CN118922437A - Methods of treating fibrosis and arrhythmia with neuregulin-1 fusion proteins - Google Patents

Abstract

The present invention relates to a recombinant fusion protein comprising a fragment of the neuroprotective protein neuregulin-1 (NRG-1) fused to the backbone of a HER3 monoclonal antibody (mAb), and to a method of treating atrial tremor and/or cardiac fibrosis in a subject in need thereof comprising administering a therapeutically effective amount of a recombinant fusion protein disclosed herein or a pharmaceutical composition comprising a recombinant fusion protein.

Description

Translated fromChinese

用神经调节蛋白-1融合蛋白治疗纤维化及心律失常的方法Methods for treating fibrosis and arrhythmias using neuregulin-1 fusion proteins

相关申请案的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本申请案主张于2022年3月15日提交的美国临时申请案第63/319,886号及于2022年12月1日提交的美国临时申请案第63/385,705号的优先权及权益，该等临时申请案各自的内容以全文引用的方式并入本发明中。This application claims priority to and the benefits of U.S. Provisional Application No. 63/319,886, filed on March 15, 2022, and U.S. Provisional Application No. 63/385,705, filed on December 1, 2022, the contents of each of which are incorporated herein by reference in their entirety.

序列表以引用的方式并入Sequence Listing Incorporated by Reference

本申请案含有序列表，其已经由EFS-Web以XML格式提交且特此以全文引用的方式并入。该XML副本创建于2023年3月12日，命名为SBTI-003-001WO_SeqList_ST26.xml，大小为24,646字节。This application contains a sequence listing, which has been submitted in XML format by EFS-Web and is hereby incorporated by reference in its entirety. This XML copy was created on March 12, 2023, named SBTI-003-001WO_SeqList_ST26.xml, and is 24,646 bytes in size.

背景技术Background Art

神经调节蛋白(NRG；heregulin，HRG)，还称为神经胶质生长因子(GGF)及新分化因子(NDF)，是一类糖蛋白，分子量为44KD。NRG蛋白家族有四个成员：NRG-1、NRG-2、NRG-3及NRG-4。NRG(包括NRG-1)在心脏发育中发挥特别重要的作用。作为ErbB家族酪氨酸激酶受体的配体，NRG-1直接与膜结合型ErbB3或ErbB4结合，诱导二聚化产生ErbB2/ErbB4、ErbB2/ErbB3、ErbB3/ErbB3及ErbB4/ErbB4复合物，以及后续细胞内信号传导。在动物模型中，NRG的表达诱导旁分泌信号传导，以在胚胎发育期间促进心脏组织的生长及分化，而缺失ErbB2、ErbB4或NRG-1中的任一种均会导致胚胎死亡。此外，阻断ErbB2受体信号传导的癌症疗法已被证明具有显著的心脏毒性副作用，表明在人类中ErbB2介导的信号传导不仅对于发育至关重要，且对于健康心脏组织的稳态也至关重要。Neuregulin (NRG; heregulin, HRG), also known as glial growth factor (GGF) and new differentiation factor (NDF), is a class of glycoproteins with a molecular weight of 44KD. The NRG protein family has four members: NRG-1, NRG-2, NRG-3 and NRG-4. NRG (including NRG-1) plays a particularly important role in cardiac development. As a ligand of the ErbB family tyrosine kinase receptor, NRG-1 directly binds to membrane-bound ErbB3 or ErbB4, inducing dimerization to produce ErbB2/ErbB4, ErbB2/ErbB3, ErbB3/ErbB3 and ErbB4/ErbB4 complexes, as well as subsequent intracellular signal transduction. In animal models, the expression of NRG induces paracrine signal transduction to promote the growth and differentiation of cardiac tissue during embryonic development, and the absence of any of ErbB2, ErbB4 or NRG-1 will lead to embryonic death. Furthermore, cancer therapies that block ErbB2 receptor signaling have been shown to have significant cardiotoxic side effects, suggesting that in humans ErbB2-mediated signaling is critical not only for development but also for homeostasis of healthy cardiac tissue.

证据还显示，NRG-1信号转导在其他器官系统的发育及功能以及人类疾病(包括精神分裂症及头颈癌)的发病机制中发挥作用。NRG-1有许多异构体。对基因突变小鼠(基因敲除小鼠)的研究表明，具有不同N端区域或EGF样结构域的异构体具有不同的体内功能。本发明是基于NRG-1βa2同功异型物。Evidence also suggests that NRG-1 signaling plays a role in the development and function of other organ systems and in the pathogenesis of human diseases, including schizophrenia and head and neck cancer. NRG-1 has many isoforms. Studies of mutant mice (knockout mice) have shown that isoforms with different N-terminal regions or EGF-like domains have different functions in vivo. The present invention is based on the NRG-1βa2 isoform.

内源性NRG-1结合于ErbB3(HER3)及ErbB4(HER4)且经由两者诱导信号传导。许多临床前及临床研究已显示NRG-1在各种心血管适应症中的治疗潜力，主要经由其与心肌细胞表达的ErbB4(HER4)的相互作用。然而，三个关键因素限制重组人类NRG-1(rhNRG-1)的临床应用及效用。第一，NRG-1经由HER3的信号传导可能会促进癌症的发展和/或进展，从而引起对需要长期给药或无严重心血管(CV)风险因素的任何应用的重大关注。第二，NRG-1过度激活HER3可能会破坏胃肠道(GI)上皮完整性及稳态，导致严重的GI毒性，从而失去NRG-1的治疗窗口。第三，rhNRG-1的两个临床阶段活性蛋白片段均显示短半衰期，表明可能需要繁重的剂量及给药方案来达成所需的治疗暴露水平。因此，需要提供一种基于NRG-1的治疗剂，其在各种心血管适应症中保持临床上显著的治疗潜力，但具有较低的致癌或促进癌症进展的风险、更好的GI耐受性及更有利的药代动力学(PK)概况。Endogenous NRG-1 binds to ErbB3 (HER3) and ErbB4 (HER4) and induces signaling through both. Many preclinical and clinical studies have shown the therapeutic potential of NRG-1 in various cardiovascular indications, primarily through its interaction with ErbB4 (HER4) expressed by cardiomyocytes. However, three key factors limit the clinical application and utility of recombinant human NRG-1 (rhNRG-1). First, NRG-1 signaling through HER3 may promote the development and/or progression of cancer, raising significant concerns for any application requiring long-term dosing or in the absence of severe cardiovascular (CV) risk factors. Second, overactivation of HER3 by NRG-1 may disrupt gastrointestinal (GI) epithelial integrity and homeostasis, leading to severe GI toxicity, thereby losing the therapeutic window of NRG-1. Third, both clinical-stage active protein fragments of rhNRG-1 display short half-lives, suggesting that onerous dosing and dosing regimens may be required to achieve the desired therapeutic exposure levels. Therefore, there is a need to provide NRG-1-based therapeutics that maintain clinically significant therapeutic potential in various cardiovascular indications but have a lower risk of causing or promoting cancer progression, better GI tolerability, and more favorable pharmacokinetic (PK) profiles.

本发明通过提供一种包含rhNRG-1活性结构域与HER3特异性拮抗剂抗体的融合物的重组蛋白来解决这些需求：HER3信号传导以减轻rhNRG-1的致癌风险及GI毒性的方式被阻断，同时抗体主链形式赋予典型单克隆抗体的分子半衰期，使产品的给药及施用更加方便。The present invention addresses these needs by providing a recombinant protein comprising a fusion of the active domain of rhNRG-1 and a HER3-specific antagonist antibody: HER3 signaling is blocked in a manner that mitigates the carcinogenic risk and GI toxicity of rhNRG-1, while the antibody backbone format confers the molecular half-life of a typical monoclonal antibody, making the product more convenient to administer and apply.

发明内容Summary of the invention

本发明提供治疗受试者的心房震颤和/或心脏纤维化的方法，其包括向受试者施用包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段的重组融合蛋白。在一些实施方案中，心脏纤维化包括心房纤维化。The present invention provides a method of treating atrial fibrillation and/or cardiac fibrosis in a subject, comprising administering to the subject a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb). In some embodiments, cardiac fibrosis comprises atrial fibrosis.

本发明提供治疗受试者的心房震颤的方法，其包括向受试者施用包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段的重组融合蛋白。在一些实施方案中，心房震颤与心房纤维化有关。The present invention provides a method of treating atrial fibrillation in a subject comprising administering to the subject a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb). In some embodiments, atrial fibrillation is associated with atrial fibrosis.

本发明提供治疗受试者的心房纤维化的方法，其包括向受试者施用包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段的重组融合蛋白。The present invention provides a method of treating atrial fibrosis in a subject comprising administering to the subject a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

本发明提供一种重组融合蛋白，其包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段，用于治疗心房震颤和/或心脏纤维化的方法中。在一些实施方案中，心脏纤维化包括心房纤维化。The present invention provides a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for use in a method for treating atrial fibrillation and/or cardiac fibrosis. In some embodiments, cardiac fibrosis comprises atrial fibrosis.

在本发明使用的方法或重组融合蛋白的一些实施方案中，NRG-1片段包含NRG-1的活性结构域。在一些实施方案中，NRG-1片段包含ERBB3/4结合结构域。在一些实施方案中，NRG-1片段结合于ErbB4(HER4)且经由其诱导信号传导。在一些实施方案中，mAb抑制经由ErbB3(HER3)的NRG-1信号传导。在一些实施方案中，NRG-1片段包含NRG-1β2a同功异型物。In some embodiments of the methods or recombinant fusion proteins used in the present invention, the NRG-1 fragment comprises an active domain of NRG-1. In some embodiments, the NRG-1 fragment comprises an ERBB3/4 binding domain. In some embodiments, the NRG-1 fragment binds to ErbB4 (HER4) and induces signal transduction therethrough. In some embodiments, the mAb inhibits NRG-1 signal transduction via ErbB3 (HER3). In some embodiments, the NRG-1 fragment comprises an NRG-1β2a isoform.

在本发明使用的方法或重组融合蛋白的一些实施方案中，NRG-1片段经由其N端氨基酸使用连接子与抗体重链的C端融合。在一些实施方案中，连接子包含至少一个拷贝的SEQ ID NO:5所示的Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser连接子。在一些实施方案中，抗体重链的C端包含抗体的Fc结构域。在一些实施方案中，单克隆抗体是糖基化的。In some embodiments of the methods or recombinant fusion proteins used in the present invention, the NRG-1 fragment is fused to the C-terminus of the antibody heavy chain via its N-terminal amino acid using a linker. In some embodiments, the linker comprises at least one copy of the Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser linker shown in SEQ ID NO:5. In some embodiments, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody. In some embodiments, the monoclonal antibody is glycosylated.

在本发明使用的方法或重组融合蛋白的一些实施方案中，NRG-1片段包含SEQ IDNO:4的氨基酸序列。在一些实施方案中，mAb包含SEQ ID NO:2的重链氨基酸序列。在一些实施方案中，mAb包含SEQ ID NO:3的轻链氨基酸序列。在一些实施方案中，mAb在SEQ ID NO:2的氨基酸234、239及434中的至少一个中包含取代突变。在一些实施方案中，至少一个取代突变包含L234F突变、S239A突变、N434A突变或其组合。在一些实施方案中，重组融合蛋白包含SEQ ID NO:3及SEQ ID NO:14的氨基酸序列。In some embodiments of the methods or recombinant fusion proteins used in the present invention, the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the mAb comprises the heavy chain amino acid sequence of SEQ ID NO: 2. In some embodiments, the mAb comprises the light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the mAb comprises a substitution mutation in at least one of amino acids 234, 239, and 434 of SEQ ID NO: 2. In some embodiments, at least one substitution mutation comprises an L234F mutation, an S239A mutation, an N434A mutation, or a combination thereof. In some embodiments, the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 14.

在本发明使用的方法或重组融合蛋白的一些实施方案中，相对于重组NRG-1的信号诱导潜力，重组融合蛋白促进HER2/4信号传导超过HER2/3信号传导。在一些实施方案中，重组融合蛋白促进受试者的心肌细胞或心脏组织的增殖、分化及存活。在一些实施方案中，相对于重组NRG-1，重组融合蛋白减弱肿瘤或癌症细胞的增殖。In some embodiments of the methods or recombinant fusion proteins used in the present invention, the recombinant fusion protein promotes HER2/4 signaling over HER2/3 signaling relative to the signal induction potential of recombinant NRG-1. In some embodiments, the recombinant fusion protein promotes proliferation, differentiation and survival of cardiomyocytes or cardiac tissue in a subject. In some embodiments, the recombinant fusion protein attenuates the proliferation of tumor or cancer cells relative to recombinant NRG-1.

在本发明使用的方法或重组融合蛋白的一些实施方案中，施用重组融合蛋白减少心房震颤发作的持续时间，或降低心房震颤发生的频率。在一些实施方案中，施用重组融合蛋白减少心房震颤或心房纤维化的体征或症状。在一些实施方案中，心房震颤的症状包括心跳不规则、心悸、头晕、极度疲劳、呼吸短促、胸痛或其组合。在一些实施方案中，心房纤维化的体征包括胶原蛋白沉积，而施用重组融合蛋白减少心房组织中的胶原蛋白沉积。In some embodiments of the methods or recombinant fusion proteins used in the present invention, the administration of the recombinant fusion protein reduces the duration of atrial fibrillation episodes, or reduces the frequency of atrial fibrillation. In some embodiments, the administration of the recombinant fusion protein reduces the signs or symptoms of atrial fibrillation or atrial fibrosis. In some embodiments, the symptoms of atrial fibrillation include irregular heartbeat, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, or a combination thereof. In some embodiments, the signs of atrial fibrosis include collagen deposition, and the administration of the recombinant fusion protein reduces collagen deposition in atrial tissue.

在一些实施方案中，NRG-1结合于ErbB4(HER4)且经由其诱导信号传导。在一些实施方案中，mAb抑制经由ErbB3(HER3)的NRG-1信号传导。In some embodiments, NRG-1 binds to and induces signaling through ErbB4 (HER4). In some embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3).

本发明提供一种药盒，其包含有效量的本发明的重组融合蛋白或包含本发明的重组融合蛋白的药物组合物。The present invention provides a medicine kit, which comprises an effective amount of the recombinant fusion protein of the present invention or a pharmaceutical composition comprising the recombinant fusion protein of the present invention.

本发明提供一种重组融合蛋白，其包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段，用于制造治疗心房震颤和/或心脏纤维化的药物。The present invention provides a recombinant fusion protein, which comprises a neuregulin-1 (NRG-1) fragment fused with a monospecific ErbB3 (HER3) monoclonal antibody (mAb), and is used for manufacturing a drug for treating atrial fibrillation and/or cardiac fibrosis.

本发明的其他特征及优点将从以下详细描述的实例及附图中显而易见。但是，应当理解的是，虽然详细描述和具体实例指示本发明实施例，但仅以举例说明的方式给出，因为本领域的技术人员从这一详细描述中将显而易见在本发明的精神和范围内的各种变化和修改。Other features and advantages of the present invention will be apparent from the examples and drawings described in detail below. However, it should be understood that although the detailed description and specific examples indicate embodiments of the present invention, they are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

参照以下详细描述及所附权利要求并结合附图，本发明的各种目的及优点以及更完整的理解将显而易见且更容易理解，其中：Various objects and advantages of the present invention and a more complete understanding will become apparent and more readily appreciated by referring to the following detailed description and appended claims taken in conjunction with the accompanying drawings, in which:

图1显示用于表达本发明所公开的重组融合蛋白的表达质粒的构建。FIG. 1 shows the construction of an expression plasmid for expressing the recombinant fusion protein disclosed in the present invention.

图2A显示本发明的抗HER3 mAb/NRG-1融合蛋白的分子示意图。FIG2A shows a molecular schematic diagram of the anti-HER3 mAb/NRG-1 fusion protein of the present invention.

图2B显示SDS-PAGE分析生成的代表性数据。Figure 2B shows representative data generated by SDS-PAGE analysis.

图2C显示通过对包含HER3/4结合域的NRG-1的61个氨基酸活性片段(“NRG-1”，R&DSystems,Minneapolis,MN)具有特异性的一抗检测到的蛋白质印迹结果。FIG. 2C shows the results of Western blot detection by a primary antibody specific for a 61 amino acid active fragment of NRG-1 containing the HER3/4 binding domain ("NRG-1", R&D Systems, Minneapolis, MN).

图2D显示通过对IgG具有特异性的一抗检测到的蛋白质印迹结果。FIG2D shows the results of Western blot detection by a primary antibody specific for IgG.

图3示出了结合分析，显示本发明所公开的重组融合蛋白结合于HER3蛋白(曲线1，步骤2)且可同时结合抗NRG-1抗体(曲线1，步骤3)。请注意，Fc突变被引入本发明所公开的重组融合蛋白中，以敲除编码HER3特异性抗体的亲本抗体序列的Fc效应功能，从而可以减轻对表达HER3受体的正常组织的不期望的细胞毒性。Figure 3 shows a binding analysis showing that the recombinant fusion protein disclosed in the present invention binds to the HER3 protein (curve 1, step 2) and can simultaneously bind to the anti-NRG-1 antibody (curve 1, step 3). Please note that the Fc mutation is introduced into the recombinant fusion protein disclosed in the present invention to knock out the Fc effector function of the parent antibody sequence encoding the HER3-specific antibody, thereby reducing the undesirable cytotoxicity to normal tissues expressing the HER3 receptor.

图4A至4D示出了代表性图表，显示用抗HER3 mAb/NRG-1融合蛋白或对照处理的不同癌细胞株的平均相对生长率±SEM(n＝3)。图4A显示NCI-N87胃癌细胞株的平均相对生长率。图4B显示MCF-7乳癌细胞株的平均相对生长率。图4C显示RT-112膀胱癌细胞株的平均相对生长率。图4D显示T47D乳癌细胞株的平均相对生长率。与对照NRG-1肽及GP120 mAb/NRG-1融合蛋白相比，本发明提供的重组融合蛋白在促进癌细胞增殖方面表现出明显较低的活性。Figures 4A to 4D show representative graphs showing the mean relative growth rate ± SEM (n = 3) of different cancer cell lines treated with anti-HER3 mAb/NRG-1 fusion protein or control. Figure 4A shows the mean relative growth rate of NCI-N87 gastric cancer cell line. Figure 4B shows the mean relative growth rate of MCF-7 breast cancer cell line. Figure 4C shows the mean relative growth rate of RT-112 bladder cancer cell line. Figure 4D shows the mean relative growth rate of T47D breast cancer cell line. Compared with the control NRG-1 peptide and GP120 mAb/NRG-1 fusion protein, the recombinant fusion protein provided by the present invention exhibits significantly lower activity in promoting cancer cell proliferation.

图5A至5B显示尽管癌细胞生长潜力降低，但本发明提供的重组融合蛋白完全保留在心肌细胞中诱导PI3K/AKT信号传导的能力-显示与重组NRG-1及GP120 mAb/NRG-1融合蛋白的活性相当。图5A为显示用本发明的重组融合蛋白及对照处理的人类心肌细胞中磷酸-AKT(pAKT)与总AKT(tAKT)的相对比率与抗体浓度(以nM为单位)的图。图5B为用本发明的重组融合蛋白及对照处理的人类心肌细胞中AKT磷酸化的蛋白质印迹分析。Figures 5A to 5B show that despite the reduced cancer cell growth potential, the recombinant fusion proteins provided by the present invention fully retain the ability to induce PI3K/AKT signaling in cardiomyocytes - showing comparable activity to recombinant NRG-1 and GP120 mAb/NRG-1 fusion proteins. Figure 5A is a graph showing the relative ratio of phospho-AKT (pAKT) to total AKT (tAKT) versus antibody concentration (in nM) in human cardiomyocytes treated with the recombinant fusion proteins of the present invention and controls. Figure 5B is a Western blot analysis of AKT phosphorylation in human cardiomyocytes treated with the recombinant fusion proteins of the present invention and controls.

图6A至6C显示在本发明所公开的重组融合蛋白及对照存在下HER2/4及HER2/3二聚化的直接比较。图6A显示用于检测配体诱导的二聚化的分析原理。由EurofinsDiscoverX(Fremont,CA)开发的PathHunter Dimerization Assay用于检测配体诱导的受体-二聚体对的两个亚基的二聚化。β-gal酶分成两个片段，即ProLink(PK)及酶受体(EA)。细胞经过工程改造以共表达与酶供体PK融合的靶蛋白1及与酶受体EA融合的靶蛋白2。配体与一个靶蛋白的结合诱导其与另一个靶蛋白相互作用，迫使两个酶片段互补且导致酶反应释放化学发光信号，该信号以相对荧光单位或RFU检测。图6B为显示本发明提供的重组融合蛋白可与NRG-1效力相当地诱导HER2/HER4二聚化的图。图6C为显示本发明提供的重组融合蛋白诱导HER2/HER3二聚化的效力显著低于NRG-1的图。这些发现进一步验证本发明提供的重组融合蛋白保留NRG-1的全部HER2/4信号传导潜力，同时显著减少HER2/3信号传导诱导。Figures 6A to 6C show a direct comparison of HER2/4 and HER2/3 dimerization in the presence of the recombinant fusion protein disclosed in the present invention and a control. Figure 6A shows the analytical principle for detecting ligand-induced dimerization. The PathHunter Dimerization Assay developed by Eurofins DiscoverX (Fremont, CA) is used to detect ligand-induced dimerization of two subunits of the receptor-dimer pair. The β-gal enzyme is divided into two fragments, namely ProLink (PK) and the enzyme receptor (EA). Cells are engineered to co-express target protein 1 fused to the enzyme donor PK and target protein 2 fused to the enzyme receptor EA. The binding of a ligand to one target protein induces its interaction with another target protein, forcing the two enzyme fragments to complement and causing the enzyme reaction to release a chemiluminescent signal, which is detected in relative fluorescence units or RFU. Figure 6B is a diagram showing that the recombinant fusion protein provided by the present invention can induce HER2/HER4 dimerization with comparable efficacy to NRG-1. Figure 6C is a diagram showing that the efficacy of the recombinant fusion protein provided by the present invention in inducing HER2/HER3 dimerization is significantly lower than that of NRG-1. These findings further verify that the recombinant fusion protein provided by the present invention retains the full HER2/4 signaling potential of NRG-1 while significantly reducing HER2/3 signaling induction.

图7显示本发明的抗HER3 mAb/NRG-1融合蛋白对包括人类、猴、大鼠及小鼠的不同物种的HER3抗原的结合亲和力。通过BIAcore分析确定的平衡解离速率(KD)分别为3.13×10^-10(人类)、3.97×10^-10(猴)、2.68×10^-9(大鼠)及2.77×10^-9(小鼠)。这些数据表明，本发明的重组融合蛋白对人类及猴HER3具有相似的结合亲和力，而其对啮齿类动物(大鼠及小鼠)HER3的亲和力降低大约一个数量级。Figure 7 shows the binding affinity of the anti-HER3 mAb/NRG-1 fusion protein of the present invention to HER3 antigens of different species including humans, monkeys, rats and mice. The equilibrium dissociation rates (KD) determined by BIAcore analysis were 3.13×^10-10 (human), 3.97×^10-10 (monkey), 2.68×^10-9 (rat) and 2.77×^10-9 (mouse), respectively. These data indicate that the recombinant fusion protein of the present invention has similar binding affinity to human and monkey HER3, while its affinity to rodent (rat and mouse) HER3 is reduced by about one order of magnitude.

图8为显示重组融合蛋白对冠状动脉结扎诱导的收缩性心脏衰竭大鼠模型中对射血分数(EF)的影响的图。FIG8 is a graph showing the effect of the recombinant fusion protein on ejection fraction (EF) in a rat model of systolic heart failure induced by coronary artery ligation.

图9A至9F为一是列6张影像，显示冠状动脉结扎诱导的收缩性心脏衰竭大鼠模型中心肌结构的组织病理学变化。收集手术部位附近的心脏组织且在4％甲醛中固定，随后制备石蜡切片且用H&E染色。图9A显示假手术对照大鼠的心脏组织。图9B显示用溶媒对照处理的收缩性心脏衰竭模型大鼠的心脏组织。图9C显示用GP120 mAb/NRG-1(10mg/kg)处理的收缩性心脏衰竭模型大鼠的心脏组织。图9D显示用抗HER3 mAb/NRG-1(1mg/kg)处理的收缩性心脏衰竭模型大鼠的心脏组织。图9E显示用抗HER3 mAb/NRG-1(3mg/kg)处理的收缩性心脏衰竭模型大鼠的心脏组织。图9F显示用抗HER3 mAb/NRG-1(10mg/kg)处理的收缩性心脏衰竭模型大鼠的心脏组织。Figures 9A to 9F are a series of 6 images showing histopathological changes in myocardial structure in a rat model of systolic heart failure induced by coronary artery ligation. Heart tissues near the surgical site were collected and fixed in 4% formaldehyde, followed by preparation of paraffin sections and staining with H&E. Figure 9A shows heart tissues of sham-operated control rats. Figure 9B shows heart tissues of systolic heart failure model rats treated with vehicle control. Figure 9C shows heart tissues of systolic heart failure model rats treated with GP120 mAb/NRG-1 (10 mg/kg). Figure 9D shows heart tissues of systolic heart failure model rats treated with anti-HER3 mAb/NRG-1 (1 mg/kg). Figure 9E shows heart tissues of systolic heart failure model rats treated with anti-HER3 mAb/NRG-1 (3 mg/kg). Figure 9F shows heart tissues of systolic heart failure model rats treated with anti-HER3 mAb/NRG-1 (10 mg/kg).

图10为显示使用NOD/SCID小鼠皮下FaDu癌异种移植模型评估活体内抗肿瘤活性的图。FIG. 10 is a graph showing the evaluation of in vivo anti-tumor activity using a NOD/SCID mouse subcutaneous FaDu cancer xenograft model.

图11为显示用本发明的重组融合蛋白及对照处理的肿瘤小鼠的体重变化的图。FIG. 11 is a graph showing changes in body weight of tumor-bearing mice treated with the recombinant fusion protein of the present invention and a control.

图12为显示重组融合蛋白在食蟹猕猴(猕猴)中的药代动力学曲线的图。FIG. 12 is a graph showing the pharmacokinetic profile of the recombinant fusion protein in cynomolgus monkeys (Macaca fascicularis).

图13A显示描述体外诱导大鼠心房组织样本纤维化的纤维化分析的图。FIG. 13A shows a graph depicting fibrosis analysis of in vitro induced fibrosis in rat atrial tissue samples.

图13B显示描述在图13A中所描述的分析中在不存在例示性NRG-1/HER3抗体融合蛋白的情况下诱导I型胶原蛋白的图。x轴描述取样日。y轴描述I型胶原蛋白的mRNA表达(I型胶原蛋白α1链或Col1a1，mRNA)，表示为存在的Col1a1 mRNA相较于在分析第1天存在的Col1a1 mRNA水平的倍数变化。Figure 13B shows a graph depicting the induction of type I collagen in the absence of an exemplary NRG-1/HER3 antibody fusion protein in the assay depicted in Figure 13A. The x-axis depicts the sampling day. The y-axis depicts mRNA expression of type I collagen (type I collagen alpha 1 chain or Col1a1, mRNA), expressed as the fold change of Col1a1 mRNA present compared to the level of Col1a1 mRNA present on day 1 of the assay.

图13C显示描述在图13A中所描述的分析中在不存在例示性NRG-1/HER3抗体融合蛋白的情况下诱导III型胶原蛋白的图。x轴描述取样日。y轴描述III型胶原蛋白的mRNA表达(III型胶原蛋白α链1或Col3a1i，mRNA)，表示为存在的Col3a1 mRNA相较于在分析第1天存在的Col3a1mRNA水平的倍数变化。Figure 13C shows a graph depicting the induction of type III collagen in the absence of an exemplary NRG-1/HER3 antibody fusion protein in the assay depicted in Figure 13A. The x-axis depicts the sampling day. The y-axis depicts the mRNA expression of type III collagen (type III collagen alpha chain 1 or Col3a1i, mRNA), expressed as the fold change of the Col3a1 mRNA present compared to the Col3a1 mRNA level present on day 1 of the assay.

图14A显示描述在图13A中所描述的分析中NRG-1/HER3抗体融合蛋白(NRG-1/HER3)对I型胶原蛋白诱导的影响的图。x轴描述取样日。y轴描述在不存在NRG-1/HER3的情况下I型胶原蛋白的mRNA表达，表示为存在的Col1a1 mRNA相较于在分析第2天存在的Col1a1mRNA水平的倍数变化。Figure 14A shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein (NRG-1/HER3) on type I collagen induction in the assay depicted in Figure 13A. The x-axis depicts the sampling day. The y-axis depicts the mRNA expression of type I collagen in the absence of NRG-1/HER3, expressed as the fold change of Col1a1 mRNA present compared to the Col1a1 mRNA level present on day 2 of the assay.

图14B显示描述在图13A中所描述的分析中NRG-1/HER3抗体融合蛋白(NRG-1/HER3)对III型胶原蛋白诱导的影响的图。x轴描述取样日。y轴描述在不存在NRG-1/HER3的情况下III型胶原蛋白的mRNA表达，表示为存在的Col3a1 mRNA相较于在分析第2天存在的Col3a1mRNA水平的倍数变化。Figure 14B shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein (NRG-1/HER3) on type III collagen induction in the assay depicted in Figure 13A. The x-axis depicts the sampling day. The y-axis depicts the mRNA expression of type III collagen in the absence of NRG-1/HER3, expressed as the fold change of Col3a1 mRNA present compared to the Col3a1 mRNA level present on day 2 of the assay.

图15显示描述在图16及图18中所描述的体内实验模型中使用经颈静脉八极导管进行程序化电刺激(PES)量测心房震颤(AF)诱导的图。15 shows graphs depicting the measurement of atrial fibrillation (AF) induction using programmed electrical stimulation (PES) using a transjugular octopole catheter in the in vivo experimental model depicted in FIGS. 16 and 18 .

图16显示描述使用血管收缩素II(Ang-II)诱导的高血压小鼠模型测量体内心房震颤的图。FIG. 16 shows graphs depicting the measurement of atrial fibrillation in vivo using angiotensin II (Ang-II)-induced hypertensive mouse model.

图17A显示描述NRG-1/HER3抗体融合蛋白对图16所描述的模型中总AF持续时间的影响的图。x轴描述处理条件：CTRL，假处理对照；ANG II+溶媒，仅溶媒对照，ANG II+NRG-1/HER3，用NRG-1/HER3抗体融合蛋白处理的患有ANG II诱导的高血压的动物。y轴描述以秒(s)为单位的时间。Figure 17A shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on total AF duration in the model depicted in Figure 16. The x-axis depicts treatment conditions: CTRL, sham control; ANG II+vehicle, vehicle only control, ANG II+NRG-1/HER3, animals with ANG II-induced hypertension treated with NRG-1/HER3 antibody fusion protein. The y-axis depicts time in seconds (s).

图17B显示描述NRG-1/HER3抗体融合蛋白对图16所描述的模型中AF诱导的影响的图。x轴描述处理。y轴描述患有AF的小鼠的百分比。Figure 17B shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on AF induction in the model depicted in Figure 16. The x-axis depicts the treatment. The y-axis depicts the percentage of mice with AF.

图18显示描述第二心房震颤(AF)模型的图，其中小鼠被饲喂高脂肪饮食。FIG. 18 shows graphs depicting a second atrial fibrillation (AF) model in which mice are fed a high fat diet.

图19A显示描述图18所描述的模型中三组小鼠随时间推移的体重的图。x轴描述以周(W)为单位的时间。y轴描述体重。CRTL：未饲喂高脂肪饮食且用单独溶媒对照处理的小鼠；HFD+溶媒：饲喂高脂肪饮食且用单独溶媒对照的小鼠；HFD+NRG-1/HER3，饲喂高脂肪饮食且用NRG-1/HER3抗体融合蛋白处理的小鼠。Figure 19A shows a graph depicting body weight over time for the three groups of mice in the model depicted in Figure 18. The x-axis depicts time in weeks (W). The y-axis depicts body weight. CRTL: mice not fed a high-fat diet and treated with vehicle alone control; HFD+vehicle: mice fed a high-fat diet and treated with vehicle alone control; HFD+NRG-1/HER3, mice fed a high-fat diet and treated with NRG-1/HER3 antibody fusion protein.

图19B显示描述NRG-1/HER3抗体融合蛋白对图18所描述的代谢综合征模型随时间推移的总体重的影响的图。x轴描述以周(W)为单位的时间。y轴描述体重的变化。Figure 19B shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on total body weight over time in the metabolic syndrome model depicted in Figure 18. The x-axis depicts time in weeks (W). The y-axis depicts the change in body weight.

图19C显示描述NRG-1/HER3抗体融合蛋白对图18所描述的模型中葡萄糖耐量的影响的图。x轴描述注射20％葡萄糖后的时间。y轴描述以mg/dL为单位的血糖浓度。Figure 19C shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on glucose tolerance in the model depicted in Figure 18. The x-axis depicts the time after injection of 20% glucose. The y-axis depicts the blood glucose concentration in mg/dL.

图20A显示描述NRG-1/HER3抗体融合蛋白对图18所描述的代谢综合征模型中不规则房性心律失常的总持续时间的影响的图。x轴描述处理条件：WT，野生型，无高脂肪饮食且仅注射溶媒；HFD+溶媒：饲喂高脂肪饮食且用单独溶媒对照的小鼠；HFD+NRG-1/HER3，饲喂高脂肪饮食且用NRG-1/HER3抗体融合蛋白处理的小鼠。y轴描述以秒(s)为单位的时间。Figure 20A shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on the total duration of irregular atrial arrhythmias in the metabolic syndrome model depicted in Figure 18. The x-axis depicts treatment conditions: WT, wild type, no high fat diet and vehicle only injection; HFD+vehicle: mice fed a high fat diet and controlled with vehicle alone; HFD+NRG-1/HER3, mice fed a high fat diet and treated with NRG-1/HER3 antibody fusion protein. The y-axis depicts time in seconds (s).

图20B显示描述NRG-1/HER3抗体融合蛋白对图18所描述的代谢综合征模型中心律失常诱导的影响的图。x轴描述处理条件：CRTL：未饲喂高脂肪饮食且用单独溶媒对照处理的小鼠；HFD+溶媒：饲喂高脂肪饮食且用单独溶媒对照的小鼠；HFD+NRG-1/HER3，饲喂高脂肪饮食且用NRG-1/HER3抗体融合蛋白处理的小鼠。y轴描述诱发AF的小鼠的百分比。Figure 20B shows a graph depicting the effect of NRG-1/HER3 antibody fusion protein on arrhythmia induction in the metabolic syndrome model depicted in Figure 18. The x-axis depicts the treatment conditions: CRTL: mice not fed a high-fat diet and treated with vehicle alone control; HFD+vehicle: mice fed a high-fat diet and treated with vehicle alone control; HFD+NRG-1/HER3, mice fed a high-fat diet and treated with NRG-1/HER3 antibody fusion protein. The y-axis depicts the percentage of mice with induced AF.

图21A至21F为一是列影像，显示来自收缩性心脏衰竭(或射血分数降低的心脏衰竭，HFrEF)模型大鼠的心脏组织的胶原蛋白I免疫组织化学染色(实施例7)。大鼠用假手术(图21A)处理，或用手术及单独的溶媒(图21B)、GP120-NRG-1抗体融合蛋白(图21C)、1mg/kgNRG-1/HER3抗体融合蛋白(图21D)、3mg/kg NRG-1/HER3抗体融合蛋白(图21E)或10mg/kgNRG-1/HER3抗体融合蛋白(图21F)处理。Figures 21A to 21F are a series of images showing collagen I immunohistochemical staining of cardiac tissue from rats with a systolic heart failure (or heart failure with reduced ejection fraction, HFrEF) model (Example 7). Rats were treated with sham surgery (Figure 21A), surgery and vehicle alone (Figure 21B), GP120-NRG-1 antibody fusion protein (Figure 21C), 1 mg/kg NRG-1/HER3 antibody fusion protein (Figure 21D), 3 mg/kg NRG-1/HER3 antibody fusion protein (Figure 21E), or 10 mg/kg NRG-1/HER3 antibody fusion protein (Figure 21F).

图22为显示用于测试NRG-1/HER3抗体融合蛋白在乙酸脱氧皮质固酮(醛固酮促效剂)诱导的高血压及心房纤维化Aachener小型猪模型中的效果的实验设计的图。22 is a diagram showing the experimental design used to test the effect of NRG-1/HER3 antibody fusion protein in the Aachener minipig model of hypertension and atrial fibrosis induced by deoxycorticosterone acetate (aldosterone agonist).

图23显示来自DOCA模型小型猪的一对心电图(ECG)及一个方程式。顶部ECG为50Hz短阵快速起搏诱导心房震颤的代表性ECG。底部ECG为50Hz短阵快速起搏未诱导心房震颤的代表性ECG。该方程式显示ECG数据如何用于计算心房震颤诱导。Figure 23 shows a pair of electrocardiograms (ECGs) from a DOCA model minipig and an equation. The top ECG is a representative ECG of atrial fibrillation induced by 50Hz short burst rapid pacing. The bottom ECG is a representative ECG of atrial fibrillation not induced by 50Hz short burst rapid pacing. The equation shows how the ECG data is used to calculate atrial fibrillation induction.

图24为显示对照、DOCA+溶媒(VEH)及DOCA+NRG-1/HER3小型猪的平均心房压力(以mmHg为单位，y轴)的图。24 is a graph showing mean atrial pressure (in mmHg, y-axis) for control, DOCA+Vehicle (VEH), and DOCA+NRG-1/HER3 minipigs.

图25为显示对照、DOCA+溶媒及DOCA+NRG-1/HER3小型猪的心房震颤(AF)诱导的图。AF诱导性的计算如图23中所示。25 is a graph showing atrial fibrillation (AF) induction in control, DOCA+vehicle, and DOCA+NRG-1/HER3 minipigs. AF inducibility was calculated as shown in FIG23 .

图26为显示对照、DOCA+溶媒及DOCA+NRG-1/HER3小型猪的心房纤维化程度的图(左)及三个代表性影像(右)。FIG. 26 is a graph (left) and three representative images (right) showing the extent of atrial fibrosis in control, DOCA+vehicle, and DOCA+NRG-1/HER3 minipigs.

具体实施方式DETAILED DESCRIPTION

本发明利用一种重组融合蛋白，其包含单克隆抗体与神经调节蛋白-1蛋白同功异型物的活性片段融合的融合物，适用于多种心血管及中枢神经系统(CNS)适应症。The present invention utilizes a recombinant fusion protein, which comprises a fusion product of a monoclonal antibody and an active fragment of a neuregulin-1 protein isoform, and is suitable for a variety of cardiovascular and central nervous system (CNS) indications.

定义definition

除非另外定义，否则本发明中所用的技术及科学术语具有与本发明所属领域的普通技术人员通常理解的相同含义。Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

为了解释本说明书，以下定义将适用，且只要适当，单数使用的术语还包括复数，反之亦然。若下述任何定义与以引用的方式并入本发明的任何文件相冲突，则以下列定义为准。For purposes of interpreting this specification, the following definitions shall apply and, wherever appropriate, terms used in the singular shall also include the plural and vice versa. In the event of a conflict between any of the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

“神经调节蛋白或神经调节蛋白类似物”为可激活ErbB2/ErbB4或ErbB2/ErbB3异二聚体蛋白质酪氨酸激酶的分子，诸如所有神经调节蛋白同功异型物、单独的神经调节蛋白EGF结构域、神经调节蛋白突变体及还激活上述受体的任何种类的神经调节蛋白样基因产物。本发明中使用的优选“神经调节蛋白”为含有EGF样结构域及受体结合结构域的人类神经调节蛋白1β2同功异型物的多肽片段。在一个实施例中，神经调节蛋白片段为活性片段。神经调节蛋白-1(NRG-1)及其同功异型物在本领域中还称为神经调节蛋白1(NRG1)、神经胶质生长因子(GGF)、Heregulin(HGL)、HRG、新分化因子(NDF)、ARIA、GGF2、HRG1、HRGA、SMDF、MST131、MSTP131及NRG1内含子转录物2(NRG1-IT2)。"Neuregulin or neuregulin analogs" are molecules that can activate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimeric protein tyrosine kinases, such as all neuregulin isoforms, individual neuregulin EGF domains, neuregulin mutants, and any type of neuregulin-like gene products that also activate the above receptors. Preferred "neuregulin" used in the present invention is a polypeptide fragment of a human neuregulin 1β2 isoform containing an EGF-like domain and a receptor binding domain. In one embodiment, the neuregulin fragment is an active fragment. Neuregulin-1 (NRG-1) and its isoforms are also known in the art as neuregulin 1 (NRG1), glial growth factor (GGF), Heregulin (HGL), HRG, new differentiation factor (NDF), ARIA, GGF2, HRG1, HRGA, SMDF, MST131, MSTP131, and NRG1 intron transcript 2 (NRG1-IT2).

术语“ErbB3”、“ErbB3(HER3)”、“HER3”是指相同蛋白质(或在提及时指相同基因)且在本发明中可互换使用。在一些实施方案中，重组融合物包含对ErbB3具有特异性的单克隆抗体部分。ErbB3(erb-b2受体酪氨酸激酶3)在本领域中还称为FERLK、LCCS2、ErbB-3、c-erbB3、erbB3-S、MDA-BF-1、c-erbB-3、p180-ErbB3、p45-sErbB3及p85-sErbB3。The terms "ErbB3", "ErbB3 (HER3)", "HER3" refer to the same protein (or, when referred to, the same gene) and are used interchangeably in the present invention. In some embodiments, the recombinant fusion comprises a monoclonal antibody portion that is specific for ErbB3. ErbB3 (erb-b2 receptor tyrosine kinase 3) is also known in the art as FERLK, LCCS2, ErbB-3, c-erbB3, erbB3-S, MDA-BF-1, c-erbB-3, p180-ErbB3, p45-sErbB3, and p85-sErbB3.

在一个实施例中，术语“ErbB4”、“ErbB4(HER4)”、“HER4”是指相同蛋白质(或在提及时指相同基因)且在本发明中可互换使用。ErbB4(erb-b2受体酪氨酸激酶4)在本领域中还称为ALS19及p180erbB4。In one embodiment, the terms "ErbB4", "ErbB4 (HER4)", "HER4" refer to the same protein (or the same gene when mentioned) and are used interchangeably in the present invention. ErbB4 (erb-b2 receptor tyrosine kinase 4) is also known in the art as ALS19 and p180erbB4.

在一个实施例中，术语“ErbB2”、“ErbB2(HER2)”、“HER2”是指相同蛋白质(或在提及时指相同基因)且在本发明中可互换使用。ErbB2(erb-b2受体酪氨酸激酶2)在本领域中还称为NEU、NGL、TKR1、CD340、HER-2、MLN 19及HER-2/neu。In one embodiment, the terms "ErbB2", "ErbB2 (HER2)", "HER2" refer to the same protein (or, when referred to, the same gene) and are used interchangeably in the present invention. ErbB2 (erb-b2 receptor tyrosine kinase 2) is also known in the art as NEU, NGL, TKR1, CD340, HER-2, MLN 19, and HER-2/neu.

如本发明所用，术语“活性”是指具有生物活性或生物功能的片段。在一些实施方案中，活性等于或接近于野生型蛋白质的活性。As used herein, the term "activity" refers to a fragment having biological activity or biological function. In some embodiments, the activity is equal to or close to the activity of the wild-type protein.

如本发明所用，术语“受试者”包括但不限于哺乳动物，包括例如人类、非人类灵长类动物(例如猴)、小鼠、猪、牛、山羊、兔、大鼠、天竺鼠、仓鼠、马、猴、羊或其他非人类哺乳动物；非哺乳动物，包括例如非哺乳动物脊椎动物，诸如鸟(例如鸡或鸭)或鱼类；及非哺乳动物无脊椎动物。在一些实施方案中，本发明的方法及组合物用于治疗(预防性和/或治疗性)非人类动物。术语“受试者”还可指患者，即等待或接受医疗护理的个体。As used herein, the term "subject" includes, but is not limited to, mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals, including, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish; and non-mammalian invertebrates. In some embodiments, the methods and compositions of the present invention are used to treat (preventatively and/or therapeutically) non-human animals. The term "subject" may also refer to a patient, i.e., an individual awaiting or receiving medical care.

本发明中的术语“药物组合物”是指适用于受试者(包括动物或人类)的医药用途的组合物。药物组合物通常包含有效量的活性剂(例如本发明的重组融合蛋白)及医药学上可接受的载剂、稀释剂或赋形剂(例如缓冲剂、佐剂或其类似物)。The term "pharmaceutical composition" in the present invention refers to a composition suitable for medical use in a subject (including an animal or a human). A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., a recombinant fusion protein of the present invention) and a pharmaceutically acceptable carrier, diluent or excipient (e.g., a buffer, an adjuvant or the like).

术语“有效量”是指足以产生预期结果的剂量或量。预期结果可包括该剂量或量的接受者的客观或主观改善(例如长期存活、肿瘤的数目和/或尺寸减少、疾病状态的有效预防等)。The term "effective amount" refers to a dose or amount sufficient to produce the desired result. The desired result may include an objective or subjective improvement in the recipient of the dose or amount (e.g., long-term survival, reduction in the number and/or size of tumors, effective prevention of a disease state, etc.).

“预防性治疗”为向未显示疾病、病理或医学病症的体征或症状或仅显示疾病、病理或病症的早期体征或症状的受试者施用的治疗，使得治疗是出于减少、预防或降低罹患疾病、病理或医学病症的风险的目的施用。预防性治疗起针对疾病或病症的预防性治疗的作用。“预防活性”为药剂(诸如本发明的重组融合蛋白或其组合物)的活性，当向未显示病理、疾病或病症的体征或症状(或仅显示病理、疾病或病症的早期体征或症状)的受试者施用时，减少、预防或降低受试者罹患病理、疾病或病症的风险。“预防上有用的”药剂或化合物(例如本发明的重组融合蛋白)是指可用于减少、预防、治疗或降低病理、疾病或病症的发展的药剂或化合物。"Prophylactic treatment" is a treatment administered to a subject who does not show signs or symptoms of a disease, pathology, or medical condition, or who only shows early signs or symptoms of a disease, pathology, or medical condition, such that the treatment is administered for the purpose of reducing, preventing, or reducing the risk of developing a disease, pathology, or medical condition. Prophylactic treatment acts as a prophylactic treatment for a disease or condition. "Prophylactic activity" is the activity of an agent (such as a recombinant fusion protein of the present invention, or a composition thereof) that, when administered to a subject who does not show signs or symptoms of a pathology, disease, or condition (or who only shows early signs or symptoms of a pathology, disease, or condition), reduces, prevents, or reduces the risk of a subject developing a pathology, disease, or condition. A "prophylactically useful" agent or compound (e.g., a recombinant fusion protein of the present invention) refers to an agent or compound that can be used to reduce, prevent, treat, or reduce the development of a pathology, disease, or condition.

“治疗性治疗”为向显示出病理、疾病或病症的症状或体征的受试者施用的治疗，其中向受试者施用治疗的目的为减少或消除病理、疾病或病症的这些体征或症状。“治疗活性”为药剂(诸如本发明的重组融合蛋白或其组合物)的活性，当向罹患病理、疾病或病症的体征或症状的受试者施用时，消除或减轻此类体征或症状。“治疗上有用的”药剂或化合物(例如本发明的重组融合蛋白)指示药剂或化合物可用于减轻、治疗或消除病理、疾病或病症的此类体征或症状。"Therapeutic treatment" is a treatment administered to a subject who exhibits symptoms or signs of a pathology, disease, or condition, wherein the purpose of administering the treatment to the subject is to reduce or eliminate these signs or symptoms of the pathology, disease, or condition. "Therapeutically active" is the activity of an agent (such as a recombinant fusion protein of the invention or a composition thereof) that, when administered to a subject suffering from signs or symptoms of a pathology, disease, or condition, eliminates or alleviates such signs or symptoms. A "therapeutically useful" agent or compound (e.g., a recombinant fusion protein of the invention) indicates that the agent or compound can be used to reduce, treat, or eliminate such signs or symptoms of a pathology, disease, or condition.

除非另外指明，否则如本发明所用，术语“治疗癌症”是指部分或完全逆转、缓解、抑制进展或防止受试者的肿瘤生长、肿瘤转移或其他致癌或肿瘤细胞。除非另外指明，否则如本发明所用，术语“治疗”是指治疗的行为。Unless otherwise indicated, as used herein, the term "treating cancer" refers to partially or completely reversing, alleviating, inhibiting the progression of, or preventing a subject's tumor growth, tumor metastasis, or other carcinogenic or tumor cells. Unless otherwise indicated, as used herein, the term "treating" refers to the act of treating.

除非另外指明，否则如本发明所用，术语“治疗心血管疾病”是指部分或完全预防、抑制、遏制、延迟、逆转或缓解受试者的心血管疾病或病况的发作，或受试者预先存在的心血管疾病或病况或其症状的进展。可通过本发明方法治疗的心血管疾病的非限制性实例包括慢性心脏衰竭/充血性心脏衰竭(CHF)、急性心脏衰竭/心肌梗塞(MI)、左心室收缩功能障碍、与MI相关的再灌注损伤、化学疗法诱导的心脏毒性(成人或儿童)、放射诱发的心脏毒性、儿童先天性心脏病手术干预的辅助治疗及心房纤维化。心血管疾病症状的非限制性实例包括呼吸短促、咳嗽、体重迅速增加、腿部、踝部及腹部肿胀、头晕、疲劳、虚弱、眩晕、胸痛、昏厥(晕厥)、心动过速、心动过缓及心律失常，诸如心房震颤。判定心血管疾病进展及治疗有效性的方法对与本领域的普通技术人员来说是显而易见的。例如，各种心血管疾病的进展可通过射血分数、心电图(ECG)、动态心电图、超声心动图、压力测试、心脏导管插入术、心脏计算机断层扫瞄(CT)扫描及心脏磁共振成像(MRI)来判定。Unless otherwise indicated, as used in the present invention, the term "treating cardiovascular disease" refers to partially or completely preventing, inhibiting, curbing, delaying, reversing or alleviating the onset of a subject's cardiovascular disease or condition, or the progression of a subject's pre-existing cardiovascular disease or condition or its symptoms. Non-limiting examples of cardiovascular diseases that can be treated by the methods of the present invention include chronic heart failure/congestive heart failure (CHF), acute heart failure/myocardial infarction (MI), left ventricular systolic dysfunction, reperfusion injury associated with MI, chemotherapy-induced cardiotoxicity (adult or child), radiation-induced cardiotoxicity, adjuvant therapy for surgical intervention of congenital heart disease in children, and atrial fibrosis. Non-limiting examples of cardiovascular disease symptoms include shortness of breath, cough, rapid weight gain, swelling of the legs, ankles and abdomen, dizziness, fatigue, weakness, vertigo, chest pain, fainting (syncope), tachycardia, bradycardia and arrhythmia, such as atrial fibrillation. The method for determining the progression of cardiovascular disease and the effectiveness of treatment is obvious to those of ordinary skill in the art. For example, the progression of various cardiovascular diseases can be determined by ejection fraction, electrocardiogram (ECG), Holter, echocardiogram, stress test, cardiac catheterization, cardiac computed tomography (CT) scan, and cardiac magnetic resonance imaging (MRI).

如本发明所用，“纤维化”是指细胞外基质(ECM)组分例如胶原蛋白的不适度形成及沉积。ECM通常围绕实质细胞，且支持其迁移、分化、增殖及正常功能。纤维化ECM会损害组织稳态，且可能由于结构完整性的丧失及异常重塑而导致器官功能障碍。纤维化的特征在于纤维母细胞的增殖，其可分化为分泌ECM蛋白的肌纤维母细胞。“心脏纤维化”一般是指心脏的纤维化，且包括心房纤维化，以及影响心脏其他区域的纤维化，诸如但不限于心室、心肌、心包、心内膜及瓣膜。As used herein, "fibrosis" refers to the inappropriate formation and deposition of extracellular matrix (ECM) components such as collagen. ECM usually surrounds parenchymal cells and supports their migration, differentiation, proliferation and normal function. Fibrotic ECM can impair tissue homeostasis and may cause organ dysfunction due to loss of structural integrity and abnormal remodeling. Fibrosis is characterized by the proliferation of fibroblasts, which can differentiate into myofibroblasts that secrete ECM proteins. "Cardiac fibrosis" generally refers to fibrosis of the heart, and includes atrial fibrosis, as well as fibrosis affecting other areas of the heart, such as but not limited to the ventricles, myocardium, pericardium, endocardium and valves.

在心脏中，置换性或修复性纤维化发生在心脏损伤后，且与心肌细胞死亡及用纤维化疤痕组织置换坏死性心肌区域相关。在反应性纤维化中，胶原蛋白及其他ECM蛋白在围绕心脏细胞及血管的间质空间中的沉积增加，导致此空间扩大，而没有置换受伤或死亡的心肌细胞。In the heart, replacement or reparative fibrosis occurs after cardiac injury and is associated with cardiomyocyte death and replacement of necrotic myocardial areas with fibrotic scar tissue. In reactive fibrosis, deposition of collagen and other ECM proteins in the interstitial space surrounding cardiac cells and blood vessels increases, leading to enlargement of this space without replacement of injured or dead cardiomyocytes.

如本发明所用，“心房纤维化”是指心房的纤维化。心房纤维化与心房震颤(AF)密切相关，心房震颤是人类最常见的心律失常的一。在不希望受理论束缚的情况下，认为心房纤维化会导致通过心房的异常电传导，从而导致心房震颤。纤维化可通过本领域中已知的任何适合手段检测，包括但不限于DE-MR成像(MRI)、循环生物标志物(例如泌乳素-3、MMP-3、MMP-9、高敏心肌肌钙蛋白T、骨桥蛋白、抑制致瘤性2、结缔组织生长因子(CTGF)、抵抗素(RETN)、骨膜蛋白及中段心房利钠肽前体，以及微RNA，诸如miRNA-15、miR-21、miR-29c、miR-328、miR-30a、miR-214、miR-503及miR-133a)及电解剖电压图。As used in the present invention, "atrial fibrosis" refers to fibrosis of the atrium. Atrial fibrosis is closely related to atrial fibrillation (AF), which is one of the most common arrhythmias in humans. Without wishing to be bound by theory, it is believed that atrial fibrosis can cause abnormal electrical conduction through the atrium, thereby causing atrial fibrillation. Fibrosis can be detected by any suitable means known in the art, including but not limited to DE-MR imaging (MRI), circulating biomarkers (e.g., prolactin-3, MMP-3, MMP-9, high-sensitivity cardiac troponin T, osteopontin, inhibition of tumorigenicity 2, connective tissue growth factor (CTGF), resistin (RETN), periostin and mid-segment atrial natriuretic peptide precursor, and microRNA, such as miRNA-15, miR-21, miR-29c, miR-328, miR-30a, miR-214, miR-503 and miR-133a) and electroanatomical voltage maps.

如本发明所用，“心律失常”是指不规则的心跳，且包括心动过速(异常快速的心跳)及心动过缓(异常缓慢的心跳)。心房震颤(AF)为一种不规则且通常非常快速的心律，可导致心脏血栓。在AF中，心房的正常跳动为不规则的，阻碍自心房至心室的血液流动。AF可为急性或慢性的。AF可依据AF发作的持续时间及在给定单位时间内发生的发作次数(例如AF/发作/天、周或月)来评定。阵发性AF突然开始且在7天内自发结束。相反，持续性AF发生超过7天，且自发或通过治疗结束。长期持续性AF是指一年以上不间断的AF。永久性AF是指尽管进行恢复正常窦性心律的治疗，但AF仍持续存在。AF的症状包括心跳不规则、心悸、头晕、极度疲劳、呼吸短促及胸痛。As used herein, "arrhythmia" refers to an irregular heartbeat and includes tachycardia (abnormally fast heartbeat) and bradycardia (abnormally slow heartbeat). Atrial fibrillation (AF) is an irregular and usually very fast heart rhythm that can lead to blood clots in the heart. In AF, the normal beats of the atria are irregular, obstructing the flow of blood from the atria to the ventricles. AF can be acute or chronic. AF can be assessed based on the duration of the AF episode and the number of episodes that occur in a given unit of time (e.g., AF/episode/day, week, or month). Paroxysmal AF begins suddenly and ends spontaneously within 7 days. In contrast, persistent AF occurs for more than 7 days and ends spontaneously or with treatment. Long-term persistent AF refers to uninterrupted AF for more than one year. Permanent AF refers to AF that persists despite treatment to restore normal sinus rhythm. Symptoms of AF include irregular heartbeat, palpitations, dizziness, extreme fatigue, shortness of breath, and chest pain.

除非另外指明，否则如本发明所用，术语“治疗中枢神经系统(CNS)相关疾病”是指部分或完全预防、抑制、遏制、延迟、逆转或缓解受试者的CNS相关疾病或病况发作的方法。术语“治疗CNS相关疾病”还可是指逆转、减缓或以其他方式缓解预先存在的CNS相关疾病或病况或其症状。可用本发明方法治疗的CNS相关疾病或病况的例示性但非限制性实例包括肌萎缩性脊髓侧索硬化症(ALS)、帕金森氏病(Parkinson’s disease)、阿兹海默氏病(Alzheimer's Disease)、贝尔氏麻痹(Bell's Palsy)、癫痫及癫痫发作、格林-巴利症候群(Guillain-Barre Syndrome)、中风、创伤性脑损伤、多发性硬化症或组合。治疗CNS相关疾病可改善或预防症状，诸如震颤、运动迟缓、肌肉僵硬、失去平衡、姿势受损、语言改变、运动控制丧失、瘫痪、吞咽困难、肌肉痉挛、癫痫发作、记忆力减退和混乱等症状。Unless otherwise indicated, as used herein, the term "treating a central nervous system (CNS)-related disease" refers to a method of partially or completely preventing, inhibiting, suppressing, delaying, reversing, or alleviating the onset of a CNS-related disease or condition in a subject. The term "treating a CNS-related disease" may also refer to reversing, slowing down, or otherwise alleviating a pre-existing CNS-related disease or condition or a symptom thereof. Exemplary but non-limiting examples of CNS-related diseases or conditions that can be treated with the methods of the present invention include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's Disease, Bell's Palsy, epilepsy and epileptic seizures, Guillain-Barre Syndrome, stroke, traumatic brain injury, multiple sclerosis, or a combination. Treatment of CNS-related disorders can improve or prevent symptoms such as tremors, bradykinesia, muscle rigidity, loss of balance, impaired posture, speech changes, loss of motor control, paralysis, difficulty swallowing, muscle spasms, seizures, memory loss, and confusion.

术语“相同”或“同一性百分比”在两个或更多个核酸或多肽序列的上下文中，是指两个或更多个序列或子序列在比较及比对以获得最大对应性时相同或具有特定百分比的核苷酸或氨基酸残基相同。为了确定同一性百分比，为了最佳比较目的对序列进行比对(例如，可在第一氨基酸或核酸序列的序列中引入间隙以与第二氨基酸或核酸序列进行最佳比对)。随后比较相应的氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置处相同。两个序列间的同一性百分比为序列共享的相同位置数的函数(即同一性％＝相同位置数/总位置数(例如重叠位置)×100)。在一些实施方案中，两个序列长度相同。The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of nucleotides or amino acid residues that are identical when compared and aligned for maximum correspondence. In order to determine the percent identity, the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences are the same length.

术语“基本相同”在两个核酸或多肽的上下文中，是指两个或更多个序列或子序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％同一性或至少99％同一性(例如，使用下文所述方法之一判定)。The term "substantially identical" in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical, or at least 99% identical (e.g., as determined using one of the methods described below).

如本发明所用，术语“结合”、“特异性结合于”或“特异性”是指可测量及可再现的相互作用，诸如靶标与抗体间的结合，其决定了在包括生物分子在内的异质分子群体存在情况下靶标的存在。例如，特异性结合于靶标(可以是表位)的抗体为与结合其他靶标相比以更大的亲和力、亲合力、更容易和/或更长的持续时间结合此靶标的抗体。在一个实施例中，抗体与不相关靶标的结合程度小于该抗体与靶标的结合的约10％，例如通过放射免疫分析法(RIA)所测量。在某些实施例中，特异性结合于靶标的抗体的解离常数(Kd)<1μΜ、<100nM、<10nM、<1nM或<0.1nM。As used herein, the terms "binding", "specifically binding to" or "specificity" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily and/or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, for example as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is <1 μM, <100 nM, <10 nM, <1 nM or <0.1 nM.

在某些实施例中，抗体特异性结合于蛋白质上的表位，该表位在来自不同物种的蛋白质之间是保守的。在另一个实施例中，特异性结合可包括但不要求排他性结合。In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding.

如在本说明书中所用，单数形式“一(a)”、“一(an)”及“该(the)”包括复数指代，除非上下文另外明确规定。因此，例如，提及“神经调节蛋白”或“神经调节蛋白肽”包括此类神经调节蛋白、神经调节蛋白同功异型物和/或神经调节蛋白样多肽的混合物。提及“制剂”或“方法”包括本发明所述类型的一种活多种制剂、方法和/或步骤，和/或在本领域技术人员阅读本发明后将变得显而易见的制剂、方法和/或步骤。As used in this specification, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a neuregulin" or "a neuregulin peptide" includes mixtures of such neuregulins, neuregulin isoforms, and/or neuregulin-like polypeptides. Reference to a "formulation" or "method" includes one or more formulations, methods, and/or steps of the type described herein, and/or formulations, methods, and/or steps that will become apparent to one skilled in the art upon reading this disclosure.

术语“多肽”是指氨基酸的聚合物及其等效物，而非指特定长度的产物；因此，“肽”及“蛋白质”均包括在多肽的定义内。多肽的定义中还包括如本发明所定义的“抗体”。“多肽区”是指多肽的片段，该片段可含有例如一个或多个结构域或基序(例如，抗体的多肽区可含有例如一个或多个互补决定区(CDR))。术语“片段”是指多肽的一部分，优选具有多肽的至少20个连续的或至少50个连续的氨基酸。The term "polypeptide" refers to a polymer of amino acids and their equivalents, rather than a specific length of the product; therefore, "peptide" and "protein" are included in the definition of polypeptide. The definition of polypeptide also includes "antibodies" as defined in the present invention. "Polypeptide region" refers to a fragment of a polypeptide, which may contain, for example, one or more domains or motifs (for example, a polypeptide region of an antibody may contain, for example, one or more complementarity determining regions (CDRs)). The term "fragment" refers to a portion of a polypeptide, preferably having at least 20 consecutive or at least 50 consecutive amino acids of a polypeptide.

除非上下文另外指明，否则“衍生物”为相对于第二多肽具有一个或多个非保守或保守氨基酸取代的多肽或其片段(还称为“变体”)；或通过共价连接第二分子，例如通过连接异源多肽，或通过糖基化、乙酰化、磷酸化及其类似物而经修饰的多肽或其片段。“衍生物”的定义中进一步包括例如含有一个或多个氨基酸类似物(例如非天然氨基酸及其类似物)的多肽、具有未取代键的多肽，以及本领域中已知的天然及非天然存在的其他修饰的多肽。Unless the context indicates otherwise, a "derivative" is a polypeptide or fragment thereof (also referred to as a "variant") having one or more non-conservative or conservative amino acid substitutions relative to a second polypeptide; or a polypeptide or fragment thereof modified by covalent attachment to a second molecule, such as by attachment to a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, and the like. Further included in the definition of "derivative" are, for example, polypeptides containing one or more amino acid analogs (e.g., non-natural amino acids and their analogs), polypeptides with unsubstituted bonds, and other modified polypeptides known in the art, both naturally and non-naturally occurring.

“经分离”多肽为已经鉴别且自其自然环境的组分中分离和/或回收的多肽。其自然环境的污染组分为会干扰多肽的诊断或治疗用途的物质，且可包括酶、激素及其他蛋白质或非蛋白质溶质。经分离多肽包括经分离抗体或其片段或衍生物。An "isolated" polypeptide is one that has been identified and separated and/or recovered from components of its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated polypeptides include isolated antibodies or fragments or derivatives thereof.

如本发明所用，术语“约”在数量上是指加或减5％，或在另一个实施例中，加或减10％，或在另一个实施例中，加或减15％，或在另一个实施例中，加或减20％。As used herein, the term "about" in relation to a quantity refers to plus or minus 5%, or in another embodiment, plus or minus 10%, or in another embodiment, plus or minus 15%, or in another embodiment, plus or minus 20%.

除非另有定义，本文中使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。尽管任何与本文所述相似或等效的方法和材料都可以用于本发明的实践或测试，但现在描述了优选的方法和材料。本文提及的所有出版物均通过引用并入本文，目的是披露和描述引用所依据的材料。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the materials on which the references are based.

重组融合蛋白-抗体Recombinant fusion protein-antibody

本发明利用一种重组融合蛋白，其包含单克隆抗体与神经调节蛋白-1蛋白质同功异型物的片段融合的融合物，用于多种心血管及神经适应症。在典型实施例中，抗体对ERBB3(HER3)具有特异性。The present invention utilizes a recombinant fusion protein comprising a monoclonal antibody fused to a fragment of a neuregulin-1 protein isoform for use in a variety of cardiovascular and neurological indications. In typical embodiments, the antibody is specific for ERBB3 (HER3).

如本发明所用，“抗体”是指包含一个或多个基本上或部分由免疫球蛋白基因或免疫球蛋白基因的片段编码的多肽的蛋白质。公认的免疫球蛋白基因包括κ、λ、α、γ、δ、ε及μ恒定区基因，以及无数免疫球蛋白可变区基因。轻链分类为κ或λ。重链分类为γ、μ、α、δ或ε，其继而分别定义免疫球蛋白类别IgG、IgM、IgA、IgD及IgE。典型的免疫球蛋白(例如抗体)结构单元包含四聚体。各四聚体由两对相同的多肽链构成，各对具有一条“轻”链(约25kD)及一条“重”链(约50 -70kD)。各链的N端定义约100至110个或更多氨基酸的可变区，主要负责抗原识别。术语可变轻链(VL)及可变重链(VH)分别是指这些轻链及重链。As used herein, "antibody" refers to a protein comprising one or more polypeptides that are substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typical immunoglobulin (e.g., antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50 -70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light chains and heavy chains, respectively.

抗体以完整免疫球蛋白的形式存在，或以各种肽酶消化产生的许多特征明确的片段形式存在。因此，例如，胃蛋白酶消化铰链区二硫键以下的抗体，产生F(ab′)2，即Fab的二聚体，其本身为通过二硫键接合至VH-CH1的轻链。F(ab′)2可在温和条件下还原以破坏铰链区中的二硫键，从而将F(ab′)2二聚体转化为Fab′单体。Fab′单体本质上为具有铰链区的一部分的Fab(参见Fundamental Immunology,W.E.Paul编,Raven Press,New York(1999)，关于其他抗体片段的更详细描述)。尽管各种抗体片段是根据完整抗体的消化而定义，但本领域技术人员应了解，此类Fab′片段等可以化学方式或通过使用重组DNA方法来从头合成。因此，如本发明所用，术语抗体还包括通过修饰全抗体产生或使用重组DNA方法从头合成的抗体片段。抗体包括单链抗体，包括单链Fv(sFv或scFv)抗体，其中可变重链及可变轻链接合在一起(直接或经由肽连接子)以形成连续多肽。抗体包括单域抗体，其包含由能够选择性地结合于抗原域的单个单体可变抗体域组成的抗体片段。例示性单域抗体包括VHH片段，其最初是自骆驼科动物中分离。Antibodies exist in the form of complete immunoglobulins, or in the form of many well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region to produce F(ab')2, a dimer of Fab, which itself is a light chain joined to VH-CH1 by a disulfide bond. F(ab')2 can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. Fab' monomers are essentially Fabs with a portion of the hinge region (see Fundamental Immunology, W.E.Paul, Raven Press, New York (1999), for a more detailed description of other antibody fragments). Although various antibody fragments are defined based on the digestion of complete antibodies, it will be appreciated by those skilled in the art that such Fab' fragments, etc., can be synthesized de novo chemically or by using recombinant DNA methods. Therefore, as used herein, the term antibody also includes antibody fragments produced by modifying whole antibodies or synthesized de novo using recombinant DNA methods. Antibodies include single chain antibodies, including single chain Fv (sFv or scFv) antibodies, in which the variable heavy chain and the variable light chain are linked together (directly or via a peptide linker) to form a continuous polypeptide. Antibodies include single domain antibodies, which comprise antibody fragments consisting of a single monomeric variable antibody domain capable of selectively binding to an antigenic domain. Exemplary single domain antibodies include VHH fragments, which were originally isolated from camelids.

融合蛋白的抗体结构域可选地包含免疫球蛋白分子的全部或一部分，且可选地含有免疫球蛋白可变区(即，疾病相关抗原的特异性区域)的全部或一部分，且可选地包含由V基因和/或D基因和/或J基因编码的区域。The antibody domain of the fusion protein optionally comprises all or a portion of an immunoglobulin molecule, and optionally contains all or a portion of an immunoglobulin variable region (i.e., a region specific for a disease-associated antigen), and optionally comprises regions encoded by V genes and/or D genes and/or J genes.

如上文所阐述(参见上文定义)，本发明所用的抗体可选地包含F(ab)2、F(ab′)2、Fab、Fab′、scFv、单域抗体等，视实施例的具体要求而定。一些实施方案利用包含IgG域的融合蛋白。然而，其他实施例包含替代性免疫球蛋白，诸如IgM、IgA、IgD及IgE。此外，各种免疫球蛋白的所有可能的同型还涵盖在当前实施例内。因此，IgG1、IgG2、IgG3等均为本发明中使用的抗体-免疫刺激剂融合蛋白的抗体域中的所有可能分子。除了在选择免疫球蛋白的类型及同型方面的选择外，本发明的不同实施例包含各种铰链区(或其功能等效物)。此类铰链区提供抗体-免疫刺激剂融合蛋白的不同域之间的可挠性。参见例如Penichet等人2001“Antibody-cytokine fusion proteins for the therapy of cancer”J ImmunolMethods 248:91-101。As described above (see above definition), the antibodies used in the present invention optionally include F(ab)2, F(ab')2, Fab, Fab', scFv, single domain antibodies, etc., depending on the specific requirements of the embodiment. Some embodiments utilize fusion proteins comprising IgG domains. However, other embodiments include alternative immunoglobulins, such as IgM, IgA, IgD and IgE. In addition, all possible isotypes of various immunoglobulins are also included in the current embodiment. Therefore, IgG1, IgG2, IgG3, etc. are all possible molecules in the antibody domain of the antibody-immunostimulator fusion protein used in the present invention. In addition to the selection of the type and isotype of immunoglobulins, different embodiments of the present invention include various hinge regions (or their functional equivalents). Such hinge regions provide flexibility between different domains of antibody-immunostimulator fusion proteins. See, for example, Penichet et al. 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248: 91-101.

在一些实施方案中，本发明的重组融合蛋白所包含的mAb对ErbB3(HER3)具有单特异性。In some embodiments, the mAb comprised by the recombinant fusion protein of the present invention is monospecific for ErbB3 (HER3).

人类HER3(ErbB-3、ERBB3、c-erbB-3、c-erbB3、受体酪氨酸蛋白激酶erbB-3)编码受体酪氨酸激酶表皮生长因子受体(EGFR)家族的成员，其还包括HER1(还称为EGFR)、HER2及HER4(Kraus,M.H.等人,PNAS 86(1989)9193-9197；Plowman,G.D.等人,PNAS 87(1990)4905-4909；Kraus,M.H.等人,PNAS 90(1993)2900-2904)。与原型表皮生长因子受体一样，跨膜受体HER3由细胞外配体结合域(ECD)、ECD内的二聚化域、跨膜域、细胞内蛋白酪氨酸激酶域(TKD)及C端磷酸化域组成。此膜结合的HER3蛋白在细胞外域内具有Heregulin(HRG)结合域，但没有活性激酶域。因此，其可结合此配体，但不能经由蛋白质磷酸化将信号传送至细胞中。然而，其确实与其他具有激酶活性的HER家族成员形成异二聚体。异二聚化导致受体介导的信号传导路径的激活及其细胞内域的转磷酸化。HER家族成员之间的二聚体形成扩大HER3的信号传导潜力，不仅为信号多样化的手段，且还为信号放大的手段。例如，HER2/HER3异二聚体经由HER家族成员中的PI3K及AKT路径诱导最重要的细胞分裂信号之一(Sliwkowski M.X.等人,J.Biol.Chem.269(1994)14661-14665；Alimandi M等人,Oncogene.10(1995)1813-1821；Hellyer,N.J.,J.Biol.Chem.276(2001)42153-4261；Singer,E.,J.Biol.Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which also includes HER1 (also known as EGFR), HER2 and HER4 (Kraus, M.H. et al., PNAS 86 (1989) 9193-9197; Plowman, G.D. et al., PNAS 87 (1990) 4905-4909; Kraus, M.H. et al., PNAS 90 (1993) 2900-2904). Like the prototype epidermal growth factor receptor, the transmembrane receptor HER3 consists of an extracellular ligand binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. This membrane-bound HER3 protein has a Heregulin (HRG) binding domain within the extracellular domain, but no active kinase domain. Therefore, it can bind this ligand, but cannot transmit signals into the cell via protein phosphorylation. However, it does form heterodimers with other HER family members that have kinase activity. Heterodimerization leads to activation of receptor-mediated signaling pathways and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3, not only as a means of signal diversification, but also as a means of signal amplification. For example, HER2/HER3 heterodimers induce one of the most important cell division signals through the PI3K and AKT pathways in HER family members (Sliwkowski M.X. et al., J.Biol.Chem.269 (1994) 14661-14665; Alimandi M et al., Oncogene.10 (1995) 1813-1821; Hellyer, N.J., J.Biol.Chem.276 (2001) 42153-4261; Singer, E., J.Biol.

在一个实施例中，人类ERBB3蛋白包含GenBank AAH02706.1中提供且在SEQ IDNO:1中所列的以下氨基酸序列：In one embodiment, the human ERBB3 protein comprises the following amino acid sequence provided in GenBank AAH02706.1 and set forth in SEQ ID NO: 1:

MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF(SEQ ID NO:1)。应理解，本发明方法及组合物的抗体所靶向的ERBB3(HER3)序列可为SEQ ID NO:1的异构体、同源物或变体。MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF (SEQ ID NO: 1). It should be understood that the ERBB3 (HER3) sequence targeted by the antibodies of the methods and compositions of the present invention may be an isomer, homolog or variant of SEQ ID NO: 1.

在一个实施例中，本发明提供的重组融合蛋白的mAb为抗Her3 mAb，其抑制经由ErbB3(HER3)的NRG-1信号传导。In one embodiment, the mAb of the recombinant fusion protein provided by the present invention is an anti-Her3 mAb, which inhibits NRG-1 signaling via ErbB3 (HER3).

在另一特定实施例中，本发明的重组融合蛋白所包含的mAb包含抗HER3 mAb。此类抗HER3抗体及其序列为本领域中已知的，且可包括但不限于以下：帕曲妥单抗(patritumab)、司里班妥单抗(seribantumab)(全人mAb)、LJM716、KTN3379、AV-203、REGN1400、GSK2849330或MM-141。此类抗体还可选自以下形式中的任一项，包括嵌合、双特异性、非人、全人或人源化形式，只要其结合于人类ERBB3(HER3)且抑制其信号传导即可。在一些实施方案中，抗HER3抗体为人源化的。In another specific embodiment, the mAb included in the recombinant fusion protein of the present invention includes an anti-HER3 mAb. Such anti-HER3 antibodies and their sequences are known in the art and may include, but are not limited to, the following: patritumab, seribantumab (all-human mAb), LJM716, KTN3379, AV-203, REGN1400, GSK2849330 or MM-141. Such antibodies may also be selected from any of the following forms, including chimeric, bispecific, non-human, all-human or humanized forms, as long as they bind to human ERBB3 (HER3) and inhibit its signal transduction. In some embodiments, the anti-HER3 antibody is humanized.

在一些实施方案中，术语“抗体”涵盖各种形式的抗体结构，包括但不限于全抗体及抗体片段。根据本发明的抗体优选为人抗体、人源化抗体、嵌合抗体或进一步经基因工程改造的抗体，只要保留本发明的特性即可。“抗体片段”包含全长抗体的一部分，优选为其可变域，或至少其抗原结合位点。抗体片段的实例包括由抗体片段形成的双功能抗体、单链抗体分子及多特异性抗体。scFv抗体描述于例如Huston,J.S.,Methods in Enzymol.203(1991)46-88中。此外，抗体片段包含单链多肽，其具有V_H域的特征，即能够与V_L域组装在一起，或具有结合于相应抗原的V_L域的特征，即能够与V_H域组装在一起形成功能性抗原结合位点，从而提供根据本发明的抗体的性质。如本发明所用，术语“单克隆抗体”或“单克隆抗体组合物”是指具有单一氨基酸组成的抗体分子的制剂。In some embodiments, the term "antibody" encompasses various forms of antibody structures, including but not limited to whole antibodies and antibody fragments. The antibody according to the present invention is preferably a human antibody, a humanized antibody, a chimeric antibody or an antibody further genetically engineered, as long as the characteristics of the present invention are retained. "Antibody fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include bifunctional antibodies, single-chain antibody molecules and multispecific antibodies formed by antibody fragments. ScFv antibodies are described in, for example, Huston, JS, Methods in Enzymol. 203 (1991) 46-88. In addition, the antibody fragment comprises a single-chain polypeptide having the characteristics of a_VH domain, i.e., being able to be assembled with a_VL domain, or having the characteristics of a_VL domain that is bound to a corresponding antigen, i.e., being able to be assembled with a_VH domain to form a functional antigen binding site, thereby providing the properties of the antibody according to the present invention. As used in the present invention, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of an antibody molecule having a single amino acid composition.

在一些实施方案中，嵌合抗体可用于本发明提供的组合物及方法中。在一个实施例中，术语“嵌合抗体”是指包含来自小鼠的可变区(即，结合区)及来源于不同来源或物种的恒定区的至少一部分的单克隆抗体，通常通过重组DNA技术制备。包含小鼠可变区及人类恒定区的嵌合抗体尤其优选。此类大鼠/人类嵌合抗体为包含编码大鼠免疫球蛋白可变区的DNA片段及编码人类免疫球蛋白恒定区的DNA片段的免疫球蛋白基因的表达产物。本发明所涵盖的其他形式的“嵌合抗体”为类别或子类别已自原始抗体修饰或改变的抗体。此类“嵌合”抗体还称为“类别转换抗体”。用于产生嵌合抗体的方法涉及本领域目前熟知的常规重组DNA及基因转染技术。参见例如Morrison,S.L.等人,Proc.Natl.Acad Sci.USA 81(1984)6851-6855；US 5,202,238及US 5,204,244。In some embodiments, chimeric antibodies can be used in the compositions and methods provided by the present invention. In one embodiment, the term "chimeric antibody" refers to a monoclonal antibody comprising a variable region (i.e., a binding region) from a mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA technology. Chimeric antibodies comprising mouse variable regions and human constant regions are particularly preferred. Such rat/human chimeric antibodies are the expression products of immunoglobulin genes comprising DNA fragments encoding rat immunoglobulin variable regions and DNA fragments encoding human immunoglobulin constant regions. Other forms of "chimeric antibodies" encompassed by the present invention are antibodies whose classes or subclasses have been modified or changed from the original antibodies. Such "chimeric" antibodies are also referred to as "class switching antibodies". Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques currently known in the art. See, for example, Morrison, S.L. et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

在一个实施例中，人源化抗体可用于本发明提供的组合物及方法中。在一些实施方案中，术语“人源化抗体”或“抗体的人源化形式”是指抗体的框架或“互补决定区”(CDR)已经修饰以包含与亲本免疫球蛋白相比具有不同特异性的免疫球蛋白的CDR。在其他实施例中，将VH及VL的CDR移植至人类抗体的框架区中以制备“人源化抗体”。参见例如Riechmann,L.等人,Nature 332(1988)323-327；及Neuberger,M.S.等人,Nature 314(1985)268-270。重链及轻链可变框架区可来源于相同或不同的人抗体序列。人抗体序列可为天然存在的人抗体序列。人重链及轻链可变框架区列于例如Lefranc,M.-P.,CurrentProtocols in Immunology(2000)-Appendix IP A.1P.1-A.1P.37中，且可经由IMGT，即international ImMunoGeneTics information(http://imgt.cines.fr)或经由http://vbase.mrc-cpe.cam.ac.uk获得。框架区可选择地通过其他突变来修饰。特别优选的CDR对应于代表识别上文关于嵌合抗体提及的抗原的序列。如本发明所用，术语“人源化抗体”还包含此类在恒定区中经修饰以产生根据本发明的性质的抗体，尤其在补体组分1q(Clq)结合和/或Fc受体(FcR)结合方面，例如通过“类别转换”，即Fc部分的变化或突变(例如IgGl至IgG4和/或IgGl/IgG4突变)。如本发明所用，术语“人抗体”意欲包括具有来源于人种系免疫球蛋白序列的可变区及恒定区的抗体。人抗体为目前本领域中众所周知的(vanDijk,M.A.及van de Winkel,J.G.,Curr.Opin.Chem.Biol.5(2001)368-374)。人抗体还可在转殖基因动物(例如小鼠)中产生，这些动物在免疫后能够在不存在内源性免疫球蛋白产生的情况下产生全谱是或精选的人抗体。将人类种系免疫球蛋白基因阵列转移至此类种系突变小鼠体内将导致在抗原攻击时产生人抗体(参见例如Jakobovits,A.等人,Proc.Natl.Acad.Sci.USA 90(1993)2551-2555；Jakobovits,A.等人,Nature 362(1993)255-258；Brueggemann,M.D.等人,Year Immunol.7(1993)33-40)。人抗体还可在噬菌体展示库中产生(Hoogenboom,H.R.及Winter,G.,J.Mol.Biol.227(1992)381-388；Marks,J.D.等人,J.Mol.Biol.222(1991)581-597)。Cole,A.等人及Boerner,P.等人的技术还可用于制备人单克隆抗体(Cole,A.等人,Monoclonal Antibodies and Cancer Therapy,Liss,A.L.,第77页(1985)；及Boerner,P.等人,J.Immunol.147(1991)86-95)。如已关于根据本发明的人源化抗体所提及，如本发明所用的术语“人抗体”还包含此类在恒定区中经修饰以产生根据本发明的性质的抗体。In one embodiment, humanized antibodies can be used in the compositions and methods provided by the present invention. In some embodiments, the term "humanized antibody" or "humanized form of an antibody" refers to an antibody framework or "complementarity determining region" (CDR) that has been modified to include a CDR of an immunoglobulin with different specificity compared to the parent immunoglobulin. In other embodiments, the CDRs of VH and VL are transplanted into the framework region of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, MS et al., Nature 314 (1985) 268-270. The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. The human antibody sequence may be a naturally occurring human antibody sequence. Human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix IP A.1P.1-A.1P.37 and can be obtained through IMGT, the international ImMunoGeneTics information (http://imgt.cines.fr) or obtained via http://vbase.mrc-cpe.cam.ac.uk. The framework region may be optionally modified by other mutations. Particularly preferred CDRs correspond to sequences representing the recognition of the antigens mentioned above for chimeric antibodies. As used herein, the term "humanized antibody" also includes such antibodies modified in the constant region to produce the properties according to the present invention, especially in complement component 1q (Clq) binding and/or Fc receptor (FcR) binding, for example, by "class switching", i.e., changes or mutations in the Fc portion (e.g., IgG1 to IgG4 and/or IgG1/IgG4 mutations). As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are currently well known in the art (van Dijk, MA and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full spectrum or selected human antibodies in the absence of endogenous immunoglobulin production after immunization. Transferring the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Brueggemann, MD et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A. et al. and Boerner, P. et al. can also be used to prepare human monoclonal antibodies (Cole, A. et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985); and Boerner, P. et al., J. Immunol. 147 (1991) 86-95). As already mentioned with respect to humanized antibodies according to the present invention, the term "human antibody" as used in the present invention also includes such antibodies that have been modified in the constant region to produce the properties according to the present invention.

在本发明的一个特定实施例中，本发明提供的重组融合蛋白所包含的mAb在Fc域或区中包含至少一个突变。In a specific embodiment of the present invention, the mAb contained in the recombinant fusion protein provided by the present invention comprises at least one mutation in the Fc domain or region.

如本发明所用，术语“重组人抗体”意欲包括通过重组手段制备、表达、产生或分离的所有人抗体，诸如自宿主细胞例如NS0或CHO细胞或自转基因人类免疫球蛋白基因的动物(例如小鼠)分离的抗体，或使用转染至宿主细胞中的重组表达载体表达的抗体。此类重组人抗体具有重排形式的可变区及恒定区。根据本发明的重组人抗体已经历体内体细胞超突变。因此，重组抗体的VH及VL区的氨基酸序列为虽然来源于人种系VH及VL序列且与其相关，但可能并非天然存在于体内人抗体种系库中的序列。As used in the present invention, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells such as NS0 or CHO cells or from animals (such as mice) of transgenic human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable regions and constant regions in rearranged form. The recombinant human antibodies according to the present invention have undergone in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that may not naturally exist in the human antibody germline library in vivo, although they are derived from and related to human germline VH and VL sequences.

在一些实施方案中，术语“结合于人类HER3”、“特异性结合于人类HER3”或“抗HER3抗体”可互换，且是指特异性结合于人类HER3抗原的抗体，其在25℃下的KD值为约4.81×^-10mol/L或更低。结合亲和力是在25℃下用标准结合分析确定，诸如表面等离子体共振技术(,GE-Healthcare Uppsala,Sweden)。因此，如本发明所用的“结合于人类HER3的抗体”是指特异性结合于人类HER3抗原的抗体或其部分，其在25℃下的结合亲和力在KD1.0×10^-8mol/L-1.0×10^-13mol/L范围内，且优选在25℃下的KD值为4.81×^-10mol/L或更低。In some embodiments, the terms "bind to human HER3", "specifically bind to human HER3" or "anti-HER3 antibody" are interchangeable and^{refer to an antibody that specifically binds to the human HER3 antigen with a KD value of about 4.81× -10mol} /L or less at 25°C. Binding affinity is determined at 25°C using a standard binding assay, such as surface plasmon resonance (SPR) technology. , GE-Healthcare Uppsala, Sweden). Therefore, as used in the present invention, "antibodies that bind to human HER3" refers to antibodies or portions thereof that specifically bind to human HER3 antigens, and whose binding affinity at 25°C is in the range of KD 1.0×10^-8 mol/L-1.0×10^-13 mol/L, and preferably has a KD value of 4.81×^{-10 -13} mol/L or less at 25°C.

在另一实施例中，本发明所公开的重组融合蛋白所包含的抗HER3抗体包含可变区重(VH)链及可变区轻(VL)链。在一个实施例中，该抗体包含分别为SEQ ID NO:2及SEQ IDNO:3的VH及VL序列；且具有以下特性中的一个或多者：抑制肿瘤细胞中的HER3磷酸化、抑制肿瘤细胞中的AKT磷酸化、抑制经由ErbB3(HER3)的信号传导及抑制肿瘤细胞的增殖。In another embodiment, the anti-HER3 antibody included in the recombinant fusion protein disclosed in the present invention comprises a variable region heavy (VH) chain and a variable region light (VL) chain. In one embodiment, the antibody comprises VH and VL sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and has one or more of the following properties: inhibiting HER3 phosphorylation in tumor cells, inhibiting AKT phosphorylation in tumor cells, inhibiting signal transduction through ErbB3 (HER3), and inhibiting the proliferation of tumor cells.

在一个实施例中，本发明提供的抗HER3 mAb包含SEQ ID NO:2中所列的VH氨基酸序列:In one embodiment, the anti-HER3 mAb provided by the present invention comprises the VH amino acid sequence listed in SEQ ID NO: 2:

重链：Heavy Chain:

QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYNPSLKSRVTIS VETSKNQFSL KLSSVTAADT AVYYCARDKW TWYFDLWGRG TLVTVSSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APEFLGGPAV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHAHYTQKS LSLSPGK(SEQ ID NO:2)。QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYNPSLKSRVTIS VETSKNQFSL KLSSVTAADT AVYYCARDKW TWYFDLWGRG TLVTVSSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APEFLGGPAV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHAHYTQKS LSLSPGK (SEQ ID NO: 2).

在一个实施例中，本发明提供的抗HER3 mAb包含SEQ ID NO:3的VL氨基酸序列：轻链：In one embodiment, the anti-HER3 mAb provided by the present invention comprises the VL amino acid sequence of SEQ ID NO: 3: Light chain:

DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNYLA WYQQNPGQPP KLLIYWASTRESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFPPSDEQLKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYACEVTHQGLSSP VTKSFNRGEC(SEQ ID NO:3)。DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNYLA WYQQNPGQPP KLLIYWASTRESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFPPSDEQLKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 3).

在一个实施例中，本发明的抗HER3抗体在Fc区中包含至少一个突变。在另一个实施例中，本发明的成熟抗HER3抗体(即缺乏信号肽)在氨基酸234、239、434或其组合中包含至少一个突变，而在其他实施例中，氨基酸突变包含以下取代突变中的至少一者：L234F、S239A、N434A或其组合。在另一个实施例中，氨基酸234和/或239的突变减弱抗HER3抗体的效应功能。在另一个实施例中，氨基酸434的突变延长抗体在受试者体内的半衰期。In one embodiment, the anti-HER3 antibody of the present invention comprises at least one mutation in the Fc region. In another embodiment, the mature anti-HER3 antibody of the present invention (i.e., lacking a signal peptide) comprises at least one mutation in amino acids 234, 239, 434, or a combination thereof, and in other embodiments, the amino acid mutation comprises at least one of the following substitution mutations: L234F, S239A, N434A, or a combination thereof. In another embodiment, the mutation of amino acids 234 and/or 239 reduces the effector function of the anti-HER3 antibody. In another embodiment, the mutation of amino acid 434 extends the half-life of the antibody in a subject.

在一些实施方案中，Fc区中的一个或多个突变降低效应功能。在一些实施方案中，效应功能降低包括抗HER3抗体对一个或多个Fc受体的亲和力降低。FcR可为FcγRI、FcγRIIa、FcγRIIb、FcγRIIIa(158F)、FcγRIIIa(158V)及C1q。在一些实施方案中，亲和力降低包括解离常数增加约1个数量级或更大。在一些实施方案中，引入一个或多个Fc突变使包含其的融合蛋白的抗HER3抗体对FcγRI的KD自2.81×10^-9M增加至1.03×10^-8M。在一些实施方案中，引入一个或多个Fc突变使包含其的融合蛋白的抗HER3抗体对FcγRIIa的KD自3.95×10^-7M增加至1.35×10^-6M。在一些实施方案中，引入一个或多个Fc突变使包含其的融合蛋白的抗HER3抗体对FcγRIIb的KD自1.03×10^-7M增加至1.52×10^-6M。在一些实施方案中，引入一个或多个Fc突变使包含其的融合蛋白的抗HER3抗体对FcγRIIIa(158F)的KD自6.37×10^-8M增加至1.18×10^-7M。在一些实施方案中，引入一个或多个Fc突变使包含其的融合蛋白的抗HER3抗体对FcγRIIIa(158V)的KD自3.41×10^-8M增加至9.10×10^-8M。In some embodiments, one or more mutations in the Fc region reduce effector function. In some embodiments, the reduced effector function includes a reduced affinity of the anti-HER3 antibody for one or more Fc receptors. FcR may be FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa (158F), FcγRIIIa (158V) and C1q. In some embodiments, the reduced affinity includes an increase in the dissociation constant by about 1 order of magnitude or greater. In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody comprising the fusion protein thereof to FcγRI from 2.81×^10-9 M to 1.03×^10-8 M. In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody comprising the fusion protein thereof to FcγRI from 3.95×^10-7 M to 1.35×^10-6 M. In some embodiments, the introduction of one or more Fc mutations increases the KD of an anti-HER3 antibody comprising a fusion protein thereof for FcγRIIb from 1.03×^10-7 M to 1.52×^10-6 M. In some embodiments, the introduction of one or more Fc mutations increases the KD of an anti-HER3 antibody comprising a fusion protein thereof for FcγRIIIa(158F) from 6.37×^10-8 M to 1.18×^10-7 M. In some embodiments, the introduction of one or more Fc mutations increases the KD of an anti-HER3 antibody comprising a fusion protein thereof for FcγRIIIa(158V) from 3.41×^10-8 M to 9.10×^10-8 M.

在一些实施方案中，抗HER3抗体或包含其的重组融合蛋白结合于FcγRI，其平衡解离常数(KD)高于或等于1.03×10^-8M。在一些实施方案中，抗HER3抗体或包含其的重组融合蛋白包含一个或多个Fc突变且以高于或等于1.35×10^-6M的KD结合于FcγRIIa。在一些实施方案中，抗HER3抗体或包含其的重组融合蛋白包含一个或多个Fc突变且以高于或等于1.5×10^-6M的KD结合于FcγRIIb。在一些实施方案中，抗HER3抗体或包含其的重组融合蛋白包含一个或多个Fc突变且以高于或等于1.18×10^-7M的KD结合于FcγRIIIa(158F)。在一些实施方案中，抗HER3抗体或包含其的重组融合蛋白包含一个或多个Fc突变且以高于或等于9.10×10^-8M的KD结合于FcγRIIIa(158V)。In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same binds to FcγRI with an equilibrium dissociation constant (KD) greater than or equal to 1.03×^10-8 M. In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIa with a KD greater than or equal to 1.35×^10-6 M. In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIb with a KD greater than or equal to 1.5×^10-6 M. In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIIa(158F) with a KD greater than or equal to 1.18×^10-7 M. In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIIa(158V) with a KD greater than or equal to 9.10×^10-8 M.

如本发明所用，术语“抗体效应功能”是指由Ig的Fc区贡献的功能。此类功能可通过例如Fc效应区与具有吞噬或溶解活性的免疫细胞上的Fc受体结合或通过Fc效应区与补体系统的组分结合来实现。As used herein, the term "antibody effector function" refers to the function contributed by the Fc region of Ig. Such functions can be achieved by, for example, binding of the Fc effector region to an Fc receptor on an immune cell having phagocytic or lytic activity or by binding of the Fc effector region to a component of the complement system.

在一个实施例中，抗HER3抗体不诱导抗体依赖性细胞毒性(ADCC)。术语“抗体依赖性细胞毒性(ADCC)”是指根据本发明的抗体在效应细胞存在下对人类靶细胞的裂解。In one embodiment, the anti-HER3 antibody does not induce antibody-dependent cellular cytotoxicity (ADCC).The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody according to the present invention in the presence of effector cells.

在本发明的一个实施例中，根据本发明的抗体是糖基化的。在一些实施方案中，糖基化为N-糖基化。在其他实施例中，糖基化为O-糖基化。In one embodiment of the invention, the antibody according to the invention is glycosylated. In some embodiments, the glycosylation is N-glycosylation. In other embodiments, the glycosylation is O-glycosylation.

在本发明提供且根据本发明的重组融合蛋白的上下文中，重组融合蛋白所包含的抗体可经由重组手段产生。此类方法在现有技术中广泛已知且包括在原核及真核细胞中表达蛋白质，随后分离抗体多肽且通常纯化至药学上可接受的纯度。对于蛋白质表达，编码轻链及重链的核酸或其片段通过标准方法插入表达载体中。在适当的原核或真核宿主细胞中进行表达，诸如CHO细胞、NS0细胞、SP2/0细胞、HEK293细胞、COS细胞、酵母或大肠杆菌细胞，且自细胞(上清液或细胞裂解后)回收抗体。抗体的重组产生为现有技术中熟知的且描述于例如综述论文Makrides,S.C.,Protein Expr.Purif.17(1999)183-202；Geisse,S.等人,Protein Expr.Purif.8(1996)271-282；Kaufman,R.J.,Mol.Biotechnol.16(2000)151-161；Werner,R.G.,Drug Res.48(1998)870-880中。抗体可存在于全细胞、细胞裂解物中或以部分纯化或基本纯化形式存在。进行纯化以消除其他细胞组分或其他污染物，例如其他细胞核酸或蛋白质，通过标准技术，包括柱层析及本领域中熟知的其他技术(参见Ausubel,F.等人编,Current Protocols in Molecular Biology,Greene Publishing and WileyInterscience,New York(1987))。NSO细胞中的表达由例如Barnes,L.M.等人,Cytotechnology 32(2000)109-123；Barnes,L.M.等人,Biotech.Bioeng.73(2001)261-270描述。瞬时表达由例如Durocher,Y.等人,Nucl.Acids.Res.30(2002)E9描述。可变结构域的克隆由Orlandi,R.等人,Proc.Natl.Acad.Sci.USA 86(1989)3833-3837；Carter,P.等人,Proc.Natl.Acad.Sci.USA 89(1992)4285-4289；Norderhaug,L.等人,J.Immunol.Methods204(1997)77-87描述。优选的瞬时表达系统(HEK 293)由Schlaeger,E.-J.及Christensen,K.,在Cytotechnology 30(1999)71-83中及由Schlaeger,E.-J.,在J.Immunol.Methods194(1996)191-199中描述。单克隆抗体适合通过常规免疫球蛋白纯化程序自培养基中分离，诸如蛋白A-Sepharose、羟磷灰石层析、凝胶电泳、透析或亲和层析。使用常规程序，可以容易地分离和测序编码单克隆抗体的DNA及RNA。杂交瘤细胞可充当此类DNA及RNA的来源。一旦分离，DNA可插入表达载体中，随后转染至不另外产生免疫球蛋白蛋白质的宿主细胞中，诸如HEK 293细胞、CHO细胞或骨髓瘤细胞，以在宿主细胞中获得重组单克隆抗体的合成。In the context of the recombinant fusion protein provided by the present invention and according to the present invention, the antibody contained in the recombinant fusion protein can be produced via recombinant means. Such methods are widely known in the prior art and include expressing proteins in prokaryotic and eukaryotic cells, followed by separation of antibody polypeptides and generally purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expressed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast or Escherichia coli cells, and antibodies are recovered from cells (after supernatant or cell lysis). Recombinant production of antibodies is well known in the art and is described in, for example, review articles Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880. The antibodies may be present in whole cells, cell lysates, or in partially purified or substantially purified form. Purification is performed to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including column chromatography and other techniques well known in the art (see Ausubel, F. et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Expression in NSO cells is described, for example, by Barnes, L.M. et al., Cytotechnology 32 (2000) 109-123; Barnes, L.M. et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, by Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L. et al., J. Immunol. Methods 204 (1997) 77-87. Preferred transient expression systems (HEK 293) are described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199. Monoclonal antibodies are suitably isolated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector and subsequently transfected into a host cell that does not otherwise produce immunoglobulin protein, such as HEK 293 cells, CHO cells, or myeloma cells, to obtain synthesis of recombinant monoclonal antibodies in the host cell.

根据本发明的重链及轻链可变结构域与启动子序列、翻译起始序列、恒定区序列、3'非翻译区序列、多聚腺苷酸化序列及转录终止序列组合以形成表达载体构建体。重链及轻链表达构建体可组合成单个载体，共转染、连续转染或分别地转染至宿主细胞，其随后融合形成表达重链及轻链二者的单个宿主细胞。The heavy chain and light chain variable domains according to the present invention are combined with a promoter sequence, a translation initiation sequence, a constant region sequence, a 3' untranslated region sequence, a polyadenylation sequence, and a transcription termination sequence to form an expression vector construct. The heavy chain and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into a host cell, which is then fused to form a single host cell expressing both the heavy chain and the light chain.

不言而喻的是，抗体以治疗有效量向受试者施用，治疗有效量为引发研究人员、兽医、医师或其他临床医师所寻求的组织、系统、动物或人类的生物或医学反应的本发明化合物或组合的量。It is understood that the antibody is administered to the subject in a therapeutically effective amount, which is an amount of a compound or combination of the invention that elicits the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, physician or other clinician.

重组融合蛋白-神经调节蛋白Recombinant fusion protein-Neuregulin

在一个实施例中，本发明提供的重组融合蛋白包含NRG-1蛋白的片段。NRG蛋白可结合于心肌细胞表面上的ErbB4受体，持续激活细胞中的PI3K/AKT信号通路，且改变心肌细胞的结构，从而改善心肌细胞的功能。In one embodiment, the recombinant fusion protein provided by the present invention comprises a fragment of NRG-1 protein. NRG protein can bind to the ErbB4 receptor on the surface of cardiomyocytes, continuously activate the PI3K/AKT signaling pathway in the cells, and change the structure of cardiomyocytes, thereby improving the function of cardiomyocytes.

如本发明所用，“神经调节蛋白”或“NRG”是指可结合且激活ErbB3、ErbB4或其异二聚体或同二聚体的蛋白质或肽，包括神经调节蛋白同功异型物、神经调节蛋白EGF样结构域、包含神经调节蛋白EGF样结构域的多肽、神经调节蛋白突变体或衍生物，以及可激活上述受体的任何种类的神经调节蛋白样基因产物。神经调节蛋白还包括具有神经调节蛋白功能的NRG-1、NRG-2、NRG-3及NRG-4蛋白、肽、片段及化合物。在优选实施例中，神经调节蛋白为一种可结合且激活ErbB2/ErbB4或ErbB2/ErbB3异二聚体的蛋白质或肽，例如但并非出于限制目的，本发明的肽包括NRG-1β2同功异型物的片段，即177-237氨基酸片段，其含有具有以下氨基酸序列的EGF样结构域域：SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ(SEQ ID NO:4)。本发明的NRG蛋白可激活上述受体且调节其生物功能，例如刺激骨骼肌细胞中乙酰胆碱受体的合成、促进心肌细胞的分化及存活以及DNA合成。本领域技术人员熟知，非关键区域中单个氨基酸的突变一般不会改变所得蛋白质或多肽的生物活性(参见例如Watson等人,Molecular Biology of the Gene,第4版,1987,TheBejacmin/Cummings Pub.co.,第224页)。本发明的NRG蛋白可自天然来源分离，可经由重组技术、人工合成或其他手段修饰。As used in the present invention, "neuregulin" or "NRG" refers to a protein or peptide that can bind to and activate ErbB3, ErbB4 or their heterodimers or homodimers, including neuregulin isoforms, neuregulin EGF-like domains, polypeptides containing neuregulin EGF-like domains, neuregulin mutants or derivatives, and any type of neuregulin-like gene products that can activate the above receptors. Neuregulin also includes NRG-1, NRG-2, NRG-3 and NRG-4 proteins, peptides, fragments and compounds that have neuregulin functions. In a preferred embodiment, the neuregulin is a protein or peptide that can bind to and activate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers, for example but not for the purpose of limitation, the peptides of the present invention include fragments of NRG-1β2 isoforms, i.e., 177-237 amino acid fragments, which contain an EGF-like domain having the following amino acid sequence: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 4). The NRG proteins of the present invention can activate the above receptors and regulate their biological functions, such as stimulating the synthesis of acetylcholine receptors in skeletal muscle cells, promoting differentiation and survival of cardiomyocytes, and DNA synthesis. It is well known to those skilled in the art that mutations of single amino acids in non-critical regions generally do not change the biological activity of the resulting protein or polypeptide (see, for example, Watson et al., Molecular Biology of the Gene, 4th edition, 1987, The Bejacmin/Cummings Pub.co., page 224). The NRG protein of the present invention can be isolated from natural sources, and can be modified through recombinant technology, artificial synthesis or other means.

如本发明所用，“表皮生长因子样结构域”或“EGF样结构域”是指由神经调节蛋白基因编码的多肽片段，该片段结合且激活ErbB3、ErbB4或其异二聚体或同二聚体且包括与ErbB2的异二聚体，且结构上类似于EGF受体结合区，如WO 00/64400，Holmes等人,Science,256:1205-1210(1992)；美国专利第5,530,109号及第5,716,930号；Hijazi等人,Int.J.Oncol.,13:1061-1067(1998)；Chang等人,Nature,387:509-512(1997)；Carraway等人,Nature,387:512-516(1997)；Higashiyama等人,J.Biochem.,122:675-680(1997)；及WO97/09425中所述，其内容全部以引用的方式并入本发明中。在某些实施例中，EGF样结构域结合且激活ErbB2/ErbB4或ErbB2/ErbB3异二聚体。在某些实施例中，EGF样结构域包含NRG-1的受体结合域的氨基酸序列。在一些实施方案中，EGF样结构域是指NRG-1的氨基酸残基177-226、177-237或177-240。在某些实施例中，EGF样结构域包含神经调节蛋白-2(NRG-2，本领域中还称为DON1、HRG2及NTAK)的受体结合域的氨基酸序列。在某些实施例中，NRG-2的EGF样结构域包含HARKCNETAKSYCVNGGVCYYIEGINQLSCKCPNGFFGQRCL(SEQ ID NO:15)的序列。在某些实施例中，EGF样结构域包含神经调节蛋白3(NRG-3，本领域中还称为HRG3及pro-NRG3)的受体结合域的氨基酸序列。在某些实施例中，NRG-3的EGF样结构域包含HFKPCRDKDLAYCLNDGECFVIETLTGSHKHCRCKEGYQGVRCD(SEQ ID NO:16)的序列。在某些实施例中，EGF样结构域包含神经调节蛋白4(NRG-4，本领域中还称为HER4)的受体结合域的氨基酸序列。在某些实施例中，NRG-4的EGF样结构域包含HEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCE(SEQ ID NO:17)的序列。在某些实施例中，EGF样结构域包含Ala Glu Lys Glu Lys ThrPhe Cys Val Asn Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro(SEQ ID NO:18)的氨基酸序列，如美国专利第5,834,229号中所述。As used herein, "epidermal growth factor-like domain" or "EGF-like domain" refers to a polypeptide fragment encoded by the neuregulin gene, which fragment binds to and activates ErbB3, ErbB4, or heterodimers or homodimers thereof and includes heterodimers with ErbB2, and is structurally similar to the EGF receptor binding region, such as WO 00/64400, Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol., 13:1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997); Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO97/09425, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the EGF-like domain binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of NRG-1. In some embodiments, the EGF-like domain refers to amino acid residues 177-226, 177-237, or 177-240 of NRG-1. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin-2 (NRG-2, also known in the art as DON1, HRG2, and NTAK). In certain embodiments, the EGF-like domain of NRG-2 comprises the sequence of HARKCNETAKSYCVNGGVCYYIEGINQLSCKCPNGFFGQRCL (SEQ ID NO: 15). In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin 3 (NRG-3, also known in the art as HRG3 and pro-NRG3). In certain embodiments, the EGF-like domain of NRG-3 comprises the sequence of HFKPCRDKDLAYCLNDGECFVIETLTGSHKHCRCKEGYQGVRCD (SEQ ID NO: 16). In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin 4 (NRG-4, also known in the art as HER4). In certain embodiments, the EGF-like domain of NRG-4 comprises the sequence of HEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCE (SEQ ID NO: 17). In certain embodiments, the EGF-like domain comprises the amino acid sequence of Ala Glu Lys Glu Lys ThrPhe Cys Val Asn Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro (SEQ ID NO: 18), as described in U.S. Pat. No. 5,834,229.

在一个实施例中，本发明所公开的重组融合蛋白中提供的NRG-1蛋白为NRG-1β2a同功异型物。In one embodiment, the NRG-1 protein provided in the recombinant fusion protein disclosed in the present invention is an NRG-1β2a isoform.

在一些实施方案中，活性NRG-1片段包含ERBB3/4结合域。在另一相关实施例中，NRG-1结合于ErbB4(HER4)且经由其诱导信号传导。在其他实施例中，mAb抑制经由ErbB3(HER3)的NRG-1信号传导。在一些实施方案中，NRG-1的活性蛋白质片段包含NRG-1的活性结构域。In some embodiments, the active NRG-1 fragment comprises an ERBB3/4 binding domain. In another related embodiment, NRG-1 binds to ErbB4 (HER4) and induces signaling therethrough. In other embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3). In some embodiments, the active protein fragment of NRG-1 comprises an active domain of NRG-1.

在一些实施方案中，NRG-1片段包含SEQ ID NO:4或与其具有至少70％、至少80％、至少90％或至少95％同一性的序列，能够结合于ErbB4且经由其诱导信号传导。In some embodiments, the NRG-1 fragment comprises SEQ ID NO: 4, or a sequence at least 70%, at least 80%, at least 90%, or at least 95% identical thereto, and is capable of binding to ErbB4 and inducing signaling therethrough.

重组融合蛋白-组合物Recombinant fusion protein-composition

在一个实施例中，在本发明所公开的重组融合蛋白中，NRG-1使用连接子与抗HER3抗体重链的C端融合。在另一相关方面，NRG-1经由NRG-1的N端上的第一个(1^st)氨基酸连接至连接子，该氨基酸在一个实施例中为丝胺酸(S或Ser)氨基酸。本发明中所用的特定重组融合蛋白可选择地通过本领域中已知的任何方法(包括自市售来源购买)获得或形成。例如，编码适当抗体框架的核酸序列可选地克隆且连接至适当载体(例如用于例如原核或真核生物的表达载体)。此外，编码NRG-1β2a同功异型物分子的核酸序列可选地以适当的方向及位置克隆至同一载体中，以便自载体表达产生抗体-NRG-1β2a同功异型物融合蛋白。一些可选择的实施例还需要表达后修饰，例如抗体亚基的组装等。上述(及类似)操作的技术及工艺为本领域技术人员熟知的。相关说明可见于例如Sambrook等人,Molecular Cloning-ALaboratory Manual(第2版),第1-3卷,Cold Spring Harbor Laboratory,Cold SpringHarbor,N.Y.,1989及Current Protocols in Molecular Biology,F.M.Ausubel等人编,Current Protocols,a joint venture between Greene Publishing Associates,Inc.and John Wiley&Sons,Inc.(增补至1999)。在一些替代实施例中，抗体结构域及NRG-1β2a同功异型物经由例如化学手段在表达后组装。在一个实施例中，本发明提供一种组合物，例如包含本发明的重组融合蛋白的药物组合物。In one embodiment, in the recombinant fusion protein disclosed in the present invention, NRG-1 is fused to the C-terminus of the heavy chain of the anti-HER3 antibody using a linker. In another related aspect, NRG-1 is connected to the linker via the first (^1st ) amino acid on the N-terminus of NRG-1, which in one embodiment is a serine (S or Ser) amino acid. The specific recombinant fusion protein used in the present invention can be optionally obtained or formed by any method known in the art (including purchase from commercial sources). For example, a nucleic acid sequence encoding an appropriate antibody framework is optionally cloned and connected to an appropriate vector (e.g., an expression vector for, for example, prokaryotes or eukaryotes). In addition, a nucleic acid sequence encoding an NRG-1β2a isoform molecule is optionally cloned into the same vector in an appropriate direction and position so that the antibody-NRG-1β2a isoform fusion protein is expressed from the vector. Some optional embodiments also require post-expression modifications, such as assembly of antibody subunits, etc. The techniques and processes for the above (and similar) operations are well known to those skilled in the art. Related descriptions can be found in, for example, Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented to 1999). In some alternative embodiments, the antibody domain and NRG-1β2a isoform are assembled after expression, for example, by chemical means. In one embodiment, the present invention provides a composition, for example, a pharmaceutical composition comprising a recombinant fusion protein of the present invention.

在一个实施例中，重组融合蛋白减少心房震颤发作的持续时间。在一个实施例中，重组融合蛋白降低心房震颤发生的频率。心房震颤的症状包括但不限于心跳不规则、心悸、头晕、极度疲劳、呼吸短促、胸痛及其组合。在一个实施例中，重组融合蛋白减少心脏组织中的胶原蛋白含量或沉积。In one embodiment, the recombinant fusion protein reduces the duration of atrial fibrillation episodes. In one embodiment, the recombinant fusion protein reduces the frequency of atrial fibrillation. Symptoms of atrial fibrillation include, but are not limited to, irregular heartbeat, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, and combinations thereof. In one embodiment, the recombinant fusion protein reduces collagen content or deposition in cardiac tissue.

在一个实施例中，重组融合蛋白促进心肌细胞增殖、分化及存活。在另一个实施例中，重组融合蛋白促进心脏组织的增殖、分化及存活。在一个实施例中，重组融合蛋白促进心肌细胞增殖、分化及存活而不促进癌症和/或肿瘤生长。在另一个实施例中，重组融合蛋白促进心脏组织的增殖、分化及存活而不促进癌症或肿瘤生长。In one embodiment, the recombinant fusion protein promotes cardiomyocyte proliferation, differentiation and survival. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation and survival of cardiac tissue. In one embodiment, the recombinant fusion protein promotes the proliferation, differentiation and survival of cardiomyocytes without promoting cancer and/or tumor growth. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation and survival of cardiac tissue without promoting cancer or tumor growth.

在一个实施例中，癌症为肾上腺皮质癌、AIDS相关癌症、AIDS相关淋巴瘤、肛门癌、肛门直肠癌、肛管癌、阑尾癌、儿童小脑星形细胞瘤、儿童脑星形细胞瘤、基底细胞癌、皮肤癌(非黑色素瘤)、胆道癌、肝外胆管癌、肝内胆管癌、膀胱癌、膀胱癌、骨关节癌、骨肉瘤及恶性纤维组织细胞瘤、脑癌、脑瘤、脑干神经胶质瘤、小脑星形细胞瘤、脑星形细胞瘤/恶性神经胶质瘤、室管膜瘤、髓母细胞瘤、幕上原始神经外胚层肿瘤、视觉路径及下丘脑神经胶质瘤、乳癌、支气管腺瘤/类癌、类癌瘤、胃肠道癌、神经系统癌、神经系统淋巴瘤、中枢神经系统癌、中枢神经系统淋巴瘤、子宫颈癌、儿童癌症、慢性淋巴细胞性白血病、慢性骨髓性白血病、慢性骨髓增生病、结肠癌、结肠直肠癌、皮肤T细胞淋巴瘤、淋巴赘瘤、蕈状肉芽肿、塞扎里氏症候群(Seziary Syndrome)、子宫内膜癌、食道癌、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、肝外胆管癌、眼癌、眼内黑色素瘤、视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌瘤、胃肠道间质瘤(GIST)、生殖细胞肿瘤、卵巢生殖细胞肿瘤、妊娠滋养细胞肿瘤神经胶质瘤、头颈癌、肝细胞(肝)癌、霍奇金淋巴瘤(Hodgkin lymphoma)、下咽癌、眼内黑色素瘤、眼癌、胰岛细胞瘤(内分泌胰脏)、卡波西肉瘤(Kaposi Sarcoma)、肾癌、肾癌、肾癌、喉癌、急性淋巴母细胞性白血病、急性骨髓性白血病、慢性淋巴细胞性白血病、慢性骨髓性白血病、毛细胞白血病、唇及口腔癌、肝癌、肺癌、非小细胞肺癌、小细胞肺癌、AIDS相关淋巴瘤、非霍奇金淋巴瘤、原发性中枢神经系统淋巴瘤、华氏巨球蛋白血症(Waldenstroemmacroglobulinemia)、神经管胚细胞瘤、黑色素瘤、眼内(眼睛)黑色素瘤、梅克尔细胞癌(merkel cell carcinoma)、恶性间皮瘤、间皮瘤、转移性鳞状颈癌、口腔癌、舌癌、多发性内分泌瘤症候群、蕈状肉芽肿、骨髓发育不良症候群、骨髓发育不良/骨髓增生性疾病、慢性骨髓性白血病、急性骨髓性白血病、多发性骨髓瘤、慢性骨髓增生病、鼻咽癌、神经母细胞瘤、口腔癌、口腔癌、口咽癌、卵巢癌、卵巢上皮癌、卵巢低度恶性潜能肿瘤、胰脏癌、胰岛细胞胰脏癌、副鼻窦及鼻腔癌症、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体母细胞瘤及幕上原始神经外胚层肿瘤、垂体肿瘤、浆细胞瘤/多发性骨髓瘤、胸膜肺母细胞瘤、前列腺癌、直肠癌、肾盂及输尿管癌、移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、尤文氏肉瘤家族肿瘤(Ewing family of sarcoma tumor)、卡波西肉瘤、软组织肉瘤、上皮样肉瘤、滑膜肉瘤、子宫癌、子宫肉瘤、皮肤癌(非黑色素瘤)、皮肤癌(黑色素瘤)、梅克尔细胞皮肤癌、小肠癌、软组织肉瘤、鳞状细胞癌、胃癌、幕上原始神经外胚层肿瘤、睪丸癌、咽喉癌、胸腺瘤、胸腺瘤及胸腺癌、甲状腺癌、肾盂及输尿管及其他泌尿器官的移行细胞癌、妊娠滋养细胞肿瘤、尿道癌、子宫内膜子宫癌、子宫肉瘤、子宫体癌、阴道癌、外阴癌或威尔姆氏肿瘤(Wilm’sTumor)。In one embodiment, the cancer is adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood brain astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, brain astrocytoma/malignant glioma, ventricular Tubuloma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma, breast cancer, bronchial adenoma/carcinoid, carcinoid tumor, gastrointestinal cancer, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Sezary syndrome Syndrome), endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, eye cancer, islet cell tumor (endocrine pancreas), Kaposi sarcoma Sarcoma), kidney cancer, renal cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstroemmacroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma carcinoma), malignant mesothelioma, mesothelioma, metastatic squamous neck cancer, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disease, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disease, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low-grade malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors tumor), Kaposi's sarcoma, soft tissue sarcoma, epithelioid sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, or Wilm's Tumor.

在另一个实施例中，重组融合蛋白促进中枢神经系统(CNS)细胞的增殖、分化及存活。在另一个实施例中，重组融合蛋白促进中枢神经系统(CNS)细胞的增殖、分化及存活而不促进癌症和/或肿瘤生长。在另一个实施例中，重组融合蛋白具有降低的诱导抗体依赖性细胞毒性(ADCC)的能力。In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation and survival of central nervous system (CNS) cells. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation and survival of central nervous system (CNS) cells without promoting cancer and/or tumor growth. In another embodiment, the recombinant fusion protein has a reduced ability to induce antibody-dependent cellular cytotoxicity (ADCC).

在一些实施方案中，相对于重组NRG-1的信号诱导潜力，重组融合蛋白促进HER2/4信号传导超过HER2/3信号传导。In some embodiments, the recombinant fusion protein promotes HER2/4 signaling over HER2/3 signaling relative to the signal induction potential of recombinant NRG-1.

在本发明的另一特定实施例中，重组融合蛋白包含与SEQ ID NO:4的NRG-1β2a同功异型物融合或经由GGGGSGGGGS(G4S)连接子(SEQ ID NO:5)可操作地连接于抗体重链C端的抗HER3mAb。在一些实施方案中，可使用连接子的一个或多个拷贝。在其他实施例中，本发明中可使用G4S连接子或本领域中已知适用于本发明所公开的组合物的任何其他连接子的2、3、4或5个拷贝。In another specific embodiment of the invention, the recombinant fusion protein comprises an anti-HER3 mAb fused to the NRG-1β2a isoform of SEQ ID NO: 4 or operably linked to the C-terminus of the antibody heavy chain via a GGGGSGGGGS (G4S) linker (SEQ ID NO: 5). In some embodiments, one or more copies of the linker may be used. In other embodiments, 2, 3, 4, or 5 copies of the G4S linker or any other linker known in the art to be suitable for use in the compositions disclosed herein may be used in the present invention.

术语“连接子”为本领域中公认的且是指连接两个化合物，诸如两个多肽的分子(包括但不限于未经修饰或经修饰的核酸或氨基酸)或分子群(例如2个或更多，例如2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100个或更多)。连接子可由单个连接分子构成，或可包含连接分子及至少一个间隔子分子，其意欲使该连接分子及化合物分开特定距离。The term "linker" is recognized in the art and refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or a group of molecules (e.g., 2 or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) that connects two compounds, such as two polypeptides. A linker may consist of a single linker molecule, or may comprise a linker molecule and at least one spacer molecule, which is intended to separate the linker molecule and the compound by a specific distance.

核酸在与另一核酸序列处于功能区时为“可操作地连接的”。举例而言，若核酸前序列或分泌性前导序列表达为参与多肽分泌的前蛋白，则其与编码该多肽的核酸可操作地连接；若启动子或增强子影响编码序列的转录，则其与该序列可操作地连接；或若核糖体结合位点经定位以便有助于翻译，则其与编码序列可操作地连接。一般而言，“可操作地连接”是指连接的核酸序列相邻，且在分泌性前导序列的情况下，相邻且在阅读框中。然而，增强子可选地为相邻的。连接可例如通过在适宜限制性位点处接合来实现。若此类位点不存在，则可使用合成寡核苷酸衔接子、连接子或本领域中已知的其他方法。在另一个实施例中，“可操作地连接”还指不同氨基酸序列、肽或蛋白质的功能配对，如本发明所述的抗体及NRG-1片段经由本发明还描述的连接子序列配对。A nucleic acid is "operably linked" when it is in a functional region with another nucleic acid sequence. For example, if a nucleic acid presequence or secretory leader sequence is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to a nucleic acid encoding the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to the sequence; or if a ribosome binding site is positioned so as to facilitate translation, it is operably linked to a coding sequence. In general, "operably linked" means that the linked nucleic acid sequences are adjacent, and in the case of a secretory leader sequence, adjacent and in the reading frame. However, enhancers are optionally adjacent. Connection can be achieved, for example, by joining at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adapters, linkers, or other methods known in the art can be used. In another embodiment, "operably linked" also refers to the functional pairing of different amino acid sequences, peptides or proteins, such as the antibodies and NRG-1 fragments described in the present invention are paired via linker sequences also described in the present invention.

在另一个实施例中，本发明提供的重组融合蛋白所包含的抗HER3 mAb重链是由SEQ ID NO:6编码：In another embodiment, the anti-HER3 mAb heavy chain contained in the recombinant fusion protein provided by the present invention is encoded by SEQ ID NO: 6:

ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCGCCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAAGTCCCGGGTGACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCGTGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGATCTGTGGGGAAGGGGCACCCTGGTGACAGTGTCTTCCGCCTCTACAAAGGGCCCCTCCGTGTTTCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGAAGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGAGTGCATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGTGCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTGAATCACAAGCCCTCCAATACAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGTCCAGCTCCTGAGCTGCTGGGAGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACACCCTGATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATCCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCTAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCTAGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTATAAGACCACACCACCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGCAGCGTGATGCACGAGGCCCTGCACAATCATTATACACAGAAATCTCTGTCCCTGAGCCCAGGCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGCAGCTCTCATCTGGTGAAGTGTGCTGAGAAGGAGAAGACCTTCTGCGTGAACGGCGGCGAGTGTTTTATGGTGAAGGACCTGTCTAATCCATCCAGATACCTGTGCAAGTGTCCCAACGAGTTCACAGGCGATCGCTGCCAGAATTACGTGATGGCCTCTTTTTATAAGGCTGAGGAGCTGTACCAGTAA(SEQ ID NO:6)。在一个实施例中，SEQ ID NO:6中所示的序列不包含Fc突变。在一个实施例中，SEQ ID NO:6还称为“NPCF”。ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCGCCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAAGTCCCGGGT GACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCGTGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGATCTGTGGGGAAGGGGCACCCTGGTGACAGTGTC TTCCGCCTCTACAAAGGGCCCCTCCGTGTTTCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGAAGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGAGTGCATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGTGCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTG AATCACAAGCCCTCCAATACAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGTCCAGCTCCTGAGCTGCTGGGAGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACACCCT GATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATCCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCTAGGGAGGAGCAGTACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCGAGAAGACCATC TCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCTAGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCC (SEQ ID NO: 6). In one embodiment, the sequence shown in SEQ ID NO: 6 does not contain an Fc mutation. In one embodiment, SEQ ID NO: 6 is also referred to as "NPCF".

在一个实施例中，本发明提供的重组融合蛋白包含抗HER3 mAb的重链。在另一个实施例中，抗HER3 mAb重链是由SEQ ID NO:7编码：In one embodiment, the recombinant fusion protein provided by the present invention comprises the heavy chain of anti-HER3 mAb. In another embodiment, the heavy chain of anti-HER3 mAb is encoded by SEQ ID NO:7:

ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCGCCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAAGTCCCGGGTGACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCGTGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGATCTGTGGGGAAGGGGCACCCTGGTGACAGTGTCTTCCGCCTCTACAAAGGGCCCCTCCGTGTTTCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGAAGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCTGGAGTGCATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGTGCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTGAATCACAAGCCCTCCAATACAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGTCCAGCTCCTGAGTTCCTGGGAGGACCTGCCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACACCCTGATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATCCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCTAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCTAGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTACCCATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTATAAGACCACACCACCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGCAGCGTGATGCACGAGGCCCTGCACGCTCATTATACACAGAAATCTCTGTCCCTGAGCCCAGGCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGCAGCTCTCATCTGGTGAAGTGTGCTGAGAAGGAGAAGACCTTCTGCGTGAACGGCGGCGAGTGTTTTATGGTGAAGGACCTGTCTAATCCATCCAGATACCTGTGCAAGTGTCCCAACGAGTTCACAGGCGATCGCTGCCAGAATTACGTGATGGCCTCTTTTTATAAGGCTGAGGAGCTGTACCAGTAA(SEQ ID NO:7)。在一个实施例中，SEQ ID NO:7还称为“NPCFA”。在一个实施例中，SEQ ID NO:7包含编码本发明提供的抗HER3 mAb的恒定(Fc)区中的一个或多个突变的一个或多个突变。在一个实施例中，本发明的成熟抗HER3抗体包含氨基酸234、239、434中的至少一个突变或其组合。在另一个实施例中，氨基酸突变包括以下取代突变中的至少一者：L234F、S239A、N434A或其组合。ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCGCCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAAGTCCCGGGT GACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCGTGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGATCTGTGGGGAAGGGGCACCCTGGTGACAGTGTC TTCCGCCTCTACAAAGGGCCCCTCCGTGTTTCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGAAGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCTGGAGTGCATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGTGCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTG AATCACAAGCCCTCCAATACAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGTCCAGCTCCTGAGTTCCTGGGAGGACCTGCCGTGTTCGTTTCCTCCAAAGCCAAAGGACACCCT GATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATCCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCTAGGGAGGAGCAGTACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCGAGAAGACCATC TCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCTAGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTACCCATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCC AGCCCGAGAACAATTATAAGACCACACCACCCGTGCTGGACTCCGATGGCAGCTTCTTTCGTACTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTTCCTGCAGCGTGATGCACGAGGCCCTGCACGCTCATTATACACAGAAATCTCTGTCCCTGAGCCCAGGCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGCAGCTCTCATCTGGTGAAGTGTGCTGAGAAGGAGAAGACCTTCTGCGT GAACGGCGGCGAGTGTTTTATGGTGAAGGACCTGTCTAATCCATCCAGATACCTGTGCAAGTGTCCCAACGAGTTCACAGGCGATCGCTGCCAGAATTACGTGATGGCCTCTTTTTATAAGGCTGAGGAGCTGTACCAGTAA (SEQ ID NO: 7). In one embodiment, SEQ ID NO:7 is also referred to as "NPCFA." In one embodiment, SEQ ID NO:7 comprises one or more mutations encoding one or more mutations in the constant (Fc) region of an anti-HER3 mAb provided herein. The anti-HER3 antibody comprises at least one mutation in amino acids 234, 239, 434, or a combination thereof. In another embodiment, the amino acid mutation comprises at least one of the following substitution mutations: L234F, S239A, N434A, or a combination thereof.

在一个实施例中，本发明提供的重组融合蛋白包含抗HER3 mAb的轻链序列。在另一个实施例中，轻链序列是由(SEQ ID NO:8)编码：In one embodiment, the recombinant fusion protein provided by the present invention comprises the light chain sequence of anti-HER3 mAb. In another embodiment, the light chain sequence is encoded by (SEQ ID NO: 8):

ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCGAGATGACCCAGTCTCCAGATTCCCTGGCCGTGAGCCTGGGAGAGAGGGCTACAATCAACTGCCGGTCCAGCCAGTCTGTGCTGTACTCTTCCAGCAACAGGAATTACCTGGCCTGGTATCAGCAGAATCCCGGCCAGCCCCCTAAGCTGCTGATCTATTGGGCTAGCACCAGAGAGTCTGGAGTGCCTGACCGCTTCTCTGGATCCGGAAGCGGCACAGACTTCACCCTGACAATCTCTTCCCTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTATTACTCTACCCCTAGGACATTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACAGTGGCCGCTCCATCCGTGTTCATCTTTCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCAAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGAGCGGCAATTCTCAGGAGTCCGTGACCGAGCAGGACAGCAAGGATTCTACATATTCCCTGAGCTCTACCCTGACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACAAAGAGCTTCAACCGCGGCGAGTGTTAA(SEQ ID NO:8)。在一个实施例中，SEQ ID NO:8还称为“PAL”。ATGGTGTTGCAGACCCAGGTCTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCGAGATGACCCAGTCTCCAGATTCCCTGGCCGTGAGCCTGGGAGAGAGGGCTACAATCAACTGCCGGTCCAGCCAGTCTGTGCTGTACTCTTCCAGCAACAGGAATTACCTGGCCTGGTATCAGCAGAATCCCGGCCAGCCCCCTAAGCTGCTGATCTATTGGGCTAGCACCAGAGAGTCTGGAGTGCCTG ACCGCTTCTCTGGATCCGGAAGCGGCACAGACTTCACCCTGACAATCTCTTCCCTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTATTACTCTACCCCT AGGACATTCGCCAGGGGCACCAAGGTGGAGATCAAGCGGACAGTGGCCGCTCCATCCGTGTTCATCTTTCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCAAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGAGCGGCAATTCTCAGGAGTCCGTGACCGAGCAGGACAGCAAGGATTCTACATATTCCCTGAGCTCTACCCTGACACT GTCCAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACAAAGAGCTTCAACCGCGGCGAGTGTTAA (SEQ ID NO: 8). In one embodiment, SEQ ID NO:8 is also referred to as "PAL."

在一个实施例中，本发明提供的重组融合蛋白所包含的抗HER3抗体的重链包含以下氨基酸序列：In one embodiment, the heavy chain of the anti-HER3 antibody contained in the recombinant fusion protein provided by the present invention comprises the following amino acid sequence:

MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ(SEQ ID NO:9)。MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 9).

在一个实施例中，本发明提供的重组融合蛋白所包含的抗HER3抗体的重链包含以下氨基酸序列：In one embodiment, the heavy chain of the anti-HER3 antibody contained in the recombinant fusion protein provided by the present invention comprises the following amino acid sequence:

MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFLGGPAVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ(SEQ ID NO:10)。MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFLGGPAVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLS LSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 10).

在一个实施例中，抗HER3 mAb重链序列包含信号肽序列。在另一个实施例中，抗HER3mAb重链信号肽序列包含MEFGLSWVFLVAIIKGVQC(SEQ ID NO:11)的氨基酸序列。In one embodiment, the anti-HER3 mAb heavy chain sequence comprises a signal peptide sequence. In another embodiment, the anti-HER3 mAb heavy chain signal peptide sequence comprises an amino acid sequence of MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 11).

在一个实施例中，重组融合蛋白所包含的抗HER3抗体的轻链包含以下氨基酸序列：MVLQTQVFISLLLWISGAYGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:12)。In one embodiment, the light chain of the anti-HER3 antibody comprised by the recombinant fusion protein comprises the following amino acid sequence: MVLQTQVFISLLLWISGAYGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

在一个实施例中，抗HER3 mAb轻链序列包含信号肽序列。在另一个实施例中，抗HER3mAb轻链信号肽序列包含MVLQTQVFISLLLWISGAYG(SEQ ID NO:13)的氨基酸序列。在一个实施例中，本发明所公开的成熟多肽诸如抗体重链或轻链氨基酸序列缺乏信号肽。In one embodiment, the anti-HER3 mAb light chain sequence comprises a signal peptide sequence. In another embodiment, the anti-HER3 mAb light chain signal peptide sequence comprises an amino acid sequence of MVLQTQVFISLLLWISGAYG (SEQ ID NO: 13). In one embodiment, the mature polypeptide disclosed in the present invention, such as an antibody heavy chain or light chain amino acid sequence, lacks a signal peptide.

在一个实施例中，重组融合蛋白包含以下氨基酸序列：In one embodiment, the recombinant fusion protein comprises the following amino acid sequence:

重链Heavy chain

(SEQ ID NO:14，其中粗斜体指示连接子，且粗体指示NRG-1片段)；及 (SEQ ID NO: 14, wherein bold italics indicate a linker, and bold indicates an NRG-1 fragment); and

轻链Light chain

DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:3)。DIEST HQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

在一个实施例中，成熟重组融合蛋白中的重链序列及轻链序列均缺乏信号肽氨基酸序列。In one embodiment, both the heavy chain sequence and the light chain sequence in the mature recombinant fusion protein lack a signal peptide amino acid sequence.

在本发明的另一特定实施例中，本发明提供的抗HER3抗体的重链经由C端连接子序列与本发明提供的NRG-1β2a同功异型物融合。在另一个实施例中，抗体重链的C端包含抗体的Fc结构域。In another specific embodiment of the present invention, the heavy chain of the anti-HER3 antibody provided by the present invention is fused to the NRG-1β2a isoform provided by the present invention via a C-terminal linker sequence. In another embodiment, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody.

在一些实施方案中，提供药物组合物，其包含与医药载剂一起调配的本发明所公开的重组融合蛋白。In some embodiments, a pharmaceutical composition is provided, which comprises a recombinant fusion protein disclosed herein formulated together with a pharmaceutical carrier.

在一些实施方案中，本发明所述的抗HER3抗体及NRG-1片段经由连接子以重组方式或以化学方式融合/可操作地连接以形成融合蛋白。“融合蛋白”、“融合多肽”、“重组融合蛋白”或“重组多肽”是指包含来自至少两个不同多肽的多肽部分的混合多肽。如本发明所定义的“融合蛋白”为包含例如本发明的NRG-1β2a同功异型物的第一氨基酸序列(蛋白质)经由连接子与包含特异性结合于ERBB3(HER3)的抗体重链的第二氨基酸序列的C端接合的融合物。In some embodiments, the anti-HER3 antibody and NRG-1 fragment described in the present invention are fused/operably linked via a linker in a recombinant or chemical manner to form a fusion protein. "Fusion protein", "fusion polypeptide", "recombinant fusion protein" or "recombinant polypeptide" refers to a mixed polypeptide comprising polypeptide portions from at least two different polypeptides. As defined in the present invention, a "fusion protein" is a fusion comprising, for example, a first amino acid sequence (protein) of an NRG-1β2a isoform of the present invention joined to the C-terminus of a second amino acid sequence of an antibody heavy chain that specifically binds to ERBB3 (HER3) via a linker.

在一个实施例中，融合蛋白以重组方式编码及产生。在一些实施方案中，重组融合蛋白由编码本发明抗体的核酸序列编码，该核酸序列经由编码连接子的核酸序列可操作地连接至编码本发明的NRG-1β2a同功异型物的核酸序列。In one embodiment, the fusion protein is encoded and produced in a recombinant manner. In some embodiments, the recombinant fusion protein is encoded by a nucleic acid sequence encoding an antibody of the invention, which is operably linked to a nucleic acid sequence encoding an NRG-1β2a isoform of the invention via a nucleic acid sequence encoding a linker.

在一个实施例中，重组融合蛋白氨基酸序列与SEQ ID NO:14融合至SEQ ID NO:3同源。术语“同源性”可指与重组融合蛋白序列(例如SEQ ID NO:1-18中的任一个)的同一性大于70％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于72％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于75％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个人的同一性大于78％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于80％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于82％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于83％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于85％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于87％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于88％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于90％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于92％。在另一个实施例中，“同源性”是指与SEQID NO:1-18中的任一个的同一性大于93％。在另一个实施例中，“同源性”是指与SEQ IDNO:1-18中的任一个的同一性大于95％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于96％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于97％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于98％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性大于99％。在另一个实施例中，“同源性”是指与SEQ ID NO:1-18中的任一个的同一性为100％。In one embodiment, the recombinant fusion protein amino acid sequence is homologous to SEQ ID NO: 14 fused to SEQ ID NO: 3. The term "homology" may refer to an identity greater than 70% with a recombinant fusion protein sequence (e.g., any one of SEQ ID NOs: 1-18). In another embodiment, "homology" refers to an identity greater than 72% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 75% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 78% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 80% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 82% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 83% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 85% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 87% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 88% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 90% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 92% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 93% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 95% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 96% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 97% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 98% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity greater than 99% with any one of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to an identity of 100% with any one of SEQ ID NOs: 1-18.

两个序列之间的同一性百分比的确定可使用数学算法来实现。用于比较两个序列的数学算法的非限制性实例为Karlin及Altschul,1990,Proc.Natl.Acad.Sci.USA 87:2264-2268中的算法，如Karlin and Altschul,1993,Proc.Natl.Acad.Sci.USA 90:5873-5877中进行修改。此类算法并入Altschul et al.,1990,J.Mol.Biol.215:403-410的NBLAST及XBLAST程序中。BLAST核苷酸检索可用NBLAST程序(得分＝100，字长＝12)执行以获得与编码所关注蛋白质的核酸同源的核苷酸序列。BLAST蛋白质检索可用XBLAST程序(得分＝50，字长＝3)执行以获得与所关注蛋白质同源的氨基酸序列。为了获得用于比较目的的间隙比对，可如Altschul等人,1997,Nucleic Acids Res.25:3389-3402中所述使用间隙BLAST。或者，可使用PSI-Blast进行迭代搜寻，检测分子间的距离关系(id.)。当利用BLAST、间隙BLAST及PSI-BLAST程序时，可使用各自程序(例如XBLAST及NBLAST)的默认参数。用于序列比较的数学算法的另一个非限制性实例为Myers及Miller,CABIOS(1989)的算法。此类算法并入ALIGN程序(版本2.0)中，该程序为GCG序列比对软件包的一部分。当利用ALIGN程序来比较氨基酸序列时，可使用PAM120权重残基表、间隙长度罚分12及间隙罚分4。用于序列分析的其他算法为本领域中已知的且包括如Torellis及Robotti,1994,Comput.Appl.Biosci.10:3-5中所述的ADVANCE及ADAM；以及Pearson及Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444-8中所述的FASTA。在FASTA中，ktup是设定搜寻敏感度和速度的控制选项。若ktup＝2，则通过观察成对的比对残基发现所比较的两个序列中的相似区域；若ktup＝1，则检查单一比对氨基酸。ktup对于蛋白质序列可设定为2或1，或对于DNA序列可设定为1至6。若未指定ktup，则默认值对于蛋白质为2且对于DNA为6。或者，可使用CLUSTAL W算法进行蛋白质序列比对，如Higgins等人,1996,Methods Enzymol.266:383-402所述。The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to nucleic acids encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to a protein of interest. In order to obtain gap alignments for comparison purposes, gap BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used for iterative searches to detect distance relationships (id.) between molecules. When using BLAST, gap BLAST, and PSI-BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Other algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by observing pairs of aligned residues; if ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or to 6 for DNA sequences. If ktup is not specified, the default value is 2 for proteins and 6 for DNA. Alternatively, the CLUSTAL W algorithm can be used for protein sequence alignment, as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

在一些实施方案中，本发明的多核苷酸是使用PCR技术，使用本领域技术人员已知的程序及方法制备。在一些实施方案中，该程序涉及两个不同DNA序列的接合(参见例如“Current Protocols in Molecular Biology”,Ausubel等人编,John Wiley&Sons,1992)。In some embodiments, the polynucleotides of the present invention are prepared using PCR technology, using procedures and methods known to those skilled in the art. In some embodiments, the procedure involves the joining of two different DNA sequences (see, for example, "Current Protocols in Molecular Biology", Ausubel et al., ed., John Wiley & Sons, 1992).

在一个实施例中，将本发明的多核苷酸插入表达载体(即核酸构建体)中，以促成重组多肽的表达。在一个实施例中，本发明的表达载体包括额外序列，其使得此载体适合在原核生物中复制及整合。在一个实施例中，本发明的表达载体包括额外序列，其使得此载体适合在真核生物中复制及整合。在一个实施例中，本发明的表达载体包括穿梭载体，其使得此载体适合在原核生物及真核生物中复制及整合。在一些实施方案中，克隆载体包含转录及翻译起始序列(例如启动子、增强子)以及转录及翻译终止子(例如多聚腺苷酸化信号)。In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to facilitate the expression of a recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences that make this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences that make this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector that makes this vector suitable for replication and integration in prokaryotes and eukaryotes. In some embodiments, the cloning vector comprises transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

在一个实施例中，多种原核或真核细胞可用作宿主表达系统，以表达本发明的多肽。在一些实施方案中，这些包括但不限于微生物，诸如用含有多肽编码序列的重组噬菌体DNA、质粒DNA或黏性质粒DNA表达载体转化的细菌；用含有多肽编码序列的重组酵母表达载体转化的酵母。In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host expression systems to express the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing polypeptide coding sequences; yeast transformed with recombinant yeast expression vectors containing polypeptide coding sequences.

在一些实施方案中，使用非细菌表达系统(例如哺乳动物表达系统，诸如CHO细胞)来表达本发明的多肽。在一个实施例中，用于在哺乳动物细胞中表达本发明的多核苷酸的表达载体为包含CMV启动子及新霉素抗性基因的pCI-DHFR载体。In some embodiments, a non-bacterial expression system (e.g., a mammalian expression system, such as CHO cells) is used to express the polypeptides of the invention. In one embodiment, the expression vector used to express the polynucleotides of the invention in mammalian cells is a pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene.

在一些实施方案中，在本发明的细菌系统中，可根据所表达多肽的预期用途有利地选择多种表达载体。在一个实施例中，需要大量多肽。在一个实施例中，需要载体引导蛋白质产物高水平的表达，可能表达成与疏水性信号序列的融合物，该疏水性信号序列引导所表达的产物至细菌周质或培养基中，其中蛋白质产物容易被纯化。在一个实施例中，某些融合蛋白经工程改造以具有特定裂解位点，从而帮助回收多肽。在一个实施例中，可适应于此类操作的载体包括但不限于pET系列的大肠杆菌表达载体[Studier等人,Methods inEnzymol.185:60-89(1990)]。In some embodiments, in the bacterial system of the present invention, a variety of expression vectors can be advantageously selected according to the intended use of the expressed polypeptide. In one embodiment, a large amount of polypeptide is required. In one embodiment, a carrier is required to guide the expression of a high level of protein product, which may be expressed as a fusion with a hydrophobic signal sequence, which guides the expressed product to the bacterial periplasm or culture medium, where the protein product is easily purified. In one embodiment, some fusion proteins are engineered to have specific cleavage sites to help recover the polypeptide. In one embodiment, carriers that can be adapted to such operations include, but are not limited to, the E. coli expression vectors of the pET series [Studier et al., Methods in Enzymol. 185: 60-89 (1990)].

在一个实施例中，使用酵母表达系统。在一个实施例中，许多含有组成型或诱导型启动子的载体可用于酵母中，如美国专利第5,932,447号中所公开。在另一个实施例中，使用促进外来DNA序列整合至酵母染色体中的载体。In one embodiment, a yeast expression system is used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast, as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, a vector that promotes integration of foreign DNA sequences into yeast chromosomes is used.

在一个实施例中，本发明的表达载体可进一步包括额外多核苷酸序列，允许例如自单个mRNA翻译数种蛋白质，诸如内部核糖体进入位点(IRES)及用于启动子嵌合多肽的基因组整合的序列。In one embodiment, the expression vector of the present invention may further include additional polynucleotide sequences allowing, for example, translation of several proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter chimeric polypeptide.

在一些实施方案中，本发明的表达载体包括增加本发明的重组融合蛋白的表达的元件。此类特征包括但不限于启动子及多聚腺苷酸化的选择。在一些实施方案中，多聚腺苷酸化序列为牛生长激素(BGH)多聚腺苷酸化序列。在一些实施方案中，启动子包括组成型活性启动子。在一些实施方案中，启动子包括巨细胞病毒启动子(pCMV)。In some embodiments, the expression vector of the present invention includes elements that increase the expression of the recombinant fusion protein of the present invention. Such features include, but are not limited to, the selection of promoters and polyadenylation. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence. In some embodiments, the promoter includes a constitutively active promoter. In some embodiments, the promoter includes a cytomegalovirus promoter (pCMV).

在一些实施方案中，哺乳动物表达载体包括但不限于pcDNA3、pcDNA3.1(+/-)、pGL3、pZeoSV2(+/-)、pSecTag2、pDisplay、pEF/myc/cyto、pCMV/myc/cyto、pCR3.1、pSinRep5、DH26S、DHBB、pNMT1、pNMT41、pNMT81，其可购自Invitrogen；pCI，其可购自Promega；pMbac、pPbac、pBK-RSV及pBK-CMV，其可购自Strategene；pTRES，其可购自Clontech；及其衍生物。In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which can be purchased from Invitrogen; pCI, which can be purchased from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which can be purchased from Strategene; pTRES, which can be purchased from Clontech; and derivatives thereof.

在一些实施方案中，本发明使用含有来自真核病毒诸如反转录病毒的调控元件的表达载体。SV40载体包括pSVT7及pMT2。在一些实施方案中，来源于牛乳头状瘤病毒的载体包括pBV-1MTHA，且来源于埃-巴二氏病毒(Epstein Barr virus)的载体包括pHEBO及p205。其他例示性载体包括pMSG、pAV009/A⁺\pMTO10/A⁺、pMAMneo-5、杆状病毒pDSVE以及允许蛋白在SV-40早期启动子、SV-40晚期启动子、金属硫蛋白启动子、鼠乳房肿瘤病毒启动子、劳斯肉瘤病毒(Rous sarcoma virus)启动子、多角体蛋白启动子或显示为对于在真核细胞中的表达有效的其他启动子的指导下表达的任何其他载体。In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Barr virus include pHEBO and p205. Other exemplary vectors include pMSG, pAV009/A⁺ \pMTO10/A⁺ , pMAMneo-5, baculovirus pDSVE, and any other vectors that allow protein expression under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

在一些实施方案中，重组病毒载体可用于本发明的多肽的体内表达，因为其提供诸如横向感染及靶向特异性的优点。在一个实施例中，横向感染为例如反转录病毒的生命周期中所固有的，且为单个受感染细胞产生许多后代病毒粒子的过程，该等病毒粒子出芽且感染邻近的细胞。在一个实施例中，结果为大片区域被迅速感染，其中大部分区域最初未被原始病毒粒子感染。在一个实施例中，产生不能横向传播的病毒载体。在一个实施例中，若预期目的是将特定基因仅引入局部数目的靶细胞中，则此特征可为有用的。In some embodiments, recombinant viral vectors can be used for in vivo expression of polypeptides of the present invention because they provide advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, retroviruses, and is the process by which a single infected cell produces many progeny virions that bud and infect adjacent cells. In one embodiment, the result is that large areas are rapidly infected, most of which were not initially infected by the original virions. In one embodiment, a viral vector that cannot spread laterally is produced. In one embodiment, this feature may be useful if the intended purpose is to introduce a specific gene only into a local number of target cells.

在一个实施例中，可使用各种方法将编码本发明的重组融合蛋白的表达载体引入细胞中。此类方法一般描述于Sambrook等人,Molecular Cloning:A Laboratory Manual,Cold Springs Harbor Laboratory,New York(1989,1992)；Ausubel等人,CurrentProtocols in Molecular Biology,John Wiley and Sons,Baltimore,Md.(1989)；Chang等人,Somatic Gene Therapy,CRC Press,Ann Arbor,Mich.(1995)；Vega等人,GeneTargeting,CRC Press,Ann Arbor Mich.(1995),Vectors:ASurvey of MolecularCloning Vectors and Their Uses,Butterworths,Boston Mass.(1988)及Gilboa等人[Biotechniques 4(6):504-512,1986]中，且包括例如稳定或瞬时转染、脂质转染、电穿孔及用重组病毒载体感染。另外，关于正向-负向选择方法，参见美国专利第5,464,764号及第5,487,992号。In one embodiment, various methods can be used to introduce expression vectors encoding recombinant fusion proteins of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, regarding the positive-negative selection method, see US Pat. Nos. 5,464,764 and 5,487,992.

在一些实施方案中，通过病毒感染引入核酸提供相较于其他方法，诸如脂质转染及电穿孔的若干优点，因为可由于病毒的感染性质而获得较高转染效率。In some embodiments, introduction of nucleic acids by viral infection offers several advantages over other methods, such as lipofection and electroporation, because higher transfection efficiencies can be obtained due to the infectious nature of the virus.

在一个实施例中，应了解，本发明的多肽还可由采用上文所描述的任何适合的施用模式向个体施用的核酸构建体表达(即体内基因疗法)。在一个实施例中，核酸构建体经由适当的基因递送载体/方法(转染、转导、同源重组等)及表达系统(视需要)引入适合的细胞中，且随后将经修饰的细胞在培养中扩增且返回至个体(即体外基因疗法)。In one embodiment, it should be understood that the polypeptides of the present invention can also be expressed by nucleic acid constructs administered to individuals using any suitable mode of administration described above (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into suitable cells via appropriate gene delivery vectors/methods (transfection, transduction, homologous recombination, etc.) and expression systems (optionally), and the modified cells are subsequently amplified in culture and returned to the individual (i.e., in vitro gene therapy).

应了解，除含有所插入编码序列(编码多肽)的转录及翻译所必需的元件以外，本发明的表达构建体还可包括经工程改造以使所表达多肽的稳定性、产生、纯化、产量或活性优化的序列。It will be appreciated that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

在一些实施方案中，经转化细胞是在允许表达大量重组融合蛋白或多肽的有效条件下培养。在一些实施方案中，有效培养条件包括但不限于允许蛋白质产生的有效培养基、生物反应器、温度、pH及氧气条件。在一个实施例中，有效培养基是指其中培养细胞以产生本发明的重组多肽的任何培养基。在一些实施方案中，培养基通常包括具有可吸收的碳、氮及磷酸盐源以及适当盐、矿物质、金属及其他营养素诸如维生素的水溶液。在一些实施方案中，本发明的细胞可在常规发酵生物反应器、摇瓶、试管、微量滴定盘及培养皿中培养。在一些实施方案中，培养在适于重组细胞的温度、pH及氧气含量下进行。在一些实施方案中，培养条件在本领域普通技术人员的专业知识内。In some embodiments, the transformed cells are cultured under effective conditions that allow expression of a large amount of recombinant fusion proteins or polypeptides. In some embodiments, effective culture conditions include but are not limited to effective culture medium, bioreactor, temperature, pH and oxygen conditions that allow protein production. In one embodiment, effective culture medium refers to any culture medium in which cells are cultured to produce recombinant polypeptides of the present invention. In some embodiments, culture medium generally includes an aqueous solution with absorbable carbon, nitrogen and phosphate sources and appropriate salts, minerals, metals and other nutrients such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates and culture dishes. In some embodiments, culture is carried out at a temperature, pH and oxygen content suitable for recombinant cells. In some embodiments, culture conditions are within the professional knowledge of those of ordinary skill in the art.

在一些实施方案中，视用于生产的载体及宿主系统而定，本发明的所得多肽保留在重组细胞内、分泌至发酵培养基中、分泌至两个细胞膜之间的空间(诸如大肠杆菌的周质空间)中；或保留在细胞或病毒膜的外表面。In some embodiments, depending on the vector and host system used for production, the resulting polypeptide of the present invention is retained within the recombinant cell, secreted into the fermentation medium, secreted into the space between two cell membranes (such as the periplasmic space of E. coli); or retained on the outer surface of the cell or viral membrane.

在一个实施例中，在预定的培养时间后，实现重组多肽的回收。In one embodiment, after a predetermined incubation time, recovery of the recombinant polypeptide is achieved.

在一个实施例中，本发明所用的词组“回收重组多肽”是指收集含有多肽的整个发酵培养基，且无需暗示额外的分离或纯化步骤。In one embodiment, the phrase "recovering the recombinant polypeptide" as used herein refers to collecting the entire fermentation medium containing the polypeptide, without implying additional separation or purification steps.

在一个实施例中，本发明的多肽使用多种标准蛋白纯化技术纯化，诸如但不限于亲和层析、离子交换层析、过滤、电泳、疏水性相互作用层析、凝胶过滤层析、反相层析、刀豆球蛋白A层析、层析聚焦及差异溶解。In one embodiment, the polypeptides of the invention are purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubility.

在一个实施例中，为了促进回收，所表达的编码序列可经工程改造以编码本发明的多肽及融合可裂解部分。在一个实施例中，融合蛋白可经设计以使得多肽可容易地通过亲和层析分离；例如，通过固定于对可裂解部分具有特异性的柱上。在一个实施例中，裂解位点经工程改造在多肽与可裂解部分之间，且多肽可通过用在此位点特异性裂解融合蛋白的适当酶或药剂处理而自层析柱释放[例如参见Booth等人,Immunol.Lett.19:65-70(1988)；及Gardella等人,J.Biol.Chem.265:15854-15859(1990)]。In one embodiment, to facilitate recovery, the expressed coding sequence may be engineered to encode a polypeptide of the invention and a fused cleavable moiety. In one embodiment, the fusion protein may be designed so that the polypeptide can be easily separated by affinity chromatography; for example, by being immobilized on a column specific for the cleavable moiety. In one embodiment, the cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [see, for example, Booth et al., Immunol. Lett. 19: 65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].

在一个实施例中，本发明的多肽以“基本上纯的”的形式回收。In one embodiment, a polypeptide of the invention is recovered in a "substantially pure" form.

在一个实施例中，词组“基本上纯的”是指允许蛋白质在本发明所述的应用中有效使用的纯度。In one embodiment, the phrase "substantially pure" refers to a degree of purity that allows the protein to be effectively used in the applications described herein.

在一个实施例中，本发明的多肽还可使用体外表达系统合成。在一个实施例中，体外合成方法为本领域中所熟知，且系统的组分为可商购的。In one embodiment, the polypeptides of the present invention can also be synthesized using an in vitro expression system. In one embodiment, in vitro synthesis methods are well known in the art, and the components of the system are commercially available.

在一些实施方案中，重组多肽经合成及纯化；其治疗功效可在体内或体外加以分析。In some embodiments, recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be analyzed in vivo or in vitro.

在一个实施例中，包含本发明的重组融合蛋白的本发明提供的药物组合物与医药载剂一起进一步调配。如本发明所用，“医药载剂”包括生理学上兼容的任何及所有溶剂、分散介质、包衣、抗细菌剂及抗真菌剂、等张剂及吸收延迟剂及其类似物。优选地，载剂适用于静脉内、肌肉内、皮下、肠胃外、脊椎或表皮给药(例如通过注射或输注)。In one embodiment, the pharmaceutical composition provided by the present invention comprising the recombinant fusion protein of the present invention is further formulated with a pharmaceutical carrier. As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

治疗方法Treatment

在一个实施例中，本发明提供一种治疗有需要的受试者的疾病或病况的方法，该方法包括施用治疗有效量的重组融合蛋白或包含本发明所公开的重组融合蛋白的药物组合物。In one embodiment, the present invention provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein disclosed herein.

在一个实施例中，本发明提供一种治疗有需要的受试者的心血管疾病或病况的方法，该方法包括施用治疗有效量的重组融合蛋白或包含其的药物组合物。In one embodiment, the present invention provides a method of treating a cardiovascular disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the same.

在一个实施例中，本发明提供一种预防、抑制、遏制或延缓受试者的心血管疾病或病况发作的方法，该方法包括施用有效量的本发明所述的重组融合蛋白或药物组合物。In one embodiment, the present invention provides a method for preventing, inhibiting, suppressing or delaying the onset of a cardiovascular disease or condition in a subject, the method comprising administering an effective amount of the recombinant fusion protein or pharmaceutical composition of the present invention.

在一些实施方案中，心血管疾病或病况包括心房震颤。In some embodiments, the cardiovascular disease or condition comprises atrial fibrillation.

在一些实施方案中，心血管疾病或病况包括心脏纤维化。In some embodiments, the cardiovascular disease or condition comprises cardiac fibrosis.

在一些实施方案中，心血管疾病或病况包括心房震颤及心脏纤维化。In some embodiments, cardiovascular diseases or conditions include atrial fibrillation and cardiac fibrosis.

在一些实施方案中，心血管疾病包括慢性心脏衰竭/充血性心脏衰竭(CHF)、急性心脏衰竭/心肌梗塞(MI)、左心室收缩功能障碍、与MI相关的再灌注损伤、化学疗法诱导的心脏毒性(成人或儿童)、放射诱导的心脏毒性、儿童先天性心脏病的手术干预的辅助治疗。In some embodiments, cardiovascular diseases include chronic heart failure/congestive heart failure (CHF), acute heart failure/myocardial infarction (MI), left ventricular systolic dysfunction, reperfusion injury associated with MI, chemotherapy-induced cardiotoxicity (adults or children), radiation-induced cardiotoxicity, adjunctive treatment with surgical intervention for congenital heart disease in children.

在一些实施方案中，其中化学疗法诱导的心脏毒性由接受用作化学疗法的蒽环霉素、烷基化剂、抗微管剂及抗代谢物药剂的受试者产生。In some embodiments, wherein chemotherapy-induced cardiotoxicity results from a subject receiving anthracyclines, alkylating agents, anti-microtubule agents, and antimetabolite agents as chemotherapy.

在一些实施方案中，心血管病况是由于受试者接受癌症疗法而导致的心脏毒性。在其他实施例中，癌症疗法为HER-2靶向疗法。在其他实施例中，HER-2靶向疗法包含使用曲妥珠单抗、阿多曲妥珠单抗、恩他新、拉帕替尼、来那替尼及帕妥株单抗、任何抗HER2抗体、任何抗HER2剂或其组合。In some embodiments, the cardiovascular condition is cardiotoxicity caused by the subject receiving cancer therapy. In other embodiments, the cancer therapy is a HER-2 targeted therapy. In other embodiments, the HER-2 targeted therapy comprises the use of trastuzumab, adotrastuzumab, emtansine, lapatinib, neratinib and pertuzumab, any anti-HER2 antibody, any anti-HER2 agent, or a combination thereof.

在另一方面中，本发明是关于一种诱导肌细胞肌节及细胞骨架结构重塑或细胞-细胞黏附的方法，该方法包括用本发明所公开的重组融合蛋白处理细胞。In another aspect, the present invention relates to a method for inducing sarcomere and cytoskeleton structure remodeling or cell-cell adhesion in muscle cells, which comprises treating cells with the recombinant fusion protein disclosed in the present invention.

在一个实施例中，治疗方法是针对治疗由哺乳动物中心肌细胞-细胞黏附的分离和/或肌节结构紊乱引起的心脏衰竭。In one embodiment, the method of treatment is directed to treating heart failure caused by dissociation of cardiomyocyte-cell adhesions and/or sarcomere structural disturbances in a mammal.

在另一方面中，本发明提供一种用于预防、治疗或延缓人类的射血分数保留型心脏衰竭的方法，该方法包括施用包含本发明所公开的重组融合蛋白的药物组合物。In another aspect, the present invention provides a method for preventing, treating or delaying heart failure with preserved ejection fraction in humans, the method comprising administering a pharmaceutical composition comprising the recombinant fusion protein disclosed in the present invention.

如本发明所用，术语“射血分数”是指射血分数(EF)，即左心室在每次收缩时泵出多少血液的测量值，通常表示为百分比。例如，50％的射血分数意味着左心室中血液总量的50％在每次心跳时被排出。As used herein, the term "ejection fraction" refers to the ejection fraction (EF), which is a measurement of how much blood the left ventricle pumps out each time it contracts, usually expressed as a percentage. For example, an ejection fraction of 50% means that 50% of the total amount of blood in the left ventricle is ejected in each heartbeat.

本发明是关于治疗患有或有风险罹患心脏病及相关病况例如心脏衰竭的受试者。The present invention relates to treating subjects suffering from or at risk for heart disease and related conditions such as heart failure.

术语“心脏衰竭”是指心脏功能异常，其中心脏不能以代谢组织所需的速率泵血。心脏衰竭包括各种疾病状态，诸如充血性心脏衰竭、心肌梗塞、心动过速、家族性肥厚性心肌病、缺血性心脏病、特发性扩张型心肌病及心肌炎。心脏衰竭可由多种因素引起，包括缺血性、先天性、风湿性或特发性形式。慢性心脏肥大为一种严重的患病状态，其为充血性心脏衰竭及心跳骤停的先兆。The term "heart failure" refers to abnormal cardiac function in which the heart cannot pump blood at the rate required by metabolic tissues. Heart failure includes various disease states, such as congestive heart failure, myocardial infarction, tachycardia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. Heart failure can be caused by a variety of factors, including ischemic, congenital, rheumatic, or idiopathic forms. Chronic cardiac hypertrophy is a serious disease state that is a precursor to congestive heart failure and cardiac arrest.

在一个实施例中，“治疗”是指治疗性治疗及预防性或防治性措施，其目的为预防或减缓(减轻)心脏肥大。需要治疗者包括已患有病症者以及易患有病症者或需要预防病症者。心脏肥大可来自对视黄酸起反应的任何病因，包括先天性、病毒性、特发性、心脏营养性或肌营养性病因，或由于局部缺血或局部缺血损伤，诸如心肌梗塞。通常，进行治疗以阻止或减缓肥大进展，尤其在已发生诸如来自局部缺血的心脏损伤之后。优选地，对于治疗心肌梗塞，本发明提供的药物组合物在心肌梗塞后立即给予以防止或减轻肥大。In one embodiment, "treatment" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) cardiac hypertrophy. Those in need of treatment include those who already have the disease and those who are susceptible to the disease or need to prevent the disease. Cardiac hypertrophy can come from any cause that responds to retinoic acid, including congenital, viral, idiopathic, cardiotrophic or myotrophic causes, or due to ischemia or ischemic injury, such as myocardial infarction. Typically, treatment is performed to prevent or slow the progression of hypertrophy, especially after cardiac damage such as from ischemia has occurred. Preferably, for the treatment of myocardial infarction, the pharmaceutical composition provided by the present invention is administered immediately after the myocardial infarction to prevent or reduce hypertrophy.

在一些实施方案中，用本发明所述的重组融合蛋白或包含其的药物组合物治疗患有心房震颤或心脏纤维化的受试者可导致减少心房震颤或纤维化的体征或症状。举例而言，用重组融合蛋白治疗受试者可减少心房震颤的持续时间和/或频率，或减少心房震颤的体征或症状，诸如减少心跳不规则、心悸、头晕、极度疲劳、呼吸短促、胸痛或其组合。在一些实施方案中，用重组融合蛋白治疗受试者减少心脏组织，例如心房组织中的胶原蛋白含量或沉积。In some embodiments, treating a subject with atrial fibrillation or cardiac fibrosis with the recombinant fusion protein of the present invention or a pharmaceutical composition comprising the same can result in reducing the signs or symptoms of atrial fibrillation or fibrosis. For example, treating a subject with the recombinant fusion protein can reduce the duration and/or frequency of atrial fibrillation, or reduce the signs or symptoms of atrial fibrillation, such as reducing irregular heartbeat, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, or a combination thereof. In some embodiments, treating a subject with the recombinant fusion protein reduces the content or deposition of collagen in cardiac tissue, such as atrial tissue.

在一些实施方案中，相较于接受非本发明化合物的药物或其医药学上可接受的盐、溶剂合物、类似物或衍生物的单一疗法的群体，用包含本发明提供的重组融合蛋白的药物组合物治疗受试者可使得经治疗的受试者群体的平均存活时间增加。优选地，在用本发明所提供的策略、治疗方式、方法、组合及组合物治疗后，平均存活时间增加超过30天；更优选超过60天；更优选超过90、120或365天；更优选超过365天。群体平均存活时间的增加可通过任何可再现的手段来测量。群体平均存活时间的增加可例如通过计算群体在开始用活性化合物治疗后的平均存活时间长度测来量。群体平均存活时间的增加还可例如通过计算群体在完成本发明所公开的药物组合物的第一轮治疗后的平均存活时间长度来测量。In some embodiments, compared to a group receiving a monotherapy of a drug that is not a compound of the present invention or a pharmaceutically acceptable salt, solvate, analog or derivative thereof, treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided by the present invention can increase the average survival time of the treated subject group. Preferably, after treatment with the strategies, treatment methods, methods, combinations and compositions provided by the present invention, the average survival time increases by more than 30 days; more preferably more than 60 days; more preferably more than 90, 120 or 365 days; more preferably more than 365 days. The increase in the average survival time of the group can be measured by any reproducible means. The increase in the average survival time of the group can be measured, for example, by calculating the average survival time length of the group after starting treatment with an active compound. The increase in the average survival time of the group can also be measured, for example, by calculating the average survival time length of the group after completing the first round of treatment of the pharmaceutical composition disclosed in the present invention.

在一些实施方案中，用包含本发明提供的重组融合蛋白的药物组合物治疗受试者相比于仅接受载剂的群体可使得经治疗的受试者群体的死亡率降低。治疗癌症可使得经治疗的受试者群体与未治疗的群体相比死亡率降低。与接受非本发明化合物的药物或其医药学上可接受的盐、溶剂合物、类似物或衍生物的单一疗法的群体相比，治疗癌症可导致经治疗的受试者群体的死亡率降低。优选地，在用本发明所提供的策略、治疗方式、方法、组合及组合物治疗后，死亡率降低超过2％；更优选超过5％；更优选超过10％；且最优选超过25％。经治疗的受试者群体的死亡率降低可通过任何可再现的手段来测量。群体死亡率的降低可例如通过计算群体在开始用活性化合物治疗后每单位时间的疾病相关的平均死亡数来测量。群体死亡率的降低还可例如通过计算群体在完成本发明所公开的药物组合物的第一轮治疗后每单位时间的疾病相关的平均死亡数来测量。In some embodiments, treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided by the present invention can reduce the mortality rate of the treated subject population compared to the population receiving only the carrier. Treating cancer can reduce the mortality rate of the treated subject population compared to the untreated population. Compared with a population receiving a monotherapy of a drug or a pharmaceutically acceptable salt, solvate, analog or derivative of a non-compound of the present invention, treating cancer can result in a reduced mortality rate in the treated subject population. Preferably, after treatment with the strategies, treatment methods, methods, combinations and compositions provided by the present invention, the mortality rate is reduced by more than 2%; more preferably more than 5%; more preferably more than 10%; and most preferably more than 25%. The reduction in the mortality rate of the treated subject population can be measured by any reproducible means. The reduction in the mortality rate of the population can be measured, for example, by calculating the average number of deaths related to the disease per unit time after the population begins treatment with the active compound. The reduction in the mortality rate of the population can also be measured, for example, by calculating the average number of deaths related to the disease per unit time after the first round of treatment of the pharmaceutical composition disclosed by the present invention.

在一个实施例中，本发明提供一种治疗有需要的受试者的中枢神经系统(CNS)相关疾病或病况的方法，该方法包括施用治疗有效量的本发明所述的重组融合蛋白或药物组合物。In one embodiment, the present invention provides a method for treating a central nervous system (CNS)-related disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant fusion protein or pharmaceutical composition of the present invention.

在一个实施例中，本发明提供一种预防、抑制、遏制或延缓个体的CNS相关疾病或病况发作的方法，该方法包括施用有效量的本发明所述的重组融合蛋白或药物组合物。In one embodiment, the present invention provides a method for preventing, inhibiting, suppressing or delaying the onset of a CNS-related disease or condition in an individual, the method comprising administering an effective amount of the recombinant fusion protein or pharmaceutical composition of the present invention.

在一些实施方案中，CNS相关疾病或病况为肌萎缩性脊髓侧索硬化症(ALS)、帕金森氏病、阿兹海默氏病、贝尔氏麻痹、癫痫及癫痫发作、格林-巴利综合征、中风、创伤性脑损伤、多发性硬化症或组合。In some embodiments, the CNS-related disease or condition is amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Bell's palsy, epilepsy and seizures, Guillain-Barre syndrome, stroke, traumatic brain injury, multiple sclerosis, or a combination.

给药、剂量Administration, dosage

本发明的组合物可肠胃外方式施用于有需要的受试者，或可通过本领域中已知的各种方法施用。如本领域技术人员所了解，施用途径和/或模式将视期望的结果而变化。为了通过某些施用途径施用本发明化合物，可能需要用材料包覆该化合物或将该化合物与材料共同施用以防止其失活。举例而言，化合物可于适当载剂(例如脂质体或稀释剂)中向受试者施用。医药学上可接受的稀释剂包括盐水及水性缓冲溶液。医药载剂包括无菌水溶液或分散液及用于临时制备无菌可注射溶液或分散液的无菌散剂。此类介质及药剂用于医药活性物质的用途为本领域中已知的。The composition of the present invention can be administered to a subject in need parenterally, or can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route of administration and/or mode will vary depending on the desired result. In order to administer the compounds of the present invention by certain routes of administration, it may be necessary to coat the compound with a material or co-administer the compound with a material to prevent its inactivation. For example, the compound can be administered to a subject in an appropriate carrier (e.g., liposomes or diluents). Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is known in the art.

在典型实施例中，向受试者施用的制剂包括无菌水性或非水性溶液、悬浮液及乳液。一些实施方案包括非水性溶剂，诸如丙二醇、聚乙二醇、植物油(例如橄榄油)、有机酯(例如油酸乙酯)及本领域技术人员已知的其他溶剂。生理学上可接受的载剂(或赋形剂)可选地用于本发明的某些实施例中。此类的实例包括例如生理盐水、PBS、林格氏溶液、乳酸林格氏溶液等。另外，防腐剂及添加剂可选地添加至组合物中，以帮助确保稳定性及无菌性。举例而言，抗生素及其他杀细菌剂、抗氧化剂、螯合剂及其类似物可选地均存在于本发明组合物的各种实施例中。In typical embodiments, the preparations administered to the subject include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Some embodiments include non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), organic esters (e.g., ethyl oleate) and other solvents known to those skilled in the art. Physiologically acceptable carriers (or excipients) are optionally used in certain embodiments of the present invention. Such examples include, for example, normal saline, PBS, Ringer's solution, lactated Ringer's solution, etc. In addition, preservatives and additives are optionally added to the composition to help ensure stability and sterility. For example, antibiotics and other bactericides, antioxidants, chelating agents and the like are optionally present in various embodiments of the present composition.

如本发明所用，词组“肠胃外给药”及“肠胃外给予”是指除经肠及局部给药以外的给药模式，通常通过注射，且包括但不限于静脉内、肌肉内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛膜下、脊柱内、硬膜外及胸骨内注射及输注。As used herein, the phrases "parenteral administration" and "parenteral administration" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

无论选择何种给药途径，可以适合的水合形式使用的本发明化合物和/或本发明的药物组合物是通过本领域技术人员已知的常规方法调配成医药学上可接受的剂型。Regardless of the administration route selected, the compound of the present invention and/or the pharmaceutical composition of the present invention, which can be used in a suitable hydrated form, is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

重组融合蛋白或包含其的药物组合物可选地在任何适当的无菌医药载剂中向需要治疗(治疗性或预防性)的受试者施用。此类医药载剂用以维持融合蛋白的可溶性及作用。在一些实施方案中，可能需要结合融合蛋白施用额外组分。举例而言，在一些治疗方案中，化学治疗剂、抗生素、包含本发明的重组融合蛋白的额外调配物及一个或多种护理标准剂等全部可选地包括在本发明的组合物中。The recombinant fusion protein or a pharmaceutical composition comprising it is optionally administered to a subject in need of treatment (therapeutic or preventive) in any suitable sterile pharmaceutical carrier. Such pharmaceutical carriers are used to maintain the solubility and effect of the fusion protein. In some embodiments, additional components may need to be administered in conjunction with the fusion protein. For example, in some treatment regimens, chemotherapeutic agents, antibiotics, additional formulations comprising the recombinant fusion protein of the present invention, and one or more standard of care agents, etc. are all optionally included in the composition of the present invention.

如本发明所用，术语“组合治疗”、“组合疗法”及“共同疗法”可互换使用，且一般是指以重组融合蛋白或包含本发明提供的重组融合蛋白及额外治疗剂的药物组合物为特征的治疗方式。通常，组合治疗方式为意欲自治疗剂组合的共同作用提供有益效果的特定治疗方案的一部分。组合的有益效果可包括但不限于由治疗剂组合产生的药代动力学或药效学协同作用。组合施用这些治疗剂通常在规定时间段(视所选组合而定，通常数分钟、数小时、数天或数周)内进行。在一些实施方案中，组合治疗包括以依序方式施用两种或更多种治疗剂，其中各治疗剂在不同时间施用，以及以基本上同时的方式施用这些治疗剂或至少两种治疗剂。基本上同时施用可例如通过向受试者施用具有固定比率的各治疗剂的单一剂型或治疗剂的多个单独剂型来实现。依序或基本上同时施用各治疗剂可通过任何适当途径实现，包括但不限于经口途径、静脉内途径、肌肉内途径及经由黏膜组织直接吸收。治疗剂可通过相同途径或通过不同途径施用。治疗剂可按照相同或不同的给药时间间隔给予。例如，所选择的组合的第一治疗剂可通过静脉内注射给予，而该组合的其他治疗剂可经口给予。或者，例如，所有治疗剂可经口给予或所有治疗剂可通过静脉内注射给予。As used in the present invention, the terms "combination therapy", "combination therapy" and "co-therapy" are used interchangeably and generally refer to a treatment method characterized by a recombinant fusion protein or a pharmaceutical composition comprising a recombinant fusion protein provided by the present invention and an additional therapeutic agent. Typically, a combination therapy is intended to be part of a specific treatment regimen that provides a beneficial effect from the combined action of a combination of therapeutic agents. The beneficial effects of the combination may include, but are not limited to, pharmacokinetic or pharmacodynamic synergy produced by a combination of therapeutic agents. The combined administration of these therapeutic agents is usually carried out within a specified time period (depending on the selected combination, usually minutes, hours, days or weeks). In some embodiments, the combination therapy includes administering two or more therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at different times, and administering these therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration can be achieved, for example, by administering to a subject a single dosage form of each therapeutic agent having a fixed ratio or a plurality of separate dosage forms of the therapeutic agent. Sequentially or substantially simultaneously administering each therapeutic agent can be achieved by any appropriate route, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption via mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. The therapeutic agents can be administered at the same or different dosing intervals. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally or all therapeutic agents can be administered by intravenous injection.

在一些实施方案中，组合疗法还涵盖如上文所述的治疗剂与其他生物活性成分及非药物疗法(例如手术或放射治疗)进一步组合施用。在组合疗法进一步包含非药物疗法的情况下，非药物疗法可在任何适合的时间执行，只要治疗剂与非药物疗法的组合的协同作用达成有益的效果。举例而言，在适当情况下，当非药物治疗暂时自治疗剂给予中移除(可能隔数天或甚至数周)时，仍达成有益效果。In some embodiments, the combination therapy also encompasses the further combined administration of therapeutic agents as described above with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation therapy). Where the combination therapy further comprises non-drug therapy, the non-drug therapy may be performed at any suitable time, as long as the synergistic effect of the combination of the therapeutic agent and the non-drug therapy achieves a beneficial effect. For example, in appropriate cases, when the non-drug therapy is temporarily removed from the administration of the therapeutic agent (perhaps for several days or even weeks), a beneficial effect is still achieved.

在一些实施方案中，额外治疗剂为化学治疗剂(还称为抗肿瘤剂或抗增殖剂)，例如，烷基化剂；抗生素；抗代谢物；解毒剂；干扰素；多克隆或单克隆抗体；EGFR抑制剂；HER2抑制剂；组蛋白去乙酰酶抑制剂；激素；有丝分裂抑制剂；MTOR抑制剂；多激酶抑制剂；丝氨酸/苏氨酸激酶抑制剂；酪氨酸激酶抑制剂；VEGF/VEGFR抑制剂；紫杉烷或紫杉烷衍生物、芳香酶抑制剂、蒽环霉素、微管靶向药物、拓朴异构酶毒药、分子靶标或酶的抑制剂(例如激酶或蛋白质甲基转移酶)、胞苷类似物药物或任何化学治疗剂、免疫检查点抑制剂、铂类抗肿瘤剂、CDK抑制剂、PARP抑制剂或本领域技术人员已知的任何抗肿瘤剂或抗增殖剂。In some embodiments, the additional therapeutic agent is a chemotherapeutic agent (also known as an anti-neoplastic agent or anti-proliferative agent), for example, an alkylating agent; an antibiotic; an antimetabolite; an antidote; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR inhibitor; a multikinase inhibitor; a serine/threonine kinase inhibitor; a tyrosine kinase inhibitor; a VEGF/VEGFR inhibitor; a taxane or a taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule-targeted drug, a topoisomerase poison, an inhibitor of a molecular target or enzyme (e.g., a kinase or protein methyltransferase), a cytidine analog drug, or any chemotherapeutic agent, an immune checkpoint inhibitor, a platinum anti-neoplastic agent, a CDK inhibitor, a PARP inhibitor, or any anti-neoplastic agent or anti-proliferative agent known to those skilled in the art.

适合根据本发明提供的组合治疗方式使用的例示性烷基化剂包括但不限于环磷酰胺(Cytoxan；Neosar)；苯丁酸氮芥(Leukeran)；美法仑(Alkeran)；卡莫司汀(BiCNU)；白消安(Busulfex)；洛莫司汀(CeeNU)；达喀尔巴仁(DTIC-Dome)；奥沙利铂(Eloxatin)；卡莫司汀(Gliadel)；异环磷酰胺(Ifex)；二氯甲基二乙胺(Mustargen)；白消安(Myleran)；卡铂(Paraplatin)；顺铂(CDDP；Platinol)；替莫唑胺(Temodar)；噻替派(Thioplex)；苯达莫司汀(Treanda)；或链佐星(Zanosar)。Exemplary alkylating agents suitable for use in the combination therapy provided herein include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbarin (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mustargen; busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).

例示性适合的蒽环霉素包括但不限于阿霉素(Adriamycin)；阿霉素脂质粒(Doxil)；米托蒽醌(Novantrone)；博莱霉素(Blenoxane)；道诺霉素(Cerubidine)；道诺霉素脂质粒(DaunoXome)；更生霉素(Cosmegen)；表柔比星(Ellence)；伊达比星(Idamycin)；普卡霉素(Mithracin)；丝裂霉素(Mutamycin)；喷司他丁(Nipent)；或戊柔比星(Valstar)。Exemplary suitable anthracyclines include, but are not limited to, Adriamycin; Doxil; Novantrone; Blenoxane; Cerubidine; Daunoxome; Cosmegen; Ellence; Idamycin; Mithracin; Mutamycin; Nipent; or Valstar.

例示性抗代谢物包括但不限于氟尿嘧啶(Adrucil)；卡培他滨(Xeloda)；羟基脲(Hydrea)；巯基嘌呤(Purinethol)；培美曲塞(Alimta)；氟达拉滨(Fludara)；奈拉滨(Arranon)；克拉屈滨(Cladribine Novaplus)；氯法拉滨(Clolar)；阿糖胞苷(Cytosar-U)；地西他滨(Dacogen)；阿糖胞苷脂质粒(DepoCyt)；羟基脲(Droxia)；普拉曲沙(Folotyn)；氟尿苷(FUDR)；吉西他滨(Gemzar)；克拉屈滨(Leustatin)；氟达拉滨(Oforta)；甲氨蝶呤(MTX；Rheumatrex)；甲氨蝶呤(Trexall)；硫鸟嘌呤(Tabloid)；TS-1或阿糖胞苷(TarabinePFS)。Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposome (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

例示性解毒剂包括但不限于阿米福汀(Ethyol)或美司钠(Mesnex)。Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

例示性干扰素包括但不限于干扰素α-2b(内含子A)或干扰素α-2a(Roferon-A)。Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

例示性多克隆或单克隆抗体包括但不限于曲妥珠单抗(Herceptin)；奥法木单抗(Arzerra)；贝伐单抗(Avastin)；利妥昔单抗(Rituxan)；西妥昔单抗(Erbitux)；帕尼单抗(Vectibix)；托西莫单抗/碘-131托西莫单抗(Bexxar)；阿仑单抗(Campath)；替伊莫单抗(Zevalin；In-111；Y-90Zevalin)；吉妥珠单抗(Mylotarg)；依库珠单抗(Soliris)或德诺单抗。Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodine-131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab tiuxetan (Zevalin; In-111; Y-90Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris) or denosumab.

例示性EGFR抑制剂包括但不限于吉非替尼(Iressa)；拉帕替尼(Tykerb)；西妥昔单抗(Erbitux)；厄洛替尼(Tarceva)；帕尼单抗(Vectibix)；PKI-166；卡奈替尼(CI-1033)；马妥珠单抗(EMD 72000)或EKB-569。Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (EMD 72000), or EKB-569.

例示性HER2抑制剂包括但不限于曲妥珠单抗(Herceptin)；拉帕替尼(Tykerb)或AC-480。Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

组蛋白去乙酰酶抑制剂包括但不限于伏立诺他(Zolinza)。Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

例示性激素包括但不限于他莫昔芬(Soltamox；Nolvadex)；雷洛昔芬(Evista)；甲地孕酮(Megace)；亮丙瑞林(Lupron；Lupron Depot；Eligard；Viadur)；氟维司群(Faslodex)；来曲唑(Femara)；曲普瑞林(Trelstar LA；Trelstar Depot)；依西美坦(Aromasin)；戈舍瑞林(Zoladex)；比卡鲁胺(Casodex)；阿那曲唑(Arimidex)；氟甲睾酮(Androxy；Halotestin)；甲羟孕酮(Provera；Depo-Provera)；雌莫司汀(Emcyt)；氟他胺(Eulexin)；托瑞米芬(Fareston)；地加瑞克(Firmagon)；尼鲁胺(Nilandron)；阿巴瑞克(Plenaxis)；或睾内酯(Teslac)。Exemplary sex hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol acetate (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot; exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

例示性有丝分裂抑制剂包括但不限于紫杉醇(Taxol；Onxol；Abraxane)；多西他赛(Taxotere)；长春新碱(Oncovin；Vincasar PFS)；长春碱(Velban)；依托泊苷(Toposar；Etopophos；VePesid)；替尼泊苷(Vumon)；伊沙匹隆(Ixempra)；诺考达唑；埃博霉素；长春瑞宾(Navelbine)；喜树碱(CPT)；伊立替康(Camptosar)；拓朴替康(Hycamtin)；安吖啶或片螺素D(LAM-D)。Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellaemin D (LAM-D).

例示性MTOR抑制剂包括但不限于依维莫司(Afinitor)或替西罗莫司(Torisel)；雷帕鸣、地磷莫司；或AP23573。Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, riboflavin; or AP23573.

例示性多激酶抑制剂包括但不限于索拉非尼(Nexavar)；舒尼替尼(Sutent)；BIBW2992；E7080；Zd6474；PKC-412；莫替沙尼；或AP24534。Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

例示性丝胺酸/苏胺酸激酶抑制剂包括但不限于鲁伯斯塔；厄瑞尔/法舒地尔盐酸盐；夫拉平度；塞利希布(CYC202；Roscovitine)；SNS-032(BMS-387032)；Pkc412；苔藓虫素；KAI-9803；SF1126；VX-680；Azd1152；Arry-142886(AZD-6244)；SCIO-469；GW681323；CC-401；CEP-1347或PD 332991。Exemplary serine/threonine kinase inhibitors include, but are not limited to, rubustard; oerel/fasudil hydrochloride; flavopiridol; celecoxib (CYC202; Roscovitine); SNS-032 (BMS-387032); Pkc412; bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

例示性酪氨酸激酶抑制剂包括但不限于厄洛替尼(Tarceva)；吉非替尼(Iressa)；伊马替尼(Gleevec)；索拉非尼(Nexavar)；舒尼替尼(Sutent)；曲妥珠单抗(Herceptin)；贝伐单抗(Avastin)；利妥昔单抗(Rituxan)；拉帕替尼(Tykerb)；西妥昔单抗(Erbitux)；帕尼单抗(Vectibix)；依维莫司(Afinitor)；阿仑单抗(Campath)；吉妥珠单抗(Mylotarg)；替西罗莫司(Torisel)；帕唑帕尼(Votrient)；达沙替尼(Sprycel)；尼罗替尼(Tasigna)；瓦他拉尼(Ptk787；ZK222584)；CEP-701；SU5614；MLN518；XL999；VX-322；Azd0530；BMS-354825；SKI-606CP-690；AG-490；WHI-P154；WHI-P131；AC-220；或AMG888。Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

例示性VEGF/VEGFR抑制剂包括但不限于贝伐单抗(Avastin)、索拉非尼(Nexavar)、舒尼替尼(Sutent)、兰尼单抗、派加替尼或凡德替尼。Exemplary VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegatinib, or vandetinib.

例示性微管靶向药物包括但不限于紫杉醇、多西他赛、长春新碱、长春碱、诺考达唑、埃博霉素及温诺平。Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilone, and vinpoline.

例示性拓朴异构酶毒药包括但不限于替尼泊苷、依托泊苷、阿霉素、喜树碱、道诺霉素、更生霉素、米托蒽醌、安吖啶、表柔比星及伊达比星。Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunomycin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

例示性紫杉烷或紫杉烷衍生物包括但不限于紫杉醇及多烯紫杉醇。Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

例示性免疫检查点抑制剂包括程序性细胞死亡1(PD-1)、CD274分子(PD-L1)及细胞毒性T淋巴细胞相关蛋白4(CTLA4)抑制剂。例示性PD-1抑制剂包括派姆单抗、纳武单抗及西米普利单抗。例示性PD-L1抑制剂包括阿特珠单抗、阿维鲁单抗及德瓦鲁单抗。例示性CLTA4抑制剂包括伊匹单抗。Exemplary immune checkpoint inhibitors include programmed cell death 1 (PD-1), CD274 molecule (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors. Exemplary PD-1 inhibitors include pembrolizumab, nivolumab, and cemiplimab. Exemplary PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. Exemplary CLTA4 inhibitors include ipilimumab.

例示性铂类抗肿瘤剂包括顺铂及卡铂。Exemplary platinum anti-tumor agents include cisplatin and carboplatin.

例示性周期蛋白依赖性激酶(CDK)抑制剂包括阿贝西尼、帕博西尼及瑞博西尼。Exemplary cyclin-dependent kinase (CDK) inhibitors include abemaciclib, palbociclib, and ribociclib.

例示性聚(ADP-核糖)聚合酶(PARP)抑制剂包括塔拉佐帕瑞、奥拉帕尼、卢卡帕尼、尼拉帕尼及维利帕尼。Exemplary poly (ADP-ribose) polymerase (PARP) inhibitors include talazopari, olaparib, rucaparib, niraparib, and veliparib.

例示性通用化学治疗剂、抗肿瘤剂、抗增殖剂包括但不限于六甲蜜胺(Hexalen)；异维甲酸(Accutane；Amnesteem；Claravis；Sotret)；维甲酸(Vesanoid)；阿扎胞苷(Vidaza)；硼替佐米(Velcade)天冬酰胺酶(Elspar)；左旋咪唑(Ergamisol)；米托坦(Lysodren)；丙卡巴肼(Matulane)；培门冬酶(Oncaspar)；地尼白介素(Ontak)；卟吩姆(Photofrin)；阿地介白素(Proleukin)；来那度胺(Revlimid)；贝沙罗汀(Targretin)；沙利度胺(Thalomid)；替西罗莫司(Torisel)；三氧化二砷(Trisenox)；维替泊芬(Visudyne)；含羞草碱(Leucenol)；(1M喃氟啶-0.4M 5-氯-2,4-二羟基吡啶-1M氧嗪酸钾)或洛伐他汀。Exemplary general chemotherapeutic agents, anti-tumor agents, anti-proliferative agents include, but are not limited to, hexamethylmelamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane) ; pegaspargase (Oncaspar); denileukin (Ontak); porphin (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosapine (Leucenol); (1M fluazifop-0.4M 5-chloro-2,4-dihydroxypyridine-1M potassium oxadiazine) or lovastatin.

在一些实施方案中，提供组合治疗方式，其中额外治疗剂为细胞因子，例如G-CSF(粒细胞集落刺激因子)。在另一方面中，本发明所提供的药物组合物可与放射疗法组合施用。放射疗法还可与本发明所提供的药物组合物及本发明中描述为另一化学治疗剂组合施用，作为多药剂疗法的一部分。在另一方面中，本发明所提供的药物组合物可与标准化学疗法组合施用，诸如但不限于CMF(环磷酰胺、甲氨蝶呤及5-氟尿嘧啶)、CAF(环磷酰胺、阿霉素及5-氟尿嘧啶)、AC(阿霉素及环磷酰胺)、FEC(5-氟尿嘧啶、表柔比星及环磷酰胺)、ACT或ATC(阿霉素、环磷酰胺及紫杉醇)、利妥昔单抗、Xeloda(卡培他滨)、顺铂(CDDP)、卡铂、TS-1(喃氟啶、吉美司特及奥他司特钾，摩尔比为1:0.4:1)、喜树碱-11(CPT-11、伊立替康或Camptosar^TM)、CHOP(环磷酰胺、羟基道诺霉素、安可平及普赖松或普赖苏秾)、R-CHOP(利妥昔单抗、环磷酰胺、羟基道诺霉素、安可平、普赖松或普赖苏秾)或CMFP(环磷酰胺、甲氨蝶呤、5-氟尿嘧啶及普赖松)。In some embodiments, a combination therapy is provided, wherein the additional therapeutic agent is a cytokine, such as G-CSF (granulocyte colony stimulating factor). In another aspect, the pharmaceutical composition provided by the present invention can be administered in combination with radiotherapy. Radiotherapy can also be administered in combination with the pharmaceutical composition provided by the present invention and another chemotherapeutic agent described in the present invention as part of a multi-agent therapy. In another aspect, the pharmaceutical compositions provided by the present invention can be administered in combination with standard chemotherapy, such as but not limited to CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF (cyclophosphamide, doxorubicin and 5-fluorouracil), AC (doxorubicin and cyclophosphamide), FEC (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC (doxorubicin, cyclophosphamide and paclitaxel), rituximab, Xeloda (capecitabine), cisplatin (CDDP), carboplatin, TS-1 (flunisal, gimerastat and ortalast potassium, in a molar ratio of 1:0.4:1), camptothecin-11 (CPT-11, irinotecan or Camptosar^™ ), CHOP (cyclophosphamide, hydroxydaunomycin, enclosing and pralidone or pralidone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunomycin, enclosing, pralidone or pralidone) or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil and pralidone).

在一些优选实施例中，本发明所提供的药物组合物可与酶(诸如受体或非受体激酶)的抑制剂一起施用。受体及非受体激酶为例如酪氨酸激酶或丝氨酸/苏氨酸激酶。本发明所述的激酶抑制剂为小分子、多核酸、多肽或抗体。In some preferred embodiments, the pharmaceutical composition provided by the present invention can be administered together with an inhibitor of an enzyme (such as a receptor or non-receptor kinase). Receptor and non-receptor kinases are, for example, tyrosine kinases or serine/threonine kinases. The kinase inhibitors described in the present invention are small molecules, polynucleic acids, polypeptides or antibodies.

例示性激酶抑制剂包括但不限于贝伐单抗(靶向VEGF)、BIBW 2992(靶向EGFR及Erb2)、西妥昔单抗/Erbitux(靶向Erb1)、伊马替尼/Gleevec(靶向Bcr-Abl)、曲妥珠单抗(靶向Erb2)、吉非替尼/Iressa(靶向EGFR)、兰尼单抗(靶向VEGF)、派加替尼(靶向VEGF)、厄洛替尼/Tarceva(靶向Erb1)、尼罗替尼(靶向Bcr-Abl)、拉帕替尼(靶向Erb1及Erb2/Her2)、GW-572016/二甲苯磺酸拉帕替尼(靶向HER2/Erb2)、帕尼单抗/Vectibix(靶向EGFR)、凡德替尼(靶向RET/VEGFR)、E7080(多目标，包括RET及VEGFR)、Herceptin(靶向HER2/Erb2)、PKI-166(靶向EGFR)、卡奈替尼/CI-1033(靶向EGFR)、舒尼替尼/SU-11464/Sutent(靶向EGFR及FLT3)、马妥珠单抗/Emd7200(靶向EGFR)、EKB-569(靶向EGFR)、Zd6474(靶向EGFR及VEGFR)、PKC-412(靶向VEGR及FLT3)、瓦他拉尼/Ptk787/ZK222584(靶向VEGR)、CEP-701(靶向FLT3)、SU5614(靶向FLT3)、MLN518(靶向FLT3)、XL999(靶向FLT3)、VX-322(靶向FLT3)、Azd0530(靶向SRC)、BMS-354825(靶向SRC)、SKI-606(靶向SRC)、CP-690(靶向JAK)、AG-490(靶向JAK)、WHI-P154(靶向JAK)、WHI-P131(靶向JAK)、索拉非尼/Nexavar(靶向RAF激酶、VEGFR-1、VEGFR-2、VEGFR-3、PDGFR-β、KIT、FLT-3及RET)、达沙替尼/Sprycel(BCR/ABL及Src)、AC-220(靶向Flt3)、AC-480(靶向所有HER蛋白，“panHER”)、二磷酸莫替沙尼(靶向VEGF1-3、PDGFR及c-kit)、德诺单抗(靶向RANKL，抑制SRC)、AMG888(靶向HER3)及AP24534(多靶，包括Flt3)。Exemplary kinase inhibitors include, but are not limited to, bevacizumab (targeting VEGF), BIBW 2992 (targeting EGFR and Erb2), cetuximab/Erbitux (targeting Erb1), imatinib/Gleevec (targeting Bcr-Abl), trastuzumab (targeting Erb2), gefitinib/Iressa (targeting EGFR), ranibizumab (targeting VEGF), pegatinib (targeting VEGF), erlotinib/Tarceva (targeting Erb1), nilotinib (targeting Bcr-Abl), lapatinib (targeting Erb1 and Erb2/Her2), GW-572016/lapatinib ditosylate (targeting HER2/Erb2), panitumumab /Vectibix (targeting EGFR), vandetinib (targeting RET/VEGFR), E7080 (multi-target, including RET and VEGFR), Herceptin (targeting HER2/Erb2), PKI-166 (targeting EGFR), canertinib/CI-1033 (targeting EGFR), sunitinib/SU-11464/Sutent (targeting EGFR and FLT3), matuzumab/Emd7200 (targeting EGFR), EKB-569 (targeting EGFR), Zd6474 (targeting EGFR and VEGFR), PKC-412 (targeting VEGR and FLT3), vatalanib/Ptk787/ZK222584 (targeting VEGR), CEP-701 (targeting FLT3), SU5614 (targeting FLT3), MLN518 (targeting FLT3), XL999 (targeting FLT3), VX-322 (targeting FLT3), Azd0530 (targeting SRC), BMS-354825 (targeting SRC), SKI-606 (targeting SRC), CP-690 (targeting JAK), AG-490 (targeting JAK), WHI-P154 (targeting JAK), WHI-P131 (targeting JAK) , sorafenib/Nexavar (targeting RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3 and RET), dasatinib/Sprycel (BCR/ABL and Src), AC-220 (targeting Flt3), AC-480 (targeting all HER proteins, "panHER"), motesanib diphosphate (targeting VEGF1-3, PDGFR and c-kit), denosumab (targeting RANKL, inhibiting SRC), AMG888 (targeting HER3) and AP24534 (multi-target, including Flt3).

在一些实施方案中，例如施用重组融合蛋白以治疗心房震颤的这些实施例，组合疗法可包括施用β阻断剂(例如比索洛尔或琥珀酸美托洛尔)、钙通道阻断剂(例如地尔硫卓或维拉帕米)、地高辛、抗心律失常药物(例如普罗帕酮、氟卡尼、索他洛尔、多非利特、胺碘酮及屈奈达隆)或血液稀释剂(例如华法林、阿哌沙布、达比加群、依度沙班或利伐沙班)。在一些实施方案中，组合疗法可包括心脏复律疗法，以重置心脏窦性心律(例如电复律或药物复律)。在一些实施方案中，组合疗法可包括消融(ablation)，例如AV结节消融或Maze手术。消融使用手术刀、热疗或冷疗在心脏上形成小疤痕，阻断错误的电信号，恢复正常的心律。In some embodiments, such as those embodiments where a recombinant fusion protein is administered to treat atrial fibrillation, the combination therapy may include administering a beta blocker (e.g., bisoprolol or metoprolol succinate), a calcium channel blocker (e.g., diltiazem or verapamil), digoxin, an antiarrhythmic drug (e.g., propafenone, flecainide, sotalol, dofetilide, amiodarone, and dronedarone), or a blood thinner (e.g., warfarin, apixaban, dabigatran, edoxaban, or rivaroxaban). In some embodiments, the combination therapy may include cardioversion therapy to reset the heart's sinus rhythm (e.g., electrical or drug cardioversion). In some embodiments, the combination therapy may include ablation, such as AV node ablation or Maze surgery. Ablation uses a scalpel, heat therapy, or cold therapy to form a small scar on the heart, blocking erroneous electrical signals and restoring normal heart rhythm.

在另一个实施例中，包含本发明所公开的相同多肽的重组融合蛋白或药物组合物每天向受试者施用一次。在一些实施方案中，重组融合蛋白或包含其的药物组合物每两天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每三天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每四天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每五天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每六天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每周向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每7-14天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每10-20天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每5-15天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物每15-30天向受试者施用一次。In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same polypeptide disclosed in the present invention is administered to a subject once a day. In some embodiments, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every two days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every three days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every four days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every five days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every six days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once a week. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every 7-14 days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every 10-20 days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every 5-15 days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject once every 15-30 days.

在一个实施例中，本发明的重组融合蛋白的剂量在可注射溶液中包含0.005至0.1mg/kg。在另一个实施例中，剂量包含0.005至0.5mg/kg重组融合蛋白。在另一个实施例中，剂量包含0.05至0.1微克重组融合蛋白。在另一个实施例中，剂量包含0.005至0.1mg/kg的可注射溶液中的重组融合蛋白。In one embodiment, the dosage of the recombinant fusion protein of the present invention comprises 0.005 to 0.1 mg/kg in an injectable solution. In another embodiment, the dosage comprises 0.005 to 0.5 mg/kg of the recombinant fusion protein. In another embodiment, the dosage comprises 0.05 to 0.1 micrograms of the recombinant fusion protein. In another embodiment, the dosage comprises 0.005 to 0.1 mg/kg of the recombinant fusion protein in an injectable solution.

在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.0001mg至0.6mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.001mg至0.005mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.005mg至0.01mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.01mg至0.3mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.2mg至0.6mg范围内的剂量向受试者施用。In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.0001 mg to 0.6 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.001 mg to 0.005 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.005 mg to 0.01 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.01 mg to 0.3 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.2 mg to 0.6 mg.

在另一个实施例中，重组融合蛋白或包含其的药物组合物以1-100mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以10-80mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以20-60mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以10-50mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以40-80mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以10-30mcg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以30-60mcg/kg范围内的剂量向受试者施用。In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 1-100mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 10-80mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 20-60mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 10-50mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 40-80mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 10-30mcg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 30-60mcg/kg.

在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至100mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至50mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至25mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至10mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至5mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至1mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.1mcg/kg至0.1mg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以10mg/kg至60mg/kg范围内的剂量向受试者施用。In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 100mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 50mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 25mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 10mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 5mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 1mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 0.1mcg/kg to 0.1mg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 10 mg/kg to 60 mg/kg.

在另一个实施例中，重组融合蛋白或包含其的药物组合物以约0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、约1mg/kg、约2mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg、约10mg/kg、约20mg/kg、约30mg/kg、约40mg/kg、约50mg/kg、约60mg/kg或约70mg/kg的剂量向受试者施用。在一些实施方案中，重组融合蛋白或包含其的药物组合物以约0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、0.6mg/kg、0.7mg/kg、0.8mg/kg、0.9mg/kg或1mg/kg的剂量向受试者施用。In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg or about 70 mg/kg. In some embodiments, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg or 1 mg/kg.

在另一个实施例中，重组融合蛋白或包含其的药物组合物以0.2mg至2mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以2mg至6mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以4mg至10mg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以5mg至15mg范围内的剂量向受试者施用。In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 0.2 mg to 2 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 2 mg to 6 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 4 mg to 10 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage in the range of 5 mg to 15 mg.

在一个实施例中，重组融合蛋白或包含其的药物组合物以10μg/kg-1000μg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以25μg/kg-600μg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以50μg/kg-400μg/kg范围内的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约25μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约50μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约100μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约200μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约300μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约400μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约500μg/kg的剂量向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物以约600μg/kg的剂量向受试者施用。In one embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 10 μg/kg-1000 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 25 μg/kg-600 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage within the range of 50 μg/kg-400 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage of about 25 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage of about 50 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage of about 100 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage of about 200 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising it is administered to a subject at a dosage of about 300 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 400 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 500 μg/kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 600 μg/kg.

在一个实施例中，向受试者施用单次剂量的重组融合蛋白或包含其的药物组合物。在另一个实施例中，向受试者施用总共两次剂量。在另一个实施例中，向受试者施用总共两次或更多次剂量。In one embodiment, a single dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject. In another embodiment, a total of two doses are administered to the subject. In another embodiment, a total of two or more doses are administered to the subject.

在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每两天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每两天或更多天向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每周、每两周或每三周向受试者施用。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每周向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每两周向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每三周向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每三周或更多周向受试者施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每周向受试者施用两次或更多次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每月向受试者施用两次或更多次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每年向受试者施用两次或更多次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每两年向受试者施用两次或更多次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量每两年或更多年向受试者施用两次或更多次。In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once a day. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every two days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every two days or more. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject weekly, every two weeks or every three weeks. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once a week. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every two weeks. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every three weeks. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every three weeks or more weeks. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject twice or more per week. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject twice or more per month. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject twice or more per year. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject twice or more every two years. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject twice or more every two years or more.

在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每36小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每48小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每60小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每72小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每84小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每96小时施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每5天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每6天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每7天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每8-10天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每10-12天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每12-15天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每15-25天施用一次。在另一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每20-30天施用一次。In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 36 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 48 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 60 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 72 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 84 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 96 hours. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 5 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 6 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 7 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 8-10 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 10-12 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 12-15 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 15-25 days. In another embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 20-30 days.

在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每1个月向受试者施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每2个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每3个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每4个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每5个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每6个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量至少每6-12个月施用一次。在一个实施例中，重组融合蛋白或包含其的药物组合物的剂量是每季度施用。在另一个实施例中，剂量是每日、每周、每两周、每月或每年施用。在另一个实施例中，剂量是一天、一周、一个月或一年施用一次、两次或更多次。在另一个实施例中，剂量是每两年、三年、四年或至少五年施用。In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered to the subject at least once every 1 month. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 2 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 3 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 4 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 5 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 6 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered at least once every 6-12 months. In one embodiment, the dose of the recombinant fusion protein or a pharmaceutical composition comprising it is administered quarterly. In another embodiment, the dose is administered daily, weekly, biweekly, monthly or annually. In another embodiment, the dose is administered once, twice or more in one day, one week, one month or one year. In another embodiment, the dose is administered every two years, three years, four years or at least five years.

在一个实施例中，本发明的组合物的重复施用(给药)可在第一个疗程后立即进行，或在间隔数天、数周或数年后进行，以达到本发明进一步提供的预期效果(例如预防或治疗心血管疾病或病况，或CNS相关疾病或病况)。In one embodiment, repeated administration (dosing) of the composition of the present invention can be carried out immediately after the first course of treatment, or after an interval of several days, weeks or years, to achieve the desired effect further provided by the present invention (e.g., prevention or treatment of cardiovascular diseases or conditions, or CNS-related diseases or conditions).

在一个实施例中，药物组合物通过静脉内、动脉内、皮下或肌肉内注射液体制剂来施用。在另一个实施例中，液体制剂包括溶液、悬浮液、分散液、乳液、油及其类似物。在一个实施例中，药物组合物是静脉内施用，且因此以适用于静脉内施用的形式调配。在另一个实施例中，药物组合物是动脉内施用，且因此以适用于动脉内施用的形式调配。In one embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, subcutaneous or intramuscular injection of a liquid formulation. In another embodiment, the liquid formulation comprises a solution, a suspension, a dispersion, an emulsion, an oil and the like. In one embodiment, the pharmaceutical composition is administered intravenously and is therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intraarterially and is therefore formulated in a form suitable for intraarterial administration.

在一些实施方案中，用于本发明所公开的方法的组合物包含溶液或乳液，其在一些实施方案中为包含安全且有效量的本发明所公开的化合物及可选地其他化合物的水溶液或乳液，意欲用于静脉内或皮下施用。In some embodiments, the compositions used in the methods disclosed herein comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of a compound disclosed herein and optionally other compounds, intended for intravenous or subcutaneous administration.

在一些实施方案中，组合物的各种成分是预测量和/或预封装和/或无需额外测量即使用等。本发明还可选地包含用于进行/使用本发明的方法和/或组合物的药盒。特别地，这些药盒可选地包括例如适当的重组融合蛋白(及可选地若干此类蛋白质的混合物，用于进行协同治疗，参见上文)以及可选地适当的疾病相关抗原)。另外，此类药盒还可包含适当的赋形剂(例如医药学上可接受的赋形剂)，用于进行本发明的治疗性和/或预防性治疗。此类药盒可选地含有用于组装和/或使用本发明的组合物的额外组分，包括但不限于例如稀释剂等。In some embodiments, the various components of the composition are pre-measured and/or pre-packaged and/or used without additional measurement, etc. The present invention also optionally includes a kit for performing/using the method and/or composition of the present invention. In particular, these kits optionally include, for example, suitable recombinant fusion proteins (and optionally a mixture of several such proteins, for synergistic therapy, see above) and optionally suitable disease-related antigens). In addition, such kits may also include suitable excipients (e.g., pharmaceutically acceptable excipients) for therapeutic and/or preventive treatments of the present invention. Such kits optionally contain additional components for assembling and/or using the composition of the present invention, including, but not limited to, for example, diluents, etc.

本发明所述的组合物可选地封装成包括用于进行本发明的方法或用于使用本发明的组合物的所有(或几乎所有)必要组分(可选地包括例如本发明的方法/组合物的使用的书面说明)。举例而言，药盒可选地包括诸如缓冲液、试剂、血清蛋白、抗体、底物等的组分。在预封装试剂的情况下，药盒可选地包括预测量或预给药的量，无需测量即可并入方法中，例如预测量的流体等分试样；或预称重或预测量的固体试剂，其可容易由药盒的最终使用者复原。The compositions described herein are optionally packaged to include all (or substantially all) of the necessary components for performing the methods of the invention or for using the compositions of the invention (optionally including, for example, written instructions for use of the methods/compositions of the invention). For example, kits optionally include components such as buffers, reagents, serum proteins, antibodies, substrates, etc. In the case of prepackaged reagents, the kits optionally include pre-measured or pre-dosed amounts that can be incorporated into the method without measurement, such as pre-measured aliquots of fluids; or pre-weighed or pre-measured solid reagents that can be easily reconstituted by the end user of the kit.

此类药盒通常还包括执行本发明的方法和/或使用本发明的组合物的适当说明书。在一些实施方案中，药盒/封装的组分是以稳定形式提供，以便防止在长期储存期间发生降解或其他损失，例如渗漏。多种稳定方法/药剂广泛用于待储存的试剂等，诸如包括化学稳定剂(即酶抑制剂、杀微生物剂/抑菌剂、抗凝血剂)等。本发明的药物组合物中活性成分的实际剂量浓度可变化，以便获得能有效达成特定受试者、组合物及给药方式的所要治疗反应而对受试者无毒的一定量的活性成分。所选剂量浓度将视多种药代动力学因素而定，包括所用本发明的特定组合物的活性；给药途径；给药时间；所采用的特定化合物的排泄速率；治疗持续时间；与所采用的特定组合物组合使用的其他药物、化合物和/或物质；所治疗的受试者的年龄、性别、体重、病状、一般健康状况及先前病史；及医学领域中熟知的类似因素。Such kits also typically include appropriate instructions for performing the methods of the present invention and/or using the compositions of the present invention. In some embodiments, the components of the kit/package are provided in a stable form to prevent degradation or other losses, such as leakage, during long-term storage. Various stabilization methods/agents are widely used for reagents to be stored, etc., such as chemical stabilizers (i.e., enzyme inhibitors, microbicides/bacteriostatics, anticoagulants), etc. The actual dosage concentration of the active ingredient in the pharmaceutical composition of the present invention may vary so as to obtain a certain amount of active ingredient that is effective in achieving the desired therapeutic response of a specific subject, composition, and administration method without being toxic to the subject. The selected dosage concentration will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention used; route of administration; time of administration; excretion rate of the specific compound used; duration of treatment; other drugs, compounds, and/or substances used in combination with the specific composition used; age, sex, weight, condition, general health, and previous medical history of the subject being treated; and similar factors well known in the medical field.

组合物必须为无菌的，且流动性达到组合物可通过注射器递送的程度。除水之外，载剂优选为等张缓冲盐水溶液。举例而言，可通过使用诸如卵磷脂的包衣、通过在分散液的情况下维持所需粒度及通过使用界面活性剂来维持适当流动性。在许多情况下，组合物中优选包括等张剂，例如糖、多元醇(诸如甘露糖醇或山梨糖醇)及氯化钠。The composition must be sterile and fluid to the extent that the composition can be delivered by a syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol (such as mannitol or sorbitol), and sodium chloride in the composition.

本发明的药物组合物中活性成分的实际剂量浓度可变化，以便获得能有效达成特定受试者、组合物及给药方式的所要治疗反应而对受试者无毒的一定量的活性成分。所选剂量浓度将视多种药代动力学因素而定，包括所用本发明的特定组合物的活性；给药途径；给药时间；所采用的特定化合物的排泄速率；治疗持续时间；与所采用的特定组合物组合使用的其他药物、化合物和/或物质；所治疗的受试者的年龄、性别、体重、病状、一般健康状况及先前病史；及医学领域中熟知的类似因素。The actual dosage concentration of the active ingredient in the pharmaceutical composition of the present invention may be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject. The selected dosage concentration will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed; the route of administration; the time of administration; the rate of excretion of the particular compound employed; the duration of treatment; other drugs, compounds, and/or substances used in combination with the particular composition employed; the age, sex, weight, condition, general health, and previous medical history of the subject being treated; and similar factors well known in the medical field.

尽管本发明已描述及显示若干本发明实施例，本领域普通技术人员将容易设想到各种其他方法和/或结构以执行功能和/或获得本发明所述的结果和/或该等优点的一个或多者，且此类变化和/或修饰中的每一者被视为处于本发明所述的本发明实施例的范围中。更一般而言，本领域技术人员将容易理解，本发明所述的所有参数、尺寸、材料及组态是意欲为例示性，且实际参数、尺寸、材料和/或组态将视本发明教导所使用/所使用特定应用或应用而定。本领域的技术人员将认识到，或能够仅使用常规实验来确定本文所述的特定发明实施例的许多等效物。因此，应理解，前述实施例仅作为实施例且在其所附权利要求及其等效物范围中呈现，本发明实施例可不如具体描述及主张来实施。本发明的发明实施例是关于本发明中描述的各个特征、系统、物品、材料、药盒和/或方法。此外，当这些特征、系统、物品、材料、药盒和/或方法不相互矛盾时，将两种或更多种此类特征、系统、材料、药盒和/或方法的任何组合纳入本发明的范围中。Although the present invention has described and shown several embodiments of the present invention, a person of ordinary skill in the art will readily conceive of various other methods and/or structures to perform the functions and/or obtain the results and/or one or more of the advantages described in the present invention, and each of such changes and/or modifications is considered to be within the scope of the embodiments of the present invention described in the present invention. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials and configurations described in the present invention are intended to be exemplary, and actual parameters, dimensions, materials and/or configurations will depend on the specific application or application used/used by the teachings of the present invention. A person skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are presented only as embodiments and in the scope of the claims attached thereto and their equivalents, and embodiments of the present invention may not be implemented as specifically described and claimed. The inventive embodiments of the present invention are about the various features, systems, articles, materials, kits and/or methods described in the present invention. In addition, any combination of two or more such features, systems, articles, materials, kits and/or methods is included within the scope of the present invention when such features, systems, articles, materials, kits and/or methods are not mutually inconsistent.

如本发明所定义及使用的所有定义应理解为控制在辞典定义、以引用的方式并入的文献中的定义和/或所定义术语的通常含义内。All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

本发明所公开的全部参考文献、专利及专利申请案根据各自所引述的主题、以引用的方式并入，在一些情况下可涵盖整个文献。All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter for which they are cited, and in some cases may be incorporated in their entirety.

除非明确相反指示，否则如在本发明说明书及权利要求中使用的不定冠词“一(a)”及“一(an)”应理解为是指“至少一个”。Unless expressly indicated to the contrary, the indefinite articles "a" and "an" as used in the present specification and claims should be understood to mean "at least one".

如本发明在说明书及权利要求中所用的词组“和/或”应理解为是指如此结合的要素(即，在一些情况下结合存在且在其他情况下分离存在的要素)中的“任一项或两者”。使用“和/或”列出的多个要素应以相同方式解释，即，如此结合的“一个或多个”要素。除由“和/或”条款具体识别的要素外，可可选地存在其他要素，无论与具体识别的这些要素相关抑或不相关。因此，作为非限制性实例，指代“A和/或B”在结合诸如“包含”等开放式措辞使用时，在一个实施例中，可仅指A(可选地包括除了B以外的要素)；在另一个实施例中，可仅指B(可选地包括除了A以外的要素)；在另一个实施例中，可指A及B两者(可选地包括其他要素)；等。The phrase "and/or" as used in the specification and claims of the present invention should be understood to refer to "any or both" of the elements so combined (i.e., elements that exist in combination in some cases and separately in other cases). Multiple elements listed using "and/or" should be interpreted in the same manner, that is, "one or more" elements so combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with an open-ended phrase such as "comprising", may refer to A alone (optionally including elements other than B) in one embodiment; to B alone (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in another embodiment; etc.

如说明书及权利要求中所用，词组“至少一个”在提及一个或多个组件的列表时，应该理解为是指选自组件列表的任一个或多个组件中的至少一个组件，但不一定包括组件列表内具体所列的每一个组件中的至少一者且不排除组件列表中的任何组件组合。此定义还允许可可选地存在除词组“至少一个”所指的要素的清单内具体识别的要素之外的要素，而无论与具体识别的这些要素相关抑或不相关。由此，作为非限制性实例，“至少一个A及B”(或等效地“至少一个A或B”或等效地“至少一个A和/或B”)可在一个实施例中指至少一个(可选地包括超过一个)A而不存在B(且可选地包括除B以外的要素)；在另一实施例中，指至少一个(可选地包括超过一个)B而不存在A(且可选地包括除A的外的要素)；在又一实施例中，指至少一个(可选地包括超过一个)A及至少一个(可选地包括超过一个)B(且可选地包括其他要素)；等等。As used in the specification and claims, the phrase "at least one" when referring to a list of one or more components should be understood to refer to at least one component selected from any one or more components in the list of components, but does not necessarily include at least one of each component specifically listed in the list of components and does not exclude any combination of components in the list of components. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one A and B" (or equivalently "at least one A or B" or equivalently "at least one A and/or B") may refer to at least one (optionally including more than one) A without B (and optionally including elements other than B) in one embodiment; to at least one (optionally including more than one) B without A (and optionally including elements other than A) in another embodiment; to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements) in yet another embodiment; and so on.

本发明进一步提供一种用于预防、治疗或延缓人类的心血管疾病或病况的药盒，其中该药盒包含一个或多个剂量的包含本发明所公开的重组融合蛋白的药物组合物，用于预防、治疗或延缓心血管疾病或病况，及如何使用医药制剂或组合物的说明书。The present invention further provides a kit for preventing, treating or delaying cardiovascular diseases or conditions in humans, wherein the kit comprises one or more doses of a pharmaceutical composition comprising the recombinant fusion protein disclosed in the present invention, for preventing, treating or delaying cardiovascular diseases or conditions, and instructions on how to use the pharmaceutical preparation or composition.

本发明进一步提供一种用于预防、治疗或延缓人类的CNS相关疾病或病况的药盒，其中该药盒包含一个或多个剂量的包含本发明所公开的重组融合蛋白的药物组合物，用于预防、治疗或延缓心血管疾病或病况，及如何使用医药制剂或组合物的说明书。The present invention further provides a kit for preventing, treating or delaying CNS-related diseases or conditions in humans, wherein the kit comprises one or more doses of a pharmaceutical composition comprising the recombinant fusion protein disclosed in the present invention, for preventing, treating or delaying cardiovascular diseases or conditions, and instructions on how to use the pharmaceutical preparation or composition.

本发明进一步提供一种用于预防、治疗或延缓人类的射血分数保留型心脏衰竭的药盒，其中该药盒包含一个或多个剂量的包含本发明所公开的重组融合蛋白的药物组合物，用于预防、治疗或延缓射血分数保留型心脏衰竭，及如何使用医药制剂或组合物的说明书。The present invention further provides a drug kit for preventing, treating or delaying heart failure with preserved ejection fraction in humans, wherein the drug kit comprises one or more doses of a pharmaceutical composition comprising the recombinant fusion protein disclosed in the present invention, for preventing, treating or delaying heart failure with preserved ejection fraction, and instructions on how to use the pharmaceutical preparation or composition.

提供以下实施例以更充分地例示本发明的优选实施例。然而，不应以任何方式将其解释为限制本发明的广泛范围。The following examples are provided to more fully illustrate the preferred embodiments of the invention. However, they should not be construed in any way as limiting the broad scope of the invention.

实施例Example

实施例1-表现质粒的克隆及构建Example 1 - Cloning and construction of expression plasmid

编码重组融合蛋白的重链(分别命名为NPCFA及NPCF，表示有或没有Fc突变的序列)及轻链(命名为PAL)的DNA序列由GENEWIZ(Suzhou,China)合成。表达载体pCHOGUN是根据许可协议自Horizon Discovery(Cambridge,UK)获得。表达质粒的构建如图1中所概述进行。简而言之，pCHOGUN载体通过限制酶BfuAI线性化，且在通过NcoI及AscI进行双重限制酶消化后纯化基因插入片段，诸如NPCF、NPCFA及PAL。经线性化的pCHOGUN/BfuAI及经纯化的基因插入片段根据标准方案连接，且随后转化至大肠杆菌DH5α感受态细胞中。将DH5α细胞接种且在37℃下孵育过夜。分离出质粒pCHOGUN-NPCF、pCHOGUN-NPCFA及pCHOGUN-PAL，且通过限制酶消化或PCR确认。含有重链插入物的质粒(pCHOGUN-NPCF或pCHOGUN-NPCFA)用限制酶BspEI及PciI消化，而含有轻链插入物的质粒(pCHOGUN-PAL)用限制酶NgoMIV及PciI消化。在限制酶消化后，将具有重链或轻链插入物的片段纯化，连接且随后转化到DH5α细胞中。含有重链及轻链插入物的质粒构建体(pCHOGUN-NPCF+PAL或pCHOGUN-NPCFA+PAL)通过限制酶消化及DNA测序加以鉴别及确认。The DNA sequences encoding the heavy chain (named NPCFA and NPCF, respectively, indicating the sequence with or without Fc mutation) and the light chain (named PAL) of the recombinant fusion protein were synthesized by GENEWIZ (Suzhou, China). The expression vector pCHOGUN was obtained from Horizon Discovery (Cambridge, UK) under a license agreement. The construction of the expression plasmid was performed as outlined in Figure 1. In short, the pCHOGUN vector was linearized by the restriction enzyme BfuAI, and the gene inserts, such as NPCF, NPCFA and PAL, were purified after double restriction enzyme digestion by NcoI and AscI. The linearized pCHOGUN/BfuAI and the purified gene inserts were connected according to standard protocols and then transformed into Escherichia coli DH5α competent cells. The DH5α cells were inoculated and incubated overnight at 37°C. Plasmids pCHOGUN-NPCF, pCHOGUN-NPCFA and pCHOGUN-PAL were isolated and confirmed by restriction enzyme digestion or PCR. The plasmid containing the heavy chain insert (pCHOGUN-NPCF or pCHOGUN-NPCFA) was digested with restriction enzymes BspEI and PciI, while the plasmid containing the light chain insert (pCHOGUN-PAL) was digested with restriction enzymes NgoMIV and PciI. After restriction enzyme digestion, the fragments with heavy or light chain inserts were purified, ligated and subsequently transformed into DH5α cells. The plasmid constructs containing heavy and light chain inserts (pCHOGUN-NPCF+PAL or pCHOGUN-NPCFA+PAL) were identified and confirmed by restriction enzyme digestion and DNA sequencing.

实施例2-抗体产生、纯化及表征Example 2 - Antibody Production, Purification and Characterization

HD-BIOP3，一种衍生自CHO K1细胞的谷氨酰胺酸合成酶缺失(GS^-/-)细胞株，是根据许可协议自Horizon Discovery(Cambridge,UK)获得。质粒DNA是使用市售Qiagen质粒药盒分离。使用来自Lonza的市售电穿孔系统将质粒DNA转染至HD-BIOP3细胞中。将经转染细胞接种于96孔板中，且使用标准程序进行池选择。来自选定池的细胞在125-mL摇瓶中培养10-14天，且收获培养基进行抗体纯化。通过蛋白A亲和层析纯化抗体蛋白，接着进行尺寸排阻层析纯化，然后根据标准方案用SDS-PAGE及蛋白质印迹分析。HD-BIOP3, a glutamine synthetase-deficient (GS^-/- ) cell line derived from CHO K1 cells, was obtained from Horizon Discovery (Cambridge, UK) under a license agreement. Plasmid DNA was isolated using a commercially available Qiagen plasmid kit. Plasmid DNA was transfected into HD-BIOP3 cells using a commercially available electroporation system from Lonza. Transfected cells were seeded in 96-well plates and pool selection was performed using standard procedures. Cells from the selected pools were cultured in 125-mL shake flasks for 10-14 days, and the culture medium was harvested for antibody purification. Antibody proteins were purified by protein A affinity chromatography, followed by size exclusion chromatography purification, and then analyzed by SDS-PAGE and Western blotting according to standard protocols.

图2A显示本发明所公开的重组融合蛋白的示意性结构。图2B显示SDS-PAGE分析生成的代表性数据。通过对包含HER3/4结合域的NRG-1的61个氨基酸活性片段(“NRG-1”，R&DSystems,Minneapolis,MN)具有特异性的一抗或对IgG具有特异性的一抗检测到的蛋白质印迹结果分别显示于图2C及2D中。Figure 2A shows a schematic structure of the recombinant fusion protein disclosed in the present invention. Figure 2B shows representative data generated by SDS-PAGE analysis. Western blot results detected by a primary antibody specific for a 61 amino acid active fragment of NRG-1 containing a HER3/4 binding domain ("NRG-1", R&D Systems, Minneapolis, MN) or a primary antibody specific for IgG are shown in Figures 2C and 2D, respectively.

实施例3-通过基于SPR的结合分析评定分子完整性Example 3 - Assessment of molecular integrity by SPR-based binding analysis

本发明所公开的重组融合蛋白的分子结构完整性是通过评估其与HER3蛋白及抗NRG-1抗体的同时结合能力来评定。带His标签的HER3重组蛋白(Sino Biological,Beijing,China)被捕获在固定有抗His抗体(Thermo Fisher,Waltham,MA)的传感器芯片上(步骤1)，然后注入样品(包括本发明所公开的重组融合蛋白、无Fc突变的本发明所公开的重组融合蛋白、抗HER3 mAb(步骤2)及抗NRG-1抗体(R&D Systems,Minneapolis,MN)(步骤3)。His-HER3附接在传感器芯片上可经由步骤1中所有6个信道上的信号增加来可视化。本发明所公开的重组融合蛋白及无Fc突变的本发明所公开的重组融合蛋白均在步骤2通过结合于HER3及在步骤3通过结合于注入的抗NRG-1抗体产生显著反应(Ch1,3)，表明在本发明所公开的重组融合蛋白上存在HER3结合表位及NRG-1。相反，抗HER3 mAb在步骤2仅与His-HER3结合，而不与抗NRG-1抗体及缓冲液(步骤3)结合(Ch 4,5)，从而验证抗HER3mAb分子没有NRG-1结合活性。缓冲液在步骤2及3被注入作为空白对照。因此，HER3结合表位及NRG-1均存在于本发明的重组融合蛋白中。The molecular structural integrity of the recombinant fusion protein disclosed in the present invention is assessed by evaluating its ability to simultaneously bind to HER3 protein and anti-NRG-1 antibody. The His-tagged HER3 recombinant protein (Sino Biological, Beijing, China) was captured on a sensor chip immobilized with an anti-His antibody (Thermo Fisher, Waltham, MA) (step 1), and then the sample (including the recombinant fusion protein disclosed in the present invention, the recombinant fusion protein disclosed in the present invention without Fc mutation, anti-HER3 mAb (step 2), and anti-NRG-1 antibody (R&D Systems, Minneapolis, MN) (step 3) was injected. The attachment of His-HER3 to the sensor chip can be visualized by the increase in signal on all 6 channels in step 1. The recombinant fusion protein disclosed in the present invention and the recombinant fusion protein disclosed in the present invention without Fc mutation both produced significant responses by binding to HER3 in step 2 and by binding to the injected anti-NRG-1 antibody in step 3 (Ch1, 3), indicating the presence of HER3 binding epitopes and NRG-1 on the recombinant fusion protein disclosed in the present invention. In contrast, the anti-HER3 The mAb only binds to His-HER3 in step 2, but not to the anti-NRG-1 antibody and buffer (step 3) (Ch 4, 5), thereby verifying that the anti-HER3 mAb molecule has no NRG-1 binding activity. The buffer was injected as a blank control in steps 2 and 3. Therefore, both the HER3 binding epitope and NRG-1 are present in the recombinant fusion protein of the present invention.

结合传感器图及样品注入顺序显示于图3中。The combined sensor diagram and sample injection sequence are shown in FIG3 .

实施例4-对体外肿瘤细胞株增殖的影响Example 4 - Effect on proliferation of tumor cell lines in vitro

肿瘤细胞以每孔2,500-20,000个细胞接种于96孔板中，视各细胞株的生长动力学而定。随后用本发明所公开的重组融合蛋白、抗体或对照蛋白以逐步1:4连续稀释处理细胞5天。细胞活力是使用来自Dojindo Molecular Technologies的细胞计数药盒-8(Kumamoto,Japan)根据制造商说明书来评定。用GraphPad Prism软件分析数据，且以相对于未处理对照的生长率表示。Tumor cells were seeded in 96-well plates at 2,500-20,000 cells per well, depending on the growth kinetics of each cell line. Cells were then treated with the recombinant fusion proteins, antibodies, or control proteins disclosed in the present invention at a stepwise 1:4 serial dilution for 5 days. Cell viability was assessed using a cell counting kit-8 from Dojindo Molecular Technologies (Kumamoto, Japan) according to the manufacturer's instructions. Data were analyzed using GraphPad Prism software and expressed as growth rate relative to untreated controls.

图4包括显示不同癌细胞株的平均相对生长率±SEM(n＝3)的代表图：(A)NCI-N87，胃；(B)MCF-7，乳房；(C)RT-112，膀胱；及(D)T47D，乳房。与对照NRG-1及GP120 mAb/NRG-1融合蛋白相比，本发明所公开的重组融合蛋白在促进癌细胞增殖方面表现出明显较低的活性。Figure 4 includes representative graphs showing the mean relative growth rate ± SEM (n=3) of different cancer cell lines: (A) NCI-N87, stomach; (B) MCF-7, breast; (C) RT-112, bladder; and (D) T47D, breast. Compared with the control NRG-1 and GP120 mAb/NRG-1 fusion proteins, the recombinant fusion proteins disclosed in the present invention exhibit significantly lower activity in promoting cancer cell proliferation.

实施例5-人类心肌细胞中PI3K/AKT信号传导路径的激活Example 5 - Activation of the PI3K/AKT signaling pathway in human cardiomyocytes

将从Cellular Dynamics(Madison,WI)获得的人类心肌细胞接种于经0.1％明胶包被的96孔板中，且在铺板培养基(Cellular Dynamics)中恢复4小时。随后将细胞在维持培养基(Cellular Dynamics)中培养96小时，然后用于实验。为了检验本发明的重组融合蛋白激活心肌细胞中HER2:HER4信号传导路径的能力，细胞首先在无血清培养基中饥饿4小时，且随后用重组融合蛋白或对照剂(NRG-1、GP120 mAb/NRG-1、抗HER3 mAb或GP120 mAb)以逐步1:4连续稀释处理细胞15分钟。在处理结束时，裂解细胞且使用Abcam的磷酸-AKT/总AKT ELISA药盒(Cambridge,MA)根据制造商说明书分析AKT磷酸化。用GraphPad Prism软件分析数据，且以相对于未处理对照的磷酸-AKT与总AKT的比率表示。Human cardiomyocytes obtained from Cellular Dynamics (Madison, WI) were seeded in 96-well plates coated with 0.1% gelatin and recovered in plating medium (Cellular Dynamics) for 4 hours. The cells were then cultured in maintenance medium (Cellular Dynamics) for 96 hours before being used in experiments. To test the ability of the recombinant fusion protein of the present invention to activate the HER2:HER4 signaling pathway in cardiomyocytes, the cells were first starved for 4 hours in serum-free medium, and then treated with recombinant fusion protein or control agent (NRG-1, GP120 mAb/NRG-1, anti-HER3 mAb or GP120 mAb) for 15 minutes in a stepwise 1:4 serial dilution. At the end of the treatment, the cells were lysed and AKT phosphorylation was analyzed using Abcam's phospho-AKT/total AKT ELISA kit (Cambridge, MA) according to the manufacturer's instructions. The data were analyzed with GraphPad Prism software and expressed as the ratio of phospho-AKT to total AKT relative to the untreated control.

对于蛋白质印迹分析，将细胞接种于6孔板中且用16nM单一浓度的本发明的重组融合蛋白或对照剂处理。在处理结束时，将细胞溶解于含有蛋白酶及磷酸酶抑制剂的RIPA裂解缓冲液中。SDS-PAGE及蛋白质印迹是根据标准方案进行。总AKT及磷酸-AKT分别用AKT兔抗体及p-AKT(S473)兔抗体(Cell Signaling；Danvers,MA)进行印迹。For Western blot analysis, cells were seeded in 6-well plates and treated with a single concentration of 16 nM of the recombinant fusion protein of the present invention or a control agent. At the end of the treatment, cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. SDS-PAGE and Western blotting were performed according to standard protocols. Total AKT and phospho-AKT were blotted with AKT rabbit antibody and p-AKT (S473) rabbit antibody (Cell Signaling; Danvers, MA), respectively.

图5显示人类心肌细胞中响应于刺激的AKT磷酸化。结果表明，本发明所公开的重组融合蛋白可激活心肌细胞中的HER2:HER4信号传导路径，其效力与NRG-1相当。Figure 5 shows AKT phosphorylation in human cardiomyocytes in response to stimulation. The results show that the recombinant fusion protein disclosed in the present invention can activate the HER2:HER4 signaling pathway in cardiomyocytes with an efficacy comparable to that of NRG-1.

实施例6：HER2:HER3二聚化与HER2:HER4二聚化的诱导Example 6: Induction of HER2:HER3 dimerization and HER2:HER4 dimerization

由Eurofins DiscoverX(Fremont,CA)开发的PathHunter Dimerization Assay检测配体诱导的受体-二聚体对的两个亚基的二聚化。分析原理显示于图6A中。β-gal酶分成两个片段，即ProLink(PK)及酶受体(EA)。细胞经过工程改造以共表达与酶供体PK融合的靶蛋白1及与酶受体EA融合的靶蛋白2。配体与一个靶蛋白的结合诱导其与另一个靶蛋白相互作用，迫使两个酶片段互补且导致酶反应释放化学发光信号，该信号以相对荧光单位或RFU检测。The PathHunter Dimerization Assay developed by Eurofins DiscoverX (Fremont, CA) detects the dimerization of two subunits of the receptor-dimer pair induced by ligands. The analysis principle is shown in Figure 6A. The β-gal enzyme is divided into two fragments, namely ProLink (PK) and enzyme receptor (EA). Cells are engineered to co-express target protein 1 fused to the enzyme donor PK and target protein 2 fused to the enzyme receptor EA. The binding of a ligand to a target protein induces it to interact with another target protein, forcing the two enzyme fragments to complement each other and causing the enzyme reaction to release a chemiluminescent signal, which is detected in relative fluorescence units or RFU.

PathHunter U2OS ErbB2/ErbB4二聚化细胞株及ErbB2/ErbB3二聚化细胞株是获自Eurofins DiscoverX。细胞以4,000个细胞/孔接种于384孔板中，且在37℃/5％ CO₂下孵育过夜。测试剂自28.8nM开始以逐步1:4连续稀释系列制备，且随后添加至384孔板中的细胞。孵育4小时后，根据制造商说明书分析细胞的受体二聚化。用GraphPad Prism软件分析数据，且以平均RFU±SEM(n＝3)表示。PathHunter U2OS ErbB2/ErbB4 dimerization cell line and ErbB2/ErbB3 dimerization cell line were obtained from Eurofins DiscoverX. Cells were seeded at 4,000 cells/well in 384-well plates and incubated overnight at 37°C/5%_CO2 . Test agents were prepared in a stepwise 1:4 serial dilution series starting from 28.8 nM and then added to the cells in 384-well plates. After 4 hours of incubation, cells were analyzed for receptor dimerization according to the manufacturer's instructions. Data were analyzed with GraphPad Prism software and are presented as mean RFU±SEM (n=3).

如图6B及6C所示，本发明所公开的重组融合蛋白可诱导HER2/HER4二聚化，其效力与NRG-1相当；而其诱导HER2/HER3二聚化的能力则弱得多。作为研究的阴性对照，同型对照抗体GP120 mAb及抗HER3 mAb均不诱导受体二聚化。As shown in Figures 6B and 6C, the recombinant fusion protein disclosed in the present invention can induce HER2/HER4 dimerization with an efficacy comparable to that of NRG-1, while its ability to induce HER2/HER3 dimerization is much weaker. As negative controls for the study, the isotype control antibody GP120 mAb and anti-HER3 mAb did not induce receptor dimerization.

在通过参考随附附图描述本发明的实施例后，应理解本发明不限于精确实施例，且本领域技术人员可在其中进行各种改变及修改，而不偏离如随附权利要求中所定义的本发明范围或精神。Having described the embodiments of the present invention by referring to the accompanying drawings, it should be understood that the present invention is not limited to the precise embodiments and that various changes and modifications may be made therein by one skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

实施例7：重组融合蛋白在收缩性心脏衰竭大鼠模型中的体内功效Example 7: In vivo efficacy of recombinant fusion protein in a rat model of systolic heart failure

为了评估重组融合蛋白在疾病模型中恢复心脏功能的能力，采用心肌梗塞及收缩性心脏衰竭的史泊格多利(Sprague Dawley)大鼠模型。为了建立疾病模型，在外科手术中使用6-0丝质缝合线结扎左心耳下方3-4mm处的左前降支冠状动脉(LAD)。结扎后四周，通过M型超声波心电图(ECG)多普勒超声波记录射血分数(EF)，以相对于手术前的基线EF测量心脏功能。纳入后续研究时，使用最低下降30％的EF阈值。假性对照动物进行相同的手术，但没有进行LAD结扎。To evaluate the ability of the recombinant fusion protein to restore cardiac function in a disease model, the Sprague Dawley rat model of myocardial infarction and systolic heart failure was used. To establish the disease model, the left anterior descending coronary artery (LAD) was ligated 3-4 mm below the left atrial appendage using a 6-0 silk suture during surgery. Four weeks after ligation, the ejection fraction (EF) was recorded by M-mode echocardiography (ECG) Doppler ultrasound to measure cardiac function relative to the baseline EF before surgery. When included in the subsequent study, a minimum EF threshold of 30% decrease was used. Sham control animals underwent the same surgery, but without LAD ligation.

建立疾病模型后，将动物分为五组，每组十一只大鼠，另外十只假手术大鼠在第六组。研究设计为各组每周接受两次尾部静脉注射，为期四周，或总共八次注射。假手术组及阴性对照溶媒组均接受生理盐水，三组接受1、3或10mg/kg的重组融合蛋白，且最后一组接受阳性对照GP120 mAb/NRG-1融合蛋白(10mg/kg)。After the disease model was established, the animals were divided into five groups of eleven rats each, and ten sham-operated rats were in the sixth group. The study design was that each group received two tail vein injections per week for four weeks, or a total of eight injections. The sham-operated group and the negative control vehicle group all received normal saline, three groups received 1, 3 or 10 mg/kg of the recombinant fusion protein, and the last group received the positive control GP120 mAb/NRG-1 fusion protein (10 mg/kg).

由于在研究期间观察到体重减轻，在接受3mg/kg及10mg/kg的重组融合蛋白组中进行完整顺序的八次注射之前停止治疗，这些组分别仅接受六次及三次注射。所有其他组则接受完整八次注射。Due to weight loss observed during the study, treatment was stopped before the full sequence of eight injections in the groups receiving 3 mg/kg and 10 mg/kg of the recombinant fusion protein, which received only six and three injections, respectively. All other groups received the full eight injections.

第一次处理后四周，再次通过M型ECG量测EF。如图8所示，相对于基线，重组融合蛋白显著增加所有三个剂量组的EF。具体而言，1、3及10mg/kg组分别观察到14.7％(P<0.001)、26.9％(P<0.001)及36.6％(P<0.001)的增加。GP120 mAb/NRG-1阳性对照在匹配的时间点使EF增加28.8％(P<0.001)。生理盐水在假性对照组或溶媒对照组中均未显示效果。Four weeks after the first treatment, EF was measured again by M-mode ECG. As shown in Figure 8, the recombinant fusion protein significantly increased EF in all three dose groups relative to baseline. Specifically, increases of 14.7% (P<0.001), 26.9% (P<0.001), and 36.6% (P<0.001) were observed in the 1, 3, and 10 mg/kg groups, respectively. The GP120 mAb/NRG-1 positive control increased EF by 28.8% (P<0.001) at the matched time points. Saline showed no effect in either the sham or vehicle control groups.

在处理后28天收集ECG值后，对小鼠进行安乐死，收集手术部位旁的心脏组织，在4％甲醛中固定且包埋于石蜡中。用苏木精及伊红染料对心脏组织的五微米厚的石蜡切片进行染色，且在光学显微镜下观察组织病理学变化。如图9A-9F所示，在假手术组中，心肌细胞以有序方式排列，且细胞质及心肌纤维被均匀染色。在间质空间中未观察到炎性细胞浸润，且未发现心肌坏死。相比之下，在溶媒对照组中，心肌梗塞边缘区展现心肌细胞之间的间隙变宽；细胞核被缩合并碎裂，且心肌纤维排列失去其有序结构；细胞大小增大且注意到间质水肿。用重组融合蛋白进行的处理部分缓解心肌梗塞区的病理变化，包括坏死细胞显著减少、心肌细胞之间的间质空间变窄及心肌纤维排列恢复至正常结构。After collecting ECG values 28 days after treatment, mice were euthanized, and cardiac tissues next to the surgical site were collected, fixed in 4% formaldehyde and embedded in paraffin. Five-micron-thick paraffin sections of cardiac tissue were stained with hematoxylin and eosin dyes, and histopathological changes were observed under an optical microscope. As shown in Figures 9A-9F, in the sham group, cardiomyocytes were arranged in an orderly manner, and the cytoplasm and myocardial fibers were uniformly stained. Inflammatory cell infiltration was not observed in the interstitial space, and no myocardial necrosis was found. In contrast, in the vehicle control group, the myocardial infarction edge zone showed widening of the gaps between myocardial cells; the nuclei were condensed and fragmented, and the myocardial fiber arrangement lost its orderly structure; the cell size increased and interstitial edema was noted. Treatment with the recombinant fusion protein partially alleviated the pathological changes in the myocardial infarction area, including a significant reduction in necrotic cells, narrowing of the interstitial space between myocardial cells, and restoration of myocardial fiber arrangement to a normal structure.

实施例8：重组融合蛋白减弱NOD/SCID小鼠皮下FaDu癌异种移植模型的肿瘤生长Example 8: Recombinant fusion protein attenuates tumor growth in the NOD/SCID mouse subcutaneous FaDu cancer xenograft model

为了评估重组融合蛋白促进肿瘤生长的潜在风险，在FaDu癌异种移植模型中进行体内研究。NOD/SCID小鼠(Beijing AK Bio-Technology Co.Ltd.)根据机构指南在CrownBio国际研发中心(中国北京)的SPF设施中饲养。所有实验均按照实验室动物护理评定及认证协会(AAALAC)的要求及在CrownBio IACUC委员会的许可下进行。To evaluate the potential risk of recombinant fusion proteins promoting tumor growth, in vivo studies were performed in a FaDu cancer xenograft model. NOD/SCID mice (Beijing AK Bio-Technology Co. Ltd.) were housed in an SPF facility at CrownBio International Research and Development Center (Beijing, China) according to institutional guidelines. All experiments were performed in accordance with the requirements of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and with the permission of the CrownBio IACUC Committee.

7-10周龄的雌性NOD/SCID小鼠在右腹皮下接种悬浮于0.1ml PBS中的FaDu肿瘤细胞(3×10⁶)。当肿瘤达到约150mm³时，将小鼠随机分为6个研究组，每组8只动物。测试样品通过尾部静脉注射静脉内施用，一周两次，连续三周，共6次处理。通过卡尺测量来监测肿瘤生长。研究在处理后21天终止。Female NOD/SCID mice, 7-10 weeks old, were inoculated subcutaneously in the right flank with FaDu tumor cells (3×10⁶ ) suspended in 0.1 ml PBS. When tumors reached approximately 150 mm³ , mice were randomized into 6 study groups of 8 animals each. Test samples were administered intravenously via tail vein injection twice a week for three consecutive weeks for a total of 6 treatments. Tumor growth was monitored by caliper measurement. The study was terminated 21 days after treatment.

响应于不同处理的肿瘤生长汇总于图10中。10mg/kg的抗HER3 mAb显示出显著的抗肿瘤活性，在研究结束时肿瘤生长抑制(TGI)为93.5％(与溶媒组相比p<0.001)。重组融合蛋白在研究结束时还表现出统计学上显著的TGI：在1mg/kg剂量下为19.2％(与溶媒组相比p＝0.048)及在10mg/kg剂量下为56.2％(与溶媒组相比p<0.001)。对照分子GP120 mAb/NRG-1融合蛋白在高或低剂量下均未显示出抗肿瘤活性。研究期间未发生动物死亡。肿瘤小鼠对所有测试剂的耐受性良好。在任何实验组中均未观察到显著的体重减轻(图11)。这些数据表明，在体内肿瘤生长活跃的条件下，重组融合蛋白以剂量依赖性方式表现出肿瘤生长抑制，且表明重组融合蛋白体内增强或加速肿瘤生长的风险低于天然NRG-1蛋白。Tumor growth in response to different treatments is summarized in Figure 10. Anti-HER3 mAb at 10 mg/kg showed significant antitumor activity, with a tumor growth inhibition (TGI) of 93.5% at the end of the study (p<0.001 compared to the vehicle group). The recombinant fusion protein also showed statistically significant TGI at the end of the study: 19.2% at a dose of 1 mg/kg (p=0.048 compared to the vehicle group) and 56.2% at a dose of 10 mg/kg (p<0.001 compared to the vehicle group). The control molecule GP120 mAb/NRG-1 fusion protein did not show antitumor activity at high or low doses. No animal deaths occurred during the study. Tumor mice tolerated all test agents well. No significant weight loss was observed in any experimental group (Figure 11). These data indicate that under conditions of active tumor growth in vivo, the recombinant fusion protein exhibits tumor growth inhibition in a dose-dependent manner, and that the risk of recombinant fusion protein enhancing or accelerating tumor growth in vivo is lower than that of natural NRG-1 protein.

实施例9：在给予重组融合蛋白的食蟹猕猴中未观察到显著的胃肠道毒性Example 9: No significant gastrointestinal toxicity was observed in cynomolgus monkeys administered the recombinant fusion protein

先前报道指出，在一项一期临床研究(NCT01258387)中，受试者接受安慰剂或单剂量给予的西马格勒明(cimaglermin)(全长重组NRG-1β3)，恶心及腹泻为第二及第四常见的治疗突发不良事件，分别发生在40％及27％的合计高剂量群组中(Lenihan等人J Am CollCardiol Basic Trans Science.2016；1(7):576-86)。同样，在重组NRG-1肽片段(纽卡定(neucardin))的二期研究中，恶心为最常观察到的治疗相关不良事件，见于20％的研究受试者中(Jabbour等人European Journal of Heart Failure(2011)13:83-92)。最后，在纽卡定的第二个二期研究(ChiCTR-TRC-00000414)中，公布的结果显示，观察到48.4％的不良事件是胃肠道性质的，是本研究中最常观察到的不良事件类型且与剂量水平相关(Gao等人J Am Coll Cardiol 2010；55:1907-14)。It has been previously reported that in a phase I clinical study (NCT01258387) in which subjects received placebo or a single dose of cimaglermin (full-length recombinant NRG-1β3), nausea and diarrhea were the second and fourth most common treatment-emergent adverse events, occurring in 40% and 27% of the combined high-dose group, respectively (Lenihan et al. J Am Coll Cardiol Basic Trans Science. 2016; 1(7):576-86). Similarly, in a phase II study of a recombinant NRG-1 peptide fragment (neucardin), nausea was the most commonly observed treatment-related adverse event, occurring in 20% of the study subjects (Jabbour et al. European Journal of Heart Failure (2011) 13:83-92). Finally, in the second Phase 2 study of necardin (ChiCTR-TRC-00000414), published results showed that 48.4% of adverse events observed were gastrointestinal in nature, the most commonly observed type of adverse event in this study and were related to dose level (Gao et al. J Am Coll Cardiol 2010;55:1907-14).

进行两项研究，以评估重组融合蛋白在食蟹猴(Macaca fascicularis)中的安全性及耐受性：一项单剂量非GLP(良好实验室规范)研究及一项重复剂量GLP研究。密切监测胃肠道毒性。在单剂量研究中，在10、30及60mg/kg的剂量下，与溶媒对照相比评估重组融合蛋白的安全性及耐受性，各群组中包括一只雄性及一只雌性动物。在此单剂量研究中，在整个两周的处理后评估期间，没有出现与测试剂有关的对体重或食物定性评估的影响，也未观察到呕吐或腹泻。在重复剂量GLP研究中，在以3、10及30mg/kg的剂量连续每周四次给予后，与溶媒对照相比评估重组融合蛋白的安全性及耐受性，在主要的28天研究期间，各群组中包括三只雄性和三只雌性，在随后的28天恢复期后，评估30mg/kg及溶媒对照群组中的另外两只雄性及两只雌性。在此重复剂量研究中未观察到与测试剂有关的对摄食量的影响。虽然在此重复剂量研究中观察到与测试剂有关的呕吐，但临床观察到的呕吐仅与输注反应相关，仅在10mg/kg群组的一只动物(17％)及30mg/kg群组的两只动物(20％)中观察到，并且是短暂的。腹泻仅在溶媒对照群组及30mg/kg重组融合蛋白群组中观察到，分别在仅一只(10％)及三只(30％)动物中观察到，且被视为此类型程序的正常现象，与重组融合蛋白无关。最后，在此重复剂量研究中，仅在10mg/kg及30mg/kg剂量下，平均体重相对于基线减少>10％，且仅在10mg/kg群组的第四次给药及30mg/kg群组的第三及第四次给药后才出现。总之，除了在急性输注反应期间以外，用重组融合蛋白处理没有导致任何与食物摄入、呕吐或腹泻有关的临床上显著发现，且胃肠道发现对两个研究中无不良事件程度的判定没有影响。这些结果表明，重组融合蛋白的设计减轻NRG-1重组蛋白对胃肠道的不良影响。Two studies were conducted to evaluate the safety and tolerability of the recombinant fusion protein in cynomolgus monkeys (Macaca fascicularis): a single-dose non-GLP (good laboratory practice) study and a repeated-dose GLP study. Gastrointestinal toxicity was closely monitored. In a single-dose study, the safety and tolerability of the recombinant fusion protein were evaluated at doses of 10, 30 and 60 mg/kg compared to vehicle controls, with one male and one female animal in each group. In this single-dose study, no effects on body weight or food qualitative assessments related to the test agent occurred during the entire two-week post-treatment assessment period, and no vomiting or diarrhea was observed. In a repeated-dose GLP study, the safety and tolerability of the recombinant fusion protein was evaluated compared to vehicle controls after continuous administration four times a week at doses of 3, 10 and 30 mg/kg. During the main 28-day study period, three males and three females were included in each group, and after a subsequent 28-day recovery period, two other males and two females in the 30 mg/kg and vehicle control groups were evaluated. No test agent-related effects on food intake were observed in this repeated dose study. Although test agent-related vomiting was observed in this repeated dose study, clinically observed vomiting was only associated with infusion reactions, observed in only one animal (17%) in the 10 mg/kg group and two animals (20%) in the 30 mg/kg group, and was transient. Diarrhea was observed only in the vehicle control group and the 30 mg/kg recombinant fusion protein group, observed in only one (10%) and three (30%) animals, respectively, and was considered a normal phenomenon for this type of procedure and was not related to the recombinant fusion protein. Finally, in this repeated dose study, only at the 10 mg/kg and 30 mg/kg doses, the mean body weight decreased by >10% relative to baseline, and only after the fourth dose of the 10 mg/kg group and the third and fourth doses of the 30 mg/kg group. In summary, except during acute infusion reactions, treatment with the recombinant fusion protein did not result in any clinically significant findings related to food intake, vomiting or diarrhea, and gastrointestinal findings had no effect on the determination of the degree of no adverse events in both studies. These results suggest that the design of recombinant fusion protein alleviates the adverse effects of NRG-1 recombinant protein on the gastrointestinal tract.

在单剂量给予60mg/kg的重组融合蛋白后的不同时间点自食蟹猴采集血液样本(～1ml)，提取血清且储存于-80℃下直至测试。血清样本中重组融合蛋白的浓度是通过根据标准程序捕获ELISA来分析。简而言之，96孔板包被有重组人类HER3蛋白(R&D System)，用BSA封闭且与测试样品一起孵育。在多次洗涤后，将板与HRP偶联的抗人IgG Fc抗体一起孵育，且随后用TMB底物检测。图12显示，重组融合蛋白的药代动力学曲线与IgG抗体相似。Blood samples (~1 ml) were collected from cynomolgus monkeys at different time points after a single dose of 60 mg/kg of the recombinant fusion protein, and serum was extracted and stored at -80°C until testing. The concentration of the recombinant fusion protein in the serum samples was analyzed by capturing ELISA according to standard procedures. In brief, 96-well plates were coated with recombinant human HER3 protein (R&D System), blocked with BSA and incubated with the test samples. After multiple washings, the plates were incubated with HRP-coupled anti-human IgG Fc antibodies and subsequently detected with TMB substrate. Figure 12 shows that the pharmacokinetic profile of the recombinant fusion protein is similar to that of the IgG antibody.

实施例10：Fc受体结合动力学常数的总结Example 10: Summary of Fc receptor binding kinetic constants

使用无标记SPR技术来测量重组抗HER3 mAb/NRG-1融合蛋白与Fc受体的结合亲和力。分别针对重组融合蛋白、无Fc突变的重组融合蛋白及抗HER3抗体分析总共六种Fc受体(各自与His标签融合)，包括FcγRI(Abcam)、FcγRIIa、FcγRIIb、FcγRIIIa(158F)、FcγRIIIa(158V)及C1q(Sino Biological)。所有Fc受体及测试样品均通过亲和层析纯化。所有实验均在Biacore 8K系统(GE Healthcare)上进行，使用HBS-EP+(10mM HEPES、150mMNaCl、3mM EDTA及0.05％v/vSurfactant P20)作为运行缓冲液。具体而言，抗His抗体是通过胺偶联方法在CM5传感器芯片的活性及参考流动槽中偶联。经纯化的带His标签的Fc受体经由与固定的抗His抗体结合而在各单个通道的活性流动槽上被捕获。各Fc受体的捕获水平维持在80-120RU之间。对于动力学分析，将重组融合蛋白及所有其他样品连续稀释总共6个浓度，在0.3nM至30nM范围内，且连续稀释液依次经由各通道的两个流动槽注入。通过在多个通道上同时注入样品，在同一次运行中完成多项分析。The binding affinity of recombinant anti-HER3 mAb/NRG-1 fusion protein to Fc receptors was measured using label-free SPR technology. A total of six Fc receptors (each fused to a His tag), including FcγRI (Abcam), FcγRIIa, FcγRIIb, FcγRIIIa (158F), FcγRIIIa (158V) and C1q (Sino Biological) were analyzed for recombinant fusion proteins, recombinant fusion proteins without Fc mutations and anti-HER3 antibodies, respectively. All Fc receptors and test samples were purified by affinity chromatography. All experiments were performed on a Biacore 8K system (GE Healthcare) using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v Surfactant P20) as the running buffer. Specifically, the anti-His antibody was coupled in the active and reference flow cells of a CM5 sensor chip by an amine coupling method. Purified His-tagged Fc receptors were captured on the active flow cell of each individual channel via binding to an immobilized anti-His antibody. Capture levels for each Fc receptor were maintained between 80-120 RU. For kinetic analysis, recombinant fusion proteins and all other samples were serially diluted to a total of 6 concentrations ranging from 0.3 nM to 30 nM, and the serial dilutions were injected sequentially through two flow cells of each channel. Multiple analyses were completed in the same run by injecting samples simultaneously on multiple channels.

使用Biacore 8K评估软件，用两态结合模型拟合所得传感器图谱以提取动力学常数。所有分析的平衡解离率(KD)汇总于下表1中。重组融合蛋白与FcγRI、FcγRIIa及FcγRIIb结合的动力学推导的KD值比无Fc突变的重组融合蛋白及抗HER3抗体的KD值高10倍以上，表明由于重组融合蛋白Fc区内的特定突变而使亲和力低得多。对于FcγRIIIa(158F)及FcγRIIIa(158V)，Fc突变分别导致重组融合蛋白的结合亲和力下降2至3倍。与C1q的结合太弱，无法在所有样品中检测到。The obtained sensor spectra were fitted with a two-state binding model using Biacore 8K evaluation software to extract kinetic constants. The equilibrium dissociation rates (KD) of all analyses are summarized in Table 1 below. The kinetically derived KD values for the binding of the recombinant fusion proteins to FcγRI, FcγRIIa and FcγRIIb were more than 10 times higher than the KD values of the recombinant fusion proteins without Fc mutations and the anti-HER3 antibody, indicating that the affinity is much lower due to specific mutations in the Fc region of the recombinant fusion proteins. For FcγRIIIa (158F) and FcγRIIIa (158V), the Fc mutations resulted in a 2- to 3-fold decrease in the binding affinity of the recombinant fusion proteins, respectively. Binding to C1q was too weak to be detected in all samples.

为了证实重组融合蛋白具有有限的Fc效应功能，使用来自Promega(Madison,WI)的ADCC Reporter Bioassay检查抗体依赖性细胞毒性(ADCC)。该分析使用经工程改造的Jurkat细胞株作为效应细胞，其稳定表达FcγRIIIa(V158)受体及驱动萤火虫荧光素酶表达的NFAT反应元件。利妥昔单抗作为该分析的阳性对照，显示出针对CD20阳性Raji细胞的强ADCC活性；而重组融合蛋白没有可检测到的针对HER3阳性靶细胞(MCF7或BT474)的ADCC(数据未显示)。To confirm that the recombinant fusion protein has limited Fc effector function, antibody-dependent cellular cytotoxicity (ADCC) was examined using the ADCC Reporter Bioassay from Promega (Madison, WI). The analysis used an engineered Jurkat cell line as effector cells, which stably expresses the FcγRIIIa (V158) receptor and the NFAT response element driving the expression of firefly luciferase. Rituximab, as a positive control for the analysis, showed strong ADCC activity against CD20-positive Raji cells; while the recombinant fusion protein had no detectable ADCC against HER3-positive target cells (MCF7 or BT474) (data not shown).

表1：Fc受体结合动力学常数总结Table 1: Summary of Fc receptor binding kinetic constants

*<LOD-由于弱结合而低于检测极限(LOD)*<LOD - below the limit of detection (LOD) due to weak binding

实施例11：NRG-1-HER3重组融合蛋白在体外心房组织中显示出抗纤维化作用及在体内小鼠模型中显示出AF预防作用Example 11: NRG-1-HER3 recombinant fusion protein shows anti-fibrotic effect in atrial tissue in vitro and AF prevention effect in mouse model in vivo

简介.心房震颤(AF)是由心房的电性和结构性重塑所造成，炎症及纤维化在其中发挥作用。目前的疗法限于抗心律失常药物及消融术，但并不针对结构性问题。最近的研究表明，神经调节蛋白-1(NRG1)，一种表皮生长因子家族成员，在心肌中具有抗纤维化及抗炎作用。Introduction. Atrial fibrillation (AF) is caused by electrical and structural remodeling of the atria, in which inflammation and fibrosis play a role. Current therapies are limited to antiarrhythmic drugs and ablation, but do not target the structural problem. Recent studies have shown that neuregulin-1 (NRG1), a member of the epidermal growth factor family, has antifibrotic and anti-inflammatory effects in the myocardium.

目的.测试例示性NRG-1/HER3抗体融合蛋白对心房纤维化及AF诱导的影响。NRG-1/HER3抗体融合蛋白包含NRG-1活性片段及拮抗性HER3(ERBB3)抗体，且选择性地经由ERBB4优先于ERBB3传导信号。Objective. Test the effects of exemplary NRG-1/HER3 antibody fusion proteins on atrial fibrosis and AF induction. The NRG-1/HER3 antibody fusion protein comprises an NRG-1 active fragment and an antagonistic HER3 (ERBB3) antibody, and selectively signals through ERBB4 over ERBB3.

方法.如图13A中所描述的体外纤维化分析用于体外诱导大鼠组织样本的纤维化。自雄性大鼠(Wistar Han，10周龄)采集心房样本，切成小块(1-2mm²)且在NRG-1/HER3融合蛋白(5nM浓度)存在或不存在的情况下保存在低血清培养基中。24-72小时后对Col1a1及Col3a1 mRNA进行量化，分别如图14A及14B中所描述。Methods. The in vitro fibrosis assay as described in FIG. 13A was used to induce fibrosis in rat tissue samples in vitro. Atrial samples were collected from male rats (Wistar Han, 10 weeks old), cut into small pieces (1-2 mm² ) and stored in low serum medium in the presence or absence of NRG-1/HER3 fusion protein (5 nM concentration). Col1a1 and Col3a1 mRNA were quantified after 24-72 hours as described in FIGS. 14A and 14B , respectively.

在两个模型中测试AF诱导。在第一AF模型中，雄性小鼠(C57BL/6N，12-15周龄)用血管收缩素-II(Ang-II，4周，渗透微型泵，3000ng/kg/min)处理，其实验大纲描述于图16中。在图18中所描述的第二AF模型中，给小鼠喂食高脂肪饮食(HFD，8周，60％ Kcal脂肪)，诱导体重严重增加(增加56±3％，相较于常规饮食增加23±4％)，如图19A中所示。在两个模型中，AF诱导是通过用经颈静脉八极导管进行5次程序化电刺激(PES)来测试，如图15中所描述。记录AF诱导(通过≥3次PES诱导的小鼠％)及PES诱导的AF的持续时间(AF持续时间)。将小鼠随机分组以用溶媒或示例性NRG-1/HER3融合蛋白处理(2次/周，1mg/kg，IV，n＝5-7/组)。AF induction was tested in two models. In the first AF model, male mice (C57BL/6N, 12-15 weeks old) were treated with angiotensin-II (Ang-II, 4 weeks, osmotic minipump, 3000 ng/kg/min), the experimental outline of which is described in Figure 16. In the second AF model described in Figure 18, mice were fed a high-fat diet (HFD, 8 weeks, 60% Kcal fat), inducing severe weight gain (an increase of 56±3%, an increase of 23±4% compared to a regular diet), as shown in Figure 19A. In both models, AF induction was tested by 5 programmed electrical stimulations (PES) using a transjugular octopole catheter, as described in Figure 15. AF induction (% of mice induced by ≥3 PES) and the duration of PES-induced AF (AF duration) were recorded. Mice were randomized to treatment with vehicle or exemplary NRG-1/HER3 fusion protein (2 times/week, 1 mg/kg, IV, n=5-7/group).

结果.在培养的心房样本中，Col1a1及Col3a1 mRNA表达经3天逐渐增加至2-3倍，分别如图13A及13B中所示。NRG-1/HER3融合蛋白将此效应有力地减弱59±17％(p<0.05)，如图14A及14B中所示。在小鼠中，Ang-II及HFD均显著增加AF诱导及AF持续时间。在Ang-II小鼠中，NRG-1/HER3融合蛋白减弱AF诱导(自57％至20％)，如图17B中所示，及AF持续时间(自33.3±15.1至1.5±1s)，如图17A中所示。在HFD小鼠中，NRG-1/HER3融合蛋白显著减弱AF诱导(自57％至0％)，如图20B中所示，及AF持续时间(自10.9±3.2s至0.76±0.5s，p<0.05)，如图20A中所示。Results. In cultured atrial samples, Col1a1 and Col3a1 mRNA expression gradually increased 2-3 fold over 3 days, as shown in Figures 13A and 13B, respectively. NRG-1/HER3 fusion protein strongly attenuated this effect by 59±17% (p<0.05), as shown in Figures 14A and 14B. In mice, both Ang-II and HFD significantly increased AF induction and AF duration. In Ang-II mice, NRG-1/HER3 fusion protein attenuated AF induction (from 57% to 20%), as shown in Figure 17B, and AF duration (from 33.3±15.1 to 1.5±1 s), as shown in Figure 17A. In HFD mice, NRG-1/HER3 fusion protein significantly attenuated AF induction (from 57% to 0%), as shown in Figure 20B, and AF duration (from 10.9±3.2s to 0.76±0.5s, p<0.05), as shown in Figure 20A.

结论.这些结果显示体外用NRG-1/HER3融合蛋白对心房组织进行选择性ERBB4刺激的抗纤维化作用，同时在两种不相关小鼠模型中具有AF预防作用。Conclusions. These results demonstrate an antifibrotic effect of selective ERBB4 stimulation of atrial tissue with NRG-1/HER3 fusion protein in vitro and a protective effect against AF in two unrelated mouse models.

实施例12：NRG-1/HER3抗体融合蛋白减少与心肌僵硬相关的I型胶原蛋白Example 12: NRG-1/HER3 antibody fusion protein reduces type I collagen associated with myocardial stiffness

实施例7中采用的心肌梗塞及收缩性心脏衰竭(还称为射血分数降低的心脏衰竭，或HFrEF)的史泊格多利大鼠模型用于分析I型胶原蛋白及III型胶原蛋白表达。处理四周后，如实施例7中所述取出心脏组织样本，固定及切片，且使用免疫组织化学对I型胶原蛋白或III型胶原蛋白表达进行染色。The Sprague-Dawley rat model of myocardial infarction and systolic heart failure (also known as heart failure with reduced ejection fraction, or HFrEF) employed in Example 7 was used to analyze type I and type III collagen expression. After four weeks of treatment, heart tissue samples were removed, fixed and sectioned as described in Example 7, and stained for type I or type III collagen expression using immunohistochemistry.

I型胶原蛋白的代表性结果显示于图21A至21F中。如图21A至21F中可见，NRG-1/HER3抗体融合蛋白以1mg/kg、3mg/kg及10mg/k施用，与溶媒对照相比，分别使I型胶原蛋白的表达下调6.6％、37.1％及40.5％。此外，如下表2所示，相对于溶媒对照，胶原蛋白I/III比率分别降低4.7％、40.8％及36.6％。Representative results for type I collagen are shown in Figures 21A to 21F. As can be seen in Figures 21A to 21F, NRG-1/HER3 antibody fusion protein administered at 1 mg/kg, 3 mg/kg, and 10 mg/kg downregulated type I collagen expression by 6.6%, 37.1%, and 40.5%, respectively, compared to vehicle control. In addition, as shown in Table 2 below, the collagen I/III ratio was reduced by 4.7%, 40.8%, and 36.6%, respectively, relative to vehicle control.

表2.心肌胶原蛋白的变化Table 2. Changes of myocardial collagen

△△△p<0.01表示与假对照相比的统计显著性△△△p<0.01 indicates statistical significance compared with sham control

*p<0.05，**p<0.01表示与溶媒对照相比的统计显著性*p<0.05, **p<0.01 indicate statistical significance compared with vehicle control

实施例13：NRG-1/HER3抗体融合蛋白预防猪DOCA模型的心房纤维化及心房震颤诱导Example 13: NRG-1/HER3 antibody fusion protein prevents atrial fibrosis and atrial fibrillation induction in pig DOCA model

心房纤维化是心房颤动(AF)复发的底物，纤维化负担越重，治疗耐药性和预后越差。纤维化程度预测预后及疗法难治性。目前，不存在靶向心房纤维化的疗法。NRG-1/HER3融合蛋白为一种长效的神经调节蛋白融合蛋白，已被证明在心脏衰竭动物模型中经由选择性刺激ErbB4受体来减少心室纤维化(即，具有抗纤维化特性)。在此实施例中，测试NRG-1/HER3融合蛋白在乙酸脱氧皮质固酮(DOCA，一种醛固酮促效剂)诱导的高血压的小型猪模型中减少心房纤维化及AF诱导的能力。Atrial fibrosis is a substrate for recurrence of atrial fibrillation (AF), and the heavier the fibrosis burden, the worse the treatment resistance and prognosis. The degree of fibrosis predicts prognosis and refractory to therapy. Currently, there is no therapy targeting atrial fibrosis. NRG-1/HER3 fusion protein is a long-acting neuregulin fusion protein that has been shown to reduce ventricular fibrosis (i.e., has anti-fibrotic properties) in animal models of heart failure via selective stimulation of the ErbB4 receptor. In this example, the ability of NRG-1/HER3 fusion protein to reduce atrial fibrosis and AF induction in a minipig model of hypertension induced by deoxycorticosterone acetate (DOCA, an aldosterone agonist) was tested.

方法：18只Aachener小型猪被随机分为3组：对照(CTRL)、DOCA+溶媒(DOCA+VEH)及DOCA+NRG-1/HER3融合蛋白。对照组并未进行治疗性干预。实验图显示于图22中。为了诱导高血压及心房纤维化，在DOCA+VEH和DOCA+NRG-1/HER3融合蛋白组的小型猪中植入释放10mg/kg DOCA的颗粒，持续60天。植入DOCA的动物自植入当天开始每周用NRG-1/HER3融合蛋白(0.3mg/kg；DOCA+NRG-1/HER3融合蛋白)或其溶媒(DOCA+VEH)进行处理(共9次给药)。60天后，侵入性测量动脉血压，且将十极导管置于右心房中。AF诱导是通过进行50次短阵快速起搏发作来测试，且量化为50次尝试中“成功”发作的百分比，其中在短阵快速起搏后可诱导AF运行≥5秒。AF诱导测试的示例性结果显示于图23中。使用ImageJ软件对动物安乐死后分离的左心房标本的Masson三色染色进行心房纤维化的量化。Methods: Eighteen Aachener miniature pigs were randomly divided into three groups: control (CTRL), DOCA+vehicle (DOCA+VEH), and DOCA+NRG-1/HER3 fusion protein. The control group did not receive therapeutic intervention. The experimental figures are shown in Figure 22. To induce hypertension and atrial fibrosis, particles releasing 10 mg/kg DOCA were implanted in miniature pigs in the DOCA+VEH and DOCA+NRG-1/HER3 fusion protein groups for 60 days. Animals implanted with DOCA were treated weekly with NRG-1/HER3 fusion protein (0.3 mg/kg; DOCA+NRG-1/HER3 fusion protein) or its vehicle (DOCA+VEH) starting from the day of implantation (9 doses in total). After 60 days, arterial blood pressure was measured invasively and a decapolar catheter was placed in the right atrium. AF induction was tested by performing 50 bursts of rapid pacing and quantified as the percentage of "successful" episodes out of 50 attempts, where AF could be induced to run for ≥5 seconds after the burst of rapid pacing. Exemplary results of the AF induction test are shown in Figure 23. Quantification of atrial fibrosis was performed using ImageJ software using Masson's trichrome staining of left atrial specimens isolated after animal euthanasia.

结果：DOCA+VEH(142±10mmHg)及DOCA+NRG-1/HER3融合蛋白(132±15mmHg)组的平均动脉压显著高于CTRL组(105±8mmHg，p<0.01)，而无NRG-1/HER3融合蛋白的影响(图24)。Results: The mean arterial pressure in the DOCA+VEH (142±10 mmHg) and DOCA+NRG-1/HER3 fusion protein (132±15 mmHg) groups was significantly higher than that in the CTRL group (105±8 mmHg, p<0.01), but was not affected by NRG-1/HER3 fusion protein (Figure 24).

如图25中可见，DOCA+VEH组的AF诱导显著高于CTRL组(66/250对比9/300，p<0.001)及DOCA+NRG-1/HER3融合蛋白组(66/250对比8/300，p<0.001)。同样，与CTRL(14.18±1.81对比8.30±2.52，p<0.001)相比及与DOCA+NRG-1/HER3融合蛋白(14.18±1.81对比10.65±1.59，p＝0.0049)相比，DOCA+VEH组的心房纤维化程度显著更高，如图26中可见。As can be seen in Figure 25, AF induction in the DOCA+VEH group was significantly higher than that in the CTRL group (66/250 vs. 9/300, p<0.001) and the DOCA+NRG-1/HER3 fusion protein group (66/250 vs. 8/300, p<0.001). Similarly, the degree of atrial fibrosis in the DOCA+VEH group was significantly higher compared to CTRL (14.18±1.81 vs. 8.30±2.52, p<0.001) and compared to DOCA+NRG-1/HER3 fusion protein (14.18±1.81 vs. 10.65±1.59, p=0.0049), as can be seen in Figure 26.

结论：NRG-1/HER3融合蛋白预防猪DOCA模型的心房纤维化及AF诱导。NRG-1/HER3融合蛋白的作用与对血压的影响无关，但可能与减少心房纤维化有关。Conclusion: NRG-1/HER3 fusion protein prevents atrial fibrosis and AF induction in the porcine DOCA model. The effects of NRG-1/HER3 fusion protein are not related to the effect on blood pressure, but may be related to the reduction of atrial fibrosis.

Claims (27)

Translated fromChinese

1.一种治疗受试者的心房震颤和/或心脏纤维化的方法，其包括向该受试者施用重组融合蛋白，该重组融合蛋白包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段。1. A method for treating atrial fibrillation and/or cardiac fibrosis in a subject, comprising administering to the subject a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).2.一种重组融合蛋白的用途，其包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段，用于治疗受试者的心房震颤和/或心脏纤维化的方法中。2. Use of a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for use in a method for treating atrial fibrillation and/or cardiac fibrosis in a subject.3.如权利要求1的方法或权利要求2的重组融合蛋白的用途，其中，该心脏纤维化包括心房纤维化。3. The method of claim 1 or the use of the recombinant fusion protein of claim 2, wherein the cardiac fibrosis comprises atrial fibrosis.4.如权利要求1至3中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段包含NRG-1的活性结构域。4. The method or use of the recombinant fusion protein according to any one of claims 1 to 3, wherein the NRG-1 fragment comprises the active domain of NRG-1.5.如权利要求1至4中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段包含ERBB3/4结合域。5. The method or use of the recombinant fusion protein according to any one of claims 1 to 4, wherein the NRG-1 fragment comprises an ERBB3/4 binding domain.6.如权利要求1至5中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段结合于ErbB4(HER4)且经由其诱导信号传导。6. The method or use of the recombinant fusion protein according to any one of claims 1 to 5, wherein the NRG-1 fragment binds to ErbB4 (HER4) and induces signal transduction therethrough.7.如权利要求1至6中任一项的方法或重组融合蛋白的用途，其中，该mAb抑制经由ErbB3(HER3)的NRG-1信号传导。7. Use of the method or recombinant fusion protein according to any one of claims 1 to 6, wherein the mAb inhibits NRG-1 signaling via ErbB3 (HER3).8.如权利要求1至7中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段包含NRG-1β2a同功异型物。8. The method or use of the recombinant fusion protein according to any one of claims 1 to 7, wherein the NRG-1 fragment comprises an NRG-1 β2a isoform.9.如权利要求1至8中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段是使用连接子经由其N端氨基酸融合至该抗体重链的C端。9. The method or use of the recombinant fusion protein according to any one of claims 1 to 8, wherein the NRG-1 fragment is fused to the C-terminus of the antibody heavy chain via its N-terminal amino acid using a linker.10.如权利要求9的方法或重组融合蛋白的用途，其中，该连接子包含至少一个拷贝的SEQ ID NO:5中所列的Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser连接子。10. The method or use of the recombinant fusion protein according to claim 9, wherein the linker comprises at least one copy of the Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser linker listed in SEQ ID NO:5.11.如权利要求1至10中任一项的方法或重组融合蛋白的用途，其中，该抗体重链的C端包含该抗体的Fc域。11. The method or use of the recombinant fusion protein according to any one of claims 1 to 10, wherein the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody.12.如权利要求1至11中任一项的方法或重组融合蛋白的用途，其中，该单克隆抗体是糖基化的。12. The method or use of the recombinant fusion protein according to any one of claims 1 to 11, wherein the monoclonal antibody is glycosylated.13.如权利要求1至12中任一项的方法或重组融合蛋白的用途，其中，该NRG-1片段包含SEQ ID NO:4的氨基酸序列。13. The method or use of the recombinant fusion protein according to any one of claims 1 to 12, wherein the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO: 4.14.如权利要求1至13中任一项的方法或重组融合蛋白的用途，其中，该mAb包含SEQ IDNO:2的重链氨基酸序列。14. The method or use of the recombinant fusion protein according to any one of claims 1 to 13, wherein the mAb comprises the heavy chain amino acid sequence of SEQ ID NO: 2.15.如权利要求1至14中任一项的方法或重组融合蛋白的用途，其中，该mAb包含SEQ IDNO:3的轻链氨基酸序列。15. The method or use of the recombinant fusion protein according to any one of claims 1 to 14, wherein the mAb comprises the light chain amino acid sequence of SEQ ID NO: 3.16.如权利要求15的方法或重组融合蛋白的用途，其中，该mAb包含SEQ ID NO:2的氨基酸234、239及434中的至少一个的取代突变。16. The method or use of the recombinant fusion protein of claim 15, wherein the mAb comprises a substitution mutation of at least one of amino acids 234, 239 and 434 of SEQ ID NO:2.17.如权利要求16的方法或重组融合蛋白的用途，其中，该至少一个取代突变包含L234F突变、S239A突变、N434A突变或其组合。17. The method or use of the recombinant fusion protein according to claim 16, wherein the at least one substitution mutation comprises L234F mutation, S239A mutation, N434A mutation or a combination thereof.18.如权利要求1至8中任一项的方法或重组融合蛋白的用途，其中，该重组融合蛋白包含SEQ ID NO:3及SEQ ID NO:14的氨基酸序列。18. The method or use of the recombinant fusion protein according to any one of claims 1 to 8, wherein the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 14.19.如权利要求1至18中任一项的方法或重组融合蛋白的用途，其中，相对于重组NRG-1的信号诱导潜力，该重组融合蛋白促进HER2/4信号传导超过HER2/3信号传导。19. The method or use of the recombinant fusion protein according to any one of claims 1 to 18, wherein the recombinant fusion protein promotes HER2/4 signaling over HER2/3 signaling relative to the signal induction potential of recombinant NRG-1.20.如权利要求1至19中任一项的方法或重组融合蛋白的用途，其中，该重组融合蛋白促进该受试者的心肌细胞或心脏组织的增殖、分化及存活。20. The method or use of the recombinant fusion protein according to any one of claims 1 to 19, wherein the recombinant fusion protein promotes the proliferation, differentiation and survival of the cardiomyocytes or cardiac tissue of the subject.21.如权利要求1至20中任一项的方法或重组融合蛋白的用途，其中，该重组融合蛋白相对于重组NRG-1减弱肿瘤或癌症细胞的增殖。21. The method or use of the recombinant fusion protein of any one of claims 1 to 20, wherein the recombinant fusion protein attenuates the proliferation of tumor or cancer cells relative to recombinant NRG-1.22.如权利要求1至21中任一项的方法或重组融合蛋白的用途，其中，施用该重组融合蛋白减少心房震颤发作的持续时间，或降低心房震颤发生的频率。22. The method or use of the recombinant fusion protein according to any one of claims 1 to 21, wherein administration of the recombinant fusion protein reduces the duration of an atrial fibrillation episode, or reduces the frequency of atrial fibrillation occurrence.23.如权利要求1至22中任一项的方法或重组融合蛋白的用途，其中，施用该重组融合蛋白减少心房震颤或心脏纤维化的体征或症状。23. The method or use of the recombinant fusion protein of any one of claims 1 to 22, wherein administration of the recombinant fusion protein reduces signs or symptoms of atrial fibrillation or cardiac fibrosis.24.如权利要求23的方法或重组融合蛋白的用途，其中，该心房震颤的症状包括心跳不规则、心悸、头晕、极度疲劳、呼吸短促、胸痛或其组合。24. The method of claim 23 or the use of the recombinant fusion protein, wherein the symptoms of atrial fibrillation include irregular heartbeat, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, or a combination thereof.25.如权利要求23的方法或重组融合蛋白的用途，其中，该心脏纤维化的体征包括胶原蛋白含量或沉积，且其中施用该重组融合蛋白减少心脏组织的胶原蛋白含量或沉积。25. The method or use of a recombinant fusion protein according to claim 23, wherein the signs of cardiac fibrosis include collagen content or deposition, and wherein administration of the recombinant fusion protein reduces collagen content or deposition in cardiac tissue.26.如权利要求1至25中任一项的方法或重组融合蛋白的用途，其包括向该受试者施用0.1mcg/kg至5mg/kg的该重组融合蛋白。26. The method or use of the recombinant fusion protein according to any one of claims 1 to 25, comprising administering to the subject 0.1 mcg/kg to 5 mg/kg of the recombinant fusion protein.27.一种药盒，其包括治疗有效量的重组融合蛋白，该重组融合蛋白包含与单特异性ErbB3(HER3)单克隆抗体(mAb)融合的神经调节蛋白-1(NRG-1)片段，用于治疗受试者的心房震颤和/或心脏纤维化。27. A drug kit comprising a therapeutically effective amount of a recombinant fusion protein comprising a neuregulin-1 (NRG-1) fragment fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for treating atrial fibrillation and/or cardiac fibrosis in a subject.