 4848 Hu-28A2E11-14Hu-28A2E11-14 3838 4949 Hu-28A2E11-15Hu-28A2E11-15 3939 4343 Hu-28A2E11-16Hu-28A2E11-16 3939 4444 Hu-28A2E11-17Hu-28A2E11-17 3939 4545 Hu-28A2E11-18Hu-28A2E11-18 3939 4646

Hu-28A2E11-19Hu-28A2E11-19 3939 4747 Hu-28A2E11-20Hu-28A2E11-20 3939 4848 Hu-28A2E11-21Hu-28A2E11-21 4040 4343 Hu-28A2E11-22Hu-28A2E11-22 4040 4444 Hu-28A2E11-23Hu-28A2E11-23 4040 4545 Hu-28A2E11-24Hu-28A2E11-24 4040 4646 Hu-28A2E11-25Hu-28A2E11-25 4040 4747 Hu-28A2E11-26Hu-28A2E11-26 4141 4343 Hu-28A2E11-27Hu-28A2E11-27 4141 4444 Hu-28A2E11-28Hu-28A2E11-28 4141 4545 Hu-28A2E11-29Hu-28A2E11-29 4141 4646 Hu-28A2E11-31Hu-28A2E11-31 4242 4343 Hu-28A2E11-32Hu-28A2E11-32 4242 4444 Hu-28A2E11-33Hu-28A2E11-33 4242 4545 Hu-28A2E11-34Hu-28A2E11-34 4242 4646 Hu-35E2C3-1Hu-35E2C3-1 5151 5858 Hu-35E2C3-2Hu-35E2C3-2 5151 5959 Hu-35E2C3-3Hu-35E2C3-3 5151 6060 Hu-35E2C3-4Hu-35E2C3-4 5252 5858 Hu-35E2C3-5Hu-35E2C3-5 5252 5959 Hu-35E2C3-6Hu-35E2C3-6 5252 6060 Hu-35E2C3-7Hu-35E2C3-7 5353 5858 Hu-35E2C3-8Hu-35E2C3-8 5353 5959 Hu-35E2C3-9Hu-35E2C3-9 5353 6060 Hu-35E2C3-10Hu-35E2C3-10 5454 5858 Hu-35E2C3-11Hu-35E2C3-11 5454 5959 Hu-35E2C3-12Hu-35E2C3-12 5454 6060 Hu-35E2C3-13Hu-35E2C3-13 5555 5858 Hu-35E2C3-14Hu-35E2C3-14 5555 5959

Hu-35E2C3-15 55 60 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60 。 Hu-35E2C3-15 55 60 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60 .

在另一优选例中，所述人源化抗体的重链可变区序列如SEQ ID NO:57、51、52、53、54、55、或56所示；并且所述人源化抗体的轻链可变区序列如SEQ ID NO:58、59、或60所示。In another preferred embodiment, the heavy chain variable region sequence of the humanized antibody is as set forth in SEQ ID NO: 57, 51, 52, 53, 54, 55, or 56; and the humanized antibody The light chain variable region sequence is set forth in SEQ ID NO: 58, 59, or 60.

在另一优选例中，所述的人源化抗体还包括重链恒定区和/或轻链恒定区。In another preferred embodiment, the humanized antibody further comprises a heavy chain constant region and/or a light chain constant region.

在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。In another preferred embodiment, the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.

在另一优选例中，所述人源化抗体为单克隆抗体。In another preferred embodiment, the humanized antibody is a monoclonal antibody.

在另一优选例中，所述人源化抗体为双链抗体、单链抗体、单域抗体、或单区抗体。In another preferred embodiment, the humanized antibody is a diabody, a single chain antibody, a single domain antibody, or a single region antibody.

在另一优选例中，所述人源化抗体为抗体全长蛋白、或抗原结合片段。In another preferred embodiment, the humanized antibody is an antibody full length protein, or an antigen binding fragment.

在另一优选例中，所述人源化抗体为双特异性抗体、或多特异性抗体。In another preferred embodiment, the humanized antibody is a bispecific antibody, or a multispecific antibody.

在另一优选例中，所述人源化抗体的重链可变区还包括人源的框架区，和/或所述人源化抗体的轻链可变区还包括人源的框架区。In another preferred embodiment, the heavy chain variable region of the humanized antibody further comprises a human framework region, and/or the light chain variable region of the humanized antibody further comprises a human framework region.

在另一优选例中，所述人源化抗体的重链可变区上的重链FR序列，来源于人种系重链序列包含1)VH1-f或VH4-59的FR1、FR2、FR3区，和2)JH1或JH6的FR4区的人抗体重链构架区的组合。In another preferred embodiment, the heavy chain FR sequence on the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain sequence comprising 1) VH1-f or VH4-59 FR1, FR2, FR3 The region, and 2) the combination of the human antibody heavy chain framework regions of the FR4 region of JH1 or JH6.

在另一优选例中，所述人源化抗体的轻链可变区上的轻链FR序列，来源于人种系轻链包含1)L1、7B或O12的FR1、FR2、FR3区，和2)JK4或JL1的FR4区的人抗体轻链构架区的组合。In another preferred embodiment, the light chain FR sequence on the light chain variable region of the humanized antibody is derived from the FR1, FR2, FR3 region of the human germline light chain comprising 1) L1, 7B or O12, and 2) Combination of human antibody light chain framework regions of the FR4 region of JK4 or JL1.

在另一优选例中，所述人源化抗体的重链框架区选自种系：DP3(VH1-f)、DP4、DP7、DP8、DP9、DP10、DP31、DP33、DP35(VH3-11)、DP45、DP46、DP47、 DP48、DP49(VH3-30)、DP50、DP51(VH3-48)、DP53、DP54、DP65、DP66、DP67、DP68、DP69或DP71(VH4-59)；和/或In another preferred embodiment, the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11) , DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59); and/or

所述人源化抗体的轻链框架区选自种系：L1、O2、O12、DPK1(O18)、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12(A2)、DPK13、DPK15、DPK16、DPKI8、DPK19、DPK20、DPK21、DPK22、DPK23、DPK24(B3)、DPK25、DPK26、DPK28、DPL1、DPL7、DPL6、DPL2、DPL3、DPL5、DPL12、DPL11、DPL13、DPL10、D9、DPL16、DPL24、DPL18、DPL19(7b)、DPL21、DPL22或DPL20。The light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9 , DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20.

在另一优选例中，所述人源化抗体的重链框架区选自种系：DP3(VH1-f)、DP4、DP7、DP8、DP9、DP10、DP31、DP33、DP35(VH3-11)、DP45、DP46、DP47、DP48、DP49(VH3-30)、DP50、DP51(VH3-48)、DP53、DP54、DP65、DP66、DP67、DP68、DP69或DP71(VH4-59)的FR1、FR2、FR3；以及JH片段JH1、JH2、JH3、JH4、JH4b、JH5和JH6，特别是这些种系的FR4编码的序列，或重链框架区的共有序列。In another preferred embodiment, the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11) FR1, FR2 of DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59) FR3; and JH fragments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these lines, or the consensus sequence of the heavy-chain framework regions.

在另一优选例中，所述人源化抗体的轻链框架区选自种系：L1、O2、O12、DPK1(O18)、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12(A2)、DPK13、DPK15、DPK16、DPKI8、DPK19、DPK20、DPK21、DPK22、DPK23、DPK24(B3)、DPK25、DPK26、DPK28、DPL1、DPL7、DPL6、DPL2、DPL3、DPL5、DPL12、DPL11、DPL13、DPL10、D9、DPL16、DPL24、DPL18、DPL19(7b)、DPL21、DPL22或DPL20，优选为这些种系的FR1、FR2、FR3；以及由Jk片段Jk1、Jk2、Jk3、Jk4和JK5，特别是这些种系的FR4编码的序列。In another preferred embodiment, the light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9, DPL16, DPL24, DPL18, DPL19(7b), DPL21, DPL22 or DPL20, preferably FR1, FR2, FR3 of these germplasms; and Jk segments Jk1, Jk2, Jk3, Jk4 and JK5 In particular, the FR4 encoded sequences of these lines.

在另一优选例中，所述人源化抗体的重链和轻链可变区序列如表B所示：In another preferred embodiment, the heavy and light chain variable region sequences of the humanized antibody are shown in Table B:

表BTable B

抗体编号重链可变区SEQ ID NO. 轻链可变区SEQ ID NO. Hu-35E2C3-19 57 58 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 。 Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. Hu-35E2C3-19 57 58 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 .

在另一优选例中，所述人源化抗体的重链和轻链可变区序列如表C所示：In another preferred embodiment, the heavy and light chain variable region sequences of the humanized antibody are shown in Table C:

表CTable C

抗体编号重链可变区SEQ ID NO. 轻链可变区SEQ ID NO. Hu-29D9H8-10 28 34 Hu-29D9H8-19 30 35 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-20 39 48 。 Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. Hu-29D9H8-10 28 34 Hu-29D9H8-19 30 35 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-20 39 48 .

在另一优选例中，所述的抗体为药物偶联物形式。In another preferred embodiment, the antibody is in the form of a drug conjugate.

在本发明的第二方面，提供了一种重组蛋白，所述的重组蛋白包括：In a second aspect of the invention, a recombinant protein is provided, the recombinant protein comprising:

(i)表2所示的任一重链可变区序列和任一轻链可变区序列；以及(i) any of the heavy chain variable region sequences and any of the light chain variable region sequences set forth in Table 2;

(ii)任选的协助表达和/或纯化的标签序列；(ii) an optional tag sequence that facilitates expression and/or purification;

表2Table 2

抗体编号Antibody number 重链可变区SEQ ID NO.Heavy chain variable region SEQ ID NO. 轻链可变区SEQ ID NO.Light chain variable region SEQ ID NO. c29D9H8c29D9H8 11 55 Hu-29D9H8-1Hu-29D9H8-1 2626 3333 Hu-29D9H8-2Hu-29D9H8-2 2626 3434 Hu-29D9H8-3Hu-29D9H8-3 2626 3535 Hu-29D9H8-4Hu-29D9H8-4 2727 3232 Hu-29D9H8-5Hu-29D9H8-5 2727 3333 Hu-29D9H8-6Hu-29D9H8-6 2727 3434 Hu-29D9H8-7Hu-29D9H8-7 2727 3535 Hu-29D9H8-8Hu-29D9H8-8 2828 3232 Hu-29D9H8-9Hu-29D9H8-9 2828 3333 Hu-29D9H8-10Hu-29D9H8-10 2828 3434 Hu-29D9H8-11Hu-29D9H8-11 2828 3535 Hu-29D9H8-12Hu-29D9H8-12 2929 3232 Hu-29D9H8-13Hu-29D9H8-13 2929 3333 Hu-29D9H8-14Hu-29D9H8-14 2929 3434 Hu-29D9H8-15Hu-29D9H8-15 2929 3535

Hu-29D9H8-16Hu-29D9H8-16 3030 3232 Hu-29D9H8-17Hu-29D9H8-17 3030 3333 Hu-29D9H8-18Hu-29D9H8-18 3030 3434 Hu-29D9H8-19Hu-29D9H8-19 3030 3535 Hu-29D9H8-20Hu-29D9H8-20 3131 3232 Hu-29D9H8-21Hu-29D9H8-21 3131 3333 Hu-29D9H8-22Hu-29D9H8-22 3131 3434 Hu-29D9H8-23Hu-29D9H8-23 3131 3535 c28A2E11c28A2E11 99 1313 Hu-28A2E11-1Hu-28A2E11-1 3737 4343 Hu-28A2E11-2Hu-28A2E11-2 3737 4444 Hu-28A2E11-3Hu-28A2E11-3 3737 4545 Hu-28A2E11-4Hu-28A2E11-4 3737 4646 Hu-28A2E11-5Hu-28A2E11-5 3737 4747 Hu-28A2E11-6Hu-28A2E11-6 3737 4848 Hu-28A2E11-7Hu-28A2E11-7 3737 4949 Hu-28A2E11-8Hu-28A2E11-8 3838 4343 Hu-28A2E11-9Hu-28A2E11-9 3838 4444 Hu-28A2E11-10Hu-28A2E11-10 3838 4545 Hu-28A2E11-11Hu-28A2E11-11 3838 4646 Hu-28A2E11-12Hu-28A2E11-12 3838 4747 Hu-28A2E11-13Hu-28A2E11-13 3838 4848 Hu-28A2E11-14Hu-28A2E11-14 3838 4949 Hu-28A2E11-15Hu-28A2E11-15 3939 4343 Hu-28A2E11-16Hu-28A2E11-16 3939 4444 Hu-28A2E11-17Hu-28A2E11-17 3939 4545 Hu-28A2E11-18Hu-28A2E11-18 3939 4646 Hu-28A2E11-19Hu-28A2E11-19 3939 4747 Hu-28A2E11-20Hu-28A2E11-20 3939 4848

Hu-28A2E11-21Hu-28A2E11-21 4040 4343 Hu-28A2E11-22Hu-28A2E11-22 4040 4444 Hu-28A2E11-23Hu-28A2E11-23 4040 4545 Hu-28A2E11-24Hu-28A2E11-24 4040 4646 Hu-28A2E11-25Hu-28A2E11-25 4040 4747 Hu-28A2E11-26Hu-28A2E11-26 4141 4343 Hu-28A2E11-27Hu-28A2E11-27 4141 4444 Hu-28A2E11-28Hu-28A2E11-28 4141 4545 Hu-28A2E11-29Hu-28A2E11-29 4141 4646 Hu-28A2E11-31Hu-28A2E11-31 4242 4343 Hu-28A2E11-32Hu-28A2E11-32 4242 4444 Hu-28A2E11-33Hu-28A2E11-33 4242 4545 Hu-28A2E11-34Hu-28A2E11-34 4242 4646 c35E2C3c35E2C3 1717 21twenty one Hu-35E2C3-1Hu-35E2C3-1 5151 5858 Hu-35E2C3-2Hu-35E2C3-2 5151 5959 Hu-35E2C3-3Hu-35E2C3-3 5151 6060 Hu-35E2C3-4Hu-35E2C3-4 5252 5858 Hu-35E2C3-5Hu-35E2C3-5 5252 5959 Hu-35E2C3-6Hu-35E2C3-6 5252 6060 Hu-35E2C3-7Hu-35E2C3-7 5353 5858 Hu-35E2C3-8Hu-35E2C3-8 5353 5959 Hu-35E2C3-9Hu-35E2C3-9 5353 6060 Hu-35E2C3-10Hu-35E2C3-10 5454 5858 Hu-35E2C3-11Hu-35E2C3-11 5454 5959 Hu-35E2C3-12Hu-35E2C3-12 5454 6060 Hu-35E2C3-13Hu-35E2C3-13 5555 5858 Hu-35E2C3-14Hu-35E2C3-14 5555 5959 Hu-35E2C3-15Hu-35E2C3-15 5555 6060

Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60 。 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60 .

在另一优选例中，所述的重组蛋白包括：In another preferred embodiment, the recombinant protein comprises:

(i)SEQ ID NO:57、28、37、17、1或9所示的重链可变区，SEQ ID NO:58、34、48、21、5或13所示的轻链可变区；以及(i) a heavy chain variable region of SEQ ID NO: 57, 28, 37, 17, 1 or 9, a light chain variable region of SEQ ID NO: 58, 34, 48, 21, 5 or ;as well as

(ii)任选的协助表达和/或纯化的标签序列。(ii) an optional tag sequence that facilitates expression and/or purification.

在本发明的第三方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：In a third aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:

(1)表2所示的任一重链可变区序列和任一轻链可变区序列；以及(1) any of the heavy chain variable region sequences and any of the light chain variable region sequences shown in Table 2;

(2)本发明的第二方面所述的重组蛋白。(2) The recombinant protein of the second aspect of the invention.

在另一优选例中，所述多核苷酸如表4所示。In another preferred embodiment, the polynucleotide is as shown in Table 4.

在另一优选例中，所述多核苷酸编码选自下组的多肽：In another preferred embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of:

(1)SEQ ID NO:57、28、37、17、1或9所示的重链可变区，SEQ ID NO:58、34、48、21、5或13所示的轻链可变区；或(1) The heavy chain variable region of SEQ ID NO: 57, 28, 37, 17, 1 or 9, the light chain variable region of SEQ ID NO: 58, 34, 48, 21, 5 or ;or

在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:117、88、97、77、61或69所示；和/或，In another preferred embodiment, the polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 117, 88, 97, 77, 61 or 69; and/or

编码所述轻链可变区的多核苷酸如SEQ ID NO:118、94、108、81、65或73所示。The polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 118, 94, 108, 81, 65 or 73.

在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:117所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:118所示；或In another preferred embodiment, the polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 117, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 118; or

编码所述重链可变区的多核苷酸如SEQ ID NO:88所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:94所示；The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 88, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 94;

或or

编码所述重链可变区的多核苷酸如SEQ ID NO:97所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:108所示；The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 97, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 108;

或or

编码所述重链可变区的多核苷酸如SEQ ID NO:77所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:81所示；The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 77, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 81;

或or

编码所述重链可变区的多核苷酸如SEQ ID NO:61所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:65所示；The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 61, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 65;

或or

编码所述重链可变区的多核苷酸如SEQ ID NO:69所示，并且编码所述轻链可变区的多核苷酸如SEQ ID NO:73所示。The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 69, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO:73.

在本发明的第四方面，提供了一种载体，所述载体含有本发明本发明的第三方面中任一项所述的多核苷酸。In a fourth aspect of the invention, there is provided a vector comprising the polynucleotide of any one of the third aspect of the invention.

在本发明的第五方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有本发明的第四方面所述的载体或基因组中整合有本发明的第三方面中任一项所述的多核苷酸。In a fifth aspect of the invention, a genetically engineered host cell comprising the vector or genome of the fourth aspect of the invention, wherein any one of the third aspects of the invention is integrated Polynucleotides as described.

在本发明的第六方面，提供了一种抗体偶联物，该抗体偶联物含有：In a sixth aspect of the invention, there is provided an antibody conjugate comprising:

(a)抗体部分，所述抗体部分为本发明的第一方面中任一项所述的人源化抗体；和(a) an antibody portion, which is a humanized antibody of any one of the first aspects of the invention;

(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。(b) a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

在本发明的第七方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明的第一方面中任一项所述的人源化抗体。In a seventh aspect of the invention, there is provided an immune cell which is expressed or exposed to the humanized antibody of any one of the first aspects of the invention.

在本发明的第八方面，提供了一种药物组合物，所述药物组合物含有：In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising:

(i)活性成分，所述活性成分选自下组：本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合；以及(i) an active ingredient, which is selected from the group consisting of the humanized antibody of any one of the first aspect of the invention, the recombinant protein of the second aspect of the invention, and the sixth aspect of the invention The antibody conjugate of the aspect, the immune cell of the seventh aspect of the invention, or a combination thereof;

(ii)药学上可接受的载体。(ii) a pharmaceutically acceptable carrier.

在另一优选例中，所述的药物组合物包括0.01～99.99％的本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the humanized antibody of any one of the first aspects of the invention, the recombinant protein of the second aspect of the invention, and the present invention The antibody conjugate according to the sixth aspect of the invention, the immune cell of the seventh aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is the mass of the pharmaceutical composition percentage.

在本发明的第九方面，提供了一种活性成分的用途，所述活性成分选自下组：本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合，其中所述活性成分被用于(a)制备诊断试剂；和/或(b)制备预防和/或治疗IL-13相关的疾病的药物。In a ninth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of the humanized antibody of any one of the first aspect of the invention, the second aspect of the invention The recombinant protein, the antibody conjugate of the sixth aspect of the invention, the immune cell of the seventh aspect of the invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a diagnostic reagent And/or (b) preparing a medicament for preventing and/or treating an IL-13-related disease.

在另一优选例中，所述诊断试剂包括药剂、试剂、检测板或试剂盒。In another preferred embodiment, the diagnostic reagent comprises a medicament, a reagent, a test plate or a kit.

在另一优选例中，所述IL-13相关的疾病选自下组：支气管哮喘。In another preferred embodiment, the IL-13 associated disease is selected from the group consisting of bronchial asthma.

在本发明的第十方面，提供了一种体外检测样品中IL-13蛋白的方法，所述方法包括步骤：In a tenth aspect of the invention, there is provided a method of detecting IL-13 protein in a sample in vitro, the method comprising the steps of:

(1)在体外，将所述样品与本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合接触；(1) The humanized antibody according to any one of the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, or the sixth aspect of the present invention Contacting the antibody conjugate, the immune cell of the seventh aspect of the invention, or a combination thereof;

(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-13蛋白。(2) Detecting whether an antigen-antibody complex is formed, wherein formation of a complex means that IL-13 protein is present in the sample.

在本发明的第十一方面，提供了一种体外检测样品中IL-13蛋白的组合物，其包括本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合作为活性成分。In an eleventh aspect of the invention, there is provided a composition for detecting IL-13 protein in a sample in vitro, comprising the humanized antibody of any one of the first aspects of the invention, the second of the invention The recombinant protein according to aspect 6, the antibody conjugate according to the sixth aspect of the invention, the immune cell of the seventh aspect of the invention, or a combination thereof, as an active ingredient.

在本发明的第十二方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞、或其组合。In a twelfth aspect of the invention, there is provided a test board comprising: a substrate (support plate) and a test strip, the test strip comprising any one of the first aspects of the invention The humanized antibody, the recombinant protein of the second aspect of the invention, the antibody conjugate according to the sixth aspect of the invention, the immune cell of the seventh aspect of the invention, or a combination thereof.

在本发明的第十三方面，提供了一种重组多肽的制备方法，该方法包括：In a thirteenth aspect of the invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

(a)在适合表达的条件下，培养本发明的第五方面所述的宿主细胞；(a) cultivating the host cell of the fifth aspect of the invention under conditions suitable for expression;

(b)从培养物中分离出重组多肽，所述的重组多肽是本发明的第一方面中任一项所述的人源化抗体或本发明的第二方面所述的重组蛋白。(b) The recombinant polypeptide is isolated from the culture, the recombinant polypeptide of the humanized antibody of any one of the first aspect of the invention or the recombinant protein of the second aspect of the invention.

在本发明的第十四方面，提供了一种治疗支气管哮喘的方法，包括：使用本发明的第一方面中任一项所述的人源化抗体、本发明的第二方面所述的重组蛋白、本发明的第六方面所述的抗体偶联物、本发明的第七方面所述的免疫细胞。In a fourteenth aspect of the invention, a method of treating bronchial asthma, comprising: using the humanized antibody of any one of the first aspects of the invention, the recombination of the second aspect of the invention The antibody, the antibody conjugate according to the sixth aspect of the invention, and the immune cell of the seventh aspect of the invention.

应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

图1为TARC分泌试验检验重组IL-13蛋白的生物学活性的结果。Figure 1 shows the results of examining the biological activity of recombinant IL-13 protein by the TARC secretion assay.

图2为ELISA检测免疫原A免疫后小鼠血清抗体效价的结果。Figure 2 shows the results of ELISA for detecting serum antibody titers in mice after immunization with immunogen A.

图3为ELISA(酶联免疫吸附实验)中IL-13先导抗体与人IL-13反应活性的结果。Figure 3 shows the results of the reactivity of IL-13 leader antibody with human IL-13 in an ELISA (enzyme-linked immunosorbent assay).

图4为ELISA(酶联免疫吸附实验)中IL-13先导抗体与人IL-13变体(IL-13R130Q变体)反应活性的结果。Figure 4 shows the results of the reactivity of the IL-13 leader antibody with the human IL-13 variant (IL-13R130Q variant) in an ELISA (enzyme-linked immunosorbent assay).

图5为ELISA(酶联免疫吸附实验)中IL-13先导抗体与猴IL-13变体反应活性的结果。Figure 5 is a graph showing the results of reactivity of IL-13 leader antibody with monkey IL-13 variant in ELISA (enzyme-linked immunosorbent assay).

图6为ELISA(酶联免疫吸附实验)中IL-13先导抗体与鼠IL-13变体反应活性的结果。Figure 6 shows the results of the reactivity of IL-13 leader antibody with murine IL-13 variant in ELISA (enzyme-linked immunosorbent assay).

图7为流式细胞分析方法检测过表达全长人IL-13Ra1的HEK293细胞株中hIL-13Ra1蛋白的表达水平的结果。其中，抗体指羊抗人IL-13Ra1抗体(购自RnD systems)；阴性对照指羊IgG对照。Figure 7 is a graph showing the results of flow cytometric analysis of the expression level of hIL-13Ra1 protein in a HEK293 cell line overexpressing full-length human IL-13Ra1. Among them, the antibody refers to a goat anti-human IL-13Ra1 antibody (purchased from RnD systems); the negative control refers to a goat IgG control.

图8为流式细胞分析方法检测过表达全长人IL-4Ra的HEK293细胞株中hIL-4Ra蛋白的表达水平的结果。其中，抗体指鼠抗人IL-4Ra抗体(购自RnD systems)；阴性对照指鼠IgG对照。Figure 8 is a graph showing the results of flow cytometric analysis of the expression level of hIL-4Ra protein in a HEK293 cell line overexpressing full-length human IL-4Ra. Among them, the antibody refers to a murine anti-human IL-4Ra antibody (purchased from RnD systems); the negative control refers to a murine IgG control.

图9为流式细胞分析方法检测过表达全长人IL-13Ra2的HEK293细胞株中hIL-13Ra2蛋白的表达水平的结果。其中，抗体指羊抗人IL-13Ra2抗体(购自RnD systems)；阴性对照指羊IgG对照。Figure 9 is a graph showing the results of flow cytometric analysis of the expression level of hIL-13Ra2 protein in a HEK293 cell line overexpressing full-length human IL-13Ra2. Among them, the antibody refers to a goat anti-human IL-13Ra2 antibody (purchased from RnD systems); the negative control refers to a goat IgG control.

图10为FACS检测IL-13先导抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 10 is a graph showing the results of FACS detection of IL-13 leader antibody blocking IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图11为FACS检测IL-13先导抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 11 shows the results of FACS detection of IL-13 leader antibody blocking IL-13 binding to the cell surface receptor IL-13Ra2.

图12为IL-13先导抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 12 is a graph showing the results of IL-13 leader antibody neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion.

图13为ELISA检测IL-13嵌合抗体与免疫原A的反应活性的结果。Figure 13 is a result of ELISA detecting the reactivity of an IL-13 chimeric antibody with immunogen A.

图14为ELISA检测IL-13嵌合抗体与IL-13R130Q变体的反应活性的结果。Figure 14 is a graph showing the results of ELISA assay for the reactivity of IL-13 chimeric antibodies with IL-13R130Q variants.

图15为ELISA检测IL-13嵌合抗体与猴IL-13变体的反应活性的结果。Figure 15 is the result of ELISA detecting the reactivity of IL-13 chimeric antibodies with monkey IL-13 variants.

图16为FACS检测IL-13嵌合抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结果。Figure 16 shows the results of FACS detection of IL-13 chimeric antibodies blocking IL-13 and the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图17为为FACS检测IL-13嵌合抗体阻滞IL-13与细胞表面受体IL-13Ra2结合的结果。Figure 17 is a graph showing the results of FACS detection of IL-13 chimeric antibody blocking IL-13 binding to the cell surface receptor IL-13Ra2.

图18为IL-13嵌合抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 18 is a graph showing the results of IL-13 chimeric antibodies neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion.

图19为IL-13嵌合抗体中和IL-13诱导的骨膜蛋白分泌的结果。Figure 19 is a graph showing the results of IL-13 chimeric antibody neutralizing IL-13-induced secretion of periostin.

图20为IL-13嵌合抗体中和IL-13诱导的血管细胞粘附分子-1表达的结果。Figure 20 is a graph showing the results of IL-13 chimeric antibody neutralizing IL-13-induced expression of vascular cell adhesion molecule-1.

图21为IL-13嵌合抗体抑制人IL-13诱导的小鼠呼吸炎症实验的结果。Figure 21 is a graph showing the results of an IL-13 chimeric antibody inhibiting human IL-13-induced respiratory inflammation in mice.

图22为人源化抗IL-13抗体Hu-29D9H8重链可变区Hu-29D9H8.VH1及其变体与c29D9H8嵌合抗体VH及人种系VH外显子hVH1-F/JH1的序列比较。Figure 22 is a sequence comparison of the humanized anti-IL-13 antibody Hu-29D9H8 heavy chain variable region Hu-29D9H8.VH1 and its variants with the c29D9H8 chimeric antibody VH and the human germline VH exon hVH1-F/JH1.

图23为人源化抗IL-13抗体Hu-29D9H8轻链可变区Hu-29D9H8.V _K1及其变体与c29D9H8嵌合抗体V _K及人种系V _K外显子L1/JK4的序列比较。 Figure 23 is a sequence of humanized anti-IL-13 antibody Hu-29D9H8 light chain variable region Hu-29D9H8.V _K 1 and its variant and c29D9H8 chimeric antibody V _K and human germline V _K exon L1/JK4 Comparison.

图24为人源化抗IL-13抗体Hu-28A2E11重链可变区Hu-28A2E11.VH1及其变体与c28A2E11嵌合抗体VH及人种系VH外显子hVH1-F/JH1的序列比较。Figure 24 is a sequence comparison of the humanized anti-IL-13 antibody Hu-28A2E11 heavy chain variable region Hu-28A2E11.VH1 and its variants with the c28A2E11 chimeric antibody VH and the human germline VH exon hVH1-F/JH1.

图25为人源化抗IL-13抗体Hu-28A2E11轻链可变区Hu-28A2E11.V _L1及其变体与c28A2E11嵌合抗体V _L及人种系V _L外显子7B/JL1的序列比较。 FIG 25 is a humanized anti-IL-13 antibody Hu-28A2E11 light chain variable region Hu-28A2E11.V _L 1 and variants thereof and chimeric antibodies c28A2E11 human germline V _L and V _L exon 7B / JL1 sequence Comparison.

图26为人源化抗IL-13抗体Hu-35E2C3重链可变区Hu-35E2C3.VH1及其变体与c35E2C3嵌合抗体VH及人种系VH外显子hVH4-59/JH6的序列比较。Figure 26 is a sequence comparison of the humanized anti-IL-13 antibody Hu-35E2C3 heavy chain variable region Hu-35E2C3.VH1 and its variants with the c35E2C3 chimeric antibody VH and the human germline VH exon hVH4-59/JH6.

图27为人源化抗IL-13抗体Hu-35E2C3轻链可变区Hu-35E2C3.V _K1及其变体与c35E2C3嵌合抗体V _K及人种系V _K外显子O12/JK4的序列比较。 Figure 27 is a sequence of humanized anti-IL-13 antibody Hu-35E2C3 light chain variable region Hu-35E2C3.V _K 1 and its variant and c35E2C3 chimeric antibody V _K and human germline V _K exon O12/JK4 Comparison.

图28为ELISA(酶联免疫吸附实验)中人源化抗IL-13抗体与免疫原A的反应活性的结果。Figure 28 is a graph showing the results of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).

图29为ELISA(酶联免疫吸附实验)中人源化抗IL-13抗体与免疫原A的反应活性的结果。Figure 29 shows the results of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).

图30为ELISA(酶联免疫吸附实验)中人源化抗IL-13抗体与免疫原A的反应活性的结果。Figure 30 is the result of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).

图31-1流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 31-1 Flow cytometry assay detects the binding of humanized anti-IL-13 antibody to block the binding of IL-13 to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图31-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 31-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图32-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 32-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图32-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 32-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图33-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 33-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图33-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 33-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图34-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 34-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图34-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 34-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图35-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 35-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图35-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 35-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图36-1流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 36-1 Flow cytometric analysis method detects the binding of humanized anti-IL-13 antibody to block the binding of IL-13 to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图36-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 36-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图37-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 37-1 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图37-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 37-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图38-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 38-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图38-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 38-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图39-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 39-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图39-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 39-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图40-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 40-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图40-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 40-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图41-1为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合的结果。Figure 41-1 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.

图41-2为流式细胞分析方法检测检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合的结果。Figure 41-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.

图42-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 41-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图42-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 42-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图43-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 43-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图43-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 43-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图44-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 44-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图44-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 44-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图45-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 45-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图45-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 45-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图46-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 46-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图46-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 46-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图47-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 47-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图47-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 47-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图48-1为人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 48-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.

图48-2为人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子(TARC)分泌的结果。Figure 48-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.

图49-1为IL-13人源化抗体抑制人IL-13诱导的小鼠肺杯状细胞化生及黏液过度分泌实验的结果。Figure 49-1 shows the results of IL-13 humanized antibody inhibition of human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice.

图49-2为IL-13人源化抗体抑制人IL-13诱导的小鼠肺杯状细胞化生及黏液过度分泌实验的结果。Figure 49-2 shows the results of IL-13 humanized antibody inhibition of human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice.

Detailed ways

本发明人通过广泛而深入的研究，以重组人IL-13蛋白作为免疫原，采用传统的杂交瘤制备技术(参见Kohler and Milstein，Nature，1975，256:495)，通过一系列的调整和改进获得IL-13抗体的先导抗体。再通过对先导抗体的初步生产、纯化和鉴定，获得具备与人IL-13蛋白等蛋白具有高度亲和力的IL-13抗体。并且通过分子生物学方法测序获知所得的IL-13抗体的重链可变区和IL-13抗体的轻链可变区的氨基酸序列。The present inventors conducted a series of adjustments and improvements through extensive and intensive research using recombinant human IL-13 protein as an immunogen using conventional hybridoma preparation techniques (see Kohler and Milstein, Nature, 1975, 256:495). A lead antibody to the IL-13 antibody is obtained. Further, an IL-13 antibody having a high affinity to a protein such as human IL-13 protein is obtained by preliminary production, purification and identification of the leader antibody. And the amino acid sequence of the heavy chain variable region of the obtained IL-13 antibody and the light chain variable region of the IL-13 antibody was obtained by sequencing by molecular biological methods.

更进一步，本发明利用经过详细分析的小鼠抗体，取其与抗原结合的CDR区与人的抗体框架嫁接，经亲和力重塑，形成CDR移植抗体，即人源化抗体，维持其特异性和大部分的亲和力，同时几乎完全去除免疫原性和毒副作用。具体地，本发明经过大量筛选，意外地最终获得一种抗IL-13人源化单克隆抗体Hu-35E2C3-19，实验结果表明，所述的人源化单克隆抗体能够高特异性地结合IL-13抗原，其具有很高的亲和力(KD<1×10 ^-8M)并且，所述的抗体具有显著的抑制小鼠呼吸炎症的活性，而对于哺乳动物本身没有可见的毒副作用。在此基础上完成了本发明。 Furthermore, the present invention utilizes a mouse antibody which has been analyzed in detail, and the CDR region bound to the antigen is grafted with a human antibody framework, and is reshaped by affinity to form a CDR-grafted antibody, ie, a humanized antibody, to maintain its specificity and Most of the affinity, while almost completely removing immunogenicity and side effects. Specifically, the present invention has been extensively screened and unexpectedly finally obtained an anti-IL-13 humanized monoclonal antibody Hu-35E2C3-19, and the experimental results show that the humanized monoclonal antibody can bind with high specificity. The IL-13 antigen, which has a high affinity (KD < 1 × 10 ^-8 M), and the antibody has a remarkable activity of inhibiting respiratory inflammation in mice, and has no visible side effects to mammals themselves. The present invention has been completed on this basis.

本发明通过阻断IL-13信号通路，可以很好地缓解IL-13导致的一系列哮喘症状，从而起到治疗哮喘的作用。尤其是针对严重哮喘起到辅助常规治疗方式甚至替代常规疗法的治疗作用。By blocking the IL-13 signaling pathway, the present invention can well alleviate a series of asthma symptoms caused by IL-13, thereby playing a role in treating asthma. Especially for severe asthma, it can help to treat conventional treatments or even replace conventional treatments.

术语the term

人源化抗IL-13抗体Humanized anti-IL-13 antibody

本发明提供一种人源化抗IL-13抗体，其包括：The present invention provides a humanized anti-IL-13 antibody comprising:

1)所述的人源化抗IL-13抗体包含人抗体框架区(构架区)残基的框架区。1) The humanized anti-IL-13 antibody comprises a framework region of a human antibody framework region (framework region) residue.

所述的人抗体框架区包括重链框架区和轻链框架区，人抗体轻链框架区残基可以包含种系L1、O2、O12、DPK1(O18)、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12(A2)、DPK13、DPK15、DPK16、DPKI8、 DPK19、DPK20、DPK21、DPK22、DPK23、DPK24(B3)、DPK25、DPK26、DPK28、DPL1、DPL7、DPL6、DPL2、DPL3、DPL5、DPL12、DPL11、DPL13、DPL10、D9、DPL16、DPL24、DPL18、DPL19(7b)、DPL21、DPL22或DPL20，特别是这些种系的FR1、FR2、FR3；以及由Jk片段Jk1、Jk2、Jk3、Jk4和JK5，特别是这些种系的FR4编码的序列。人抗体重链框架区残基可以包含种系DP3(VH1-f)、DP4、DP7、DP8、DP9、DP10、DP31、DP33、DP35(VH3-11)、DP45、DP46、DP47、DP48、DP49(VH3-30)、DP50、DP51(VH3-48)、DP53、DP54、DP65、DP66、DP67、DP68、DP69或DP71(VH4-59)，特别是这些种系的FR1、FR2、FR3；以及JH片段JH1、JH2、JH3、JH4、JH4b、JH5和JH6，特别是这些种系的FR4编码的序列，或重链框架区的共有序列。此类框架区序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在"VBase"人种系序列数据库(www.mrcco8.com.ac.uk/vbase)获得，以及在Kabat，E.A等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。The human antibody framework region comprises a heavy chain framework region and a light chain framework region, and the human antibody light chain framework region residues may comprise germline L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6 , DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2 , DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9, DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20, in particular FR1, FR2, FR3 of these species; and Jk segment Jk1 Jk2, Jk3, Jk4 and JK5, in particular the FR4-encoded sequences of these lines. Human antibody heavy chain framework region residues may comprise germline DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 ( VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59), especially FR1, FR2, FR3 of these lines; and JH fragment JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these lines, or the consensus sequence of the heavy-chain framework regions. Such framework region sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. The germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (www.mrcco8.com.ac.uk/vbase), and in Kabat, EA et al., 1991. Sequences of Proteins of Immunological Interest, found in the 5th edition.

所述人源化抗体的轻链框架区选自种系：人的抗体可变区框架经过选择，其中所述抗体轻链可变区上的轻链FR序列，来源于人种系轻链包含1)L1、7B或O12的FR1、FR2、FR3区，和2)JK4或JL1的FR4区的人抗体轻链框架区的组合；所述抗体的重链可变区上的重链FR序列，来源于人种系重链序列包含1)VH1-f或VH4-59的FR1、FR2、FR3区，和2)JH1或JH6的FR4区的人抗体重链框架区的组合。一般而言，人受体框架区的选择应类似于供体抗体的框架区，或最类似于可变区亚家族的共有序列进行选择。移植后，可以在供体和/或受体序列中进行序列突变，以优化抗原结合、功能性、密码子使用、表达水平等，包括将非人残基引入框架区内。The light chain framework region of the humanized antibody is selected from the group consisting of: a human antibody variable region framework selected, wherein the light chain FR sequence on the antibody light chain variable region is derived from a human germline light chain comprising 1) a combination of the FR1, FR2, FR3 regions of L1, 7B or O12, and 2) the human antibody light chain framework region of the FR4 region of JK4 or JL1; the heavy chain FR sequence on the heavy chain variable region of the antibody, A combination of human antibody heavy chain framework regions derived from the human germline heavy chain sequence comprising 1) FR1, FR2, FR3 regions of VH1-f or VH4-59, and 2) FR4 regions of JH1 or JH6. In general, the selection of the human acceptor framework region should be similar to the framework region of the donor antibody, or the consensus sequence most similar to the variable region subfamily. Following transplantation, sequence mutations can be made in the donor and/or acceptor sequences to optimize antigen binding, functionality, codon usage, expression levels, and the like, including introduction of non-human residues into the framework regions.

2)所述的人源化抗IL-13抗体中，CDR是小鼠的CDR，选自SEQ ID NO.5、13或21所示的轻链可变区的一个或多个CDRs，或SEQ ID NO.1、9或17所示的重链可变区的一个或多个CDRs。2) In the humanized anti-IL-13 antibody, the CDR is a CDR of a mouse selected from one or more CDRs of the light chain variable region set forth in SEQ ID NO. 5, 13 or 21, or SEQ One or more CDRs of the heavy chain variable region of ID NO. 1, 9 or 17.

较佳地，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10或SEQ ID No.18所示，更佳地，编码所述重链CDR1的氨基酸序列的核苷酸序列如序列表中SEQ ID No.62、SEQ ID No.70或SEQ ID No.78所示；Preferably, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2, SEQ ID No. 10 or SEQ ID No. 18 of the Sequence Listing, and more preferably, the amino acid sequence encoding the heavy chain CDR1. The nucleotide sequence is shown as SEQ ID No. 62, SEQ ID No. 70 or SEQ ID No. 78 in the sequence listing;

所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11或SEQ ID No.19所示，更佳地，编码所述重链CDR2的氨基酸序列的核苷酸序列如序列表中SEQ SEQ ID No.63、SEQ ID No.71或SEQ ID No.79所示；The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 19 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain CDR2 is as SEQ SEQ ID No. 63, SEQ ID No. 71 or SEQ ID No. 79 in the sequence listing;

所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12或SEQ ID No.20所示，更佳地，编码所述重链CDR3的氨基酸序列的核苷酸序列如序列表中SEQ ID No.64、SEQ ID No.72或SEQ ID No.80所示；The amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4, SEQ ID No. 12 or SEQ ID No. 20 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain CDR3. As shown in SEQ ID No. 64, SEQ ID No. 72 or SEQ ID No. 80 in the Sequence Listing;

所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14或SEQ ID No.22所示，更佳地，编码所述轻链CDR1的氨基酸序列的核苷酸序列如序列表中SEQ ID No.66、SEQ ID No.74或SEQ ID No.82所示；The amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6, SEQ ID No. 14 or SEQ ID No. 22 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR1. As shown in SEQ ID No. 66, SEQ ID No. 74 or SEQ ID No. 82 in the sequence listing;

所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15或SEQ ID No.23所示，更佳地，编码所述轻链CDR2的氨基酸序列的核苷酸序列如序列表中SEQ ID No.67、SEQ ID No.75或SEQ ID No.83所示；The amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7, SEQ ID No. 15 or SEQ ID No. 23 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR2. As shown in SEQ ID No. 67, SEQ ID No. 75 or SEQ ID No. 83 in the Sequence Listing;

所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16或SEQ ID No.24所示；更佳地，编码所述轻链CDR3的氨基酸序列的核苷酸序列如序列表中SEQ ID No.68、SEQ ID No.76或SEQ ID No.84所示；The amino acid sequence of the light chain CDR3 is set forth in SEQ ID No. 8, SEQ ID No. 16 or SEQ ID No. 24 of the Sequence Listing; more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR3 As shown in SEQ ID No. 68, SEQ ID No. 76 or SEQ ID No. 84 in the sequence listing;

较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3. As shown in the Sequence Listing SEQ ID No. 4; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 11 of the Sequence Listing. The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is SEQ ID: And the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, and the light chain CDR2 The amino acid sequence is shown in SEQ ID No. 7 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 14 of the Sequence Listing. The amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 15 of the Sequence Listing and the amino acid of the light chain CDR3 The sequence is shown in SEQ ID No. 16 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the CDR2 of the light chain is shown in SEQ ID No. 23 of the Sequence Listing and The amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 24 of the Sequence Listing.

较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；和，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；和，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；和，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3. As shown in the Sequence Listing SEQ ID No. 4; and, the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 7 of the Sequence Listing. And the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the sequence listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the sequence listing, and the amino acid sequence of the heavy chain CDR2 is as in the sequence listing. SEQ ID No. 11 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the Sequence Listing; and the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 14 of the Sequence Listing. The amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 15 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 16 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is SEQ ID No. As shown in .18, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 19 of the sequence listing and the heavy chain CDR3 The amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is SEQ ID No. The amino acid sequence of the light chain CDR3 shown in Figure 23 is shown in SEQ ID No. 24 of the Sequence Listing.

3)所述的人源化抗IL-13抗体包括至少一个重链可变区和/或至少一个轻链可变区。3) The humanized anti-IL-13 antibody comprises at least one heavy chain variable region and/or at least one light chain variable region.

所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.26、SEQ ID No.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.36、SEQ ID No.37、SEQ ID No.38、SEQ ID No.39、SEQ ID No.40、SEQ ID No.41、SEQ ID No.42、SEQ ID No.50、SEQ ID No.51、SEQ ID No.52、SEQ ID No.53、SEQ ID No.54、SEQ ID No.55、SEQ ID No.56或SEQ ID No.57所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.32、SEQ ID No.33、SEQ ID No.34、SEQ ID No.35、SEQ ID No.43、SEQ ID No.44、SEQ ID No.45、SEQ ID No.46、SEQ ID No.47、SEQ ID No.48、SEQ ID No.49、SEQ ID No.58、SEQ ID No.59或SEQ ID No.60所示；更佳地，编码所述重链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.61、SEQ ID No.69、 SEQ ID No.77、SEQ ID No.85、SEQ ID No.86、SEQ ID No.87、SEQ ID No.88、SEQ ID No.89、SEQ ID No.90、SEQ ID No.91、SEQ ID No.96、SEQ ID No.97、SEQ ID No.98、SEQ ID No.99、SEQ ID No.100、SEQ ID No.101、SEQ ID No.102、SEQ ID No.110、SEQ ID No.111、SEQ ID No.112、SEQ ID No.113、SEQ ID No.114、SEQ ID No.115、SEQ ID No.116或SEQ ID No.117所示；编码所述的轻链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.92、SEQ ID No.93、SEQ ID No.94、SEQ ID No.95、SEQ ID No.103、SEQ ID No.104、SEQ ID No.105、SEQ ID No.106、SEQ ID No.107、SEQ ID No.108、SEQ ID No.109、SEQ ID No.118、SEQ ID No.119或SEQ ID No.120所示。The amino acid sequence of the heavy chain variable region is SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56 or SEQ ID No. 57; the amino acid sequence of the light chain variable region is SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID in the sequence listing. No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47. SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 58, SEQ ID No. 59 or SEQ ID No. 60; more preferably, an amino acid sequence encoding the heavy chain variable region The nucleotide sequences are respectively SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85, SEQ in the Sequence Listing. ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. .98, SEQ ID No. 99, SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 112, SEQ ID No. 113 SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116 or SEQ ID No. 117; the nucleotide sequence encoding the amino acid sequence of the light chain variable region is as shown in the sequence listing ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 103, SEQ ID No. .104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119 or SEQ ID No. 120 Shown.

或者，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.26、SEQ ID No.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.36、SEQ ID No.37、SEQ ID No.38、SEQ ID No.39、SEQ ID No.40、SEQ ID No.41、SEQ ID No.42、SEQ ID No.50、SEQ ID No.51、SEQ ID No.52、SEQ ID No.53、SEQ ID No.54、SEQ ID No.55、SEQ ID No.56或SEQ ID No.57所示的氨基酸序列至少有80％序列同源的氨基酸序列；所述轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.32、SEQ ID No.33、SEQ ID No.34、SEQ ID No.35、SEQ ID No.43、SEQ ID No.44、SEQ ID No.45、SEQ ID No.46、SEQ ID No.47、SEQ ID No.48、SEQ ID No.49、SEQ ID No.58、SEQ ID No.59或SEQ ID No.60所示的氨基酸序列至少有80％序列同源的氨基酸序列；较佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85、SEQ ID No.86、SEQ ID No.87、SEQ ID No.88、SEQ ID No.89、SEQ ID No.90、SEQ ID No.91、SEQ ID No.96、SEQ ID No.97、SEQ ID No.98、SEQ ID No.99、SEQ ID No.100、SEQ ID No.101、SEQ ID No.102、SEQ ID No.110、SEQ ID No.111、SEQ ID No.112、SEQ ID No.113、SEQ ID No.114、SEQ ID No.115、SEQ ID No.116或SEQ ID No.117所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.92、SEQ ID No.93、SEQ ID No.94、SEQ ID No.95、SEQ ID No.103、SEQ ID No.104、SEQ ID No.105、SEQ ID No.106、SEQ ID No.107、SEQ ID No.108、SEQ ID No.109、SEQ ID No.118、SEQ ID No.119或SEQ ID No.120所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列。Alternatively, the amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54. The amino acid sequence of SEQ ID No. 55, SEQ ID No. 56 or SEQ ID No. 57 having at least 80% sequence homology; the amino acid sequence of the light chain variable region is, and SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 58, SEQ ID No. 59 or SEQ ID An amino acid sequence represented by No. 60 having at least 80% sequence homology; preferably, The amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. .87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99 SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 112, SEQ ID No. 113, SEQ ID No. 114, SEQ The amino acid sequence encoded by the nucleotide sequence represented by ID No. 115, SEQ ID No. 116 or SEQ ID No. 117 has an amino acid sequence of 80% sequence homology; the amino acid sequence of the light chain variable region is And SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No in the Sequence Listing .103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119 Or the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 120 has 8 0% sequence homologous amino acid sequence.

较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.26所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.86所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing; more preferably The nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. sequence;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.86所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.86所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.27所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.87所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.92所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 32 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.27所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.87所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.27所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.87所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.27所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.87所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.88所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.92所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.88所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.88所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.88所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.29所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.89所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.92所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.29所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.89所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.29所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.89所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.29所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.89所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.90所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.92所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.90所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 33 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.90所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.90所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.31所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.91所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.92所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.31所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.33所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.91所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.93所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 33 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.31所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.91所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.94所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 34 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.31所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.35所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.91所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.95所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.47所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.107所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 47 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.48所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.108所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 48 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.49所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.97所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.109所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 49 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 109 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.47所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.107所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 47 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.48所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.108所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 48 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.49所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.98所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.109所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 49 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 109 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.47所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.107所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 47 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.48所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.99所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.108所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 48 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.100所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.100所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.100所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.100所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.47所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.100所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.107所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 47 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.101所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.101所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.101所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.101所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.43所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.102所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.103所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 43 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 103 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.102所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.104所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.102所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.105所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID No. 105 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.102所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.106所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.51所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.111所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.51所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.111所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.51所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.111所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.52所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.112所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.52所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.112所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.52所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.112所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.53所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.113所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.53所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.113所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.53所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.113所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.54所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.114所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.54所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.114所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.54所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.114所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.55所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.115所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.55所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.115所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.55所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.115所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.56所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.116所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.56所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.116所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.56所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.116所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.58所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.117所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.118所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 118 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.59所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.117所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.119所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;

或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.60所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.117所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.120所示的序列；Alternatively, the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing; Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 120 of the Sequence Listing. Sequence shown;

综上所述，本发明的较佳实例抗体氨基酸序列的编号如表1和2所示：In summary, the numbering of the preferred example antibody amino acid sequences of the present invention is shown in Tables 1 and 2:

表1-1 IL-13抗体的氨基酸序列编号Table 1-1 Amino acid sequence number of IL-13 antibody

表1-2 IL-13抗体的氨基酸序列编号Table 1-2 Amino acid sequence number of IL-13 antibody

表1-3 IL-13抗体的氨基酸序列编号Table 1-3 Amino acid sequence number of IL-13 antibody

表1-4 IL-13抗体的氨基酸序列编号Table 1-4 Amino acid sequence number of IL-13 antibody

表2.人源化IL-13抗体蛋白序列编号Table 2. Humanized IL-13 antibody protein sequence numbering

抗体编号Antibody number 重链可变区SEQ ID NO.Heavy chain variable region SEQ ID NO. 轻链可变区SEQ ID NO.Light chain variable region SEQ ID NO. c29D9H8c29D9H8 11 55 Hu-29D9H8-1Hu-29D9H8-1 2626 3333 Hu-29D9H8-2Hu-29D9H8-2 2626 3434 Hu-29D9H8-3Hu-29D9H8-3 2626 3535 Hu-29D9H8-4Hu-29D9H8-4 2727 3232 Hu-29D9H8-5Hu-29D9H8-5 2727 3333 Hu-29D9H8-6Hu-29D9H8-6 2727 3434

Hu-29D9H8-7Hu-29D9H8-7 2727 3535 Hu-29D9H8-8Hu-29D9H8-8 2828 3232 Hu-29D9H8-9Hu-29D9H8-9 2828 3333 Hu-29D9H8-10Hu-29D9H8-10 2828 3434 Hu-29D9H8-11Hu-29D9H8-11 2828 3535 Hu-29D9H8-12Hu-29D9H8-12 2929 3232 Hu-29D9H8-13Hu-29D9H8-13 2929 3333 Hu-29D9H8-14Hu-29D9H8-14 2929 3434 Hu-29D9H8-15Hu-29D9H8-15 2929 3535 Hu-29D9H8-16Hu-29D9H8-16 3030 3232 Hu-29D9H8-17Hu-29D9H8-17 3030 3333 Hu-29D9H8-18Hu-29D9H8-18 3030 3434 Hu-29D9H8-19Hu-29D9H8-19 3030 3535 Hu-29D9H8-20Hu-29D9H8-20 3131 3232 Hu-29D9H8-21Hu-29D9H8-21 3131 3333 Hu-29D9H8-22Hu-29D9H8-22 3131 3434 Hu-29D9H8-23Hu-29D9H8-23 3131 3535 c28A2E11c28A2E11 99 1313 Hu-28A2E11-1Hu-28A2E11-1 3737 4343 Hu-28A2E11-2Hu-28A2E11-2 3737 4444 Hu-28A2E11-3Hu-28A2E11-3 3737 4545 Hu-28A2E11-4Hu-28A2E11-4 3737 4646 Hu-28A2E11-5Hu-28A2E11-5 3737 4747 Hu-28A2E11-6Hu-28A2E11-6 3737 4848 Hu-28A2E11-7Hu-28A2E11-7 3737 4949 Hu-28A2E11-8Hu-28A2E11-8 3838 4343 Hu-28A2E11-9Hu-28A2E11-9 3838 4444 Hu-28A2E11-10Hu-28A2E11-10 3838 4545 Hu-28A2E11-11Hu-28A2E11-11 3838 4646

Hu-28A2E11-12Hu-28A2E11-12 3838 4747 Hu-28A2E11-13Hu-28A2E11-13 3838 4848 Hu-28A2E11-14Hu-28A2E11-14 3838 4949 Hu-28A2E11-15Hu-28A2E11-15 3939 4343 Hu-28A2E11-16Hu-28A2E11-16 3939 4444 Hu-28A2E11-17Hu-28A2E11-17 3939 4545 Hu-28A2E11-18Hu-28A2E11-18 3939 4646 Hu-28A2E11-19Hu-28A2E11-19 3939 4747 Hu-28A2E11-20Hu-28A2E11-20 3939 4848 Hu-28A2E11-21Hu-28A2E11-21 4040 4343 Hu-28A2E11-22Hu-28A2E11-22 4040 4444 Hu-28A2E11-23Hu-28A2E11-23 4040 4545 Hu-28A2E11-24Hu-28A2E11-24 4040 4646 Hu-28A2E11-25Hu-28A2E11-25 4040 4747 Hu-28A2E11-26Hu-28A2E11-26 4141 4343 Hu-28A2E11-27Hu-28A2E11-27 4141 4444 Hu-28A2E11-28Hu-28A2E11-28 4141 4545 Hu-28A2E11-29Hu-28A2E11-29 4141 4646 Hu-28A2E11-31Hu-28A2E11-31 4242 4343 Hu-28A2E11-32Hu-28A2E11-32 4242 4444 Hu-28A2E11-33Hu-28A2E11-33 4242 4545 Hu-28A2E11-34Hu-28A2E11-34 4242 4646 c35E2C3c35E2C3 1717 21twenty one Hu-35E2C3-1Hu-35E2C3-1 5151 5858 Hu-35E2C3-2Hu-35E2C3-2 5151 5959 Hu-35E2C3-3Hu-35E2C3-3 5151 6060 Hu-35E2C3-4Hu-35E2C3-4 5252 5858 Hu-35E2C3-5Hu-35E2C3-5 5252 5959 Hu-35E2C3-6Hu-35E2C3-6 5252 6060

Hu-35E2C3-7Hu-35E2C3-7 5353 5858 Hu-35E2C3-8Hu-35E2C3-8 5353 5959 Hu-35E2C3-9Hu-35E2C3-9 5353 6060 Hu-35E2C3-10Hu-35E2C3-10 5454 5858 Hu-35E2C3-11Hu-35E2C3-11 5454 5959 Hu-35E2C3-12Hu-35E2C3-12 5454 6060 Hu-35E2C3-13Hu-35E2C3-13 5555 5858 Hu-35E2C3-14Hu-35E2C3-14 5555 5959 Hu-35E2C3-15Hu-35E2C3-15 5555 6060 Hu-35E2C3-16Hu-35E2C3-16 5656 5858 Hu-35E2C3-17Hu-35E2C3-17 5656 5959 Hu-35E2C3-18Hu-35E2C3-18 5656 6060 Hu-35E2C3-19Hu-35E2C3-19 5757 5858 Hu-35E2C3-20Hu-35E2C3-20 5757 5959 Hu-35E2C3-21Hu-35E2C3-21 5757 6060

其中，表1\2中的数字即为序列表“SEQ ID No.”编号，如c29D9H8的重链蛋白可变区的氨基酸序列为序列表SEQ ID No.1，而c29D9H8的轻链蛋白可变区的氨基酸序列为序列表SEQ ID No.5。Wherein, the number in Table 1\2 is the sequence number "SEQ ID No.", and the amino acid sequence of the heavy chain protein variable region of c29D9H8 is SEQ ID No. 1 of the sequence listing, and the light chain protein of c29D9H8 is variable. The amino acid sequence of the region is SEQ ID No. 5 of the Sequence Listing.

4)所述的人源化抗IL-13抗体，较佳地为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(Single chain antibodyfragment，scFv)、单域抗体(Single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。本发明还包括超人源化抗体、双抗体(diabody)等。4) The humanized anti-IL-13 antibody, preferably an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multi-specific antibody, a single chain antibody (Single chain antibody fragment, scFv) One or more of a single domain antibody (sdAb) and a single-region antibody (Signle-domain antibody), and a monoclonal antibody or a polyclonal antibody produced by the above antibody. The monoclonal antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology. The invention also includes superhumanized antibodies, diabody, and the like.

所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。The antibody full-length protein is a conventional full-length antibody protein of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.

较佳地，所述的人源化抗IL-13抗体还包括人源抗体重链恒定区和/或人源抗体轻链恒定区。所述的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成人源化抗体全长蛋白。其中所述的人源化抗体重链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源IgG1，IgG2，IgG3，IgG4或其变体的重链恒定区；所述的人源化抗体轻链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源κ、λ链或其变体的轻链恒定区。Preferably, the humanized anti-IL-13 antibody further comprises a human antibody heavy chain constant region and/or a human antibody light chain constant region. The heavy chain variable region and the light chain variable region and the human heavy chain constant region and the human light chain constant region constitute a humanized antibody full length protein. Wherein the humanized antibody heavy chain constant region is routine in the art and may comprise a constant region derived from a human constant region, further comprising a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof; The humanized antibody light chain constant region is routine in the art and may comprise a constant region derived from a human constant region, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.

所述的单链抗体为本领域常规的单链抗体，其包括所述的重链可变区、轻链可变区和15～20个氨基酸的短肽。The single-chain antibody is a conventional single-chain antibody in the art, which includes the heavy chain variable region, the light chain variable region, and a short peptide of 15-20 amino acids.

所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)2。The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F(ab')2.

所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。The single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.

所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。The single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.

其中，所述人源化抗IL-13抗体的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该人源化抗IL-13抗体的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该人源化抗IL-13抗体的表达转化体中分离获得优选如下方法：将编码所述人源化抗IL-13抗体的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述人源化抗IL-13抗体。本发明的代表性人源化抗IL-13抗体的制备在实施例10中得到描述。Wherein, the preparation method of the humanized anti-IL-13 antibody is a preparation method conventional in the art. The preparation method is preferably obtained by isolating the expression transformant recombinantly expressing the humanized anti-IL-13 antibody or by artificially synthesizing the protein sequence. The method for isolating the expression transformant recombinantly expressing the humanized anti-IL-13 antibody preferably obtains a method of cloning the nucleic acid molecule encoding the humanized anti-IL-13 antibody into a recombinant vector, and the resulting recombinant The vector is transformed into a transformant to obtain a recombinant expression transformant, and the recombinant expression transformant obtained by culturing can be isolated and purified to obtain the humanized anti-IL-13 antibody. The preparation of representative humanized anti-IL-13 antibodies of the invention is described in Example 10.

人源化抗体广义上也是一类嵌合抗体，其中负责抗原结合的可变区残基，包括衍生自非人物种的互补决定区、缩短的互补决定区、或参与抗原结合的任何其它残基；而其余可变区残基如，框架区的残基和恒定区至少部分衍生自人抗体序列。人源化抗体的框架区残基和恒定区残基的一个子集可以衍生自非人来源。人源化抗体的可变区也描述为人源化的轻链可变区和/或重链可变区。非人物种一般是用于由抗原免疫接种的物种，例如小鼠、大鼠、兔、非人灵长类、或其他非人哺乳动物物种。人源化抗体一般比传统嵌合抗体的免疫原性小，并且在给人施用后显示出改善的稳定性。Humanized antibodies are also broadly a class of chimeric antibodies in which the variable region residues responsible for antigen binding, including complementarity determining regions derived from non-human species, shortened complementarity determining regions, or any other residue involved in antigen binding. Whereas the remaining variable region residues, eg, the residues and constant regions of the framework regions, are at least partially derived from human antibody sequences. A subset of the framework region residues and constant region residues of the humanized antibody can be derived from a non-human source. The variable region of a humanized antibody is also described as a humanized light chain variable region and/or heavy chain variable region. Non-human species are generally species used for immunization with antigens, such as mice, rats, rabbits, non-human primates, or other non-human mammal species. Humanized antibodies are generally less immunogenic than traditional chimeric antibodies and exhibit improved stability upon administration to humans.

互补决定区(CDRs)是参与抗原结合的抗体可变区的残基。用于标识CDRs的几种编号系统是常用的，包括例如Kabat定义、Chothia定义和AbM定义。概括地说，Kabat定义是基于序列的变异性、Chothia定义是基于结构环区的位置，AbM定义是Kabat和Chothia方法之间的折中。根据Kabat、Chothia或AbM算法，轻链可变区有三个CDR区，其CDR1位于由第24-34位氨基酸(CDR1-L)、CDR2位于第50-56位氨基酸(CDR2-L)、CDR3位于第89-97位氨基酸(CDR3-L)。由于可变区的长度变化，在不同的中枢或不同的亚群中，第27位可有1-6个氨基酸，第95位也可有1-6个氨基酸，它们在原编号的基础上加上英文字母进行编位，如：27A、27B、95A、95B等。根据Kabat定义，重链可变区的CDRs由在第31和35B位(CDR1-H)、第50和65位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。根据Chothia定义，重链可变区的CDRs由第26和32位(CDR1-H)、第52和56位(CDR2-H)、以及第95和102位(CDR3-H)上的残基界定(根据Chothia编号)。根据AbM定义，重链可变区的CDRs由26和35B位(CDR1-H)、第50和58位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。与轻链可变区类似，在第35位、52位、82位及100位也可有多个氨基酸，以A、B、C……等编号。参见Martin等人(1989)Proc.Nat l.Acad.Sci.USA 86:9268-9272；Martin等人(1991)Methods Enzymol.203:121-153；Pedersen等人(1992)lmmunomethods 1:126；Protein Structure Prediction,Oxford University Press,Oxford,第141-172页。Complementarity determining regions (CDRs) are residues of antibody variable regions involved in antigen binding. Several numbering systems for identifying CDRs are commonly used, including, for example, Kabat definitions, Chothia definitions, and AbM definitions. In summary, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of the structural loop region, and the AbM definition is a compromise between the Kabat and Chothia methods. According to the Kabat, Chothia or AbM algorithm, the light chain variable region has three CDR regions, the CDR1 is located at amino acids 24-34 (CDR1-L), the CDR2 is at amino acids 50-56 (CDR2-L), and the CDR3 is located. Amino acids 89-97 (CDR3-L). Due to the change in the length of the variable region, in the different central or different subgroups, the 27th position may have 1-6 amino acids, and the 95th position may have 1-6 amino acids, which are added to the original number. English letters are numbered, such as: 27A, 27B, 95A, 95B, etc. According to the Kabat definition, the CDRs of the heavy chain variable region are defined by residues at positions 31 and 35B (CDR1-H), positions 50 and 65 (CDR2-H), and positions 95 and 102 (CDR3-H). (according to Kabat numbering). According to the definition of Chothia, the CDRs of the heavy chain variable region are defined by residues at positions 26 and 32 (CDR1-H), positions 52 and 56 (CDR2-H), and positions 95 and 102 (CDR3-H) (according to the Chothia number). According to the definition of AbM, the CDRs of the heavy chain variable region are defined by the residues at positions 26 and 35B (CDR1-H), at positions 50 and 58 (CDR2-H), and at positions 95 and 102 (CDR3-H) (according to Kabat number). Similar to the light chain variable region, a plurality of amino acids may be present at positions 35, 52, 82, and 100, and are numbered A, B, C, and the like. See Martin et al. (1989) Proc. Natl. Acad. Sci. USA 86:9268-9272; Martin et al. (1991) Methods Enzymol. 203: 121-153; Pedersen et al. (1992) lmmunomethods 1:126; Structure Prediction, Oxford University Press, Oxford, pp. 141-172.

在本发明的优选例中，各CDR区是用Kabat定义的。In a preferred embodiment of the invention, each CDR region is defined by Kabat.

特异性决定区(SDRs)是与抗原直接相互作用的CDRs内的残基。SDRs对应高变残基。参见Padlan等人(1995)FASEB J.9:133-139)。Specificity determining regions (SDRs) are residues within CDRs that interact directly with antigen. SDRs correspond to hypervariable residues. See Padlan et al. (1995) FASEB J. 9: 133-139).

框架(构架)残基是轻链可变区或重链可变区的一部分，是除高变残基(高变残基多指互补决定区或CDR)或CDR残基外的抗体可变区残基，其用作该可变结构域的抗原结合环(CDR)的支架。构架残基可以衍生自天然存在的人的抗体，还可以使用代表个体序列之间的共有序列的人工框架区序列。当选择用于人源化的框架区时，在人类中广泛呈现的序列可能优于较不常见的序列。可以制备人构架受体序列的另外突变，以恢复被认为涉及抗原接触的鼠类残基和/或涉及抗原结合位点的结构完整性的残基，或改善抗体表达。肽结构预测可以用于分析人源化重链可变区和轻链可变区序列，以鉴定和避免由人源化设计引入的翻译后蛋白质修饰位点。A framework (framework) residue is part of a light chain variable region or a heavy chain variable region and is an antibody variable region other than a hypervariable residue (a hypervariable residue multi-finger complementarity determining region or CDR) or a CDR residue A residue that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. The framework residues can be derived from naturally occurring human antibodies, and artificial framework region sequences representing consensus sequences between individual sequences can also be used. When a framework region for humanization is selected, sequences that are widely present in humans may be superior to less common sequences. Additional mutations in the human framework receptor sequence can be made to restore murine residues believed to be involved in antigen contact and/or residues involved in the structural integrity of the antigen binding site, or to improve antibody expression. Peptide structure prediction can be used to analyze humanized heavy chain variable region and light chain variable region sequences to identify and avoid post-translational protein modification sites introduced by humanized design.

所述的人源化抗体可以使用各种方法中的任何一种进行制备，包括互补决定区(CDRs)的镶饰、移植、缩短的CDRs的移植、特异性决定区(SDRs)的移植、和Frankenstein装配。The humanized antibodies can be prepared using any of a variety of methods, including inlays of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, transplantation of specificity determining regions (SDRs), and Frankenstein assembly.

所述的人源化抗体还包括超人源化抗体，它是一种人源化抗体的制备方法，此方法不依赖于将人构架序列作为分析点，而是依赖于比较非人抗体的规范CDR结构类型和人抗体的CDR结构类型，尤其是人胚系序列所编码的人抗体，从中识别出可以得到适宜的人构架序列的候选的人抗体序列。例如，人残基可以置换CDRs中的非人残基，其中一个或多个变化已引入CDRs中。镶饰(veneering)的一个前提是鼠源抗体可变区的免疫原性起源于它的表面残基，且残基的运动性和溶剂的可及性是其成为抗原决定簇的基本条件。根据对现有的抗体晶体结构数据的分析结果统计，在序列配对位置上，人和鼠的抗体可变区残基的相对溶剂可及性分布的保真度达98％，这说明在异种间诱导免疫反应的残基是由其余的种特异性溶剂可及表面残基引起的。因此将鼠特异性表面残基换成人源性的，就可以模拟人源抗体的表面轮廓，逃避人体免疫系统的识别，达到人源化的目的。简单来说，镶饰基于通过用人氨基酸序列重建抗体的溶剂可及的表面来减少在啮齿类动物或其他非人抗体中的潜在免疫原性的氨基酸序列的概念。参见Padlan(1991)Mol.Immunol.28:489-980.通过鉴定非人抗体中暴露在表面的溶剂可及性残基的外部框架区残基(所述残基不同于人抗体的框架区中相同位置上的那些残基)，并用占据人抗体中的那些相同位置的氨基酸替换所鉴定的残基，以进行镶饰，即镶饰的抗体，其表面残基主要是人源序列，而包裹在内部的残基主要是最初的鼠源序列。CDRs的移植通过用供体抗体(如，非人抗体)的CDRs替换受体抗体(例如，包含所需构架残基的人抗体或其他抗体)的一个或多个CDRs来进行。受体抗体可以基于在候选受体抗体和供体抗体之间的构架残基的相似性进行选择。例如，根据Frankenstein方法，鉴定与相关的非人抗体的各框架区具有实质上的序列同源性的人框架区，并且将非人抗体的CDRs移植到这些不同的人框架区的复合物上。可以结合上述方法，以产生任何所需序列的抗IL-13抗体。The humanized antibody also includes a superhumanized antibody, which is a method for preparing a humanized antibody, which does not rely on the human framework sequence as an analysis point, but relies on comparing the normative CDRs of the non-human antibody. The structural type and the CDR structure type of the human antibody, particularly the human antibody encoded by the human germline sequence, from which a candidate human antibody sequence from which a suitable human framework sequence can be obtained is identified. For example, a human residue can replace a non-human residue in a CDR, wherein one or more changes have been introduced into the CDRs. One premise of veneering is that the immunogenicity of the murine antibody variable region originates from its surface residues, and the mobility of the residues and the accessibility of the solvent are essential conditions for its becoming an antigenic determinant. According to the analysis results of the existing antibody crystal structure data, the fidelity of the relative solvent accessibility distribution of the human and mouse antibody variable region residues is 98% at the sequence pairing position, which indicates that among the heterogeneous species The residues that induce an immune response are caused by the remaining species-specific solvent accessible surface residues. Therefore, by changing the mouse-specific surface residue to adult-derived, the surface profile of the human antibody can be simulated, and the recognition of the human immune system can be avoided to achieve the purpose of humanization. Briefly, the inlay is based on the concept of reducing the amino acid sequence of potential immunogenicity in rodents or other non-human antibodies by reconstituting the solvent-accessible surface of the antibody with a human amino acid sequence. See Padlan (1991) Mol. Immunol. 28: 489-980. Identification of the outer framework region residues of solvent accessible residues exposed to the surface in non-human antibodies (the residues are different from the framework regions of human antibodies) Those residues at the same position) and replacing the identified residues with amino acids occupying the same position in the human antibody for inlaying, ie, the embedded antibody, the surface residues of which are predominantly human sequences, and The internal residues are primarily the original murine sequences. Transplantation of CDRs is performed by replacing one or more CDRs of a receptor antibody (eg, a human antibody or other antibody comprising the desired framework residues) with CDRs of a donor antibody (eg, a non-human antibody). The acceptor antibody can be selected based on the similarity of the framework residues between the candidate acceptor antibody and the donor antibody. For example, according to the Frankenstein method, a human framework region having substantial sequence homology to each framework region of a related non-human antibody is identified, and CDRs of non-human antibodies are grafted onto the complex of these different human framework regions. The above methods can be combined to produce an anti-IL-13 antibody of any desired sequence.

5)本发明还提供一种核酸，其编码上述的人源化抗IL-13抗体，包括编码所述重链可变区的核酸，和/或编码所述轻链链可变区的核酸。5) The invention also provides a nucleic acid encoding the above-described humanized anti-IL-13 antibody, comprising a nucleic acid encoding the heavy chain variable region, and/or a nucleic acid encoding the light chain variable region.

较佳地，所述重链可变区的核酸编码的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.26、SEQ ID No.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.36、SEQ ID No.37、SEQ ID No.38、SEQ ID No.39、SEQ ID No.40、SEQ ID No.41、SEQ ID No.42、SEQ ID No.50、SEQ ID No.51、SEQ ID No.52、SEQ ID No.53、SEQ ID No.54、SEQ ID No.55、SEQ ID No.56或SEQ ID No.57所示；所述轻链可变区的核酸编码的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.32、SEQ ID No.33、SEQ ID No.34、SEQ ID No.35、SEQ ID No.43、SEQ ID No.44、SEQ ID No.45、SEQ ID No.46、SEQ ID No.47、SEQ ID No.48、SEQ ID No.49、SEQ ID No.58、SEQ ID No.59或SEQ ID No.60所示；更佳地，编码所述重链可变区的核酸的核苷酸序列分别如序列表中SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85、SEQ ID No.86、SEQ ID No.87、SEQ ID No.88、SEQ ID No.89、SEQ ID No.90、SEQ ID No.91、SEQ ID No.96、SEQ ID No.97、SEQ ID No.98、SEQ ID No.99、SEQ ID No.100、SEQ ID No.101、SEQ ID No.102、SEQ ID No.110、SEQ ID No.111、SEQ ID No.112、SEQ ID No.113、SEQ ID No.114、SEQ ID No.115、SEQ ID No.116或SEQ ID No.117所示；编码所述的轻链可变区的核酸的核苷酸序列分别如序列表中SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.92、SEQ ID No.93、SEQ ID No.94、SEQ ID No.95、SEQ ID No.103、SEQ ID No.104、SEQ ID No.105、SEQ ID No.106、SEQ ID No.107、SEQ ID No.108、SEQ ID No.109、SEQ ID No.118、SEQ ID No.119或SEQ ID No.120所示。Preferably, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26 in the sequence listing. SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. .54, SEQ ID No. 55, SEQ ID No. 56 or SEQ ID No. 57; the amino acid sequence encoded by the nucleic acid of the light chain variable region is SEQ ID No. 5, SEQ ID No. in the sequence listing. 13. SEQ ID No. 21, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 58, SEQ ID No. 59 or SEQ ID No. 60; more preferably, the coding house The nucleotide sequences of the nucleic acids of the variable region of the heavy chain are respectively SEQ ID No. 61, SEQ ID No. 69, SEQ I in the sequence listing. D No. 77, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. .96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99, SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111 , SEQ ID No. 112, SEQ ID No. 113, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116 or SEQ ID No. 117; nucleic acid encoding the light chain variable region The nucleotide sequences are respectively SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. in the Sequence Listing. 95, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119 or SEQ ID No. 120.

进一步更佳地，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.26所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.86所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Further preferably, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is SEQ ID No. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region As shown in the sequence listing SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.26所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.86所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.26所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.86所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.27所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 32 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 92;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.27所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.27所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.27所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.28所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 32 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 92;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.28所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 33 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.28所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.28所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.29所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 32 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.29所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.29所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.29所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.30所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.30所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.30所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.30所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.33所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.35所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.47所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 107;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.48所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.108所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 48 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 108;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.37所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.49所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.109所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 109;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.47所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 107;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.48所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.108所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is shown in SEQ ID No. 48 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 108;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.49所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.109所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 109;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.47所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 47 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 107;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.48所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.108所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 48 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 108;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.47所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 107;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.42所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.43所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.102所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.103所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.42所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.102所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.104所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.42所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.45所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.102所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.42所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.102所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.51所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.51所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.51所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.52所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.112所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.52所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.112所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.52所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.112所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.53所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.53所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.53所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.54所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.114所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.54所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.114所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.54所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.114所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.56所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.116所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is set forth in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.56所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.116所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.56所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.116所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.58所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.59所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;

或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.60所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；Alternatively, the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing. Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;

上述抗体的核苷酸序列的编号如表3-4所示：The nucleotide sequences of the above antibodies are numbered as shown in Table 3-4:

表3-1 IL-13抗体的核苷酸序列编号Table 3-1 Nucleotide sequence number of IL-13 antibody

表3-2 IL-13抗体的核苷酸序列编号Table 3-2 Nucleotide sequence number of IL-13 antibody

表3-3 IL-13抗体的核苷酸序列编号Table 3-3 Nucleotide sequence number of IL-13 antibody

表3-4 IL-13抗体的核苷酸序列编号Table 3-4 Nucleotide sequence numbers of IL-13 antibodies

表4.人源化IL-13抗体核苷酸序列编号Table 4. Humanized IL-13 antibody nucleotide sequence numbering

抗体编号Antibody number 重链可变区SEQ ID NO.Heavy chain variable region SEQ ID NO. 轻链可变区SEQ ID NO.Light chain variable region SEQ ID NO. c29D9H8c29D9H8 6161 6565 Hu-29D9H8-1Hu-29D9H8-1 8686 9393 Hu-29D9H8-2Hu-29D9H8-2 8686 9494 Hu-29D9H8-3Hu-29D9H8-3 8686 9595 Hu-29D9H8-4Hu-29D9H8-4 8787 9292 Hu-29D9H8-5Hu-29D9H8-5 8787 9393 Hu-29D9H8-6Hu-29D9H8-6 8787 9494 Hu-29D9H8-7Hu-29D9H8-7 8787 9595

Hu-29D9H8-8Hu-29D9H8-8 8888 9292 Hu-29D9H8-9Hu-29D9H8-9 8888 9393 Hu-29D9H8-10Hu-29D9H8-10 8888 9494 Hu-29D9H8-11Hu-29D9H8-11 8888 9595 Hu-29D9H8-12Hu-29D9H8-12 8989 9292 Hu-29D9H8-13Hu-29D9H8-13 8989 9393 Hu-29D9H8-14Hu-29D9H8-14 8989 9494 Hu-29D9H8-15Hu-29D9H8-15 8989 9595 Hu-29D9H8-16Hu-29D9H8-16 9090 9292 Hu-29D9H8-17Hu-29D9H8-17 9090 9393 Hu-29D9H8-18Hu-29D9H8-18 9090 9494 Hu-29D9H8-19Hu-29D9H8-19 9090 9595 Hu-29D9H8-20Hu-29D9H8-20 9191 9292 Hu-29D9H8-21Hu-29D9H8-21 9191 9393 Hu-29D9H8-22Hu-29D9H8-22 9191 9494 Hu-29D9H8-23Hu-29D9H8-23 9191 9595 c28A2E11c28A2E11 6969 7373 Hu-28A2E11-1Hu-28A2E11-1 9797 103103 Hu-28A2E11-2Hu-28A2E11-2 9797 104104 Hu-28A2E11-3Hu-28A2E11-3 9797 105105 Hu-28A2E11-4Hu-28A2E11-4 9797 106106 Hu-28A2E11-5Hu-28A2E11-5 9797 107107 Hu-28A2E11-6Hu-28A2E11-6 9797 108108 Hu-28A2E11-7Hu-28A2E11-7 9797 109109 Hu-28A2E11-8Hu-28A2E11-8 9898 103103 Hu-28A2E11-9Hu-28A2E11-9 9898 104104 Hu-28A2E11-10Hu-28A2E11-10 9898 105105 Hu-28A2E11-11Hu-28A2E11-11 9898 106106 Hu-28A2E11-12Hu-28A2E11-12 9898 107107

Hu-28A2E11-13Hu-28A2E11-13 9898 108108 Hu-28A2E11-14Hu-28A2E11-14 9898 109109 Hu-28A2E11-15Hu-28A2E11-15 9999 103103 Hu-28A2E11-16Hu-28A2E11-16 9999 104104 Hu-28A2E11-17Hu-28A2E11-17 9999 105105 Hu-28A2E11-18Hu-28A2E11-18 9999 106106 Hu-28A2E11-19Hu-28A2E11-19 9999 107107 Hu-28A2E11-20Hu-28A2E11-20 9999 108108 Hu-28A2E11-21Hu-28A2E11-21 100100 103103 Hu-28A2E11-22Hu-28A2E11-22 100100 104104 Hu-28A2E11-23Hu-28A2E11-23 100100 105105 Hu-28A2E11-24Hu-28A2E11-24 100100 106106 Hu-28A2E11-25Hu-28A2E11-25 100100 107107 Hu-28A2E11-26Hu-28A2E11-26 101101 103103 Hu-28A2E11-27Hu-28A2E11-27 101101 104104 Hu-28A2E11-28Hu-28A2E11-28 101101 105105 Hu-28A2E11-29Hu-28A2E11-29 101101 106106 Hu-28A2E11-31Hu-28A2E11-31 102102 103103 Hu-28A2E11-32Hu-28A2E11-32 102102 104104 Hu-28A2E11-33Hu-28A2E11-33 102102 105105 Hu-28A2E11-34Hu-28A2E11-34 102102 106106 c35E2C3c35E2C3 7777 8181 Hu-35E2C3-1Hu-35E2C3-1 111111 118118 Hu-35E2C3-2Hu-35E2C3-2 111111 119119 Hu-35E2C3-3Hu-35E2C3-3 111111 120120 Hu-35E2C3-4Hu-35E2C3-4 112112 118118 Hu-35E2C3-5Hu-35E2C3-5 112112 119119 Hu-35E2C3-6Hu-35E2C3-6 112112 120120 Hu-35E2C3-7Hu-35E2C3-7 113113 118118

Hu-35E2C3-8Hu-35E2C3-8 113113 119119 Hu-35E2C3-9Hu-35E2C3-9 113113 120120 Hu-35E2C3-10Hu-35E2C3-10 114114 118118 Hu-35E2C3-11Hu-35E2C3-11 114114 119119 Hu-35E2C3-12Hu-35E2C3-12 114114 120120 Hu-35E2C3-13Hu-35E2C3-13 115115 118118 Hu-35E2C3-14Hu-35E2C3-14 115115 119119 Hu-35E2C3-15Hu-35E2C3-15 115115 120120 Hu-35E2C3-16Hu-35E2C3-16 116116 118118 Hu-35E2C3-17Hu-35E2C3-17 116116 119119 Hu-35E2C3-18Hu-35E2C3-18 116116 120120 Hu-35E2C3-19Hu-35E2C3-19 117117 118118 Hu-35E2C3-20Hu-35E2C3-20 117117 119119 Hu-35E2C3-21Hu-35E2C3-21 117117 120120

其中，表3-4中的数字即为序列表“SEQ ID No.”编号，如编码c29D9H8的重链蛋白可变区的核苷酸序列为序列表SEQ ID No.61，而编码c29D9H8的轻链蛋白可变区的核苷酸序列为序列表SEQ ID No.65。Wherein, the numbers in Table 3-4 are the sequence number "SEQ ID No.", and the nucleotide sequence of the heavy chain protein variable region encoding c29D9H8 is SEQ ID No. 61 of the sequence listing, and the light encoding c29D9H8 is light. The nucleotide sequence of the variable region of the streptavidin is SEQ ID No. 65 of the Sequence Listing.

所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述人源化抗IL-13抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述人源化抗IL-13抗体的核酸分子。The preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above humanized anti-IL-13 antibody by gene cloning technology, or by artificial full sequence synthesis method. A nucleic acid molecule encoding the above humanized anti-IL-13 antibody is obtained.

本领域技术人员知晓，编码上述人源化抗IL-13抗体的核酸可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该人源化抗IL-13抗体的核酸的一个或多个核苷酸在保持抗体活性范围内进行替换、缺失或增加来制得。Those skilled in the art will recognize that a nucleic acid encoding a humanized anti-IL-13 antibody as described above may be suitably introduced with a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. A homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more nucleotides of a nucleic acid encoding the humanized anti-IL-13 antibody while maintaining antibody activity.

本发明还提供一种包含所述核酸的重组表达载体。其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。The invention also provides a recombinant expression vector comprising the nucleic acid. Wherein the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors. The expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.

本发明还提供一种包含上述重组表达载体的重组表达转化体。其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。The present invention also provides a recombinant expression transformant comprising the above recombinant expression vector. Wherein, the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell. The host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed. Preferably, the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody). The recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell. Wherein the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.

本发明提供一种人源化抗IL-13抗体的制备方法，其包括如下步骤：培养上述的重组表达转化体、或者细胞、或者细胞系，从培养物中获得人源化抗IL-13抗体。The present invention provides a method for producing a humanized anti-IL-13 antibody, which comprises the steps of culturing the above recombinant expression transformant, or a cell, or a cell line, and obtaining a humanized anti-IL-13 antibody from the culture. .

本发明还提供一种检测过表达IL-13蛋白的细胞的方法，包括如下的步骤：上述的蛋白质与待检样品在体外接触，检测上述的蛋白质与所述待检样品的结合即可。The present invention also provides a method for detecting cells overexpressing IL-13 protein, comprising the steps of: contacting the above-mentioned protein with a sample to be tested in vitro, and detecting the binding of the above protein to the sample to be tested.

所述的过表达的含义为本领域常规，指IL-13蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在底物的酶水解作用增加的情况下)。The meaning of the overexpression is conventional in the art, and refers to overexpression of RNA or protein of IL-13 protein in a sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation), and Local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (as in the case of increased enzymatic hydrolysis of the substrate).

所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。The detection mode of the combination is a conventional detection method in the art, preferably FACS detection.

本发明提供一种检测过表达IL-13蛋白的细胞的组合物，其包括上述的蛋白质作为活性成分。较佳地，其还包括上述的蛋白质的功能片段组成的化合物作为活性成分。The present invention provides a composition for detecting cells overexpressing IL-13 protein, which comprises the above-described protein as an active ingredient. Preferably, it further comprises a compound consisting of functional fragments of the above proteins as an active ingredient.

本发明提供上述蛋白质在制备药物中的应用。较佳地，所述的药物是用于预防或治疗支气管哮喘的药物。The present invention provides the use of the above proteins for the preparation of a medicament. Preferably, the medicament is a medicament for preventing or treating bronchial asthma.

本发明还提供一种药物组合物，其活性成分包括上述的蛋白质。较佳地，所述的药物组合物是用于预防或治疗支气管哮喘的药物组合物。The present invention also provides a pharmaceutical composition comprising the above-mentioned protein as an active ingredient. Preferably, the pharmaceutical composition is a pharmaceutical composition for preventing or treating bronchial asthma.

本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。The administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally. The administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. The pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.

较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。Preferably, the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutically acceptable carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.

较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。Preferably, the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought. One skilled in the art can determine the effective amount by using the above factors such as the individual basis and using no more than conventional experiments.

本发明提供上述蛋白质在制备预防或治疗IL-13表达或功能异常相关疾病的药物中的应用。The present invention provides the use of the above protein for the preparation of a medicament for preventing or treating a disease associated with IL-13 expression or dysfunction.

本发明中，所述IL-13表达或功能异常相关疾病为本领域常规的IL-13表达或功能异常相关疾病。较佳地，为支气管哮喘。In the present invention, the IL-13 expression or dysfunction-associated disease is a conventional IL-13 expression or dysfunction-related disease in the art. Preferably, it is bronchial asthma.

本发明提供上述药物组合物在制备预防或治疗IL-13表达或功能异常相关疾病的药物中的应用。The present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for preventing or treating a disease associated with IL-13 expression or dysfunction.

下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件(例如商品说明书)。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。The present invention will be further described in detail below with reference to specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples, which do not specify the detailed conditions, are generally subjected to the conditions described in the "Molecular Cloning Laboratory Guide" (translation by Huang Peitang et al., Beijing: Science Press, 2002) in accordance with conventional conditions such as Sambrook.J. Or according to the conditions recommended by the manufacturer (such as the product specification). Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are available from commercially available sources unless otherwise specified.

实施例中所述的室温为本领域常规的室温，一般为20-25℃。The room temperature described in the examples is room temperature conventional in the art, generally 20-25 °C.

实施例中若无特别说明，所述的PBS缓冲液为PBS磷酸缓冲液，pH7.4。In the examples, unless otherwise specified, the PBS buffer was PBS phosphate buffer, pH 7.4.

本发明的主要优点在于：The main advantages of the invention are:

(1)本发明所述的蛋白质，即提供的IL-13抗体亲和力高，与人IL-13的亲和力达到KD<1×10 ^-8M； (1) The protein of the present invention, that is, the IL-13 antibody provided has high affinity, and the affinity with human IL-13 reaches KD<1×10 ^-8 M;

(2)在IL-13诱导胸腺活化调节趋化因子(TARC)分泌试验中，能明显抑制IL-13诱导的分泌；(2) In IL-13-induced thymic activation-regulated chemokine (TARC) secretion assay, IL-13-induced secretion was significantly inhibited;

(3)在IL-13诱导的骨膜蛋白(Periostin)分泌试验中，能明显抑制IL-13诱导的分泌；(3) IL-13-induced secretion can be significantly inhibited in IL-13-induced periostin secretion assay;

(4)在IL-13诱导的人脐带静脉内皮细胞表达血管细胞粘附分子-1(VCAM-1)，能明显抑制IL-13诱导的表达；(4) Expression of vascular cell adhesion molecule-1 (VCAM-1) in IL-13-induced human umbilical vein endothelial cells significantly inhibited IL-13-induced expression;

(5)在以流式细胞分析建立的受体-配体结合阻滞实验中，抑制IL-13与其配体IL-13Ra1/IL-4Ra异源二聚体的结合；(5) inhibiting the binding of IL-13 to its ligand IL-13Ra1/IL-4Ra heterodimer in a receptor-ligand binding block assay established by flow cytometry;

(6)在以流式细胞分析建立的受体-配体结合阻滞实验中，抑制IL-13与其配体IL-13Ra2的结合；(6) inhibiting the binding of IL-13 to its ligand IL-13Ra2 in a receptor-ligand binding block assay established by flow cytometry;

(7)在IL-13诱导的小鼠气道炎症的动物模型中，能明显抑制小鼠气道呼吸炎症，抑制IL-13诱导的小鼠气道高反应性；(7) In the animal model of IL-13-induced airway inflammation in mice, it can significantly inhibit airway respiratory inflammation in mice and inhibit IL-13-induced airway hyperresponsiveness in mice;

(8)在IL-13诱导的小鼠肺杯状细胞化生及黏液过度分泌的动物模型中，能明显抑制小鼠肺中杯状细胞化生及，抑制IL-13诱导的小鼠肺部粘液分泌；(8) In the IL-13-induced mouse goblet cell metaplasia and mucus hypersecretion animal model, it can significantly inhibit goblet cell metaplasia in mouse lung and inhibit IL-13 induced mouse lung Mucus secretion;

(9)本发明所述的蛋白质，有望提供的IL-13抗体能够应用于制备预防或治疗支气管哮喘的药物。(9) The protein of the present invention, which is expected to be provided, can be applied to a medicament for preventing or treating bronchial asthma.

实施例1：人IL-13和人IL-13R130Q变体的表达与纯化Example 1: Expression and purification of human IL-13 and human IL-13R130Q variants

1.免疫原A和人IL-13R130Q变体的制备1. Preparation of immunogen A and human IL-13R130Q variants

将编码人IL-13蛋白氨基酸序列(参见GenBank数据库，登录号：AAK53823.1)中Met1-Asn132的核苷酸序列的3’端加入编码六个组氨酸的核苷酸，得编码his标签重组人IL-13蛋白的核苷酸序列(如序列表SEQ ID No.121所示)。The nucleotide sequence encoding Met1-Asn132 in the amino acid sequence encoding human IL-13 protein (see GenBank database, accession number: AAK53823.1) was added to the nucleotide encoding six histidine to obtain the his tag. The nucleotide sequence of the recombinant human IL-13 protein (as shown in SEQ ID No. 121 of the Sequence Listing).

或者，将编码人IL-13蛋白变体IL-13R130Q氨基酸序列中Met1-Asn132的核苷酸序列的3’端加入编码六个组氨酸的核苷酸，得编码his标签的重组人IL-13R130Q变体的核苷酸序列(如序列表SEQ ID No.122所示)。Alternatively, the 3' end of the nucleotide sequence encoding Met1-Asn132 in the amino acid sequence of human IL-13 protein variant IL-13R130Q is added to a nucleotide encoding six histidines to obtain a recombinant human IL-encoded with his tag. The nucleotide sequence of the 13R130Q variant (as shown in SEQ ID No. 122 of the Sequence Listing).

将编码his标签重组人IL-13蛋白的核苷酸序列和编码his标签的重组人IL-13R130Q变体的核苷酸序列分别克隆到PCP载体(购自Invitrogen)并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,NewYork:Cold Spring Harbor Laboratory Press)。对FreeStyle 293F细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle ^TM 293(Invitrogen)在37℃下进行扩大培养。5-7天后收集细胞培养液，离心去除细胞成分，得含his标签的人IL-13的培养上清液，或含his标签的人IL-13R130Q变体的培养上清液。将上述培养上清液分别上样到Ni-NTA亲和层析柱(购自GE)，纯化上清液中his标签的人IL-13(即免疫原A)和含his标签的人IL-13R130Q变体，再使用分子筛柱(购自GE)进行进一步纯化，除去大分子聚合体等杂质。纯化后的免疫原A和含his标签的人IL-13R130Q变体保存在PBS磷酸盐缓冲液(pH7.4)中，经0.22μm无菌滤膜过滤后分装于-80℃保存。 The nucleotide sequence encoding the his-tag recombinant human IL-13 protein and the nucleotide sequence of the recombinant human IL-13R130Q variant encoding the his tag were cloned into the PCP vector (purchased from Invitrogen) and established according to the established standard molecular organisms. Methods For the preparation of plasmids, see Sambrook, J., Fritsch, EF, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). Of FreeStyle 293F cells (purchased from Invitrogen) for transient transfection (PEI, Polysciences), using FreeStyle ^TM 293 (Invitrogen) were expanded at 37 ℃. After 5-7 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant of human-containing IL-13 containing his-tag or a culture supernatant of a human-containing IL-13R130Q variant containing his-tag. The above culture supernatants were separately loaded onto a Ni-NTA affinity chromatography column (purchased from GE), and the his-tagged human IL-13 (i.e., immunogen A) and his-tagged human IL- in the supernatant were purified. The 13R130Q variant was further purified using a molecular sieve column (purchased from GE) to remove impurities such as macromolecular aggregates. Purified immunogen A and his-tagged human IL-13R130Q variant were stored in PBS phosphate buffer (pH 7.4), filtered through a 0.22 μm sterile filter, and stored at -80 °C.

将纯化的免疫原A进行TARC分泌试验。TARC分泌试验的具体方法参照Miller et al.2008,J Immunol Methods 334(1-2):134-41。The purified immunogen A was subjected to a TARC secretion test. Specific methods for TARC secretion assays are described in Miller et al. 2008, J Immunol Methods 334(1-2): 134-41.

将A549细胞(购自ATCC)培养在含有10％(w/w)胎牛血清F-12k培养基(购自Gibco)中，待其在T-175细胞培养瓶中扩大培养至75-90％汇合度时，弃去培养基，用PBS缓冲液洗涤1-2次，然后用胰蛋白酶溶液(Trypsin-EDTA，购自Life technology)消化并收集细胞。将收集到的细胞重悬与培养基中，计数后将细胞稀释至2×10 ⁶个细胞/mL，按照每孔细胞100μL加到96孔细胞培养板中(每孔2×10 ⁵个细胞)，将培养板置于37℃，5％(v/v)CO ₂培养箱培养过夜。第二日，将梯度稀释的免疫原A与重组人TNFα(购自Peprotech)混合得混合物Ⅰ(其中，TNFα占混合物Ⅰ的终浓度为200ng/mL)。将过夜培养的细胞培养板弃去培养上清，加入上述混合物Ⅰ，将细胞培养板放在37℃、5％(v/v)CO ₂培养箱培养过夜。20小时后，吸出板中的培养上清液，离心去除细胞，用TARC ELISA试剂盒(购自RnD systems)检定培养上清液中TARC的浓度。实验操作按照试剂盒说明书要求进行。 A549 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum F-12k medium (purchased from Gibco), and expanded to 75-90% in T-175 cell culture flasks. At the confluency, the medium was discarded, washed 1-2 times with PBS buffer, and then digested with trypsin-EDTA (purchased from Life Technology) and the cells were collected. The collected cells were resuspended in the medium, and after counting, the cells were diluted to 2 × 10 ⁶ cells/mL, and 100 μL of each well was added to a 96-well cell culture plate (2 × 10 ⁵ cells per well). The plates were incubated overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. On the second day, the gradient diluted immunogen A was mixed with recombinant human TNFα (purchased from Peprotech) to obtain a mixture I (wherein TNFα accounted for 200 ng/mL of the final concentration of the mixture I). The culture supernatant of the overnight cultured cell culture plate was discarded, and the above mixture I was added, and the cell culture plate was cultured overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. After 20 hours, the culture supernatant in the plate was aspirated, the cells were removed by centrifugation, and the concentration of TARC in the culture supernatant was determined using a TARC ELISA kit (purchased from RnD systems). The experimental procedure was carried out in accordance with the instructions of the kit.

具体实验简述如下：将鼠抗人TARC抗体用PBS稀释至2μg/mL，然后按照100μL每孔加到96孔酶标板。4℃孵育过夜；第二天用洗板液[含有0.05％(w/w)Tween20的PBS缓冲液]洗板2次后，加入样品稀释液[含有1％(w/w)BSA的PBS缓冲液]300μL每孔室温封闭1小时；弃去封闭液，将标准品用样品稀释液稀释至500pg/mL，再倍比稀释六个点，用样品稀释液作为空白对照，同时将培养上清液用样品稀释液稀释8倍。按照每孔100μL将标准品和样品加入酶标板，室温孵育2小时。用洗板液洗板2-3次。将生物素标记的羊抗人TARC抗体用样品稀释液稀释至终浓度0.5ng/mL，按照每孔100微升加入酶标板。室温孵育2小时；将辣根过氧化物酶标记(HRP)的链霉亲和素用样品稀释液稀释至体积比为1:200后按照每孔100μL加入酶标板，室温孵育30分钟后，用洗板液洗板2-3次。加入TMB底物(购自湖州英创)每孔100μL，室温孵育15分钟后，每孔加入50μL终止液(1.0N HCl)。用ELISA读板机(SpectraMax M5e，购自Molecular Device)读取OD _450nm值，并以OD _540nm值作为背景计算光吸值，计算出培养上清液中的TARC浓度。部分实验结果见图1和表5。表5说明，免疫原A可以刺激A549细胞分泌胞胸腺活化调节趋化因子(TARC)，且免疫原A所具有的生物学活性与商业化蛋白的生物学活性基本一致。 The specific experiment is briefly described as follows: The murine anti-human TARC antibody was diluted to 2 μg/mL with PBS, and then added to a 96-well microtiter plate according to 100 μL per well. Incubate overnight at 4 ° C; wash the plate 2 times with the washing solution [PBS buffer containing 0.05% (w/w) Tween 20], and add the sample dilution [PBS buffer containing 1% (w/w) BSA] Liquid] 300 μL per well at room temperature for 1 hour; discard the blocking solution, dilute the standard dilution to 500 pg/mL with the sample dilution, dilute the dilution by six points, use the sample dilution as a blank control, and simultaneously cultivate the supernatant Dilute 8 times with sample dilution. Standards and samples were added to the plate according to 100 μL per well and incubated for 2 hours at room temperature. Wash the plate 2-3 times with the washing solution. The biotin-labeled goat anti-human TARC antibody was diluted with a sample dilution to a final concentration of 0.5 ng/mL, and the microtiter plate was added at 100 microliters per well. Incubate for 2 hours at room temperature; dilute horseradish peroxidase-labeled (HRP) streptavidin with a sample dilution to a volume ratio of 1:200, add 100 μL per well to the plate, and incubate for 30 minutes at room temperature. Wash the plate 2-3 times with the washing solution. After adding 100 μL of TMB substrate (purchased from Huzhou Yingchuang) per well for 15 minutes at room temperature, 50 μL of stop solution (1.0 N HCl) was added to each well. The OD _{450 nm} value was read by an ELISA plate reader (SpectraMax M5e, available from Molecular Device), and the light absorption value was calculated using the OD _{540 nm} value as a background, and the TARC concentration in the culture supernatant was calculated. Some experimental results are shown in Figures 1 and 5. Table 5 shows that immunogen A can stimulate the secretion of thymocyte activation chemokine (TARC) by A549 cells, and the biological activity of immunogen A is basically consistent with the biological activity of commercial proteins.

表5 TARC分泌试验检验IL-13蛋白的生物学活性Table 5 TARC secretion test to test the biological activity of IL-13 protein

其中，IL-13(Sino生物)指商业化的IL-13蛋白，购自Sino biological，作为阳性对照；IL-13(批次1)即为上述免疫原A。Among them, IL-13 (Sino Bio) refers to the commercial IL-13 protein, which is purchased from Sino biological as a positive control; IL-13 (Batch 1) is the above-mentioned immunogen A.

2.免疫原A的生物素标记2. Biotin labeling of immunogen A

将Biotin-X-X-NHS(购自SIGMA-ALDRICH)和实施例1制备的纯化的免疫原A按摩尔比3:1的比例混合，室温下振荡反应30分钟后，加入终浓度为50mM的NH ₄Cl终止反应，得生物素标记的免疫原A。然后将生物素标记的免疫原A透析到PBS缓冲液(pH7.4)中，用Broadford试剂(购自Pierce)，以BSA作为标准品测定浓度(具体操作方法参见Bradford，1976，Anal Biochem 72:248-54)。实验结果见表6。利用标准品拟合曲线，生物素标记的免疫原A测定的OD _595nm值为0.49。经计算，生物素标记的免疫原A的浓度为0.319mg/mL。 Biotin-XX-NHS (purchased from SIGMA-ALDRICH) and the purified immunogen A prepared in Example 1 were mixed in a ratio of 3:1, and the reaction was shaken at room temperature for 30 minutes, and then a final concentration of 50 mM of NH _{4 was added.} The reaction was stopped by Cl to obtain biotin-labeled immunogen A. The biotin-labeled immunogen A was then dialyzed into PBS buffer (pH 7.4), and the concentration was determined using Broadford reagent (purchased from Pierce) using BSA as a standard (for details, see Bradford, 1976, Anal Biochem 72: 248-54). The experimental results are shown in Table 6. The OD ₅₉₅ nm value determined by biotin-labeled immunogen A was 0.49 using a standard fit curve. The concentration of biotin-labeled immunogen A was calculated to be 0.319 mg/mL.

表6 生物素标记的免疫原A的浓度的测定Table 6 Determination of the concentration of biotinylated immunogen A

将生物素标记的免疫原A用0.22μm无菌滤膜过滤后无菌分装，再置于-80℃保存。The biotin-labeled immunogen A was filtered through a 0.22 μm sterile filter, aseptically dispensed, and stored at -80 °C.

实施例2：利用杂交瘤技术获得先导抗体Example 2: Obtaining a lead antibody using hybridoma technology

1.小鼠免疫Mouse immunity

使用免疫原A免疫。采用的是6-8周龄的Balb/c，SJL/J小鼠(上海斯莱克提供)。小鼠接收后在Specific pathogen Free(SPF)条件下饲养。初次免疫剂量为每只小鼠50μg免疫原A。将蛋白用弗氏完全佐剂乳化后尾部皮下注射0.25mL。初次免疫2周后，加强免疫。免疫原A(每只小鼠25μg)用弗氏不完全佐剂乳化后腹腔注射0.25mL。以后每次加强免疫间隔3周。每次加强免疫一周后采集血清样品，用ELISA检测血清中抗体效价并用受体配体结合阻滞实验检测血清中抗体的活性。血清效价较高且可以更好阻滞免疫原A与受体结合的小鼠将优先选择用于细胞融合和杂交瘤细胞制备，剩余小鼠继续加强免疫备用。部分实验结果见图2和表7所示。表7说明，经免疫原A免疫的小鼠的血清对免疫原A均有不同程度的结合，呈现抗原抗体反应。其中，血清的最高稀释度(即稀释倍数)在十万左右。表7中空白对照指1％(w/w)BSA，批次TB2指第二次加强免疫后第七天的小鼠血清，表中的数据为OD _450nm值。 Immunization with immunogen A. Balb/c, SJL/J mice (supplied by Shanghai Slack), 6-8 weeks old, were used. Mice were housed under Specific pathogen Free (SPF) conditions after receipt. The initial immunization dose was 50 μg of immunogen A per mouse. The protein was emulsified with Freund's complete adjuvant and 0.25 mL was injected subcutaneously into the tail. After 2 weeks of initial immunization, boost the immunization. Immunogen A (25 μg per mouse) was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 mL. Each time the booster interval is 3 weeks. Serum samples were collected one week after each boost, and antibody titers in serum were measured by ELISA and antibody activity in serum was measured by receptor ligand binding block assay. Mice with higher serum titers and better blocking of binding of immunogen A to the receptor will be preferred for cell fusion and hybridoma cell preparation, and the remaining mice will continue to boost the immunization reserve. Some experimental results are shown in Figure 2 and Table 7. Table 7 shows that the sera of mice immunized with immunogen A have different degrees of binding to immunogen A, exhibiting an antigen-antibody reaction. Among them, the highest dilution of serum (ie, dilution factor) is about 100,000. The blank control in Table 7 refers to 1% (w/w) BSA, and the batch TB2 refers to the serum of the mice on the seventh day after the second boost, and the data in the table is the OD _{450 nm} value.

表7 ELISA检测免疫原A免疫后小鼠血清抗体效价Table 7 ELISA detection of serum antibody titer in mice after immunization with immunized A

使用基因免疫。将实施例1中构建的表达重组人IL-13蛋白的PCP表达载体包被到1.0μm金胶体子弹上，用Helios基因枪(Bio-rad)免疫。金胶体子弹制备和免疫程序根据Helios基因枪说明书进行制定。6-8周龄雌性SJL/J小鼠(上海斯莱克提供)接收后在SPF条件下饲养。所有小鼠经腹部用基因枪免疫3-4次，每次3-4枪，每枪1.0μg质粒。初次免疫与第一次加强免疫之间隔2周，以后每次加强免疫间隔3周。每次加强免疫7天后采集血清样品，用ELISA检测血清中抗体效价并用受体配体结合阻滞实验检测血清中抗体的活性。血清效价较高且可以更好阻滞重组人IL-13蛋白与受体结合的小鼠将优先选择用于细胞融合和杂交瘤细胞制备，剩余小鼠继续加强免疫备用。Use genetic immunization. The PCP expression vector expressing the recombinant human IL-13 protein constructed in Example 1 was coated onto a 1.0 μm gold colloidal bullet and immunized with a Helios gene gun (Bio-rad). Gold colloidal bullet preparation and immunization procedures were developed according to the Helios gene gun instructions. Female SJL/J mice (supplied by Shanghai Slack), 6-8 weeks old, were housed under SPF conditions after receiving. All mice were immunized 3-4 times with a gene gun through the abdomen, 3-4 shots each time, 1.0 μg of plasmid per shot. The interval between the initial immunization and the first booster immunization was 2 weeks, and each booster interval was 3 weeks later. Serum samples were collected 7 days after booster immunization, antibody titers in serum were detected by ELISA and antibody activity in serum was measured by receptor ligand binding block assay. Mice with higher serum titers and better blocking of binding of recombinant human IL-13 protein to the receptor will be preferred for cell fusion and hybridoma cell preparation, and the remaining mice will continue to boost the immunization reserve.

2.杂交瘤细胞的制备和先导抗体筛选2. Preparation of hybridoma cells and screening of lead antibodies

通常大部分使用免疫原A免疫的小鼠经2-3次免疫后效价可达到1:1000以上，即可用于收集淋巴细胞进行细胞融合和杂交瘤制备。Usually, most mice immunized with immunogen A can achieve a titer of 1:1 or more after 2-3 immunizations, and can be used to collect lymphocytes for cell fusion and hybridoma preparation.

细胞融合前，小鼠最后一次免疫用每只50-100μg免疫原A腹腔注射。3-5天后处死小鼠，收集脾细胞。加入NH ₄OH至终浓度1％(w/w)以裂解脾细胞悬液中的红细胞，用DMEM基础培养基离心清洗细胞2-3次，然后按细胞数5:1的比率与小鼠骨髓瘤细胞SP2/0混合(购自ATCC)，采用传统的PEG细胞融合方法或高效电融合方法进行细胞融合(参见METHODS IN ENZYMOLOGY,VOL.220)，得融合后的细胞，即为杂交瘤细胞。 Prior to cell fusion, the mice were immunized with 50-100 μg of immunogen A per patient for the last immunization. Mice were sacrificed 3-5 days later and spleen cells were collected. Add NH ₄ OH to a final concentration of 1% (w/w) to lyse red blood cells in spleen cell suspension, wash the cells 2-3 times with DMEM basal medium, and then mix the mouse bone marrow at a ratio of 5:1. Tumor cells SP2/0 mixed (purchased from ATCC), cell fusion using traditional PEG cell fusion method or high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY, VOL.220), the obtained cells are hybridoma cells.

将融合后的细胞稀释到含20％(w/w)胎牛血清、1×HAT的DMEM选择性培养基中，按1×10 ⁵/20μL每孔加入到96孔细胞培养板中，放入5％(v/v)CO ₂、37℃培养箱中。10-14天后用ELISA筛选细胞融合板上清，将ELISA中OD _450nm>1.0的阳性克隆扩增到24孔板扩大培养，2-3天后对24孔板上清复检，包括利用ELISA检测上清中的抗体对免疫原A的结合活性、利用流式细胞技术检测上清中的抗体阻滞人IL-13与受体结合活性以及利用A549胸腺活化调节趋化因子(TARC)分泌试验检验上清中的抗体中和免疫原A的生物学活性。挑选ELISA实验中OD _450nm>1.0、受体配体结合阻滞实验中杂交瘤细胞培养上清对人IL-13与hIL-13Ra/hIL-4R异源二聚体和/或人IL-13与hIL-13Ra2受体结合的阻滞率达到60％，和/或A549胸腺活化调节趋化因子(TARC)分泌试验中杂交瘤上清中和人IL-13诱导TARC分泌，抑制率达到60％的杂交瘤细胞为符合条件的阳性克隆。 The fused cells were diluted into DMEM selective medium containing 20% (w/w) fetal bovine serum and 1×HAT, and added to a 96-well cell culture plate at 1×10 ⁵ /20 μL per well, and placed. 5% (v/v) CO ₂ in a 37 ° C incubator. After 10-14 days, the cell fusion plate was screened by ELISA, and the positive clone with OD _450nm >1.0 in the ELISA was amplified into 24-well plate to expand the culture. After 2-3 days, the 24-well plate was retested, including detection by ELISA. The binding activity of the antibodies in Qingqing to immunogen A, the detection of antibody blocking IL-13 and receptor binding activity by supernatant using flow cytometry, and the use of A549 thymic activation-regulating chemokine (TARC) secretion test The antibodies in the serum neutralize the biological activity of immunogen A. Selection of hybridoma cell culture supernatants in ELISA experiments with OD _450nm >1.0, receptor ligand binding block assay for human IL-13 and hIL-13Ra/hIL-4R heterodimer and/or human IL-13 The blockade of hIL-13Ra2 receptor binding reached 60%, and/or the A549 thymic activation-regulating chemokine (TARC) secretion test in which the hybridoma supernatant and human IL-13 induced TARC secretion, the inhibition rate reached 60%. Hybridoma cells are eligible positive clones.

根据24孔板样品的筛选结果，选择符合条件的阳性克隆用有限稀释法在96孔板进行亚克隆，即在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃，5％(v/v)CO ₂的条件下培养上述阳性克隆。亚克隆后7-10天用ELISA进行初步筛选，挑选3-4个阳性单克隆扩增到24孔板继续培养。2-3天后对上清按照亚克隆之间的检验方法进行复检，包括ELISA，受体配体结合阻滞实验，胸腺活化调节趋化因子分泌试验。根据24孔板样品检测结果，挑选最优亚克隆进行扩大培养、液氮冻存、抗体生产和纯化，作为先导抗体。这些先导抗体的克隆号分别为29D9H8、28A2E11和35E2C3。 According to the screening results of 24-well plate samples, selected positive clones were subcloned in a 96-well plate by limiting dilution, ie, in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 °C. The above positive clones were cultured under conditions of 5% (v/v) CO ₂ . Primary screening was performed by ELISA 7-10 days after subcloning, and 3-4 positive monoclonal clones were selected and expanded into 24-well plates for further culture. After 2-3 days, the supernatant was retested according to the test method between subclones, including ELISA, receptor ligand binding inhibition assay, and thymic activation regulating chemokine secretion assay. According to the results of 24-well plate sample detection, the optimal subclones were selected for expansion culture, liquid nitrogen cryopreservation, antibody production and purification as lead antibodies. The clone numbers of these lead antibodies were 29D9H8, 28A2E11 and 35E2C3, respectively.

实施例3：小鼠杂交瘤细胞单克隆抗体的生产和纯化Example 3: Production and purification of monoclonal antibodies against mouse hybridoma cells

杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/mL，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5mg)抗体生产纯化。Hybridoma cells produce a lower concentration of antibodies, which are only about 1-10 μg/mL, and the concentration varies greatly. Moreover, the various proteins produced by the cell culture in the medium and the fetal bovine serum components contained in the medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1-5 mg) antibody production purification is required.

将实施例2制备的杂交瘤细胞扩增到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen)驯化传代2-3代。待杂交瘤细胞生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入200-500mL生产培养基，接种细胞密度为0.5-1.0×10 ⁵个/mL。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，调到转速3转/分钟。连续旋转培养10-14天后，收集细胞培养液，离心或过滤去除细胞，并用0.22-0.45μm的滤膜过滤。处理后的细胞培养上清可马上进行纯化或-30℃冻存。 The hybridoma cells prepared in Example 2 were expanded into T-75 cell culture flasks and domesticated for 2-3 passages using a production medium (Hybridoma serum free medium, purchased from Invitrogen). The hybridoma cells are in good growth state, and the cell culture spinner is inoculated. 200-500 mL of production medium was added to each 2 liter culture spinner flask, and the seed density was 0.5-1.0×10 ⁵ /mL. Cap the cap tightly and place the spinner on a roller shaker in a 37 ° C incubator and adjust to a speed of 3 rpm. After 10-14 days of continuous spin culture, the cell culture medium was collected, centrifuged or filtered to remove the cells, and filtered through a 0.22-0.45 μm filter. The treated cell culture supernatant can be immediately purified or frozen at -30 °C.

杂交瘤细胞培养上清中的单克隆抗体可用蛋白G亲和层析(Protein G，蛋白G柱)柱进行纯化。根据样品量的大小，准备相应的体积的层析柱。对于200-300mL的小体积纯化，需要1-2mL的蛋白G柱。蛋白G柱先用平衡缓冲液(PBS缓冲液，pH7.4)平衡，然后将培养上清上样至蛋白G柱，控制流速在3-4mL/分钟。上样完毕，用平衡缓冲液清洗层析柱3-5柱床体积。用洗脱液(0.1M甘氨盐酸缓冲液，pH2.5)洗脱结合在柱上的抗体，用紫外检测器监测洗脱情况。收集洗脱的抗体(根据A280紫外吸收峰)，加入10％(v/v)的1.0M Tris-HCl缓冲液中和pH，然后立即用PBS缓冲液透析过夜，第二天换液一次，并继续透析2-3小时。收集透析后的抗体，用0.22μm的滤器进行无菌过滤，即得IL-13单克隆抗体，将其无菌保存。分装小样进行蛋白浓度、纯度、内毒等检测分析。检测发现，IL-13单克隆抗体的内毒素浓度小于3.0EU/mg。它们的部分检测分析结果参见表8。The monoclonal antibody in the hybridoma cell culture supernatant can be purified by Protein G affinity chromatography (Protein G, Protein G column) column. According to the size of the sample, prepare a corresponding volume of the column. For small volume purification of 200-300 mL, 1-2 mL of Protein G column is required. The Protein G column was first equilibrated with equilibration buffer (PBS buffer, pH 7.4) and the culture supernatant was applied to a Protein G column with a flow rate of 3-4 mL/min. After loading, wash the column with 3-5 column volumes with equilibration buffer. The antibody bound to the column was eluted with an eluent (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution was monitored with a UV detector. Collect the eluted antibody (according to the A280 UV absorption peak), add 10% (v/v) 1.0 M Tris-HCl buffer and neutralize the pH, then immediately dialyze overnight with PBS buffer, and change the solution once a day, and Continue dialysis for 2-3 hours. The dialyzed antibody was collected and sterile-filtered using a 0.22 μm filter to obtain an IL-13 monoclonal antibody, which was aseptically stored. Packing samples for protein concentration, purity, and endotoxin detection and analysis. The endotoxin concentration of the IL-13 monoclonal antibody was found to be less than 3.0 EU/mg. See Table 8 for the results of their partial detection analysis.

表8 抗体检测分析Table 8 Antibody detection and analysis

实施例4：先导抗体的检定Example 4: Assay of lead antibody

将实施例3获得的先导抗体分别进行如下的检定试验。The lead antibodies obtained in Example 3 were each subjected to the following assay.

A、酶联免疫吸附实验(ELISA)检测先导抗体与免疫原A、IL-13R130Q变体、猴IL-13以及鼠IL-13的结合A. Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of lead antibody to immunogen A, IL-13R130Q variant, monkey IL-13 and murine IL-13.

将链霉亲和素用PBS稀释到终浓度1.0μg/mL，然后按照100微升每孔加到96孔酶标板。4℃孵育过夜；第二天用洗板液[含有0.05％(w/w)Tween20的PBS缓冲液]洗板2次，加入封闭液[含有0.05％(w/w)Tween20和2％(w/w)BSA的PBS缓冲液]37℃封闭1-2小时。弃去封闭液，将实施例1制备的生物素标记的免疫原A、IL-13R130Q变体(实施例1制备)、猴IL-13(购自sino biological)和鼠IL-13(购自sino biological)分别用样品稀释液[含有0.05％(w/w)Tween20和0.2％(w/w)BSA的PBS缓冲液]稀释至 0.5μg/mL，按照每孔50-100μL加入酶标板，37℃孵育1小时。用洗板液[含有0.01％(w/w)Tween20的PBS缓冲液]洗板2-3次。分别加入梯度稀释的实施例3制备的先导抗体每孔50-100μL，37℃孵育1小时后，用洗板液洗板2-3次。加入辣根过氧化物酶标记(HRP)的人或小鼠IgG二抗(购自Sigma)，37℃孵育1小时后，再用洗板液[含有0.05％(w/w)Tween20的PBS缓冲液]洗板2-3次。加入100μL/孔TMB底物，室温孵育15分钟后，每孔加入50μL 1.0N HCl终止。用ELISA读板机(SpectraMax M5e，购自Molecular Device)读取OD _450nm值。部分实验结果见图3～6和表9～11所示。表9～11说明，先导抗体与重组人IL-13蛋白，重组人IL-13R130Q变体，重组猴IL-13在ELISA水平结合。但与小鼠IL-13没有结合。其中IgG对照为鼠IgG，表中的数据为OD _450nm值。 Streptavidin was diluted with PBS to a final concentration of 1.0 μg/mL, and then added to a 96-well microtiter plate according to 100 μl per well. Incubate overnight at 4 ° C; wash the plate 2 times with a washing solution [PBS containing 0.05% (w/w) Tween 20], add blocking solution [containing 0.05% (w/w) Tween 20 and 2% (w /w) BSA in PBS buffer] blocked at 37 ° C for 1-2 hours. The blocking solution was discarded, and the biotin-labeled immunogen A, IL-13R130Q variant (prepared in Example 1), monkey IL-13 (purchased from sino biological) and murine IL-13 (purchased from sino) prepared in Example 1 were discarded. Biological) Dilute to 0.5 μg/mL with sample diluent [PBS buffer containing 0.05% (w/w) Tween20 and 0.2% (w/w) BSA, respectively, add 50-100 μL per well to the plate, 37 Incubate for 1 hour at °C. The plate was washed 2-3 times with a washing solution [PBS buffer containing 0.01% (w/w) Tween20]. The gradient-diluted leader antibody prepared in Example 3 was added to 50-100 μL per well, and after incubation at 37 ° C for 1 hour, the plate was washed 2-3 times with a washing solution. Horseradish peroxidase-labeled (HRP) human or mouse IgG secondary antibody (purchased from Sigma) was added, and after incubation at 37 ° C for 1 hour, the plate was washed with PBS buffer containing 0.05% (w/w) Tween20. Liquid] Wash the plate 2-3 times. 100 μL/well of TMB substrate was added and incubated for 15 minutes at room temperature, and 50 μL of 1.0 N HCl was added to each well to terminate. The OD _{450 nm} value was read using an ELISA plate reader (SpectraMax M5e, available from Molecular Device). Some experimental results are shown in Figures 3-6 and Tables 9-11. Tables 9-11 illustrate that the leader antibody binds to recombinant human IL-13 protein, recombinant human IL-13R130Q variant, and recombinant monkey IL-13 at the ELISA level. However, it did not bind to mouse IL-13. The IgG control was murine IgG and the data in the table is the OD _{450 nm} value.

表9 ELISA检测先导抗体与免疫原A的反应活性Table 9 ELISA detection of lead antibody and immunogen A reactivity

表10 ELISA检测先导抗体与猴IL-13变体的反应活性Table 10 ELISA detection of the reactivity of lead antibody with monkey IL-13 variant

表11 ELISA检测先导抗体与鼠IL-13变体的反应活性Table 11 ELISA detection of the reactivity of the lead antibody with murine IL-13 variant

B、受体配体结合阻滞实验B, receptor ligand binding block experiment

1.稳定表达细胞株的构建：1. Construction of stable expression cell lines:

将人IL-13Ra1、人IL-13Ra2和人IL-4Ra全长基因的核苷酸序列(如序列表SEQ ID No.123～125所示)分别克隆到pIRES表达载体上，并包装成慢病毒(pIRES表达载体和慢病毒均购自上海吉玛制药技术有限公司，并按照说明书进行操作)，对HEK293细胞分别进行慢病毒感染。人IL-13Ra1和人IL-4Ra基因被共同转染至同一HEK293细胞株，以在其表面形成IL-13Ra1/IL-4Ra异原二聚体，感染后将细胞在含100μg/mL Hygromycin B(购自MILLIPORE)和0.25μg/mL Puromycin(购自Invitrogen)中的一种或两种和10％(w/w)胎牛血清的DMEM培养基中，37℃、5％(v/v)CO ₂下选择培养2周。2周后用有限稀释法将感染后的细胞亚克隆到96孔培养板中。待克隆长大后，将单克隆孔细胞扩增到6孔板中或培养瓶中，得过表达全长人IL-13Ra1和人IL-4Ra的HEK293细胞株。对扩增后的克隆用抗每种受体对应的特异性抗体(hIL-13Ra1抗体、hIL-4Ra抗体、hIL-13Ra2抗体均购自RnD systems)经流式细胞分析法检测受体表达水平，及与配体IL-13蛋白的结合能力。选择长势较好、表达水平较高、结合强的单克隆的细胞系继续扩大培养并液氮冻存。用包含人IL-13Ra2基因的慢病毒颗粒感染HEK293细胞，按照同样的方法筛选选择长势较好、表达水平较高、结合强的单克隆的细胞系扩大培养并于液氮中保存，得过表达全长人IL-13Ra2的HEK293细胞株。部分实验结果见图7～9和表12～14。表12～14的结果说明，HEK293细胞克隆4C1表面同时表达hIL-13Ra1和hIL-4Ra两个受体，HEK293细胞克隆1A1表达hIL-13Ra2受体，且细胞表面受体表达量均较高，可以用作后续试验使用。 The nucleotide sequences of human IL-13Ra1, human IL-13Ra2 and human IL-4Ra full-length genes (shown in SEQ ID No. 123-125 of the Sequence Listing) were cloned into pIRES expression vector and packaged into lentiviruses. (The pIRES expression vector and lentivirus were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. and operated according to the instructions), and the HEK293 cells were each subjected to lentivirus infection. The human IL-13Ra1 and human IL-4Ra genes were co-transfected into the same HEK293 cell line to form IL-13Ra1/IL-4Ra heterodimers on the surface, and the cells were infected with 100 μg/mL Hygromycin B ( 37 mg, 5% (v/v) CO in DMEM medium purchased from MILLIPORE) and 0.25 μg/mL Puromycin (purchased from Invitrogen) in one or two and 10% (w/w) fetal bovine serum. ₂ cultures were selected for 2 weeks. After 2 weeks, the infected cells were subcloned into 96-well culture plates by limiting dilution. After the clones were grown, the monoclonal well cells were expanded into 6-well plates or culture flasks to obtain HEK293 cell lines expressing full-length human IL-13Ra1 and human IL-4Ra. The amplified clones were assayed for receptor expression levels by flow cytometry using specific antibodies against each receptor (hIL-13Ra1 antibody, hIL-4Ra antibody, hIL-13Ra2 antibody purchased from RnD systems). And the ability to bind to the ligand IL-13 protein. Cell lines with better growth, higher expression levels, and stronger binding were selected to continue to expand culture and cryopreservation of liquid nitrogen. HEK293 cells were infected with lentiviral particles containing human IL-13Ra2 gene, and the cell lines with good growth, high expression level and strong binding were selected and expanded in the same manner and stored in liquid nitrogen to obtain overexpression. HEK293 cell line of full length human IL-13Ra2. Some experimental results are shown in Figures 7-9 and Tables 12-14. The results of Tables 12 to 14 indicate that HEK293 cell clone 4C1 surface expresses both hIL-13Ra1 and hIL-4Ra receptors, HEK293 cell clone 1A1 expresses hIL-13Ra2 receptor, and cell surface receptor expression is high, Used as a follow-up test.

表12流式细胞分析方法检测过表达全长人IL-13Ra1的HEK293细胞株中hIL-13Ra1蛋白的表达水平Table 12 Flow cytometry analysis for expression of hIL-13Ra1 protein in HEK293 cell line overexpressing full-length human IL-13Ra1

表13流式细胞分析方法检测过表达全长人IL-4Ra的HEK293细胞株中hIL-4Ra蛋白的表达水平Table 13 Flow cytometric analysis of hIL-4Ra protein expression in HEK293 cell lines overexpressing full-length human IL-4Ra

表14流式细胞分析方法检测过表达全长人IL-13Ra2的HEK293细胞株中hIL-13Ra2蛋白的表达水平Table 14 Flow cytometric analysis of hIL-13Ra2 protein expression in HEK293 cell lines overexpressing full-length human IL-13Ra2

2.利用流式细胞技术的受体配体结合阻滞实验2. Receptor ligand binding block experiment using flow cytometry

将实施例4实验B中步骤1.所得的过表达全长人IL-13Ra1和人IL-4Ra的HEK293细胞株克隆4C1和过表达全长人IL-13Ra2的HEK293细胞株克隆1A1在T-175细胞培养瓶中扩大培养至75-90％汇合度弃去培养基。其中，扩大培养的培养基为含100μg/mL Hygromycin B(购自MILLIPORE)和0.25μg/mL Puromycin(购自Invitrogen)中的一种或两种和10％(w/w)胎牛血清的DMEM培养基。扩大培养的条件为37℃、5％(v/v)CO ₂下培养。用PBS缓冲液洗涤1-2次，然后用重组酶细胞解离液(TrypLE，购自Life technology)消化并收集细胞；用PBS缓冲液洗涤细胞1-2次，进行细胞计数后将细胞用封闭液[含有2％(w/w)胎牛血清的PBS缓冲液]稀释至1-2×10 ⁶个细胞/mL，冰上孵育20-30分钟，然后用封闭液[含有2％(w/w)胎牛血清的PBS缓冲液]洗涤2次。将收集的细胞用封闭液悬浮至1×10 ⁶个细胞/mL，按每孔100μL加入到96孔FACS反应板中(即每孔1×10 ⁵个细胞)。 HEK293 cell line clone 4C1 overexpressing full-length human IL-13Ra1 and human IL-4Ra obtained in step 1 of Example 4 in Experiment B, and HEK293 cell line clone 1A1 overexpressing full-length human IL-13Ra2 in T-175 Incubate the culture in a cell culture flask to 75-90% confluence and discard the medium. Among them, the culture medium to be expanded is DMEM containing 100 μg/mL Hygromycin B (purchased from MILLIPORE) and 0.25 μg/mL Puromycin (purchased from Invitrogen), one or two, and 10% (w/w) fetal bovine serum. Medium. The conditions for the expanded culture were cultured at 37 ° C and 5% (v/v) CO ₂ . Wash 1-2 times with PBS buffer, then digest and collect cells with recombinant enzyme cell dissociation solution (TrypLE, purchased from Life technology); wash cells with PBS buffer 1-2 times, perform cell counting and block cells Dilute [PBS containing 2% (w/w) fetal bovine serum] to 1-2×10 ⁶ cells/mL, incubate on ice for 20-30 minutes, then use blocking solution [containing 2% (w/) w) fetal bovine serum in PBS buffer] washed twice. The collected cells were suspended in a blocking solution to 1 × 10 ⁶ cells/mL, and added to a 96-well FACS reaction plate (i.e., 1 × 10 ⁵ cells per well) at 100 μL per well.

将梯度稀释的实施例3制备的先导抗体(以下统称“先导抗体”)与实施例1制备的生物素标记的免疫原A混合后，按照每孔100μL加入细胞中，冰上孵育1-2小时。其中，对实施例4试验B步骤1.所得的过表达全长人IL-13Ra1和人IL-4Ra的HEK293细胞株，加入梯度稀释的先导抗体和终浓度为30ng/mL的生物素标记的免疫原A；对过表达全长人IL-13Ra2的HEK293细胞株，加入梯度稀释的先导抗体和终浓度为20ng/mL的生物素标记的免疫原A。用封闭液离心洗涤2次，加入每孔100μL荧光(Alexa 488)标记的链霉亲和素(购自Life technology，货号S11223)，冰上孵育0.5-1.0小时。用封闭液离心洗涤2-3次，加入每孔100μL PBS缓冲液悬浮细胞，用FACS(FACS Verse，购自BD)检测和分析结果。部分实验结果见图10～11和表15～16，表15～16说明，IL-13抗体与人IL-13结合后，能阻滞人IL-13与细胞表面的受体人IL-13Ra1/hIL-4Ra异源二聚体的结合，或者能阻滞IL-13与细胞表面人IL-13Ra2受体的结合。其中IgG对照为鼠IgG，表格中数据为平均荧光强度。The gradient-diluted leader antibody prepared in Example 3 (hereinafter collectively referred to as "lead antibody") was mixed with the biotin-labeled immunogen A prepared in Example 1, and then added to the cells in 100 μL per well, and incubated on ice for 1-2 hours. . Among them, the HEK293 cell line overexpressing the full-length human IL-13Ra1 and human IL-4Ra obtained in the step B of Example 4, Test B, was added with a gradient dilution of the lead antibody and a biotin-labeled immunization with a final concentration of 30 ng/mL. Pro-A; For HEK293 cell lines overexpressing full-length human IL-13Ra2, a gradient-diluted leader antibody and biotin-labeled immunogen A at a final concentration of 20 ng/mL were added. The mixture was washed twice with a blocking solution, and 100 μL of fluorescent (Alexa 488)-labeled streptavidin (purchased from Life Technology, Cat. No. S11223) per well was added and incubated on ice for 0.5-1.0 hours. The cells were washed 2-3 times with a blocking solution, and the cells were suspended by adding 100 μL of PBS buffer per well, and the results were detected and analyzed by FACS (FACS Verse, available from BD). Some experimental results are shown in Figures 10-11 and Tables 15-16. Tables 15-16 show that IL-13 antibody binds to human IL-13 and blocks human IL-13 and cell surface receptor IL-13Ra1/ Binding of hIL-4Ra heterodimer, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor. The IgG control was murine IgG and the data in the table is the mean fluorescence intensity.

表15 FACS检测检测先导抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 15 FACS detection detection of lead antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表16 FACS检测检测先导抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 16 FACS detection of lead antibody blocking IL-13 binding to cell surface receptor IL-13Ra2

C、胸腺活化调节趋化因子(TARC)分泌试验C, thymic activation regulating chemokine (TARC) secretion test

将A549细胞(购自ATCC)培养在含有10％(w/w)胎牛血清F-12k培养基(购自Gibco)中于37℃、5％(v/v)CO ₂培养，待其在T-175细胞培养瓶中扩大培养至75-90％汇合度时，弃去培养基，用PBS缓冲液洗涤1-2次，然后用胰蛋白酶溶液(Trypsin-EDTA，购自Life technology)消化并收集细胞。将收集到的细胞重悬于培养基中，计数后将细胞稀释至2×10 ⁶个细胞/mL，按照每孔细胞100μL加到96孔细胞培养板中(每孔2×10 ⁵个细胞)，将培养板置于37℃、5％(v/v)CO ₂培养箱培养过夜。第二日，将梯度稀释的实施例3制备的先导抗体与实施例1制备的免疫原A混合得混合物A；将重组人TNFα(购自Peprotech)用含有10％(w/w)胎牛血清F-12k培养基等体积混合得混合物B于培养基中。其中，混合后的重组人TNFα在混合物B的终浓度为200ng/mL。弃去培养过夜的细胞培养板的培养上清后，向该细胞培养板中按照1:1体积比例加入上述混合物A和混合物B。其中，免疫原A占混合物A与混合物B的总体积的终浓度为5ng/mL，重组人TNFα占混合物A和混合物B的总体积的终浓度为200ng/mL。将细胞培养板放在37℃、5％(v/v)CO ₂培养箱培养过夜。20小时后，吸出板中的培养上清液，离心去除细胞，用TARC ELISA试剂盒(购自RnD systems)检定培养上清液中TARC的浓度。实验操作按照试剂盒说明书要求进行(详见实施例1)。部分实验结果见图12和表17。表17说明，先导抗体通过与人IL-13的结合，能够中和由IL-13和TNFα共同刺激条件下诱导产生的A549细胞分泌TARC。表17的数据为培养上清中TARC的浓度(pg/ml)，其中IgG对照为鼠IgG。 A549 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum F-12k medium (purchased from Gibco) at 37 ° C, 5% (v/v) CO _{2 , and} cultured at When the culture was expanded to 75-90% confluence in T-175 cell culture flask, the medium was discarded, washed 1-2 times with PBS buffer, and then digested with trypsin solution (Trypsin-EDTA, purchased from Life technology). Collect cells. The collected cells were resuspended in the medium, and after counting, the cells were diluted to 2 × 10 ⁶ cells/mL, and 100 μL of each well was added to a 96-well cell culture plate (2 × 10 ⁵ cells per well). The plates were incubated overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. On the second day, the gradient-diluted lead antibody prepared in Example 3 was mixed with the immunogen A prepared in Example 1 to obtain a mixture A; recombinant human TNFα (purchased from Peprotech) was used to contain 10% (w/w) fetal bovine serum. The F-12k medium was mixed in an equal volume to obtain a mixture B in the medium. Among them, the final concentration of the mixed recombinant human TNFα in the mixture B was 200 ng/mL. After discarding the culture supernatant of the overnight cultured cell culture plate, the above mixture A and mixture B were added to the cell culture plate in a 1:1 volume ratio. Here, the final concentration of the immunogen A in the total volume of the mixture A and the mixture B was 5 ng/mL, and the final concentration of the recombinant human TNFα in the total volume of the mixture A and the mixture B was 200 ng/mL. The cell culture plates were incubated overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. After 20 hours, the culture supernatant in the plate was aspirated, the cells were removed by centrifugation, and the concentration of TARC in the culture supernatant was determined using a TARC ELISA kit (purchased from RnD systems). The experimental procedure was carried out in accordance with the instructions of the kit (see Example 1 for details). Some experimental results are shown in Figure 12 and Table 17. Table 17 shows that the leader antibody can neutralize the secretion of TARC by A549 cells induced by IL-13 and TNFα co-stimulation by binding to human IL-13. The data in Table 17 is the concentration (pg/ml) of TARC in the culture supernatant, wherein the IgG control is murine IgG.

表17 先导抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 17 Leading antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

实施例5：轻重链可变区氨基酸序列测定Example 5: Determination of amino acid sequence of light and heavy chain variable region

总RNA分离：将实施例3制得的先导抗体所对应的杂交瘤细胞复苏，培养，离心收集1-5×10 ⁷个细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H ₂O溶解(55℃水浴促溶10分钟)，即得总RNA。 Total RNA isolation: The hybridoma cells corresponding to the lead antibody prepared in Example 3 were resuscitated, cultured, and 1-5×10 ⁷ cells were collected by centrifugation, mixed with 1 mL of Trizol and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature. Set for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed. After standing at room temperature for 10 minutes, it was centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was discarded. Add 1 mL of 75% (v/v) ethanol, gently wash the precipitate, centrifuge at 12000g for 5 minutes at 4 ° C, discard the supernatant, and dry the precipitate. Add DEPC-treated H ₂ O to dissolve (55 ° C water bath to dissolve 10 Minutes), that is, total RNA.

逆转录与PCR：取1μg总RNA，配置20μL体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物(其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492)。Reverse transcription and PCR: 1 μg of total RNA was taken, 20 μL of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated by reaction at 7 ° C for 10 minutes. A 50 μL PCR system was configured, including 1 μL of cDNA, 25 pmol of each primer, 1 μL of DNA polymerase, and a matching buffer system, 250 μmol dNTPs. The PCR program was set up, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 72 ° C for 35 seconds, and 35 cycles and then extended at 72 ° C for 5 minutes to obtain a PCR product (for reverse transcription). The kit was PrimeScript RT Master Mix, available from Takara, item number RR036; the kit used for PCR was Q5 super-fidelity enzyme, available from NEB, item number M0492).

克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化(其中回收试剂盒为

Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609)。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物(其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402)；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于 42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μL PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health,Bethesda,Md.(1991)。 Cloning and sequencing: 5 μL of PCR product was detected by agarose gel electrophoresis, and the positive sample was purified by column recovery kit (the recovery kit was

Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609). The ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 μL of ligase, 1 μL of buffer, 10 μL of reaction system, and reacted at 16 ° C for half an hour to obtain a ligation product (the linked kit was T4 DNA ligase, purchased from NEB, article number M0402). 5 μL of the ligation product was added to 100 μL of competent cells (Ecos 101competent cells, purchased from Yeastern, Cat. No. FYE607), and ice-bathed for 5 minutes. Then, heat-shock in a 42 ° C water bath for 1 minute, put back on ice for 1 minute, add 650 μL of antibiotic-free SOC medium, resuscitate at 200 RPM for 30 minutes on a 37 ° C shaker, and take out 200 μL of LB solid medium coated with antibiotics. Incubate overnight at 37 ° C in an incubator. On the next day, 30 μL of the PCR system was configured using the primers M13F and M13R on the T vector, colony PCR was performed, and the colony was pipetted by a pipette tip to suck in the PCR reaction system, and 0.5 μL of the spot was taken up to another block containing 100 nM ampicillin. The strain was preserved on an LB solid culture dish. After the end of the PCR reaction, 5 μL was taken for agarose gel electrophoresis detection, and the positive samples were sequenced. Among them, the steps of sequencing are described in Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991).

结果显示：The results show:

所述的29D9H8重链可变区的氨基酸序列如序列表中SEQ ID NO.1所示，轻链可变区序列如序列表中SEQ ID NO.5所示。The amino acid sequence of the 29D9H8 heavy chain variable region is set forth in SEQ ID NO. 1 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 5 of the Sequence Listing.

其中重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.1中的第31位至第35位所示，CDR2的氨基酸序列如序列表SEQ ID No.1中的第50位至第66位所示，CDR3的氨基酸序列如序列表SEQ ID No.1中的第99位至第109位所示；The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 1, and the amino acid sequence of CDR2 is from position 50 to SEQ ID No. 1 in the sequence listing. As indicated at position 66, the amino acid sequence of CDR3 is shown in positions 99 to 109 of SEQ ID No. 1 of the Sequence Listing;

所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.5中的第24位至第34位所示，CDR2的氨基酸序列如序列表SEQ ID No.5中的第50位至第56位所示，CDR3的氨基酸序列如序列表SEQ ID No.5中的第89位至第97位所示。The amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 24 to 34 of SEQ ID No. 5 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 5 of the Sequence Listing. As shown in positions 50 to 56, the amino acid sequence of CDR3 is shown in positions 89 to 97 of SEQ ID No. 5 of the Sequence Listing.

所述28A2E11重链可变区的氨基酸序列如序列表中SEQ ID NO.9所示，轻链可变区序列如序列表中SEQ ID NO.13所示。The amino acid sequence of the 28A2E11 heavy chain variable region is set forth in SEQ ID NO. 9 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 13 of the Sequence Listing.

其中重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.9中的第31位至第35位所示，CDR2的氨基酸序列如序列表SEQ ID No.9中的第50位至第66位所示，CDR3的氨基酸序列如序列表SEQ ID No.9中的第99位至第107位所示；The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of CDR2 is the 50th to the SEQ ID No. 9 of the Sequence Listing. As indicated at position 66, the amino acid sequence of CDR3 is shown in positions 99 to 107 of SEQ ID No. 9 of the Sequence Listing;

所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.13中的第23位至第36位所示，CDR2的氨基酸序列如序列表SEQ ID No.13中的第52位至第58位所示，CDR3的氨基酸序列如序列表SEQ ID No.13中的第91位至第99位所示。The amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 23 to 36 of SEQ ID No. 13 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 13 of the Sequence Listing. As shown in positions 52 to 58, the amino acid sequence of CDR3 is shown in positions 91 to 99 of SEQ ID No. 13 of the Sequence Listing.

所述35E2C3重链可变区的氨基酸序列如序列表中SEQ ID NO.17所示，轻链可变区序列如序列表中SEQ ID NO.21所示。The amino acid sequence of the 35E2C3 heavy chain variable region is set forth in SEQ ID NO. 17 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 21 of the Sequence Listing.

其中重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.17中的第31位至第35位所示，CDR2的氨基酸序列如序列表SEQ ID No.17中的第50位至第65位所示，CDR3的氨基酸序列如序列表SEQ ID No.17中的第98位至第107位所示；The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 17 of the Sequence Listing, and the amino acid sequence of CDR2 is from position 50 to SEQ ID No. 17 of the Sequence Listing. As indicated at position 65, the amino acid sequence of CDR3 is shown in positions 98 to 107 of SEQ ID No. 17 of the Sequence Listing;

所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.21中的第24位至第34位所示，CDR2的氨基酸序列如序列表SEQ ID No.21中的第50位至第56位所示，CDR3的氨基酸序列如序列表SEQ ID No.21中的第89位至第97位所示。The amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 24 to 34 of SEQ ID No. 21 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 21 of the Sequence Listing. As shown in positions 50 to 56, the amino acid sequence of CDR3 is shown in positions 89 to 97 of SEQ ID No. 21 of the Sequence Listing.

核苷酸测序结果：Nucleotide sequencing results:

29D9H8的重链可变区的核苷酸序列如SEQ ID NO.61所示，轻链可变区的核苷酸序列如SEQ ID NO.65所示；The nucleotide sequence of the heavy chain variable region of 29D9H8 is set forth in SEQ ID NO. 61, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.

28A2E11的重链可变区的核苷酸序列如SEQ ID NO.69所示，轻链可变区的核苷酸序列如SEQ ID NO.73所示；The nucleotide sequence of the heavy chain variable region of 28A2E11 is set forth in SEQ ID NO. 69, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.

35E2C3的重链可变区的核苷酸序列如SEQ ID NO.77所示，轻链可变区的核苷酸序列如SEQ ID NO.81所示；The nucleotide sequence of the heavy chain variable region of 35E2C3 is set forth in SEQ ID NO. 77, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.

其中，编码29D9H8的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.61中的第91位至第105位；Wherein, the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 29D9H8 is the 91st to 105th positions in SEQ ID No. 61 of the Sequence Listing;

编码29D9H8的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.61中的第148位至第198位；The nucleotide sequence of the CDR2 domain in the heavy chain protein variable region encoding 29D9H8 is from positions 148 to 198 of the sequence listing SEQ ID No. 61;

编码29D9H8的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.61中的第295位至第327位；The nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 29D9H8 is from positions 295 to 327 in SEQ ID No. 61 of the Sequence Listing;

编码29D9H8的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.65中的第70位至第102位；The nucleotide sequence of the CDR1 domain in the light chain protein variable region encoding 29D9H8 is from positions 70 to 102 of SEQ ID No. 65 of the Sequence Listing;

编码29D9H8的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.65中的第148位至第168位；The nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 29D9H8 is from positions 148 to 168 of SEQ ID No. 65 of the Sequence Listing;

编码29D9H8的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.65中的第265位至第291位；The nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 29D9H8 is position 265 to 291 of SEQ ID No. 65 of the Sequence Listing;

编码28A2E11的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.69中的第91位至第105位；The nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 28A2E11 is position 91 to 105 in SEQ ID No. 69 of the Sequence Listing;

编码28A2E11的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.69中的第148位至第198位；The nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 28A2E11 is from positions 148 to 198 of SEQ ID No. 69 of the Sequence Listing;

编码28A2E11的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.69中的第295位至第321位；The nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 28A2E11 is from positions 295 to 321 of SEQ ID No. 69 of the Sequence Listing;

编码28A2E11的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.73 中的第67位至第108位；The nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 28A2E11 is from position 67 to position 108 of SEQ ID No. 73 of the Sequence Listing;

编码28A2E11的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.73中的第154位至第174位；The nucleotide sequence of the CDR2 domain in the light chain protein variable region encoding 28A2E11 is from positions 154 to 174 of SEQ ID No. 73 of the Sequence Listing;

编码28A2E11的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.73中的第271位至第297位；The nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 28A2E11 is from positions 271 to 297 of SEQ ID No. 73 of the Sequence Listing;

编码35E2C3的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.77中的第91位至第105位；The nucleotide sequence of the CDR1 domain of the heavy chain protein variable region encoding 35E2C3 is position 91 to 105 of SEQ ID No. 77 of the Sequence Listing;

编码35E2C3的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.77中的第148位至第195位；The nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 35E2C3 is 148th to 195th in SEQ ID No. 77 of the Sequence Listing;

编码35E2C3的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.77中的第292位至第321位；The nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 35E2C3 is position 292 to 321 in SEQ ID No. 77 of the Sequence Listing;

编码35E2C3的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID No.81中的第70位至第102位；The nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 35E2C3 is from positions 70 to 102 of SEQ ID No. 81 of the Sequence Listing;

编码35E2C3的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID No.81中的第148位至第168位；The nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 35E2C3 is from positions 148 to 168 of SEQ ID No. 81 of the Sequence Listing;

编码35E2C3的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID No.81中的第265位至第291位；The nucleotide sequence of the CDR3 domain in the light chain protein variable region encoding 35E2C3 is from position 265 to position 291 in SEQ ID No. 81 of the Sequence Listing;

实施例6：小鼠-人嵌合IL-13抗体的制备Example 6: Preparation of mouse-human chimeric IL-13 antibody

实施例3已从杂交瘤细胞的培养上清液中获得了纯化的IL-13抗体(先导抗体)。其中，按照本实施例记载的步骤进行操作，能够从实施例3所得的先导抗体制备出小鼠-人嵌合IL-13抗体。Example 3 A purified IL-13 antibody (lead antibody) was obtained from the culture supernatant of hybridoma cells. Among them, the mouse-human chimeric IL-13 antibody can be prepared from the lead antibody obtained in Example 3 by following the procedure described in the present example.

1、质粒构建与准备：1. Plasmid construction and preparation:

根据实施例5的测序结果明确了IL-13抗体重链可变区和轻链可变区序列。将实施例3所得的先导抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购自Invitrogen)中，将IL-13抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区或Lambda恒定区的表达载体(其中表达载体购自Invitrogen)当中，得重组质粒(上述质粒重组的实验原理及步骤的出处见《分子克隆实验指南(第三版)》，(美)J.萨姆布鲁克等著)并经测序验证(测序方法与实施例5中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。The IL-13 antibody heavy chain variable region and light chain variable region sequences were clarified according to the sequencing results of Example 5. The heavy chain variable region sequence of the leader antibody obtained in Example 3 was recombined into an expression vector comprising a signal peptide and a human heavy chain antibody IgG1 constant region (in which the expression vector was purchased from Invitrogen), and the light chain of the IL-13 antibody was The variable region sequence was recombined into an expression vector comprising the signal peptide and the human antibody light chain kappa constant region or Lambda constant region (wherein the expression vector was purchased from Invitrogen) to obtain a recombinant plasmid (see above for the experimental principle and procedure of plasmid recombination) "Molecular Cloning Experiment Guide (Third Edition)", (US) J. Sambrook et al.) and verified by sequencing (sequencing method is the same as the sequencing method in Example 5). The recombinant plasmid having a purity increased by mass spectrometry using an alkali lysis kit (purchased from MACHEREY-NAGEL) was used at a mass of 500 μg or more, and filtered through a 0.22 μm filter (purchased from Millopore) for transfection.

2、细胞转染：2. Cell transfection:

在培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM和8％CO ₂(v/v)。Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒(即重链重组质粒和轻链重组质粒按常规等比例混合的混合重组质粒)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 ⁶个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). The shaker was set to 37 ° C, 130 RPM and 8% CO ₂ (v/v). Freestyle 293 expression medium was added with 10% (v/v) F68 (purchased from Invitrogen) to a final concentration of 0.1% (v/v) of F68 during transfection to obtain Freestyle 293 expression culture containing 0.1% (v/v) F68. Base, medium A. 5 mL of Medium A and 200 μg/mL of PEI (purchased from Sigma) were mixed to obtain Medium B. 5 mL of the medium A and 100 μg/mL of the recombinant plasmid obtained in the step (1) (that is, the recombinant plasmid in which the heavy chain recombinant plasmid and the light chain recombinant plasmid were mixed in a usual equal ratio) were mixed to obtain a medium C. After 5 minutes, the medium B and the medium C were combined and mixed, and allowed to stand for 15 minutes to obtain a mixture D. 10 mL of the mixture D was slowly added to 100 mL of the 293E-containing medium Freestyle 293 expression medium until the cell density of 293E was 1.5×10 ⁶ /mL, and the mixture was shaken while shaking to avoid excessive concentration of PEI and placed in a shaker culture. Peptone was added the next day to a final concentration of 0.5% (w/v). On the 5th to 7th day, the antibody titer of the culture solution was measured. On the 6th to 7th day, the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 μm filter to obtain a filtered cell supernatant for purification.

3、抗体纯化：3. Antibody purification:

对于连续生产的无内毒素的层析柱和Protein A填料(购自GE)，使用5个柱体积的0.5M NaOH冲洗。然后用5个柱体积的PBS(PBS缓冲液，pH7.4)进行平衡至中性后，将步骤(2)所得过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH 3.0的Glycine-HCl进行洗脱，收集洗脱液，并立刻在洗脱液中加入0.1倍体积的pH 8.5的1M Tris-HCl(1.5M NaCl)中和IL-13抗体。上述所用溶液均需要新配置。收获IL-13抗体后，在1╳PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。并对所获IL-13抗体进行特性鉴定。用29D9H8、28A2E11和35E2C3先导抗体分别制得嵌合抗体c29D9H8、c28A2E11和c35E2C3，克隆号前端字母c(chimera)表示小鼠-人嵌合抗体(以下简称嵌合抗体)。For a continuously produced endotoxin-free chromatography column and Protein A filler (available from GE), rinse with 5 column volumes of 0.5 M NaOH. Then, after equilibrating to neutral with 5 column volumes of PBS (PBS buffer, pH 7.4), the filtered cell supernatant obtained in the step (2) was applied to the column, and the flow-through was collected as necessary. After the upper column was completed, it was washed with 5 column volumes of PBS. Elution was carried out with 5 column volumes of 0.1 M Glycine-HCl, pH 3.0, and the eluate was collected and immediately neutralized with 0.1 volume of 1 M Tris-HCl (1.5 M NaCl) at pH 8.5 in the eluate. -13 antibody. All of the solutions used above require a new configuration. After harvesting the IL-13 antibody, it was dialyzed against 1 PBS for 4 hours to avoid endotoxin contamination. After the end of dialysis, the concentration was measured using a spectrophotometer or a kit, the purity of the antibody was determined using HPLC-SEC, and the endotoxin content of the antibody was detected using an endotoxin test kit (purchased from Lonza). The obtained IL-13 antibody was characterized. The chimeric antibodies c29D9H8, c28A2E11 and c35E2C3 were prepared using the 29D9H8, 28A2E11 and 35E2C3 leader antibodies, respectively. The cloned front-end letter c (chimera) represents a mouse-human chimeric antibody (hereinafter referred to as chimeric antibody).

实施例7:小鼠-人嵌合IL-13抗体的检定Example 7: Assay of mouse-human chimeric IL-13 antibody

将实施例6获得的小鼠-人嵌合IL-13抗体进行如下的检定试验(操作步骤同实施例4)。The mouse-human chimeric IL-13 antibody obtained in Example 6 was subjected to the following assay (the procedure is the same as in Example 4).

A、酶联免疫吸附实验(ELISA)检测嵌合抗体与免疫原A、IL-13R130Q变体以及猴IL-13的结合A, enzyme-linked immunosorbent assay (ELISA) to detect the binding of chimeric antibodies to immunogen A, IL-13R130Q variant and monkey IL-13

具体操作步骤同实施例4试验A。部分实验结果见图13～15和表18～20所示。表18～20说明，上述嵌合抗体与重组人IL-13蛋白，重组人IL-13 R130Q变体，重组猴IL-13在ELISA水平结合。其中IgG对照为鼠IgG，表中的数据为OD _450nm值。 The specific procedure is the same as that of Example 4, Test A. Some experimental results are shown in Figures 13-15 and Tables 18-20. Tables 18-20 illustrate that the chimeric antibodies were conjugated to recombinant human IL-13 protein, recombinant human IL-13 R130Q variant, and recombinant monkey IL-13 at the ELISA level. The IgG control was murine IgG and the data in the table is the OD _{450 nm} value.

表18 ELISA检测IL-13嵌合抗体与免疫原A的反应活性Table 18 ELISA to detect the reactivity of IL-13 chimeric antibody with immunogen A

表19 ELISA检测IL-13嵌合抗体与IL-13R130Q变体的反应活性Table 19 ELISA for the reactivity of IL-13 chimeric antibodies with IL-13R130Q variants

表20 ELISA检测IL-13嵌合抗体与猴IL-13变体的反应活性Table 20 ELISA for the reactivity of IL-13 chimeric antibodies with monkey IL-13 variants

B、受体配体结合阻滞实验B, receptor ligand binding block experiment

具体试验步骤请参见实施例4试验B部分。部分实验结果见图16～17和表21～22，表21～22说明，嵌合抗体与人IL-13结合后，能阻滞人IL-13与细胞表面的受体人IL-13Ra1/hIL-4Ra异源二聚体的结合，或者能阻滞IL-13与细胞表面人IL-13Ra2受体的结合。其中IgG对照为鼠IgG，表格中数据为平均荧光强度。For the specific test procedure, please refer to the test part B of Example 4. Some of the experimental results are shown in Figures 16-17 and Tables 21-22. Tables 21-22 illustrate that chimeric antibodies bind to human IL-13 and block human IL-13 and cell surface receptor IL-13Ra1/hIL. -4Ra heterodimer binding, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor. The IgG control was murine IgG and the data in the table is the mean fluorescence intensity.

表21 FACS检测嵌合抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 21 FACS detection of chimeric antibodies blocking the binding of IL-13 to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表22 FACS检测嵌合抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 22 FACS detection of chimeric antibodies blocking the binding of IL-13 to the cell surface receptor IL-13Ra2

具体操作步骤请参见实施例4的C试验部分。部分实验结果见图18和表23。表23说明，嵌合抗体通过与人IL-13的结合，能够中和由IL-13和TNFα共同刺激条件下诱导产生的A549细胞分泌TARC。表23的数据为培养上清中TARC的浓度(pg/ml)，其中IgG对照为鼠IgG。For the specific operation steps, please refer to the C test part of Example 4. Some experimental results are shown in Figure 18 and Table 23. Table 23 shows that chimeric antibodies can neutralize the secretion of TARC by A549 cells induced by IL-13 and TNFα co-stimulation by binding to human IL-13. The data in Table 23 is the concentration (pg/ml) of TARC in the culture supernatant, wherein the IgG control is murine IgG.

表23嵌合抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 23 Chimeric antibodies neutralize IL-13-induced thymic activation and regulate chemokine secretion

D、骨膜蛋白(periostin)分泌试验D, periostin secretion test

将MRC5细胞(购自ATCC)培养在含有10％(w/w)胎牛血清EMEM培养基(购自Gibco)中，于37℃、5％(v/v)CO ₂条件下培养。待其在T-175细胞培养瓶中扩大培养至75-90％汇合度，弃去培养基，用PBS缓冲液洗涤1-2次，然后用胰蛋白酶溶液(Trypsin-EDTA，购自Life technology)消化并收集细胞。计数后将细胞用培养液稀释至1×10 ⁵个细胞/mL，按照每孔细胞100μL加到96孔细胞培养板中(每孔1×10 ⁴个细胞)，将培养板置于37℃、5％(v/v)CO ₂培养箱培养过夜；第二天，弃去培养上清，将梯度稀释的实施例6制备的嵌合抗体与实施例1制备的免疫原A混合得混合物C(免疫原A在混合物C中的终浓度为5ng/mL)，加入混合物C于培养板中，将细胞培养板放在37℃、5％(v/v)CO ₂培养箱培养过夜。20小时后，用骨膜蛋白ELISA试剂盒(购自RnD systems)检定上清中骨膜蛋白的浓度。实验操作按照试剂盒说明书要求进行。部分实验结果见图19和表24。表24说明，嵌合抗体与人IL-13结合，能够中和由人IL-13诱导下MRC5细胞分泌骨膜蛋白。表24中数据为培养上清中骨膜蛋白的浓度(pg/ml)，IgG对照为鼠IgG。 MRC5 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum EMEM medium (purchased from Gibco) and cultured at 37 ° C under 5% (v/v) CO ₂ . After being expanded to 75-90% confluence in a T-175 cell culture flask, the medium was discarded, washed 1-2 times with PBS buffer, and then trypsin-EDTA (purchased from Life Technology). Digest and collect cells. After counting, the cells were diluted to 1 × 10 ⁵ cells/mL with a culture solution, and 100 μL of each well was added to a 96-well cell culture plate (1 × 10 ⁴ cells per well), and the plate was placed at 37 ° C. The 5% (v/v) CO ₂ incubator was cultured overnight; the next day, the culture supernatant was discarded, and the gradient-diluted chimeric antibody prepared in Example 6 was mixed with the immunogen A prepared in Example 1 to obtain a mixture C ( The final concentration of immunogen A in mixture C was 5 ng/mL), mixture C was added to the culture plate, and the cell culture plate was incubated overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. After 20 hours, the concentration of periostin in the supernatant was determined using a periostin ELISA kit (purchased from RnD systems). The experimental procedure was carried out in accordance with the instructions of the kit. Some experimental results are shown in Figures 19 and 24. Table 24 shows that the chimeric antibody binds to human IL-13 and is capable of neutralizing the secretion of periostin from MRC5 cells induced by human IL-13. The data in Table 24 is the concentration of periostin (pg/ml) in the culture supernatant, and the IgG control is murine IgG.

表24嵌合抗体中和IL-13诱导的骨膜蛋白分泌Table 24 Chimeric antibodies neutralize IL-13-induced secretion of periostin

E、血管细胞粘附分子-1(VCAM-1)表达实验E, vascular cell adhesion molecule-1 (VCAM-1) expression experiment

将人脐带静脉内皮细胞(HUVEC)(购自AllCells)培养在HUVEC完全培养基中，置于37℃、5％(v/v)CO ₂条件下培养。待其在T-175扩大培养至75-90％汇合度，弃去培养基，用PBS缓冲液洗涤1-2次，然后用胰蛋白酶溶液(Trypsin-EDTA，购自Life technology)消化并收集细胞。计数后将细胞用培养液稀释至1.5×10 ⁵个细胞/mL，按照每孔HUVEC细胞3000个细胞(20μL)加到384孔细胞培养板中；将梯度稀释的实施例6制备的抗体与实施例1制备的免疫原A以及重组人TNFα按体积比1:1:2混合得混合物D。将混合物D按照每孔20μL的添加量添加到培养板中得混合物D，使混合物D与培养板中HUVEC细胞的体积比为1:1。此时重组人TNFα占混合物D’的终浓度为25ng/mL，免疫原A占混合物D’的终浓度为0.5ng/mL)。 Human umbilical vein endothelial cells (HUVEC) (purchased from AllCells) were cultured in HUVEC complete medium and cultured at 37 ° C under 5% (v/v) CO ₂ . After it was expanded to T-175 to 75-90% confluence, the medium was discarded, washed 1-2 times with PBS buffer, and then digested and collected with trypsin-EDTA (purchased from Life Technology). . After counting, the cells were diluted to 1.5×10 ⁵ cells/mL with the culture medium, and 3000 cells (20 μL) per HUVEC cells were added to the 384-well cell culture plate; the antibody prepared in Example 6 was diluted with the implementation. The immunogen A prepared in Example 1 and recombinant human TNFα were mixed at a volume ratio of 1:1:2 to obtain a mixture D. The mixture D was added to the culture plate in an amount of 20 μL per well to obtain a mixture D such that the volume ratio of the mixture D to the HUVEC cells in the culture plate was 1:1. At this time, the recombinant human TNFα accounted for a final concentration of 25 ng/mL of the mixture D', and the immunogen A accounted for 0.5 ng/mL of the final concentration of the mixture D'.

将细胞培养板放在37℃、5％(v/v)CO ₂培养箱培养过夜。20小时后，弃去培养液上清，将鼠抗人VCAM-1(CD106)抗体(购自Biolegend)用培养基稀释至终浓度为2μg/mL，按照每孔20μL加入培养板中，冰上孵育2小时。用FACS缓冲液[含有2％(w/w)BSA的PBS缓冲液]洗涤3次，加入每孔20μL荧光(Alexa 488)标记的驴抗鼠二抗(购自invitrogen)，冰上孵育0.5-1.0小时。再加入每孔20μL固定液[4％(w/w)多聚甲醛]，5-10分钟后用PBS缓冲液洗涤3次。用PBS缓冲液将PI(购自invitrogen) 和RNAseA(购自Qiagen)稀释至PI和RNAseA的终浓度分别为9nM和200ng/mL，按照每孔20μL加入384孔板中，在37℃孵育30分钟后，用Acumen(微孔板细胞检测法)检验并分析细胞表面上血管细胞粘附分子-1(VCAM-1)的表达水平。部分实验结果见图20和表25。表25说明，嵌合抗体与人IL-13结合，能够中和由人IL-13和TNFα共同刺激条件下诱导产生的HUVEC细胞表面表达血管粘附因子-1。表25中数据为平均荧光强度；IgG对照为鼠IgG；TNFα为单独用TNFα刺激，而无IL-13情况下的背景值。 The cell culture plates were incubated overnight at 37 ° C in a 5% (v/v) CO ₂ incubator. After 20 hours, the culture supernatant was discarded, and the mouse anti-human VCAM-1 (CD106) antibody (purchased from Biolegend) was diluted with the medium to a final concentration of 2 μg/mL, and added to the plate at 20 μL per well on ice. Incubate for 2 hours. Wash 3 times with FACS buffer [PBS buffer containing 2% (w/w) BSA], add 20 μL of fluorescent (Alexa 488)-labeled donkey anti-mouse secondary antibody (purchased from invitrogen) per well, and incubate 0.5-on ice. 1.0 hour. Further, 20 μL of fixative [4% (w/w) paraformaldehyde per well) was added, and after 5-10 minutes, it was washed 3 times with PBS buffer. The final concentrations of PI (purchased from invitrogen) and RNAseA (purchased from Qiagen) to PI and RNAseA were 9 nM and 200 ng/mL, respectively, in PBS buffer, and added to 384-well plates at 20 μL per well, and incubated at 37 ° C for 30 minutes. Thereafter, the expression level of vascular cell adhesion molecule-1 (VCAM-1) on the cell surface was examined and analyzed by Acumen (microplate assay). Some experimental results are shown in Figure 20 and Table 25. Table 25 shows that the chimeric antibody binds to human IL-13 and is capable of neutralizing the expression of vascular adhesion factor-1 on the surface of HUVEC cells induced by co-stimulation of human IL-13 and TNFα. The data in Table 25 is the mean fluorescence intensity; the IgG control is murine IgG; TNFα is the background value stimulated with TNFα alone, without IL-13.

表25嵌合抗体中和IL-13诱导的血管细胞粘附分子-1表达Table 25 Chimeric antibodies neutralize IL-13-induced vascular cell adhesion molecule-1 expression

实施例8：IL-13嵌合抗体抑制人IL-13诱导的小鼠呼吸炎症实验Example 8: IL-13 chimeric antibody inhibits human IL-13-induced respiratory inflammation in mice

雌性Balb/c小鼠(8-12周龄，购自上海灵畅生物科技有限公司)接收后在SPF级饲养，适应1周后，开始实验。小鼠在第一天及第三天腹腔注射实施例6制备的嵌合抗体，克隆号分别为c29D9H8、c28A2E11和c35E2C3，每只动物200μL(3mg抗体每公斤体重)。第二天及第四天用实施例1制备的免疫原A进行刺激诱导，剂量为1mg/mL，每只动物给予气道喷雾25μL。第五天将所有动物用FinePointe全身体积描记系统(即DSI

FinePointe WBP系统，购自DSI)进行肺功能检测。动物在清醒无约束状态下通过雾化给药方式给予甲基乙酰胆碱，同时通过仪器自带软件记录气道缩窄指数Penh(增强呼气间歇)。(检测的具体操作步骤按照仪器的说明手册中记载的方法进行) Female Balb/c mice (8-12 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were received at the SPF level after receiving, and the experiment was started after 1 week of adaptation. Mice were injected intraperitoneally with the chimeric antibodies prepared in Example 6 on

days

1 and 3, clone numbers c29D9H8, c28A2E11 and c35E2C3, respectively, 200 μL per animal (3 mg antibody per kg body weight). On the second and fourth days, the immunogen A prepared in Example 1 was used for stimulation induction at a dose of 1 mg/mL, and each animal was given an airway spray of 25 μL. On the fifth day, all animals were treated with the FinePointe Whole Body Plethysmography System (DSI).

The FinePointe WBP system, purchased from DSI, was tested for lung function. Animals were given methyl acetylcholine by aerosolization in a conscious, unconstrained state, and the airway narrowing index Penh (enhanced expiratory interval) was recorded by the instrument's own software. (The specific operation steps of the test are carried out according to the method described in the instruction manual of the instrument)

部分实验结果见图21和表26。表26说明，小鼠在人IL-13，或者说免疫原A的刺激之下会诱发气道的高反应性，使其对甲基乙酰胆碱的刺激更为敏感，使penh读数增大。嵌合抗体会与人IL-13结合，并中和由人IL-13诱导产生的气道高反应性。表中数据为气道缩窄指数Penh，代表气道高反应性；IgG对照为人IgG。Some experimental results are shown in Figure 21 and Table 26. Table 26 shows that mice stimulated high airway responsiveness under stimulation with human IL-13, or immunogen A, making them more sensitive to methyl acetylcholine stimulation and increasing penh readings. The chimeric antibody binds to human IL-13 and neutralizes airway hyperresponsiveness induced by human IL-13. The data in the table is the airway narrowing index Penh, which represents airway hyperresponsiveness; the IgG control is human IgG.

表26嵌合抗体抑制人IL-13诱导的小鼠呼吸炎症实验Table 26 Chimeric antibodies inhibit human IL-13 induced respiratory inflammation in mice

实施例9：IL-13嵌合抗体亲和力检测试验Example 9: IL-13 chimeric antibody affinity test

用氨基偶联方法将抗人Fc IgG(购自Geneway)偶联固定在CM5芯片(购自GE)表面至6000-10000RU，FC1作为参比通道。偶联固定过程如下：用新鲜配置的摩尔比为1:1的50mM N-羟基琥珀酰亚胺(NHS)和200mM 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)的混合物活化芯片7分钟。然后注入10-50μg/mL稀释在10mM醋酸纳缓冲液(pH5.0)中的抗人Fc IgG。剩余的活化位点用1M乙醇胺封闭。然后，用含HBS-EP缓冲液将实施例6制备的嵌合抗体稀释至5μg/mL(可根据捕获水平适当调整)。以10μL/分钟的流速捕获到芯片上，得到大约100～300RU的响应值。接着将实施例1制备的纯化的免疫原A稀释至100nM(即最高浓度暂定100nM)，以30μL/分钟的流速流经芯片表面。若得到足够的信号值，就将实施例1制备的纯化的免疫原A倍比稀释几个浓度梯度，分别流经芯片表面。在每个循环结束后，芯片表面用10mM，pH 1.5的甘氨酸进行再生。动力学速率常数需减去空白对照，用global fit分析方法1：1结合模型进行数据拟合(参照Biacore操作手册进行操作)。解离平衡速率常数(KD)用以下公式计算：KD＝kd/ka，其中，Kd为解离常数，Ka为结合常数。部分实验结果见表27。表27说明，实施例6制备的嵌合抗体与人IL-13的亲和力KD<9×10 ^-8M。 Anti-human Fc IgG (purchased from Geneway) was coupled to the surface of a CM5 chip (purchased from GE) by an amino coupling method to 6000-10000 RU, and FC1 was used as a reference channel. The coupling fixation procedure was as follows: 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a freshly prepared molar ratio of 1:1 The mixture of hydrochloride (EDC) activated the chip for 7 minutes. Then, 10-50 μg/mL of anti-human Fc IgG diluted in 10 mM sodium acetate buffer (pH 5.0) was injected. The remaining activation sites were blocked with 1 M ethanolamine. Then, the chimeric antibody prepared in Example 6 was diluted to 5 μg/mL with HBS-EP-containing buffer (which can be appropriately adjusted depending on the capture level). The chip was captured at a flow rate of 10 μL/min to obtain a response value of approximately 100 to 300 RU. The purified immunogen A prepared in Example 1 was then diluted to 100 nM (i.e., the highest concentration tentatively 100 nM) and flowed through the surface of the chip at a flow rate of 30 μL/min. If sufficient signal values are obtained, the purified immunogen A prepared in Example 1 is diluted by several concentration gradients and flowed through the surface of the chip, respectively. At the end of each cycle, the surface of the chip was regenerated with 10 mM glycine at pH 1.5. The kinetic rate constants were subtracted from the blanks and the data fit was performed using the global fit analysis method 1:1 binding model (refer to the Biacore operating manual for operation). The dissociation equilibrium rate constant (KD) is calculated by the following formula: KD = kd / ka, where Kd is the dissociation constant and Ka is the binding constant. Some experimental results are shown in Table 27. Table 27 shows that the affinity of the chimeric antibody prepared in Example 6 to human IL-13 is KD < 9 x 10 ^-8 M.

表27嵌合抗体与人IL-13亲和力检测结果Table 27: Detection results of chimeric antibodies and human IL-13 affinity

克隆号Clone number ka(1/Ms)Ka(1/Ms) kd(1/s)Kd(1/s) KD(M)KD(M) c29D9H8c29D9H8 2.51×10 ⁷ 2.51×10 ⁷ 8.13×10 ^-4 8.13×10 ^-4 3.24×10 ^-11 3.24×10 ^-11 c28A2E11c28A2E11 5.49×10 ⁵ 5.49×10 ⁵ 4.38×10 ^-3 4.38×10 ^-3 7.98×10 ^-9 7.98×10 ^-9 c35E2C3c35E2C3 3.00×10 ⁵ 3.00×10 ⁵ 2.60×10 ^-4 2.60×10 ^-4 8.63×10 ^-10 8.63×10 ^-10

实施例10:人源化抗IL-13抗体的制备Example 10: Preparation of humanized anti-IL-13 antibody

在Germline数据库中选取与上述嵌合抗体c29D9H8、c28A2E11和c35E2C3的非CDR区匹配最好的人种系抗体重链和轻链可变区模板。人源化IL-13抗体的人接受序列选自人种系外显子V _H、J _H、V _k、J _k、V _L和J _L序列。其中c29D9H8抗体重链可变区的模板为人种系抗体重链V _H外显子的VH1-F，J _H外显子的J _H-1，轻链可变区的模板为人种系抗体轻链V _K外显子的L1，J _K外显子的J _K-4。其中c28A2E11抗体重链可变区的模板为人种系抗体重链V _H外显子的VH1-F，J _H外显子的J _H-1，轻链可变区的模板为人种系抗体轻链V _L外显子的7B，J _L外显子的J _L-1。其中c35E2C3抗体重链可变区的模板为人种系抗体重链V _H外显子的VH4-59，J _H外显子的J _H-6，轻链可变区的模板为人种系抗体轻链V _K外显子的O12，J _K外显子的J _K-4。 The human germline antibody heavy and light chain variable region templates that best match the non-CDR regions of the chimeric antibodies c29D9H8, c28A2E11 and c35E2C3 described above were selected in the Germline database. Humanized IL-13 human antibody sequences receiving exon _{_{_{V H, J H, V k}}} , J k, V L and J _L germline sequence selected from outside. Wherein c29D9H8 template antibody heavy chain variable region of an antibody heavy chain human germline outer V _H exon VH1-F, an outer J _H exon J _H -1, the template for the light chain variable region of human germline antibody outer light chain V _K exon L1, J _K exon J _K -4. Wherein c28A2E11 template antibody heavy chain variable region of the outer V _H human germline antibody heavy chain exon VH1-F, an outer J _H exon J _H -1, the template for the light chain variable region of human germline antibody outer light chain V _L exon 7B, J _L exon J _L -1. Wherein c35E2C3 template antibody heavy chain variable region of an antibody heavy chain human germline outer V _H exon VH4-59, template peripheral J _H exon J _H -6, light chain variable region of human germline antibody Light chain V _K exon O12, J _K exon J _K -4.

根据Kabat定义确定的嵌合抗体c29D9H8、c28A2E11和c35E2C3的重链和轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，得到人源化的抗体。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的构架区的残基进行回复突变，得到人源化之后的抗体。简要说，产生跨越人源化V _H或V _L结构域的合成重叠寡核苷酸、并利用PCR重叠延伸来组装各结构域。利用掺入PCR产物的限制性位点将V _H结构域定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体，将V _L结构域定向克隆到包含信号肽和人源抗体轻链kappa恒定区或Lambda恒定区的表达载体中，得到的重组质粒经过测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。 The heavy and light chain CDRs of the chimeric antibodies c29D9H8, c28A2E11 and c35E2C3, which were determined according to the Kabat definition, were each transplanted into a selected human germline template, replacing the CDR regions of the human germline template to obtain a humanized antibody. Then, based on the three-dimensional structure of the murine antibody, the residues of the framework region, which have an important interaction with the CDR region and the CDR region, and the residues of the framework region which have an important influence on the conformation of VL and VH are subjected to back mutation. Humanized antibody. Briefly, humanized generated across the V _H or V _L domains of synthetic overlapping oligonucleotides and assembled by PCR overlap extension of each domain. Using the restriction sites incorporated into the V _H domain directional cloning of PCR products into an expression vector comprising a signal peptide and a human antibody heavy chain IgG1 constant region of the V _L domain containing the signal peptide was cloned into human antibody light and In the expression vector of the kappa constant region or the Lambda constant region, the obtained recombinant plasmid was verified by sequencing, and the recombinant plasmid having a purity of 500 μg or more was obtained by using an alkali lysis kit (purchased from MACHEREY-NAGEL). The μm filter (purchased from Millopore) was filtered for transfection.

人源化抗IL-13抗体变体的重链和轻链可变区与人种系重链和轻链可变区及嵌合抗体的重链和轻链可变区的序列比对如图22-27所示，其中图22为人源化抗IL-13抗体Hu-29D9H8重链可变区Hu-29D9H8.VH1及其变体与c29D9H8嵌合抗体VH及人种系VH外显子hVH1-F/JH1的序列比较；图23为人源化抗IL-13抗体Hu-29D9H8轻链可变区Hu-29D9H8.V _K1及其变体与c29D9H8嵌合抗体V _K及人种系V _K外显子L1/JK4的序列比较；图24为人源化抗IL-13抗体Hu-28A2E11重链可变区Hu-28A2E11.VH1及其变体与c28A2E11嵌合抗体VH及人种系VH外显子hVH1-F/JH1的序列比较；图25为人源化抗IL-13抗体Hu-28A2E11轻链可变区Hu-28A2E11.V _L1及其变体与c28A2E11嵌合抗体V _L及人种系V _L外显子7B/JL1的序列比较；图26为人源化抗IL-13抗体Hu-35E2C3重链可变区Hu-35E2C3.VH1及其变体与c35E2C3嵌合抗体VH及人种系VH外显子hVH4-59/JH6的序列比较；图27为人源化抗IL-13抗体Hu-35E2C3轻链可变区Hu-35E2C3.V _K1及其变体与c35E2C3嵌合抗体V _K及人种系V _K外显子O12/JK4的序列比较。 Sequence alignment of the heavy and light chain variable regions of humanized anti-IL-13 antibody variants with the human heavy and light chain variable regions and the heavy and light chain variable regions of chimeric antibodies 22-27, wherein Figure 22 shows the humanized anti-IL-13 antibody Hu-29D9H8 heavy chain variable region Hu-29D9H8.VH1 and its variants and c29D9H8 chimeric antibody VH and human germline VH exon hVH1- Sequence comparison of F/JH1; Figure 23 shows humanized anti-IL-13 antibody Hu-29D9H8 light chain variable region Hu-29D9H8.V _K 1 and its variants and c29D9H8 chimeric antibody V _K and human germline V _K Sequence comparison of the exon L1/JK4; Figure 24 shows the humanized anti-IL-13 antibody Hu-28A2E11 heavy chain variable region Hu-28A2E11.VH1 and its variants and c28A2E11 chimeric antibody VH and human germline VH exons / comparison of sequences hVH1-F JH1; Figure 25 is a humanized anti-IL-13 antibody Hu-28A2E11 light chain variable region Hu-28A2E11.V _L 1 and variants thereof and chimeric antibodies c28A2E11 human germline V _L and V Sequence comparison of _L exon 7B/JL1; Figure 26 shows humanized anti-IL-13 antibody Hu-35E2C3 heavy chain variable region Hu-35E2C3.VH1 and its variants and c35E2C3 chimeric antibody VH and human germline VH Sequence comparison of the exon hVH4-59/JH6; Figure 27 shows the humanized anti-IL-13 antibody Hu-35E2C3 light Sequence comparison of the variable region Hu-35E2C3.V _K 1 and its variants with the c35E2C3 chimeric antibody V _K and the human germline V _K exon O12/JK4.

选择数个框架位置以重新引入小鼠供体残基。可通过在VH结构域中掺入小鼠框架供体残基或在VL结构域中掺入人CDR残基的不同组合产生数种人源化 Hu-29D9H8、Hu-28A2E11和Hu-35E2C3变体。下表28-30小结了这些变体。其中，表28-30显示的是这些变体的可变区，不包括恒定区。表28-30中，c开头表示嵌合抗体，Hu开头表示人源化抗体；其中供体构架残基及回复突变栏中显示的：例如各人源化抗IL-13抗体Hu-29D9H8重链可变区Hu-29D9H8.VH1及其变体中“T73K”表明如图22中所示的第73位氨基酸由“T”谷氨酸突变为“K”精氨酸，回复突变的位点位于构架区，以此为例，不一一说明。Several framework positions were selected to reintroduce mouse donor residues. Several humanized Hu-29D9H8, Hu-28A2E11 and Hu-35E2C3 variants can be generated by incorporating mouse framework donor residues in the VH domain or by incorporating different combinations of human CDR residues in the VL domain. . These variants are summarized in Tables 28-30 below. Among them, Tables 28-30 show the variable regions of these variants, excluding the constant regions. In Tables 28-30, c begins with a chimeric antibody, and Hu begins with a humanized antibody; wherein the donor framework residues and the back mutations are shown: for example, each humanized anti-IL-13 antibody Hu-29D9H8 heavy chain "T73K" in the variable region Hu-29D9H8.VH1 and its variants indicates that the 73rd amino acid shown in Figure 22 is mutated from "T" glutamate to "K" arginine, and the site of the back mutation is located. The framework area, as an example, will not be explained one by one.

表28Table 28

表29Table 29

表30Table 30

注：表中的“/”代表的含义是“和”，为并列关系。Note: The “/” in the table means “and” and is a side-by-side relationship.

根据各人源化抗体轻链可变区和重链可变区的氨基酸序列合成cDNA(即分别为序列表中的SEQ ID NO.86，87，88，89，90，91，92，93，94，95，96，97，98，99，100，101，102，103，104，105，106，107，108，109，111，112，113，114，115，116，118，119和120所示序列)，重链cDNA用FspAI和AfeI消化，轻链cDNA用FspAI和BsiwI或者FspAI和HindIII后，将cDNA片段通过FspAI/AfeI，FspAI/BsiwI或者FspAI/HindIII酶切位点分别插入到包含信号肽和人源重链抗体IgG1恒定区的表达载体及包含信号肽和人源抗体轻链kappa/Lambda恒定区的表达载体当中(其中表达载体购自Invitrogen)中，重组质粒并经测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。cDNA was synthesized based on the amino acid sequences of the light chain variable region and the heavy chain variable region of each humanized antibody (ie, SEQ ID NO. 86, 87, 88, 89, 90, 91, 92, 93 in the sequence listing, respectively). 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 118, 119 and 120 The sequence of the heavy chain cDNA was digested with FspAI and AfeI. After the light chain cDNA was FspAI and BsiwI or FspAI and HindIII, the cDNA fragment was inserted into the containing signal by FspAI/AfeI, FspAI/BsiwI or FspAI/HindIII cleavage sites, respectively. An expression vector for the IgG1 constant region of the peptide and human heavy chain antibody and an expression vector comprising the signal peptide and the human antibody light chain kappa/Lambda constant region (in which the expression vector is purchased from Invitrogen), the recombinant plasmid is verified by sequencing, and used The alkaline lysis kit (purchased from MACHEREY-NAGEL) was used to extract a recombinant plasmid having a purity of 500 μg or more, and was filtered through a 0.22 μm filter (purchased from Millopore) for transfection.

转染前，用培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。转染时在Freestyle 293 expression medium中添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg重链和轻链重组质粒(重链重组质粒与轻链重组质粒的质量比为1:1-1:3范围)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 ⁶个/mL，边加边振荡，避免PEI过度集中，放入摇床培养，摇床设置为37℃、130RPM和8％CO ₂(v/v)。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。 Prior to transfection, 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). When transfected, 10% (v/v) F68 (purchased from Invitrogen) was added to Freestyle 293 expression medium to a final concentration of 0.1% (v/v) of F68 to obtain Freestyle 293 expression containing 0.1% (v/v) F68. Medium, medium A. 5 mL of Medium A and 200 μg/mL of PEI (purchased from Sigma) were mixed to obtain Medium B. 5 mL of medium A and 100 μg of the heavy and light chain recombinant plasmid (the mass ratio of the heavy chain recombinant plasmid to the light chain recombinant plasmid was in the range of 1:1 to 1:3) were mixed to obtain a medium C. After 5 minutes, the medium B and the medium C were combined and mixed, and allowed to stand for 15 minutes to obtain a mixture D. 10 mL of the mixture D was slowly added to 100 mL of the 293E-containing medium Freestyle 293 expression medium until the cell density of 293E was 1.5×10 ⁶ /mL, and the mixture was shaken while shaking to avoid excessive concentration of PEI and placed in a shaker culture. The shaker was set to 37 ° C, 130 RPM and 8% CO ₂ (v/v). Peptone was added the next day to a final concentration of 0.5% (w/v). On the 5th to 7th day, the antibody titer of the culture solution was measured. On the 6th to 7th day, the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 μm filter to obtain a filtered cell supernatant for purification.

抗体纯化时，对于连续生产的无内毒素的层析柱和蛋白A填料(购自GE)，使用5个柱体积的0.5M NaOH冲洗。然后用5个柱体积的PBS(PBS缓冲液，pH7.4)进行平衡至中性后，将过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH 3.0的Glycine-HCl进行洗脱，收集洗脱液，并立刻在洗脱液中加入0.1倍体积的pH 8.5的1MTris-HCl(1.5M NaCl)中和IL-13抗体。上述所用溶液均需要新鲜配制。收获IL-13抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。并对所获IL-13抗体进行特性鉴定(操作步骤如下述实施例4和7所述)。For antibody purification, for a continuously produced endotoxin-free chromatography column and protein A filler (purchased from GE), rinse with 5 column volumes of 0.5 M NaOH. Then, after equilibrating to neutral with 5 column volumes of PBS (PBS buffer, pH 7.4), the filtered cell supernatant was applied to the column, and the flow-through was collected as necessary. After the upper column was completed, it was washed with 5 column volumes of PBS. Elution was carried out with 5 column volumes of 0.1 M Glycine-HCl, pH 3.0, and the eluate was collected and immediately neutralized with 0.1 volume of 1 M Tris-HCl (1.5 M NaCl) at pH 8.5 in the eluate. 13 antibodies. All of the solutions used above need to be freshly prepared. After harvesting the IL-13 antibody, it was dialyzed against 1 x PBS for 4 hours to avoid endotoxin contamination. After the end of dialysis, the concentration was measured using a spectrophotometer or a kit, the purity of the antibody was determined using HPLC-SEC, and the endotoxin content of the antibody was detected using an endotoxin test kit (purchased from Lonza). The obtained IL-13 antibody was characterized (the procedure is as described in Examples 4 and 7 below).

实施例11:人源化抗IL-13抗体的检定Example 11: Verification of humanized anti-IL-13 antibody

将实施例10制备的人源化抗IL-13抗体分别进行如下的检定试验(操作步骤同实施例4和7)。The humanized anti-IL-13 antibodies prepared in Example 10 were subjected to the following assays (the procedure is the same as in Examples 4 and 7).

A、酶联免疫吸附实验(ELISA)检测抗体与免疫原A的结合A, enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to immunogen A

具体操作步骤同实施例4和7试验A。部分实验结果见图28-30和表31-33所示。表31-33说明，上述人源化抗IL-13抗体与重组人IL-13蛋白在ELISA水平结合。其中IgG对照为人IgG，表中的数据为OD _450nm值。 The specific procedure is the same as Tests A of Examples 4 and 7. Some experimental results are shown in Figure 28-30 and Table 31-33. Tables 31-33 illustrate that the above humanized anti-IL-13 antibody binds to recombinant human IL-13 protein at the ELISA level. The IgG control was human IgG and the data in the table is the OD _{450 nm} value.

表31-1人源化抗IL-13抗体与免疫原A的反应活性Table 31-1 Reactivity of humanized anti-IL-13 antibody with immunogen A

表31-2人源化抗IL-13抗体与免疫原A的反应活性Table 31-2 Reactivity of humanized anti-IL-13 antibody with immunogen A

表31-3人源化抗IL-13抗体与免疫原A的反应活性Table 31-3 Reactivity of humanized anti-IL-13 antibody with immunogen A

表31-4人源化抗IL-13抗体与免疫原A的反应活性Table 31-4 Reactivity of humanized anti-IL-13 antibody with immunogen A

表31-5人源化抗IL-13抗体与免疫原A的反应活性Table 31-5 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-1人源化抗IL-13抗体与免疫原A的反应活性Table 32-1 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-2人源化抗IL-13抗体与免疫原A的反应活性Table 32-2 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-3人源化抗IL-13抗体与免疫原A的反应活性Table 32-3 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-4人源化抗IL-13抗体与免疫原A的反应活性Table 32-4 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-5人源化抗IL-13抗体与免疫原A的反应活性Table 32-5 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-6人源化抗IL-13抗体与免疫原A的反应活性Table 32-6 Reactivity of humanized anti-IL-13 antibody with immunogen A

表32-7人源化抗IL-13抗体与免疫原A的反应活性Table 32-7 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-1人源化抗IL-13抗体与免疫原A的反应活性Table 33-1 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-2人源化抗IL-13抗体与免疫原A的反应活性Table 33-2 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-3人源化抗IL-13抗体与免疫原A的反应活性Table 33-3 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-4人源化抗IL-13抗体与免疫原A的反应活性Table 33-4 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-5人源化抗IL-13抗体与免疫原A的反应活性Table 33-5 Reactivity of humanized anti-IL-13 antibody with immunogen A

表33-6人源化抗IL-13抗体与免疫原A的反应活性Table 33-6 Reactivity of humanized anti-IL-13 antibody with immunogen A

B、受体配体结合阻滞实验B, receptor ligand binding block experiment

具体试验步骤请参见实施例4和7试验B部分。部分实验结果见图31～41和表34～44，表34～44说明，部分人源化抗IL-13抗体与人IL-13结合后，能阻滞人IL-13与细胞表面的受体人IL-13Ra1/hIL-4Ra异源二聚体的结合，或者能阻滞IL-13与细胞表面人IL-13Ra2受体的结合。其中IgG对照为人IgG，表格中数据为平均荧光强度。See Sections B and B of Examples 4 and 7 for specific test procedures. Some experimental results are shown in Figures 31-41 and Tables 34-44. Tables 34-44 show that some humanized anti-IL-13 antibodies bind to human IL-13 and block human IL-13 and cell surface receptors. Binding of human IL-13Ra1/hIL-4Ra heterodimer, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor. The IgG control was human IgG and the data in the table was the mean fluorescence intensity.

表34-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 34-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表34-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结Table 34-2 FACS detection of humanized anti-IL-13 antibody blocking IL-13 and cell surface receptor IL-13Ra2

表35-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 35-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表35-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 35-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表36-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 36-1 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表36-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 36-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表37-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 37-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表37-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 37-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表38-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 38-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表38-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 38-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表39-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 39-1 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表39-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 39-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表40-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 40-1 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表40-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 40-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表41-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 41-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表41-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 41-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表42-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 41-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表42-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 42-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表43-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 43-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表43-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 43-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

表44-1 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra1/IL-4Ra异源二聚体的结合Table 44-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer

表44-2 FACS检测人源化抗IL-13抗体阻滞IL-13与细胞表面受体IL-13Ra2的结合Table 44-2 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra2

具体操作步骤请参见实施例4的C试验部分。部分实验结果见图42-48和表45-51。表45-51说明，人源化抗IL-13抗体通过与人IL-13/人IL-13R130Q变体的结合，能够中和由人IL-13/人IL-13R130Q变体和TNFα共同刺激条件下诱导产生的A549细胞分泌TARC。表45-51的数据为培养上清中TARC的浓度(pg/ml)，其中IgG对照为人IgG。For the specific operation steps, please refer to the C test part of Example 4. Some experimental results are shown in Figures 42-48 and Tables 45-51. Tables 45-51 illustrate that humanized anti-IL-13 antibodies are capable of neutralizing co-stimulation conditions by human IL-13/human IL-13R130Q variant and TNFα by binding to human IL-13/human IL-13R130Q variant. The under-induced A549 cells secrete TARC. The data in Tables 45-51 are the concentration of TARC (pg/ml) in the culture supernatant, where the IgG control is human IgG.

表45-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 45-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表45-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 45-2 Humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant induced thymic activation regulating chemokine secretion

表46-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 46-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表46-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 46-2 Humanized anti-IL-13 antibody neutralizes human IL-13R130Q variant-induced thymic activation-regulated chemokine secretion

表47-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 47-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表47-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 47-2 Humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant induced thymic activation regulating chemokine secretion

表48-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 48-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表48-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 48-2 Humanized anti-IL-13 antibody neutralizes human IL-13R130Q variant-induced thymic activation-regulated chemokine secretion

表49-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 4-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表49-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 49-2 Humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant induced thymic activation regulating chemokine secretion

表50-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 50-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表50-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 50-2 Humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant induced thymic activation regulating chemokine secretion

表51-1人源化抗IL-13抗体中和IL-13诱导的胸腺活化调节趋化因子分泌Table 51-1 Humanized anti-IL-13 antibody neutralizes IL-13-induced thymic activation and regulates chemokine secretion

表51-2人源化抗IL-13抗体中和人IL-13R130Q变体诱导的胸腺活化调节趋化因子分泌Table 51-2 Humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant-induced thymic activation regulating chemokine secretion

实施例12：人源化抗IL-13抗体的表征分析(Biacore)Example 12: Characterization analysis of humanized anti-IL-13 antibody (Biacore)

为了评估人源化抗IL-13抗体的结合特异性和亲和力，使用固定在CM5芯片上的人IL-13抗原进行Biacore分析。Biacore技术利用抗体与固定在表层上的IL-13抗原结合后表层上的折射率改变。结合通过从表面折射的激光的表面等离子体共振(SPR)进行检测。信号动力学结合速率和解离速率的分析允许区分非特异性和特异性相互作用。To assess the binding specificity and affinity of the humanized anti-IL-13 antibody, Biacore analysis was performed using human IL-13 antigen immobilized on a CM5 chip. The Biacore technique utilizes a change in the refractive index of the surface layer of the antibody after binding to the IL-13 antigen immobilized on the surface layer. The detection is performed in combination with surface plasmon resonance (SPR) of a laser refracted from the surface. Analysis of signal kinetics binding rate and dissociation rate allows for the discrimination of non-specific and specific interactions.

具体操作步骤同实施例9，部分实验结果见表52。表52说明，实施例10制备的人源化抗IL-13抗体与人IL-13的亲和力KD<9×10 ^-8M。 The specific operation steps are the same as those in Embodiment 9, and some experimental results are shown in Table 52. Table 52 shows that the affinity of the humanized anti-IL-13 antibody prepared in Example 10 to human IL-13 was KD < 9 x 10 ^-8 M.

表52人源化抗IL-13抗体与人IL-13亲和力检测结果Table 52: Detection results of humanized anti-IL-13 antibody and human IL-13 affinity

AntigenAntigen AntiboyAntiboy ka(1/Ms)Ka(1/Ms) kd(1/s)Kd(1/s) KD(M)KD(M) hIL-13hIL-13 c29D9H8c29D9H8 3.90E+073.90E+07 9.62E-049.62E-04 2.47E-112.47E-11 hIL-13hIL-13 hu-29D9H8-19hu-29D9H8-19 5.11E+075.11E+07 1.44E-031.44E-03 2.81E-112.81E-11 hIL-13hIL-13 hu-29D9H8-3hu-29D9H8-3 6.42E+076.42E+07 3.33E-033.33E-03 5.18E-115.18E-11 hIL-13hIL-13 hu-29D9H8-10hu-29D9H8-10 4.46E+074.46E+07 2.56E-032.56E-03 5.74E-115.74E-11 hIL-13hIL-13 hu-28A2E11-6hu-28A2E11-6 1.28E+061.28E+06 3.90E-043.90E-04 3.03E-103.03E-10 hIL-13hIL-13 c28A2E11c28A2E11 8.10E+058.10E+05 3.29E-043.29E-04 4.06E-104.06E-10 hIL-13hIL-13 hu-35E2C3-21hu-35E2C3-21 3.02E+053.02E+05 2.21E-042.21E-04 7.33E-107.33E-10 hIL-13hIL-13 c35E2C3c35E2C3 2.89E+052.89E+05 2.48E-042.48E-04 8.60E-108.60E-10 hIL-13hIL-13 hu-35E2C3-20hu-35E2C3-20 3.22E+053.22E+05 3.29E-043.29E-04 1.02E-091.02E-09 hIL-13hIL-13 hu-35E2C3-19hu-35E2C3-19 2.96E+052.96E+05 3.19E-043.19E-04 1.08E-091.08E-09 hIL-13hIL-13 hu-35E2C3-9hu-35E2C3-9 4.40E+054.40E+05 1.37E-031.37E-03 3.11E-093.11E-09 hIL-13hIL-13 hu-35E2C3-10hu-35E2C3-10 5.06E+055.06E+05 1.71E-031.71E-03 3.37E-093.37E-09 hIL-13hIL-13 hu-35E2C3-11hu-35E2C3-11 5.30E+055.30E+05 1.79E-031.79E-03 3.38E-093.38E-09

实施例13：IL-13人源化抗体抑制人IL-13诱导的小鼠肺杯状细胞化生及黏液过度分泌实验Example 13: IL-13 humanized antibody inhibits human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice

雌性Balb/c小鼠(8-12周龄，购自上海灵畅生物科技有限公司)接收后在SPF级饲养，适应1周后，开始实验。小鼠在第一天及第三天腹腔注射制备的人源化抗体，克隆号分别为：IgG对照，Tab2(根据专利US 8,088,618B2中克隆228B/C氨基酸序列表达纯化完成)、Tab3(根据专利US7935343B2中克隆BAK0502G9氨基酸序列表达纯化完成)、Hu-35E2C3-19，每只动物200μL(3mg或0.3mg抗体每公斤体重)。第二天及第四天用实施例1制备的免疫原A进行刺激诱导，剂量为1mg/mL，每只动物给予气道喷雾25μL。另有一组老鼠不给抗体，也不给免疫原A，最为阴性对照组。第六天进行安乐死，取肺组织，放入10％(v/v)福尔马林固定。将固定好的肺组织进行石蜡包埋，切片，通过PAS染色法进行染色，观察杯状细胞化生(粘液分泌)状况。Female Balb/c mice (8-12 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were received at the SPF level after receiving, and the experiment was started after 1 week of adaptation. The humanized antibodies prepared by intraperitoneal injection of mice on the first day and the third day were: IgG control, Tab2 (completed according to the amino acid sequence of clone 228B/C in US Pat. No. 8,088,618B2), Tab3 (according to patent In US7935343B2, the cloned BAK0502G9 amino acid sequence was expressed and purified), Hu-35E2C3-19, 200 μL per animal (3 mg or 0.3 mg antibody per kg body weight). On the second and fourth days, the immunogen A prepared in Example 1 was used for stimulation induction at a dose of 1 mg/mL, and each animal was given an airway spray of 25 μL. Another group of mice did not give antibodies, nor immunogen A, the most negative control group. On the sixth day, euthanasia was performed and lung tissue was taken and placed in 10% (v/v) formalin. The fixed lung tissue was paraffin-embedded, sectioned, stained by PAS staining, and the goblet cell metaplasia (mucus secretion) was observed.

部分实验结果见图49和表53。表53说明，小鼠在人IL-13，或者说免疫原A的刺激之下会诱发肺组织内的杯状细胞化生及粘液过度分泌，使PAS染色阳性区域占比增大。人源化抗体会与人IL-13结合，并中和由人IL-13诱导产生的杯状细胞化生及粘液过度分泌。表中数据为每组中不同老鼠肺组织中PAS染色阳性区域占比的平均值；IgG对照为人IgG。Some experimental results are shown in Figure 49 and Table 53. Table 53 shows that mice stimulated goblet cell metaplasia and excessive mucus secretion in lung tissue under the stimulation of human IL-13, or immunogen A, and increased the proportion of PAS staining positive regions. Humanized antibodies bind to human IL-13 and neutralize goblet cell metaplasia and mucus hypersecretion induced by human IL-13. The data in the table is the average of the proportion of PAS staining positive areas in the lung tissue of different mice in each group; the IgG control is human IgG.

表53 IL-13人源化抗体抑制人IL-13诱导的小鼠肺杯状细胞化生实验Table 53 IL-13 humanized antibody inhibits human IL-13 induced mouse goblet cell metaplasia experiment

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

本发明涉及的序列信息如下：The sequence information related to the present invention is as follows:

IL-13抗体的氨基酸序列编号Amino acid sequence number of IL-13 antibody

表54Table 54

表55Table 55

表56Table 56

表57Table 57

IL-13抗体的氨基酸序列编号对应的氨基酸序列：The amino acid sequence corresponding to the amino acid sequence number of the IL-13 antibody:

表58Table 58

IL-13抗体编码氨基酸的核苷酸序列编号：The nucleotide sequence number of the amino acid encoding the IL-13 antibody:

表59Table 59

表60Table 60

表61Table 61

表62Table 62

IL-13抗体的编码氨基酸序列对应的核苷酸序列Nucleotide sequence corresponding to the amino acid sequence of the IL-13 antibody

表63Table 63

Claims

A humanized antibody comprising a heavy chain variable region and/or a light chain variable region;

Wherein the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

CDR1 set forth in SEQ ID NO: 18, SEQ ID NO: 2 or SEQ ID NO: 10,

CDR2 of SEQ ID NO: 19, SEQ ID NO: 3 or SEQ ID NO: 11, and

CDR3 of SEQ ID NO:20, SEQ ID NO:4 or SEQ ID NO:12;

The light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

CDR1 of SEQ ID NO:22, SEQ ID NO:6 or SEQ ID NO:14,

CDR2 of SEQ ID NO: 23, SEQ ID NO: 7 or SEQ ID NO: 15, and

CDR3 of SEQ ID NO:24, SEQ ID NO:8 or SEQ ID NO:16;

Wherein, any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining IL-13 binding affinity.

The humanized antibody of claim 1, wherein the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO: 18,

CDR2 shown in SEQ ID NO: 19, and

CDR3 shown in SEQ ID NO:20;

and / or

CDR1 shown in SEQ ID NO: 22,

CDR2 shown in SEQ ID NO: 23, and

CDR3 shown in SEQ ID NO:24.

The humanized antibody of claim 1, wherein the heavy chain and light chain variable region sequences of the humanized antibody are as shown in Table A:

Table A

Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. Hu-29D9H8-1 26 33 Hu-29D9H8-2 26 34

Hu-29D9H8-3 26 35 Hu-29D9H8-4 27 32 Hu-29D9H8-5 27 33 Hu-29D9H8-6 27 34 Hu-29D9H8-7 27 35 Hu-29D9H8-8 28 32 Hu-29D9H8-9 28 33 Hu-29D9H8-10 28 34 Hu-29D9H8-11 28 35 Hu-29D9H8-12 29 32 Hu-29D9H8-13 29 33 Hu-29D9H8-14 29 34 Hu-29D9H8-15 29 35 Hu-29D9H8-16 30 32 Hu-29D9H8-17 30 33 Hu-29D9H8-18 30 34 Hu-29D9H8-19 30 35 Hu-29D9H8-20 31 32 Hu-29D9H8-21 31 33 Hu-29D9H8-22 31 34 Hu-29D9H8-23 31 35 Hu-28A2E11-1 37 43 Hu-28A2E11-2 37 44 Hu-28A2E11-3 37 45 Hu-28A2E11-4 37 46 Hu-28A2E11-5 37 47 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-8 38 43

Hu-28A2E11-9 38 44 Hu-28A2E11-10 38 45 Hu-28A2E11-11 38 46 Hu-28A2E11-12 38 47 Hu-28A2E11-13 38 48 Hu-28A2E11-14 38 49 Hu-28A2E11-15 39 43 Hu-28A2E11-16 39 44 Hu-28A2E11-17 39 45 Hu-28A2E11-18 39 46 Hu-28A2E11-19 39 47 Hu-28A2E11-20 39 48 Hu-28A2E11-21 40 43 Hu-28A2E11-22 40 44 Hu-28A2E11-23 40 45 Hu-28A2E11-24 40 46 Hu-28A2E11-25 40 47 Hu-28A2E11-26 41 43 Hu-28A2E11-27 41 44 Hu-28A2E11-28 41 45 Hu-28A2E11-29 41 46 Hu-28A2E11-31 42 43 Hu-28A2E11-32 42 44 Hu-28A2E11-33 42 45 Hu-28A2E11-34 42 46 Hu-35E2C3-1 51 58 Hu-35E2C3-2 51 59 Hu-35E2C3-3 51 60 Hu-35E2C3-4 52 58

Hu-35E2C3-5 52 59 Hu-35E2C3-6 52 60 Hu-35E2C3-7 53 58 Hu-35E2C3-8 53 59 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 Hu-35E2C3-12 54 60 Hu-35E2C3-13 55 58 Hu-35E2C3-14 55 59 Hu-35E2C3-15 55 60 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60

.

The humanized antibody of claim 1, wherein said humanized antibody further comprises a heavy chain constant region and/or a light chain constant region.

The humanized antibody of claim 1, wherein the heavy chain variable region of the humanized antibody further comprises a framework region of human origin, and/or the light chain of the humanized antibody is variable The district also includes a framework area for human resources.

The humanized antibody according to claim 1, wherein the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59); and/or

The light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9 , DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20.

The humanized antibody according to claim 1, wherein the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 FR1, FR2, FR3 of (VH4-59); and JH fragments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these germ lines, or the consensus sequence of the heavy-chain framework regions.

The humanized antibody according to claim 1, wherein the light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9, DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20, preferably FR1, FR2, FR3 of these germplasms; Fragments Jk1, Jk2, Jk3, Jk4 and JK5, in particular the FR4-encoded sequences of these lines.

The humanized antibody of claim 1, wherein the heavy chain and light chain variable region sequences of the humanized antibody are as shown in Table B:

Table B

Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. Hu-35E2C3-19 57 58 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59

.

The humanized antibody of claim 1, wherein the heavy chain and light chain variable region sequences of the humanized antibody are as shown in Table C:

Table C

Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. Hu-29D9H8-10 28 34 Hu-29D9H8-19 30 35

Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-20 39 48

.

A recombinant protein characterized in that the recombinant protein comprises:

(i) any of the heavy chain variable region sequences and any of the light chain variable region sequences set forth in Table 2;

(ii) an optional tag sequence that facilitates expression and/or purification;

Table 2

Antibody number Heavy chain variable region SEQ ID NO. Light chain variable region SEQ ID NO. c29D9H8 1 5 Hu-29D9H8-1 26 33 Hu-29D9H8-2 26 34 Hu-29D9H8-3 26 35 Hu-29D9H8-4 27 32 Hu-29D9H8-5 27 33 Hu-29D9H8-6 27 34 Hu-29D9H8-7 27 35 Hu-29D9H8-8 28 32 Hu-29D9H8-9 28 33 Hu-29D9H8-10 28 34 Hu-29D9H8-11 28 35 Hu-29D9H8-12 29 32 Hu-29D9H8-13 29 33 Hu-29D9H8-14 29 34 Hu-29D9H8-15 29 35 Hu-29D9H8-16 30 32 Hu-29D9H8-17 30 33 Hu-29D9H8-18 30 34 Hu-29D9H8-19 30 35 Hu-29D9H8-20 31 32

Hu-29D9H8-21 31 33 Hu-29D9H8-22 31 34 Hu-29D9H8-23 31 35 c28A2E11 9 13 Hu-28A2E11-1 37 43 Hu-28A2E11-2 37 44 Hu-28A2E11-3 37 45 Hu-28A2E11-4 37 46 Hu-28A2E11-5 37 47 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-8 38 43 Hu-28A2E11-9 38 44 Hu-28A2E11-10 38 45 Hu-28A2E11-11 38 46 Hu-28A2E11-12 38 47 Hu-28A2E11-13 38 48 Hu-28A2E11-14 38 49 Hu-28A2E11-15 39 43 Hu-28A2E11-16 39 44 Hu-28A2E11-17 39 45 Hu-28A2E11-18 39 46 Hu-28A2E11-19 39 47 Hu-28A2E11-20 39 48 Hu-28A2E11-21 40 43 Hu-28A2E11-22 40 44 Hu-28A2E11-23 40 45 Hu-28A2E11-24 40 46 Hu-28A2E11-25 40 47

Hu-28A2E11-26 41 43 Hu-28A2E11-27 41 44 Hu-28A2E11-28 41 45 Hu-28A2E11-29 41 46 Hu-28A2E11-31 42 43 Hu-28A2E11-32 42 44 Hu-28A2E11-33 42 45 Hu-28A2E11-34 42 46 c35E2C3 17 twenty one Hu-35E2C3-1 51 58 Hu-35E2C3-2 51 59 Hu-35E2C3-3 51 60 Hu-35E2C3-4 52 58 Hu-35E2C3-5 52 59 Hu-35E2C3-6 52 60 Hu-35E2C3-7 53 58 Hu-35E2C3-8 53 59 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 Hu-35E2C3-12 54 60 Hu-35E2C3-13 55 58 Hu-35E2C3-14 55 59 Hu-35E2C3-15 55 60 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59

Hu-35E2C3-21 57 60

.

A polynucleotide, characterized in that the polynucleotide encodes a polypeptide selected from the group consisting of:

(1) any of the heavy chain variable region sequences and any of the light chain variable region sequences shown in Table 2;

(2) The recombinant protein of claim 11.

The polynucleotide according to claim 12, wherein the polynucleotide encoding the heavy chain variable region is represented by SEQ ID NO: 117, 88, 97, 77, 61 or 69; and/or

The polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 118, 94, 108, 81, 65 or 73.

The polynucleotide according to claim 13, wherein the polynucleotide encoding the heavy chain variable region is represented by SEQ ID NO: 117, and the polynucleotide encoding the light chain variable region is as SEQ ID NO: 118; or

The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 88, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 94;

or

The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 97, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 108;

or

The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 77, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 81;

or

The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 61, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 65;

or

The polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 69, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO:73.

A vector comprising the polynucleotide of any one of claims 12-14 of the present invention.

A genetically engineered host cell, comprising the vector of claim 15 or a polynucleotide in which the polynucleotide of any one of claims 12-14 is integrated.

An antibody conjugate, characterized in that the antibody conjugate comprises:

(a) an antibody portion, which is a humanized antibody of any one of claims 1 to 10;

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

An immune cell characterized in that the immune cell expresses or is exposed to the humanized antibody of any one of claims 1 to 10 outside the cell membrane.

A pharmaceutical composition, characterized in that the pharmaceutical composition comprises:

(i) an active ingredient, which is selected from the group consisting of the humanized antibody of any one of claims 1 to 10, the recombinant protein of claim 11, the antibody of claim 17, a conjugate, the immune cell of claim 18, or a combination thereof;

(ii) a pharmaceutically acceptable carrier.

Use of an active ingredient, wherein the active ingredient is selected from the group consisting of the humanized antibody of any one of claims 1 to 10, the recombinant protein of claim 11, according to the claims The antibody conjugate of claim 18, the immune cell of claim 18, or a combination thereof, wherein the active ingredient is used to (a) prepare a diagnostic reagent; and/or (b) prepare a prophylactic and/or therapeutic IL -13 related diseases of the drug.

A method for detecting IL-13 protein in a sample in vitro, characterized in that the method comprises the steps of:

(1) In vitro, the sample, the humanized antibody according to any one of claims 1 to 10, the recombinant protein according to claim 11, the antibody conjugate according to claim 17, The immune cell of claim 18, or a combination thereof;

(2) Detecting whether an antigen-antibody complex is formed, wherein formation of a complex means that IL-13 protein is present in the sample.

A composition for detecting IL-13 protein in a sample in vitro, comprising the humanized antibody of any one of claims 1 to 10, the recombinant protein of claim 11, according to the claims The antibody conjugate according to 17, the immune cell of claim 18, or a combination thereof as an active ingredient.

A test panel, comprising: a substrate (support plate) and a test strip, the test strip comprising the humanized antibody of any one of claims 1 to 10, such as The recombinant protein according to claim 11, the antibody conjugate according to claim 17, the immune cell according to claim 18, or a combination thereof.

A method for preparing a recombinant polypeptide, the method comprising:

(a) cultivating the host cell of claim 16 under conditions suitable for expression;

(b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide of the humanized antibody of any one of claims 1 to 10 or the recombinant protein of claim 11.

A method of treating bronchial asthma, comprising: using the humanized antibody of any one of claims 1 to 10, the recombinant protein of claim 11, the method of claim 17, An antibody conjugate, the immune cell of claim 18.