WO2023046116A1 - Polypeptide complex of interleukin 15 and receptor thereof - Google Patents

Polypeptide complex of interleukin 15 and receptor thereof Download PDF

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WO2023046116A1
WO2023046116A1 PCT/CN2022/121068 CN2022121068W WO2023046116A1 WO 2023046116 A1 WO2023046116 A1 WO 2023046116A1 CN 2022121068 W CN2022121068 W CN 2022121068W WO 2023046116 A1 WO2023046116 A1 WO 2023046116A1
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antigen
il15rα
antibody
seq
mutation
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PCT/CN2022/121068
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芦迪
霍永庭
路力生
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广东菲鹏制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Abstract

The present invention relates to the field of biomedicine, and in particular to a polypeptide complex of interleukin 15 and a receptor thereof, and a use thereof. A non-natural interchain bond is present between IL15 and IL15Rα in the polypeptide complex of the present invention.

Description

一种白介素15及其受体的多肽复合物A kind of polypeptide complex of interleukin 15 and its receptor
本申请要求申请日为2021年9月24日的中国专利申请(申请号：202111123534.X，发明名称：一种白介素15及其受体的多肽复合物)以及申请日为2021年9月24日的中国专利申请(申请号：202111121937.0，发明名称：双特异性多功能融合多肽)的优先权，这2件中国专利申请的全文通过引用方式整体并入到本申请。This application requires a Chinese patent application with an application date of September 24, 2021 (application number: 202111123534.X, invention name: a polypeptide complex of interleukin 15 and its receptor) and an application date of September 24, 2021 The priority of the Chinese patent application (application number: 202111121937.0, invention name: bispecific multifunctional fusion polypeptide), the full text of these two Chinese patent applications is incorporated by reference in its entirety into this application.
技术领域technical field
本发明涉及生物医药领域，具体而言，涉及一种包含白细胞介素15及其受体的二硫键改造多肽复合物。The invention relates to the field of biomedicine, in particular to a disulfide bond modified polypeptide complex comprising interleukin 15 and its receptor.
背景技术Background technique
细胞因子在人体免疫调节中起重要作用，同时也参与肿瘤的免疫调控，与肿瘤的发生、发展密切相关。在免疫疗法中，细胞因子可直接作用于肿瘤微环境中的免疫效应细胞，增强肿瘤抑制效果。通过临床研究以及动物实验，许多细胞因子已被证明具有显著的抗肿瘤活性，已有多个细胞因子获得FDA批准上市。Cytokines play an important role in human immune regulation, and also participate in the immune regulation of tumors, which are closely related to the occurrence and development of tumors. In immunotherapy, cytokines can directly act on immune effector cells in the tumor microenvironment to enhance the tumor suppressive effect. Through clinical research and animal experiments, many cytokines have been proven to have significant anti-tumor activity, and several cytokines have been approved by the FDA for marketing.
白细胞介素15(IL-15)是Grabstein等人于1994年发现的一种约为12-14kD的细胞因子，可在机体正常的免疫应答中发挥作用，如促进T细胞、B细胞、自然杀伤(NK)细胞的增殖。Interleukin 15 (IL-15) is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994, which can play a role in the normal immune response of the body, such as promoting T cells, B cells, natural killing Proliferation of (NK) cells.
IL-15属于四个小α螺旋束细胞因子家族(Small fourα-helix bundle familyof cytokines)中的成员。IL-15需通过与其受体结合发挥生物学活性。IL-15受体由三个受体亚基组成：IL-15受体α(IL-15Rα)、IL-2受体β(IL-2Rβ，也称IL-15Rβ或CD122)和γc(也称CD132)。IL-15Rα内含一个Sushi结构域，能与IL-15结合，并且是使结合后的IL-15发挥生物学功能所必需的。IL-15 is a member of the small four α-helix bundle family of cytokines. IL-15 exerts its biological activity through binding to its receptor. The IL-15 receptor consists of three receptor subunits: IL-15 receptor α (IL-15Rα), IL-2 receptor β (IL-2Rβ, also known as IL-15Rβ or CD122) and γc (also known as CD132). IL-15Rα contains a Sushi domain, which can bind IL-15 and is necessary for the biological function of IL-15 after binding.
近年来，IL15及IL15受体更多地用于构建融合蛋白，为了提高融合蛋白的稳定性，本申请在白介素15及其受体之间引入二硫键。In recent years, IL15 and IL15 receptors have been more used to construct fusion proteins. In order to improve the stability of fusion proteins, this application introduces a disulfide bond between interleukin 15 and its receptor.
发明内容Contents of the invention
本发明提供一种IL15/IL15Rα多肽复合物，所述IL15和IL15Rα之间具有一个非天然的链间键，所述非天然链间键形成于IL15的第一突变残基和IL15Rα的第二突变残基之间，所述IL15第一突变残基为第90位的E突变为C，IL15Rα第二突变残基为第67位的P突变为C。所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点，所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述IL15和IL15Rα特异性结合；在一些实施方式中，其中，The present invention provides an IL15/IL15Rα polypeptide complex, there is a non-natural interchain bond between IL15 and IL15Rα, and the non-natural interchain bond is formed by the first mutation residue of IL15 and the second mutation of IL15Rα Among the residues, the first mutated residue of IL15 is E at position 90 to C, and the second mutated residue of IL15Rα is P at position 67 to C. The amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO:26, and the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27. In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the above, the IL15 and IL15Rα specifically bind; In some embodiments, wherein,
A)所述IL15第61位的D突变为N，第64位的E突变为Q,和/或第65位的N突变位D；和/或A) the D mutation at position 61 of the IL15 is N, the E mutation at position 64 is Q, and/or the N mutation at position 65 is D; and/or
B)所述IL15至少一个N糖基化位点不存在；在一些实施方式中，所述N糖基化位点选自N71、N79和/或N112；在一些实施方式中，所述IL15包含以下氨基酸突变：N71Q、N79Q和/或N112Q；和/或所述IL15Rα至少一个O糖基化位点不存在；在一些实施方式中，所述O糖基化位点选自T2、T81和/或T86；在一些实施方式中，所述IL15Rα包含以下氨基酸突 变：T2A、T81A和/或T86A；B) at least one N-glycosylation site of the IL15 is absent; in some embodiments, the N-glycosylation site is selected from N71, N79 and/or N112; in some embodiments, the IL15 comprises The following amino acid mutations: N71Q, N79Q and/or N112Q; and/or at least one O-glycosylation site of IL15Rα does not exist; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or or T86; in some embodiments, the IL15Rα comprises the following amino acid mutations: T2A, T81A and/or T86A;
所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点，所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。The amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO:26, and the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述IL15为SEQ ID NO.84所示的氨基酸序列或其突变序列；在一些实施方式中，所述突变序列包括选自D61N、E64Q和/或N65D氨基酸突变，和/或选自N71Q、N79Q和/或N112Q氨基酸突变；所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点。In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the above, the IL15 is the amino acid sequence shown in SEQ ID NO.84 or its mutant sequence; in some embodiments, the mutant sequence includes Amino acid mutation selected from D61N, E64Q and/or N65D, and/or amino acid mutation selected from N71Q, N79Q and/or N112Q; the amino acid residue mutation site of IL15 refers to the natural sequence numbering position corresponding to SEQ ID NO:26 point.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述IL15Rα包含SEQ ID NO.28或其突变序列、SEQ ID NO.77或其突变序列、SEQ ID NO.78或其突变序列、SEQ ID NO.79或其突变序列、SEQ ID NO.80或其突变序列、或SEQ ID NO.81或其突变序列；在一些实施方式中，所述突变序列包括选自T2A、T81A和/或T86A的氨基酸突变；在一些实施方式中，所述突变序列包括P67C氨基酸突变；在一些实施方式中，所述突变序列包括P67C氨基酸突变，还包括选自T2A、T81A和/或T86A的氨基酸突变；所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the above, said IL15Rα comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or Its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; Amino acid mutations of T81A and/or T86A; in some embodiments, the mutant sequence includes a P67C amino acid mutation; in some embodiments, the mutant sequence includes a P67C amino acid mutation, and also includes an amino acid mutation; the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述IL15Rα为SEQ ID NO.77或其突变序列、SEQ ID NO.78或其突变序列、SEQ ID NO.79或其突变序列、SEQ ID NO.80或其突变序列、或SEQ ID NO.81或其突变序列；在一些实施方式中，所述突变序列包括P67C氨基酸突变；在一些实施方式中，所述突变序列包括P67C氨基酸突变，还包括选自T2A、T81A和/或T86A的氨基酸突变；在一些实施方式中，所述突变序列包括选自T2A、T81A和/或T86A的氨基酸突变；所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the above, the IL15Rα is SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or Its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; In some embodiments, the mutant sequence includes P67C amino acid mutation; In some embodiments, the mutant sequence Including P67C amino acid mutation, also includes amino acid mutation selected from T2A, T81A and/or T86A; in some embodiments, the mutant sequence includes amino acid mutation selected from T2A, T81A and/or T86A; the amino acid residue of IL15Rα The base mutation site refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，其还包含抗体Fc恒定区；在一些实施方式中，所述抗体Fc恒定区是异源二聚体；在一些实施方式中，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体；在一些实施方式中，所述IL15或IL15Rα的C端与Fc恒定区的N端连接。In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the foregoing further comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments In this way, the Fc constant region of the antibody is based on KiH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction and/or increased flexibility to associate into a heterodimer; in some embodiments, the IL15 Or the C-terminus of IL15Rα is connected to the N-terminus of the Fc constant region.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述多肽复合物为双特异性融合多肽，所述双特异性融合多肽包含第一抗原结合部分，其中，In some embodiments, the IL15/IL15Rα polypeptide complex according to any one of the above, the polypeptide complex is a bispecific fusion polypeptide, and the bispecific fusion polypeptide comprises a first antigen-binding moiety, wherein,
(A)所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至IL15；和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至IL15Rα；或(A) said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15Rα; or
(B)所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至IL15Rα；和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至IL15。(B) said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15Rα; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述多肽复合物还包含第二抗原结合部分，其中，所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域 VL2，其可操作地连接至抗体轻链恒定区CL。In some embodiments, the IL15/IL15Rα polypeptide complex according to any one of the above, the polypeptide complex further comprises a second antigen-binding moiety, wherein the second antigen-binding moiety comprises: a third polypeptide, wherein The third polypeptide comprises from the N-terminus to the C-terminus the second heavy chain variable domain VH2 of the second antibody, which is operably linked to the antibody heavy chain constant region CH1, and a fourth polypeptide, the fourth The polypeptide comprises from N-terminus to C-terminus the second light chain variable domain VL2 of the second antibody operably linked to the antibody light chain constant region CL.
在一些实施方式中，前面任一项所述的IL15/IL15Rα多肽复合物，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；In some embodiments, for the IL15/IL15Rα polypeptide complex described in any one of the foregoing, the first antigen-binding portion and the second antigen-binding portion bind to different antigens or bind to different epitopes of the same antigen;
在一些实施方式中，，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；In some embodiments, the first antigen-binding moiety targets immune cells and the second antigen-binding moiety targets tumor cells;
在一些实施方式中，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；In some embodiments, both the first antigen binding portion and the second antigen binding portion target tumor cells;
在一些实施方式中，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞；In some embodiments, both the first antigen binding moiety and the second antigen binding moiety target immune cells;
在一些实施方式中，所述第一抗原结合部分靶向人PD-L1，第二抗原结合部分靶向人TIGIT；或者所述第一抗原结合部分靶向人TIGIT，第二抗原结合部分靶向人PD-L1。In some embodiments, the first antigen-binding portion targets human PD-L1, and the second antigen-binding portion targets human TIGIT; or the first antigen-binding portion targets human TIGIT, and the second antigen-binding portion targets Human PD-L1.
本发明还涉及分离的核酸，其编码前面任一项所述的IL15/IL15Rα多肽复合物。本发明还涉及含有如上所述核酸的载体。The present invention also relates to an isolated nucleic acid encoding the IL15/IL15Rα polypeptide complex according to any one of the preceding items. The present invention also relates to a vector comprising a nucleic acid as described above.
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。The present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
本发明还涉及制备所述的IL15/IL15Rα多肽复合物的方法，包括步骤：用如上所述的载体转化宿主细胞；培养所转化的宿主细胞；和收集宿主细胞中表达的IL15/IL15Rα多肽复合物。The present invention also relates to a method for preparing the IL15/IL15Rα polypeptide complex, comprising the steps of: transforming a host cell with the above-mentioned vector; cultivating the transformed host cell; and collecting the IL15/IL15Rα polypeptide complex expressed in the host cell .
本发明还涉及药物组合物，其包含如上所述IL15/IL15Rα多肽复合物和药学上可接受的载体、赋形剂或稳定剂。The present invention also relates to a pharmaceutical composition, which comprises the above-mentioned IL15/IL15Rα polypeptide complex and a pharmaceutically acceptable carrier, excipient or stabilizer.
本发明还涉及如上所述IL15/IL15Rα多肽复合物或药物组合物在制备用于治疗疾病的药物中的应用。The present invention also relates to the application of the aforementioned IL15/IL15Rα polypeptide complex or pharmaceutical composition in the preparation of medicines for treating diseases.
本发明还涉及用作药物的前面任一项所述的IL15/IL15Rα多肽复合物或药物组合物，所述药物用于治疗疾病或病症。The present invention also relates to the IL15/IL15Rα polypeptide complex or pharmaceutical composition described in any one of the preceding items for use as a medicament for treating diseases or conditions.
本发明还涉及一种治疗疾病的方法，所述方法包含向有需要的对象施与治疗有效量的前面任一项所述的IL15/IL15Rα多肽复合物或药物组合物。The present invention also relates to a method for treating diseases, the method comprising administering a therapeutically effective amount of the IL15/IL15Rα polypeptide complex or pharmaceutical composition described above to a subject in need.
附图说明Description of drawings
图1为4种经典的双抗平台：图1A为KiH异源二聚Fc改造技术；图1B为CrossMab双特异性抗体技术；图1C为武汉友芝友YBody双抗技术(非对称型scFv双抗)；图1D为对称型scFv双抗；Figure 1 shows four classic double antibody platforms: Figure 1A shows the KiH heterodimerization Fc engineering technology; Figure 1B shows the CrossMab bispecific antibody technology; Figure 1C shows Wuhan Youzhiyou YBody double antibody technology (asymmetric scFv double antibody technology) anti); Figure 1D is a symmetric scFv double antibody;
图2为本发明提供的一种新型双特性抗体FiBody，由具有特异性亲和力的配受体替换一侧Fab的CH1、CL；Fig. 2 is a novel bispecific antibody FiBody provided by the present invention, in which CH1 and CL of Fab on one side are replaced by ligand receptors with specific affinity;
图3为示例性的展示FiBody的4中可行方案：图3-1为改造的配受体，配受体之间具有非天然存在的链间键；图3-2为两侧Fab的CH1、CL均被受体、配体取代，两侧选自不同的配受体；图3-3为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH3段也被配受体替换；图3-4为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH2也被配受体替换；其他的可行性改造方式还有很多；Figure 3 is an exemplary display of the 4 feasible schemes of FiBody: Figure 3-1 is a modified ligand receptor with non-naturally occurring interchain bonds between ligand receptors; Figure 3-2 is CH1, Fab on both sides. CL is replaced by receptors and ligands, and both sides are selected from different ligand receptors; Figure 3-3 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and the CH3 segment in the Fc dimer is also replaced by ligand receptors. Receptor replacement; Figure 3-4 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and CH2 in the Fc dimer is also replaced by ligand receptors; there are many other feasible transformation methods;
图4为示例性的当本发明的双特异性抗体用于治疗肿瘤时，双特异性抗体的抗原结合部分的靶向结合包括示例性的3种类型：图4-A第一抗原结合部分靶向T细胞，第二抗原结合部分靶向肿瘤细胞；图4-B第一抗原结合部分和第二抗原结合部分均靶向肿瘤细胞；图4-C第一 抗原结合部分与第二抗原结合部分均靶向T细胞；图4-D示例性的体现本发明双特性抗体可选的为三功能融合蛋白，除发挥不同的抗原结合，还能激活配受体通路，激发配受体生物学活性；Figure 4 is an example of when the bispecific antibody of the present invention is used to treat tumors, the targeted binding of the antigen-binding part of the bispecific antibody includes exemplary three types: Figure 4-A The first antigen-binding part target To T cells, the second antigen-binding part targets tumor cells; Figure 4-B The first antigen-binding part and the second antigen-binding part both target tumor cells; Figure 4-C The first antigen-binding part and the second antigen-binding part Both target T cells; Figure 4-D exemplarily reflects that the bispecific antibody of the present invention can optionally be a trifunctional fusion protein, which can not only play different antigen binding, but also activate the ligand receptor pathway and stimulate the biological activity of the ligand receptor ;
图5为白细胞介素及其受体的立体构像图，可以分为四类：A类为托举型，B类为蝴蝶结型，C类为棒球手型，D类为钳型；Figure 5 is a three-dimensional conformation diagram of interleukin and its receptors, which can be divided into four categories: Type A is lifting type, Type B is bow-tie type, Type C is baseball player type, and Type D is pincer type;
图6为四类立体构像的白介素及其受体的举例，A类托举型为IL2/IL2R，B类蝴蝶结型为IL22/IL22R,C类蝴蝶结型为IL18/IL18R,D类钳型为IL21/IL21R；Figure 6 is an example of four types of interleukins and their receptors in three-dimensional configurations. Class A holds IL2/IL2R, B bow-tie IL22/IL22R, C bow-tie IL18/IL18R, and D clamps IL21/IL21R;
图7为本发明实施例中基于IL15(配体)与IL15RA(受体)FiBody设计，第二抗原结合区VH与IL15RA相连，第二抗原结合区VL与IL15相连；Figure 7 is the FiBody design based on IL15 (ligand) and IL15RA (receptor) in the embodiment of the present invention, the second antigen-binding region VH is connected to IL15RA, and the second antigen-binding region VL is connected to IL15;
图8为二硫键改造优化结构示意图；Figure 8 is a schematic diagram of the optimized structure of disulfide bond reformation;
图9为IL15/IL15RA与IL2/15Rβ/γC复合物相互作用立体结构示意图；Figure 9 is a schematic diagram of the three-dimensional structure of the interaction between IL15/IL15RA and IL2/15Rβ/γC complex;
图10为本发明实施例中错配分子R1042/R1124结构示意图；Figure 10 is a schematic diagram of the structure of the mismatch molecule R1042/R1124 in the embodiment of the present invention;
图11为本发明实施例中样品R0951的HPLC-SEC检测结果；Fig. 11 is the HPLC-SEC detection result of sample R0951 in the embodiment of the present invention;
图12为本发明实施例中样品R1042的HPLC-SEC检测结果；Fig. 12 is the HPLC-SEC detection result of sample R1042 in the embodiment of the present invention;
图13为本发明实施例中样品R0809的HPLC-SEC检测结果；Fig. 13 is the HPLC-SEC detection result of sample R0809 in the embodiment of the present invention;
图14为本发明实施例中样品R1110的HPLC-SEC检测结果；Fig. 14 is the HPLC-SEC detection result of sample R1110 in the embodiment of the present invention;
图15为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；Figure 15 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody TIGIT end and CHO-Tigit cells (R0950, R0951, R0952, R0954, R0955, R0960);
图16为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1123/R1119/R1120/R1124)；Figure 16 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R1123/R1119/R1120/R1124);
图17为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1042/R1043)；Figure 17 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R1042/R1043);
图18为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R0810)；Figure 18 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R0810);
图19为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；Figure 19 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R0950, R0951, R0952, R0954, R0955, R0960);
图20为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1115-R1120、R1123-R1124)；Figure 20 is the detection of the binding activity of double antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1115-R1120, R1123-R1124) by FCM method in the embodiment of the present invention;
图21为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R0950、R1042、R1043)；Figure 21 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R0950, R1042, R1043);
图22为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1081-R1086)；Figure 22 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end and CHO-PD-L1 cells (R1072, R1081-R1086);
图23为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1109-R1111)；Figure 23 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R1072, R1109-R1111);
图24为本发明实施例中样品受配体复合物(IL15/IL15R)的结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；Figure 24 is the binding activity (R0950, R0951, R0952, R0954, R0955, R0960) of the sample receptor complex (IL15/IL15R) in the example of the present invention;
图25为本发明实施例中样品受配体复合物(IL15/IL15R)的结合活性(R1042、R1043)；Figure 25 is the binding activity (R1042, R1043) of the sample receptor ligand complex (IL15/IL15R) in the embodiment of the present invention;
图26为本发明实施例中二硫键改造优化样品凝胶电泳检测结果(R1072、R1081、R1082、R0954、R1084-R1086)Figure 26 is the results of gel electrophoresis detection of disulfide bond transformation optimization samples in the embodiment of the present invention (R1072, R1081, R1082, R0954, R1084-R1086)
图27为IL15(配体)与IL15RA(受体)分子间二硫键突变配对立体结构示意图；Figure 27 is a schematic diagram of the three-dimensional structure of the disulfide bond mutation pairing between IL15 (ligand) and IL15RA (receptor) molecules;
图28为示例性的IL15/IL15Rα复合物结构示意图；Figure 28 is a schematic diagram of the structure of an exemplary IL15/IL15Rα complex;
图29为复合物1-4凝胶电泳检测结果；Figure 29 is the gel electrophoresis detection result of complex 1-4;
图30为IL15/IL15Rα复合物对靶向区结合力(@TIGIT)的检测结果；Figure 30 is the detection result of IL15/IL15Rα complex binding to the targeting region (@TIGIT);
图31为IL15/IL15Rα复合物对靶向区结合力(@PD-L1)的检测结果。Figure 31 shows the detection results of the IL15/IL15Rα complex binding to the targeting region (@PD-L1).
具体实施方式Detailed ways
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。在本发明中引用的所有文献，包括公开出版物、专利和专利申请，都通过引用的方式全文并入本文。Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment. All documents, including publications, patents, and patent applications, cited in this application are hereby incorporated by reference in their entirety.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
术语“抗原结合部分”或“抗原结合结构域”意指对抗原决定簇赋予其结合特异性的抗原结合分子的部分。在一些实施方案中，所述“抗原结合部分”为抗体功能片段。The term "antigen-binding portion" or "antigen-binding domain" means the portion of an antigen-binding molecule that confers its binding specificity for an antigenic determinant. In some embodiments, the "antigen-binding portion" is a functional fragment of an antibody.
术语“野生型或WT”意指在自然界发现的氨基酸序列或核苷酸序列，包含等位变异。WT蛋白质具有未经过有意修饰的氨基酸序列或核苷酸序列。The term "wild type or WT" means the amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
术语“抗体”涵盖任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体、双特异性(双价)抗体或双特异性融合多肽。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一个可变区(“HCVR”或VH)以及第一、第二和第三恒定区(分别为CH1、CH2、CH3)组成，每条轻链由一个可变区(“LCVR”或VL)以及一个恒定区(CL)组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。The term "antibody" encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody or bispecific fusion polypeptide that binds a particular antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR” or VH) and first, second, and third constant regions (CH1, CH2, CH3, respectively), and each light chain consists of a variable region (“LCVR” " or VL) and a constant region (CL). Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains can be classified as λ or κ.
抗体呈“Y”型，主干由两条重链的第二(CH2)、第三(CH3)以及任选地第四(CH4)恒定区组成，其通过二硫键结合。“Y”型结构的每条臂包含其中一条重链的可变区(VH)和第一恒定区(CH1)，其与一条轻链的可变区(VL)和恒定区(CL)结合。轻链和重链的可变区负责抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)，(轻(L)链的CDR包含LCDR1、LCDR2、LCDR3，重(H)链的CDR包含HCDR1、HCDR2、HCDR3。其中，三个CDR由被称为框架区(FR)的部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。HCVR和LCVR各包含4个FR，并且CDR和FR自氨基端至羧基端依以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。Antibodies have a "Y" shape, with a backbone consisting of the second (CH2), third (CH3), and optionally fourth (CH4) constant regions of the two heavy chains, joined by disulfide bonds. Each arm of the "Y"-shaped structure contains the variable region (VH) and the first constant region (CH1) of one of the heavy chains, which are combined with the variable region (VL) and constant region (CL) of one of the light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR), (the CDR of the light (L) chain includes LCDR1, LCDR2, LCDR3, and the CDR of the heavy (H) chain includes HCDR1, HCDR2, HCDR3. Among them, the three CDRs are separated by parts called framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops. HCVR and LCVR each contain 4 FRs, and the CDRs and FRs Arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
重链和轻链的恒定区不参与抗原结合，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异形体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。The constant regions of the heavy and light chains are not involved in antigen binding, but have various effector functions. Antibodies can be classified into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms based on the presence or absence of alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively. Several major antibody classes can be further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain) and so on.
高变区通常包含来自轻链可变区中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)以及重链可变区中的31-35B(HCDR1)、50-65(HCDR2)和95-102(HCDR3)的氨基酸残基(Kabat等人，《免疫学相关蛋白质的序列(SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST)》，第5版.马里兰州贝塞斯达美国国家卫生研究院公共卫生服务部(Public Health Service，National Institutes of Health，Bethesda，Md.)(1991))，或那些形成高变环的残基，例如轻链可变区中的残基26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)以及重链可变区中的26-32(HCDR1)、53-55(HCDR2)和96-101(HCDR3)(Chothia和Lesk(1987)《分子生物学杂志(J.Mol.Biol.)》196：901-917)。The hypervariable region typically comprises amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the variable region of the light chain and 31-35B (HCDR1) in the variable region of the heavy chain , 50-65 (HCDR2) and 95-102 (HCDR3) amino acid residues (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed. Bethesda, MD National Institutes of Health Public Health Service (Public Health Service, National Institutes of Health, Bethesda, Md.) (1991)), or those residues that form hypervariable loops, such as residue 26 in the light chain variable region -32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917).
在一些实施方式中，所述抗体为双特异抗体(BiAb)。术语“双特异性”在本文中是指两种不同抗原，或者当这两者是相同抗原时，它们每一个都具有针对不同表位的结合特异性。所述表位可以源自不同抗原或相同抗原。术语“双特异性融合多肽”和“双特异性抗体”在本文中是指所有制得的具有全长抗体或带抗原结合位点的片段的产物。所述抗体可以是人抗体，非人抗体(如小鼠来源抗体)，人源化抗体，或嵌合抗体(如人-小鼠嵌合抗体或不同亚型抗体的嵌合)。在一些情况下，抗体的变体是在本发明所提供的抗体序列上发生保守修饰或保守置换或取代所得到的。“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。所属领域技术人员将能够使用熟知的技术确定如本文所阐明的抗原结合分子的合适变体。对于核苷酸和氨基酸序列，术语“同一性”表明当具有适当的插入或缺失的情况下最佳比对和比较时两个核酸或两个氨基酸序列之间的同一性程度。In some embodiments, the antibody is a bispecific antibody (BiAb). The term "bispecific" herein refers to two different antigens, or when the two are the same antigen, each of them has binding specificities for different epitopes. The epitopes may be derived from different antigens or from the same antigen. The terms "bispecific fusion polypeptide" and "bispecific antibody" herein refer to all products made with full-length antibodies or fragments with antigen-binding sites. The antibody can be a human antibody, a non-human antibody (such as a mouse-derived antibody), a humanized antibody, or a chimeric antibody (such as a human-mouse chimeric antibody or a chimeric antibody of different subtypes). In some cases, antibody variants are obtained by conservative modifications or conservative substitutions or substitutions on the antibody sequences provided by the present invention. "Conservative modification" or "conservative substitution or substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Those of skill in the art will be able to determine suitable variants of the antigen binding molecules as set forth herein using well known techniques. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
术语“Fab”为免疫球蛋白中不含或含一小部分残余Fc片段的Fab片段，例如，Fab片断包括重链和轻链的可变区、以及所有或部分的第一恒定域。The term "Fab" is a Fab fragment of an immunoglobulin that contains no or a small portion of the residual Fc fragment, eg, a Fab fragment includes the variable domains of the heavy and light chains, and all or part of the first constant domain.
术语“Fc”或“Fc区”或“Fc结构域”意指包含抗体的恒定区，在一些情况下排除第一恒定区免疫球蛋白结构域(例如CH1)的全部或一部分，并且在一些情况下进一步排除铰链的全部或一部分的多肽。因此，Fc可指IgA、IgD和IgG的最后两个恒定区免疫球蛋白结构域(例如CH2和CH3)，IgE和IgM的最后三个恒定区免疫球蛋白结构域(例如CH2、CH3和CH4)，以及任选地这些结构域的柔性铰链N端的全部或一部分。对于IgA和IgM，Fc可以包含J链。对于IgG来说，Fc结构域包含免疫球蛋白结构域CH2和CH3(Cγ2和Cγ3)以及位于CH1(Cγ1)与CH2(Cγ2)之间的较低铰链区。尽管Fc区的边界可以变化，但是人类IgG重链Fc区通常被定义为包括其羧基端的残基E216、C226或A231，其中编号根据如Kabat中的EU索引。在一些实施方案中，如下文更全面地描述，对Fc区进行氨基酸修饰，例如所述Fc为异源二聚体。The term "Fc" or "Fc region" or "Fc domain" means comprising the constant region of an antibody, in some cases excluding all or a portion of the first constant region immunoglobulin domain (e.g. CH1), and in some cases Polypeptides that are all or part of the hinge are further excluded. Thus, Fc can refer to the last two constant region immunoglobulin domains (eg CH2 and CH3) of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM (eg CH2, CH3 and CH4) , and optionally all or a portion of the flexible hinge N-terminus of these domains. For IgA and IgM, Fc may contain a J chain. For IgG, the Fc domain comprises the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and a lower hinge region located between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined to include residues E216, C226 or A231 at its carboxy-terminus, with numbering according to the EU index as in Kabat. In some embodiments, as described more fully below, amino acid modifications are made to the Fc region, eg, the Fc is a heterodimer.
本文的“修饰”是指多肽序列中的氨基酸取代、插入和/或缺失或与蛋白质化学连接的部分的改变。本文的“氨基酸修饰”是指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起 见，除非另外指出，否则氨基酸修饰是由DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20个氨基酸。"Modification" herein refers to amino acid substitutions, insertions and/or deletions in the polypeptide sequence or changes in the part chemically linked to the protein. "Amino acid modification" herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are amino acids encoded by DNA, e.g., the 20 amino acids with codons in DNA and RNA.
“表位”在本文中意指与特定抗原结合结构域，例如抗体分子的可变区(称为互补位)相互作用的决定子。表位是例如氨基酸或糖侧链的分子的分组，并且通常具有特定的结构特征以及特定的电荷特征。单个分子可具有超过一个表位。表位可以包含直接参与结合的氨基酸残基(也称为表位的免疫显性组分)和不直接参与结合的其它氨基酸残基，例如被特异性抗原结合肽有效阻断的氨基酸残基；换句话说，氨基酸残基在特异性抗原结合肽的覆盖面积内。表位可以是构形的也可以是线性的。构形表位是由来自线性多肽链的不同区段的氨基酸空间并置而产生。线性表位是由多肽链中的相邻氨基酸残基产生的表位。构形和非构形表位的区别可以在于在变性溶剂存在下，与前者而非后者的结合丧失。表位通常包括独特空间构象中的至少3个，并且更通常至少5个或8-10个氨基酸。识别相同表位的抗原结合分子可以在简单的免疫分析中验证，显示一种抗原结合分子阻断另一种抗原结合分子与靶抗原结合的能力。如下所概述，本发明不仅包括本文中所列举的抗原结合分子和抗原结合结构域，还包括与所列举的抗原结合分子或抗原结合结构域结合的表位竞争结合的抗原结合分子和抗原结合结构域。"Epitope" means herein a determinant of interaction with a particular antigen-binding domain, eg, the variable region of an antibody molecule, called a paratope. An epitope is a grouping of molecules such as amino acids or sugar side chains, and usually has specific structural characteristics as well as specific charge characteristics. A single molecule may have more than one epitope. An epitope may comprise amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; In other words, the amino acid residues are within the coverage area of the specific antigen binding peptide. Epitopes can be either conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. A linear epitope is one that arises from contiguous amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished by the loss of binding to the former but not to the latter in the presence of denaturing solvents. An epitope usually includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Antigen-binding molecules that recognize the same epitope can be validated in a simple immunoassay showing the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to the target antigen. As outlined below, the present invention includes not only the antigen-binding molecules and antigen-binding domains recited herein, but also antigen-binding molecules and antigen-binding structures that compete for binding with epitopes that bind to the recited antigen-binding molecules or antigen-binding domains. area.
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指有指向的、能被相应物质竞争阻断的某种配基在体外或体内与特异结构位点相互作用的生物结合过程。如抗原和抗体或受体和配体之间的结合。The terms "specifically binding", "selectively binding", "selectively binding" and "specifically binding" refer to a certain ligand that is directed and can be competitively blocked by the corresponding substance in vitro or in vivo. Biological binding processes of structural site interactions. Such as the binding between antigen and antibody or receptor and ligand.
特异性结合的强度或亲和力可以根据相互作用的解离常数(KD)表示，其中较小的KD表示较大的亲和力，较大的KD表示较低的亲和力。例如KD为至少约10 -4M、至少约10 -5M、至少约10 -6M、至少约10 -7M、至少约10 -8M、至少约10 -9M、替代地至少约10 -10M、至少约10 -11M、至少约10 -12M、或更大的抗原结合力来展现。结合特性可以通过所属领域众所周知的方法，例如生物层干涉测量法和基于表面等离振子共振的方法来确定。一种这样的方法需要测量抗原结合位点/抗原或受体/配体复合物缔合和解离的速率，其中速率取决于复合物搭配物的浓度、相互作用的亲和力以及在两个方向上同等地影响速率的几何参数。因此，可以确定缔合速率(ka)和解离速率(kd)，并且kd/ka的比例等于解离常数KD(《自然(Nature)》361：186-187(1993)和Davies等人(1990)《生物化学年鉴(Annual RevBiochem)》59：439-473)。 The strength or affinity of specific binding can be expressed in terms of the dissociation constant (KD) of the interaction, where a smaller KD indicates greater affinity and a larger KD indicates lower affinity. For example, KD is at least about 10 −4 M, at least about 10 −5 M, at least about 10 −6 M, at least about 10 −7 M, at least about 10 −8 M, at least about 10 −9 M, alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or greater antigen binding. Binding properties can be determined by methods well known in the art, such as biolayer interferometry and surface plasmon resonance based methods. One such method entails measuring the rates of association and dissociation of antigen-binding site/antigen or receptor/ligand complexes, where the rates depend on the concentration of the complex partner, the affinity of the interaction, and the equivalent in both directions. Geometric parameters that affect the velocity. Therefore, the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio of kd/ka is equal to the dissociation constant KD ("Nature (Nature)" 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473).
术语“免疫细胞”包括参与保护机体抵抗传染性疾病和外来物质二者的免疫系统的细胞。免疫细胞可以包括例如嗜中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，淋巴细胞，如B细胞和T细胞，和单核细胞。T细胞可以包括例如，CD4+、CD8+、T辅助细胞、细胞毒性T细胞、γδT细胞、调节性T细胞、抑制性T细胞和天然杀伤细胞。The term "immune cell" includes cells of the immune system involved in protecting the body against both infectious disease and foreign substances. Immune cells can include, for example, neutrophils, eosinophils, basophils, lymphocytes, such as B cells and T cells, and monocytes. T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, γδ T cells, regulatory T cells, suppressor T cells, and natural killer cells.
术语“多功能融合多肽”意指设计来靶向两个或更多个抗原的非天然存在的结合分子。本文所述的“多功能融合多肽”通常是遗传工程化的融合蛋白，例如其经设计以将两个不同的所需的生物学功能带入单个结合分子。例如，多功能融合多肽可以是多功能结合分子。The term "multifunctional fusion polypeptide" means a non-naturally occurring binding molecule designed to target two or more antigens. A "multifunctional fusion polypeptide" as described herein is typically a genetically engineered fusion protein, eg, designed to bring two different desired biological functions into a single binding molecule. For example, a multifunctional fusion polypeptide can be a multifunctional binding molecule.
术语“FiBody”,是利用配体及其受体特异性亲和力取代双特异性抗体部分或全部恒定区，从而得到的双特异性融合多肽或多功能融合蛋白。本发明中提到的“YBody”技术由武汉友芝友公司于2012年开发，该技术是在“Knob-into-Holes”技术的基础上，形成异源二聚体的 其中一条为正常重链，另外一条为Fc功能区的N端链接scFv，形成了不对称的双特异性抗体。The term "FiBody" refers to a bispecific fusion polypeptide or a multifunctional fusion protein obtained by substituting part or all of the constant region of a bispecific antibody with specific affinity for a ligand and its receptor. The "YBody" technology mentioned in this invention was developed by Wuhan Youzhiyou Company in 2012. This technology is based on the "Knob-into-Holes" technology, and one of the heterodimers formed is a normal heavy chain , and the other one is the N-terminal link scFv of the Fc functional region to form an asymmetric bispecific antibody.
“IL15/IL15Rα多肽复合物”是指包含IL15和IL15Rα的多肽复合物，所述IL15与IL15Rα特异性结合形成多肽复合物。"IL15/IL15Rα polypeptide complex" refers to a polypeptide complex comprising IL15 and IL15Rα, and the IL15 specifically binds to IL15Rα to form a polypeptide complex.
术语“约”或“大约”是指与参照定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度相差30、25、20、25、10、9、8、7、6、5、4、3、2或1％的定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度。在特定实施方式中，当术语“约”或“大约”位于数值之前时，表示所述值加上或减去15％、10％、5％或1％的范围。The term "about" or "approximately" refers to a difference of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% quantification, level, value, quantity, frequency, percentage, dimension, size, volume, weight or length. In particular embodiments, when the term "about" or "approximately" precedes a numerical value, it means a range of plus or minus 15%, 10%, 5% or 1% of the stated value.
除非上下文另有规定，词语“包含”、“包括”和“含有”将被理解为表示包括所述的步骤或要素或一组步骤或要素，但不排除任何其他步骤或要素或一组步骤或要素。“由……组成”所表示的是包括并且限于短语“由……组成”所接的内容。因此，短语“由……组成”表示所列出的要素是需要的或必需的，并且没有其他要素可存在。“基本由……组成”所表示的是包括列于此短语之后的任意要素，并且限于有助于或不妨碍所列的要素的如在本发明中详述的活性或作用的其他要素。因此，短语“基本由……组成”表示所列出的要素是需要的或必需的，但其他要素是可选地并可取决于其是否影响所列出的要素的活性或作用而存在或不存在。Unless the context dictates otherwise, the words "comprising", "including" and "containing" will be understood to mean the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and being limited to what the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the listed elements are required or required and that no other elements may be present. "Consisting essentially of" means including any element listed after the phrase, and being limited to other elements that contribute to or do not interfere with the activity or effect of the listed element as specified in the present invention. Thus, the phrase "consisting essentially of" means that the listed elements are required or essential, but that other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements. exist.
在本发明全文中提及的“一个实施方式”、“实施方式”、“特定实施方式”、“相关实施方式”、“某种实施方式”、“另外的实施方式”或“进一步的实施方式”或其组合表示所描述的与所述实施方式相关的特定特征、结构或特性包含于本发明的至少一个实施方式中。因此，在本说明书全文各处出现前述用语未必都指同一实施方式。此外，所述特定特征、结构或特性可在一个或多个实施方式中以任意适宜方式组合。References throughout this disclosure to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "another embodiment," or "further embodiments" ” or a combination thereof means that a particular feature, structure or characteristic described in relation to said embodiment is included in at least one embodiment of the present invention. Therefore, appearances of the foregoing terms in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。The term "optionally" is used for descriptive purposes only and is not to be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may be expressly or implicitly included or excluded.
在说明书和权利要求中的术语“第一”、“第二”用于区分相似元素，而不一定用于描述顺序或时间次序。应当理解，如此使用的术语在合适环境下是可互换的，并且本文描述的本发明的实施方案能够以与本文描述或举例说明不同的其他顺序操作。The terms "first" and "second" in the description and claims are used to distinguish similar elements, not necessarily to describe a sequence or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
双特异性融合多肽bispecific fusion polypeptide
本发明提供了新的双特异性融合多肽，其包含配体(或其片段)及其受体(或其片段)，所述配体(或其片段)及其受体(或其片段)分别独立地替换抗体一侧Fab的CH1和CL，具体地，所述双特异性融合多肽包含第一抗原结合部分，所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段；和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段；其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。The present invention provides a novel bispecific fusion polypeptide, which comprises a ligand (or a fragment thereof) and a receptor (or a fragment thereof), and the ligand (or a fragment thereof) and a receptor (or a fragment thereof) are respectively independently replacing CH1 and CL of Fab on one side of the antibody, specifically, the bispecific fusion polypeptide comprises a first antigen-binding portion, and the first antigen-binding portion comprises: a first polypeptide, the first polypeptide is from N-terminus to C-terminus comprising the first heavy chain variable domain VH1 of the first antibody, which is operably linked to the first conjugated fragment; and a second polypeptide from N-terminus to C-terminus The end comprises the first light chain variable domain VL1 of the first antibody, which is operably linked to a second conjugated fragment; wherein the first conjugated fragment is a receptor and the second conjugated fragment is a ligand; or the first conjugated fragment is a ligand and the second conjugated fragment is a receptor.
在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL15]-[连接子2]-[CH2]-[CH3],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL15RA]；在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N 端到C端依次为：[VH1]-[连接子1]-[IL15RA]-[连接子2]-[CH2]-[CH3],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL15]。其中，所述CH2和CH3为重链恒定区亚基，所述连接子1、连接子2和连接子3为连接多肽的连接子，其可以相同，也可以不相同；在一些实施方案中，所述连接子1、连接子2和连接子3独立选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。In some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [CH2]-[CH3], the second polypeptide, from N-terminus to C-terminus: [VL1]-[Linker 3]-[[IL15RA]; In some embodiments, the bispecific fusion The polypeptide has: the first polypeptide, which is in order from N-terminal to C-terminal: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[CH2]-[CH3], the second polypeptide , which from N-terminus to C-terminus are: [VL1]-[Linker 3]-[[IL15]. Wherein, the CH2 and CH3 are heavy chain constant region subunits, and the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments, The linker 1, linker 2 and linker 3 are independently selected from (GxS)y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6.
在一些实施方式中，所述双特异性融合多肽还包含第二抗原结合部分，所述第二抗原结合部分与第一抗原结合部分不同；所述第二抗原结合部分可选自：In some embodiments, the bispecific fusion polypeptide further comprises a second antigen-binding moiety that is different from the first antigen-binding moiety; the second antigen-binding moiety may be selected from:
1.抗体另一侧Fab的CH1和CL被另一种配体(或其片段)及其受体(或其片段)替换，即所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；其中，所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；所述第三缀合片段/第四缀合物片段与所述第一缀合物片段/第二缀合物片段选自不同的受体/配体，所述第三缀合片段和所述第四缀合物片段能够特异性结合；或者1. CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or its fragment) and its receptor (or its fragment), that is, the second antigen binding part includes: the third polypeptide, the The third polypeptide comprises from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the third conjugated fragment, and a fourth polypeptide which The second light chain variable domain VL2 comprising the second antibody from the N-terminus to the C-terminus is operably linked to a fourth conjugated fragment; wherein the third conjugated fragment is a receptor, and the second conjugated fragment is The four conjugated fragments are ligands; or the third conjugated fragment is a ligand, and the fourth conjugated fragment is a receptor; the third conjugated fragment/fourth conjugated fragment is the same as the first conjugated fragment a conjugate fragment/second conjugate fragment are selected from different receptors/ligands, said third conjugate fragment and said fourth conjugate fragment are capable of specific binding; or
2.抗体另一侧Fab保留原来的CH1和CL，即，所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。2. The Fab on the other side of the antibody retains the original CH1 and CL, that is, the second antigen-binding part includes: a third polypeptide, and the third polypeptide includes the second heavy chain of the second antibody from the N-terminus to the C-terminus Chain variable domain VH2, which is operably linked to antibody heavy chain constant region CH1, and a fourth polypeptide comprising from N-terminus to C-terminus the second light chain variable region of the second antibody. Domain VL2, which is operably linked to the antibody light chain constant region CL.
本发明利用配体及其受体本身特有的特异性结合力，将其创造性地与抗原结合区(抗体可变区VH/VL)可操作性地连接，所述连接包括与其中之一抗原结合区连接，另一抗原结合区仍与CH1和CL连接；或者两种抗原结合区都与配体/受体连接，但两种抗原结合区连接不同种类的配体/受体，从而避免不同抗原结合区的轻重链发生错配。The present invention utilizes the specific binding force unique to the ligand and its receptor itself, and creatively connects it to the antigen-binding region (antibody variable region VH/VL), and the connection includes binding to one of the antigens region, another antigen-binding region is still connected to CH1 and CL; or both antigen-binding regions are connected to ligands/receptors, but the two antigen-binding regions are connected to different kinds of ligands/receptors, thereby avoiding different antigens The light and heavy chains in the binding region are mismatched.
在一些实施方式中，本发明提供的双特异性融合多肽是一种多功能融合多肽，其包含2种Fab,其中一个Fab的CH1和CL独立地被配体及其受体所取代，另一个Fab的CH1和CL未被取代，所述受体既包含识别并结合配体的活性部位，也包含产生应答反应的功能活性部位；所述第一抗原结合部分的轻链不会与所述第二抗原结合部分的重链错配。在一些实施方式中，其中一个Fab的CH1和CL独立地被第一配体及其受体所取代，另一侧Fab的CH1和CL独立地被第二配体及其受体取代，所述第一配体及其受体与所述第二配体及其受体不同。In some embodiments, the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide comprising two Fabs, wherein CH1 and CL of one Fab are independently replaced by a ligand and its receptor, and the other The CH1 and CL of the Fab are not substituted, and the receptor includes not only the active site for recognizing and binding the ligand, but also the functional active site for generating a response; the light chain of the first antigen-binding part will not interact with the second Heavy chain mismatch of two antigen-binding portions. In some embodiments, CH1 and CL of one Fab are independently replaced by a first ligand and its receptor, and CH1 and CL of the other Fab are independently replaced by a second ligand and its receptor, said The first ligand and its receptor are different from the second ligand and its receptor.
所述多功能融合蛋白不仅能发挥双靶点特异性，且能发挥配体/受体传导的生物学活性。例如，在某个特定的实施方式中，所述配体及其受体为IL15和IL15Rα，所述多功能融合多肽除具有双靶点靶向作用外，IL15Rα还能将IL-15递呈给IL-2/15Rβγ二聚体形成三元复合物，激活JAK和STAT型号通路，促进靶细胞增殖与活化、IFN-γ、TNF-α分泌水平提升；JAK/STAT，Ras/MAPK—增强增殖信号；Bcl-2、Bcl-XL(抗凋亡蛋白)的上调、Bim、Puma(促凋亡蛋白)的下调--减弱凋亡信号。The multifunctional fusion protein can not only exert dual-target specificity, but also exert ligand/receptor conduction biological activity. For example, in a specific embodiment, the ligand and its receptors are IL15 and IL15Rα, and the multifunctional fusion polypeptide not only has a dual-target targeting effect, IL15Rα can also present IL-15 to IL-2/15Rβγ dimer forms a ternary complex, activates JAK and STAT pathways, promotes target cell proliferation and activation, increases IFN-γ, TNF-α secretion levels; JAK/STAT, Ras/MAPK—enhances proliferation signals ; Up-regulation of Bcl-2, Bcl-XL (anti-apoptotic proteins), down-regulation of Bim, Puma (pro-apoptotic proteins) -- weakening of apoptotic signals.
在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL15]-[连接子2]-[Fc1],第二多肽，其从N端到C端依次为：[VL1]-[连接子 3]-[[IL15RA],第三多肽，其从N端到C端依次为：[VH2]--[Fc2],和第四多肽，其从N端到C端依次为：[VL2]-[CL]；在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL15RA]-[连接子2]-[Fc1],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL15],第三多肽，其从N端到C端依次为：[VH2]--[Fc2],和第四多肽，其从N端到C端依次为：[VL2]-[CL]。其中，所述Fc1和Fc2为重链恒定区Fc的2个亚基，可以相同，也可以不相同，优选的所述Fc恒定区是异源二聚体(异二聚体Fc融合蛋白)；在一些实施方案中，所述Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体。所述连接子1、连接子2和连接子3为连接多肽的连接子，其可以相同，也可以不相同；在一些实施方案中，所述连接子1、连接子2和连接子3独立选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。In some embodiments, the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [Fc1], the second polypeptide, its sequence from N-terminal to C-terminal is: [VL1]-[Linker 3]-[[IL15RA], the third polypeptide, its sequence from N-terminal to C-terminal is: [ VH2]--[Fc2], and the fourth polypeptide, which are: [VL2]-[CL] from N-terminus to C-terminus; in some embodiments, the bispecific fusion polypeptide has: the first polypeptide Peptide, its order from N-terminal to C-terminal is: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[Fc1], the second polypeptide, its order from N-terminal to C-terminal is : [VL1]-[Linker 3]-[[IL15], the third polypeptide, from the N-terminal to the C-terminal: [VH2]--[Fc2], and the fourth polypeptide, from the N-terminal To the C terminal: [VL2]-[CL]. Wherein, the Fc1 and Fc2 are two subunits of the heavy chain constant region Fc, which may be the same or different, and preferably the Fc constant region is a heterodimer (heterodimer Fc fusion protein); In some embodiments, the Fc constant regions are associated into heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility. The linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments, the linker 1, linker 2 and linker 3 are independently selected From (GxS)y linker, wherein, x is selected from the integer of 1-5, and y is selected from the integer of 0-6.
在一些实施方式中，所VH1和VL1配合形成特异性结合TIGIT的抗原结合位点，所VH2和VL2配合形成特异性结合PD-L1的抗原结合位点。在一些实施方式中，所VH1和VL1配合形成特异性结合PD-L1的抗原结合位点，所VH2和VL2配合形成特异性结合TIGIT的抗原结合位点。在一些实施方式中，所述结合TIGIT的抗原结合部分包括重链可变区和轻链可变区，其中重链可变区包括SEQ ID NO.73或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括SEQ ID NO.74或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。在一些实施方式中，其中所述结合TIGIT的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.73中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.74中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.36或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.37或与其具有至少80％序列同一性的序列；在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.71或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.72或与其具有至少80％序列同一性的序列；在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.75或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.76或与其具有至少80％序列同一性的序列；所述具有至少80％序列同一性的序列，可以是例如具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列。在一些实施方式中，所述结合PD-L1的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，轻链可变区包括LCDR1、LCDR2和LCDR3区，其中：1)所述HCDR1、 HCDR2和HCDR3分别包括SEQ ID NO.36中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.37中的LCDR1、LCDR2和LCDR3；2)所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.71中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.72中的LCDR1、LCDR2和LCDR3；或者3)所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.75中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.76中的LCDR1、LCDR2和LCDR3。在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。In some embodiments, the VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT, and the VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1. In some embodiments, the VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1, and the VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT. In some embodiments, the antigen-binding portion that binds to TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO. 73 or has at least 80% (e.g., at least 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity, the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, wherein the heavy chain variable region of the antigen-binding portion in conjunction with TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.73, respectively , in some embodiments, wherein said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising LCDR1, LCDR2 and LCDR3 in SEQ ID NO.74; In some embodiments , the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds to PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.36 or has at least 80% sequence therewith A sequence of identity, the light chain variable region comprising SEQ ID NO.37 or a sequence having at least 80% sequence identity thereto; In some embodiments, the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO.72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.75 or A sequence having at least 80% sequence identity with it, the light chain variable region includes SEQ ID NO.76 or a sequence having at least 80% sequence identity therewith; said sequence having at least 80% sequence identity can be, for example, having At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region of the antigen-binding portion that binds to PD-L1 includes HCDR1, HCDR2, and HCDR3 regions, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions, wherein: 1) the HCDR1 , HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36, and said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37; 2) said HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71, and said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72; or 3) said HCDR1, HCDR2 and HCDR3 comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75 respectively, and said LCDR1, LCDR2 and LCDR3 comprise LCDR1, LCDR2 and LCDR3 in SEQ ID NO.76 respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system system definition.
在一些实施方式中，所述IL15和IL15Rα之间具有一个非天然的链间键，所述非天然链间键形成于IL15的第一突变残基和IL15Rα的第二突变残基之间，在一些技术方案中，所述IL15第一突变残基为第90位的E突变为C，所述IL15Rα第二突变残基为第67位的P突变位C；在一些技术方案中，所述IL15第一突变残基为第93位的E突变为C，所述IL15Rα第二突变残基为第35位的R突变位C；所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点，所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。在一些实施方式中，所述IL15为SEQ ID NO.84所示的氨基酸序列或其突变序列，所述突变序列例如包括选自D61N、E64Q和/或N65D氨基酸突变，和/或选自N71Q、N79Q和/或N112Q氨基酸突变。所述IL15Rα包含SEQ ID NO.28或其突变序列、SEQ ID NO.77或其突变序列、SEQ ID NO.78或其突变序列、SEQ ID NO.79或其突变序列、SEQ ID NO.80或其突变序列、或SEQ ID NO.81或其突变序列；在一些实施方式中，所述突变序列包括选自T2A、T81A和/或T86A的氨基酸突变；在一些实施方式中，所述突变序列包括P67C氨基酸突变；在一些实施方式中，所述突变序列包括P67C氨基酸突变，还包括选自T2A、T81A和/或T86A的氨基酸突变。在一些实施方式中，所述IL15Rα为SEQ ID NO.77或其突变序列、SEQ ID NO.78或其突变序列、SEQ ID NO.79或其突变序列、SEQ ID NO.80或其突变序列、或SEQ ID NO.81或其突变序列；在一些实施方式中，所述突变为P67C氨基酸突变；在一些实施方式中，所述突变为P67C以及选自T2A、T81A和/或T86A的氨基酸突变；在一些实施方式中，所述突变为选自T2A、T81A和/或T86A的氨基酸突变。前述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。在一些实施方式中，所述IL15选自与SEQ ID NO.84序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，所述IL15RA选自与SEQ ID NO.77-81任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。In some embodiments, there is a non-natural interchain bond between IL15 and IL15Rα, and the non-natural interchain bond is formed between the first mutated residue of IL15 and the second mutated residue of IL15Rα, at In some technical solutions, the first mutation residue of IL15 is the E mutation at the 90th position to C, and the second mutation residue of IL15Rα is the P mutation position C at the 67th position; in some technical solutions, the IL15 The first mutation residue is the E mutation at position 93 to C, and the second mutation residue of IL15Rα is the R mutation position C at position 35; the amino acid residue mutation site of IL15 refers to SEQ ID NO: 26 The corresponding natural sequence numbering site, the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27. In some embodiments, the IL15 is the amino acid sequence shown in SEQ ID NO.84 or its mutation sequence, the mutation sequence includes, for example, an amino acid mutation selected from D61N, E64Q and/or N65D, and/or selected from N71Q, N79Q and/or N112Q amino acid mutations. The IL15Rα comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or Its mutant sequence, or SEQ ID NO.81 or its mutant sequence; In some embodiments, the mutant sequence includes an amino acid mutation selected from T2A, T81A and/or T86A; In some embodiments, the mutant sequence includes P67C amino acid mutation; in some embodiments, the mutant sequence includes a P67C amino acid mutation, and also includes an amino acid mutation selected from T2A, T81A and/or T86A. In some embodiments, the IL15Rα is SEQ ID NO.77 or a mutant sequence thereof, SEQ ID NO.78 or a mutant sequence thereof, SEQ ID NO.79 or a mutant sequence thereof, SEQ ID NO.80 or a mutant sequence thereof, Or SEQ ID NO.81 or its mutant sequence; In some embodiments, the mutation is a P67C amino acid mutation; In some embodiments, the mutation is P67C and an amino acid mutation selected from T2A, T81A and/or T86A; In some embodiments, the mutation is an amino acid mutation selected from T2A, T81A and/or T86A. The aforementioned amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27. In some embodiments, the IL15 is selected from the group that has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the sequence of SEQ ID NO.84) %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, said IL15RA is selected from the sequence with SEQ ID NO .77-81 any one of the indicated sequences has at least 80% (e.g. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
ⅰ.配体和受体i. Ligands and Receptors
“受体(receptor)”是细胞膜上或细胞内能识别生物活性分子并与之结合的物质，能与受体结合的生物活性物质统称为“配体(ligand)”。"Receptor" is a substance on the cell membrane or in the cell that can recognize and bind to bioactive molecules, and the bioactive substances that can bind to receptors are collectively referred to as "ligands".
根据受体在细胞中的位置，将其分为细胞表面受体和细胞内受体两大类。受体本身至少 含有两个活性部位：一个是识别并结合配体的活性部位；另一个是负责产生应答反应的功能活性部位，这一部位只有在与配体结合形成二元复合物并变构后才能产生应答反应，由此启动一系列的生化反应，最终导致靶细胞产生生物效应。According to the location of the receptors in the cell, they are divided into two categories: cell surface receptors and intracellular receptors. The receptor itself contains at least two active sites: one is the active site that recognizes and binds to the ligand; the other is the functional active site that is responsible for generating a response. Only after that can a response reaction be generated, thereby initiating a series of biochemical reactions, which eventually lead to biological effects in target cells.
受体一般为糖蛋白，野生型受体与配体之间的结合不通过共价键介导，主要靠离子键、氢键、范德华力和疏水作用而相互结合。受体在与配体结合时，具有饱和性、高亲和性、专一性等特性。Receptors are generally glycoproteins, and the binding between wild-type receptors and ligands is not mediated by covalent bonds, but mainly by ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. When the receptor binds to the ligand, it has the characteristics of saturation, high affinity and specificity.
互相配合的受体和配体具有相对特异结合的亲和力，以及任选的生物学效应。在一些实施方式中，所述受体仅包含识别并结合配体的活性部位，不包含产生应答反应的功能活性部位(例如激活下游信号通路的生物学效应的功能)。在一些实施方式中，所述受体和/或配体为天然的受配体结构，所述受体既包含识别结合配体的活性部位，又包含负责产生应答反应的功能活性部位，能够发挥相应的生物学功能，所述双特异性融合蛋白是一种多功能融合蛋白，不仅具有双特异性，而且能发挥配受体功能。Cooperating receptors and ligands have relatively specific binding affinities, and optionally biological effects. In some embodiments, the receptor only includes an active site that recognizes and binds a ligand, and does not include a functional active site that produces a response (such as the function of activating a biological effect of a downstream signaling pathway). In some embodiments, the receptor and/or ligand is a natural ligand structure, and the receptor not only includes an active site that recognizes and binds a ligand, but also includes a functional active site that is responsible for generating a response, and can exert According to the corresponding biological function, the bispecific fusion protein is a multifunctional fusion protein, which not only has bispecificity, but also can exert the function of ligand receptor.
在一些实施方式中，所述受体和/或配体在天然序列的基础上做了修饰，所述修饰包括但不限于:截短、插入和/或突变；这些修饰的目的包括但不限于：增加或降低配体和受体的结合力；增强、降低或消除配体受体的生物学功能；增加、减少或消除受体和或配体蛋白中的糖基化位点；降低或消除受配体毒性。In some embodiments, the receptor and/or ligand has been modified on the basis of the natural sequence, and the modification includes but is not limited to: truncation, insertion and/or mutation; the purpose of these modifications includes but is not limited to : increase or decrease the binding force between ligand and receptor; enhance, decrease or eliminate the biological function of ligand receptor; increase, decrease or eliminate the glycosylation site in receptor and or ligand protein; reduce or eliminate Ligand toxicity.
在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由10～1000个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由20～800个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由30～600个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由40～400个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由50～300个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由55～260个氨基酸组成。例如，受体和/或配体的氨基酸序列也可以独立地选自20、30、40、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900个氨基酸。In some embodiments, the amino acid sequences of the receptors and/or ligands are each independently composed of 10 to 1000 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands are each independently It consists of 20-800 amino acids; in some embodiments, the amino acid sequence of the receptor and/or ligand is independently composed of 30-600 amino acids; in some embodiments, the receptor and/or The amino acid sequences of the ligands each independently consist of 40-400 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands each independently consist of 50-300 amino acids; in some embodiments , the amino acid sequences of the receptors and/or ligands each independently consist of 55-260 amino acids. For example, the amino acid sequence of the receptor and/or ligand can also be independently selected from 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 amino acids.
在一些实施方式中，所述受体和/或配体分子量各自独立地选自1KD～100KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自2KD～80KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自3KD～70KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～60KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～50KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～40KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自5KD～30KD。例如，受体和/或配体的分子量可以独立地选自1KD、2KD、3KD、4KD、4.5KD、5KD、6KD、7KD、8KD、9KD、10KD、11KD、15KD、18KD、20KD、25KD、30KD、35KD、40KD、45KD、50KD、60KD、70KD、80KD、90KD、100KD。In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 1KD to 100KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 2KD to 80KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 3KD to 70KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 60KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 50KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 40KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 5KD to 30KD. For example, the molecular weight of the receptor and/or ligand can be independently selected from 1KD, 2KD, 3KD, 4KD, 4.5KD, 5KD, 6KD, 7KD, 8KD, 9KD, 10KD, 11KD, 15KD, 18KD, 20KD, 25KD, 30KD , 35KD, 40KD, 45KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD.
受体(或其片段)和其相应的配体(或其片段)的结合方式可以是共价结合、非共价相互作用或其组合；非共价键的例子包括，但不限于，氢键、疏水键、离子键、和范德华键。在一些实施方式中，当被插入或替换的缀合片段之间的亲和力低于预期时(例如不能拉近抗原结合部分中的两个可变区以使其获得特异性识别抗原的功能，或者不能防止2个或多个重链 恒定区之间的重链错配，或者不能防止抗原结合部分之间错配以实现特定VL-VH部分的组合)，可以通过对抗体所述配体和/或受体进行改造以增加亲和力。在一些实施方式中，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；在一些实施方式中，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间；在一些实施方式中，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基；在一些实施方式中，所述非天然链间键为二硫键。The receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) can be bound by covalent binding, non-covalent interaction, or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonds , hydrophobic, ionic, and van der Waals bonds. In some embodiments, when the affinity between the inserted or replaced conjugated fragments is lower than expected (for example, the two variable regions in the antigen-binding moiety cannot be brought together to allow them to specifically recognize the antigen, or Inability to prevent heavy chain mismatches between two or more heavy chain constant regions, or inability to prevent mismatches between antigen-binding moieties to achieve specific VL-VH moiety combinations), can be achieved by targeting the ligand and/or or receptors engineered to increase affinity. In some embodiments, at least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments wherein the non-native interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; in some embodiments, the first and the second mutated residue at least one of the radicals is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
“非天然的链间键”是指在野生型多肽聚合物中未发现的链间键。例如，非天然链间键可以在一条多肽的突变的氨基酸残基和另一条多肽的突变氨基酸残基之间形成。"Non-natural interchain linkages" refers to interchain linkages not found in wild-type polypeptide polymers. For example, a non-natural interchain bond can be formed between a mutated amino acid residue of one polypeptide and a mutated amino acid residue of another polypeptide.
在一些实施方式中，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。In some embodiments, at least one native glycosylation site is absent in said receptor and/or ligand.
在一些实施方式中，所述受体和配体选自白细胞介素及其受体。In some embodiments, the receptor and ligand are selected from interleukins and their receptors.
发明人对大量的白细胞介素及其受体进行了立体构像研究，发现大量的白细胞介素或IFN类分子立体构像可以分为4类：A类-托举型、B类-蝴蝶结型、C-棒球手型、D类-钳型，如表1所示：The inventors studied the three-dimensional conformation of a large number of interleukins and their receptors, and found that the three-dimensional conformations of a large number of interleukins or IFN molecules can be divided into 4 categories: A-type lifting type, B type-bow-tie type , C-baseball hand type, D type-clamp type, as shown in Table 1:
表1.立体构像分类Table 1. Stereo conformation classification
Figure PCTCN2022121068-appb-000001
Figure PCTCN2022121068-appb-000001
在一些实施例中，所述配体及其受体选自A类白细胞介素及其受体，例如IL15/IL15R、IL2/IL2R、IL4/IL-4Rα+Rγ、IL-6/IL-6R、IL-11/IL-11R、IL-13/IL-13R1、IL-20/IL20Rα+IL20Rβ、IL24/IL20Rα+IL20Rβ。In some embodiments, the ligand and its receptor are selected from class A interleukins and their receptors, such as IL15/IL15R, IL2/IL2R, IL4/IL-4Rα+Rγ, IL-6/IL-6R , IL-11/IL-11R, IL-13/IL-13R1, IL-20/IL20Rα+IL20Rβ, IL24/IL20Rα+IL20Rβ.
在一些实施例中，所述配体及其受体选自D类白细胞介素及其受体，例如IL7/IL7R、IL21/IL21R、IL23A/IL12B。In some embodiments, the ligand and its receptor are selected from class D interleukins and their receptors, such as IL7/IL7R, IL21/IL21R, IL23A/IL12B.
在一些实施方式中，所述白细胞介素及其受体具有如下表2氨基酸序列：In some embodiments, the interleukin and its receptor have the following amino acid sequences in Table 2:
表2.白细胞介素及其受体的氨基酸序列Table 2. Amino acid sequences of interleukins and their receptors
Figure PCTCN2022121068-appb-000002
Figure PCTCN2022121068-appb-000002
在一些实施方式中，所述受体-配体的组合选自IL15与IL15Rα，在本发明“IL15Rα”和“IL15RA”可以互换。在一些实施方式中，所述IL15与IL15Rα具有如下表氨基酸序列：In some embodiments, the receptor-ligand combination is selected from IL15 and IL15Rα, and “IL15Rα” and “IL15RA” are interchangeable in the present invention. In some embodiments, the IL15 and IL15Rα have the following amino acid sequences:
表3.IL15与IL15Rα的氨基酸序列Table 3. Amino acid sequences of IL15 and IL15Rα
Figure PCTCN2022121068-appb-000003
Figure PCTCN2022121068-appb-000003
Figure PCTCN2022121068-appb-000004
Figure PCTCN2022121068-appb-000004
备注：表中，例如“IL-15Rαsushi(人，sushi domain氨基酸序列)-77a.a.”意指人IL-15Rα的sushi结构域中N末端开始第1至第77位氨基酸残基的多肽片段，也标示为IL15Rαsushi-77a.a.，其它依此类推。Remarks: In the table, for example, "IL-15Rα sushi (human, sushi domain amino acid sequence)-77a.a." means the polypeptide fragment of amino acid residues 1 to 77 from the N-terminus in the sushi domain of human IL-15Rα , also marked as IL15Rαsushi-77a.a., and so on.
在一些实施方式中，所述IL15和IL15Rα包含至少一个非天然的链间键，在一些实施方式中，所述非天然链间键为二硫键，所述IL15和/或所述IL15Rα至少包含一个氨基酸突变为半胱氨酸，在一些实施方式中，所述突变位于IL-15和IL15Rα的接触界面上，在一些实施方式中，所述IL15的半胱氨酸突变选自E90C,所述IL15Rα的半胱氨酸突变选自P67C；在一些技术方案中，所述IL15半胱氨酸突变选自E93C，所述IL15Rα的半胱氨酸突变选自R35C。在一些实施方式中，所述IL15为SEQ ID NO.26的突变序列，所述突变包括E90C突变和/或E93C突变；在一些实施方式中，所述IL15Rα为SEQ ID NO.27、29、30或31的突变序列，所述突变包括P67C突变和/或R35C突变。In some embodiments, the IL15 and IL15Rα comprise at least one non-natural interchain bond, in some embodiments, the non-natural interchain bond is a disulfide bond, and the IL15 and/or the IL15Rα comprise at least one One amino acid is mutated to cysteine. In some embodiments, the mutation is located at the contact interface of IL-15 and IL15Rα. In some embodiments, the cysteine mutation of IL15 is selected from E90C, the The cysteine mutation of IL15Rα is selected from P67C; in some technical solutions, the cysteine mutation of IL15 is selected from E93C, and the cysteine mutation of IL15Rα is selected from R35C. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation includes E90C mutation and/or E93C mutation; in some embodiments, the IL15Rα is SEQ ID NO.27, 29, 30 Or the mutated sequence of 31, said mutation includes P67C mutation and/or R35C mutation.
本发明中，所述IL15氨基酸残基突变位置参照成熟形式人IL-15的氨基酸序列(SEQ ID NO.26)对应的自然顺序编号，所述IL15Rα氨基酸残基突变位置参照人IL-15Rα的sushi结构域(SEQ ID NO.27)对应的自然顺序编号。In the present invention, the IL15 amino acid residue mutation position refers to the natural sequence numbering corresponding to the amino acid sequence (SEQ ID NO.26) of the mature form of human IL-15, and the IL15Rα amino acid residue mutation position refers to the sushi of human IL-15Rα. The natural order number corresponding to the structural domain (SEQ ID NO.27).
在一些实施方式中，前面任一所述IL15第61位的D突变为N，第64位的E突变为Q,和/或第65位的N突变位D。在一些实施方案中，所述IL15为SEQ ID NO.26的突变序列，所述突变包括选自D61N、E64Q和/或N65D突变。在一些实施方式中，所述IL15为SEQ ID NO.26的突变序列，所述突变包括E90C突变，还包括D61N、E64Q和/或N65D突变。In some embodiments, the D mutation at position 61 of IL15 described above is N, the E mutation at position 64 is Q, and/or the N mutation at position 65 is D. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation comprises a mutation selected from D61N, E64Q and/or N65D. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation includes the E90C mutation, and also includes the D61N, E64Q and/or N65D mutation.
在一些实施方式中，前面任一所述IL15至少一个N糖基化位点不存在,在一些实施方式中，所述N糖基化位点选自N71、N79和/或N112；在一些实施方式中，所述IL15包含以下氨基酸突变：N71Q、N79Q和/或N112Q。在一些实施方案中，所述IL15为SEQ ID NO.26的突变序列，所述突变包括选自N71Q、N79Q和/或N112Q突变。在一些实施方式中，所述IL15 为SEQ ID NO.26的突变序列，所述突变包括E90C突变，还包括N71Q、N79Q和/或N112Q突变，和/或还包括N71Q、N79Q和/或N112Q突变。In some embodiments, at least one N-glycosylation site of IL15 described above does not exist, and in some embodiments, the N-glycosylation site is selected from N71, N79 and/or N112; in some embodiments In the manner, the IL15 comprises the following amino acid mutations: N71Q, N79Q and/or N112Q. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation comprises a mutation selected from N71Q, N79Q and/or N112Q. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, the mutation includes the E90C mutation, also includes the N71Q, N79Q and/or N112Q mutation, and/or also includes the N71Q, N79Q and/or N112Q mutation .
在一些实施方式中，前面任一所述IL15Rα至少一个O糖基化位点不存在；在一些实施方式中，所述O糖基化位点选自T2、T81和/或T86；在一些实施方式中，所述IL15Rα包含以下氨基酸突变：T2A、T81A和/或T86A。在一些实施方案中，所述IL15Rα为SEQ ID NO.27-31的突变序列，所述突变包括选自T2A、T81A和/或T86A的突变；在一些实施方案中，还包括P67C突变。In some embodiments, at least one O-glycosylation site of IL15Rα described above does not exist; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or T86; in some embodiments In the manner, the IL15Rα comprises the following amino acid mutations: T2A, T81A and/or T86A. In some embodiments, the IL15Rα is a mutant sequence of SEQ ID NO.27-31, and the mutation includes a mutation selected from T2A, T81A and/or T86A; in some embodiments, it also includes a P67C mutation.
在一些实施方式中，所述IL15与IL15Rα的氨基酸序列如下表4：In some embodiments, the amino acid sequences of IL15 and IL15Rα are as follows in Table 4:
表4.IL15与IL15Rα突变的氨基酸序列Table 4. Amino acid sequences of IL15 and IL15Rα mutations
Figure PCTCN2022121068-appb-000005
Figure PCTCN2022121068-appb-000005
备注：所述IL15突变位置参照成熟形式人IL-15的氨基酸序列(SEQ ID NO.26)对应的自然顺序编号，所述IL15Rα突变位置参照人IL-15Rα的sushi结构域(SEQ ID NO.27)对应的自然顺序编号。Remarks: The IL15 mutation position is numbered according to the natural sequence corresponding to the amino acid sequence of the mature form of human IL-15 (SEQ ID NO.26), and the IL15Rα mutation position is referenced to the sushi domain of human IL-15Rα (SEQ ID NO.27 ) corresponds to the natural order number.
互相配合的受体(或其片段)以及配体(或其片段)的插入或替换位置可以位于，例如：受体或其片段插入或替换CL区，配体或其片段插入或替换CH1区；或受体或其片段插入或替换CH1区，配体或其片段插入或替换CL区。The insertion or replacement position of the mutually coordinated receptor (or its fragment) and ligand (or its fragment) can be located, for example: the receptor or its fragment inserts or replaces the CL region, and the ligand or its fragment inserts or replaces the CH1 region; Or the receptor or its fragment inserts or replaces the CH1 region, and the ligand or its fragment inserts or replaces the CL region.
ⅱ.抗原结合部分ii. Antigen-binding portion
本发明提供的双特异性融合多肽，包含第一抗原结合部分和第二抗原结合部分，具有两 种抗原特异性，第一抗原结合部分与第二抗原结合部分是不同的，可以是第一抗原结合部分与第二抗原结合部分结合不同的抗原，也可以是第一抗原结合部分与第二抗原结合部分结合相同抗原的不同表位。The bispecific fusion polypeptide provided by the present invention comprises a first antigen-binding portion and a second antigen-binding portion, and has two antigen specificities, the first antigen-binding portion and the second antigen-binding portion are different, and may be the first antigen The binding moiety and the second antigen-binding moiety may bind different antigens, or the first antigen-binding moiety and the second antigen-binding moiety may bind different epitopes of the same antigen.
在一些实施方式中，所述双特异性融合蛋白针对的靶标是肿瘤。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在肿瘤细胞表达；在一些实施方式中，第一抗原结合部分结合的靶点在肿瘤细胞，第二抗原结合部分结合的靶点在免疫细胞；在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在免疫细胞。In some embodiments, the target of the bispecific fusion protein is a tumor. In some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety bind to targets expressed on tumor cells; in some embodiments, the first antigen-binding moiety binds to tumor cells, and the second antigen-binding moiety binds to tumor cells The portion binds to an immune cell; in some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety bind to an immune cell.
T细胞重定向杀伤是许多治疗领域中理想的作用机制。在临床前和临床试验中，各种双特异性抗体形式参与T细胞重定向(May C等人(2012)Biochem Pharmacol，84(9))：1105年至1112年，第；弗兰克尔SR，和Baeuerle PA，(2013)CURR OPIN化学生物学，第17卷(3)：385-92，页)。所有T细胞重新靶向的双特异性抗体或其片段已被工程化以具有至少两个抗原结合位点，其中一个位点与靶细胞上的表面抗原结合另一个位点与T细胞表面抗原结合。在T细胞表面抗原中，源自TCR蛋白质复合物的人CD3的ε亚基最常被靶向作为重定向T细胞杀伤的靶标。T cell redirected killing is a desirable mechanism of action in many therapeutic areas. Various bispecific antibody formats are involved in T cell redirection in preclinical and clinical trials (May C et al. (2012) Biochem Pharmacol, 84(9)): 1105-1112, pp.; Frankel SR, and Baeuerle PA, (2013) CURR OPIN Chemical Biology, Vol. 17(3): 385-92, pp.). All T cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen binding sites, one of which binds to a surface antigen on the target cell and the other binds to a T cell surface antigen . Among T cell surface antigens, the ε subunit of human CD3 derived from the TCR protein complex is most commonly targeted as a target for redirected T cell killing.
可被靶向的肿瘤相关联抗原包括但不限于：α-胎蛋白(AFP)、α-辅肌动蛋白-4、A3、对A33抗体有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、碳酸酐酶IX、CASP-8/m、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA-4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p(CSAp)、CEA(CEACAM5)、CEACAM6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、成纤维细胞生长因子(FGF)、Flt-1、Flt-3、叶酸盐受体、G250抗原、Claudin18.2、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜BCMA促性腺激素(HCG)和其亚基、HER2/neu、HMGB-1、缺氧诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、PAM4抗原、胰腺癌粘蛋白、PD-1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素、存活素-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、Thomson-Friedenreich抗原、肿瘤坏死抗原、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bcl-2、bcl-6、Kras、致癌基因标记物以及致癌基因产物(参见，例如Sensi等人，Clin Cancer  Res2006，12：5023-32；Parmiani等人，JImmunol2007，178：1975-79；Novellino等人Cancer Immunol Immunother2005，54：187-207)。Tumor-associated antigens that can be targeted include, but are not limited to: α-fetoprotein (AFP), α-actinin-4, A3, antigen specific to A33 antibody, ART-4, B7, Ba 733 , BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a- e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM , fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, Claudin18.2, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorion BCMA gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN- α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, Insulin-like Growth Factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP- 1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM -3, NCA66, NCA95, NCA90, PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, Survivin, Survivin-2B, TAC, TAG-72, Tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedenreich Antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, oncogene markers, and oncogene products (see, for example, Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007, 178: 1975-79; Novellino et al. Cancer Immunol Immunother 2005, 54: 187 -207).
虽然对于效应T细胞具有特异性的抗体或其它结合分子优选地结合至CD3抗原，但是在效应T细胞上表达的其它抗原是已知的并且可由T-细胞重定向复合物靶向。示例性T-细胞抗原包括但不限于，CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69和CD90。While antibodies or other binding molecules specific for effector T cells preferably bind to the CD3 antigen, other antigens expressed on effector T cells are known and can be targeted by T-cell redirecting complexes. Exemplary T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69, and CD90.
免疫检查点是免疫系统中的抑制途径，其对维持自身耐受性和调节外周组织中生理性免疫应答的持续时间和幅度以使附带组织损伤最小化至关重要。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点均为免疫检查点或其配体，所述免疫检查点分子包括但不限于：TIGIT、PD-1、TIM-3、LAG3、GTLA4、BTLA、BTN1A1、VISTA、LAIR、CD96、PVRIG、LILRA3、LILRA4、LILRB1、LILRB2、LILRB3、LLRB4、NKG-2A、CD47、CD200R1、CD300、Dectin-1、ICOS、NKp30、CD28、CD28H、CRTAM、DNAM-1、4-1-BB、BAFF、CD27、CD30、CD40、DR3、GITR、HVEM、LIGHT、OX40、TACI、2B4、CD2、CD48、CD229、SLAM、SLAMF5、GRAAC、TIM1、TIM4、CD7、DPPIV。Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage. In some embodiments, the targets of the first antigen-binding moiety and the second antigen-binding moiety are both immune checkpoints or their ligands, and the immune checkpoint molecules include but are not limited to: TIGIT, PD-1, TIM- 3. LAG3, GTLA4, BTLA, BTN1A1, VISTA, LAIR, CD96, PVRIG, LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, LLRB4, NKG-2A, CD47, CD200R1, CD300, Dectin-1, ICOS, NKp30, CD28, CD28H, CRTAM, DNAM-1, 4-1-BB, BAFF, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, OX40, TACI, 2B4, CD2, CD48, CD229, SLAM, SLAMF5, GRAAC, TIM1, TIM4, CD7, DPPIV.
在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为PD-L1；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIGIT；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为LAG3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIM-3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为CD47；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原结合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为TIGIT。In some embodiments, the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is PD-L1; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, the target of the second antigen-binding part is TIGIT; in some embodiments, the target of the first antigen-binding part is PD-1, and the target of the second antigen-binding part is GTLA4; in some In some embodiments, the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is LAG3; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, The target that the second antigen-binding portion binds to is TIM-3; in some embodiments, the target that the first antigen-binding portion binds to is PD-1, and the target that the second antigen-binding portion binds to is CD47; in some embodiments Among them, the first antigen-binding moiety binds to PD-1, and the second antigen-binding moiety binds to GTLA4; in some embodiments, the first antigen-binding moiety binds to PD-1, and the second antigen-binding moiety binds to PD-1. The target to which the antigen-binding moiety binds is 4-1-BB; in some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is 4-1-BB; In some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is TIGIT.
在一些实施方式中，第一抗原结合部分靶向肿瘤相关抗原，第二抗原结合部分靶向免疫检查点。在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向PD-1；在一些实施方式中，第一抗原结合部分靶向VEGF,第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向Claudin18.2,第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向CTLA-4；在一些实施方式中，第一抗原结合部分靶向CD20,第二抗原结合部分靶向CD47；在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向CD47。In some embodiments, the first antigen binding moiety targets a tumor-associated antigen and the second antigen binding moiety targets an immune checkpoint. In some embodiments, the first antigen-binding portion targets HER2, and the second antigen-binding portion targets PD-1; in some embodiments, the first antigen-binding portion targets VEGF, and the second antigen-binding portion targets PD-1 L1; in some embodiments, the first antigen-binding moiety targets Claudin18.2 and the second antigen-binding moiety targets PD-L1; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets Targets CTLA-4; in some embodiments, the first antigen-binding moiety targets CD20 and the second antigen-binding moiety targets CD47; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety Targets CD47.
在一些实施方式中，第一抗原结合部分和第二抗原结合部分同时靶向肿瘤异质性。用于肿瘤的示例性共同靶标包括但不限于HGF和VEGF,IGF-1R和VEGF,Her2和VEGF,CD19和CD3,CD20和CD3,Her2和CD3,CD19和FcγRIIIa,CD20和FcγRIIIa,Her2和FcγRIIIa。本发明的双特异性融合多肽能够结合VEGF和磷脂酰丝氨酸；VEGF和ErbB3；VEGF和PLGF； VEGF和ROBO4；VEGF和BSG2；VEGF和CDCP1；VEGF和ANPEP；VEGF和c-MET；HER-2和ERB3；HER-2和BSG2；HER-2和CDCP1；HER-2和ANPEP；EGFR和CD64；EGFR和BSG2；EGFR和CDCP1；EGFR和ANPEP；IGF1R和PDGFR；IGF1R和VEGF；IGF1R和CD20；CD20和CD74；CD20和CD30；CD20和DR4；CD20和VEGFR2；CD20和CD52；CD20和CD4；HGF和c-MET；HGF和NRP1；HGF和磷脂酰丝氨酸；ErbB3和IGF1R；ErbB3和IGF1,2；c-Met和Her-2；c-Met和NRP1；c-Met和IGF1R；IGF1,2和PDGFR；IGF1,2和CD20；IGF1,2和IGF1R；IGF2和EGFR；IGF2和HER2；IGF2和CD20；IGF2和VEGF；IGF2和IGF1R；IGF1和IGF2；PDGFRa和VEGFR2；PDGFRa和PLGF；PDGFRa和VEGF；PDGFRa和c-Met；PDGFRa和EGFR；PDGFRb和VEGFR2；PDGFRb和c-Met；PDGFRb和EGFR；RON和c-Met；RON和MTSP1；RON和MSP；RON和CDCP1；VGFR1和PLGF；VGFR1和RON；VGFR1和EGFR；VEGFR2和PLGF；VEGFR2和NRP1；VEGFR2和RON；VEGFR2和DLL4；VEGFR2和EGFR；VEGFR2和ROBO4；VEGFR2和CD55；LPA和S1P；EPHB2和RON；CTLA4和VEGF；CD3和EPCAM；CD40和IL6；CD40和IGF；CD40和CD56；CD40和CD70；CD40和VEGFR1；CD40和DR5；CD40和DR4；CD40和APRIL；CD40和BCMA；CD40和RANKL；CD28和MAPG；CD80和CD40；CD80和CD30；CD80和CD33；CD80和CD74；CD80和CD2；CD80和CD3；CD80和CD19；CD80和CD4；CD80和CD52；CD80和VEGF；CD80和DR5；CD80和VEGFR2；CD22和CD20；CD22和CD80；CD22和CD40；CD22和CD23；CD22和CD33；CD22和CD74；CD22和CD19；CD22和DR5；CD22和DR4；CD22和VEGF；CD22和CD52；CD30和CD20；CD30和CD22；CD30和CD23；CD30和CD40；CD30和VEGF；CD30和CD74；CD30和CD19；CD30和DR5；CD30和DR4；CD30和VEGFR2；CD30和CD52；CD30和CD4；CD138和RANKL；CD33和FTL3；CD33和VEGF；CD33和VEGFR2；CD33和CD44；CD33和DR4；CD33和DR5；DR4和CD137；DR4和IGF1,2；DR4和IGF1R；DR4和DR5；DR5和CD40；DR5和CD137；DR5和CD20；DR5和EGFR；DR5和IGF1,2；DR5和IGFR,DR5和HER-2,以及EGFR和DLL4。其他靶标组合包括EGF/erb-2/erb-3家族的一个或多个成员。In some embodiments, the first antigen binding moiety and the second antigen binding moiety target tumor heterogeneity simultaneously. Exemplary co-targets for tumors include, but are not limited to, HGF and VEGF, IGF-IR and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and FcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. The bispecific fusion polypeptide of the present invention can bind VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON and c- Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD CD22 and CD20; CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, and EGFR and DLL4. Other target combinations include one or more members of the EGF/erb-2/erb-3 family.
此外，用于自身免疫病症和炎性病症的示例性共同靶标包括但不限于IL-1和TNFα,IL-6和TNFα,IL-6和IL-1,IgE和IL-13,IL-1和IL-13,IL-4和IL-13,IL-5和IL-13,IL-9和IL-13,CD19和FcγRIIb,以及CD79和FcγRIIb。Additionally, exemplary co-targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNFα, IL-6 and TNFα, IL-6 and IL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.
用于治疗炎性疾病的示例性靶点包括但不限于：TNF和IL-17A；TNF和RANKL；TNF和VEGF；TNF和SOST；TNF和DKK；TNF和αVβ3；TNF和NGF；TNF和IL-23p19；TNF和IL-6；TNF和SOST；TNF和IL-6R；TNF和CD-20；IgE和IL-13；IL-13和IL23p19；IgE和IL-4；IgE和IL-9；IgE和IL-9；IgE和IL-13；IL-13和IL-9；IL-13和IL-4；IL-13和IL-9；IL-13和IL-9；IL-13和IL-4；IL-13和IL-23p19；IL-13和IL-9；IL-6R和VEGF；IL-6R和IL-17A；IL-6R和RANKL；IL-17A和IL-1β；IL-1β和RANKL；IL-1β和VEGF；RANKL和CD-20；IL-1α和IL-1β；IL-1α和IL-1β。Exemplary targets for the treatment of inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-1β; IL-1β and RANKL; IL-1β and VEGF; RANKL and CD-20; IL-1α and IL-1β; IL-1α and IL-1β.
参与类风湿性关节炎(RA)的靶点包括但不限于：TNF和IL-18；TNF和IL-12；TNF和IL-23；TNF和IL-1β；TNF和MIF；TNF和IL-17；和TNF和IL-15。Targets involved in rheumatoid arthritis (RA) include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1β; TNF and MIF; TNF and IL-17 ; and TNF and IL-15.
治疗系统性红斑狼疮(SLE)的靶点包括但不限于：CD20,CD22,CD19,CD28,CD4,CD24,CD37,CD38,CD40,CD69,CD72,CD74,CD79A,CD79B,CD80,CD81,CD83,CD86,IL-4,IL-6,IL10,IL2,IL4,IL11,TNFRSF5,TNFRSF6,TNFRSF8,C5,TNFRSF7,TNFSF5,TNFSF6,TNFSF7,BLR1,HDAC4,HDAC5,HDAC7A,HDAC9,ICOSL,IGBP1,MS4A1,RGSI,SLA2,IFNB1,AICDA,BLNK,GALNAC4S-6ST,INHA,INHBA,KLF6,DPP4,FCER2,R2,ILIR2,ITGA2,ITGA3,MS4A1,ST6GALI,CDIC,CHSTIO,HLA-A,HLA-DRA,NT5E,CTLA4,B7.1,B7.2,BlyS,BAFF,IFN-α和TNF-α。Targets for the treatment of systemic lupus erythematosus (SLE) include but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6ST, INHA, INHBA, KLF6, DPP4, FCER2, R2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA, NT5E, CTLA4, B7.1, B7.2, BlyS, BAFF, IFN-α and TNF-α.
用于治疗多发性硬化症(MS)的靶点，包括但不限于：IL-12,TWEAK,IL-23,CXCL13,CD40,CD40L,IL-18,VEGF,VLA-4,TNF,CD45RB,CD200,IFNγ,GM-CSF,FGF,C5,CD52和CCR2。Targets for the treatment of multiple sclerosis (MS), including but not limited to: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200 , IFNγ, GM-CSF, FGF, C5, CD52 and CCR2.
用于治疗脓毒症的靶点包括但不限于：TNF,IL-1,MIF,IL-6,IL-8,IL-18,IL-12,IL-10,IL-23,FasL,LPS,Toll-样受体,TLR-4,组织因子，MIP-2,ADORA2A,IL-1B,CASP1,CASP4,NFκB1,PROC,TNFRSFIA,CSF3,CCR3,ILIRN,MIF,NFκB1,PTAFR,TLR2,TLR4,GPR44,HMOX1,中期因子,IRAK1,NFκB2,SERPINA1,SERPINE1,和TREM1。Targets for treating sepsis include but not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, FasL, LPS, Toll-like receptor, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NFκB1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44 , HMOX1, Midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1.
为了形成本发明的双特异性融合蛋白，可以制备针对这些抗原的任意组合的抗体；即，这些抗原中的每一个可以任选地和独立地被根据本发明的多特异性抗体包括或不包括。To form the bispecific fusion proteins of the invention, antibodies may be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by the multispecific antibody according to the invention .
在一些实施方式中，第一抗原结合部分和第二抗原结合部分靶向同一抗原的不同表位。In some embodiments, the first antigen binding portion and the second antigen binding portion target different epitopes of the same antigen.
在一些实施方式中，至少一个两个抗原结合片段还可以包括分泌信号序列。In some embodiments, at least one of the two antigen-binding fragments may also include a secretion signal sequence.
分泌信号序列是指，通过连接至编码序列位于细胞膜外侧或细胞外侧的N端而诱导所表达的蛋白或肽的分泌的序列，所述信号序列可以是由约18-30个氨基酸组成的肽序列。所有能转运到细胞膜外侧的蛋白有不同的信号序列，所述信号序列被细胞膜上的信号肽酶切割。通常，对于并非宿主细胞天然表达的外来蛋白而言，可以采用能将该蛋白分泌到细胞周质或培养基中的分泌信号序列，或采用修饰的序列。The secretory signal sequence refers to a sequence that induces the secretion of expressed protein or peptide by linking to the N-terminus of the coding sequence outside the cell membrane or outside the cell, and the signal sequence may be a peptide sequence consisting of about 18-30 amino acids . All proteins that can be transported to the outside of the cell membrane have different signal sequences that are cleaved by signal peptidases on the cell membrane. Typically, for a foreign protein that is not natively expressed by the host cell, a secretion signal sequence that enables secretion of the protein into the periplasm or culture medium, or a modified sequence, may be used.
在一些实施方式中，所VH1和VL1配合形成特异性结合TIGIT的抗原结合位点，所VH2和VL2配合形成特异性结合PD-L1的抗原结合位点。在一些实施方式中，所VH1和VL1配合形成特异性结合PD-L1的抗原结合位点，所VH2和VL2配合形成特异性结合TIGIT的抗原结合位点。在一些实施方式中，所述结合TIGIT的抗原结合部分包括重链可变区和轻链可变区，其中重链可变区包括SEQ ID NO.73或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括SEQ ID NO.74或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。在一些实施方式中，其中所述结合TIGIT的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.73中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.74中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系 统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.36或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.37或与其具有至少80％序列同一性的序列；在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.71或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.72或与其具有至少80％序列同一性的序列；在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括SEQ ID NO.75或与其具有至少80％序列同一性的序列，轻链可变区包括SEQ ID NO.76或与其具有至少80％序列同一性的序列；所述具有至少80％序列同一性的序列，可以是例如具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列。在一些实施方式中，所述结合PD-L1的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，轻链可变区包括LCDR1、LCDR2和LCDR3区，其中：1)所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.36中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.37中的LCDR1、LCDR2和LCDR3；2)所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.71中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.72中的LCDR1、LCDR2和LCDR3；或者3)所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.75中的HCDR1、HCDR2和HCDR3，所述所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.76中的LCDR1、LCDR2和LCDR3。在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。In some embodiments, the VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT, and the VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1. In some embodiments, the VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1, and the VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT. In some embodiments, the antigen-binding portion that binds to TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO. 73 or has at least 80% (e.g., at least 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity, the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, wherein the heavy chain variable region of the antigen-binding portion in conjunction with TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.73, respectively , in some embodiments, wherein said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising LCDR1, LCDR2 and LCDR3 in SEQ ID NO.74; In some embodiments , the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds to PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.36 or has at least 80% sequence therewith A sequence of identity, the light chain variable region comprising SEQ ID NO.37 or a sequence having at least 80% sequence identity thereto; In some embodiments, the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO.72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.75 or A sequence having at least 80% sequence identity with it, the light chain variable region includes SEQ ID NO.76 or a sequence having at least 80% sequence identity therewith; said sequence having at least 80% sequence identity can be, for example, having At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region of the antigen-binding portion that binds PD-L1 includes HCDR1, HCDR2, and HCDR3 regions, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions, wherein: 1) the HCDR1 , HCDR2 and HCDR3 respectively comprise HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36, and said LCDR1, LCDR2 and LCDR3 respectively comprise LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37; 2) said HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71, and said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72; or 3) said HCDR1, HCDR2 and HCDR3 comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75 respectively, and said LCDR1, LCDR2 and LCDR3 comprise LCDR1, LCDR2 and LCDR3 in SEQ ID NO.76 respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system system definition.
ⅲ.异二聚体Fc融合蛋白iii. Heterodimeric Fc fusion protein
在一些实施方式中，其包含重链恒定区Fc，所述Fc恒定区是异源二聚体(异二聚体Fc融合蛋白)。In some embodiments, it comprises a heavy chain constant region, Fc, which is a heterodimer (heterodimer Fc fusion protein).
所述Fc包括但不限于如下组合：The Fc includes but is not limited to the following combinations:
CH2；CH2;
CH2+CH3；CH2+CH3;
CH2+CH3+CH4；CH2+CH3+CH4;
所述Fc恒定区引入突变以避免重链错配。The Fc constant region was mutated to avoid heavy chain mismatches.
在一些实施方式中，所述Fc恒定区引入突变为基于KiH技术(Knob-into-Holes),即在其中Fc恒定区一条重链中引入氨基酸突变，引入的氨基酸体积大于最初氨基酸残基体积，形成一个突起的类似“杵”的型结构(Knob)，在Fc恒定区另一条链区引入另一突变，引入的氨基酸体积小于最初氨基酸残基体积，形成一个凹陷，类似“臼”的结构(Hole)，从而凸型重链更倾向和凹型重链配对，从而避免重链发生错配。该技术由基因泰克研发，记载于专利申请WO1996027011中，该专利全文引入本发明。In some embodiments, the introduction of mutations into the Fc constant region is based on KiH technology (Knob-into-Holes), that is, amino acid mutations are introduced into a heavy chain of the Fc constant region, and the volume of the introduced amino acids is greater than the volume of the original amino acid residues, A protruding "knob"-like structure (Knob) is formed, and another mutation is introduced in the other chain region of the Fc constant region. The volume of the introduced amino acid is smaller than the volume of the original amino acid residue, forming a depression, similar to the "hole" structure ( Hole), so that the convex heavy chain is more inclined to pair with the concave heavy chain, so as to avoid heavy chain mispairing. This technology was developed by Genentech and described in patent application WO1996027011, which is incorporated herein in its entirety.
在一些实施方式中，所述Fc恒定区引入突变为基于静电相互作用，例如ART-lg技术， 该技术由罗氏子公司Chugai开发，特异性的改变Fc恒定区结构域的电荷，促进异源重链的配对，相当于电荷版的KiH技术，该技术记载于专利申请WO2006106905中，该专利全文引入本发明。In some embodiments, the introduction of mutations in the Fc constant region is based on electrostatic interactions, such as ART-lg technology, which is developed by Roche subsidiary Chugai, which specifically changes the charge of the Fc constant region domain and promotes heterologous recombination. The pairing of chains is equivalent to the KiH technology of the charge version, which is described in the patent application WO2006106905, which is incorporated in the present invention in its entirety.
在一些实施方式中，所述Fc恒定区引入突变为基于SEED技术，SEED异二聚化是另一种基于空间突变的设计策略，该策略利用了从IgG和IgA CH3域(也称为AG SEED CH3和GA SEED CH3)衍生的交替序列的互补性。IgG和IgA CH3衍生物产生互补序列，因此在组装两个互补的重链异源二聚体的同时，排除了缺乏互补性的同源二聚体的组装。该技术记载于专利申请WO2007110205中，该专利全文引入本发明。In some embodiments, the introduction of mutations in the Fc constant region is based on SEED technology. SEED heterodimerization is another design strategy based on spatial mutation, which utilizes the CH3 domain of IgG and IgA (also known as AG SEED). Complementarity of alternate sequences derived from CH3 and GA SEED (CH3). IgG and IgA CH3 derivatives generate complementary sequences, thus excluding the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technology is described in patent application WO2007110205, which is incorporated in the present invention in its entirety.
在一些实施方式中，所述Fc恒定区引入突变为基于等电点改变，便于提高异源二聚体形成率以及保持Fc区域稳定性的改造，该技术记载于WO2014145806，该专利全文引入本专利。In some embodiments, the introduction of mutations into the Fc constant region is based on the change of the isoelectric point, which facilitates the modification of improving the formation rate of heterodimers and maintaining the stability of the Fc region. This technology is described in WO2014145806, which is incorporated in this patent in its entirety. .
在一些实施方式中，所述Fc恒定区基于亲水相互作用或增加的柔性而缔合成为异源二聚体。In some embodiments, the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.
在一些实施方式中，所述Fc恒定区基于以上技术的任意组合缔合成为异源二聚体，例如，在一些实施方式中，所述Fc恒定区基于KIH和静电相互作用的组合进行了突变。例如，XmAb双特异性平台方法可以通过结合静电相互作用，CH3域构象和氢键提高双特异性抗体的热稳定性。具体的，该策略将天然IgG1的Fc侧链突变交换为S364K和K370S异二聚体，以在两者之间形成氢键，然后进行L368D/K370S取代驱动盐桥相互作用以促进异二聚体的形成，专利申请WO2014145907，该专利全文引入本专利。In some embodiments, the Fc constant regions are associated into heterodimers based on any combination of the above techniques, for example, in some embodiments, the Fc constant regions are mutated based on a combination of KIH and electrostatic interactions . For example, the XmAb bispecific platform approach can improve the thermal stability of bispecific antibodies by combining electrostatic interactions, CH3 domain conformation, and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutations of native IgG1 for S364K and K370S heterodimers to form hydrogen bonds between the two, followed by L368D/K370S substitutions to drive salt bridge interactions to promote heterodimers The formation of the patent application WO2014145907, which is incorporated in this patent in its entirety.
在一些实施方式中，所述CH2、CH3或CH4区域的全长或部分被插入或替换成受体及其配体。In some embodiments, all or part of the CH2, CH3 or CH4 region is inserted or replaced with a receptor and its ligand.
在一些实施方式中，被插入或替换的区域独立地位于CH2、CH3或CH4区，或任意像个相邻的区之间的位置(如CH1-CH2交界处、CH2-CH3交界处、CH3-CH4交界处)；In some embodiments, the region to be inserted or replaced is independently located in the CH2, CH3 or CH4 region, or any position between adjacent regions (such as CH1-CH2 junction, CH2-CH3 junction, CH3- CH4 junction);
在一些实施方式中，当上述任意两个恒定区(例如CL-CH1、CH2-CH2、CH3-CH3或CH4-CH4区之任一项)被插入或替换时，替换区域两个互相配合的缀合片段之间的亲和力，KD<1×10 -3(M)，例如如x×10 -4(M)、x×10 -5(M)、x×10 -6(M)、x×10 -7(M)、x×10 -8(M)、x×10- 9(M)、x×10 -10(M)、x×10 -11(M)；x的值可选自1～9，例如1、2、3、4、5、6、7、8或9。 In some embodiments, when any two of the above-mentioned constant regions (for example, any one of CL-CH1, CH2-CH2, CH3-CH3 or CH4-CH4 regions) are inserted or replaced, the two matching suffixes of the replacement region Affinity between combined fragments, KD<1×10 -3 (M), such as x×10 -4 (M), x×10 -5 (M), x×10 -6 (M), x×10 -7 (M), x×10 -8 (M), x×10- 9 (M), x×10 -10 (M), x×10 -11 (M); the value of x can be selected from 1～ 9, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9.
在一些实施方式中，所述缀合片段的N端和/或C端通过连接肽与所述抗原结合片段连接。In some embodiments, the N-terminal and/or C-terminal of the conjugated fragment is linked to the antigen-binding fragment via a linker peptide.
术语“可操作连接”是指部件(例如两条多肽)直接地或经由一个或多个连接子(连接肽)通过共价键连接。The term "operably linked" means that parts (eg, two polypeptides) are linked by a covalent bond, either directly or via one or more linkers (connecting peptides).
在一些实施方式中，所述连接肽的氨基酸数目为1～30个；可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个；优选5～20个。In some embodiments, the number of amino acids of the connecting peptide is 1-30; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5-20.
在一些实施方式中，所述连接肽的氨基酸是不具有除连接以外的额外功能(例如蛋白定位、酶切位点等)的无意义多肽。In some embodiments, the amino acids of the linking peptide are nonsense polypeptides that do not have additional functions (such as protein localization, enzyme cleavage sites, etc.) other than linking.
在一些实施方式中，所述连接肽为柔性连接肽；In some embodiments, the connecting peptide is a flexible connecting peptide;
在一些实施方式中，所述连接肽的氨基酸序列选自Gly、Ser、Pro、Ala以及Glu中的一种或多种。In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
在一些实施方式中，所述连接肽的氨基酸序列选自(GGGGS)n、(GGGS)n、(GGS)n、(GS)n或(G)n，其中n选自1，2，3，4，5或6。In some embodiments, the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.
连接肽通常是柔性的，可以减少融合蛋白与目的蛋白之间的空间位阻，从而更有利于蛋白正确折叠。The connecting peptide is usually flexible, which can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
在另外的实施方案中，连接肽是刚性接头肽；即相对非柔性肽接头。刚性连接肽不要求完全缺乏柔性，而是比柔性接头肽如富甘氨酸肽接头柔性少。由于其相对缺乏柔性，刚性连接肽降低通过刚性连接肽连接在一起的两个蛋白结构域(在当前情况下是稳定剂蛋白和热稳定逆转录酶)的运动。提供有序链(例如α螺旋结构)的连接肽可提供刚性接头肽。例如，精氨酸、亮氨酸、谷氨酸、谷氨酰胺和甲硫氨酸都显示出相对高的螺旋接头结构倾向。然而，包含许多脯氨酸残基的非螺旋接头也可表现显著的刚性。刚性连接肽的实例包括聚赖氨酸和聚-DL-丙氨酸聚赖氨酸。刚性肽接头的进一步描述由Wriggers等，Biopolymers，80，第736-46页(2005)提供。此外，刚性接头肽在由George等，Protein Engineering，15，第871-79页(2003)描述的接头数据库中描述。优选地，刚性连接肽也是非可切割接头肽，即非可切割刚性连接肽。In additional embodiments, the connecting peptide is a rigid linker peptide; ie a relatively inflexible peptide linker. A rigid linker peptide does not require complete lack of flexibility, but is less flexible than a flexible linker peptide such as a glycine-rich peptide linker. Due to their relative lack of flexibility, the rigid linker peptide reduces the movement of two protein domains (in the present case the stabilizer protein and the thermostable reverse transcriptase) linked together by the rigid linker peptide. Linker peptides that provide ordered chains (eg, alpha helical structures) can provide rigid linker peptides. For example, arginine, leucine, glutamic acid, glutamine, and methionine all show a relatively high propensity for helical linker structures. However, non-helical linkers containing many proline residues can also exhibit significant rigidity. Examples of rigid linking peptides include polylysine and poly-DL-alanine polylysine. A further description of rigid peptide linkers is provided by Wriggers et al., Biopolymers, 80, pp. 736-46 (2005). Furthermore, rigid linker peptides are described in the linker database described by George et al., Protein Engineering, 15, pp. 871-79 (2003). Preferably, the rigid linker peptide is also a non-cleavable linker peptide, ie a non-cleavable rigid linker peptide.
分离的核酸isolated nucleic acid
本发明还涉及分离的核酸，其编码如上所述的双特异性融合多肽或多功能融合蛋白。The invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion protein as described above.
术语“分离的核酸”在本文中是指以单链或双链形式存在的脱氧核糖核酸或核糖核酸聚合物。所述分离的核酸包括RNA基因组序列，DNA(gDNA和cDNA)或从DNA转录的RNA序列，而且，除非特别指明，所述多肽还包括天然多核苷酸、糖、或碱基改变的类似物。根据本发明一个方面，所述多核苷酸是轻链多核苷酸。The term "isolated nucleic acid" refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in single- or double-stranded form. Isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, polypeptides include native polynucleotides, sugar, or base altered analogs. According to one aspect of the invention, said polynucleotide is a light chain polynucleotide.
所述分离的核酸包括编码蛋白复合物氨基酸序列的核苷酸序列，也包括与其互补的核苷酸序列。所述互补序列包括完全互补的序列和基本上互补的序列，这是指能在本领域已知的严谨条件下与编码蛋白复合物氨基酸序列的核苷酸序列杂交的序列。The isolated nucleic acid includes the nucleotide sequence encoding the amino acid sequence of the protein complex, as well as the nucleotide sequence complementary thereto. The complementary sequence includes a fully complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize to a nucleotide sequence encoding an amino acid sequence of a protein complex under stringent conditions known in the art.
而且，编码蛋白复合物氨基酸序列的核苷酸序列可以被改变或突变。所述改变包括添加、缺失、或非保守取代或保守取代。编码蛋白复合物氨基酸序列的多核苷酸可以被解释为，包括相对于该分离的核酸有实质性同一性的核苷酸序列。所述实质性同一性将该核苷酸序列与另外的随机序列以使得它们最大对应的方式进行比对，当用本领域常见的算法分析所比对的序列时，所述序列可显示大于80％的同源性，大于90％的同源性，或大于95％的同源性。Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions. A polynucleotide encoding an amino acid sequence of a protein complex may be construed to include nucleotide sequences having substantial identity relative to the isolated nucleic acid. The substantial identity is when the nucleotide sequence is aligned with another random sequence in such a way that they correspond maximally, which may show greater than 80% identity when the aligned sequences are analyzed using algorithms common in the art. % homology, greater than 90% homology, or greater than 95% homology.
载体carrier
本发明还涉及含有如上所述核酸的载体。The present invention also relates to a vector comprising a nucleic acid as described above.
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、 细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。所述载体可以包含选择标记(例如便于富集的标签，例如his tag；或便于被检测的标签，例如GFP)，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。所述载体可以是克隆载体与表达载体。在表达或是制备抗体或片段时，常涉及原核表达载体和真核表达载体，原核表达载体常用PET系列、pGEX系列，真核表达载体常用pcDNA3.1、pcDNA3.4、pcDNA4、pEGFP-N1、pEGFP-N1、pSV2等。The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40). The vector may contain a selection marker (such as a tag that is convenient for enrichment, such as his tag; or a tag that is convenient for detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector then contain regulatory elements such as enhancers, promoters, internal ribosomal entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly U sequence, etc.). The vector can be a cloning vector and an expression vector. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved. Prokaryotic expression vectors are commonly used in PET series and pGEX series, and eukaryotic expression vectors are commonly used in pcDNA3.1, pcDNA3.4, pcDNA4, pEGFP-N1, pEGFP-N1, pSV2, etc.
在本发明中，载体可以为组合物，例如为多种质粒的混合物，不同质粒负载抗体或其片段的一部分。In the present invention, the carrier can be a composition, such as a mixture of various plasmids, and different plasmids carry a part of the antibody or its fragment.
宿主细胞host cell
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。The present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
可以使用的多种培养的宿主细胞包括，例如，原核细胞、真核细胞、细菌细胞(如大肠杆菌或嗜热脂肪芽胞杆菌(Bacilisstearothermophilus))、真菌细胞(如酿酒酵母或毕赤酵母)、昆虫细胞(如包括草地夜蛾细胞在内的鳞翅目昆虫细胞)或哺乳动物细胞(如中国仓鼠卵巢(CHO)细胞、NS0细胞、小仓鼠肾(BHK)细胞、猴肾细胞、Hela细胞、人肝细胞癌细胞或293细胞等等)。A variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as Escherichia coli or Bacilis stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insect Cells (such as Lepidoptera cells including Spodoptera cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NSO cells, baby hamster kidney (BHK) cells, monkey kidney cells, Hela cells, human Hepatocellular carcinoma cells or 293 cells, etc.).
制备双特异性融合多肽或多功能融合蛋白的方法Method for preparing bispecific fusion polypeptide or multifunctional fusion protein
可采用本领域任何已知的方法制备本发明的双特异性融合多肽或多功能融合蛋白。The bispecific fusion polypeptide or multifunctional fusion protein of the present invention can be prepared by any method known in the art.
例如：用如上所述的载体转化宿主细胞；For example: transforming a host cell with a vector as described above;
培养所转化的宿主细胞；和culturing the transformed host cell; and
收集宿主细胞中表达的双特异性融合多肽或多功能融合蛋白。The bispecific fusion polypeptide or multifunctional fusion protein expressed in the host cells is collected.
特别的，可采用如下方法。In particular, the following methods can be used.
早期构建双特异性抗体的方法有化学交联法或杂合杂交瘤或四价体瘤法(例如，Staerz UD等,Nature,314:628-31,1985；Milstein C等,Nature,305:537-540,1983；Karpovsky B等,J.Exp.Med.,160:1686-1701,1984)。化学偶联法是将2个不同的单克隆抗体用化学偶联的方式连接在一起，制备出双特异性单克隆抗体。例如两种不同单克隆抗体的化学结合，或例如两个抗体片段如两个Fab片段的化学结合。杂合—杂交瘤法是通过细胞杂交法或者三元杂交瘤的方式产生双特异性单克隆抗体，这些细胞杂交瘤或者三元杂交瘤是通过建成的杂交瘤融合，或者建立的杂交瘤和从小鼠得到的淋巴细胞融合而得到的。虽然这些技术用于制造BiAb，但各种产生问题使得此类复合物难以使用，诸如产生含有抗原结合位点的不同组合的混合群体、蛋白质表现方面的困难、需要纯化目标BiAb、低产率、生产费用高等。Early methods for constructing bispecific antibodies include chemical cross-linking or hybrid hybridoma or tetravalent tumor method (for example, Staerz UD et al., Nature, 314:628-31, 1985; Milstein C et al., Nature, 305:537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160:1686-1701, 1984). The chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. For example the chemical combination of two different monoclonal antibodies, or for example the chemical combination of two antibody fragments such as two Fab fragments. The heterozygous-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas from childhood obtained from the fusion of mouse lymphocytes. Although these techniques are used to make BiAbs, various production issues make the use of such complexes difficult, such as generation of mixed populations containing different combinations of antigen-binding sites, difficulties in protein expression, need to purify the BiAb of interest, low yields, production The cost is high.
最近的方法利用经过基因工程改造的构建体，其能够产生单一BiAb的均质产物而无需彻底纯化以去除不需要的副产物。此类构建体包括串联scFv、二抗体、串联二抗体、双可变结构域抗体和使用诸如Ch1/Ck结构域或DNLTM的基元的异源二聚 (Chames&Baty,Curr.Opin.Drug.Discov.Devel.,12:276-83,2009；Chames&Baty,mAbs,1:539-47)。相关纯化技术是公知的。More recent approaches utilize genetically engineered constructs capable of producing a homogeneous product of a single BiAb without extensive purification to remove unwanted by-products. Such constructs include tandem scFv, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as Ch1/Ck domains or DNL™ (Chames & Baty, Curr. Opin. Drug. Discov. Devel., 12:276-83, 2009; Chames & Baty, mAbs, 1:539-47). The relevant purification techniques are well known.
还可以使用单淋巴细胞抗体方法通过克隆和表达由选择用于产生特异性抗体的单个淋巴细胞产生的免疫球蛋白可变区cDNA来产生抗体，例如由Babcook J等人,Proc.Natl.Acad.Sci.USA.93:7843-7848,1996；WO 92/02551；WO 2004/051268和WO 2004/106377所述的方法。Antibodies can also be produced using the monolymphocyte antibody approach by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for production of specific antibodies, e.g. by Babcook J et al., Proc. Natl. Acad. Sci.USA.93:7843-7848,1996; WO 92/02551; methods described in WO 2004/051268 and WO 2004/106377.
用于产生例如用于免疫宿主或用于淘选诸如用于噬菌体展示(或酵母细胞或细菌细胞表面表达)的抗体的抗原多肽可以通过本领域熟知的方法从包含表达系统的遗传工程改造的宿主细胞制备，或者它们可以是从天然生物来源回收。例如，可将编码双特异性抗体的一条或两条多肽链的核酸通过多种已知的方法(如转化、转染、电穿孔、用核酸包被的微粒轰击等)引入培养的宿主细胞。在一些实施方案中，编码双特异性抗体的核酸在被引入宿主细胞前可先插入至适于在宿主细胞中表达的载体中。典型的所述载体可包含使插入的核酸能够在RNA和蛋白质水平上表达的序列元件。Antigenic polypeptides for the production of antibodies, e.g., for immunization of hosts or for panning, such as for phage display (or yeast cell or bacterial cell surface expression), can be obtained from genetically engineered hosts comprising expression systems by methods well known in the art. Cells are prepared, or they may be recovered from natural biological sources. For example, nucleic acids encoding one or both polypeptide chains of bispecific antibodies can be introduced into cultured host cells by various known methods (eg, transformation, transfection, electroporation, bombardment with nucleic acid-coated particles, etc.). In some embodiments, the nucleic acid encoding the bispecific antibody can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell. Typically such vectors will contain sequence elements that enable the expression of the inserted nucleic acid at the RNA and protein levels.
本发明的双特异性抗体，或其部分可通过常规的免疫学分析方法，例如酶联免疫吸附试验(ELISA)，放射免疫分析(RIA)或组织免疫组织化学用于检测任一或所有这些抗原(例如在生物样品，如血清或血浆中)。本发明提供检测生物样品中的抗原的方法，该方法包括：使所述生物样品与本发明的可特异识别所述抗原的双特异性抗体，或抗体部分抗原相接触，并检测与抗原结合的抗体(或抗体部分)，或非结合抗体(或抗体部分)，由此检测所述生物样品中的所述抗原。所述抗体用可检测的物质进行直接或间接的标记，以便于检测结合或非结合抗体。合适的可检测物质包括多种酶，修复基团，荧光物质，发光物质和放射性物质。合适的酶的例子包括，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，乙酰胆碱酯酶；合适的修复基团复合物的例子包括链霉抗生物素蛋白/生物素和抗生物素蛋白/生物素；合适的荧光物质的例子包括7-羟基香豆素，荧光素，荧光素异硫氰酸盐，硷性蕊香红B，二氯三嗪基胺荧光素，丹磺酰氯或藻红蛋白；发光物质的例子包括3-氨基邻苯二甲酰环肼；合适的放射性物质的例子包括I125、I131、35S或3H。The bispecific antibodies of the invention, or portions thereof, can be used to detect any or all of these antigens by conventional immunological assay methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry (eg in biological samples such as serum or plasma). The present invention provides a method for detecting an antigen in a biological sample, the method comprising: contacting the biological sample with the bispecific antibody of the present invention that can specifically recognize the antigen, or part of the antigen of the antibody, and detecting the antigen-binding An antibody (or antibody portion), or a non-binding antibody (or antibody portion), thereby detecting said antigen in said biological sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include, horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase; examples of suitable repair group complexes include streptavidin/biotin and Avidin/biotin; examples of suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, basalin B, dichlorotriazinylamine fluorescein, dan Sulfonyl chloride or phycoerythrin; examples of luminescent substances include 3-aminophthaloyl cyclic hydrazine; examples of suitable radioactive substances include I125, I131, 35S or 3H.
药物组合物pharmaceutical composition
本发明的双特异性融合多肽或多功能融合蛋白或编码其的核酸可以应用于制备药物组合物或无菌组合物，例如，将双特异性融合多肽或多功能融合蛋白与药学上可接受的载体、赋形剂或稳定剂混合。药物组合物可包括一种或组合的(如两种或更多不同的)本发明的抗体其功能片段。例如，本发明的药物组合物可包含与靶抗原上的不同表位结合的具有互补活性的抗体或抗体片段(或免疫缀合物)的组合。治疗和诊断剂的制剂可通过以例如冻干粉末、浆液、水性溶液或悬浮液的形式与药学可接受的载体、赋形剂或稳定剂混合来制备。The bispecific fusion polypeptide or multifunctional fusion protein of the present invention or nucleic acid encoding it can be applied to the preparation of pharmaceutical compositions or sterile compositions, for example, combining bispecific fusion polypeptide or multifunctional fusion protein with pharmaceutically acceptable Carriers, excipients or stabilizers are mixed. A pharmaceutical composition may comprise one or a combination (eg, two or more different) functional fragments of the antibodies of the present invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen. Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.
药物组合物中的双特异性融合多肽或多功能融合蛋白可以是与第二种激活剂(功能分子)结合的形式。所述第二种激活剂可以是能预防或治疗目标疾病的随机功能分子，可包括化合物，肽，多肽，核酸，碳水化合物，脂质，或无机粒子。在所述药物组合物中，双特异性融合多肽或多功能融合蛋白可以本身具有治疗活性；但它可以发挥将所述第二种激活剂靶向特异性疾病区的功能。所述疾病区可以是与抗原特异性结合的双特异性抗体所聚集和分布的那些 器官，组织，或细胞。靶向所述疾病区的药物以高浓度存在，使得药物效应相比注射的量增加。因此，药物组合物可以用于治疗耐药性肿瘤，并可以减少因非特异性药物分布所致的副作用和不利的药物反应。The bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition can be in the form of combining with the second activator (functional molecule). The second activator may be a random functional molecule capable of preventing or treating a target disease, and may include compounds, peptides, polypeptides, nucleic acids, carbohydrates, lipids, or inorganic particles. In the pharmaceutical composition, the bispecific fusion polypeptide or multifunctional fusion protein may itself have therapeutic activity; but it may function to target the second activator to a specific disease area. The diseased areas may be those organs, tissues, or cells where the bispecific antibody specifically binding to the antigen is accumulated and distributed. The drug targeted to the diseased area is present in a high concentration such that the effect of the drug is increased compared to the amount injected. Therefore, the pharmaceutical composition can be used to treat drug-resistant tumors, and can reduce side effects and adverse drug reactions caused by non-specific drug distribution.
药物组合物中包含双特异性融合多肽或多功能融合蛋白的激活剂可以容纳在微胶囊中，或容纳在胶体性质的药物运送系统(如脂质体，白蛋白小球体，微乳剂，纳米颗粒及纳米胶囊)中，或者容纳在大乳剂(macroemulsions)中，所述微胶囊可以通过诸如凝聚(coacervation)技术或界面聚合作用来制备，例子分别有羟甲基纤维素或明胶微胶囊和聚-(异丁烯酸甲酯)微胶囊。The activator comprising bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition can be accommodated in microcapsules, or contained in colloidal drug delivery systems (such as liposomes, albumin spheres, microemulsions, nanoparticles and nanocapsules), or contained in macroemulsions (macroemulsions), the microcapsules can be prepared by techniques such as coacervation (coacervation) or interfacial polymerization, examples are hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules.
医药用途与治疗方法Medicinal uses and treatments
本发明还涉及如上所述的双特异性融合多肽或多功能融合蛋白在制备用于治疗疾病的药物中的应用。The present invention also relates to the application of the above-mentioned bispecific fusion polypeptide or multifunctional fusion protein in the preparation of medicines for treating diseases.
本发明还涉及用作药物的如上所述的双特异性融合多肽或多功能融合蛋白；所述药物用于治疗疾病。The present invention also relates to a bispecific fusion polypeptide or a multifunctional fusion protein as described above for use as a medicine; said medicine is used for the treatment of diseases.
根据本发明一个方面，所述疾病可以是例如，癌症、免疫性病症、代谢性疾病以及微生物感染。According to one aspect of the present invention, the disease may be, for example, cancer, immune disorders, metabolic diseases, and microbial infections.
术语“癌症”是指以体内异常细胞的不受控生长为特征的一大类疾病。“癌症”包括良性和恶性癌症以及休眠肿瘤或微转移。The term "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" includes benign and malignant cancers as well as dormant tumors or micrometastases.
在一些实施方式中，微生物感染中微生物可以是外源病原体或带有外源病原体例如病毒的细胞群体。本发明适用于诸如细菌、真菌、病毒、支原体和寄生虫的外源病原体。可以用本发明治疗的病原体可以是任何本领域熟知的在动物体内致病的感染性生物，包括诸如以下的生物：革兰氏阴性或革兰氏阳性球菌或杆菌的细菌、DNA病毒和RNA病毒，包括但不限于诸如乳头瘤病毒、细小病毒、腺病毒、疱疹病毒和痘苗病毒的DNA病毒、以及诸如沙粒病毒、冠形病毒、鼻病毒、呼吸道合胞病毒、流感病毒、细小核糖核酸病毒、副粘病毒、呼肠孤病毒、逆转录病毒和弹状病毒的RNA病毒。特别感兴趣的是抗生素抗性细菌，例如抗生素抗性链球菌(Streptococcus species)和葡萄球菌(Staphlococcus species)，或者是对抗生素敏感但引起用抗生素治疗的复发性感染、以致最终产生抗性生物的细菌。这类生物可以用本发明的配体-免疫原缀合物与低于正常给予患者的剂量的抗生素联合治疗，以避免产生这些抗生素抗性细菌菌株。本发明也适用于任何真菌、支原体种、寄生虫或在动物中致病的其它感染性生物。可以用本发明方法治疗的真菌的实例包括生长为霉或酵母样的真菌，包括例如引起诸如以下疾病的真菌：癣、组织胞浆菌病、芽生菌病、曲霉病、隐球菌病、孢子丝菌病、球孢子菌病、类球孢子菌病和念珠菌病。本发明可以用来治疗寄生虫感染，包括但不限于由以下寄生虫引起的感染：体绦虫、血吸虫、组织蛔虫、变形虫和疟原虫属(Plasmodium)、锥虫属(Trypanosoma)、利什曼原虫属(Leishmania)和弓形体属(Toxoplasma)种。特别感兴趣的寄生虫是表达叶酸受体并结合叶酸的寄生虫；然而，在文献中关于对感染性生物表现出高亲和性的配体有大量的参考文献。例如，已知其抗生素活性并且与细菌细胞壁前体特异性结合的青霉素和头孢菌素同样可以用作制备按照本发明使用的配体-免疫原缀合物的配体。本发明的配体-免疫原缀合物也可以针对带有内源病原体的细胞群体，其中所述病原体特异性抗原优先在带有所述病原体的细胞表面表达，并且用作与所述抗原特异性结合的配体的受体。In some embodiments, the microorganism in a microbial infection can be an exogenous pathogen or a population of cells harboring an exogenous pathogen such as a virus. The invention is applicable to exogenous pathogens such as bacteria, fungi, viruses, mycoplasma and parasites. Pathogens that may be treated by the present invention may be any infectious organisms known in the art that cause disease in animals, including organisms such as: Gram-negative or Gram-positive cocci or bacilli, bacteria, DNA viruses, and RNA viruses , including but not limited to DNA viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, and vaccinia viruses, and DNA viruses such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial virus, influenza viruses, picornaviruses , paramyxoviruses, reoviruses, retroviruses and rhabdoviruses are RNA viruses. Of particular interest are antibiotic-resistant bacteria, such as antibiotic-resistant Streptococcus species and Staphlococcus species, or those that are susceptible to antibiotics but cause recurrent infections treated with antibiotics so that resistant organisms eventually develop bacteria. Such organisms can be treated with the ligand-immunogen conjugates of the invention in combination with lower doses of antibiotics than are normally administered to patients to avoid the development of these antibiotic resistant bacterial strains. The invention is also applicable to any fungi, mycoplasma species, parasites or other infectious organisms that cause disease in animals. Examples of fungi that may be treated by the methods of the present invention include fungi that grow as molds or yeasts, including, for example, fungi that cause diseases such as: ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichia mycosis, coccidioidomycosis, coccidioidomycosis and candidiasis. The present invention may be used to treat parasitic infections including, but not limited to, infections caused by the following parasites: Taenia spp. Leishmania and Toxoplasma species. Parasites of particular interest are those that express folate receptors and bind folate; however, there are numerous references in the literature to ligands that exhibit high affinity for infectious organisms. For example, penicillins and cephalosporins, whose antibiotic activity is known and which bind specifically to bacterial cell wall precursors, can likewise be used as ligands for the preparation of ligand-immunogen conjugates used in accordance with the present invention. The ligand-immunogen conjugates of the invention can also be directed against cell populations bearing endogenous pathogens, wherein the pathogen-specific antigens are preferentially expressed on the surface of cells bearing the pathogens and used as specific agents for the antigens. Receptors for sex-binding ligands.
本发明还涉及一种预防和/或治疗和施用治疗有效量的药物组合物以预防和/或治疗如上所述疾病的方法。The present invention also relates to a method of prophylaxis and/or treatment and administration of a therapeutically effective amount of the pharmaceutical composition to prevent and/or treat diseases as described above.
本发明的方法可以用于人类临床医学和兽医学应用。因此，带有致病生物群体并且用配体-免疫原缀合物治疗的宿主动物可以是人类，或者在兽医学应用的情况下，可以是实验室动物、农用动物、驯养动物或野生动物。本发明可以适用于包括但不限于以下的宿主动物：人类；实验室动物，诸如啮齿动物(例如小鼠、大鼠、仓鼠等)、兔、猴、黑猩猩；驯养动物，例如狗、猫和兔；农用动物，例如牛、马、猪、绵羊、山羊；和关养的野生动物，例如熊、熊猫、狮、虎、豹、大象、斑马、长颈鹿、大猩猩、海豚和鲸。The methods of the invention can be used in human clinical and veterinary applications. Thus, the host animal carrying the pathogenic population and being treated with the ligand-immunogen conjugate may be a human, or in the case of veterinary applications, a laboratory animal, an agricultural animal, a domesticated animal or a wild animal. The present invention may be applicable to host animals including, but not limited to: humans; laboratory animals such as rodents (e.g. mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; domesticated animals such as dogs, cats and rabbits ; farm animals such as cattle, horses, pigs, sheep, goats; and captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.
药物组合物可通过多种途径注射至实体中，所述实体包括大鼠、小鼠、家养动物、和/或人类。所有注射方法都可以预期，例如，口服，直肠，静脉，鼻，腹部，皮下，或局部注射都是有可能的。组合物可以用本领域已知的其它方法来注射。Pharmaceutical compositions can be injected into entities, including rats, mice, domestic animals, and/or humans, by a variety of routes. All injection methods are contemplated, for example, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or local injections are all possible. The compositions can be injected by other methods known in the art.
“治疗有效量”在本文中是指，根据合理的益损比来看，能治疗疾病的足够量。治疗有效量可以因患者引起的多种原因而有不同，所述原因例如，疾病类型、严重程度、发作、实体的年龄、体重、排泄速度、反应易感性、健康状态、和/或并发症；和/或药物活性、注射途径、注射周期和注射次数、和/或药物组合；也可以由本领域普通技术人员根据治疗目的进行适当选择。例如，注射量可以随机分为多次，使得该量为约0.001-100mg/kg成人体重。A "therapeutically effective amount" herein refers to an amount sufficient to treat a disease with a reasonable benefit to loss ratio. A therapeutically effective amount may vary depending on the patient for a variety of reasons, for example, type of disease, severity, onset, age of the entity, body weight, rate of excretion, susceptibility to reactions, health status, and/or complications; And/or drug activity, injection route, injection cycle and injection times, and/or drug combination; it can also be appropriately selected by those of ordinary skill in the art according to the purpose of treatment. For example, the injection amount can be randomly divided into multiples so that the amount is about 0.001-100 mg/kg body weight of an adult.
本发明的双特异性融合多肽或多功能融合蛋白或编码本发明抗体的核酸或多核苷酸还可与例如标准癌症治疗(例如，手术、放射和化学疗法)组合施用。例如，使用本发明的组合物和/或装备了这些组合物的效应细胞的抗肿瘤疗法与化学疗法联合使用。本发明抗体组合治疗的非限制性实例包括手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。A bispecific fusion polypeptide or multifunctional fusion protein of the invention, or a nucleic acid or polynucleotide encoding an antibody of the invention can also be administered in combination with, for example, standard cancer treatments (eg, surgery, radiation, and chemotherapy). For example, antitumor therapy using the compositions of the invention and/or effector cells equipped with these compositions is used in combination with chemotherapy. Non-limiting examples of antibody combination therapy of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and combinations thereof.
下面将结合实施例对本发明的实施方案进行详细描述。Embodiments of the present invention will be described in detail below in conjunction with examples.
实施例1、FiBody设计 Embodiment 1, FiBody design
在一些实施例中，FiBody是利用配体及其受体之间特异性亲和力，取代双特异性抗体一侧的CL和CH1，重组获得的双特异性抗体，其能够避免或减少双特异性抗体轻链与重链发生错配。In some embodiments, FiBody is a recombinantly obtained bispecific antibody that uses the specific affinity between the ligand and its receptor to replace CL and CH1 on one side of the bispecific antibody, which can avoid or reduce the Mismatches between light and heavy chains.
本实施例以白细胞介素及其受体为例构建FiBody，根据白细胞介素及其受体的立体构像将其分为四类，见下表5和图5：In this example, taking interleukin and its receptor as an example to construct FiBody, it is divided into four categories according to the three-dimensional conformation of interleukin and its receptor, as shown in Table 5 and Figure 5 below:
表5.FiBody分类Table 5. FiBody classification
Figure PCTCN2022121068-appb-000006
Figure PCTCN2022121068-appb-000006
基于以上4类白细胞介素及其受体分别构建双特异性抗体。Bispecific antibodies were constructed based on the above four types of interleukins and their receptors.
实施例2、基于白细胞介素及其受体FiBody的构建Example 2, construction based on interleukin and its receptor FiBody
选取靶向第一抗体的VH通过Linker连接在受体蛋白上，再通过Hinge与抗体的Fc连 接；靶向第一抗体的VL通过Linker连接在配体蛋白上，以降低或避免轻链与重链发生错配；另一端为靶向第二抗体(抗TIGIT抗体)的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链发生错配。或The VH targeting the first antibody is selected to be connected to the receptor protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the first antibody is connected to the ligand protein through a Linker to reduce or avoid light chains and heavy chains. The chain is mismatched; the other end is the complete Fab structure of the targeting second antibody (anti-TIGIT antibody), and the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to reduce or avoid the heavy chain. match. or
选取靶向第一抗体的VH通过Linker连接在配体蛋白上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在受体蛋白(IL15)上，以降低或避免轻链与重链发生错配；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链错配。构建的一些示例性FiBody分子结构及序列表见表6及表7The VH targeting the first antibody is selected to be connected to the ligand protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the second antibody is connected to the receptor protein (IL15) through a Linker to reduce or avoid light There is a mismatch between the chain and the heavy chain; the other end is the complete Fab structure targeting the first antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatches. Some exemplary FiBody molecular structures and sequence lists constructed are shown in Table 6 and Table 7
表6.FiBody分子结构Table 6. Molecular structure of FiBody
Figure PCTCN2022121068-appb-000007
Figure PCTCN2022121068-appb-000007
表7.示例性FiBody分子序列Table 7. Exemplary FiBody Molecular Sequences
Figure PCTCN2022121068-appb-000008
Figure PCTCN2022121068-appb-000008
Figure PCTCN2022121068-appb-000009
Figure PCTCN2022121068-appb-000009
Figure PCTCN2022121068-appb-000010
Figure PCTCN2022121068-appb-000010
Figure PCTCN2022121068-appb-000011
Figure PCTCN2022121068-appb-000011
Figure PCTCN2022121068-appb-000012
Figure PCTCN2022121068-appb-000012
Figure PCTCN2022121068-appb-000013
Figure PCTCN2022121068-appb-000013
实施例3具有二硫键改造的的双特异性抗体Example 3 Bispecific antibody with disulfide bond engineering
3.1为了进一步改善双特异性抗体稳定性并且延长双特异性抗体的半衰期，对双特异性抗体进行二硫键改造，见图8。3.1 In order to further improve the stability of the bispecific antibody and prolong the half-life of the bispecific antibody, the bispecific antibody is modified with a disulfide bond, as shown in Figure 8.
配受体二硫键改造：选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对受体、配体蛋白进行突变，目的形成分子间二硫键，进一步提高分子的稳定性，具体例如下表8：Ligand receptor disulfide bond modification: select the VH targeting the second antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through the Linker, and then connect to the Fc of the antibody through the Hinge; the VL targeting the second antibody is connected to the receptor protein (IL15RA) through the Linker Linked to the ligand protein (IL15); the other end is the complete Fab structure targeting the first antibody. The Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to avoid heavy chain mismatch. At the same time, the receptor and ligand proteins are mutated to form an intermolecular disulfide bond to further improve the stability of the molecule, as shown in Table 8 below:
表8.二硫键改造的的双特异性抗体的氨基酸序列Table 8. Amino acid sequences of disulfide bond engineered bispecific antibodies
Figure PCTCN2022121068-appb-000014
Figure PCTCN2022121068-appb-000014
Figure PCTCN2022121068-appb-000015
Figure PCTCN2022121068-appb-000015
3.2在VH、VL之间设计二硫键改造，形成dsFv，帮助非共价设计的受配体重、轻链之间形成共价二硫键链接。二硫键改造位置包括但不限于以下突变位点：3.2 Design a disulfide bond modification between VH and VL to form a dsFv, and help form a covalent disulfide bond link between the non-covalently designed ligand heavy and light chains. Disulfide bond modification positions include but are not limited to the following mutation sites:
表9.VH、VL之间设计二硫键改造位置Table 9. The designed disulfide bond modification position between VH and VL
组合 combination VHVH VLVL
11 37C 37C 95C95C
22 44C 44C 100C100C
33 44C 44C 101C101C
44 44C44C 105C105C
55 45C 45C 87C87C
66 45C45C 98C98C
77 100C 100C 50C50C
88 100bC100bC 49C49C
99 98C 98C 46C46C
1010 101C101C 46C46C
1111 105C 105C 43C43C
1212 106C106C 57C57C
1313 108C108C 43C43C
表10.VH/VL二硫键改造的双特异性抗体的氨基酸序列Table 10. Amino acid sequences of bispecific antibodies engineered by VH/VL disulfide bonds
Figure PCTCN2022121068-appb-000016
Figure PCTCN2022121068-appb-000016
Figure PCTCN2022121068-appb-000017
Figure PCTCN2022121068-appb-000017
Figure PCTCN2022121068-appb-000018
Figure PCTCN2022121068-appb-000018
实施例4、消除糖基化的突变改造 Embodiment 4, the mutation transformation that eliminates glycosylation
Fab-IL15/IL15RA_Fc的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对受体、配体蛋白进行突变，目的形成分子间二硫键，进一步提高分子的稳定性；进一步的，对受体、配体蛋白上的糖基化位点进行改造，目的是消除分子的异质性，具体例如：The construction method of Fab-IL15/IL15RA_Fc specifically selects the VH targeting the second antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through a Linker, and then connects to the Fc of the antibody through a Hinge; the VL targeting the second antibody Linked to the ligand protein (IL15) through Linker; the other end is the complete Fab structure targeting the first antibody, the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch . At the same time, the receptor and ligand protein are mutated to form intermolecular disulfide bonds to further improve the stability of the molecule; further, the glycosylation sites on the receptor and ligand protein are modified to eliminate Molecular heterogeneity, for example:
表11.IL15/IL15RA改造的双特异性抗体的氨基酸序列Table 11. Amino acid sequences of IL15/IL15RA engineered bispecific antibodies
Figure PCTCN2022121068-appb-000019
Figure PCTCN2022121068-appb-000019
Figure PCTCN2022121068-appb-000020
Figure PCTCN2022121068-appb-000020
Figure PCTCN2022121068-appb-000021
Figure PCTCN2022121068-appb-000021
实施例5、具有降低IL15/IL15RA与IL2/15Rβ/γC复合物亲和力改造的IL15/IL15RA及其组成的双特异性抗体Example 5. IL15/IL15RA modified to reduce the affinity of IL15/IL15RA and IL2/15Rβ/γC complex and bispecific antibody composed of it
在某些应用中，为了避免IL15/IL15RA及其组成的双特异性抗体与IL2/15Rβ/γC复合物相互作用，引起不需要的非特异性结合，我们对IL15/IL15RA及其组成的双特异性抗体进行改造，以降低或完全丧失IL15/IL15RA与IL2/15Rβ/γC复合物亲和力。通过检查IL15/IL15RA与IL2/15Rβ/γC复合物作用界面晶体结构，以及使用MolecularOperating Environment(MOE；Chemical Computing Group,Montreal,Quebec，加拿大)软件建模，我们预测在IL15/IL15RA界面处可以进行氨酸突变改造以便降低或完全丧失IL15/IL15RA与IL2/15Rβ/γC复合物亲和力，如图9中描绘的。In some applications, in order to avoid the interaction of IL15/IL15RA and its component bispecific antibody with IL2/15Rβ/γC complex and cause unwanted non-specific binding, our bispecific antibody against IL15/IL15RA and its component Antibodies engineered to reduce or completely lose affinity for IL15/IL15RA and IL2/15Rβ/γC complexes. By examining the crystal structure of the interaction interface between IL15/IL15RA and IL2/15Rβ/γC complexes, and using Molecular Operating Environment (MOE; Chemical Computing Group, Montreal, Quebec, Canada) software modeling, we predicted that ammonia can be carried out at the IL15/IL15RA interface. Acid mutations were engineered to reduce or completely lose IL15/IL15RA and IL2/15Rβ/γC complex affinity, as depicted in FIG. 9 .
描述Fab-IL15/IL15RA_Fc的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对配体蛋白进行突变，目的是降低或失活受、配体复合物的生物学功能，具体例如：Describe the construction method of Fab-IL15/IL15RA_Fc, specifically select the VH of the targeting second antibody (anti-TIGIT) to be connected to the receptor protein (IL15RA) through Linker, and then connect to the Fc of the antibody through Hinge; VL is connected to the ligand protein (IL15) through a Linker; the other end is the complete Fab structure targeting the first antibody, and the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to avoid heavy chain misalignment match. At the same time, the purpose of mutating the ligand protein is to reduce or inactivate the biological function of the receptor-ligand complex, for example:
表12.IL15/IL15RA改造的双特异性抗体的氨基酸序列Table 12. Amino acid sequences of IL15/IL15RA engineered bispecific antibodies
Figure PCTCN2022121068-appb-000022
Figure PCTCN2022121068-appb-000022
Figure PCTCN2022121068-appb-000023
Figure PCTCN2022121068-appb-000023
实施例6、构建基于scFv、CrossMab结构的双特异性抗体作为实验对照Example 6. Construction of bispecific antibody based on scFv and CrossMab structure as experimental control
正如前文所描述，scFv和CrossMab都是常用的双特异性抗体构建技术手段，在这里作为设计对照，跟我们的分子进行对比：As described above, both scFv and CrossMab are commonly used techniques for the construction of bispecific antibodies. Here, they are used as design controls to compare with our molecules:
基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Hinge与抗体的Fc连接；另一端为靶向第一抗体的完整Fab结构，(此双抗平台为武汉友芝友开发，命名为YBody),组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如：Based on the construction method of the scFv structure double antibody, specifically select the VH of the targeting second antibody (anti-TIGIT) to connect to the VL of the second antibody through a Linker to form a scFv structure, and then connect to the Fc of the antibody through a Hinge; The complete Fab structure of the first antibody (this double antibody platform was developed by Wuhan Youzhiyou and named YBody), the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch . For example:
表13.Y-Body结构双抗分子序列Table 13. Y-Body structure double antibody molecular sequence
Figure PCTCN2022121068-appb-000024
Figure PCTCN2022121068-appb-000024
描述基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Linker与完整的靶向第一抗体的Fc的C端连接；组成一个对称的结构。具体例如：Describe the construction method of double antibodies based on the scFv structure. Specifically, the VH of the targeting second antibody (anti-TIGIT) is selected to connect to the VL of the second antibody through a Linker to form a scFv structure, and then through the Linker and the complete Fc targeting the first antibody The C-terminal connection; form a symmetrical structure. For example:
表14.scFv结构双抗分子序列Table 14. Molecular sequence of scFv structure double antibody
Figure PCTCN2022121068-appb-000025
Figure PCTCN2022121068-appb-000025
Figure PCTCN2022121068-appb-000026
Figure PCTCN2022121068-appb-000026
基于CrossMab结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH连接至CL结构域，再通过Hinge与抗体的Fc连接，靶向第二抗体(抗TIGIT)的VL连接至CH1结构域，形成轻链；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如：Based on the construction method of the CrossMab structure double antibody, the VH of the targeting second antibody (anti-TIGIT) is specifically selected to be connected to the CL domain, and then connected to the Fc of the antibody through Hinge, and the VL of the targeting second antibody (anti-TIGIT) is connected to The CH1 domain forms the light chain; the other end is the complete Fab structure targeting the first antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to avoid heavy chain mismatches. For example:
表15.CrossMab结构双抗分子序列Table 15. CrossMab structure double antibody molecular sequence
Figure PCTCN2022121068-appb-000027
Figure PCTCN2022121068-appb-000027
实施例7、抗体重-轻链错配测试Example 7, antibody heavy-light chain mismatch test
轻链错配是双抗平台面临的一个难点问题。为了验证本平台防错配性能，我们专门设计了受体、配体分布在抗体两边的Fab，故意设计错配的重、轻链结构，并进行表达验证。Light chain mismatch is a difficult problem faced by the double antibody platform. In order to verify the anti-mismatch performance of this platform, we specially designed Fabs with receptors and ligands distributed on both sides of the antibody, deliberately designed mismatched heavy and light chain structures, and performed expression verification.
描述Fab-IL15/IL15RA_Fc错配的构建方法：Describe the construction method of Fab-IL15/IL15RA_Fc mismatch:
R1042：具体选取靶向第一抗体(抗PD-L1抗体)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体(抗TIGIT抗体)的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第二抗体的VH通过常规序列连接在CH1，再通过Hinge与抗体的Fc连接，靶向第一抗体的VL通过常规序列连接在CL，两个Fc具有常规的KiH改造，以避免重链错配。具体例如：R1042: specifically select the VH targeting the first antibody (anti-PD-L1 antibody) to be connected to the receptor protein (IL15RA) through a Linker, and then connect to the Fc of the antibody through a Hinge; targeting the second antibody (anti-TIGIT antibody) The VL is connected to the ligand protein (IL15) through a Linker; the other end is connected to the VH targeting the second antibody to CH1 through a conventional sequence, and then connected to the Fc of the antibody through a Hinge, and the VL targeting the primary antibody is connected through a conventional sequence At CL, the two Fcs have conventional KiH remodeling to avoid heavy chain mismatches. For example:
表16.错配的FiBody抗体序列Table 16. Mismatched FiBody antibody sequences
分子编号molecular number R1042：PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL(图10左)R1042: PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL (Figure 10 left)
第一多肽first polypeptide @PD-L1_VH_IL15RA_Fc-Knob(SEQ ID NO.1)@PD-L1_VH_IL15RA_Fc-Knob (SEQ ID NO.1)
第二多肽second polypeptide @TIGIT_VL_IL15(SEQ ID NO.6)@TIGIT_VL_IL15(SEQ ID NO.6)
第三多肽third polypeptide @TIGIT_VH_CH1_Fc-Hole(SEQ ID NO.3)@TIGIT_VH_CH1_Fc-Hole (SEQ ID NO.3)
第四多肽The fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.8)@PD-L1_VL_CL(SEQ ID NO.8)
R1043：具体选取靶向第二抗体(抗TIGIT抗体)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第一抗体(抗PD-L1抗体)的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的VH通过常规序列连接在CH1，再通过Hinge与抗体的Fc连接，靶向第二抗体的VL通过常规序列连接在CL，两个Fc具有 常规的KiH改造，以避免重链错配。R1043: Specifically select the VH targeting the second antibody (anti-TIGIT antibody) to be connected to the receptor protein (IL15RA) through a Linker, and then connect to the Fc of the antibody through a Hinge; targeting the first antibody (anti-PD-L1 antibody) The VL is connected to the ligand protein (IL15) through a Linker; the other end is connected to the VH targeting the first antibody to CH1 through a conventional sequence, and then connected to the Fc of the antibody through a Hinge, and the VL targeting the second antibody is connected through a conventional sequence At CL, the two Fcs have conventional KiH remodeling to avoid heavy chain mismatches.
表17.KiH双抗序列Table 17. KiH double antibody sequence
分子编号molecular number R1043：PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VLR1043: PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL
第一多肽first polypeptide @TIGIT_VH_IL15RA_Fc-Knob(SEQ ID NO:5)@TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO:5)
第二多肽second polypeptide @PD-L1_VL_IL15(SEQ ID NO:2)@PD-L1_VL_IL15(SEQ ID NO:2)
第三多肽third polypeptide @PD-L1_VH_CH1_Fc-Hole(SEQ ID NO.7)@PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7)
第四多肽The fourth polypeptide @TIGIT_VL_CL(SEQ ID NO.4)@TIGIT_VL_CL(SEQ ID NO.4)
描述Fab-IL21/IL21R_Fc错配的构建方法：Describe the construction method of Fab-IL21/IL21R_Fc mismatch:
选取靶向第二抗体(抗PD-L1抗体)的VH通过Linker连接在受体蛋白(IL21R)上，再通过Hinge与抗体的Fc连接；靶向第一抗体(抗TIGIT抗体)的VL通过Linker连接在配体蛋白(IL21)上；另一端为靶向第二抗体(抗PD-L1抗体)的VL连接在CL上，靶向结构第一抗体(抗TIGIT抗体)的VH连接在CH1上，再通过Hinge与抗体的Fc连接，两端的Fc具有常规的KiH改造。具体例如：The VH targeting the second antibody (anti-PD-L1 antibody) is selected to connect to the receptor protein (IL21R) through Linker, and then connected to the Fc of the antibody through Hinge; the VL targeting the first antibody (anti-TIGIT antibody) is connected through the Linker Linked to the ligand protein (IL21); the other end is linked to the VL of the targeting second antibody (anti-PD-L1 antibody) on the CL, and the VH of the targeting structure first antibody (anti-TIGIT antibody) is linked to CH1, It is then connected to the Fc of the antibody through Hinge, and the Fc at both ends has a conventional KiH modification. For example:
表18.Fab-IL21/IL21R_Fc错配Table 18. Fab-IL21/IL21R_Fc mismatches
Figure PCTCN2022121068-appb-000028
Figure PCTCN2022121068-appb-000028
实施例8、FiBody样品的制备 Embodiment 8, the preparation of FiBody sample
蛋白瞬转表达：Protein transient expression:
将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到目的蛋白。After the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. When the plasmid is in the cell, the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
Expi293在37℃、8％二氧化碳、130rpm条件培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300ml。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀，标记为A管；将1.5mg 转染试剂PEI加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养。直到D7-D10后收样。Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection. The 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300ml. Prepare the transfection complex and prepare for transfection: first, add 750μg target plasmid to a 50ml centrifuge tube containing 15ml Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5mg transfection reagent PEI to the tube containing 15ml Opti-MEM In the 50ml centrifuge tube of the reagent, mix gently, incubate at room temperature for 5 minutes, and mark it as tube B; add the PEI dilution solution in tube B to the DNA dilution solution in tube A drop by drop, mix gently, incubate at room temperature for 15 minutes, and incubate After the completion, the PEI-target plasmid complex was added to Expi293 cells, and placed in a shaker at 37°C to continue culturing. Receive samples until D7-D10.
蛋白纯化：protein purification:
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下，使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。The transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. Purification using ProA affinity chromatography. The process is as follows, use AKTA avant 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough. After loading the sample, use at least 5CV equilibration buffer (10mM PBS) to wash the column again, then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein, and add neutralization buffer (1M Tris, pH 8.0), the added volume of the neutralization buffer is determined according to the estimated content of the eluted sample, generally 10% of the eluted volume is added.
实施例9、FiBody理化检测Embodiment 9, FiBody physical and chemical detection
样品经过一步纯化后通过HPLC-SEC进行检测(分析柱TOSOH，TSKgel G2000)纯度，各个样品的表达量及纯度结果见下表。After one-step purification, the samples were tested for purity by HPLC-SEC (analytical column TOSOH, TSKgel G2000). The expression levels and purity results of each sample are shown in the table below.
表19.双特异性抗体理化检测结果Table 19. Physicochemical test results of bispecific antibodies
Figure PCTCN2022121068-appb-000029
Figure PCTCN2022121068-appb-000029
样品R0951的HPLC-SEC进行检测结果如图11所示，样品R1042的HPLC-SEC进行检测结果如图12所示，样品R0809的HPLC-SEC进行检测结果如图13所示，样品R1110的HPLC-SEC进行检测结果如图14所示。The HPLC-SEC detection results of sample R0951 are shown in Figure 11, the HPLC-SEC detection results of sample R1042 are shown in Figure 12, the HPLC-SEC detection results of sample R0809 are shown in Figure 13, and the HPLC-SEC detection results of sample R1110 are shown in Figure 13. The results of SEC detection are shown in Figure 14.
结果显示,相比于非对称scFv(Y-Body，R0809)、对称ScFv(R0810)、CrossMab(R0959) 结构的双特异性抗体，FiBody平台制备双特异性抗体(包括各种改造优化抗体)具有更高的表达量和/或更高的纯度。The results show that compared with bispecific antibodies with asymmetric scFv (Y-Body, R0809), symmetric ScFv (R0810), and CrossMab (R0959) structures, the FiBody platform prepares bispecific antibodies (including various modified and optimized antibodies) Higher expression and/or higher purity.
意外的是具有错误配对形式的双特异性抗体(样品R1042、R1043、R1124)也能表达并具有类似正常分子的表达量，但是具有明显低的纯度。Surprisingly bispecific antibodies with mispaired forms (samples R1042, R1043, R1124) were also expressed and had expression levels similar to normal molecules, but with significantly lower purity.
实施例10、FiBody抗原亲和力检测Example 10, FiBody antigen affinity detection
TIGIT端结合活性分析TIGIT terminal binding activity assay
通过FCM实验方法检测双抗分子(TIGIT端)与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。The binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay. Prepare 3% BSA buffer: weigh 4.5gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to 800nM The initial concentration is 20 μg/mL, the volume is 300 μL, 3 times serial dilution (100+200) with a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide by 100 μL, 2E+05/well Into a 96-well V-type plate; first add 50 μL of different concentrations of antibodies to the cells, incubate at 2-8 degrees for 0.5 h, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 h; centrifuge at 350xg for 5 min, remove the supernatant, Use 200μL/well 3% BSA; centrifuge at 350xg for 5min, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) with 3% BSA, add 100μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells once with 3% BSA; Flow cytometer standard operating procedures on-machine testing.
结果如图15-图18所示；意外的是R0950(@TIGIT在Fab端)结合活性低于R0951～R0960(@TIGIT在IL15/IL15R端)；R0951～R0960结合活性与阳性对照R0226(Tigit单克隆抗体，OMP-313R12，WO2016191643)相近；A类和D类白细胞介素及其受体替换CH1和CL后，靶向区结合力并未受到影响，与阳性对照(R0226\R0774(VH如序列如SEQ ID NO:73所示，VL序列如SEQ ID NO:74所示),Tigit单克隆抗体)表现出了相当的亲和力；C类分子的替换CH1和CL后靶向区受到影响，靶向结合力明显低于阳性对照(R0226\R0774,Tigit单克隆抗体)。The results are shown in Figures 15-18; surprisingly, the binding activity of R0950 (@TIGIT at the Fab end) was lower than that of R0951~R0960 (@TIGIT at the IL15/IL15R end); the binding activity of R0951~R0960 was comparable to that of the positive control R0226 (Tigit single The cloned antibody, OMP-313R12, WO2016191643) is similar; after class A and class D interleukins and their receptors replace CH1 and CL, the binding force of the targeting region is not affected, and the positive control (R0226\R0774 (VH such as sequence As shown in SEQ ID NO: 73, the VL sequence is shown in SEQ ID NO: 74), Tigit monoclonal antibody) showed considerable affinity; after the replacement of CH1 and CL of class C molecules, the targeting region was affected, and the targeting The binding force was significantly lower than that of the positive control (R0226\R0774, Tigit monoclonal antibody).
二硫键改造优化样品R1081、R1085及糖基化样品改造分子与改造之前分子相比tigit端亲和力结果相当。Disulfide bond transformation optimization samples R1081, R1085 and glycosylation samples have similar tigit end affinity results compared with the molecules before transformation.
R1042、R1043及R1124为错配测试分子，其TIGIT结合活性显著降低；R0810是ScFv结构分子，结合活性也弱于对照分子R0226。R1042, R1043 and R1124 are mismatch test molecules, and their TIGIT binding activity is significantly reduced; R0810 is a ScFv molecule, and its binding activity is also weaker than that of the control molecule R0226.
PD-L1端结合活性分析PD-L1 terminal binding activity analysis
通过FCM实验方法检测双抗分子(PD-L1端)与CHO-PD-L1细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。The binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay. Prepare 3% BSA buffer: weigh 4.5gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to 800nM The initial concentration is 20 μg/mL, the volume is 300 μL, 3 times serial dilution (100+200) with a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide by 100 μL, 2E+05/well Into a 96-well V-type plate; first add 50 μL of different concentrations of antibodies to the cells, incubate at 2-8 degrees for 0.5 h, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 h; centrifuge at 350xg for 5 min, remove the supernatant, Use 200μL/well 3% BSA; centrifuge at 350xg for 5min, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) with 3% BSA, add 100μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells once with 3% BSA; Flow cytometer standard operating procedures on-machine testing.
结果如图19-图23所示，意外的是@PD-L1放在IL15端的活性要好于放在Fab端；R1042、R1043及R1124是对应错配分子，活性明显很弱，其他FiBody均显示了与阳性抗体 (PD-L1单克隆抗体)相当的亲和力。其中R0802为(PD-L1单抗，176F9，VH序列如SEQ ID NO:36所示，VL序列如SEQ ID NO:37所示)，R0514为(PD-L1单抗，Avelumab)，R0919为(PD-L1单抗，VH如序列如SEQ ID NO:75所示，VL序列如SEQ ID NO:76所示)，R0968为(PD-L1单抗，VH如序列如SEQ ID NO:71所示，VL序列如SEQ ID NO:72所示)The results are shown in Figure 19-Figure 23. Surprisingly, the activity of @PD-L1 placed on the IL15 end is better than that placed on the Fab end; R1042, R1043 and R1124 are corresponding mismatched molecules, and the activity is obviously weak, and other FiBody have shown Comparable affinity to positive antibody (PD-L1 monoclonal antibody). Among them, R0802 is (PD-L1 monoclonal antibody, 176F9, the VH sequence is shown in SEQ ID NO:36, and the VL sequence is shown in SEQ ID NO:37), R0514 is (PD-L1 monoclonal antibody, Avelumab), and R0919 is ( PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:75, VL sequence is shown in SEQ ID NO:76), R0968 is (PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:71 , the VL sequence is shown in SEQ ID NO:72)
实施例11、FiBody受体配体复合物(IL15/IL15R)的结合活性Example 11, the binding activity of FiBody receptor ligand complex (IL15/IL15R)
抗体稀释：用FACS buffer将所有分子稀释成初始浓度400nM,体积180μl,3倍梯度稀释(60+120)，10个浓度；细胞计数并铺板：将R0255-2(CHO-mTigit)/293T-IL15R-28细胞离心250g 5min后弃去上清，用FACS buffer调整细胞密度为2E+06，按100μL/管均分到96孔V型板中；将上述稀释好的抗体加入到细胞中，100μL/孔，2-8度孵育0.5h；取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200μL/孔，再次250g离心5min，小心去上清；用FACS buffer配制PE荧光二抗(1:500稀释)，按100μL/孔加入对于的96孔板中，重悬细胞，2-8度孵育30min；取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200μL/孔，再次250g离心5min，小心去上清；用1xPBS 100μL/孔重悬，FACS检测。Antibody dilution: use FACS buffer to dilute all molecules to an initial concentration of 400nM, volume 180μl, 3-fold serial dilution (60+120), 10 concentrations; cell counting and plating: R0255-2(CHO-mTigit)/293T-IL15R -28 cells were centrifuged at 250g for 5min, then the supernatant was discarded, the cell density was adjusted to 2E+06 with FACS buffer, and 100μL/tube was evenly divided into 96-well V-shaped plates; the above-mentioned diluted antibody was added to the cells, 100μL/ Well, incubate at 2-8 degrees for 0.5h; take out the 96-well plate, centrifuge at 250g for 5min, carefully remove the supernatant, add 200μL/well of FACS buffer, centrifuge again at 250g for 5min, carefully remove the supernatant; use FACS buffer to prepare PE fluorescent secondary antibody (1:500 dilution), add 100 μL/well to the corresponding 96-well plate, resuspend the cells, incubate at 2-8 degrees for 30 minutes; take out the 96-well plate, centrifuge at 250g for 5 minutes, carefully remove the supernatant, add FACS buffer 200 μL/ Wells were centrifuged again at 250g for 5min, and the supernatant was removed carefully; resuspended in 1xPBS 100μL/well, and detected by FACS.
结果如图24、图25所示；R0952(IL15、IL15RA换位)、R0960(减活)分子具有很低的IL15受体复合物结合活性；R0955(去糖基化)IL15活性下降；R0953、R0954共价连接后活性与R0951类似，说明对结构影响很小。The results are shown in Figure 24 and Figure 25; R0952 (IL15, IL15RA transposition), R0960 (deactivation) molecules have very low IL15 receptor complex binding activity; R0955 (deglycosylation) IL15 activity decreased; R0953, The activity of R0954 after covalent linking is similar to that of R0951, indicating that it has little effect on the structure.
错配分子R1042、R1043与R0951活性相当，说明没有产生错配，即使是错误的Fv也能表达出来；其中R0655为(IL15/IL15RFc融合蛋白,见SEQ ID NO:38)Mismatched molecules R1042, R1043 have the same activity as R0951, indicating that there is no mismatch, and even the wrong Fv can be expressed; among them, R0655 is (IL15/IL15RFc fusion protein, see SEQ ID NO:38)
实施例12、二硫键改造分子电泳检测Example 12, Electrophoretic detection of molecules modified by disulfide bonds
对二硫键双抗分子进行SDS-PAGE电泳检测，结果如图26所示，未进行二硫键改造的R1072分子在分子量25KD～35KD之间有条带，说明存在游离轻链；配受体二硫键改造分子为R0954、R1085、R1086，其中R0954、R1086电泳结果显示，仍有非共价轻链存在(25KD～35KD之间有条带)，R1085无非共价轻链存在(25KD～35KD之间无条带)，说明R1085二硫键改造成功。SDS-PAGE electrophoresis was performed on the disulfide bond double antibody molecule, and the results are shown in Figure 26. The R1072 molecule without disulfide bond modification had bands between 25KD and 35KD, indicating the presence of free light chains; ligand receptors The disulfide bond modified molecules are R0954, R1085, and R1086. The electrophoresis results of R0954 and R1086 show that there are still non-covalent light chains (bands between 25KD and 35KD), and R1085 has no non-covalent light chains (25KD ~ 35KD There is no band between them), indicating that the R1085 disulfide bond has been remodeled successfully.
轻重链二硫键改造分子为R1081、R1082、R1084,电泳结果显示无非共价轻链存在(25KD～35KD之间无条带)，说明R1081、R1082、R1084二硫键改造成功。The modified disulfide bonds of light and heavy chains were R1081, R1082, and R1084. Electrophoresis results showed that there was no non-covalent light chain (no band between 25KD and 35KD), indicating that the disulfide bonds of R1081, R1082, and R1084 were successfully modified.
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.
实施例13.IL15/IL15Rα复合物Example 13. IL15/IL15Rα complex
一.IL15、IL15Rα之间设计二硫键1. Design of disulfide bond between IL15 and IL15Rα
在IL15、IL15Rα之间设计二硫键改造，帮助非共价连接的IL15和IL15Rα之间形成共价二硫键链接，构建具有靶向性的IL15/IL15Rα复合物,IL15与IL15RA分子间二硫键突变配对立体结构示意图见附图27。示例性的，突变位点如下表：Design disulfide bond modification between IL15 and IL15Rα, help non-covalently linked IL15 and IL15Rα to form covalent disulfide bond link, construct targeted IL15/IL15Rα complex, IL15 and IL15RA intermolecular disulfide The schematic diagram of the three-dimensional structure of bond mutation pairing is shown in Figure 27. Exemplarily, the mutation site is as follows:
表20.IL15/IL15Rα二硫键改造Table 20. IL15/IL15Rα disulfide bond modification
组合 combination IL15IL15 IL15RαIL15Rα
11 E87CE87C D96+C97D96+C97
22 E90C E90C P67CP67C
33 E93CE93C R35CR35C
示例性的选取一种IL15/IL15Rα复合物的使用场景：靶向抗原的重链可变区(VH)可变区通过Linker连接在IL15Rα上，再通过Hinge与抗体的Fc连接；靶向相同抗原的轻链可变 区(VL)通过Linker连接在IL15上，制备一种具有靶向性的IL15/IL15Rα复合物。IL15/IL15Rα复合物结构示意图见附图28，示例性的，IL15/IL15Rα复合物氨基酸序列见下表21：Exemplary selection of an IL15/IL15Rα complex usage scenario: the variable region of the heavy chain variable region (VH) targeting the antigen is connected to IL15Rα through a Linker, and then connected to the Fc of the antibody through a Hinge; targeting the same antigen The variable light chain region (VL) of the light chain is connected to IL15 through the Linker to prepare a targeted IL15/IL15Rα complex. See Figure 28 for a schematic structural view of the IL15/IL15Rα complex. For example, the amino acid sequence of the IL15/IL15Rα complex is shown in Table 21 below:
表21.IL15/IL15Rα复合物氨基酸序列Table 21. IL15/IL15Rα complex amino acid sequence
Figure PCTCN2022121068-appb-000030
Figure PCTCN2022121068-appb-000030
Figure PCTCN2022121068-appb-000031
Figure PCTCN2022121068-appb-000031
二.复合物样品的制备2. Preparation of composite samples
蛋白瞬转表达：Protein transient expression:
将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到目的蛋白。After the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. When the plasmid is in the cell, the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
Expi293在37℃、8％二氧化碳、130rpm条件培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300ml。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀，标记为A管；将1.5mg转染试剂PEI加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养。直到D7-D10后收样。Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection. The 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300ml. Prepare the transfection complex and prepare for transfection: first, add 750 μg of the target plasmid to a 50ml centrifuge tube containing 15ml Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5mg transfection reagent PEI to the tube containing 15ml Opti-MEM In the 50ml centrifuge tube of the reagent, mix gently, incubate at room temperature for 5 minutes, and mark it as tube B; add the PEI dilution solution in tube B to the DNA dilution solution in tube A drop by drop, mix gently, incubate at room temperature for 15 minutes, and incubate After the completion, the PEI-target plasmid complex was added to Expi293 cells, and placed in a shaker at 37°C to continue culturing. Receive samples until D7-D10.
复合物样品的纯化：Purification of complex samples:
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下，使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。The transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. Purification using ProA affinity chromatography. The process is as follows, use AKTA avant 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough. After loading the sample, use at least 5CV equilibration buffer (10mM PBS) to wash the column again, then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein, and add neutralization buffer (1M Tris, pH 8.0), the added volume of the neutralization buffer is determined according to the estimated content of the eluted sample, generally 10% of the eluted volume is added.
三.IL15/IL15Rα复合物凝胶电泳检测3. Gel electrophoresis detection of IL15/IL15Rα complex
对二硫键改造IL15/IL15Rα复合物进行SDS-PAGE电泳检测，检测结果如图29所示，在 分子量25KD～35KD之间有条带，说明存在游离轻链；复合物1和复合物2(二硫键改造位置IL15(E90C)/IL15Rα(P67C))在分子量25KD～35KD之间无条带，说明二硫键改造成功，复合物3(二硫键改造位置IL15(E87C)/IL15Rα(D96+C97))、复合物4(二硫键改造位置IL15(E93C)/IL15Rα(R35C))在分子量25KD～35KD之间有条带，说明存在游离轻链,二硫键改造失败。SDS-PAGE electrophoresis detection was performed on the disulfide bond modified IL15/IL15Rα complex, and the detection results are shown in Figure 29. There are bands between the molecular weight of 25KD and 35KD, indicating the existence of free light chains; complex 1 and complex 2 ( Disulfide bond modified position IL15(E90C)/IL15Rα(P67C)) has no band between molecular weight 25KD～35KD, indicating that the disulfide bond modified successfully, complex 3 (disulfide bond modified position IL15(E87C)/IL15Rα(D96 +C97)) and complex 4 (disulfide bond modification position IL15(E93C)/IL15Rα(R35C)) had bands between 25KD and 35KD in molecular weight, indicating that there was a free light chain and the disulfide bond modification failed.
四.靶向部分亲和力检测4. Targeted partial affinity detection
TIGIT端结合活性分析TIGIT terminal binding activity assay
通过FCM实验方法检测双抗分子(TIGIT端)与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测，检测结果见图30与未进行二硫键改造的分子相比亲和力相当，说明二硫键改造不会影响靶向区的亲和力。The binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay. Prepare 3% BSA buffer: weigh 4.5gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to 800nM The initial concentration is 20 μg/mL, the volume is 300 μL, 3 times serial dilution (100+200) with a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide by 100 μL, 2E+05/well Into a 96-well V-type plate; first add 50 μL of different concentrations of antibodies to the cells, incubate at 2-8 degrees for 0.5 h, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 h; centrifuge at 350xg for 5 min, remove the supernatant, Use 200μL/well 3% BSA; centrifuge at 350xg for 5min, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) with 3% BSA, add 100μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells once with 3% BSA; The flow cytometer standard operating procedure was tested on the machine, and the test results are shown in Figure 30. Compared with the molecule without disulfide bond modification, the affinity is equivalent, indicating that the disulfide bond modification will not affect the affinity of the target region.
PD-L1端结合活性分析PD-L1 terminal binding activity analysis
通过FCM实验方法检测双抗分子(PD-L1端)与CHO-PD-L1细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。检测结果见图31与未进行二硫键改造的分子相比亲和力相当，说明二硫键改造不会影响靶向区的亲和力。The binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay. Prepare 3% BSA buffer: weigh 4.5gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to 800nM The initial concentration is 20 μg/mL, the volume is 300 μL, 3 times serial dilution (100+200) with a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide by 100 μL, 2E+05/well Into a 96-well V-type plate; first add 50 μL of different concentrations of antibodies to the cells, incubate at 2-8 degrees for 0.5 h, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 h; centrifuge at 350xg for 5 min, remove the supernatant, Use 200μL/well 3% BSA; centrifuge at 350xg for 5min, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) with 3% BSA, add 100μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells once with 3% BSA; Flow cytometer standard operating procedures on-machine testing. The detection results are shown in Figure 31. Compared with the molecule without disulfide bond modification, the affinity is equivalent, indicating that the disulfide bond modification will not affect the affinity of the targeting region.
本发明实施例中提及的其它相关蛋白的序列：Sequences of other related proteins mentioned in the examples of the present invention:
序列36:@PD-L1:VH(SEQ ID NO:36)Sequence 36:@PD-L1:VH (SEQ ID NO:36)
Figure PCTCN2022121068-appb-000032
Figure PCTCN2022121068-appb-000032
序列37:@PD-L1:VL(SEQ ID NO:37)Sequence 37:@PD-L1:VL (SEQ ID NO:37)
Figure PCTCN2022121068-appb-000033
Figure PCTCN2022121068-appb-000033
Figure PCTCN2022121068-appb-000034
Figure PCTCN2022121068-appb-000034
R0655氨基酸序列：(SEQ ID NO:38)R0655 amino acid sequence: (SEQ ID NO: 38)
Figure PCTCN2022121068-appb-000035
Figure PCTCN2022121068-appb-000035
R0968PD-L1VH氨基酸序列：(SEQ ID NO:71)R0968PD-L1VH amino acid sequence: (SEQ ID NO: 71)
Figure PCTCN2022121068-appb-000036
Figure PCTCN2022121068-appb-000036
R0968PD-L1VL氨基酸序列：(SEQ ID NO:72)R0968PD-L1VL amino acid sequence: (SEQ ID NO: 72)
Figure PCTCN2022121068-appb-000037
Figure PCTCN2022121068-appb-000037
R0774TIGIT VH氨基酸序列：(SEQ ID NO:73)R0774TIGIT VH amino acid sequence: (SEQ ID NO: 73)
Figure PCTCN2022121068-appb-000038
Figure PCTCN2022121068-appb-000038
R0774TIGIT VL氨基酸序列：(SEQ ID NO:74)R0774TIGIT VL amino acid sequence: (SEQ ID NO: 74)
Figure PCTCN2022121068-appb-000039
Figure PCTCN2022121068-appb-000039
R0919PD-L1VH氨基酸序列：(SEQ ID NO:75)R0919PD-L1VH amino acid sequence: (SEQ ID NO: 75)
Figure PCTCN2022121068-appb-000040
Figure PCTCN2022121068-appb-000040
R0919PD-L1VL氨基酸序列：(SEQ ID NO:76)R0919PD-L1VL amino acid sequence: (SEQ ID NO: 76)
Figure PCTCN2022121068-appb-000041
Figure PCTCN2022121068-appb-000041

Claims (13)

  1. 一种IL15/IL15Rα多肽复合物，所述IL15和IL15Rα之间具有一个非天然的链间键，所述非天然链间键形成于IL15的第一突变残基和IL15Rα的第二突变残基之间，所述IL15第一突变残基为第90位的E突变为C，所述IL15Rα第二突变残基为第67位的P突变为C；所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点，所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。An IL15/IL15Rα polypeptide complex, the IL15 and IL15Rα have a non-natural interchain bond formed between the first mutated residue of IL15 and the second mutated residue of IL15Rα Among them, the first mutation residue of IL15 is the mutation of E at position 90 to C, and the second mutation residue of IL15Rα is the mutation of P at position 67 to C; the amino acid residue mutation site of IL15 is the reference The natural sequence numbering site corresponding to SEQ ID NO: 26, the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO: 27.
  2. 根据权利要求1所述的IL15/IL15Rα多肽复合物，所述IL15和IL15Rα特异性结合；可选地，其中，IL15/IL15Rα polypeptide complex according to claim 1, said IL15 and IL15Rα specifically bind; Optionally, wherein,
    A)所述IL15第61位的D突变为N，第64位的E突变为Q,和/或第65位的N突变位D；和/或A) the D mutation at position 61 of the IL15 is N, the E mutation at position 64 is Q, and/or the N mutation at position 65 is D; and/or
    B)所述IL15至少一个N糖基化位点不存在,优选地，所述N糖基化位点选自N71、N79和/或N112；更优选地，所述IL15包含以下氨基酸突变：N71Q、N79Q和/或N112Q；和/或所述IL15Rα至少一个O糖基化位点不存在；优选地，所述O糖基化位点选自T2、T81和/或T86；更优选地，所述IL15Rα包含以下氨基酸突变：T2A、T81A和/或T86A；B) At least one N-glycosylation site of the IL15 does not exist, preferably, the N-glycosylation site is selected from N71, N79 and/or N112; more preferably, the IL15 comprises the following amino acid mutation: N71Q , N79Q and/or N112Q; and/or at least one O-glycosylation site of the IL15Rα does not exist; preferably, the O-glycosylation site is selected from T2, T81 and/or T86; more preferably, the The IL15Rα comprises the following amino acid mutations: T2A, T81A and/or T86A;
    所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点，所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。The amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO: 26, and the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO: 27.
  3. 根据权利要求1所述的IL15/IL15Rα多肽复合物，所述IL15为SEQ ID NO.84所示的氨基酸序列或其突变序列；优选地，所述突变序列包括选自D61N、E64Q和/或N65D氨基酸突变，和/或选自N71Q、N79Q和/或N112Q氨基酸突变；所述IL15的氨基酸残基突变位点为参照SEQ ID NO：26对应的自然顺序编号位点。According to the IL15/IL15Rα polypeptide complex according to claim 1, the IL15 is the amino acid sequence shown in SEQ ID NO.84 or its mutant sequence; preferably, the mutant sequence comprises a sequence selected from D61N, E64Q and/or N65D Amino acid mutation, and/or amino acid mutation selected from N71Q, N79Q and/or N112Q; the amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO:26.
  4. 根据权利要求1至3任一项所述的IL15/IL15Rα多肽复合物，所述IL15Rα包含SEQ ID NO.28或其突变序列，所述IL15Rα包含SEQ ID NO.77或其突变序列、SEQ ID NO.78或其突变序列、SEQ ID NO.79或其突变序列、SEQ ID NO.80或其突变序列、或SEQ ID NO.81或其突变序列；优选地，所述突变序列包括选自T2A、T81A和/或T86A的氨基酸突变；所述IL15Rα的氨基酸残基突变位点为参照SEQ ID NO：27对应的自然顺序编号位点。According to the IL15/IL15Rα polypeptide complex according to any one of claims 1 to 3, the IL15Rα comprises SEQ ID NO.28 or its mutant sequence, and the IL15Rα comprises SEQ ID NO.77 or its mutant sequence, SEQ ID NO .78 or a mutant sequence thereof, SEQ ID NO.79 or a mutant sequence thereof, SEQ ID NO.80 or a mutant sequence thereof, or SEQ ID NO.81 or a mutant sequence thereof; preferably, the mutant sequence comprises selected from T2A, The amino acid mutation of T81A and/or T86A; the amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  5. 根据权利要求1至4任一项所述的IL15/IL15Rα多肽复合物，其还包含抗体Fc恒定区；可选地，所述抗体Fc恒定区是异源二聚体；可选地，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体；可选地，所述IL15或IL15Rα的C端与Fc恒定区的N端连接。The IL15/IL15Rα polypeptide complex according to any one of claims 1 to 4, which further comprises an antibody Fc constant region; optionally, the antibody Fc constant region is a heterodimer; optionally, the The antibody Fc constant region is based on KiH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction and/or increased flexibility to associate as a heterodimer; optionally, the C-terminus of the IL15 or IL15Rα is associated with the Fc N-terminal linkage of the constant region.
  6. 根据权利要求1至5任一项所述的IL15/IL15Rα多肽复合物，所述多肽复合物为双特异性融合多肽，所述多肽复合物包含第一抗原结合部分，其中，The IL15/IL15Rα polypeptide complex according to any one of claims 1 to 5, wherein the polypeptide complex is a bispecific fusion polypeptide, and the polypeptide complex comprises a first antigen-binding moiety, wherein,
    (A)所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一 抗体的第一重链可变结构域VH1，其可操作性地连接至IL15；和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至IL15Rα；或(A) said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15Rα; or
    (B)所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至IL15Rα；和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至IL15。(B) said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15Rα; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15.
  7. 根据权利要求6所述的IL15/IL15Rα多肽复合物，所述多肽复合物包含第二抗原结合部分，其中，所述第二抗原结合部分包括：The IL15/IL15Rα polypeptide complex according to claim 6, said polypeptide complex comprising a second antigen-binding moiety, wherein said second antigen-binding moiety comprises:
    第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain constant region CH1, and
    第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。A fourth polypeptide comprising, from N-terminus to C-terminus, the second light chain variable domain VL2 of the second antibody, which is operably linked to the antibody light chain constant region CL.
  8. 根据权利要求6或7所述的双特异性融合多肽，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；According to the bispecific fusion polypeptide according to claim 6 or 7, the first antigen-binding portion and the second antigen-binding portion bind to different antigens or bind to different epitopes of the same antigen;
    可选地，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；Optionally, said first antigen binding moiety targets immune cells and said second antigen binding moiety targets tumor cells;
    可选地，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；Optionally, both said first antigen binding portion and said second antigen binding portion target tumor cells;
    可选地，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞；Optionally, both said first antigen binding portion and said second antigen binding portion target immune cells;
    可选地，所述第一抗原结合部分靶向人PD-L1，第二抗原结合部分靶向人TIGIT；或者所述第一抗原结合部分靶向人TIGIT，第二抗原结合部分靶向人PD-L1。Optionally, the first antigen-binding portion targets human PD-L1, and the second antigen-binding portion targets human TIGIT; or the first antigen-binding portion targets human TIGIT, and the second antigen-binding portion targets human PD -L1.
  9. 分离的核酸，其编码权利要求1至8任一项所述的IL15/IL15Rα多肽复合物。An isolated nucleic acid encoding the IL15/IL15Rα polypeptide complex of any one of claims 1-8.
  10. 含有权利要求9所述核酸的载体。A vector comprising the nucleic acid of claim 9.
  11. 含有权利要求9所述核酸或者权利要求10所述载体的宿主细胞。A host cell containing the nucleic acid of claim 9 or the vector of claim 10.
  12. 药物组合物，其包含权利要求1至8任一项所述的IL15/IL15Rα多肽复合物，和药学上可接受的载体、赋形剂或稳定剂。A pharmaceutical composition comprising the IL15/IL15Rα polypeptide complex according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier, excipient or stabilizer.
  13. 权利要求1至8任一项所述的IL15/IL15Rα多肽复合物或权利要求12所述的药物组合物在制备用于治疗疾病的药物中的应用。Use of the IL15/IL15Rα polypeptide complex according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 12 in the preparation of medicines for treating diseases.
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Citations (4)

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CN110461871A (en) * 2017-02-16 2019-11-15 索纳特生物疗法公司 Albumin binding domain fusion protein
CN113135996A (en) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 Bispecific antibody and application thereof
CN113307879A (en) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 TAA/CTLA-4/IL15 three-function fusion protein and application thereof
CN113943374A (en) * 2021-09-24 2022-01-18 广东菲鹏制药股份有限公司 Polypeptide compound of interleukin 15 and receptor thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110461871A (en) * 2017-02-16 2019-11-15 索纳特生物疗法公司 Albumin binding domain fusion protein
CN113135996A (en) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 Bispecific antibody and application thereof
CN113307879A (en) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 TAA/CTLA-4/IL15 three-function fusion protein and application thereof
CN113943374A (en) * 2021-09-24 2022-01-18 广东菲鹏制药股份有限公司 Polypeptide compound of interleukin 15 and receptor thereof

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