WO2024098939A1

WO2024098939A1 - Bifunctional fusion protein and use thereof

Info

Publication number: WO2024098939A1
Application number: PCT/CN2023/117865
Authority: WO
Inventors: 姜彦静; 王伟欢; 周景云; 田新生; 马荣; 张小锐; 刘向蒙; 张筠; 李自强
Original assignee: 北京伟德杰生物科技有限公司
Priority date: 2022-11-07
Filing date: 2023-09-08
Publication date: 2024-05-16

Abstract

Provided is a bifunctional fusion protein, comprising a first domain specifically recognizing interleukin-6 receptor (IL-6R), and a second domain specifically binding to cell surface protein CD47. Also provided is a method of using said bifunctional fusion protein to treat or prevent diseases related to IL-6R and/or CD47.

Description

双功能融合蛋白及其应用Bifunctional fusion protein and its application

技术领域Technical Field

本发明涉及融合蛋白领域，更具体地涉及针对IL-6R和CD47的双功能融合蛋白及其应用。The present invention relates to the field of fusion proteins, and more specifically to a bifunctional fusion protein targeting IL-6R and CD47 and applications thereof.

背景技术Background technique

白细胞介素-6(IL-6)是1980年发现的一种多功能细胞因子，由各种类型的细胞如T细胞、B细胞、单核细胞、成纤维细胞、成骨细胞、角质形成细胞、内皮细胞、系膜细胞和一些肿瘤细胞产生。其包含四个α-螺旋结构域，该结构域具有对于三级结构所必需的四个半胱氨酸残基的基序。人IL-6是26-kDa糖蛋白，其基因存在于7号染色体上，IL-6主要通过与受体细胞IL-6R结合后传导下游信号分子起作用。白细胞介素-6(IL-6)结合受体有两种，一种是特异性受体IL-6R(80kDa I型跨膜蛋白)，另一种是gp130，是IL-6家族细胞因子的所有成员的常见受体亚单位。gp130可以在所有细胞表达，但白细胞介素-6受体(IL-6R)的表达受到更多的限制，主要发现于肝细胞、嗜中性粒细胞、单核细胞和CD4+T细胞。白介素IL-6受体gp130二聚化会导致启动包括激活JAK-STAT3途径和ras介导的MAP激酶信号传导的细胞事件。Interleukin-6 (IL-6) is a multifunctional cytokine discovered in 1980 and produced by various types of cells such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells and some tumor cells. It contains four α-helical domains with a motif of four cysteine residues necessary for the tertiary structure. Human IL-6 is a 26-kDa glycoprotein whose gene is present on chromosome 7. IL-6 mainly acts by binding to the receptor cell IL-6R and then transmitting downstream signal molecules. There are two types of interleukin-6 (IL-6) binding receptors, one is the specific receptor IL-6R (80kDa type I transmembrane protein), and the other is gp130, which is a common receptor subunit for all members of the IL-6 family of cytokines. gp130 can be expressed in all cells, but the expression of interleukin-6 receptor (IL-6R) is more restricted and is mainly found in hepatocytes, neutrophils, monocytes and CD4+T cells. Dimerization of the interleukin 6 receptor gp130 leads to the initiation of cellular events including activation of the JAK-STAT3 pathway and ras-mediated MAP kinase signaling.

研究表明，在人类的多种肿瘤细胞上包括前髓细胞性白血病，星形细胞瘤，胶质母细胞瘤，多发性骨髓瘤、***癌，胃癌，肝癌细胞均发现IL-6R高表达，因此，靶向IL-6或IL-6R,阻断IL-6与IL-6R的结合，从而抑制IL-6通路信号转导，这种IL-6通路抑制剂作为多种疾病的潜在治疗手段开启了新历程。托珠单抗(tocilizumab，TCZ)是首个靶向白细胞介素-6受体(IL-6R)的人源化单克隆抗体，是通过将小鼠抗体PM1人源化而获得的IgG1抗体。托珠单抗可用作IL-6相关疾病的治疗剂，并且已经以商品名Actemra和RoActemra进行销售。Studies have shown that IL-6R is highly expressed in a variety of human tumor cells, including promyelocytic leukemia, astrocytoma, glioblastoma, multiple myeloma, prostate cancer, gastric cancer, and liver cancer cells. Therefore, targeting IL-6 or IL-6R, blocking the binding of IL-6 to IL-6R, and thus inhibiting IL-6 pathway signal transduction, this IL-6 pathway inhibitor has opened a new journey as a potential treatment for a variety of diseases. Tocilizumab (TCZ) is the first humanized monoclonal antibody targeting interleukin-6 receptor (IL-6R). It is an IgG1 antibody obtained by humanizing the mouse antibody PM1. Tocilizumab can be used as a therapeutic agent for IL-6-related diseases and has been sold under the trade names Actemra and RoActemra.

细胞表面蛋白CD47在许多肿瘤类型上表达或过度表达，包括急性骨髓性白血病、B细胞非霍奇金淋巴瘤的多种亚型，以及许多人类实体瘤细胞。CD47被广泛认为是一种“不要吃我”的信号，它有助于在生理条件下维持非恶性细胞的免疫耐受性，但这种分子可以帮助不同癌症类型的癌细胞存活。在许多癌症类型中，与信号调节蛋白α(SIRPα)结合的CD47启动抑制性信号通路，导致恶性细胞逃避巨噬细胞的吞噬作用。抑制癌细胞中CD47信号的疗法可能会促进巨噬细胞对肿瘤细胞的吞噬，从而限制肿瘤生长。这为抗肿瘤治疗提供了可行的免疫靶点。The cell surface protein CD47 is expressed or overexpressed on many tumor types, including acute myeloid leukemia, multiple subtypes of B-cell non-Hodgkin lymphoma, and many human solid tumor cells. CD47 is widely considered to be a "don't eat me" signal that helps maintain immune tolerance of non-malignant cells under physiological conditions, but this molecule can help cancer cells survive in different cancer types. In many cancer types, CD47 binding to signal regulatory protein alpha (SIRPα) initiates inhibitory signaling pathways that cause malignant cells to evade phagocytosis by macrophages. Therapies that inhibit CD47 signaling in cancer cells may promote phagocytosis of tumor cells by macrophages, thereby limiting tumor growth. This provides a viable immune target for anti-tumor therapy.

最近的数据表明抗CD47抗体也有助于提高免疫耐受性小鼠中有效的抗肿瘤T细胞应答。因此，抗CD47抗体是调节先天免疫***和适应性免疫***的新一类免疫检查点抑制剂。作为一个全新的靶点，CD47药物的开发并不顺利，曾因严重的血液不良反应导致研发停滞。这主要由于正常细胞如红细胞也表达CD47，凡是能与红细胞结合的CD47靶向药物，均有可能通过两种机制单独或叠加效应引起红细胞破坏，导致严重的血液毒性，引发贫血和血小板减少等副作用，同时由于“抗原沉没效应”而影响药效。一是通过偶联作用引起红细胞凝集，进而出现红细胞裂解，另一个机制则是通过Fc端介导的ADCP，激活巨噬细胞对红细胞进行吞噬而破坏红细胞。国外CD47抗体药物研发进度最快的美国的FortySeven公司的人源化CD47抗体Hu5F9与人红细胞高度结合并引起红细胞凝集，所以在临床上出现3级贫血的几率高达10％，又由于其诱导T淋巴细胞凋亡，出现3级淋巴细胞减少的几率高达15％。这些不良反应将制约该分子的长期治疗效果。Recent data indicate that anti-CD47 antibodies also help to enhance effective anti-tumor T cell responses in immune-tolerant mice. Therefore, anti-CD47 antibodies are a new class of immune checkpoint inhibitors that regulate both the innate and adaptive immune systems. As a new target, the development of CD47 drugs has not been smooth, and has been stagnant due to severe adverse blood reactions. This is mainly because normal cells such as red blood cells also express CD47. Any CD47-targeted drug that can bind to red blood cells may cause red blood cell destruction through two mechanisms alone or in combination, leading to severe blood toxicity, anemia, thrombocytopenia, etc. Side effects, and the efficacy is affected by the "antigen sinking effect". One is to cause red blood cell agglutination through coupling, and then red blood cell lysis occurs. Another mechanism is to activate macrophages to phagocytose and destroy red blood cells through Fc-terminal mediated ADCP. The humanized CD47 antibody Hu5F9 of FortySeven, an American company with the fastest CD47 antibody drug development progress abroad, is highly bound to human red blood cells and causes red blood cell agglutination. Therefore, the probability of clinical grade 3 anemia is as high as 10%. Because it induces T lymphocyte apoptosis, the probability of grade 3 lymphocytopenia is as high as 15%. These adverse reactions will restrict the long-term therapeutic effect of this molecule.

发明内容Summary of the invention

为了解决现有技术中存在的技术问题之一，本发明提供了一种新的融合蛋白，该融合蛋白能够同时激活先天性和获得性免疫反应来抑制肿瘤免疫逃逸，并通过阻断IL-6R信号通路实现协同抗肿瘤效应。In order to solve one of the technical problems existing in the prior art, the present invention provides a new fusion protein, which can simultaneously activate innate and adaptive immune responses to inhibit tumor immune escape and achieve a synergistic anti-tumor effect by blocking the IL-6R signaling pathway.

本发明的一个方面提供了一种双功能融合蛋白，所述双功能融合蛋白包括：特异性识别白细胞介素-6受体(IL-6R)的第一结构域，和特异性结合细胞表面蛋白CD47的第二结构域。One aspect of the present invention provides a bifunctional fusion protein, which includes: a first domain that specifically recognizes interleukin-6 receptor (IL-6R), and a second domain that specifically binds to cell surface protein CD47.

在一些实施方式中，所述第一结构域为特异性识别IL-6R的抗体或其功能性片段。在一些实施方式中，所述第一结构域可以包括IL-6R抗体的免疫球蛋白(Ig)结构域。In some embodiments, the first domain is an antibody or a functional fragment thereof that specifically recognizes IL-6R. In some embodiments, the first domain may include an immunoglobulin (Ig) domain of an IL-6R antibody.

在一些实施方式中，所述第二结构域为特异性结合CD47的信号调节蛋白α肽(SIRPα肽)或其功能性片段。In some embodiments, the second domain is a signal regulatory protein alpha peptide (SIRPα peptide) that specifically binds to CD47 or a functional fragment thereof.

在一些实施方式中，所述第一结构域包括特异性结合IL-6R的抗体Fab片段、Fab’片段、F(ab’)₂片段、Fv片段、scFv片段、纳米抗体、重链可变区(VH)片段或轻链可变区(VL)片段。In some embodiments, the first domain comprises an antibody Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, or light chain variable region (VL) fragment that specifically binds to IL-6R.

在一些实施方式中，所述SIRPα肽选自SIRPαV1、SIRPαV2、SIRPαV8或者与SIRPαV1、SIRPαV2、SIRPαV8的氨基酸序列相比具有1、2或3个氨基酸的***、取代或缺失的肽。In some embodiments, the SIRPα peptide is selected from SIRPαV1, SIRPαV2, SIRPαV8, or a peptide having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence of SIRPαV1, SIRPαV2, SIRPαV8.

在一些实施方式中，所述第一结构域和所述第二结构域直接连接或者通过连接子连接。In some embodiments, the first domain and the second domain are directly connected or connected through a linker.

在一些实施方式中，所述连接子具有如通式(G_nS)_m所示的氨基酸序列，n、m分别为1-10的整数；更优选地，n为1-4的整数，m为1-3的整数，或者与通式(G_nS)_m所示的氨基酸序列相比具有1、2或3个氨基酸的***、取代或缺失的氨基酸序列。在本发明的一些实施方式中，所述连接包含至少6个氨基酸。In some embodiments, the linker has an amino acid sequence as shown in the general formula (G _n S) _m , where n and m are integers of 1-10, respectively; more preferably, n is an integer of 1-4, m is an integer of 1-3, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in the general formula (G _n S) _m . In some embodiments of the present invention, the linker comprises at least 6 amino acids.

在一些实施方式中，所述双功能融合蛋白包括以任意方式连接的第一结构域和第二结构域。在一些实施方式中，所述双功能融合蛋白包括从N-末端至C-末端的第一结构域-第二结构域。In some embodiments, the bifunctional fusion protein comprises a first domain and a second domain connected in any manner. In some embodiments, the bifunctional fusion protein comprises a first domain-a second domain from the N-terminus to the C-terminus.

在一些实施方式中，所述第一结构域包括：重链，其具有如SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列；和，轻链，其具有如SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。 In some embodiments, the first domain comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:1; and a light chain having an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:2.

在一些实施方式中，对应于SEQ ID NO:1的第236、237、239、332、333位氨基酸中的一个或多个被取代。In some embodiments, one or more of the amino acids 236, 237, 239, 332, and 333 corresponding to SEQ ID NO:1 are substituted.

在一些实施方式中，对应于SEQ ID NO:1的第236位氨基酸不是亮氨酸、和/或第237位氨基酸不是亮氨酸、和/或第239位氨基酸不是甘氨酸、和/或第332位氨基酸不是丙氨酸、和/或第333位氨基酸不是脯氨酸。In some embodiments, the amino acid at position 236 corresponding to SEQ ID NO:1 is not leucine, and/or the amino acid at position 237 is not leucine, and/or the amino acid at position 239 is not glycine, and/or the amino acid at position 332 is not alanine, and/or the amino acid at position 333 is not proline.

在一些实施方式中，对应于SEQ ID NO:1的第236位氨基酸被取代为丙氨酸、和/或第237位氨基酸被替换为谷氨酸、和/或第239位氨基酸被替换为丙氨酸、和/或第332位氨基酸被替换为丝氨酸、和/或第333位氨基酸被取代为丝氨酸。In some embodiments, the amino acid at position 236 corresponding to SEQ ID NO:1 is substituted with alanine, and/or the amino acid at position 237 is substituted with glutamic acid, and/or the amino acid at position 239 is substituted with alanine, and/or the amino acid at position 332 is substituted with serine, and/or the amino acid at position 333 is substituted with serine.

在一些实施方式中，第一结构域包括如SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。In some embodiments, the first domain comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:4.

在一些实施方式中，所述第二结构域包括如SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述第二结构域包括如SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述第二结构域包括如SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述第二结构域包括如SEQ ID NO:8所示的氨基酸序列或与SEQ ID NO:8具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。In some embodiments, the second domain comprises an amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:5. In some embodiments, the second domain comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:6. In some embodiments, the second domain comprises an amino acid sequence as set forth in SEQ ID NO:7 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:7. In some embodiments, the second domain comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

在一些实施方式中，所述双功能融合蛋白包括如SEQ ID NO:9所示的氨基酸序列，或与SEQ ID NO:9所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述双功能融合蛋白包括如SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述双功能融合蛋白包括如SEQ ID NO:11所示的氨基酸序列，或与SEQ ID NO:11所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述双功能融合蛋白包括如SEQ ID NO:12所示的氨基酸序列，或与SEQ ID NO:12所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。在一些实施方式中，所述双功能融合蛋白包括如SEQ ID NO:13所示的氨基酸序列，或与SEQ ID NO:13所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。In some embodiments, the bifunctional fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO:9. In some embodiments, the bifunctional fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:10, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO:10. In some embodiments, the bifunctional fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:11, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO:11. In some embodiments, the bifunctional fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the bifunctional fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 13.

在一些实施方式中，所述双功能融合蛋白还包括如SEQ ID NO:2所示的氨基酸序列，或与SEQ ID NO:2所示的氨基酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。In some embodiments, the bifunctional fusion protein also includes an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

本发明中，针对白细胞介素-6受体(IL-6R)的单克隆抗体(206mab，其具有SEQ ID NO:1 所示的氨基酸序列)是在原研药托珠单抗(tocilizumab，TCZ)基础上，在可变区进行了5个突变，分别是重链上的Phe51、Met57和Ile103；轻链上的Gln89和Arg93，与IL6R的亲和力提高数百倍，生物学活性提高30倍左右。对206mab进行AEASS突变(L236A、L237E、G239A、A332S、P333S，其具有SEQ ID NO:4所示的氨基酸序列)，去除了Fc介导的ADCC效应对免疫细胞的杀伤，以确保临床应用的安全性。IL-6R单抗和天然SIRPα肽组成的双功能融合蛋白可通过同时激活先天性和获得性免疫反应来抑制肿瘤免疫逃逸，并通过阻断IL-6R信号通路实现协同抗肿瘤效应。在一些实施方式中，SIRPα肽选择SIRPαV2D1，SIRPαV2变体在东亚人口中占比最多(41.3％)并且天然变体V2的Domain 1可以高亲和力结合肿瘤细胞CD47，但不结合红细胞CD47，因此更加安全。In the present invention, a monoclonal antibody against interleukin-6 receptor (IL-6R) (206mab, which has SEQ ID NO: 1 The amino acid sequence shown in SEQ ID NO:4) is based on the original drug tocilizumab (TCZ), and 5 mutations were made in the variable region, namely Phe51, Met57 and Ile103 on the heavy chain; Gln89 and Arg93 on the light chain, which increased the affinity with IL6R by hundreds of times and the biological activity by about 30 times. 206mab was subjected to AEASS mutation (L236A, L237E, G239A, A332S, P333S, which has the amino acid sequence shown in SEQ ID NO:4), eliminating the Fc-mediated ADCC effect on immune cell killing to ensure the safety of clinical application. The bifunctional fusion protein composed of IL-6R monoclonal antibody and natural SIRPα peptide can inhibit tumor immune escape by activating innate and acquired immune responses at the same time, and achieve synergistic anti-tumor effects by blocking the IL-6R signaling pathway. In some embodiments, the SIRPα peptide is selected from SIRPαV2D1. The SIRPαV2 variant accounts for the largest proportion in the East Asian population (41.3%) and Domain 1 of the natural variant V2 can bind to tumor cell CD47 with high affinity but not to red blood cell CD47, and is therefore safer.

本发明的另一方面提供了一种分离的核酸分子，其编码本发明的双功能融合蛋白。Another aspect of the present invention provides an isolated nucleic acid molecule encoding the bifunctional fusion protein of the present invention.

在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:14所示的核苷酸序列，或与SEQ ID NO:14所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:15所示的核苷酸序列，或与SEQ ID NO:15所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:16所示的核苷酸序列，或与SEQ ID NO:16所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:17所示的核苷酸序列，或与SEQ ID NO:17所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:18所示的核苷酸序列，或与SEQ ID NO:18所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO:14, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the nucleotide sequence as set forth in SEQ ID NO:14. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO:15, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the nucleotide sequence as set forth in SEQ ID NO:15. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO:16, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the nucleotide sequence as set forth in SEQ ID NO:16. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 17, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 17. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 18, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 18.

在一些实施方式中，所述分离的核酸分子包括如SEQ ID NO:3所示的核苷酸序列，或与SEQ ID NO:3所示的核苷酸序列具有至少约85％、90％、95％、98％、99％或100％序列同一性的核苷酸序列。In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:3, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:3.

在一些实施方式中，本发明的融合蛋白形成二聚体。在一些实施方式中，本发明的融合蛋白形成同源二聚体。在一些实施方式中，本发明的融合蛋白形成异源二聚体。In some embodiments, the fusion protein of the present invention forms a dimer. In some embodiments, the fusion protein of the present invention forms a homodimer. In some embodiments, the fusion protein of the present invention forms a heterodimer.

本发明的又一方面提供了一种核酸递送载体，其包含本发明的分离的核酸分子。在一些实施方式中，所述核酸递送载体包括来源于腺病毒、腺相关病毒、慢病毒或其它可接受的核酸递送载体。Another aspect of the present invention provides a nucleic acid delivery vector comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the nucleic acid delivery vector comprises a nucleic acid delivery vector derived from adenovirus, adeno-associated virus, lentivirus or other acceptable nucleic acid delivery vectors.

本发明的又一方面提供了宿主细胞，其包含本发明所述的分离的核酸分子。Another aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention.

本发明的又一方面提供了一种药物组合物。在一些实施方式中，所述药物组合物包括本发明所述的双功能融合蛋白，以及药学上可接受的载体。在一些实施方式中，所述药物组合物包括本发明所述的分离的核酸分子，以及药学上可接受的载体。在一些实施方式中，所述药物组合物包括本发明所述的核酸递送载体，以及药学上可接受的载体。Another aspect of the present invention provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the bifunctional fusion protein of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the isolated nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition The composition comprises the nucleic acid delivery vector of the present invention and a pharmaceutically acceptable carrier.

在一些实施方式中，所述药物组合物为片剂、散剂、颗粒剂、丸剂、注射剂、混悬液、粉剂、乳剂、气雾剂、凝胶剂、滴眼剂、缓释剂或缓释植入体的形式。在一些实施方式中，所述药物组合物可以配制成可注射的配制品。在一些实施方式中，所述配制品适合于玻璃体内注射、皮下、皮内、肌内、静脉、鞘内或椎管内给药。In some embodiments, the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained release agents or sustained release implants. In some embodiments, the pharmaceutical composition can be formulated into an injectable formulation. In some embodiments, the formulation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal or intraspinal administration.

本发明的又一方面提供了药盒，所述药盒包括本发明所述的药物组合物，所述药物组合物被封装在容器中。在本发明的一些实施方式中，所述容器是玻璃安瓿、玻璃瓶、塑料安瓿、塑料瓶、塑料袋或预装式注射器。在一些实施方式中，本发明涉及适合于向人类肠胃外给药的药物单位剂型，该药物单位剂型包含在适合容器中的如本文所述的药物组合物。在一些实施方式中，所述适合容器是预填充的注射器。在一些实施方式中，该预填充的注射器包括注射针。Another aspect of the present invention provides a medicine box, the medicine box includes a pharmaceutical composition of the present invention, and the pharmaceutical composition is packaged in a container. In some embodiments of the present invention, the container is a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag or a prefilled syringe. In some embodiments, the present invention relates to a pharmaceutical unit dosage form suitable for parenteral administration to humans, the pharmaceutical unit dosage form is included in a pharmaceutical composition as described herein in a suitable container. In some embodiments, the suitable container is a prefilled syringe. In some embodiments, the prefilled syringe includes an injection needle.

本发明的又一方面提供了一种治疗或预防与IL-6和/或CD47相关的疾病的方法。所述方法包括向受试者施用治疗有效量的本发明所述的双功能融合蛋白，本发明所述的分离的核酸分子，或本发明所述的核酸递送载体。Another aspect of the present invention provides a method for treating or preventing diseases associated with IL-6 and/or CD47. The method comprises administering to a subject a therapeutically effective amount of the bifunctional fusion protein of the present invention, the isolated nucleic acid molecule of the present invention, or the nucleic acid delivery vector of the present invention.

在一些实施方式中，所述疾病选自癌症相关疾病。在一些实施方式中，所述疾病选自骨髓增生异常综合征。在一些实施方式中，所述疾病选自前髓细胞性白血病、急性髓系白血病、淋巴系白血病、非霍奇金淋巴瘤、黑色素瘤、平滑肌肉瘤、星形细胞瘤、胶质母细胞瘤、多发性骨髓瘤、***癌、胃癌，肝癌、乳腺癌、肾癌、卵巢癌、输卵管癌、腹膜癌、结直肠癌、非小细胞肺癌、膀胱癌、胰腺癌、肝癌和***。In some embodiments, the disease is selected from cancer-related diseases. In some embodiments, the disease is selected from myelodysplastic syndrome. In some embodiments, the disease is selected from promyelocytic leukemia, acute myeloid leukemia, lymphoid leukemia, non-Hodgkin's lymphoma, melanoma, leiomyosarcoma, astrocytoma, glioblastoma, multiple myeloma, prostate cancer, gastric cancer, liver cancer, breast cancer, kidney cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer, non-small cell lung cancer, bladder cancer, pancreatic cancer, liver cancer and cervical cancer.

本发明提供了一种可特异性结合IL-6R和CD47的双功能融合蛋白，通过将带有IL-6R抗体与SIRPα融合基因的质粒电转至CHO细胞中，获得稳定、高效表达的细胞株。本发明的单克隆细胞株可分泌表达IL-6R抗体和SIRPα双功能融合蛋白，研究表明IL-6R在红细胞上几乎不表达。本发明的双功能融合蛋白可降低与红细胞表面CD47结构域的亲合力，减少抗体对红细胞等细胞的结合，从而增加安全性，提高治疗窗口。IL-6R抗体可特异性的识别特定肿瘤细胞表面的IL-6R，使得双功能融合蛋白在目标细胞上聚集，并通过SIRPα激活先天性和获得性免疫反应来抑制肿瘤免疫逃逸。The present invention provides a bifunctional fusion protein that can specifically bind to IL-6R and CD47. By electroporating a plasmid carrying an IL-6R antibody and a SIRPα fusion gene into CHO cells, a stable and highly efficient expression cell line is obtained. The monoclonal cell line of the present invention can secrete and express the bifunctional fusion protein of IL-6R antibody and SIRPα. Studies have shown that IL-6R is hardly expressed on red blood cells. The bifunctional fusion protein of the present invention can reduce the affinity with the CD47 domain on the surface of red blood cells, reduce the binding of antibodies to cells such as red blood cells, thereby increasing safety and improving the treatment window. The IL-6R antibody can specifically recognize IL-6R on the surface of specific tumor cells, allowing the bifunctional fusion protein to aggregate on the target cells and inhibit tumor immune escape by activating innate and acquired immune responses through SIRPα.

定义definition

除非另有定义，否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数形式使用的术语也将包括复数形式，反之亦然。Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

除非上下文另有明确说明，否则本文所用的表述“一”、“一种”和“一个”包括复数指代。例如，提及“一个细胞”包括多个这样的细胞及本领域技术人员可知晓的等同物等等。Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

本文所用的术语“约”表示其后的数值的±20％的范围。在一些实施方式中，术语“约”表示其后的数值的±10％的范围。在一些实施方式中，术语“约”表示其后的数值的±5％的范围。 As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

本文所用的术语“融合蛋白”是指，使用编码蛋白的多核苷酸的遗传表达或蛋白合成方法，将两个或多个蛋白或其片段通过各自的肽骨架共线性连接。优选地，所述蛋白或其片段是不同来源。The term "fusion protein" as used herein refers to the use of genetic expression of polynucleotides encoding proteins or protein synthesis methods to connect two or more proteins or fragments thereof colinearly through their respective peptide backbones. Preferably, the proteins or fragments thereof are from different sources.

本文所用的术语“亲和力”或“结合亲和力”是指分子(例如双功能融合蛋白或者双特异性结合分子)的单个结合位点与其结合配体(例如抗原)之间的非共价相互作用的总和的强度。结合亲和力通常可以用解离常数(K_D)表示，解离常数(K_D)是解离速率(k_d)与结合速率(k_a)的比率，即K_D＝k_d/k_a。亲和力可以通过本领域已知的常规方法测量，例如表面等离子体共振(SPR)。The term "affinity" or "binding affinity" as used herein refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a bifunctional fusion protein or a bispecific binding molecule) and its binding partner (e.g., an antigen). Binding affinity can generally be expressed by a dissociation constant ( _KD ), which is the ratio of the _dissociation rate ( _kd ) to the association rate ( _ka ), i.e., _KD = _kd / _ka . Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

本文所用的术语“特异性识别”或者“特异性结合”是指融合蛋白对于受体或配体具有识别和结合选择性，并且可以与不需要的或非特异性的结合区分开来。在一个实施方式中，例如通过SPR所测得的，本发明的双功能融合蛋白，与不相关蛋白的结合程度小于与IL-6R和CD47结合程度的约10％。在某些实施方式中，本发明提供的双功能融合蛋白，与IL-6R和CD47结合的K_D均为10^-7M或更低，例如为10^-10M至10^-11M。As used herein, the term "specific recognition" or "specific binding" means that the fusion protein has recognition and binding selectivity for a receptor or ligand, and can be distinguished from unwanted or non-specific binding. In one embodiment, the degree of binding of the bifunctional fusion protein of the present invention to unrelated proteins is less than about 10% of the degree of binding to IL-6R and CD47, for example, as measured by SPR. In certain embodiments, the bifunctional fusion protein provided by the present invention binds to IL-6R and CD47 with a _KD of ^10-7 M or less, for example, ^10-10 M to ^10-11 M.

本文针对氨基酸所使用的术语“取代”是指一氨基酸序列中的至少一个氨基酸残基被另一个不同的“置换”氨基酸残基置换。本文针对氨基酸所使用的术语“***”是指将至少一个额外氨基酸掺入一氨基酸序列中。虽然***物通常由***1或2个氨基酸残基组成，但也可以制备较大的“肽***物”，例如***约3至5个或甚至最多约10、15或20个氨基酸残基。如以上所公开，***的残基可以是天然存在或非天然存在的。本文针对氨基酸所使用的术语“缺失”是指从一氨基酸序列去除至少一个氨基酸残基。The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence by another different "replacement" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. Although inserts typically consist of the insertion of 1 or 2 amino acid residues, larger "peptide inserts" can also be prepared, for example, inserting about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

本公开的融合蛋白或其片段可在一个或多个氨基酸残基处，例如在必需或非必需氨基酸残基处包含保守氨基酸取代。“保守氨基酸取代”是氨基酸残基被具有类似侧链的氨基酸残基置换。本领域中已定义具有类似侧链的氨基酸残基的家族，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链(例如苏氨酸、缬氨酸、异亮氨酸)以及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在本文中，融合蛋白或连接子中的必需或非必需氨基酸残基优选地用来自同一侧链家族的另一个氨基酸残基置换。在某些实施方式中，氨基酸链段可以用结构类似且侧链家族成员的次序和/或组成不同的链段置换。或者，在某些实施方式中，可沿编码序列的全部或一部分随机引入突变，如通过饱和诱变引入，并且由此得到的突变体可掺入本发明的融合蛋白中，并针对这些多肽结合至所希望的靶标的能力进行筛选。Fusion protein of the present disclosure or its fragment can comprise conservative amino acid substitution at one or more amino acid residues, for example, at essential or non-essential amino acid residues. "Conservative amino acid substitution" is that amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this article, the essential or non-essential amino acid residues in the fusion protein or connexon are preferably replaced with another amino acid residue from the same side chain family. In some embodiments, the amino acid segment can be replaced with a segment having a similar structure and a different order and/or composition of the side chain family members. Alternatively, in certain embodiments, mutations can be introduced randomly along all or a portion of a coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into fusion proteins of the invention and screened for the ability of these polypeptides to bind to a desired target.

本文所用的术语“抗体”包括完整的抗体和任意的抗原结合片段(即，“抗原结合部分”、“抗原结合多肽”或“免疫结合剂”)，或它们的单链。“抗体”是一种糖蛋白，其包含通过二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白，或其抗原结合部分。每条重链均包括重链可变(VH)区和重链恒定区(CH)。每条轻链均包括轻链可变(VL)区和轻链恒定区(CL)。VH区和VL区中各含有3个氨基酸组成和排列顺序高度可变的区域，称为高变区或互补决定区(complementarity determining region，CDR)，CDRl、CDR2和CDR3。VH区和VL区中除了CDR区之外的区域的氨基酸组成和排列顺序相对保守，称为骨架区(framework region，FR)。VH或VL各有四个骨架区，分别用FR1、FR2、FR3和FR4表示。The term "antibody" as used herein includes intact antibodies and any antigen-binding fragment (i.e., "antigen-binding portion,""antigen-bindingpolypeptide," or "immunobinder"), or single chains thereof. An "antibody" is a glycoprotein comprising A glycoprotein of at least two heavy chains (H) and two light chains (L) connected, or an antigen binding portion thereof. Each heavy chain includes a heavy chain variable (VH) region and a heavy chain constant region (CH). Each light chain includes a light chain variable (VL) region and a light chain constant region (CL). The VH region and the VL region each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions or complementarity determining regions (CDR), CDR1, CDR2 and CDR3. The amino acid composition and arrangement order of the regions in the VH region and the VL region except the CDR region are relatively conservative, called the framework region (FR). VH or VL each has four framework regions, represented by FR1, FR2, FR3 and FR4, respectively.

免疫球蛋白存在五种主要类别，IgA、IgD、IgE、IgG和IgM，并且这些主要类别还可以进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。不同型(κ或λ)免疫球蛋白的CL长度基本一致，但是不同类的免疫球蛋白的CH长度不同，例如IgG、IgA和IgD包括CH1、CH2和CH3，而IgM和IgE则包括CHl、CH2、CH3和CH4。铰链区(hinge region)位于CH1与CH2之间，富含脯氨酸，易伸展弯曲，从而改变抗原结合部位之间的距离，有利于抗体结合位于不同位置的抗原表位。铰链区易被木瓜蛋白酶、胃蛋白酶等水解，产生不同的水解片段。木瓜蛋白酶水解Ig的部位是在铰链区二硫键连接的两条重链的近N端，可将Ig裂解为两个完全相同的Fab段和一个Fc段。Fab段为抗原结合片段(fragment antigenbinding，Fab)，由一条完整的轻链与重链的VH和CHl结构域组成。There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and these major classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The CL lengths of different types (κ or λ) of immunoglobulins are basically the same, but the CH lengths of different classes of immunoglobulins are different, for example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The hinge region is located between CH1 and CH2, is rich in proline, and is easy to stretch and bend, thereby changing the distance between the antigen binding sites, which is conducive to the antibody binding to antigen epitopes located at different positions. The hinge region is easily hydrolyzed by papain, pepsin, etc., producing different hydrolysis fragments. Papain hydrolyzes Ig at the N-terminus of the two heavy chains connected by disulfide bonds in the hinge region, and can split Ig into two identical Fab segments and one Fc segment. The Fab segment is an antigen binding fragment (Fab), which consists of a complete light chain and the VH and CH1 domains of the heavy chain.

本文所用的术语“同源二聚体”指由本发明的融合蛋白形成，包括两条相同的本发明的融合蛋白。本文所用的术语“异源二聚体”指由本发明的融合蛋白形成，包括两条不同的本发明的融合蛋白。The term "homodimer" as used herein refers to a fusion protein of the present invention, including two identical fusion proteins of the present invention. The term "heterodimer" as used herein refers to a fusion protein of the present invention, including two different fusion proteins of the present invention.

本文所用的术语“个体”或“受试者”指哺乳动物，包括但不限于人和非人哺乳动物，例如，哺乳动物包括但不限于，驯养动物(如牛、马、犬、绵阳、山羊、猫和狗)、灵长类动物(如人和猴)和啮齿动物(如兔、小鼠和大鼠)。The term "individual" or "subject" as used herein refers to mammals, including but not limited to humans and non-human mammals, for example, mammals include but are not limited to domesticated animals (such as cows, horses, dogs, sheep, goats, cats and dogs), primates (such as humans and monkeys) and rodents (such as rabbits, mice and rats).

本文所使用的数值范围应被理解为已经列举了对该范围内的所有数字。例如，1至20的范围应当理解为包括来自下组的任何数字、数字组合或子范围：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

本文所用的术语“连接子”指天然和/或合成来源的(肽)接头，由线性氨基酸组成。本发明的双功能融合蛋白中的各结构域可以通过连接子连接，其中每个连接子与至少两个多肽或结构域融合和/或以其他方式连接(例如，经由肽键)。在一些实施方式中，存在于本发明的双功能融合蛋白中的所有连接子的氨基酸序列是相同的。在其他实施方式中，存在于本发明的双功能融合蛋白中的至少两个连接子的氨基酸序列是不同的。连接子应该具有适合以这种方式连接两个或更多个单体结构域的长度，连接子能够确保其所连接的不同结构域正确折叠并合适地呈现，从而发挥其生物学活性的功能。在不同的实施方式中，连接子具有柔性构象。合适的柔性连接子包括，例如具有甘氨酸、谷氨酰胺和/或丝氨酸残基。在一些实施方式中，连接子中的氨基酸残基可以多达5个氨基酸的小重复单元排列。The term "linker" as used herein refers to a (peptide) linker of natural and/or synthetic origin, consisting of linear amino acids. The domains in the bifunctional fusion protein of the present invention can be connected by linkers, wherein each linker is fused to at least two polypeptides or domains and/or connected in other ways (e.g., via a peptide bond). In some embodiments, the amino acid sequences of all linkers present in the bifunctional fusion protein of the present invention are identical. In other embodiments, the amino acid sequences of at least two linkers present in the bifunctional fusion protein of the present invention are different. The linker should have a length suitable for connecting two or more monomer domains in this manner, and the linker can ensure that the different domains to which it is connected are correctly folded and appropriately presented, thereby exerting their biologically active functions. In different embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, having glycine, glutamine and/or serine residues. In some embodiments, the linker The amino acid residues can be arranged in small repeating units of up to 5 amino acids.

相对于参照氨基酸序列具有“百分比(％)序列同一性”指在比对序列和(根据需要)引入缺口以获得最大百分比序列同一性后，候选序列中与参照氨基酸序列中的氨基酸残基相同的氨基酸残基的百分比，但不考虑任何保守取代为序列同一性的一部分。为了确定氨基酸序列同一性百分比，可以以本领域范围内的各种方式进行比对，例如使用BLAST、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数，包括在被比较序列全长上实现最大比对所需的任何算法。"Percent (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, after alignment of the sequences and (as required) introduction of gaps to obtain maximum percentage sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, such as using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

本文所用的术语“药学上可接受的载体”指药物制剂中除了活性成分之外的对受试者无毒性的成分。药学上可接受的载体包括，但不限于缓冲剂、赋形剂、稳定剂或防腐剂。The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

本文所使用的术语“治疗”指减轻和/或改善障碍和/或与其相关的疾病或症状，以及预防障碍症状的恶化。期望的治疗效果包括，但不限于防止疾病发生或复发，症状的缓解，疾病的任何直接或间接的病理结果的减少、防止转移、减缓疾病发展速度、改善或缓解症状、缓解或改善预后。但应当理解，治疗疾病或症状并不要求完全地消除该疾病或与其相关的症状。As used herein, the term "treat" refers to alleviating and/or ameliorating a disorder and/or a disease or symptom associated therewith, as well as preventing the deterioration of the symptoms of a disorder. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating symptoms, alleviating or improving prognosis. However, it should be understood that treating a disease or symptom does not require completely eliminating the disease or the symptoms associated therewith.

本文所用的术语“有效量”是指为了实现所需的治疗或预防效果，在必要的剂量和时间段内的有效的量。As used herein, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive effect.

下面提供实施例和附图以帮助理解本发明。但应理解，这些实施例和附图仅用于说明本发明，但不构成任何限制。本发明的实际保护范围在权利要求书中进行阐述。应理解，在不脱离本发明精神的情况下，可以进行任何修改和改变。Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1示出了本发明构建的双功能融合蛋白的结构示意图。FIG1 shows a schematic diagram of the structure of the bifunctional fusion protein constructed in the present invention.

图2示出了本发明的瞬转表达双功能融合蛋白的还原SDS-PAGE结果，其中，泳道1为PLS2124(206mab-H-SIRPαV1D1)1μg，泳道2为PLS2124(206mab-H-SIRPαV1D1)2μg，泳道3为Ladder，泳道4为PLS2125(206mab-H-SIRPαV2D1)1μg，泳道5为PLS2125(206mab-H-SIRPαV2D1)2μg，泳道6为PLS2126(206mab-H-SIRPαV1D1sg)1μg，泳道7为PLS2126(206mab-H-SIRPαV1D1sg)2μg，泳道8为PLS2127(206mab-H-SIRPαV2D1lm)1μg，泳道9为PLS2127(206mab-H-SIRPαV2D1lm)2μg。FIG2 shows the reducing SDS-PAGE results of the transiently expressed bifunctional fusion protein of the present invention, wherein lane 1 is PLS2124 (206mab-H-SIRPαV1D1) 1 μg, lane 2 is PLS2124 (206mab-H-SIRPαV1D1) 2 μg, lane 3 is Ladder, lane 4 is PLS2125 (206mab-H-SIRPαV2D1) 1 μg, lane 5 is PLS2125 (206 lane 6 is PLS2126 (206mab-H-SIRPαV1D1sg) 1 μg, lane 7 is PLS2126 (206mab-H-SIRPαV1D1sg) 2 μg, lane 8 is PLS2127 (206mab-H-SIRPαV2D1lm) 1 μg, and lane 9 is PLS2127 (206mab-H-SIRPαV2D1lm) 2 μg.

图3示出了本发明的瞬转表达双功能融合蛋白的非还原SDS-PAGE结果，其中，泳道1为PLS2124(206mab-H-SIRPαV1D1)1μg，泳道2为PLS2124(206mab-H-SIRPαV1D1)2μg，泳道3为Ladder，泳道4为PLS2125(206mab-H-SIRPαV2D1)1μg，泳道5为PLS2125(206mab-H-SIRPαV2D1)2μg，泳道6为PLS2126(206mab-H-SIRPαV1D1sg)1μg，泳道7为PLS2126(206mab-H-SIRPαV1D1sg)2μg，泳道8为PLS2127(206mab-H-SIRPαV2D1lm)1μg，泳道9为PLS2127(206mab-H-SIRPαV2D1lm)2μg。 FIG3 shows the non-reducing SDS-PAGE results of the transiently expressed bifunctional fusion protein of the present invention, wherein lane 1 is PLS2124 (206mab-H-SIRPαV1D1) 1 μg, lane 2 is PLS2124 (206mab-H-SIRPαV1D1) 2 μg, lane 3 is Ladder, lane 4 is PLS2125 (206mab-H-SIRPαV2D1) 1 μg, lane 5 is PLS2125 (20 Lane 6 is PLS2126 (206mab-H-SIRPαV1D1sg) 1 μg, lane 7 is PLS2126 (206mab-H-SIRPαV1D1sg) 2 μg, lane 8 is PLS2127 (206mab-H-SIRPαV2D1lm) 1 μg, and lane 9 is PLS2127 (206mab-H-SIRPαV2D1lm) 2 μg.

图4示出了本发明的PLS2125阻断IL6与细胞表面IL6R结合的报告基因检测结果。FIG4 shows the reporter gene assay results of PLS2125 of the present invention blocking the binding of IL6 to cell surface IL6R.

图5示出了本发明的PLS2125阻断CD47和SIRPα结合的HTRF检测结果。FIG5 shows the HTRF detection result of PLS2125 of the present invention blocking the binding between CD47 and SIRPα.

图6示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白的SDS-PAGE结果，其中M为marker，NR为非还原SDS-PAGE结果，R为还原SDS-PAGE结果。FIG6 shows the SDS-PAGE results of the protein expressed by the high-expression cell line PLT300-8#8C4 of the present invention, wherein M is a marker, NR is a non-reducing SDS-PAGE result, and R is a reducing SDS-PAGE result.

图7示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白的SEC-HPLC检测结果。FIG. 7 shows the SEC-HPLC detection results of the protein expressed by the high-expressing cell line PLT300-8#8C4 of the present invention.

图8示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白的cIEF鉴定结果。FIG. 8 shows the cIEF identification results of the protein expressed by the high-expressing cell line PLT300-8#8C4 of the present invention.

图9示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白反复冻融样品的SEC-HPLC检测结果。FIG. 9 shows the SEC-HPLC detection results of the protein expressed by the high-expressing cell line PLT300-8#8C4 of the present invention after repeated freezing and thawing.

图10示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白反复冻融样品的SDS-PAGE结果。FIG. 10 shows the SDS-PAGE results of the protein expression samples of the high-expressing cell line PLT300-8#8C4 of the present invention after repeated freezing and thawing.

图11示出了本发明的高表达细胞株PLT300-8#8C4表达蛋白在40℃加速样品的SEC-HPLC检测结果。FIG. 11 shows the SEC-HPLC detection results of the protein expressed by the high-expressing cell line PLT300-8#8C4 of the present invention at 40° C. accelerated sample.

图12示出了本发明的双功能融合蛋白结合人红细胞检测结果。FIG. 12 shows the detection results of the bifunctional fusion protein of the present invention binding to human red blood cells.

图13示出了本发明的双功能融合蛋白的巨噬细胞吞噬活性检测结果。FIG. 13 shows the results of detecting the macrophage phagocytic activity of the bifunctional fusion protein of the present invention.

具体实施方式Detailed ways

为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于构成对本发明的任何限制。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本公开的概念。In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts disclosed herein.

本发明的双功能融合蛋白IL-6R抗体与SIRPα肽，结构如图1所示，其中IL-6R抗体为206mab及其突变体206mab-mut，206mab是白介素-6受体单克隆抗体药物托珠单抗(tocilizumab，TCZ)的基因改造药物，是在托珠单抗的基础上进行改造的亲和力提高多倍的突变体。SIRPα肽为天然变体，其可以为SIRPα肽所包含SIRPαV1、SIRPαV2、SIRPαV8三种变体及在其基础上进行氨基酸经取代、缺失、和/或添加一个或几个氨基酸，IL-6R抗体与所述SIRPα肽之间设有连接肽，所述连接肽的通式为(G_nS)_m，n、m分别为1～10的整数，优选地，n为1～4的整数，m为1～3的整数，The bifunctional fusion protein IL-6R antibody and SIRPα peptide of the present invention has a structure as shown in Figure 1, wherein the IL-6R antibody is 206mab and its mutant 206mab-mut, 206mab is a genetically modified drug of the interleukin-6 receptor monoclonal antibody drug tocilizumab (TCZ), and is a mutant modified on the basis of tocilizumab with multiple times increased affinity. SIRPα peptide is a natural variant, which can be the three variants of SIRPαV1, SIRPαV2, and SIRPαV8 contained in SIRPα peptide, and amino acids are substituted, deleted, and/or one or more amino acids are added on the basis thereof. A connecting peptide is provided between the IL-6R antibody and the SIRPα peptide, and the general formula of the connecting peptide is ( _GnS ) _m , n and m are integers of 1 to 10, respectively, preferably, n is an integer of 1 to 4, and m is an integer of 1 to 3.

本发明首先构建了206mab-L和融合基因载体PLS2124(206mab-H-SIRPαV1D1)、PLS2125(206mab-H-SIRPαV2D1)、PLS2126(206mab-H-SIRPαV1D1sg)、PLS2127(206mab-H-SIRPαV2D1im)，PLS2124、PLS2125、PLS2126、PLS2127通过和206mab-L，共转HEK293瞬转表达，常规纯化获得双功能融合蛋白，所得蛋白电泳鉴定正确后，对瞬转获得的目的蛋白进行亲和力检测。综合亲和力结果和hSIRPα在不同人群中的分布情况，选择了截短了的天然SIRPαV2D1的序列，同时对206mab-H进行了AEASS氨基酸突变(L236A、L237E、G239A、A 332S、P333S)，去除了ADCC效应，用连接子(G_nS)_m连接突变的206mab-H和SIRPαV2D1，与206mab-L***同一个载体，构建用于稳转CHO细胞的质粒PLT300(206mab-mut-SIRPαV2D1)。将PLT300质粒电转至CHO细胞中，获得稳定、高效表达人源重组蛋白的CHO单克隆细胞株，对筛选到的单克隆细胞株表达的抗体进行成药性分析，蛋白稳定性强及成药性良好。The present invention first constructs 206mab-L and fusion gene vectors PLS2124 (206mab-H-SIRPαV1D1), PLS2125 (206mab-H-SIRPαV2D1), PLS2126 (206mab-H-SIRPαV1D1sg), and PLS2127 (206mab-H-SIRPαV2D1im); PLS2124, PLS2125, PLS2126, and PLS2127 are co-transfected with 206mab-L for HEK293 transient expression; a bifunctional fusion protein is obtained by conventional purification; after the obtained protein is correctly identified by electrophoresis, affinity detection is performed on the target protein obtained by transient expression. Based on the affinity results and the distribution of hSIRPα in different populations, the truncated sequence of natural SIRPαV2D1 was selected, and AEASS amino acid mutations (L236A, L237E, G239A, A 332S, P333S), the ADCC effect was removed, and the mutant 206mab-H and SIRPαV2D1 were connected with a linker (G _n S) _m , and inserted into the same vector with 206mab-L to construct the plasmid PLT300 (206mab-mut-SIRPαV2D1) for stable transfection of CHO cells. The PLT300 plasmid was electroporated into CHO cells to obtain a CHO monoclonal cell line that stably and efficiently expresses human recombinant proteins. The drugability analysis of the antibodies expressed by the screened monoclonal cell lines showed that the protein had strong stability and good drugability.

实施例1表达质粒的构建Example 1 Construction of expression plasmid

本实施例中，IL-6R抗体与SIRPα肽的双功能融合蛋白的结构如图1所示，其中IL-6R抗体为206mab(其氨基酸序列和核苷酸序列如下所示)及其突变体206mab-mut(其氨基酸序列和核苷酸序列如下所示)，206mab是白介素-6受体单克隆抗体药物托珠单抗(tocilizumab，TCZ)的基因改造药物，是在托珠单抗的基础上在可变区进行了5个突变：重链上的Phe51、Met57和Ile103，轻链上的Gln89和Arg93，使得206mab与IL-6R的亲和力提高数百倍，生物学活性提高30倍左右。SIRPα肽为天然变体，其可以为SIRPα肽所包含SIRPαV1、SIRPαV2、SIRPαV8三种变体及在其基础上进行氨基酸经取代、缺失、和/或添加一个或几个氨基酸，IL-6R抗体与所述SIRPα肽(其氨基酸序列和核苷酸序列如下所示)之间设有连接子，连接子序列在以下划线在对应序列中示出。In this embodiment, the structure of the bifunctional fusion protein of IL-6R antibody and SIRPα peptide is shown in Figure 1, wherein the IL-6R antibody is 206mab (its amino acid sequence and nucleotide sequence are shown below) and its mutant 206mab-mut (its amino acid sequence and nucleotide sequence are shown below), 206mab is a genetically modified drug of the interleukin-6 receptor monoclonal antibody drug tocilizumab (TCZ), which is based on tocilizumab and has 5 mutations in the variable region: Phe51, Met57 and Ile103 on the heavy chain, Gln89 and Arg93 on the light chain, which increases the affinity of 206mab to IL-6R by hundreds of times and the biological activity by about 30 times. The SIRPα peptide is a natural variant, which may be the three variants of SIRPα peptide, SIRPαV1, SIRPαV2, and SIRPαV8, and amino acids are substituted, deleted, and/or one or more amino acids are added thereto. A linker is provided between the IL-6R antibody and the SIRPα peptide (the amino acid sequence and nucleotide sequence of which are shown below), and the linker sequence is shown underlined in the corresponding sequence.

本实施例首先构建了206mab-L和融合基因载体PLS2124(206mab-H-SIRPαV1D1，具体序列如下所示)、PLS2125(206mab-H-SIRPαV2D1，具体序列如下所示)、PLS2126(206mab-H-SIRPαV1D1sg，具体序列如下所示)、PLS2127(206mab-H-SIRPαV2D1im，具体序列如下所示)，PLS2124、PLS2125、PLS2126、PLS2127通过和206mab-L共转HEK293瞬转表达，常规纯化获得双功能融合蛋白。In this example, 206mab-L and fusion gene vectors PLS2124 (206mab-H-SIRPαV1D1, the specific sequence is shown below), PLS2125 (206mab-H-SIRPαV2D1, the specific sequence is shown below), PLS2126 (206mab-H-SIRPαV1D1sg, the specific sequence is shown below), and PLS2127 (206mab-H-SIRPαV2D1im, the specific sequence is shown below) were first constructed. PLS2124, PLS2125, PLS2126, and PLS2127 were transiently expressed by co-transfection with 206mab-L in HEK293, and the bifunctional fusion protein was obtained by conventional purification.

进一步选择截短了的天然SIRPαV2D1的序列，同时对206mab-H进行了AEASS氨基酸突变(L236A、L237E、G239A、A332S、P333S)，获得206mab突变体206mab-mut去除了ADCC效应，用(G_nS)_m连接突变的206mab-H和SIRPαV2D1，与206mab-L***同一个载体，构建用于稳转CHO细胞的质粒PLT300(206mab-mut-SIRPαV2D1，具体序列如表1所示)。The truncated sequence of natural SIRPαV2D1 was further selected, and AEASS amino acid mutations (L236A, L237E, G239A, A332S, P333S) were performed on 206mab-H to obtain the 206mab mutant 206mab-mut that eliminated the ADCC effect. The mutant 206mab-H and SIRPαV2D1 were connected with ( _GnS ) _m and inserted into the same vector as 206mab-L to construct the plasmid PLT300 (206mab-mut-SIRPαV2D1, the specific sequence is shown in Table 1) for stable transfection of CHO cells.

表1实施例1涉及到的序列表
Table 1 Sequence Listing Related to Example 1

表1(续)
Table 1 (continued)

实施例2瞬转表达与蛋白纯化Example 2 Transient Expression and Protein Purification

转染前一天用KOP293配置50ml密度为1.2×10⁶个/ml的HEK293细胞，置于250ml三角瓶中，在135rpm，37℃、5％CO₂中培养20-25h，细胞密度达到2×10⁶个/ml左右，细胞处于对数生长期且活率在95％以上时方可用于转染实验，将50μg重组质粒加入Opti-MEM中，总体积为2ml，轻轻混匀，250μg PEI加入到Opti-MEM中，总体积为2ml，室温孵育5min。将2ml PEI稀释液，加入对应质粒稀释液中，混匀后室温孵育20min。轻轻旋转晃动含HEK293细胞的三角瓶，缓慢滴入PEI-DNA混合液，然后将转染后的细胞置于135rpm，37℃、5％CO₂摇床中培养，转染24h后，加入50×KT-Feed 1ml、500×VPA 100ul，转染5d后，3000rpm离心15min收集细胞培养液，将细胞表达上清液高速离心后，过滤去除杂质，获得的上清液用HiTrap MabSelect SuRe预装柱进行亲和层析。纯水冲洗柱子后用PB缓冲液平衡，然后将上清上样到层析柱进行结合，上样完毕后，用PB缓冲液冲洗至基线，然后用0.1M柠檬酸盐洗脱液洗脱蛋白，洗脱的蛋白用1M Tris-HCl中和后，适当浓缩，上样到PBS平衡好的Superdex200进一步纯化，将收集的蛋白浓缩至一定浓度后进行SDS-PAGE电泳鉴定结果见图2和图3。The day before transfection, use KOP293 to prepare 50ml of HEK293 cells with a density of 1.2×10 ⁶ /ml, place in a 250ml Erlenmeyer flask, and culture at 135rpm, 37℃, 5% CO ₂ for 20-25h. The cell density reaches about 2×10 ⁶ /ml. The cells are in the logarithmic growth phase and the viability is above 95% before they can be used for transfection experiments. Add 50μg of recombinant plasmid to Opti-MEM with a total volume of 2ml, mix gently, add 250μg of PEI to Opti-MEM with a total volume of 2ml, and incubate at room temperature for 5min. Add 2ml of PEI diluent to the corresponding plasmid diluent, mix well, and incubate at room temperature for 20min. Gently rotate and shake the flask containing HEK293 cells, slowly drip the PEI-DNA mixture, and then place the transfected cells in a shaker at 135rpm, 37℃, 5% _CO2 for culture. After 24h of transfection, add 50×KT-Feed 1ml and 500×VPA 100ul. After 5d of transfection, centrifuge at 3000rpm for 15min to collect the cell culture fluid. After high-speed centrifugation of the cell expression supernatant, filter to remove impurities, and use HiTrap MabSelect SuRe pre-packed column for affinity chromatography. After washing the column with pure water and balancing with PB buffer, the supernatant is loaded onto the chromatography column for binding. After loading, rinse with PB buffer to the baseline, and then elute the protein with 0.1M citrate eluent. After the eluted protein is neutralized with 1M Tris-HCl, it is appropriately concentrated and loaded onto Superdex200 balanced with PBS for further purification. After the collected protein is concentrated to a certain concentration, SDS-PAGE electrophoresis is performed to identify the results as shown in Figures 2 and 3.

实施例3亲和力测定Example 3 Affinity Determination

本实施例采用生物膜干涉技术(Bio-Layer Interferometry，BLI)测定亲和力。首先将瞬转表达双功融合能蛋白稀释至20nM，以400rpm/min的转速吸附到ProA探针上。CD47由400nM倍比稀释5个浓度梯度。设置程序，使稀释好的CD47分别在固定的时间内结合到附着了双功融合能蛋白的ProA探针上。结合完成之后，复合物的探针再转移至不含分析物的Q buffer中，使结合的分析物解离。利用gator仪器内置软件对数据进行拟合，获得KD值。结果见表2。This example uses bio-layer interferometry (BLI) to determine affinity. First, the transiently expressed dual-function fusion protein was diluted to 20nM and adsorbed onto the ProA probe at a speed of 400rpm/min. CD47 was diluted from 400nM to 5 concentration gradients. The program was set so that the diluted CD47 was bound to the ProA probe attached with the dual-function fusion protein within a fixed time. After the binding was completed, the probe of the complex was transferred to the Q buffer without the analyte to dissociate the bound analyte. The data was fitted using the built-in software of the gator instrument to obtain the KD value. The results are shown in Table 2.

表2双功能融合蛋白对CD47的亲和力
Table 2 Affinity of bifunctional fusion protein for CD47

实施例4生物学活性测定 Example 4 Biological Activity Assay

(1)206mab的生物学活性测定(1) Determination of biological activity of 206mab

采用293-IL6Res细胞/报告基因法检测206mab的生物学活性。用预热的无菌PBS润洗细胞3～5s，吸去PBS，加入胰酶消化，待细胞脱落时加基础培养基终止消化。离心弃上清，重悬并计数，将细胞密度调整为5×10⁵个细胞/ml，按照80μl/孔加入相应孔中。用基础培养基稀释IL6至50ng/ml，以10μl/孔加入到样品孔和阳性对照孔中，阴性孔加入10μl/孔基础培养基。用基础培养基稀释供试品至200μg/ml，并以5倍梯度稀释成8个浓度。以10μl/孔加入到96孔板相应的位置孔中，每个浓度设两个复孔，阳性及阴性对照组均相应加入10μl/孔基础培养基。将细胞板置于37℃、5％CO₂细胞培养箱中培养22h。将融化混匀并恢复至室温的One Lite检测试剂以100μl/孔加入到以上96孔板中，在振荡器中震荡混匀3min，静置3～5min，用多功能酶标仪读取化学发光值(RLU)。采用四参数拟合曲线对数据进行处理，以标准品或供试品浓度的对数为横坐标，以RLU值为纵坐标，计算供试品的IC50值，结果见图4，206mab的IC50值为0.1479，PLS2125(206mab-H-SIRPαV2D1)的IC50值为0.03919，206mab连接SIRPα组成双融合蛋白后，未影响206mab的抗体活性，且优于206mab参比品。The biological activity of 206mab was detected by 293-IL6Res cell/reporter gene method. Rinse the cells with preheated sterile PBS for 3-5 seconds, remove the PBS, add trypsin for digestion, and add basal medium to terminate digestion when the cells fall off. Centrifuge and discard the supernatant, resuspend and count, adjust the cell density to 5×10 ⁵ cells/ml, and add 80μl/well to the corresponding wells. Dilute IL6 to 50ng/ml with basal medium, add 10μl/well to the sample well and positive control well, and add 10μl/well basal medium to the negative well. Dilute the test sample to 200μg/ml with basal medium, and dilute it into 8 concentrations in a 5-fold gradient. Add 10μl/well to the corresponding position wells of the 96-well plate, set two replicates for each concentration, and add 10μl/well basal medium to the positive and negative control groups. Place the cell plate in a 37°C, 5% CO ₂ cell culture incubator for 22h. The melted, mixed and restored to room temperature One Lite detection reagent was added to the above 96-well plate at 100 μl/well, shaken and mixed in an oscillator for 3 minutes, left to stand for 3 to 5 minutes, and the chemiluminescence value (RLU) was read using a multifunctional microplate reader. The data was processed using a four-parameter fitting curve, with the logarithm of the concentration of the standard or test sample as the horizontal axis and the RLU value as the vertical axis to calculate the IC50 value of the test sample. The results are shown in Figure 4. The IC50 value of 206mab is 0.1479, and the IC50 value of PLS2125 (206mab-H-SIRPαV2D1) is 0.03919. After 206mab is connected to SIRPα to form a dual fusion protein, the antibody activity of 206mab is not affected, and it is better than the 206mab reference product.

(2)HTRF法测定SIRPα的生物学活性(2) HTRF method to determine the biological activity of SIRPα

96孔板中先加入4μl待测样品，顺序加入4μl Tag1-SIRPα，用移液器在加样孔内轻柔混合两次，再加入4μl Tag2-CD47，用移液器在加样孔内轻柔混合两次。将Anti-His-Eu和Anti-Fc-A2以体积1:1混合均匀，向反应体系加入8μl，用移液器在加样孔内轻柔混合两次。室温孵育2h，用多功能酶标仪(配置HTRF/TR-FRET模块)检测，激发光为320nm，检测两个波长(665nm和620nm)的发射光。将665nm的荧光值除以620nm的荧光值，获得665/620值，然后采用四参数拟合曲线对数据进行处理，以log10[样品终浓度]为横坐标，665/620的值为纵坐标，计算供试品的IC50值。结果见图5，PLS2125(206mab-H-SIRPαV2D1)的IC50值为1.883，说明SIRPα连接206mab后，基本未影响本身的生物学活性。First add 4μl of the sample to be tested to the 96-well plate, then add 4μl Tag1-SIRPα in sequence, mix gently twice in the well with a pipette, then add 4μl Tag2-CD47, mix gently twice in the well with a pipette. Mix Anti-His-Eu and Anti-Fc-A2 in a volume of 1:1, add 8μl to the reaction system, and mix gently twice in the well with a pipette. Incubate at room temperature for 2h, detect with a multifunctional microplate reader (configured with HTRF/TR-FRET module), with an excitation light of 320nm, and detect the emission light of two wavelengths (665nm and 620nm). Divide the fluorescence value of 665nm by the fluorescence value of 620nm to obtain the value of 665/620, and then use a four-parameter fitting curve to process the data, with log10 [final sample concentration] as the horizontal axis and the value of 665/620 as the vertical axis to calculate the IC50 value of the test product. The results are shown in FIG5 . The IC50 value of PLS2125 (206mab-H-SIRPαV2D1) is 1.883, indicating that the biological activity of SIRPα itself is not substantially affected after being linked to 206mab.

实施例5稳定细胞株的筛选与鉴定Example 5 Screening and identification of stable cell lines

转染前一天，将CHO-K1细胞调整密度为0.5×10⁶个细胞/mL。转染当天，准备经线性化处理、高浓度无内毒素的质粒，测定CHO-K1细胞密度及活率，保证细胞活率大于97％。CHO-K1细胞经CD CHO培养基洗涤两次后，取700μL细胞悬液，加入40μg质粒，混匀后转入4mm电极杯中，放入电转仪。设置电击参数为300V，1000μF，电击一次，将电击后的细胞悬液转入预热新鲜CD CHO培养基中，37℃孵育20min。将孵育后的细胞悬液均匀接种于96孔板中，转染24h后，进行加压，加入含有蛋氨酸亚氨基代砜(MSX)的CD CHO培养基，最终筛选压力为25～50μM MSX，5％CO₂，37℃静置培养。待96孔板中单克隆长至合适大小后，开始挑选单克隆，将所有克隆转至新的96孔板，5％CO₂，37℃静置培养。待孔内细胞长满后，取孔板中的上清进行还原电泳，检测融合蛋白表达情况，选出表达量最高细胞株进行有限稀释法筛选单克隆细胞株，按照0.3个细胞/孔接种96孔板，筛选得到3#3C9、5#5D7、6#6G4、7#7B3、8#8C4、9#9G5、10#10F9高表达细胞株，测定表达量，结果见表3；对8#8C4细胞株进行25mL体积的摇瓶流加培养，并用Mabselect sure和Superdex200对上清进行纯化。对纯化后的蛋白进行还原和非还原SDS-PAGE电泳鉴定(结果见图6)和SEC-HPLC鉴定(结果见图7)。选择稳定性好、表达量高的细胞株8#8C4进行成药性分析。One day before transfection, adjust the density of CHO-K1 cells to 0.5×10 ⁶ cells/mL. On the day of transfection, prepare linearized, high-concentration endotoxin-free plasmids, measure the density and viability of CHO-K1 cells, and ensure that the cell viability is greater than 97%. After washing CHO-K1 cells twice with CD CHO medium, take 700μL of cell suspension, add 40μg of plasmid, mix well, transfer to a 4mm electrode cup, and put it into the electroporator. Set the electroporation parameters to 300V, 1000μF, electroporate once, transfer the electroporated cell suspension to preheated fresh CD CHO medium, and incubate at 37℃ for 20min. The incubated cell suspension is evenly inoculated in a 96-well plate. After 24h of transfection, pressurize and add CD CHO medium containing methionine iminosulfone (MSX). The final screening pressure is 25-50μM MSX, 5% CO ₂ , and static culture at 37℃. After the single clones in the 96-well plate grow to a suitable size, start to select single clones and transfer all clones to a new 96-well plate and culture them at 5% CO ₂ and 37°C. After the cells in the wells are full, take the supernatant from the well plate for reduction electrophoresis to detect the expression of the fusion protein, and select the cell line with the highest expression for limiting dilution method Screen monoclonal cell lines, inoculate 96-well plates at 0.3 cells/well, screen 3#3C9, 5#5D7, 6#6G4, 7#7B3, 8#8C4, 9#9G5, 10#10F9 high-expression cell lines, measure the expression level, and the results are shown in Table 3; 8#8C4 cell line was cultured in a 25mL shake flask, and the supernatant was purified using Mabselect sure and Superdex200. The purified protein was identified by reducing and non-reducing SDS-PAGE electrophoresis (results shown in Figure 6) and SEC-HPLC (results shown in Figure 7). The cell line 8#8C4 with good stability and high expression level was selected for drugability analysis.

表3细胞株的蛋白表达量测定
Table 3 Protein expression determination of cell lines

实施例6成药性分析Example 6 Drugability Analysis

对筛选出的PLT300-8#8C4细胞株表达纯化的蛋白进行成药性分析。将蛋白分装6支，1ml/支。其中1支不处理，作为零点对照，同时对其进行cIEF鉴定，结果见图8，取另外2支，分别反复冻融3次和5次后，检测SEC-HPLC，结果见表4和图9；检测还原SDS-PAGE(R)和非还原SDS-PAGE(NR)，结果见图10。数据表明，冻融前后蛋白纯度一致，较稳定。另取3支分别于40℃水浴锅中，加速1周、2周和4周，检测SEC-HPLC，结果见表5和图11；检测206mab生物学活性，结果见表6；检测SIRPα生物学活性，结果见表7。结果表明，蛋白于40℃放置后，纯度和生物学活性均无明显变化，再次说明该蛋白有较好的稳定性。The drugability analysis of the protein expressed and purified by the screened PLT300-8#8C4 cell line was performed. The protein was divided into 6 tubes, 1 ml/tube. One of them was not treated and used as a zero-point control. At the same time, it was identified by cIEF. The results are shown in Figure 8. The other two tubes were taken and frozen and thawed 3 and 5 times respectively, and then SEC-HPLC was tested. The results are shown in Table 4 and Figure 9; the reduced SDS-PAGE (R) and non-reduced SDS-PAGE (NR) were tested, and the results are shown in Figure 10. The data showed that the purity of the protein was consistent before and after freezing and thawing, and it was relatively stable. Another 3 tubes were taken and accelerated in a 40°C water bath for 1 week, 2 weeks and 4 weeks, and SEC-HPLC was tested. The results are shown in Table 5 and Figure 11; the biological activity of 206mab was tested, and the results are shown in Table 6; the biological activity of SIRPα was tested, and the results are shown in Table 7. The results show that after the protein was placed at 40°C, there was no significant change in purity and biological activity, which once again shows that the protein has good stability.

表4冻融样品的SEC-HPLC检测结果
Table 4 SEC-HPLC test results of freeze-thaw samples

表5 40℃加速样品的SEC-HPLC检测结果
Table 5 SEC-HPLC test results of 40℃ accelerated samples

表6 40℃加速样品的Anti-IL6R生物学活性检测结果
Table 6 Anti-IL6R biological activity test results of samples accelerated at 40°C

表7 40℃加速样品的SIRPα阻断活性检测结果
Table 7 SIRPα blocking activity test results of samples accelerated at 40°C

实施例7人红细胞结合实验Example 7 Human red blood cell binding experiment

新鲜健康人血与PBS等体积混合后，300g离心5min得到细胞团，用PBS洗涤3-5次后，得到红细胞。用FACS buffer(PBS+5％BSA)重悬，调整细胞密度为2×10⁶个/ml，然后分别加入10μg/ml 206mab或10μg/ml PLT300，4℃孵育1小时，然后用FACS buffer(PBS+2％FBS)洗涤2次后，加入抗体Goat Anti-Human IgG(H+L)，冰上避光孵育30min，最后用FACS buffer洗涤2次后再重悬细胞。依次检测。FACS检测结果显示，对照抗体hu5F9对人的红细胞表面的天然CD47有较强的结合能力，双功能融合蛋白PLT300对人的红细胞表面的天然CD47几乎没有结合能力，提示上述双功能融合蛋白在安全方面的优势，结果见图12。Fresh healthy human blood was mixed with an equal volume of PBS, centrifuged at 300g for 5min to obtain a cell mass, and then washed with PBS for 3-5 times to obtain red blood cells. Resuspended with FACS buffer (PBS + 5% BSA), the cell density was adjusted to 2×10 ⁶ /ml, and then 10μg/ml 206mab or 10μg/ml PLT300 were added, incubated at 4°C for 1 hour, and then washed twice with FACS buffer (PBS + 2% FBS), and then the antibody Goat Anti-Human IgG (H+L) was added, incubated on ice in the dark for 30min, and finally washed twice with FACS buffer and resuspended. Detected in sequence. The FACS test results showed that the control antibody hu5F9 had a strong binding ability to the natural CD47 on the surface of human red blood cells, and the bifunctional fusion protein PLT300 had almost no binding ability to the natural CD47 on the surface of human red blood cells, indicating the safety advantages of the above-mentioned bifunctional fusion protein, as shown in Figure 12.

实施例8巨噬细胞吞噬活性实验Example 8 Macrophage phagocytic activity experiment

从新鲜人血中分离PBMC，利用CD14磁珠分选CD14+单核细胞。在巨噬细胞分化培养基(1640+10％FBS+50ng/ml GM-CSF)中诱导分化7天；之后再使用巨噬细胞分化培养基(1640+10％FBS+50ng/ml IFNr)处理2天。细胞刮刮取巨噬细胞，铺于6孔板中，每孔1×10⁶个细胞。实验当天，使用0.5μM的CFSE标记Raji细胞，室温15分钟。PBS清洗后，CFSE-Raji按3倍的细胞数量与巨噬细胞共孵育,同时加入PLT300(20μg/ml)、h5F9(20μg/ml)及PBS溶液。吞噬反应4小时后，移弃悬浮细胞，PBS清洗2次。细胞刮刮取细胞，离心及PBS重悬后，加入抗体Fc封闭剂以消除非特异性结合。随后加入CD14-APC抗体标记巨噬细胞，4℃反应30分钟。荧光抗体染色完成后，PBS清洗2次，使用流式细胞仪进行分析。通过APC标记的阳性细胞群圈定巨噬细胞，在此亚细胞群的基础上分析CFSE+阳性细胞的所占百分比，即为吞噬率，结果见图13。PBMCs were isolated from fresh human blood, and CD14+ monocytes were sorted using CD14 magnetic beads. Differentiation was induced in macrophage differentiation medium (1640+10% FBS+50ng/ml GM-CSF) for 7 days; then treated with macrophage differentiation medium (1640+10% FBS+50ng/ml IFNr) for 2 days. Macrophages were scraped and plated in 6-well plates, with 1×10 ⁶ cells per well. On the day of the experiment, Raji cells were labeled with 0.5μM CFSE for 15 minutes at room temperature. After washing with PBS, CFSE-Raji was incubated with macrophages at 3 times the number of cells, and PLT300 (20μg/ml), h5F9 (20μg/ml) and PBS solution were added at the same time. After phagocytosis reaction for 4 hours, the suspended cells were removed and washed twice with PBS. Cells were scraped, centrifuged and resuspended in PBS, and antibody Fc blocking agent was added to eliminate nonspecific binding. Then CD14-APC antibody was added to label macrophages and reacted at 4°C for 30 minutes. After fluorescent antibody staining, the cells were washed twice with PBS and analyzed using a flow cytometer. Macrophages were identified by the APC-labeled positive cell population, and the percentage of CFSE+ positive cells was analyzed based on this subpopulation, which was the phagocytic rate. The results are shown in Figure 13.

实施例9 IL-6R-CD47双功能融合蛋白对TF-1移植瘤人源化小鼠的药效学评价Example 9 Pharmacodynamic evaluation of IL-6R-CD47 bifunctional fusion protein on TF-1 humanized transplanted tumor mice

将人PBMC经尾静脉注射进入NSG重度免疫缺陷小鼠体内，然后在小鼠右肋部皮下接种TF-1细胞，共48只，随机分为6组(n＝8)：阴性对照组、IL-6R单抗组、CD47单抗组、IL-6R-CD47双功能融合蛋白低、中和高剂量组。以分组给药日期设为D1。给药时间为D1(细胞接种前1小鼠)、D8、D15。给药方式为静脉注射。数据记录有每周测2次瘤体积，称体重，共持续3周。各组动物体重、肿瘤体积以及瘤重均用平均值±标准误(Mean±SEM)表示，并用Graphpad Prism 9和Excel软件作图，使用单因素方差分析方法统计分析。Human PBMCs were injected into NSG severely immunodeficient mice via the tail vein, and then TF-1 cells were subcutaneously inoculated in the right rib of the mice. A total of 48 mice were randomly divided into 6 groups (n=8): negative control group, IL-6R monoclonal antibody group, CD47 monoclonal antibody group, IL-6R-CD47 bifunctional fusion protein low-dose, medium-dose and high-dose groups. The group administration date was set as D1. The administration time was D1 (1 mouse before cell inoculation), D8, and D15. The administration method was intravenous injection. The data were recorded by measuring the tumor volume and body weight twice a week for a total of 3 weeks. The body weight, tumor volume and tumor weight of each group of animals were expressed as mean ± standard error (Mean ± SEM), and graphed using Graphpad Prism 9 and Excel software, and statistically analyzed using one-way analysis of variance.

肿瘤体积(V)计算公式：V＝1/2×L_长×L_短 ² Tumor volume (V) calculation formula: V = 1/2 × L _long × L _short ²

相对体积(RTV)＝V_T/V₀ Relative volume (RTV) = _VT / _V0

抑瘤率(％)＝(C_RTV-T_RTV)/C_RTV(％)Tumor inhibition rate (%) = ( _CRTV - _TRTV )/ _CRTV (%)

其中V0、VT分别为实验开始时及结束时的肿瘤体积。C_RTV、T_RTV分别为实验结束时的空白对照组(Blank)及实验组的相对肿瘤体积。V0 and VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control group (Blank) and the experimental group at the end of the experiment, respectively.

本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。 The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

一种双功能融合蛋白，包括：特异性识别白细胞介素-6受体(IL-6R)的第一结构域，和特异性结合细胞表面蛋白CD47的第二结构域。A bifunctional fusion protein comprises: a first domain which specifically recognizes interleukin-6 receptor (IL-6R), and a second domain which specifically binds to cell surface protein CD47.
根据权利要求1所述的双功能融合蛋白，其特征在于，所述第一结构域为特异性识别IL-6R的抗体或其功能性片段，所述第二结构域为特异性结合CD47的信号调节蛋白α肽(SIRPα肽)或其功能性片段。The bifunctional fusion protein according to claim 1 is characterized in that the first domain is an antibody or a functional fragment thereof that specifically recognizes IL-6R, and the second domain is a signal regulatory protein α peptide (SIRPα peptide) or a functional fragment thereof that specifically binds to CD47.
根据权利要求1或2所述的双功能融合蛋白，其特征在于，所述第一结构域包括特异性结合IL-6R的抗体Fab片段、Fab’片段、F(ab’)2片段、Fv片段、scFv片段、纳米抗体、重链可变区(VH)片段或轻链可变区(VL)片段。The bifunctional fusion protein according to claim 1 or 2, characterized in that the first domain comprises an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment or light chain variable region (VL) fragment that specifically binds to IL-6R.
根据权利要求1至3中任一项所述的双功能融合蛋白，其特征在于，所述SIRPα肽选自SIRPαV1、SIRPαV2、SIRPαV8或者与SIRPαV1、SIRPαV2、SIRPαV8的氨基酸序列相比具有1、2或3个氨基酸的***、取代或缺失的肽。The bifunctional fusion protein according to any one of claims 1 to 3, characterized in that the SIRPα peptide is selected from SIRPαV1, SIRPαV2, SIRPαV8, or a peptide having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence of SIRPαV1, SIRPαV2, SIRPαV8.
根据权利要求1至4中任一项所所述的双功能融合蛋白，其特征在于，所述第一结构域和所述第二结构域直接连接或者通过连接子连接，The bifunctional fusion protein according to any one of claims 1 to 4, characterized in that the first domain and the second domain are directly connected or connected through a linker,

优选地，所述连接子具有如通式(GnS)m所示的氨基酸序列，n、m分别为1-10的整数；更优选地，n为1-4的整数，m为1-3的整数，或者与通式(GnS)m所示的氨基酸序列相比具有1、2或3个氨基酸的***、取代或缺失的氨基酸序列。Preferably, the linker has an amino acid sequence as shown in the general formula (GnS)m, where n and m are integers of 1-10 respectively; more preferably, n is an integer of 1-4, m is an integer of 1-3, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in the general formula (GnS)m.
根据权利要求1至5中任一项所述的双功能融合蛋白，其特征在于，所述第一结构域包括：重链，其具有如SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列；和，轻链，其具有如SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。The bifunctional fusion protein according to any one of claims 1 to 5 is characterized in that the first domain comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:1; and a light chain having an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:2.
根据权利要求6所述的双功能融合蛋白，其特征在于，对应于SEQ ID NO:1的第236、237、239、332、333位氨基酸中的一个或多个被取代；The bifunctional fusion protein according to claim 6, characterized in that one or more of the amino acids 236, 237, 239, 332, and 333 corresponding to SEQ ID NO: 1 are substituted;

优选地，对应于SEQ ID NO:1的第236位氨基酸被取代为丙氨酸、和/或第237位氨基酸被替换为谷氨酸、和/或第239位氨基酸被替换为丙氨酸、和/或第332位氨基酸被替换为丝氨酸、和/或第333位氨基酸被取代为丝氨酸；Preferably, the amino acid at position 236 corresponding to SEQ ID NO: 1 is substituted with alanine, and/or the amino acid at position 237 is substituted with glutamic acid, and/or the amino acid at position 239 is substituted with alanine, and/or the amino acid at position 332 is substituted with serine, and/or the amino acid at position 333 is substituted with serine;

优选地，第一结构域包括如SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。Preferably, the first domain comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:4.
根据权利要求1至7中任一项所述的双功能融合蛋白，其特征在于，所述第二结构域包括如SEQ ID NO:5～8中任一项所示的氨基酸序列或与SEQ ID NO:5～8中任一项具有至少约85％、90％、95％、98％、99％或100％序列同一性的氨基酸序列。 The bifunctional fusion protein according to any one of claims 1 to 7, characterized in that the second domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5 to 8, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs: 5 to 8.
一种分离的核酸分子，其编码如权利要求1至8中任一项所述的双功能融合蛋白。An isolated nucleic acid molecule encoding the bifunctional fusion protein according to any one of claims 1 to 8.
一种核酸递送载体，其包含如权利要求9所述的分离的核酸分子，优选地，所述核酸递送载体包括来源于腺病毒、腺相关病毒、慢病毒或其它可接受的核酸递送载体。A nucleic acid delivery vector comprising the isolated nucleic acid molecule according to claim 9, preferably, the nucleic acid delivery vector comprises a nucleic acid delivery vector derived from adenovirus, adeno-associated virus, lentivirus or other acceptable nucleic acid delivery vectors.
一种宿主细胞，其包含如权利要求10所述的分离的核酸分子。A host cell comprising the isolated nucleic acid molecule of claim 10.
一种药物组合物，其包括如权利要求1至8中任一项所述的双功能融合蛋白，如权利要求9所述的分离的核酸分子，或如权利要求10所述的核酸递送载体，以及药学上可接受的载体。A pharmaceutical composition comprising the bifunctional fusion protein according to any one of claims 1 to 8, the isolated nucleic acid molecule according to claim 9, or the nucleic acid delivery vector according to claim 10, and a pharmaceutically acceptable carrier.
根据权利要求12所述的药物组合物，其特征在于，所述药物组合物为片剂、散剂、颗粒剂、丸剂、注射剂、混悬液、粉剂、乳剂、气雾剂、凝胶剂、滴眼剂、缓释剂或缓释植入体的形式。The pharmaceutical composition according to claim 12, characterized in that the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained-release agents or sustained-release implants.
一种药盒，其包括如权利要求12或13所述的药物组合物，所述药物组合物被封装在容器中，所述容器优选是玻璃安瓿、玻璃瓶、塑料安瓿、塑料瓶、塑料袋或预装式注射器。A medicine kit comprising the pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutical composition is packaged in a container, and the container is preferably a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag or a prefilled syringe.
一种治疗或预防与IL-6和/或CD47相关的疾病的方法，其包括向受试者施用治疗有效量的如权利要求1至8中任一项所述的双功能融合蛋白，如权利要求9所述的分离的核酸分子，或如权利要求10所述的核酸递送载体。A method for treating or preventing a disease associated with IL-6 and/or CD47, comprising administering to a subject a therapeutically effective amount of the bifunctional fusion protein according to any one of claims 1 to 8, the isolated nucleic acid molecule according to claim 9, or the nucleic acid delivery vector according to claim 10.
根据权利要求15所述的方法，其特征在于，所述疾病选自骨髓增生异常综合征和癌症相关疾病，优选地，所述疾病选自前髓细胞性白血病、急性髓系白血病、淋巴系白血病、非霍奇金淋巴瘤、黑色素瘤、平滑肌肉瘤、星形细胞瘤、胶质母细胞瘤、多发性骨髓瘤、***癌、胃癌，肝癌、乳腺癌、肾癌、卵巢癌、输卵管癌、腹膜癌、结直肠癌、非小细胞肺癌、膀胱癌、胰腺癌、肝癌和***。 The method according to claim 15, characterized in that the disease is selected from myelodysplastic syndrome and cancer-related diseases, preferably, the disease is selected from promyelocytic leukemia, acute myeloid leukemia, lymphoid leukemia, non-Hodgkin's lymphoma, melanoma, leiomyosarcoma, astrocytoma, glioblastoma, multiple myeloma, prostate cancer, gastric cancer, liver cancer, breast cancer, kidney cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer, non-small cell lung cancer, bladder cancer, pancreatic cancer, liver cancer and cervical cancer.