CN117159685A - An amino acid sequence and its application and verification method in preparing drugs targeting Siglec molecules to treat cancer - Google Patents

本发明专利属于癌症靶向治疗的技术领域，具体公开了一种氨基酸序列及其在制备靶向SiglecE或Siglec9分子治疗胶质母细胞瘤和结直肠癌的药物中的应用，并公开了验证所述药物对癌症影响的方法。通过敲除SiglecE基因或注射SiglecE‑mFc蛋白，发现所述氨基酸序列对胶质母细胞瘤和结直肠癌产生肿瘤抑制作用。而该治疗策略，可应用于静脉注射剂型药物中，从而为免疫治疗提供新的治疗策略和药物，促进胶质母细胞瘤和结直肠癌患者的生存进展。

The patent of this invention belongs to the technical field of targeted cancer therapy. It specifically discloses an amino acid sequence and its application in preparing drugs targeting SiglecE or Siglec9 molecules to treat glioblastoma and colorectal cancer, and discloses the verification results. Describe the effects of drugs on cancer. By knocking out the SiglecE gene or injecting the SiglecE-mFc protein, the amino acid sequence was found to have a tumor suppressive effect on glioblastoma and colorectal cancer. This treatment strategy can be applied to intravenous injection drugs, thereby providing new treatment strategies and drugs for immunotherapy and promoting the survival progress of patients with glioblastoma and colorectal cancer.

一种氨基酸序列及其在制备靶向Siglec分子治疗癌症的药物 中的应用及验证方法An amino acid sequence and its use in preparing drugs targeting Siglec molecules to treat cancer Applications and verification methods in

技术领域Technical field

本发明涉及癌症靶向治疗的技术领域，更具体地，涉及一种氨基酸序列及其在制备靶向Siglec分子治疗癌症的药物中的应用。The present invention relates to the technical field of targeted cancer therapy, and more specifically, to an amino acid sequence and its application in preparing drugs targeting Siglec molecules to treat cancer.

背景技术Background technique

恶性脑肿瘤和其他中枢神经系统肿瘤虽然只占癌症病例小部分（约1%），但却是儿童和青少年中最常见的实体肿瘤，也是40岁以下男性和20岁以下女性癌症死亡的主要原因。恶性脑肿瘤最为常见的是胶质瘤，约占颅内肿瘤的40-50%，其中，胶质母细胞瘤（GBM）是发病率最高的胶质瘤。GBM是最具侵略性的脑肿瘤，起源于星形胶质细胞，其生长速度快，恶性程度高，多呈浸润性生长，患者病程短，预后差。虽然GBM近年来发病率无大幅度提升，但其患者生存率并无长足进展。经过长期努力，GBM的治疗有一定的进展，最大程度的手术切除，同时进行放疗或者放疗和辅助烷化剂化疗，2000年至2004年间和2005年至2014年间分别从34.4%提高到44.6%。尽管随着时间的推移，短期生存率逐渐提高，但5年生存率保持相对恒定，诊断后5年生存率仅为5.8%。显然需要进一步改进治疗策略，随着免疫治疗在其他肿瘤取得实质性进展，免疫治疗也进入了GBM的治疗方案候选。Malignant brain tumors and other central nervous system tumors, although accounting for a small proportion of cancer cases (about 1%), are the most common solid tumors in children and adolescents and the leading cause of cancer death in men under 40 years of age and women under 20 years of age. . The most common malignant brain tumor is glioma, accounting for approximately 40-50% of intracranial tumors. Among them, glioblastoma (GBM) is the glioma with the highest incidence rate. GBM is the most aggressive brain tumor, originating from astrocytes. It grows rapidly, is highly malignant, and mostly grows infiltratively. Patients have a short course of disease and poor prognosis. Although the incidence of GBM has not increased significantly in recent years, the survival rate of its patients has not made significant progress. After long-term efforts, the treatment of GBM has made certain progress, with maximum surgical resection and concurrent radiotherapy or radiotherapy and adjuvant alkylating agent chemotherapy, which increased from 34.4% to 44.6% between 2000 and 2004 and from 2005 to 2014 respectively. Although short-term survival rates have gradually improved over time, 5-year survival rates have remained relatively constant, with a 5-year survival rate of only 5.8% after diagnosis. There is clearly a need to further improve treatment strategies. As immunotherapy has made substantial progress in other tumors, immunotherapy has also become a candidate treatment option for GBM.

GBM是最具侵略性且高致死性的脑肿瘤，经过常规治疗，手术切除肿瘤病灶，同时辅助放疗和烷化剂化疗，中位无进展生存期是6.9个月，而中位生存期是14.6个月。肿瘤免疫疗法的出现，为脑肿瘤的治疗提供了新的方向。但GBM具有独一无二的肿瘤微环境，为其治疗大大增加了难度。GBM内存在大量的髓系细胞，报道称单核/巨噬细胞占GBM总免疫细胞群体的50%左右，易于形成免疫抑制性环境，促进肿瘤生长；因GBM患者经过常规治疗，放疗和化疗都会引起淋巴细胞的急剧减少，免疫疗法的策略是激活机体的免疫系统，大部分是激活T细胞来达到清除肿瘤的目的，淋巴细胞的减少严重影响治疗结果；因血脑屏障的存在，药物不易于进入脑组织内，又是一个疗效的限制因素。随着PD-L1/PD-1抗体在多种肿瘤取得阶段性疗效，也进入了GBM的临床试验，但结果甚不理想，在GBM III期临床试验中，与贝伐单抗（VEGF单抗）单药相比，贝伐单抗和nivolumab（PD-1抗体）联用，并未提高患者的生存期。GBM is the most aggressive and highly lethal brain tumor. After conventional treatment, surgical resection of tumor lesions, combined with adjuvant radiotherapy and alkylating agent chemotherapy, the median progression-free survival is 6.9 months, and the median survival is 14.6 months. months. The emergence of tumor immunotherapy provides a new direction for the treatment of brain tumors. However, GBM has a unique tumor microenvironment, which greatly increases the difficulty of its treatment. There are a large number of myeloid cells in GBM. It is reported that monocytes/macrophages account for about 50% of the total immune cell population in GBM, which are prone to forming an immunosuppressive environment and promoting tumor growth; because GBM patients undergo conventional treatment, radiotherapy and chemotherapy will Causes a sharp decrease in lymphocytes. The strategy of immunotherapy is to activate the body's immune system, mostly activating T cells to achieve the purpose of clearing tumors. The decrease in lymphocytes seriously affects the treatment results; due to the existence of the blood-brain barrier, drugs are not easily Entering brain tissue is another limiting factor in efficacy. As PD-L1/PD-1 antibodies have achieved phased efficacy in various tumors, they have also entered GBM clinical trials, but the results are not ideal. In the GBM phase III clinical trials, they were compared with bevacizumab (VEGF monoclonal antibody). ) Compared with single drugs, the combination of bevacizumab and nivolumab (PD-1 antibody) did not improve the survival of patients.

近年研究发现，唾液酸修饰的糖分子参与炎症反应和多种肿瘤调节过程，在多种肿瘤中都存在异常表达。唾液酸结合性免疫球蛋白样凝集素(sialic acid-bindingimmunoglobulin-like lectin，Siglec)可识别唾液酸修饰的糖蛋白或者糖脂分子，介导细胞间交流。Siglecs在多种免疫细胞表达，而大多数Siglecs成员含有免疫抑制性基序，可以调节和抑制免疫细胞的功能。在肿瘤微环境内，通过Siglecs-唾液酸轴，传递免疫抑制性信号，从而有利于肿瘤免疫逃逸。Recent studies have found that sialic acid-modified sugar molecules are involved in inflammatory responses and various tumor regulation processes, and are abnormally expressed in a variety of tumors. Sialic acid-binding immunoglobulin-like lectin (Siglec) can recognize sialic acid-modified glycoproteins or glycolipid molecules and mediate intercellular communication. Siglecs are expressed in a variety of immune cells, and most Siglecs members contain immunosuppressive motifs that can regulate and inhibit the function of immune cells. Within the tumor microenvironment, immunosuppressive signals are transmitted through the Siglecs-sialic acid axis, which is conducive to tumor immune escape.

Siglec9是Siglecs家族重要成员之一，由胞外区，跨膜区和胞内区组成，其胞内区包含两个免疫抑制性基序。主要表达于中性粒细胞，单核/巨噬细胞，而NK细胞和T细胞低表达，识别靶细胞上的a-2,3和a-2,6型唾液酸，活化其免疫抑制性基序，招募SHP1/SHP2，从而启动下游信号，抑制免疫功能。目前，Siglec9在感染性疾病和肿瘤领域都有报道：有文章报道，Siglec9参与HBV的复制，在HBV感染的患者，NK细胞上的Siglec9的水平下降。而Siglec9的表达水平与病毒的复制呈负相关。此外，阻断HBV感染者NK细胞上的Siglec9可以增加IFN-γ、TNF-α的分泌和CD107a的脱颗粒；文献报道胰腺癌上调其配体a-2,3型唾液酸的表达，通过与Siglec9的结合，调节巨噬细胞的分化，使其向M2型巨噬细胞分化，同时增强PD-L1和IL10的表达，促进胰腺癌的进展。Siglec9 is an important member of the Siglecs family. It consists of an extracellular region, a transmembrane region and an intracellular region. Its intracellular region contains two immunosuppressive motifs. Mainly expressed in neutrophils, monocytes/macrophages, and low expression in NK cells and T cells. It recognizes a-2,3 and a-2,6 sialic acid on target cells and activates its immunosuppressive genes. sequence, recruiting SHP1/SHP2, thereby initiating downstream signals and inhibiting immune function. Currently, Siglec9 has been reported in the fields of infectious diseases and tumors: some articles reported that Siglec9 is involved in the replication of HBV. In patients with HBV infection, the level of Siglec9 on NK cells decreases. The expression level of Siglec9 is negatively correlated with virus replication. In addition, blocking Siglec9 on NK cells in HBV-infected patients can increase the secretion of IFN-γ, TNF-α and the degranulation of CD107a; literature reports that pancreatic cancer upregulates the expression of its ligand a-2, type 3 sialic acid, through The binding of Siglec9 regulates the differentiation of macrophages into M2 macrophages, while enhancing the expression of PD-L1 and IL10, promoting the progression of pancreatic cancer.

此外，Siglec9在神经系统疾病也有研究，据报道，培养的小胶质细胞表达SiglecE(Siglec9的小鼠同源基因)，通过与其配体结合，降低对神经碎片的吞噬和降低了刺激后产生的超氧自由基，达到神经保护的作用。但Siglec9在GBM的研究甚少，这也是值得探讨的，因此，本方案构建了SiglecE基因敲除小鼠，旨在深入研究SiglecE在GBM中是否具有调控作用，以及是否可作为GBM的治疗靶点。In addition, Siglec9 has also been studied in neurological diseases. It is reported that cultured microglia express SiglecE (the mouse homolog of Siglec9), which reduces the phagocytosis of nerve debris and the production of nerve fragments after stimulation by binding to its ligand. Superoxide free radicals achieve neuroprotective effects. However, there are few studies on Siglec9 in GBM, which is worth exploring. Therefore, this project constructed SiglecE gene knockout mice, aiming to further study whether SiglecE has a regulatory role in GBM and whether it can be used as a therapeutic target for GBM. .

肿瘤微环境内，适应性免疫细胞之外，还存在大量固有免疫细胞，尤其是巨噬细胞，GBM亦然存在尤其多的巨噬细胞，因此系统性研究巨噬细胞的特征以期找到靶向巨噬细胞的靶点，单独或与T细胞靶点协作是GBM免疫疗法的新思路。Siglec9主要表达于巨噬细胞，在炎症性刺激或者肿瘤环境下表达升高，且在肿瘤发生过程中，其配体唾液酸表达异常增高，Siglec9是否介导巨噬细胞抑制效应免疫细胞和抑制免疫免疫细胞的抗肿瘤效应，是否可作为GBM治疗的靶点，都有待进一步研究。In the tumor microenvironment, in addition to adaptive immune cells, there are also a large number of innate immune cells, especially macrophages. GBM also has a particularly large number of macrophages. Therefore, the characteristics of macrophages are systematically studied in order to find targeted macrophages. Targets of phagocytes, alone or in collaboration with T cell targets, are new ideas in immunotherapy for GBM. Siglec9 is mainly expressed in macrophages, and its expression increases under inflammatory stimulation or tumor environment. During tumorigenesis, the expression of its ligand sialic acid increases abnormally. Does Siglec9 mediate macrophages to inhibit effector immune cells and suppress immunity? Whether the anti-tumor effect of immune cells can be used as a target for GBM treatment remains to be further studied.

综上所述，尽管免疫疗法为肿瘤患者带来福音，但仍然存在诸多疑惑和难题。如患者对肿瘤免疫治疗的反应不一、响应率较低和免疫逃逸现象等。GBM的肿瘤微环境极为特殊，免疫抑制性环境的形成、淋巴细胞的减少、血脑屏障的阻隔，为其治疗大大增加了难度。尽管目前开展了很多基于肿瘤免疫疗法的临床试验，但成功运用于临床的依然较少。目前临床上运用较多的PD-1/PD-L1通路的抗体药物因多种原因在GBM的治疗中并未取得良好疗效。Siglec家族尤其是Siglec9可能成为免疫疗法新靶点，这一新思路有待实验验证。因实验动物小鼠并无Siglec9基因，因此可以选择其同源基因SiglecE用于测试这一新思路的可行性。本方案构建了SiglecE基因敲除小鼠，旨在深入研究SiglecE在GBM中是否具有调控作用，以及是否可作为GBM的治疗靶点。In summary, although immunotherapy has brought good news to cancer patients, there are still many doubts and problems. For example, patients have varying responses to tumor immunotherapy, low response rates, and immune escape phenomena. The tumor microenvironment of GBM is extremely special. The formation of an immunosuppressive environment, the decrease of lymphocytes, and the obstruction of the blood-brain barrier greatly increase the difficulty of its treatment. Although many clinical trials based on tumor immunotherapy have been carried out, there are still few successful clinical applications. At present, antibody drugs of the PD-1/PD-L1 pathway, which are used more clinically, have not achieved good results in the treatment of GBM due to various reasons. The Siglec family, especially Siglec9, may become a new target for immunotherapy. This new idea needs to be experimentally verified. Since the experimental animal mouse does not have the Siglec9 gene, its homologous gene SiglecE can be selected to test the feasibility of this new idea. This protocol constructed SiglecE gene knockout mice, aiming to further study whether SiglecE has a regulatory role in GBM and whether it can be used as a therapeutic target for GBM.

发明内容Contents of the invention

本发明旨在克服上述现有技术至少一项的不足，提供一种氨基酸序列，用于制备靶向Siglec分子治疗癌症的药物，所述药物为静脉注射剂型，可抑制肿瘤增长，提高胶质母细胞瘤和结直肠癌的患者生存率。The present invention aims to overcome at least one of the deficiencies of the above-mentioned prior art and provide an amino acid sequence for preparing a drug targeting Siglec molecules to treat cancer. The drug is an intravenous injection dosage form, which can inhibit tumor growth and improve glioblastoma. Survival rates in patients with neoplasia and colorectal cancer.

本发明的一个目的在于提供所述氨基酸序列在制备治疗胶质母细胞瘤、结直肠癌的药物中的用途。进一步地，所述药物包括静脉注射剂型。One object of the present invention is to provide the use of the amino acid sequence in preparing drugs for treating glioblastoma and colorectal cancer. Further, the medicine includes intravenous injection dosage form.

进一步地，SiglecE-mFc氨基酸序列的设计过程和如下所示。SiglecE分子有胞外区，跨膜区和胞内区组成，胞内区含有两个免疫抑制性基序，其胞外区由2个免疫球蛋白样结构域和一个识别结合唾液酸的V结构域组成。所述胞外区和小鼠的IgG-Fc融合表达成SiglecE-mFc，其氨基酸序列为：Further, the design process of SiglecE-mFc amino acid sequence is as follows. The SiglecE molecule consists of an extracellular region, a transmembrane region and an intracellular region. The intracellular region contains two immunosuppressive motifs, and its extracellular region consists of two immunoglobulin-like domains and a V structure that recognizes and binds sialic acid. Domain composition. The extracellular region and mouse IgG-Fc are fused and expressed to form SiglecE-mFc, whose amino acid sequence is:

MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQDHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKMLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQ DHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

本发明的再一目的在于提供一种SiglecE-mFc对癌症影响的验证方法，包括步骤：Another object of the present invention is to provide a verification method for the effect of SiglecE-mFc on cancer, including the steps:

S1、以敲除SiglecE基因的小鼠为实验组，野生型小鼠为对照组，分别进行肿瘤细胞接种，构建皮下GBM模型和颅内原位GBM模型，并监测肿瘤进展，绘制生存曲线；若基因敲除小鼠肿瘤生长速度显著较慢或发病明显较晚，且生存期明显较长，则证明SiglecE基因与肿瘤生长密切相关。S1. SiglecE gene knockout mice were used as the experimental group and wild-type mice were used as the control group. Tumor cells were inoculated respectively to construct subcutaneous GBM models and intracranial orthotopic GBM models. Tumor progression was monitored and survival curves were drawn; if The tumor growth rate of gene knockout mice is significantly slower or the onset is significantly later, and the survival period is significantly longer, which proves that the SiglecE gene is closely related to tumor growth.

S2、两组野生型小鼠，构建颅内原位GBM模型、皮下GBM模型、皮下结直肠癌模型，一组注射SiglecE-mFc蛋白，另一组注射安慰剂，定期给药，并监测肿瘤进展，绘制生存曲线；若SiglecE-mFc给药组小鼠肿瘤生长速度显著减缓或颅内原位癌模型发病时间晚，且生存期显著延长，则证明SiglecE-mFc有显著抑制肿瘤生长的作用。S2. Two groups of wild-type mice were constructed to construct an intracranial orthotopic GBM model, a subcutaneous GBM model, and a subcutaneous colorectal cancer model. One group was injected with SiglecE-mFc protein, and the other group was injected with placebo. The drugs were administered regularly and tumor progression was monitored. , draw a survival curve; if the tumor growth rate of the mice in the SiglecE-mFc administration group is significantly slowed down or the intracranial carcinoma in situ model has a later onset time and the survival period is significantly prolonged, it proves that SiglecE-mFc can significantly inhibit tumor growth.

与现有技术相比，本发明的有益效果为：本发明所述的氨基酸序列SiglecE-mFc具有显著抑制肿瘤生长的作用，通过敲除SiglecE基因或注射SiglecE-mFc蛋白，证明其对胶质母细胞瘤和结直肠癌产生肿瘤抑制作用。而该治疗策略，可应用于静脉注射剂型药物中，从而为免疫治疗提供新的治疗策略和药物，促进胶质母细胞瘤和结直肠癌患者的生存进展。Compared with the existing technology, the beneficial effects of the present invention are: the amino acid sequence SiglecE-mFc described in the present invention has a significant effect on inhibiting tumor growth. By knocking out the SiglecE gene or injecting the SiglecE-mFc protein, it is proved that it has an effect on glial matrix. Cytoma and colorectal cancer produce tumor suppressive effects. This treatment strategy can be applied to intravenous injection drugs, thereby providing new treatment strategies and drugs for immunotherapy and promoting the survival progress of patients with glioblastoma and colorectal cancer.

附图说明Description of drawings

图1显示SiglecE基因敲除小鼠的构建；a. SiglecE基因敲除小鼠构建模式图；b.流式检测WT和SiglecE ^-/-小鼠脾脏各免疫细胞SiglecE的表达情况。Figure 1 shows the construction of SiglecE gene knockout mice; a. Construction model of SiglecE gene knockout mice; b. Flow cytometry detection of SiglecE expression in immune cells in the spleens of WT and SiglecE ^-/- mice.

图2显示SiglecE缺失抑制GBM生长；a.CT2A小鼠脑胶质瘤细胞在WT和SiglecE ^-/-小鼠皮下肿瘤生长曲线；b.CT2A小鼠脑胶质瘤细胞在WT和SiglecE ^-/-小鼠颅内原位接种小鼠生存率；c.CT2A小鼠脑胶质瘤细胞在WT和SiglecE ^-/-小鼠颅内原位接种小鼠肿瘤代表图；d.CT2A细胞在WT和SiglecE ^-/-小鼠颅内原位接种后，小鼠免疫细胞浸润情况。（^* p<0.05,^** p<0.01,^*** p<0.001）Figure 2 shows that SiglecE deletion inhibits GBM growth; a. Subcutaneous tumor growth curve of CT2A mouse glioma cells in WT and SiglecE ^-/- mice; b. CT2A mouse glioma cells in WT and SiglecE ^-/- Survival rate of mice in situ intracranial inoculation of mice; c. Representative images of mouse tumors inoculated intracranially with CT2A mouse glioma cells in WT and SiglecE ^-/- mice; d. CT2A cells in WT and SiglecE ^-/- Immune cell infiltration in mice after intracranial in situ inoculation. ( ^* p <0.05, ^** p <0.01, ^*** p <0.001)

图3 显示SiglecE缺失抑制GBM生长依赖T细胞和巨噬细胞；CT2A小鼠脑胶质瘤细胞在WT和SiglecE ^-/-小鼠皮下接种后，a.anti-CD4抗体剔除CD4 T细胞后，小鼠肿瘤生长曲线；b.anti-CD8抗体剔除CD8 T细胞后，小鼠肿瘤生长曲线；c.anti-CSF1R抗体剔除巨噬细胞后，小鼠肿瘤生长曲线。（^* p<0.05,^** p<0.01,^*** p<0.001）Figure 3 shows that SiglecE deletion inhibits the growth of GBM-dependent T cells and macrophages; after CT2A mouse glioma cells were inoculated subcutaneously in WT and SiglecE ^-/- mice, a. After anti-CD4 antibody deletion of CD4 T cells, small Mouse tumor growth curve; b. Mouse tumor growth curve after anti-CD8 antibody depletes CD8 T cells; c. Mouse tumor growth curve after anti-CSF1R antibody depletes macrophages. ( ^* p <0.05, ^** p <0.01, ^*** p <0.001)

图4 显示SiglecE-mFc抑制小鼠GBM和结直肠癌生长；a.WT小鼠颅内原位接种CT2A细胞，用Ctrl-FC和SiglecE-mFC蛋白治疗的小鼠生存曲线；b.WT小鼠皮下接种CT2A细胞，用Ctrl-FC和SiglecE-mFC蛋白治疗的小鼠肿瘤生长曲线；c.WT小鼠皮下接种小鼠结直肠癌MC38细胞，用Ctrl-FC和SiglecE-mFC蛋白治疗的小鼠肿瘤生长曲线。（^* p<0.05,^** p<0.01,^*** p<0.001）Figure 4 shows that SiglecE-mFc inhibits the growth of GBM and colorectal cancer in mice; a. Survival curve of WT mice inoculated intracranially with CT2A cells and treated with Ctrl-FC and SiglecE-mFC proteins; b. WT mice Tumor growth curve of mice inoculated subcutaneously with CT2A cells and treated with Ctrl-FC and SiglecE-mFC proteins; c. WT mice inoculated subcutaneously with mouse colorectal cancer MC38 cells and treated with Ctrl-FC and SiglecE-mFC proteins. Tumor growth curve. ( ^* p <0.05, ^** p <0.01, ^*** p <0.001)

具体实施方式Detailed ways

应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this application belongs.

需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular forms are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, it will be understood that when the terms "comprises" and/or "includes" are used in this specification, they indicate There are features, steps, operations, means, components and/or combinations thereof.

现结合具体实例对本发明作进一步的说明，以下实施例仅是为了解释本发明，但不构成对本发明的限制。在以下实施例中所用到的试验样本及试验过程包括以下内容（如果实施例中未注明的实验具体条件，通常按照常规条件，或按照试剂公司所推荐的条件；下述实施例中所用的试剂、耗材等，如无特殊说明，均可从商业途径得到）。The present invention will be further described with reference to specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions, or in accordance with the conditions recommended by the reagent company; the conditions used in the following examples Reagents, consumables, etc., can be obtained from commercial sources unless otherwise specified).

实施例1Example 1

一种靶向Siglec分子治疗癌症的生物大分子药物，所述药物的氨基酸序列由SiglecE分子的胞外区与小鼠IgG-Fc融合表达而成，序列为：A biological macromolecule drug targeting Siglec molecule to treat cancer. The amino acid sequence of the drug is expressed by fusion of the extracellular region of the SiglecE molecule and mouse IgG-Fc. The sequence is:

MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQDHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQ DHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

所述靶向Siglec为小鼠的SiglecE蛋白或人Siglec9蛋白。The targeted Siglec is mouse SiglecE protein or human Siglec9 protein.

所述癌症为胶质母细胞瘤或结直肠癌。The cancer is glioblastoma or colorectal cancer.

所述药物为静脉注射剂型。The medicine is in the form of intravenous injection.

实施例2Example 2

一种靶向Siglec分子治疗癌症的多肽药物，所述药物的氨基酸序列由SiglecE分子的胞外区与小鼠IgG-Fc融合表达而成，序列为：A polypeptide drug targeting Siglec molecule to treat cancer. The amino acid sequence of the drug is expressed by fusion of the extracellular region of the SiglecE molecule and mouse IgG-Fc. The sequence is:

MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQDHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQ DHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

所述靶向Siglec为小鼠的SiglecE蛋白或人Siglec9蛋白。The targeted Siglec is mouse SiglecE protein or human Siglec9 protein.

所述癌症为胶质母细胞瘤或结直肠癌。The cancer is glioblastoma or colorectal cancer.

所述药物为静脉注射剂型。The medicine is in the form of intravenous injection.

实施例3Example 3

一种靶向Siglec分子治疗癌症的蛋白质药物，所述药物的氨基酸序列由SiglecE分子的胞外区与小鼠IgG-Fc融合表达而成，序列为：A protein drug targeting Siglec molecule to treat cancer. The amino acid sequence of the drug is expressed by fusion of the extracellular region of the SiglecE molecule and mouse IgG-Fc. The sequence is:

MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQDHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。MLLLLLLLLLWGIKGVEGQNPQEVFTLNVERKVVVQEGLCVLVPCNFSYLKKRLTDWTDSDPVHGFWYREGTDRRKDSIVATNNPIRKAVKETRNRFFLLGDPWRNDCSLNIREIRKKDAGLYFFRLERGKTKYNYMWDKMTLVVTALTNTPQILLPETLEAGHPSNLTCSVPWDCGWTAPPIFSWTGTSVSFLSTNTTGSSVLTITPQPQ DHGTNLTCQVTLPGTNVSTRMTIRLNVSYAPKNLTVTIYQGADSVSTILKNGSSLPISEGQSLRLICSTDSYPPANLSWSWDNLTLCPSKLSKPGLLELFPVHLKHGGVYTCQAQHALGSQHISLSLSPQSSATLSEMMMMGTFGSENLYFQGPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

所述靶向Siglec为小鼠的SiglecE蛋白或人Siglec9蛋白。The targeted Siglec is mouse SiglecE protein or human Siglec9 protein.

所述癌症为胶质母细胞瘤或结直肠癌。The cancer is glioblastoma or colorectal cancer.

所述药物为静脉注射剂型。The medicine is in the form of intravenous injection.

实施例4Example 4

所述氨基酸序列在制备治疗癌症的药物中的应用。Application of the amino acid sequence in the preparation of drugs for treating cancer.

实施例5Example 5

一、构建基因敲除小鼠1. Construction of gene knockout mice

本方案利用CRISPR/Cas9 技术，删除SiglecE基因的1-7外显子，删除片段全长共计8.7kbp，构建SiglecE全基因敲除纯合小鼠，SiglecE基因敲除小鼠构建模式图如图1中的a所示。在该基因敲除小鼠上研究其对GBM肿瘤发生发展的影响以及治疗的潜在功能。This plan uses CRISPR/Cas9 technology to delete exons 1-7 of the SiglecE gene, with a total length of 8.7kbp of deleted fragments, to construct SiglecE full gene knockout homozygous mice. The construction model of SiglecE gene knockout mice is shown in Figure 1 As shown in a. The effect on the occurrence and development of GBM tumors and the potential function of treatment were studied in this gene knockout mouse.

二、构建成瘤模型，观测SiglecE分子对GBM生长的影响2. Construct a tumor model and observe the effect of SiglecE molecules on GBM growth.

本方案共构建了两个GBM模型以研究SiglecE敲除对GBM生长的影响：In this project, two GBM models were constructed to study the effect of SiglecE knockout on GBM growth:

1.CT2A细胞系皮下成瘤模型1. CT2A cell line subcutaneous tumor model

同年龄和性别的野生型C57BL/6J小鼠和SiglecE敲除小鼠，7-8周，小鼠右背侧皮下接种300万CT2A细胞，接种后第六天开始测量肿瘤，每三天测量一次，计算肿瘤体积：V(mm3) = L(mm) x W(mm) x H(mm)，统计野生型和基因敲除小鼠的肿瘤生长曲线，如图2中的a所示。皮下肿瘤模型的对比结果显示，基因敲除小鼠肿瘤生长速度显著慢于野生型小鼠。Wild-type C57BL/6J mice and SiglecE knockout mice of the same age and gender were inoculated subcutaneously with 3 million CT2A cells on the right dorsal side of the mice at 7-8 weeks. Tumors were measured starting on the sixth day after inoculation and measured once every three days. , calculate the tumor volume: V(mm3) = L(mm) x W(mm) x H(mm), and count the tumor growth curves of wild-type and gene knockout mice, as shown in a in Figure 2. Comparative results of subcutaneous tumor models showed that the tumor growth rate of gene knockout mice was significantly slower than that of wild-type mice.

2.CT2A细胞系颅内原位成瘤模型2. CT2A cell line intracranial tumor model in situ

同年龄和性别的野生型C57BL/6J小鼠和SiglecE敲除小鼠，7-8周，在小鼠右脑室处接种5万CT2A细胞，观测小鼠发病情况并记录小鼠死亡时间，绘制小鼠生存曲线，如图2中的b所示。颅内原位接种模型的对比结果显示，基因敲除小鼠发病明显晚于野生型小鼠，且生存期明显长于野生型小鼠。在接种肿瘤后第21天，取脑组织，进行HE染色，如图2中的c所示，显示基因敲除小鼠肿瘤明显小于野生型小鼠。Wild-type C57BL/6J mice and SiglecE knockout mice of the same age and gender were inoculated with 50,000 CT2A cells in the right ventricle of the mice at 7-8 weeks. The disease of the mice was observed and the time of death of the mice was recorded and drawn. The mouse survival curve is shown in b in Figure 2. Comparative results of the intracranial in situ inoculation model showed that the gene knockout mice developed disease significantly later than wild-type mice, and their survival period was significantly longer than that of wild-type mice. On the 21st day after tumor inoculation, brain tissue was taken and HE stained, as shown in c in Figure 2, which showed that the tumors of the knockout mice were significantly smaller than those of the wild-type mice.

3.结论3.Conclusion

综合皮下成瘤模型和颅内原位成瘤模型结果可知，SiglecE敲除后能显著抑制GBM细胞的生长和延长小鼠的生存期。Based on the results of the subcutaneous tumor model and the intracranial orthotopic tumor model, it can be seen that SiglecE knockout can significantly inhibit the growth of GBM cells and prolong the survival of mice.

三、检测肿瘤微环境，鉴定效应免疫细胞3. Detect tumor microenvironment and identify effector immune cells

CT2A细胞系颅内原位成瘤模型至接种后21天，解剖小鼠取出脑组织，分离出肿瘤组织，用肿瘤消化液制备单细胞悬液，流式检测SiglecE基因敲除小鼠肿瘤微环境内各免疫细胞浸润的差异，如图2中的d所示。结果显示，与野生型小鼠相比，基因敲除小鼠整体的免疫细胞浸润比例从5%增加到10%，巨噬细胞、CD8+T细胞和CD4+T细胞的浸润都增加了一倍。CT2A cell line intracranial tumor model was established 21 days after inoculation. The mice were dissected to remove the brain tissue, and the tumor tissue was isolated. Single cell suspension was prepared with tumor digestion fluid, and the tumor microenvironment of SiglecE gene knockout mice was detected by flow cytometry. The difference in infiltration of various immune cells within the cell is shown in d in Figure 2. The results showed that compared with wild-type mice, the overall immune cell infiltration proportion of gene knockout mice increased from 5% to 10%, and the infiltration of macrophages, CD8+T cells, and CD4+T cells doubled. .

考虑到观察的方便性，本方案效应免疫细胞的鉴定采用了皮下肿瘤模型。7-8周的同性别的野生型和SiglecE基因敲除小鼠，在接种肿瘤细胞前一天，分别用巨噬细胞和T细胞单克隆抗体CSF1R抗体、CD8+抗体和CD4+抗体，剔除巨噬细胞、CD8+T细胞和CD4+T细胞，并绘制各组小鼠肿瘤生长曲线，如图3所示。结果显示，剔除巨噬细胞或剔除T细胞后，两组小鼠的肿瘤生长趋于一致。以上结果充分表明，巨噬细胞和T细胞在GBM发挥了重要的抗肿瘤功能。Considering the convenience of observation, the subcutaneous tumor model was used for the identification of effector immune cells in this protocol. Wild-type and SiglecE gene knockout mice of the same sex at 7-8 weeks old were treated with macrophage and T cell monoclonal antibodies CSF1R antibody, CD8+ antibody and CD4+ antibody one day before inoculation of tumor cells, respectively, to eliminate macrophages, CD8+T cells and CD4+T cells, and the tumor growth curves of mice in each group were drawn, as shown in Figure 3. The results showed that after deleting macrophages or deleting T cells, the tumor growth of the two groups of mice tended to be consistent. The above results fully demonstrate that macrophages and T cells play important anti-tumor functions in GBM.

实施例6Example 6

通过注射SiglecE-Fc氨基酸序列，观测其对小鼠的GBM和结直肠癌模型是否产生肿瘤抑制作用，从而验证SiglecE-Fc功能。By injecting the SiglecE-Fc amino acid sequence, we observe whether it has a tumor inhibitory effect on GBM and colorectal cancer models in mice, thereby verifying the function of SiglecE-Fc.

1.GBM颅内成瘤模型1. GBM intracranial tumor model

7-8周的同性别的野生型小鼠，颅内原位接种CT2A细胞后第六天，尾静脉注射50μgSiglecE-mFc进行治疗，每三天给药一次，共给药四次，观察并记录小鼠死亡时间，绘制生存曲线，如图4中的a所示。结果显示，SiglecE-mFc给药组小鼠发病时间晚，且生存期显著延长。Wild-type mice of the same sex aged 7-8 weeks were treated with 50 μg SiglecE-mFc injected into the tail vein on the sixth day after intracranial inoculation of CT2A cells, once every three days for a total of four administrations, observed and recorded. The time of death of the mice was determined and a survival curve was drawn, as shown in a in Figure 4. The results showed that mice in the SiglecE-mFc administration group developed disease later and had significantly longer survival.

2.GBM/结直肠癌的皮下成瘤模型2. Subcutaneous tumorigenesis model of GBM/colorectal cancer

7-8周的同性别的野生型小鼠，皮下接种CT2A细胞或者结直肠癌MC38细胞，待肿瘤生长至100mm3左右，尾静脉注射50ug SiglecE-mFc进行治疗，每三天给药一次，共给药四次，测量并记录小鼠肿瘤的体积，绘制肿瘤生长曲线，如图4中的b、c所示。结果显示，SiglecE-mFc给药组小鼠生长速度显著减缓，且GBM存在40%左右小鼠肿瘤达到治愈或基本治愈。Wild-type mice of the same sex that are 7-8 weeks old are inoculated subcutaneously with CT2A cells or colorectal cancer MC38 cells. When the tumor grows to about 100mm3, 50ug SiglecE-mFc is injected into the tail vein for treatment. The drug is administered once every three days for a total of The drug was administered four times, the volume of mouse tumors was measured and recorded, and the tumor growth curve was drawn, as shown in b and c in Figure 4. The results showed that the growth rate of mice in the SiglecE-mFc administration group was significantly slowed down, and about 40% of mice with GBM had their tumors cured or basically cured.

显然，本发明的上述实施例仅是为清楚地说明本发明技术方案所作的举例，而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Obviously, the above-mentioned embodiments of the present invention are only examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.