CN116603062A - Preparation method and application of a ROS-responsive antibody-drug complex - Google Patents
Preparation method and application of a ROS-responsive antibody-drug complex Download PDFInfo
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- CN116603062A CN116603062A CN202310270848.5A CN202310270848A CN116603062A CN 116603062 A CN116603062 A CN 116603062A CN 202310270848 A CN202310270848 A CN 202310270848A CN 116603062 A CN116603062 A CN 116603062A
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Abstract
Description
技术领域technical field
本发明涉及肿瘤药物技术领域，特别地涉及一种ROS响应的抗体药物复合体的制备方法及其应用。The invention relates to the technical field of tumor drugs, in particular to a preparation method and application of a ROS-responsive antibody drug complex.
背景技术Background technique
肿瘤是全球日益严重的健康问题，传统的肿瘤治疗方法主要包括手术、放疗和化疗。但术中肿瘤组织不易识别，原有癌细胞易转移会导致术后易复发。化疗和放疗对正常组织也有显著的杀伤作用，毒副作用较大，肿瘤细胞也容易对化疗和放疗产生耐受。随着肿瘤免疫学的发展，免疫治疗已成为肿瘤临床治疗的重要手段。然而，随着抗PD-1(programmeddeath 1)和抗CTLA-4(cytotoxic T lymphocyte-associated antigen-4)等免疫检测点阻断抗体在肿瘤免疫治疗中得到应用，免疫系统异常激活导致的免疫相关不良反应也不可避免，比如自身反应性T细胞的异常活化引发的心肌炎、肝炎、肺炎等，特别是免疫性心肌炎和致死性心力衰竭，能够显著增加肿瘤患者的死亡率。因此，改善免疫检查点抑制剂的毒副作用且不降低其疗效是实体瘤药物研发的一个重要方向。Tumor is an increasingly serious health problem in the world. Traditional tumor treatment methods mainly include surgery, radiotherapy and chemotherapy. However, it is not easy to identify the tumor tissue during the operation, and the original cancer cells are easy to transfer, which will lead to easy recurrence after operation. Chemotherapy and radiotherapy also have a significant killing effect on normal tissues, with relatively large toxic and side effects, and tumor cells are prone to resistance to chemotherapy and radiotherapy. With the development of tumor immunology, immunotherapy has become an important means of clinical treatment of tumors. However, with the application of immune checkpoint blocking antibodies such as anti-PD-1 (programmed death 1) and anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) in tumor immunotherapy, immune-related abnormalities caused by abnormal activation of the immune system Adverse reactions are also inevitable, such as myocarditis, hepatitis, pneumonia, etc. caused by abnormal activation of autoreactive T cells, especially immune myocarditis and fatal heart failure, which can significantly increase the mortality of cancer patients. Therefore, improving the toxic and side effects of immune checkpoint inhibitors without reducing their efficacy is an important direction for the development of solid tumor drugs.
此外，如何对癌症患者进行早期诊断和规范治疗也是临床面临的困境和难题。临床肿瘤诊断的方法主要包括病理学检查、生物标志物检测和影像学诊断，其中影像学诊断技术可以鉴别多种肿瘤，突破了生物标志物检查的局限性，是目前肿瘤诊断的最佳选择。纳米材料因其独特的理化性质，也被广泛应用于肿瘤的诊疗一体化中，且在多种肿瘤的诊疗中发展极其迅速。荧光探针可以通过高灵敏度的相互作用特异性地识别或打开成像信号，可激活探针在到达目标之前保持“关闭”状态，当特定反应或刺激发生时，这些探针可以被激活，从而恢复光信号，具有广阔的应用前景。In addition, how to conduct early diagnosis and standardized treatment of cancer patients is also a clinical dilemma and difficult problem. Clinical tumor diagnosis methods mainly include pathological examination, biomarker detection and imaging diagnosis. Among them, imaging diagnosis technology can identify a variety of tumors, breaking through the limitations of biomarker examination, and is currently the best choice for tumor diagnosis. Due to their unique physical and chemical properties, nanomaterials are also widely used in the integration of diagnosis and treatment of tumors, and have developed extremely rapidly in the diagnosis and treatment of various tumors. Fluorescent probes can specifically recognize or turn on imaging signals through high-sensitivity interactions, activatable probes remain "off" until they reach their target, and can be activated when a specific response or stimulus occurs, thereby restoring Optical signals have broad application prospects.
研究表明，恶性肿瘤部位的微环境具有高ROS的特点。因此，为了减少药物对正常组织细胞的毒副作用，期望能够得到一种在体内运输过程中可以稳定存在，靶向肿瘤部位响应肿瘤微环境的高ROS，进而连接子发生解聚释放抗体激活肿瘤免疫以达到治疗肿瘤的目的，同时通过荧光信号的变化实现对肿瘤的诊断的纳米复合物，在肿瘤的诊疗中显示出巨大的应用潜力。Studies have shown that the microenvironment of malignant tumor sites is characterized by high ROS. Therefore, in order to reduce the toxic and side effects of drugs on normal tissue cells, it is expected to obtain a high ROS that can exist stably during in vivo transportation, target the tumor site and respond to the tumor microenvironment, and then depolymerize the linker to release antibodies to activate tumor immunity. In order to achieve the purpose of treating tumors and realize the diagnosis of tumors through the change of fluorescent signal, the nanocomposite has shown great application potential in the diagnosis and treatment of tumors.
发明内容Contents of the invention
针对现有技术中存在的技术问题，本发明提出了一种抗体药物复合体，包括：至少一种抗体；载体蛋白，其与所述至少一种抗体偶联，并转运所述至少一种抗体，以及；连接子，其连接所述至少一种抗体和所述载体蛋白，经配置以响应细胞微环境中的ROS浓度；其中，所述抗体药物复合体中不包括有治疗作用的非生物类化合物。Aiming at the technical problems existing in the prior art, the present invention proposes an antibody-drug complex, comprising: at least one antibody; a carrier protein, which is coupled to the at least one antibody and transports the at least one antibody , and; a linker, which connects the at least one antibody and the carrier protein, configured to respond to the ROS concentration in the cellular microenvironment; wherein, the antibody-drug complex does not include a therapeutically active abiotic compound.
在一些实施例中，其中，连接子：白蛋白：抗体的物质的量的比例为(80-120):(8-12):1，优选为100:10:1。In some embodiments, the ratio of linker: albumin: antibody is (80-120):(8-12):1, preferably 100:10:1.
在一些实施例中，所述抗体药物复合体中包括两种抗体；优选地，所述两种抗体为：抗PD1抗体和/或CTLA4抗体。In some embodiments, the antibody-drug complex includes two antibodies; preferably, the two antibodies are: anti-PD1 antibody and/or CTLA4 antibody.
在一些实施例中，其中，两种抗体的物质的量的比例为(0.01-1):1；当两种抗体为抗PD1抗体和抗CTLA4抗体时，两种抗体的物质的量的比例为1:1。。In some embodiments, wherein, the ratio of the amounts of substances of the two antibodies is (0.01-1):1; when the two antibodies are anti-PD1 antibodies and anti-CTLA4 antibodies, the ratio of the amounts of substances of the two antibodies is 1:1. .
在一些实施例中，所述连接子在ROS浓度不小于0.5mM时解聚断裂。In some embodiments, the linker is depolymerized and broken when the ROS concentration is not less than 0.5 mM.
在一些实施例中，所述连接子的分子式为C15H18N2O8S2。In some embodiments, the molecular formula of the linker is C 15 H 18 N 2 O 8 S 2 .
在一些实施例中，所述连接子的结构式为：In some embodiments, the structural formula of the linker is:
在一些实施例中，所述载体蛋白为白蛋白。In some embodiments, the carrier protein is albumin.
在一些实施例中，所述白蛋白为小鼠血清白蛋白。In some embodiments, the albumin is mouse serum albumin.
在一些实施例中，如上任一所述的抗体药物复合体进一步包括：第一荧光分子，其修饰所述载体蛋白，经配置以指示连接子对细胞微环境中的ROS浓度的响应。In some embodiments, the antibody-drug complex as described above further comprises: a first fluorescent molecule that modifies the carrier protein and is configured to indicate the response of the linker to the ROS concentration in the cellular microenvironment.
在一些实施例中，所述第一荧光分子为Cy5-马来酰亚胺、MB-马来酰亚胺、ICG-马来酰亚胺、Cy5.5-马来酰亚胺、Cy7-马来酰亚胺、DiR、DiO、DiI、DiD、FITC、SiR650、罗丹明B(Rhodamine B)、香豆素6(Coumarin 6)、GFP、EGFP、RFP、mRFP、mCherry、YFP或者mYFP。In some embodiments, the first fluorescent molecule is Cy5-maleimide, MB-maleimide, ICG-maleimide, Cy5.5-maleimide, Cy7-maleimide Laimide, DiR, DiO, DiI, DiD, FITC, SiR650, Rhodamine B, Coumarin 6, GFP, EGFP, RFP, mRFP, mCherry, YFP, or mYFP.
在一些实施例中，如上所述的抗体药物复合体进一步包括：第二荧光分子；其修饰所述载体蛋白；其中，所述第一荧光分子和所述第二荧光分子不同，经配置以指示连接子对细胞微环境中的ROS浓度的响应。In some embodiments, the antibody drug complex as described above further comprises: a second fluorescent molecule; which modifies the carrier protein; wherein the first fluorescent molecule and the second fluorescent molecule are different, configured to indicate Linker responses to ROS concentrations in the cellular microenvironment.
在一些实施例中，第一荧光分子为MB-马来酰亚胺，所述第二荧光分子为ICG-马来酰亚胺；或者所述第一荧光分子为Cy5.5-马来酰亚胺，所述第二荧光分子为Cy7-马来酰亚胺；或者所述第一荧光分子为DiO，所述第二荧光分子为DiI或罗丹明B(Rhodamine B)；或者所述第一荧光分子为香豆素6(Coumarin6)，所述第二荧光分子为DiI,；或者所述第一荧光分子为DiD，所述第二荧光分子为DiR；或者所述第一荧光分子为DiI，所述第二荧光分子为DiD。In some embodiments, the first fluorescent molecule is MB-maleimide, and the second fluorescent molecule is ICG-maleimide; or the first fluorescent molecule is Cy5.5-maleimide Amine, the second fluorescent molecule is Cy7-maleimide; or the first fluorescent molecule is DiO, the second fluorescent molecule is DiI or rhodamine B (Rhodamine B); or the first fluorescent The molecule is Coumarin 6 (Coumarin6), and the second fluorescent molecule is DiI; or the first fluorescent molecule is DiD, and the second fluorescent molecule is DiR; or the first fluorescent molecule is DiI, so The second fluorescent molecule is DiD.
一种如上任一所述的抗体药物复合体的制备方法，包括：将白蛋白溶于PBS中，获得溶液A；将抗PD1抗体溶液和抗CTLA4抗体溶液加入溶液A中，获得溶液B；将响应细胞微环境中的ROS浓度的连接子缓慢加入溶液B中，立即混匀，获得溶液C；将溶液C置于4摄氏度环境中，持续8-15小时，获得溶液D；以及获得溶液D的沉淀，所述沉淀为所述抗体药物复合体。A method for preparing an antibody-drug complex as described above, comprising: dissolving albumin in PBS to obtain solution A; adding anti-PD1 antibody solution and anti-CTLA4 antibody solution to solution A to obtain solution B; The linker that responds to the ROS concentration in the cell microenvironment is slowly added to solution B, mixed immediately to obtain solution C; solution C is placed in an environment of 4 degrees Celsius for 8-15 hours to obtain solution D; and the solution D is obtained Precipitation, the precipitation is the antibody-drug complex.
在一些实施例中，连接子：白蛋白：抗PD1抗体：抗CTLA4抗体的物质的量的比例为(160-240):(16-24):1:1，优选为200:20:1:1。In some embodiments, the ratio of linker: albumin: anti-PD1 antibody: anti-CTLA4 antibody is (160-240):(16-24):1:1, preferably 200:20:1: 1.
在一些实施例中，其中连接子的制备方法包括：将2,2'-[丙烷-2,2-二基双(硫)]二乙酸、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC和N-羟基琥珀酰亚胺NHS混合于二甲基亚砜DMSO中；室温下搅拌4-8小时，优选为6小时；以及获得连接子。In some embodiments, the preparation method of the linker includes: adding 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, 1-ethyl-(3-dimethylaminopropyl Base) carbodiimide EDC and N-hydroxysuccinimide NHS are mixed in dimethyl sulfoxide DMSO; stirred at room temperature for 4-8 hours, preferably 6 hours; and the linker is obtained.
在一些实施例中，所述连接子的分子式为C15H18N2O8S2。In some embodiments, the molecular formula of the linker is C 15 H 18 N 2 O 8 S 2 .
在一些实施例中，所述连接子的结构式为：In some embodiments, the structural formula of the linker is:
在一些实施例中，如上所述的制备方法进一步包括：获得第一荧光分子标记的白蛋白和/或获得第二荧光分子标记的白蛋白。In some embodiments, the above-mentioned preparation method further includes: obtaining albumin labeled with the first fluorescent molecule and/or obtaining albumin labeled with the second fluorescent molecule.
在一些实施例中，其中，第一荧光分子标记的白蛋白的制备方法包括：将物质的量的比值为(3-8)：1，优选为5:1的白蛋白与第一荧光分子混合；第二荧光分子标记的白蛋白的制备方法包括：将物质的量的比值为(3-8)：1，优选为5:1的白蛋白与第二荧光分子混合。In some embodiments, wherein, the preparation method of albumin labeled with the first fluorescent molecule comprises: mixing the albumin with the first fluorescent molecule at a ratio of (3-8):1, preferably 5:1 ; The preparation method of the albumin labeled with the second fluorescent molecule includes: mixing the albumin with the second fluorescent molecule at a ratio of (3-8):1, preferably 5:1.
一种治疗肿瘤的药物组合物，包括如上任一所述的抗体药物复合体，或者如上任一所述的制备方法制备的抗体药物复合体。A pharmaceutical composition for treating tumors, comprising the antibody-drug complex as described above, or the antibody-drug complex prepared by any of the above-mentioned preparation methods.
一种用于肿瘤检测的试剂盒，包括：如上任一所述的抗体药物复合体，或者如上任一所述的制备方法获得的抗体药物复合体；其中，所述载体蛋白为经第一荧光分子修饰的载体蛋白和经第二荧光分子修饰的载体蛋白。A kit for tumor detection, comprising: the antibody-drug complex as described above, or the antibody-drug complex obtained by any of the preparation methods described above; wherein, the carrier protein is A molecularly modified carrier protein and a carrier protein modified by a second fluorescent molecule.
在一些实施例中，其中，经第一荧光分子修饰的载体蛋白与经第二荧光分子修饰的载体蛋白浓度比为60:1。In some embodiments, the concentration ratio of the carrier protein modified by the first fluorescent molecule to the carrier protein modified by the second fluorescent molecule is 60:1.
如上任一所述的用于肿瘤检测的试剂盒的制备方法，包括：如上任一所述的抗体药物复合体的制备方法。The preparation method of the kit for tumor detection as described above includes: the preparation method of the antibody-drug complex described above.
一种降低抗体药物对机体的毒副作用的方法，包括：通过响应细胞微环境中的ROS浓度的连接子，将抗体连接至载体蛋白；其中，连接子的制备方法包括：将2,2'-[丙烷-2,2-二基双(硫)]二乙酸、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC和N-羟基琥珀酰亚胺NHS混合于二甲基亚砜DMSO中；室温下搅拌4-8小时，优选为6小时。A method for reducing the toxic and side effects of antibody drugs on the body, comprising: linking the antibody to a carrier protein through a linker that responds to the ROS concentration in the cell microenvironment; wherein, the preparation method of the linker includes: adding 2,2'- [Propane-2,2-diylbis(thio)]diacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were mixed in di in methyl sulfoxide DMSO; stir at room temperature for 4-8 hours, preferably 6 hours.
如上所述的方法，其中所述毒副作用包括：对无心肌炎的患者引发心肌炎症状，或者加重患有心肌炎患者的心肌炎症状。The method as above, wherein the toxic and side effects include: inducing myocarditis symptoms in patients without myocarditis, or aggravating myocarditis symptoms in patients with myocarditis.
如上任一所述的抗体药物复合体，或者如上任一所述的制备方法制备的抗体药物复合体，在制备治疗肿瘤的药物组合物方面的应用。Application of the antibody-drug complex as described above, or the antibody-drug complex prepared by any of the above-mentioned preparation methods, in the preparation of a pharmaceutical composition for treating tumors.
如上任一所述的抗体药物复合体，或者如上任一所述的制备方法制备的抗体药物复合体，在制备治疗肿瘤检测的试剂盒方面的应用。Application of the antibody-drug complex as described above, or the antibody-drug complex prepared by any of the above-mentioned preparation methods, in the preparation of a kit for tumor treatment and detection.
本申请的抗体药物复合体具有较好的生物安全性，能够靶向肿瘤部位实现诊疗一体化，激活肿瘤免疫抑制肿瘤生长，并且规避了治疗引发的免疫不良反应，是一种新型的抗肿瘤纳米药物，具有广阔的应用前景。The antibody drug complex of the present application has good biological safety, can target the tumor site to realize the integration of diagnosis and treatment, activate tumor immunity and suppress tumor growth, and avoid immune adverse reactions caused by treatment. It is a new type of anti-tumor nano Drugs have broad application prospects.
相对于现有技术，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明制备的抗体药物复合体的粒径在180nm，粒径较均一，有利于在肿瘤部位的富集，提高了药物的靶向性。另一方面，本发明所利用的ROS响应连接子能够保证复合物在肿瘤微环境高ROS的情况下解聚，释放PD1和CTLA4抗体，激活免疫系统对肿瘤细胞进行杀伤，进一步减少了对正常细胞的毒副作用。(1) The particle size of the antibody-drug complex prepared in the present invention is 180nm, and the particle size is relatively uniform, which is beneficial to the enrichment at the tumor site and improves the targeting of the drug. On the other hand, the ROS-responsive linker used in the present invention can ensure the depolymerization of the complex in the case of high ROS in the tumor microenvironment, release PD1 and CTLA4 antibodies, activate the immune system to kill tumor cells, and further reduce the damage to normal cells. toxic side effects.
(2)本发明利用FRET技术分别构建了带MB和ICG荧光的复合物，包含该复合物的试剂盒不仅可以证明该复合物解聚发生在肿瘤高ROS环境中，进一步通过荧光信号的改变还可实现对肿瘤的诊断，在实体瘤的治疗诊断中具有很大的应用潜力。(2) The present invention uses FRET technology to construct complexes with MB and ICG fluorescence, and the kit containing the complexes can not only prove that the depolymerization of the complexes occurs in the high ROS environment of the tumor, but also through the change of the fluorescent signal The diagnosis of tumors can be realized, and it has great application potential in the treatment and diagnosis of solid tumors.
(3)本发明制备的抗体药物复合体能够缓解免疫相关不良反应。考虑到研究周期，发明人构建了心肌炎小鼠模型，发现注射抗体药物复合体的小鼠的心肌炎症状不会加重，证明其能够缓解免疫相关不良反应，为实体瘤患者的临床治疗提供了更有效和安全的治疗方案。(3) The antibody-drug complex prepared in the present invention can alleviate immune-related adverse reactions. Considering the research period, the inventor constructed a mouse model of myocarditis and found that the symptoms of myocarditis in mice injected with the antibody-drug complex would not aggravate, proving that it can alleviate immune-related adverse reactions and provide a more effective clinical treatment for patients with solid tumors. and safe treatment options.
附图说明Description of drawings
下面，将结合附图对本发明的优选实施方式进行进一步详细的说明，其中：Below, preferred embodiment of the present invention will be described in further detail in conjunction with accompanying drawing, wherein:
图1显示根据本发明的一个实施例的ROS响应抗体药物复合体的结构和特征；其中：Figure 1 shows the structure and characteristics of the ROS responsive antibody-drug complex according to one embodiment of the present invention; wherein:
图1a为根据本发明一个实施例的抗体药物复合体示意图；Figure 1a is a schematic diagram of an antibody-drug complex according to an embodiment of the present invention;
图1b为根据本发明一个实施例的抗体药物复合体在肿瘤微环境中的作用机制示意图；Figure 1b is a schematic diagram of the mechanism of action of the antibody-drug complex in the tumor microenvironment according to an embodiment of the present invention;
图1c为根据本发明的一个实施例的抗体药物复合体在中性磷酸盐缓冲液中的粒径分布图；Figure 1c is a particle size distribution diagram of an antibody-drug complex in neutral phosphate buffer according to an embodiment of the present invention;
图1d为根据本发明的一个实施例的抗体药物复合体在中性磷酸盐缓冲液中的电镜形貌图；Figure 1d is an electron microscope topography of an antibody-drug complex in neutral phosphate buffer according to an embodiment of the present invention;
图1e为根据本发明的一个实施例的抗PD1和CTLA4抗体的纳米复合物的扫描电镜(STEM)图像；显示钙标记的抗CTLA4抗体(红色)和钆标记的抗PD1抗体(蓝色)；以及Figure 1e is a scanning electron microscope (STEM) image of a nanocomposite of anti-PD1 and CTLA4 antibodies according to one embodiment of the present invention; showing calcium-labeled anti-CTLA4 antibody (red) and gadolinium-labeled anti-PD1 antibody (blue); as well as
图1f为根据本发明的一个实施例的抗PD1和CTLA4抗体的纳米复合物对H2O2(0.5mM)响应的透射电镜图；Figure 1f is a transmission electron micrograph of the response of the nanocomposite of anti-PD1 and CTLA4 antibodies to H 2 O 2 (0.5mM) according to an embodiment of the present invention;
图2显示根据本发明的一个实施例的ROS响应抗体药物复合体靶向性和诊疗一体化的体内荧光成像图；其中：Figure 2 shows an in vivo fluorescence imaging diagram of ROS responsive antibody-drug complex targeting and integration of diagnosis and treatment according to an embodiment of the present invention; wherein:
图2a为根据本发明一个实施例的用Cy5修饰的PD1和CTLA4复合物或用Cy5修饰的游离PD1和CTLA4处理的小鼠器官的体内荧光成像；从左到右依次为：心、肝、脾、肺、肾、肿瘤；Figure 2a is the in vivo fluorescence imaging of mouse organs treated with Cy5-modified PD1 and CTLA4 complexes or Cy5-modified free PD1 and CTLA4 according to an embodiment of the present invention; from left to right: heart, liver, spleen , lung, kidney, tumor;
图2b为根据本发明一个实施例的肿瘤和正常组织荧光信号的荧光区域分析；Fig. 2b is the fluorescence region analysis of tumor and normal tissue fluorescence signals according to one embodiment of the present invention;
图2c为根据本发明一个实施例的不同ICG/MB配比的PD1和CTLA4纳米复合物的FRET强度；Figure 2c is the FRET intensity of PD1 and CTLA4 nanocomposites with different ICG/MB ratios according to one embodiment of the present invention;
图2d为根据本发明一个实施例的不同ICG/MB配比的PD1和CTLA4纳米复合物在经过不同时间后的FRET强度；Figure 2d is the FRET intensity of PD1 and CTLA4 nanocomposites with different ICG/MB ratios after different times according to an embodiment of the present invention;
图2e为根据本发明一个实施例的不同时间的MB/ICG荧光强度的比值；Figure 2e is the ratio of MB/ICG fluorescence intensity at different times according to one embodiment of the present invention;
图2f为根据本发明一个实施例的不同荧光通道和时间间隔的小鼠活体荧光成像结果；其中MB的激发通道为640nm，发射通道为700nm，ICG的激发通道为710nm，发射通道为800nm，FRET的激发通道为640nm，发射通道为800nm；Figure 2f is the mouse in vivo fluorescence imaging results of different fluorescence channels and time intervals according to an embodiment of the present invention; wherein the excitation channel of MB is 640nm, the emission channel is 700nm, the excitation channel of ICG is 710nm, the emission channel is 800nm, FRET The excitation channel is 640nm, and the emission channel is 800nm;
图2g-图2i为根据本发明一个实施例的肿瘤ICG、MB和FRET荧光信号的强度分析；以及Figure 2g-Figure 2i is the intensity analysis of tumor ICG, MB and FRET fluorescence signals according to one embodiment of the present invention; and
图2j为根据本发明一个实施例的体内不同时间间隔MB/ICG荧光强度比值；Figure 2j is the ratio of MB/ICG fluorescence intensity at different time intervals in vivo according to one embodiment of the present invention;
图3显示根据本发明的一个实施例的ROS响应抗体药物复合体抑制体内MC38肿瘤生长；其中：Figure 3 shows that the ROS-responsive antibody-drug complex according to one embodiment of the present invention inhibits MC38 tumor growth in vivo; wherein:
图3a-图3c为根据本发明一个实施例的给药期间肿瘤体积和重量随时间变化的生长曲线(n＝5)；给药15天后从小鼠收集的肿瘤照片(图3b)和重量(图3c)；Fig. 3a-Fig. 3c is the growth curve (n=5) of tumor volume and weight during administration according to one embodiment of the present invention; Tumor photos (Fig. 3b) and weight (Fig. 3c);
图3d-图3e为根据本发明一个实施例的移植瘤中Ki-67阳性细胞的含量；Figure 3d-Figure 3e is the content of Ki-67 positive cells in transplanted tumors according to one embodiment of the present invention;
图4显示根据本发明的一个实施例的药物复合体能够缓解肿瘤免疫抑制的微环境；其中：Figure 4 shows that the drug complex according to one embodiment of the present invention can alleviate the microenvironment of tumor immunosuppression; wherein:
图4a-图4d为根据本发明一个实施例的流式细胞术分析小鼠肿瘤中CD8+和Treg细胞的数量；Figure 4a-Figure 4d is flow cytometry analysis according to one embodiment of the present invention the number of CD8+ and Treg cells in the mouse tumor;
图4e-图4g为根据本发明一个实施例的采用免疫荧光法和ELISA法检测各组小鼠异种移植瘤中颗粒酶B、TNF-α和IFNγ的含量；Figure 4e-Figure 4g is the detection of the contents of granzyme B, TNF-α and IFNγ in mouse xenograft tumors in each group by immunofluorescence method and ELISA method according to an embodiment of the present invention;
图5为根据本发明的一个实施例的药物复合体的生物安全性评价；其中：Fig. 5 is the biosafety evaluation of the drug complex according to an embodiment of the present invention; Wherein:
图5a为根据本发明一个实施例的经过H&E染色的小鼠心脏、肝脏、脾脏、肺和肾脏切片的代表性图像，比例尺：500μm；Figure 5a is a representative image of H&E stained mouse heart, liver, spleen, lung and kidney sections according to one embodiment of the present invention, scale bar: 500 μm;
图5b-图5e为采用ELISA法检测血清小鼠肾功能和肝功能指标肌酐(CRE)、尿素氮(BUN)、谷丙转氨酶(ALT)、天冬氨酸转氨酶(AST)的水平；Figures 5b-5e are the levels of serum mouse renal function and liver function indicators creatinine (CRE), blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detected by ELISA method;
图6显示根据本发明的一个实施例的ROS响应抗体药物复合体对心肌炎小鼠心肌收缩功能的影响；其中：Figure 6 shows the effect of the ROS-responsive antibody-drug complex on myocardial contractility in mice with myocarditis according to one embodiment of the present invention; wherein:
图6a为根据本发明一个实施例将BALB/c小鼠随机分为6组(每组5只)，(1)对照组；(2)心肌炎组(EAM，模型组)；(3)心肌炎+小鼠PD-1抗体(100μg PD1抗体/小鼠/2天)；(4)心肌炎+小鼠CTLA4抗体(100μg CTLA4抗体/小鼠/2天)；(5)心肌炎+PD1和CTLA4复合物(50μgPD1抗体+50μgCTLA4抗体/小鼠/2天)；(6)心肌炎+游离PD1和CTLA4抗体(50μg PD1抗体+50μg CTLA4抗体/小鼠/2天)；除对照组外，所有小鼠分别于第0天和第7天皮下免疫小鼠α-心肌肌球蛋白重链(MyHC-α)多肽，从第7天开始，每2天给予抗小鼠PD-1、抗小鼠CTLA4、PD1和CTLA4复合物或游离PD1和CTLA4抗体，共5次；首次免疫后第21天，对小鼠进行心动超声检查；Fig. 6 a is that BALB/c mice are randomly divided into 6 groups (5 in every group) according to an embodiment of the present invention, (1) control group; (2) myocarditis group (EAM, model group); (3) myocarditis+ Mouse PD-1 antibody (100 μg PD1 antibody/mouse/2 days); (4) myocarditis+mouse CTLA4 antibody (100 μg CTLA4 antibody/mouse/2 days); (5) myocarditis+PD1 and CTLA4 complex ( 50 μg PD1 antibody + 50 μg CTLA4 antibody/mouse/2 days); (6) myocarditis + free PD1 and CTLA4 antibody (50 μg PD1 antibody + 50 μg CTLA4 antibody/mouse/2 days); On day 0 and day 7, mice were subcutaneously immunized with α-cardiac myosin heavy chain (MyHC-α) polypeptide, and anti-mouse PD-1, anti-mouse CTLA4, PD1 and CTLA4 were given every 2 days starting from day 7 Complex or free PD1 and CTLA4 antibodies, a total of 5 times; 21 days after the first immunization, echocardiography was performed on the mice;
图6b为根据本发明一个实施例的心动超声图的代表性图像；以及Figure 6b is a representative image of an echocardiogram according to one embodiment of the present invention; and
图6c为根据本发明一个实施例的射血分数(EF)和缩短分数(FS)的定量；以及Figure 6c is a quantification of ejection fraction (EF) and fraction shortening (FS) according to one embodiment of the invention; and
图7显示根据本发明的一个实施例的ROS响应抗体药物复合体对心肌炎小鼠心肌炎症、纤维化的影响；其中：Figure 7 shows the effect of a ROS-responsive antibody drug complex on myocardial inflammation and fibrosis in myocarditis mice according to an embodiment of the present invention; wherein:
图7a-图7b为根据本发明一个实施例的HE染色和Masson染色代表性图像；以及Figures 7a-7b are representative images of HE staining and Masson staining according to one embodiment of the present invention; and
图7c-图7g为根据本发明一个实施例的血清肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶-1(LDH-1)、心肌肌钙蛋白T(cTnT)、心肌肌钙蛋白I(cTnI)水平；数据以均数±标准差(mean±SD)表示，*p<0.05，**p<0.01，***p<0.001。Fig. 7c-Fig. 7g are serum creatine kinase (CK), creatine kinase isoenzyme (CK-MB), lactate dehydrogenase-1 (LDH-1), cardiac troponin T according to one embodiment of the present invention (cTnT), cardiac troponin I (cTnI) levels; data expressed as mean ± standard deviation (mean ± SD), *p<0.05, **p<0.01, ***p<0.001.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments It is a part of embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
在以下的详细描述中，可以参看作为本申请一部分用来说明本申请的特定实施例的各个说明书附图。在附图中，相似的附图标记在不同图式中描述大体上类似的组件。本申请的各个特定实施例在以下进行了足够详细的描述，使得具备本领域相关知识和技术的普通技术人员能够实施本申请的技术方案。应当理解，还可以利用其它实施例或者对本申请的实施例进行结构、逻辑或者电性的改变。In the following detailed description, reference is made to the accompanying drawings which are included in the specification and which illustrate specific embodiments of the application and which are included in this application. In the drawings, like reference numerals describe substantially similar components in different views. Various specific embodiments of the present application are described in sufficient detail below, so that those of ordinary skill in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be utilized or structural, logical or electrical changes may be made to the embodiments of the present application.
本文中涉及的名词具有以下含义：The nouns involved in this article have the following meanings:
本文所述“抗体药物复合体”是指包括载体蛋白、至少一种抗体以及一靶向连接子的复合体。在一些实施例中，抗体药物复合体进一步包括荧光分子。在一些实施例中，本申请的抗体药物复合体可实现肿瘤的诊疗一体化，也就是说，其既可以靶向肿瘤细胞，实现对肿瘤的治疗；还可以实现肿瘤的检测。The "antibody-drug complex" mentioned herein refers to a complex comprising carrier protein, at least one antibody and a targeting linker. In some embodiments, the antibody drug complex further includes a fluorescent molecule. In some embodiments, the antibody-drug complex of the present application can realize the integration of diagnosis and treatment of tumors, that is, it can not only target tumor cells, realize the treatment of tumors, but also realize the detection of tumors.
进一步地，在一些实施例中，载体蛋白可对抗体的功能起保护作用；在一些实施例中，载体蛋白为白蛋白，所述白蛋白可以为人血清白蛋白、小鼠血清白蛋白(MSA)、牛血清白蛋白(BSA)、鸡卵白蛋白(OVO)、血蓝蛋白(KLH)等；在一些实施例中，靶向连接子为可在癌细胞中特异性断裂的连接子。Further, in some embodiments, the carrier protein can protect the function of the antibody; in some embodiments, the carrier protein is albumin, and the albumin can be human serum albumin, mouse serum albumin (MSA) , bovine serum albumin (BSA), chicken ovalbumin (OVO), hemocyanin (KLH), etc.; in some embodiments, the targeting linker is a linker that can be specifically broken in cancer cells.
本文所述“诊疗一体化”是指本申请的抗体药物复合物，在结合荧光分子的前提下，既可以作为肿瘤的治疗药物，又可以用做诊断肿瘤的试剂盒。The "integration of diagnosis and treatment" mentioned in this article refers to the antibody-drug complex of the present application, which can be used not only as a drug for treating tumors but also as a kit for diagnosing tumors under the premise of combining fluorescent molecules.
本文所述“载体蛋白”在本申请中是指用于运载抗体的载体，是多回旋折叠的跨膜蛋白质，它与被传递的分子特异结合使其越过质膜。其机制是载体蛋白分子的构象可逆地变化，与被转运分子的亲和力随之改变而将分子传递过去。在一些实施例中，载体蛋白为白蛋白。进一步地，在一些实施例中，白蛋白为小鼠血清白蛋白(Mouse Serum Albumin,MSA)，其由608个氨基酸组成，分子量约68.7Kd，约占血浆总蛋白的50％，是小鼠血清中的主要蛋白成分之一，在抗体制备过程中作常为载体蛋白用于半抗原的偶联，也常作为分子量标准蛋白用于电泳或色谱层析。The "carrier protein" mentioned in this application refers to the carrier used to carry the antibody, which is a multi-turn folded transmembrane protein, which specifically binds the delivered molecule to make it cross the plasma membrane. The mechanism is that the conformation of the carrier protein molecule changes reversibly, and the affinity with the transported molecule changes accordingly, so that the molecule is passed over. In some embodiments, the carrier protein is albumin. Further, in some embodiments, the albumin is mouse serum albumin (Mouse Serum Albumin, MSA), which is composed of 608 amino acids, has a molecular weight of about 68.7Kd, accounts for about 50% of the total plasma protein, and is a mouse serum albumin One of the main protein components in the antibody preparation process, it is often used as a carrier protein for hapten coupling, and is often used as a molecular weight standard protein for electrophoresis or chromatography.
本文所述“连接子”是指可连接载体蛋白和抗体的连接子。在本申请中，连接子具有靶向性，其在肿瘤微环境中特异性断裂，进一步地，连接子为ROS浓度响应，在ROS富集的环境中断裂，而在ROS含量少的环境中不断裂。在一些实施例中，可实现连接子断裂的ROS浓度为不小于0.5mM。"Linker" as used herein refers to a linker that can link a carrier protein and an antibody. In this application, the linker is targeted, and it is broken specifically in the tumor microenvironment. Further, the linker is ROS concentration-responsive, and it breaks in an environment rich in ROS, but not in an environment with low ROS content. fracture. In some embodiments, the ROS concentration at which linker cleavage can be achieved is not less than 0.5 mM.
本文所述“毒副作用”、“副作用”是指药物在常用量时所发生的与治疗作用无关的不良反应。包括药物对机体引起的神经系统、心血管系统、呼吸系统以及消化系统等出现的各种症状，比如炎症、内脏功能衰竭、视力模糊、神志不清、头晕、胸闷、心动过速、呼吸困难、恶心、呕吐、腹泻等。例如，化疗和放疗对正常组织也有显著的杀伤作用，其毒副作用较大。抗PD-1(programmed death1)和抗CTLA-4(cytotoxic T lymphocyte-associatedantigen-4)等免疫检测点阻断抗体在肿瘤免疫治疗中，免疫系统异常激活导致的免疫相关不良反应的副作用也不可避免，比如自身反应性T细胞的异常活化引发的心肌炎、肝炎、肺炎等，特别是免疫性心肌炎和致死性心力衰竭，能够显著增加肿瘤患者的死亡率。"Toxic and side effects" and "side effects" mentioned herein refer to adverse reactions that occur when the drug is used in usual doses and have nothing to do with the therapeutic effect. Including various symptoms of the nervous system, cardiovascular system, respiratory system and digestive system caused by drugs on the body, such as inflammation, visceral failure, blurred vision, confusion, dizziness, chest tightness, tachycardia, dyspnea, Nausea, vomiting, diarrhea, etc. For example, chemotherapy and radiotherapy also have a significant killing effect on normal tissues, and their toxic and side effects are relatively large. Anti-PD-1 (programmed death1) and anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) and other immune checkpoint blocking antibodies are used in tumor immunotherapy, and the side effects of immune-related adverse reactions caused by abnormal activation of the immune system are also inevitable , such as myocarditis, hepatitis, pneumonia, etc. caused by abnormal activation of autoreactive T cells, especially immune myocarditis and fatal heart failure, can significantly increase the mortality of cancer patients.
本文所述“非生物类化合物”对应生物类化合物。在一些实施例中，生物类化合物可以用于制备生物药物，其余药物可通过非生物类化合物制备。在一些实施例中，非生物类化合物可以为化学药物，其为从天然矿物、动植物中提取的有效成分，以及经过化学合成或生物合成而制得的药物。结构明确的具有预防、治疗、诊断疾病，或为了调节人体功能、提高生活质量、保持身体健康的特殊化学品。在一些实施例中，生物类化合物制备的生物药物是指运用生物学、医学、生物化学等的研究成果，综合利用物理学、化学、生物化学、生物技术和药学等学科的原理和方法，利用生物体、生物组织、细胞、体液等制造的一类用于预防、治疗和诊断的制品。在一些实施例中，生物类药物可以为蛋白质、核酸、糖类、脂类等，这些物质的组成单元为氨基酸、核苷酸、单糖、脂肪酸等；进一步地，在一些实施例中，非生物类药物为除列举生物类药物以外的药物。The "non-biological compound" described herein corresponds to the biological compound. In some embodiments, biological compounds can be used to prepare biological drugs, and other drugs can be prepared from non-biological compounds. In some embodiments, non-biological compounds may be chemical drugs, which are active ingredients extracted from natural minerals, animals and plants, and drugs produced through chemical synthesis or biosynthesis. Special chemicals with specific structures for the prevention, treatment, and diagnosis of diseases, or for regulating human body functions, improving quality of life, and maintaining good health. In some embodiments, biopharmaceuticals prepared from biological compounds refer to the use of research results in biology, medicine, biochemistry, etc., and the comprehensive use of principles and methods in disciplines such as physics, chemistry, biochemistry, biotechnology, and pharmacy. A class of products for prevention, treatment and diagnosis made by organisms, biological tissues, cells, body fluids, etc. In some embodiments, biological drugs can be proteins, nucleic acids, sugars, lipids, etc., and the constituent units of these substances are amino acids, nucleotides, monosaccharides, fatty acids, etc.; further, in some embodiments, non- Biological drugs are drugs other than the listed biological drugs.
本文所述“荧光分子”是指连接于抗体药物复合体的荧光分子，使得抗体药物复合体的作用位点、作用机制等可被直观观察。在一些实施例中，荧光分子直接修饰载体蛋白。The "fluorescent molecule" mentioned herein refers to a fluorescent molecule linked to the antibody-drug complex, so that the action site and mechanism of the antibody-drug complex can be visually observed. In some embodiments, the fluorescent molecule directly modifies the carrier protein.
进一步地，在本申请中的荧光分子为第一荧光分子和/或第二荧光分子。在一些实施例中，第一荧光分子或者第二荧光分子，通过观察其荧光，可确认抗体药物复合体的作用位点。在一些实施例中，抗体药物复合体中包括第一荧光分子和第二荧光分子，二者分别标记载体蛋白。其中，第一荧光分子为供体荧光分子，第二荧光分子为受体荧光分子。在一些实施例中，通过对荧光分子的激活及淬灭，还可以实现肿瘤的诊断。在一些实施例中，可以作为荧光分子的荧光分子包括但不限于Cy5-马来酰亚胺、MB-马来酰亚胺、ICG-马来酰亚胺、Cy5.5-马来酰亚胺、Cy7-马来酰亚胺、DiR(细胞膜近红外荧光探针，深红色荧光)、DiO(细胞膜绿色荧光)、DiI(细胞膜橙色荧光)、DiD(细胞膜红色荧光)、FITC(异硫氰基荧光素)、SiR650(硅基罗丹明SIR(silicon rhodamine)染料)、罗丹明B(Rhodamine B)、香豆素6(Coumarin 6)、GFP、EGFP、RFP、mRFP、mCherry、YFP或者mYFP等。在一些实施例中，当第一荧光分子为MB-马来酰亚胺时，第二荧光分子为ICG-马来酰亚胺；或者当第一荧光分子为Cy5.5-马来酰亚胺时，第二荧光分子为Cy7-马来酰亚胺；或者当第一荧光分子为DiO时，第二荧光分子为DiI或罗丹明B(Rhodamine B)；或者当第一荧光分子为香豆素6(Coumarin 6)时，第二荧光分子为DiI,；或者当第一荧光分子为DiD时，第二荧光分子为DiR；或者当第一荧光分子为DiI时，所述第二荧光分子为DiD。Further, the fluorescent molecule in this application is the first fluorescent molecule and/or the second fluorescent molecule. In some embodiments, the interaction site of the antibody-drug complex can be confirmed by observing the fluorescence of the first fluorescent molecule or the second fluorescent molecule. In some embodiments, the antibody-drug complex includes a first fluorescent molecule and a second fluorescent molecule, which are respectively labeled with a carrier protein. Wherein, the first fluorescent molecule is a donor fluorescent molecule, and the second fluorescent molecule is an acceptor fluorescent molecule. In some embodiments, tumor diagnosis can also be realized by activating and quenching fluorescent molecules. In some embodiments, fluorescent molecules that can be used as fluorescent molecules include but are not limited to Cy5-maleimide, MB-maleimide, ICG-maleimide, Cy5.5-maleimide , Cy7-maleimide, DiR (membrane near-infrared fluorescent probe, deep red fluorescence), DiO (cell membrane green fluorescence), DiI (cell membrane orange fluorescence), DiD (cell membrane red fluorescence), FITC (isothiocyanate Fluorescein), SiR650 (silicon rhodamine SIR (silicon rhodamine) dye), Rhodamine B (Rhodamine B), Coumarin 6 (Coumarin 6), GFP, EGFP, RFP, mRFP, mCherry, YFP or mYFP, etc. In some embodiments, when the first fluorescent molecule is MB-maleimide, the second fluorescent molecule is ICG-maleimide; or when the first fluorescent molecule is Cy5.5-maleimide , the second fluorescent molecule is Cy7-maleimide; or when the first fluorescent molecule is DiO, the second fluorescent molecule is DiI or Rhodamine B (Rhodamine B); or when the first fluorescent molecule is coumarin 6 (Coumarin 6), the second fluorescent molecule is DiI; or when the first fluorescent molecule is DiD, the second fluorescent molecule is DiR; or when the first fluorescent molecule is DiI, the second fluorescent molecule is DiD .
本文所述“Cy5-马来酰亚胺”是指花菁类染料，是一种活性染料，其CAS登记号为146368-11-8，是用于标记肽,蛋白质,寡核苷酸的氨基基团的反应染料。在一些实施例中，使用Cy5标记载体蛋白，以追踪载体蛋白。在一些实施例中，所述载体蛋白为白蛋白。"Cy5-maleimide" mentioned in this article refers to cyanine dyes, which are a kind of reactive dyes, and their CAS registration number is 146368-11-8, which are used to label amino groups of peptides, proteins, and oligonucleotides Group reactive dyes. In some embodiments, the carrier protein is tagged with Cy5 to track the carrier protein. In some embodiments, the carrier protein is albumin.
本文所述“MB-马来酰亚胺”是指一种荧光染料，可用作荧光分子。"MB-maleimide" as used herein refers to a fluorescent dye that can be used as a fluorescent molecule.
本文所述“ICG-马来酰亚胺”是指吲哚菁绿(ICG)，是一种近红外I区荧光染料，是美国食品药品监督管理局(FDA)批准的体内应用染料。其激发和发射波长分别在785nm、810nm左右，比Cy系列(花菁类)染料(630-670nm、650-700nm)更长，可穿透更深的活体组织，可更好地用于肿瘤细胞体内成像。The "ICG-maleimide" mentioned herein refers to indocyanine green (ICG), which is a fluorescent dye in the near-infrared I region and a dye approved by the US Food and Drug Administration (FDA) for in vivo applications. Its excitation and emission wavelengths are around 785nm and 810nm respectively, which are longer than Cy series (cyanine) dyes (630-670nm, 650-700nm), can penetrate deeper living tissues, and can be better used in tumor cells imaging.
本文所述“MSA”是指小鼠血清白蛋白(mouse serum albumin)。Cy5-MSA即为Cy5修饰的小鼠血清白蛋白；MB-MSA即为MB修饰的小鼠血清白蛋白；ICG-MSA即为ICG修饰的小鼠血清白蛋白。As used herein, "MSA" refers to mouse serum albumin. Cy5-MSA is Cy5-modified mouse serum albumin; MB-MSA is MB-modified mouse serum albumin; ICG-MSA is ICG-modified mouse serum albumin.
本文所述“FRET”是指荧光共振能量转移，当一个荧光分子(又称为供体分子)的荧光光谱与另一个荧光分子(又称为受体分子)的激发光谱相重叠时，供体荧光分子的激发能诱发受体分子发出荧光，同时供体荧光分子自身的荧光强度衰减。FRET程度与供、受体分子的空间距离紧密相关，一般为7～10nm时即可发生FRET；随着距离延长，FRET信号显著减弱。在一些实施例中，FRET诊疗一体化试验中，MB为供体荧光分子，ICG为受体荧光分子。"FRET" in this paper refers to fluorescence resonance energy transfer. When the fluorescence spectrum of one fluorescent molecule (also called donor molecule) overlaps with the excitation spectrum of another fluorescent molecule (also called acceptor molecule), the donor The excitation of the fluorescent molecules can induce the acceptor molecules to emit fluorescence, while the fluorescence intensity of the donor fluorescent molecules itself decays. The degree of FRET is closely related to the spatial distance between donor and acceptor molecules. Generally, FRET can occur when the distance is 7-10 nm; as the distance increases, the FRET signal is significantly weakened. In some embodiments, in the FRET integrated diagnosis and treatment test, MB is a donor fluorescent molecule, and ICG is an acceptor fluorescent molecule.
本文所述“EAM”是指实验性自身免疫性心肌炎，EAM模型是由心肌肌球蛋白诱导的动物心肌炎模型，为机体自身免疫反应介导的心肌损伤段。EAM主要在诱导后15天发病,它是由T细胞介导的免疫紊乱。在本申请中，使用EAM小鼠模拟患有心肌炎的患者，以验证本申请的抗体药物复合体对心肌炎患者的心肌炎病程影响。The "EAM" mentioned herein refers to experimental autoimmune myocarditis, and the EAM model is an animal myocarditis model induced by myocardial myosin, which is a segment of myocardial injury mediated by the body's autoimmune response. EAM mainly occurs 15 days after induction, and it is an immune disorder mediated by T cells. In this application, EAM mice are used to simulate patients with myocarditis to verify the effect of the antibody-drug complex of the present application on the course of myocarditis in patients with myocarditis.
本申请提供一种抗肿瘤的抗体药物复合体，图1a为根据本发明一个实施例的抗体药物复合体示意图。如图1a所示，该抗体药物复合体中包括：至少一种抗体、载体蛋白以及连接子。其中，所述抗体药物复合体中不包括有治疗作用的非生物类化合物。其中，抗体可以为任何已知的抗体，本申请的实施例以抗PD1抗体和抗CTLA4抗体为例对本申请的发明内容加以阐释。在一些实施例中，载体蛋白为白蛋白；在一些实施例中，所述白蛋白为小鼠血清白蛋白。图1b为根据本发明一个实施例的抗体药物复合体在肿瘤微环境中的作用机制示意图，如图1b所示，在一些实施例中，连接子可响应细胞微环境中的ROS浓度，在ROS浓度不小于0.5mM时，连接子解聚断裂，并释放抗体。在一些实施例中，抗体药物复合体中，连接子：白蛋白：抗体的物质的量的比例为(80-120):(8-12):1，优选为100:10:1。进一步地，当抗体为两种或两种以上时，各抗体的物质的量均相同。如，在一些实施例中，抗体药物复合体中包括两种抗体，两种抗体的物质的量的比例为(0.01-1):1。当两种抗体为抗PD1抗体和抗CTLA4抗体，其中抗PD1抗体：抗CTLA4抗体的物质的量的比例为1:1。The present application provides an anti-tumor antibody-drug complex, and FIG. 1a is a schematic diagram of the antibody-drug complex according to an embodiment of the present invention. As shown in Figure 1a, the antibody drug complex includes: at least one antibody, carrier protein and linker. Wherein, the antibody-drug complex does not include non-biological compounds with therapeutic effects. Wherein, the antibody can be any known antibody, and the embodiments of the present application take anti-PD1 antibody and anti-CTLA4 antibody as examples to illustrate the content of the invention of the present application. In some embodiments, the carrier protein is albumin; in some embodiments, the albumin is mouse serum albumin. Figure 1b is a schematic diagram of the mechanism of action of the antibody-drug complex in the tumor microenvironment according to one embodiment of the present invention, as shown in Figure 1b, in some embodiments, the linker can respond to the ROS concentration in the cell microenvironment, in the ROS When the concentration is not less than 0.5mM, the linker is depolymerized and broken, and the antibody is released. In some embodiments, in the antibody-drug complex, the ratio of linker:albumin:antibody is (80-120):(8-12):1, preferably 100:10:1. Furthermore, when there are two or more kinds of antibodies, the amount of the substance of each antibody is the same. For example, in some embodiments, the antibody-drug complex includes two antibodies, and the ratio of the amounts of the two antibodies is (0.01-1):1. When the two antibodies are anti-PD1 antibody and anti-CTLA4 antibody, the ratio of anti-PD1 antibody: anti-CTLA4 antibody is 1:1.
在一些实施例中，抗体药物复合体进一步包括：第一荧光分子和/或第二荧光分子，其修饰所述载体蛋白，经配置以指示连接子对细胞微环境中的ROS浓度的响应。其中，第一荧光分子和第二荧光分子不同。在一些实施例中，第一荧光分子和第二荧光分子可以为Cy5-马来酰亚胺、MB-马来酰亚胺、ICG-马来酰亚胺、Cy5.5-马来酰亚胺、Cy7-马来酰亚胺、DiR(细胞膜近红外荧光探针，深红色荧光)、DiO(细胞膜绿色荧光)、DiI(细胞膜橙色荧光)、DiD(细胞膜红色荧光)、FITC(异硫氰基荧光素)、SiR650(硅基罗丹明SIR(siliconrhodamine)染料)、罗丹明B(Rhodamine B)、香豆素6(Coumarin 6)、GFP、EGFP、RFP、mRFP、mCherry、YFP或者mYFP。在一些实施例中，第一荧光分子为MB-马来酰亚胺，第二荧光分子为ICG-马来酰亚胺；或者当第一荧光分子为Cy5.5-马来酰亚胺时，第二荧光分子为Cy7-马来酰亚胺；或者当第一荧光分子为DiO时，第二荧光分子为DiI或罗丹明B(Rhodamine B)；或者当第一荧光分子为香豆素6(Coumarin 6)时，第二第二荧光分子为DiI,；或者当第一荧光分子为DiD时，第二荧光分子为DiR；或者当第一荧光分子为DiI时，所述第二荧光分子为DiD。在一些实施例中，本申请不限定第一荧光分子或者第二荧光分子的种类，可修饰载体蛋白，并可实现指示连接子的解聚即可。In some embodiments, the antibody-drug complex further comprises: a first fluorescent molecule and/or a second fluorescent molecule, which modifies the carrier protein and is configured to indicate the linker's response to the ROS concentration in the cellular microenvironment. Wherein, the first fluorescent molecule and the second fluorescent molecule are different. In some embodiments, the first fluorescent molecule and the second fluorescent molecule can be Cy5-maleimide, MB-maleimide, ICG-maleimide, Cy5.5-maleimide , Cy7-maleimide, DiR (membrane near-infrared fluorescent probe, deep red fluorescence), DiO (cell membrane green fluorescence), DiI (cell membrane orange fluorescence), DiD (cell membrane red fluorescence), FITC (isothiocyanate Fluorescein), SiR650 (silicon rhodamine SIR (siliconrhodamine) dye), Rhodamine B (Rhodamine B), Coumarin 6 (Coumarin 6), GFP, EGFP, RFP, mRFP, mCherry, YFP or mYFP. In some embodiments, the first fluorescent molecule is MB-maleimide, and the second fluorescent molecule is ICG-maleimide; or when the first fluorescent molecule is Cy5.5-maleimide, The second fluorescent molecule is Cy7-maleimide; or when the first fluorescent molecule is DiO, the second fluorescent molecule is DiI or Rhodamine B (Rhodamine B); or when the first fluorescent molecule is Coumarin 6 ( Coumarin 6), the second fluorescent molecule is DiI; or when the first fluorescent molecule is DiD, the second fluorescent molecule is DiR; or when the first fluorescent molecule is DiI, the second fluorescent molecule is DiD . In some embodiments, the present application does not limit the type of the first fluorescent molecule or the second fluorescent molecule, as long as the carrier protein can be modified and the depolymerization of the indicator linker can be realized.
在一些实施例中，抗体药物复合体的制备方法，包括：In some embodiments, the preparation method of antibody-drug complex includes:
将白蛋白溶于PBS中，获得溶液A；Dissolve albumin in PBS to obtain solution A;
将抗PD1抗体溶液和抗CTLA4抗体溶液加入溶液A中，获得溶液B；adding the anti-PD1 antibody solution and the anti-CTLA4 antibody solution to solution A to obtain solution B;
将响应细胞微环境中高ROS的连接子缓慢加入溶液B中，立即混匀，获得溶液C；Slowly add linkers that respond to high ROS in the cell microenvironment to solution B, and mix immediately to obtain solution C;
将溶液C置于4摄氏度环境中，持续8-15小时，获得溶液D；Place solution C in an environment of 4 degrees Celsius for 8-15 hours to obtain solution D;
获得溶液D的沉淀，该沉淀即为本申请的抗体药物复合体。A precipitate of solution D is obtained, which is the antibody-drug complex of the present application.
具体地，抗体药物复合体的制备方法包括：Specifically, the preparation method of the antibody-drug complex includes:
(1)将2,2'-[丙烷-2,2-二基双(硫)]二乙酸(5.0mg，1当量)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC(6.9mg，2当量)和N-羟基琥珀酰亚胺NHS(5.1mg，2当量)混合于200μl二甲基亚砜(DMSO)中并在室温下搅拌6h，合成ROS响应连接子。该反应的原理是EDC活化2,2'-[丙烷-2,2-二基双(硫)]二乙酸的羧基，但活化的羧基容易水解，加入NHS后形成稳定的活泼酯中间体，极大增强偶联效率。其中，ROS响应连接子的分子式为：C15H18N2O8S2，其结构式如下：(1) 2,2'-[propane-2,2-diylbis(sulfur)]diacetic acid (5.0 mg, 1 equivalent), 1-ethyl-(3-dimethylaminopropyl) carbonyl Diimine EDC (6.9 mg, 2 equivalents) and N-hydroxysuccinimide NHS (5.1 mg, 2 equivalents) were mixed in 200 μl dimethyl sulfoxide (DMSO) and stirred at room temperature for 6 h to synthesize ROS-responsive linkages son. The principle of the reaction is that EDC activates the carboxyl group of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, but the activated carboxyl group is easily hydrolyzed, and a stable active ester intermediate is formed after adding NHS, which is extremely Greatly enhance coupling efficiency. Among them, the molecular formula of the ROS responsive linker is: C 15 H 18 N 2 O 8 S 2 , and its structural formula is as follows:
(2)称取小鼠血清白蛋白(20当量)、抗PD1抗体(1当量)和抗CTLA4抗体(1当量)混合于磷酸盐缓冲液(PBS)中，然后缓慢加入步骤(1)中合成的ROS响应连接子(200当量)，在4℃下将混合物搅拌过夜，得到含有抗PD1和抗CTLA4纳米复合物溶液。(2) Weigh mouse serum albumin (20 equiv), anti-PD1 antibody (1 equiv) and anti-CTLA4 antibody (1 equiv) and mix them in phosphate buffered saline (PBS), then slowly add them to the synthesis in step (1) ROS-responsive linker (200 equivalents), the mixture was stirred overnight at 4 °C to obtain a nanocomplex solution containing anti-PD1 and anti-CTLA4.
(3)将获得的步骤(2)中的抗PD1和抗CTLA4纳米复合物溶液使用高速离心机离心，条件为20000rpm离心20min，以去除游离白蛋白或抗体，最终获得的沉淀为ROS响应的抗PD1和抗CTLA4抗体药物复合体。(3) Centrifuge the anti-PD1 and anti-CTLA4 nanocomplex solution obtained in step (2) using a high-speed centrifuge at 20,000 rpm for 20 minutes to remove free albumin or antibodies, and the final precipitate obtained is ROS-responsive anti- PD1 and anti-CTLA4 antibody-drug complex.
本发明所述的抗体药物复合体的制备方法中，步骤(1)计入NHS后形成稳定的酯中间体，增加了后续的抗体偶联效率。步骤(2)中小鼠血清白蛋白、抗PD1抗体和抗CTLA4抗体的的氨基与ROS响应连接子中胺反应性NHS酯中间物进行反应，从而在连接子两端偶联上抗体。步骤(2)中的小鼠血清白蛋白能够避免抗体被过度交联影响其激活肿瘤免疫的功能，同样的，发明人通过对白蛋白进行修饰，连接相应的荧光分子(Cy5、MB、ICG)通过步骤(2)的方法进行交联，得到的复合物能够实现靶向性验证和肿瘤诊疗一体化，进一步制备肿瘤的检测试剂盒。In the preparation method of the antibody-drug complex of the present invention, step (1) includes NHS to form a stable ester intermediate, which increases the subsequent antibody coupling efficiency. In step (2), the amino groups of mouse serum albumin, anti-PD1 antibody, and anti-CTLA4 antibody react with the amine-reactive NHS ester intermediate in the ROS-responsive linker, thereby coupling the antibodies to both ends of the linker. The mouse serum albumin in step (2) can prevent the antibody from being over-crosslinked and affecting its function of activating tumor immunity. Similarly, the inventors modified the albumin and connected the corresponding fluorescent molecules (Cy5, MB, ICG) through The method of step (2) is cross-linked, and the obtained complex can realize the integration of targeting verification and tumor diagnosis and treatment, and further prepare a tumor detection kit.
本发明涉及的荧光分子修饰白蛋白的制备方法包括以下步骤：The preparation method of fluorescent molecule modified albumin involved in the present invention comprises the following steps:
I。称取适量白蛋白溶于1.5mL PH＝8的PBS溶液中，Cy5-马来酰亚胺(Cy5-mal)、MB-马来酰亚胺(MB-mal)和ICG-马来酰亚胺(ICG-mal)溶于1.3ml PBS中。I. Weigh an appropriate amount of albumin and dissolve it in 1.5mL PBS solution of PH=8, Cy5-maleimide (Cy5-mal), MB-maleimide (MB-mal) and ICG-maleimide (ICG-mal) was dissolved in 1.3 ml PBS.
II。将反应瓶抽真空换N2保护，分别加入步骤(1)的白蛋白溶液和相应的荧光溶液，室温搅拌反应4小时。II. The reaction bottle was evacuated and replaced with N2 protection, the albumin solution and the corresponding fluorescent solution in step (1) were added respectively, and the reaction was stirred at room temperature for 4 hours.
III。4小时反应结束后，将步骤II的反应液用PBS进行透析12小时，除去游离的荧光染料，获得荧光修饰的白蛋白Cy5-MSA，MB-MSA和ICG-MSA。III. After 4 hours of reaction, the reaction solution in step II was dialyzed with PBS for 12 hours to remove free fluorescent dyes, and obtain fluorescently modified albumin Cy5-MSA, MB-MSA and ICG-MSA.
IV。将步骤III获得的产物荧光蛋白进行冻干后与连接子、抗PD1抗体和抗CTLA4抗体进行交联反应，获得对应荧光分子标记的复合体，进行后续靶向性和实体瘤诊疗一体化探究，制备肿瘤检测的试剂盒。IV. The fluorescent protein obtained in step III was lyophilized and then cross-linked with the linker, anti-PD1 antibody and anti-CTLA4 antibody to obtain a complex corresponding to the fluorescent molecular marker, and to carry out follow-up targeting and integration of solid tumor diagnosis and treatment. Prepare kits for tumor detection.
本申请的抗体药物复合体以及包含本申请抗体药物复合体的药物，可以降低抗体药物治疗肿瘤时对机体的毒副作用，该毒副作用包括：对无心肌炎的患者引发心肌炎症状，或者加重患有心肌炎的患者的心肌炎症状。The antibody-drug complex of the present application and the drug comprising the antibody-drug complex of the present application can reduce the toxic and side effects of the antibody drug on the body when treating tumors. The toxic and side effects include: causing myocarditis symptoms in patients without myocarditis, or aggravating myocarditis myocarditis symptoms in patients.
下文将通过实施例对本申请技术方案进行阐述：The technical solution of the present application will be set forth below through the embodiments:
实施例一：本发明的ROS响应抗体药物复合体的制备Example 1: Preparation of the ROS-responsive antibody-drug complex of the present invention
(1)ROS响应连接子的合成(1) Synthesis of ROS-responsive linkers
分别称取5.0mg的2,2'-[丙烷-2,2-二基双(硫)]二乙酸，6.9mg的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC和5.1mg的N-羟基琥珀酰亚胺NHS，混合于200μl二甲基亚砜DMSO中，加入磁珠放并于磁力搅拌器上室温下反应6h，得到的反应液即为ROS响应连接子。其中，ROS响应连接子的分子式为：Weigh 5.0 mg of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, 6.9 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiene Amine EDC and 5.1 mg of N-hydroxysuccinimide NHS were mixed in 200 μl dimethyl sulfoxide DMSO, added magnetic beads and reacted at room temperature on a magnetic stirrer for 6 hours. The resulting reaction solution was the ROS response connection son. Among them, the molecular formula of the ROS response linker is:
C15H18N2O8S2，其结构式如下所示：C 15 H 18 N 2 O 8 S 2 , its structural formula is as follows:
在本实施例中，2,2'-[丙烷-2,2-二基双(硫)]二乙酸购自源叶公司，EDC和NHS购自西格玛奥德里奇(Sigma-Aldrich)公司。In this example, 2,2'-[propane-2,2-diylbis(thio)]diacetic acid was purchased from Yuanye Company, and EDC and NHS were purchased from Sigma-Aldrich Company.
(2)抗体药物复合体的合成(2) Synthesis of antibody-drug complexes
以20μl的连接子为例。连接子：白蛋白：PD-1抗体：CTLA4抗体的物质的量的比例＝200：20：1：1。称取小鼠血清白蛋白14.8mg溶于20ml磷酸盐缓冲液PBS中，分别吸取1mg抗PD1抗体溶液和1mg抗CTLA4抗体溶液加入PBS。然后向其缓慢加入步骤(1)中合成的ROS响应连接子20μl，加入后立即混匀。将混合物溶液置于4℃搅拌过夜反应，得到含有抗PD1和抗CTLA4纳米复合物溶液。将获得的抗PD1和抗CTLA4纳米复合物溶液使用高速离心机离心，离心条件为4℃、20000rpm，离心20min以去除游离白蛋白或抗体，轻柔的弃去上清液，获得的沉淀即为ROS响应的抗PD1和抗CTLA4抗体药物复合体。其中，小鼠血清白蛋白购自默克公司，抗PD1抗体和抗CTLA4抗体购自BioXCell公司。Take 20 μl of linker as an example. The ratio of linker: albumin: PD-1 antibody: CTLA4 antibody amount = 200:20:1:1. Weigh 14.8 mg of mouse serum albumin and dissolve it in 20 ml of phosphate buffered saline PBS, absorb 1 mg of anti-PD1 antibody solution and 1 mg of anti-CTLA4 antibody solution and add to PBS. Then 20 μl of the ROS-responsive linker synthesized in step (1) was slowly added thereto, and mixed immediately after adding. The mixture solution was placed at 4° C. and stirred overnight to obtain a nanocomposite solution containing anti-PD1 and anti-CTLA4. Centrifuge the obtained anti-PD1 and anti-CTLA4 nanocomposite solution in a high-speed centrifuge at 4°C and 20,000 rpm for 20 minutes to remove free albumin or antibodies. Gently discard the supernatant, and the obtained precipitate is ROS Responsive anti-PD1 and anti-CTLA4 antibody-drug complexes. Among them, mouse serum albumin was purchased from Merck, and anti-PD1 antibody and anti-CTLA4 antibody were purchased from BioXCell.
(3)抗体药物复合体的材料表征(3) Material characterization of antibody-drug complexes
利用粒度仪(DLS，Zetasizer NanoZS，马尔文(Malvern))，洛伦兹场发射透射电镜(Talos F200X，赛默飞)和透射电镜(JEOL JEM-1200EX，东京(Tokyo)，日本(Japan))对得到的抗体药物复合体进行形态以及粒径表征。如图1所示，其中图1c为抗体药物复合体的粒径分布，大小约为180nm。图1d为透射电镜图，从图中可以看出，制备到的抗体药物复合体呈球形，且颗粒大小较均一，与马尔文粒度仪所测得的结果相符合。图1e为抗PD1和CTLA4抗体的纳米复合物的扫描电镜(STEM)图像，分别将CTLA4抗体进行钙离子螯合和PD1抗体进行钆离子螯合，如图所示，红色代表钙标记的抗CTLA4抗体，蓝色代表钆标记的抗PD1抗体，证实抗CLTA4抗体和抗PD1抗体均位于复合物上；图1f为抗PD1和CTLA4抗体的纳米复合物响应H2O2的透射电镜图，用0.5mM H2O2模拟体内高ROS的肿瘤微环境对复合物进行体外处理12h，电镜结果显示经过过氧化氢(H2O2)处理后，纳米复合物结构变得松散，证实连接子发生了解聚。Using a particle size analyzer (DLS, Zetasizer NanoZS, Malvern (Malvern)), a Lorentz field emission transmission electron microscope (Talos F200X, Thermo Fisher) and a transmission electron microscope (JEOL JEM-1200EX, Tokyo (Tokyo), Japan (Japan)) The morphology and particle size of the obtained antibody-drug complex were characterized. As shown in Figure 1, Figure 1c shows the particle size distribution of the antibody-drug complex, with a size of about 180nm. Figure 1d is a transmission electron microscope image. It can be seen from the figure that the prepared antibody-drug complex is spherical and the particle size is relatively uniform, which is consistent with the results measured by the Malvern particle size analyzer. Figure 1e is a scanning electron microscope (STEM) image of the nanocomposite of anti-PD1 and CTLA4 antibodies. The CTLA4 antibody was chelated with calcium ions and the PD1 antibody was chelated with gadolinium ions. As shown in the figure, the red color represents calcium-labeled anti-CTLA4 Antibody, blue represents gadolinium-labeled anti-PD1 antibody, confirming that both anti-CLTA4 antibody and anti-PD1 antibody are located on the complex; Figure 1f is the transmission electron micrograph of the nanocomposite of anti-PD1 and CTLA4 antibody in response to H 2 O 2 , with 0.5 mM H 2 O 2 simulated the tumor microenvironment with high ROS in vivo and treated the complex for 12 hours in vitro. Electron microscopy results showed that the structure of the nanocomposite became loose after hydrogen peroxide (H 2 O 2 ) treatment, confirming that the linker was decomposed. get together.
实施例二：本发明的肿瘤靶向免疫治疗复合物的靶向性和诊疗一体化测定Example 2: Targeting and integrated diagnosis and treatment of the tumor-targeted immunotherapy complex of the present invention
(1)荧光染料标记白蛋白的制备方法：(1) Preparation method of fluorescent dye-labeled albumin:
称取适量白蛋白溶于1.5mL PH＝8的PBS溶液中，荧光染料Cy5-马来酰亚胺(Cy5-mal)、MB-马来酰亚胺(MB-mal)和ICG-马来酰亚胺(ICG-mal)溶于1.3ml PBS中，PBS的PH＝8。其中Cy5-mal用于验证复合物的靶向性，MB-mal和ICG-mal用于验证FRET诊疗一体化，MB为供体荧光染料，ICG为受体荧光染料。为了保证荧光染料和蛋白的连接效率，白蛋白：荧光染料的物质的量比值为5：1。提前将反应瓶抽真空充入氮气N2保护，分别加入白蛋白溶液和相应的荧光溶液，加入磁珠，在N2保护的条件下，遮光室温搅拌反应4h。反应结束后，取出反应液用PBS进行透析以除去游离的荧光染料，透析膜大小为10KDa。透析时间为12h，每4h更换一次透析液PBS，透析时需要注意避光。透析结束获得的液体即为荧光修饰的白蛋白Cy5-MSA，MB-MSA和ICG-MSA。将获得的产物荧光蛋白进行冻干后与连接子、抗PD1抗体和抗CTLA4抗体进行交联反应，获得对应荧光染料标记的复合体，进行后续靶向性和实体瘤诊疗一体化的探究。Weigh an appropriate amount of albumin and dissolve it in 1.5mL PBS solution of PH=8, fluorescent dyes Cy5-maleimide (Cy5-mal), MB-maleimide (MB-mal) and ICG-maleimide Imine (ICG-mal) was dissolved in 1.3ml PBS, PBS pH=8. Among them, Cy5-mal is used to verify the targeting of the complex, MB-mal and ICG-mal are used to verify the integration of FRET diagnosis and treatment, MB is a donor fluorescent dye, and ICG is an acceptor fluorescent dye. In order to ensure the connection efficiency of fluorescent dyes and proteins, the ratio of albumin: fluorescent dyes was 5:1. The reaction bottle was evacuated and filled with nitrogen N2 protection in advance, albumin solution and corresponding fluorescent solution were added respectively, magnetic beads were added, and under the condition of N2 protection, the reaction was stirred at room temperature for 4 h in the dark. After the reaction, the reaction solution was taken out and dialyzed with PBS to remove free fluorescent dye, and the size of the dialyzed membrane was 10KDa. The dialysis time was 12 hours, and the dialysate PBS was replaced every 4 hours. During dialysis, care should be taken to avoid light. Fluorescence-modified albumin Cy5-MSA, MB-MSA and ICG-MSA are obtained at the end of dialysis. The obtained product fluorescent protein was lyophilized and cross-linked with the linker, anti-PD1 antibody and anti-CTLA4 antibody to obtain the corresponding fluorescent dye-labeled complex, which was then explored for subsequent targeting and integration of solid tumor diagnosis and treatment.
(2)抗体药物复合体的靶向性验证(2) Targeting verification of antibody-drug complexes
将Cy5-MSA通过实施例一的方法合成纳米复合物，对照组使用同样浓度的游离的含有Cy5-MSA的PD1和CTLA4抗体。通过小鼠腹腔注射的方式，每只小鼠的荧光蛋白注射量为0.5mg Cy5/kg，6h后通过小动物活体成像仪检测小鼠各脏器的荧光信号分布。如图2a和图2b所示，与游离Cy5修饰的PD1和CTLA4组相比，Cy5修饰的抗体药物复合体组在肿瘤中表现出非常强的荧光信号，在其他脏器中含量较低，证实抗体药物复合体对肿瘤有较好的靶向性。Cy5-MSA was used to synthesize nanocomposites by the method of Example 1, and the same concentration of free PD1 and CTLA4 antibodies containing Cy5-MSA was used in the control group. By intraperitoneal injection of mice, the amount of fluorescent protein injected into each mouse was 0.5 mg Cy5/kg, and the distribution of fluorescent signals in various organs of the mice was detected by a small animal live imager 6 hours later. As shown in Figure 2a and Figure 2b, compared with the free Cy5-modified PD1 and CTLA4 groups, the Cy5-modified antibody-drug complex group showed a very strong fluorescent signal in the tumor, and the content was lower in other organs, confirming that Antibody-drug complexes have better targeting properties to tumors.
(3)抗体药物复合体的诊疗一体化验证(3) Verification of integrated diagnosis and treatment of antibody-drug complexes
分别将MB-MSA和ICG-MSA通过实施例一的方法合成两种纳米复合物，体外探究FRET效率。图2c为根据本发明一个实施例的不同ICG/MB配比的PD1和CTLA4纳米复合物的FRET强度；图2d为根据本发明一个实施例的不同ICG/MB配比的PD1和CTLA4纳米复合物在经过不同时间后的FRET强度；图2e为根据本发明一个实施例的不同时间的MB/ICG荧光强度的比值。发明人发现当ICG:MB＝60:1时FRET效率最高，约为65.8％。通过小鼠尾静脉注射的方式，每只小鼠的荧光蛋白注射量为1mg ICG/kg，3h、6h、12h和24h后通过小动物活体成像仪检测小鼠ICG、MB荧光信号分布。如图2f-图2j所示，高ROS条件下连接子断裂发生解聚，释放出抗体，使得ICG和MB荧光信号发生改变，荧光集团MB信号/淬灭集团ICG比值上调，证明该荧光区域为高ROS环境，可借此推测该部位大概率为肿瘤部位，发挥一定的诊断作用，为肿瘤的诊疗一体化提供了新思路。MB-MSA and ICG-MSA were respectively synthesized into two nanocomposites by the method of Example 1, and the FRET efficiency was explored in vitro. Figure 2c is the FRET intensity of PD1 and CTLA4 nanocomposites with different ICG/MB ratios according to one embodiment of the present invention; Figure 2d is the PD1 and CTLA4 nanocomposites with different ICG/MB ratios according to one embodiment of the present invention FRET intensity after different times; FIG. 2e is the ratio of MB/ICG fluorescence intensity at different times according to an embodiment of the present invention. The inventors found that the FRET efficiency is the highest when ICG:MB=60:1, about 65.8%. Through the tail vein injection of mice, the injection amount of fluorescent protein per mouse was 1mg ICG/kg. After 3h, 6h, 12h and 24h, the distribution of mouse ICG and MB fluorescence signals was detected by a small animal in vivo imager. As shown in Figure 2f-Figure 2j, under high ROS conditions, the linker breaks and depolymerizes, releasing antibodies, which changes the fluorescence signals of ICG and MB, and increases the ratio of fluorescent group MB signal/quenched group ICG, which proves that the fluorescent region is The high ROS environment can be used to speculate that the site is likely to be a tumor site, which plays a certain diagnostic role and provides a new idea for the integration of tumor diagnosis and treatment.
实施例三：本发明的ROS响应抗体药物复合体的有效性测定Example 3: Determination of the effectiveness of the ROS-responsive antibody-drug complex of the present invention
将5×105MC38细胞接种于C57BL/6小鼠右侧皮下，观察免疫治疗复合物的联合治疗疗效。当肿瘤平均体积达到约100mm3时，将小鼠随机分为5组(n＝6)，分别为：对照组、抗PD-1抗体组(每次100μg)，抗CTLA4抗体组(每次100μg)，PD1和CTLA4抗体药物复合体组(50μg抗PD1和50μg CTLA4)，以及游离PD1和CTLA4组(50μg抗PD1和50μg CTLA4)。取移植瘤前测量肿瘤体积，然后取出移植瘤进行称重并将进行石蜡包埋，然后进行组织切片，将肿瘤组织切片通过免疫组化分析Ki67测定肿瘤细胞恶性增殖程度，结果如图3a-图3e所示，本申请的抗体药物复合体组的肿瘤生长缓慢，抗体药物复合体对肿瘤生长的抑制作用与游离PD1和CTLA4抗体的效果相当。抗体药物复合体组的肿瘤中，Ki67阳性细胞百分比与游离PD1和CTLA4抗体组相当。结果证实抗体药物复合体能够抑制MC38实体瘤的增殖。5×10 5 MC38 cells were inoculated subcutaneously on the right side of C57BL/6 mice, and the combined therapeutic effect of the immunotherapeutic compound was observed. When the average tumor volume reached about 100mm , the mice were randomly divided into 5 groups (n=6), namely: control group, anti-PD-1 antibody group (100 μg each time), anti-CTLA4 antibody group (100 μg each time) ), PD1 and CTLA4 antibody-drug complex group (50 μg anti-PD1 and 50 μg CTLA4), and free PD1 and CTLA4 group (50 μg anti-PD1 and 50 μg CTLA4). The tumor volume was measured before taking out the transplanted tumor, and then the transplanted tumor was taken out, weighed, embedded in paraffin, and then sliced into tissues. The tumor tissue slices were analyzed by immunohistochemical Ki67 to determine the degree of malignant proliferation of tumor cells. The results are shown in Figure 3a-figure As shown in 3e, the tumor growth of the antibody-drug complex group of the present application is slow, and the inhibitory effect of the antibody-drug complex on tumor growth is equivalent to that of free PD1 and CTLA4 antibodies. The percentage of Ki67-positive cells in tumors in the antibody-drug complex group was comparable to that in the free PD1 and CTLA4 antibody group. The results confirmed that the antibody-drug complex can inhibit the proliferation of MC38 solid tumors.
实施例四：PD1和CTLA4纳米复合物缓解肿瘤免疫抑制微环境Example 4: PD1 and CTLA4 nanocomplex alleviates tumor immunosuppressive microenvironment
为了进一步证实PD1和CTLA4纳米复合物的抗肿瘤免疫作用，申请人进一步验证了CD8+细胞毒性T细胞和Treg细胞在小鼠移植瘤中的浸润情况。将肿瘤组织制成单细胞悬液，通过流式细胞术进行检测。如图4a-图4d所示，与其他各组相比，PD1和CTLA4纳米复合物组肿瘤中CD8+T细胞浸润最明显，Treg细胞浓度最低。此外，PD1和CTLA4纳米复合物处理后，与免疫细胞活性相关的颗粒酶B、TNF-α和IFN-γ显著增加。这说明PD1和CTLA4纳米复合物通过有效改善肿瘤免疫微环境来增强抗肿瘤疗效。In order to further confirm the anti-tumor immune effect of PD1 and CTLA4 nanocomposites, the applicant further verified the infiltration of CD8+ cytotoxic T cells and Treg cells in mouse xenografts. The tumor tissue was made into a single cell suspension and detected by flow cytometry. As shown in Figures 4a-4d, compared with the other groups, the PD1 and CTLA4 nanocomplex group had the most infiltration of CD8+ T cells and the lowest concentration of Treg cells in tumors. In addition, granzyme B, TNF-α, and IFN-γ associated with immune cell activity were significantly increased after PD1 and CTLA4 nanocomplex treatment. This indicates that the PD1 and CTLA4 nanocomplex enhances the antitumor efficacy by effectively improving the tumor immune microenvironment.
实施例五：PD1和CTLA4纳米复合物的生物安全性评估Example five: Biosafety assessment of PD1 and CTLA4 nanocomposites
对PD1和CTLA4纳米复合物的生物安全性进行进一步研究。首先，申请人观察了PBS对照、抗PD1、抗CTLA4、PD1和CTLA4纳米复合物、游离PD1和CTLA4处理后的5组小鼠的心脏、肝脏、脾脏、肺和肾脏的特征，图5a证明5组小鼠脏器的H&E染色没有差异。通过ELISA试剂盒检测小鼠肝功能和肾功能的损伤程度，其中肝功能采用天门冬氨酸转氨酶(AST)和谷丙转氨酶(ALT)进行评估，肾功能采用血清肌酐(CRE)和血尿素氮(BUN)进行评估。结果如图5b-图5e所示，PD1和CTLA4纳米复合物具有较高的生物安全性。Further studies on the biosafety of PD1 and CTLA4 nanocomplexes will be conducted. First, the applicant observed the characteristics of the hearts, livers, spleens, lungs and kidneys of five groups of mice treated with PBS control, anti-PD1, anti-CTLA4, PD1 and CTLA4 nanocomplexes, free PD1 and CTLA4, and Figure 5a demonstrates that 5 There was no difference in the H&E staining of the organs of the mice in the two groups. The damage degree of liver function and renal function of mice was detected by ELISA kit, wherein the liver function was evaluated by aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and the renal function was evaluated by serum creatinine (CRE) and blood urea nitrogen (BUN) for evaluation. Results As shown in Figure 5b-5e, PD1 and CTLA4 nanocomplexes have high biosafety.
实施例六：本发明的ROS响应抗体药物复合体的毒副作用检测Example 6: Detection of toxic and side effects of the ROS-responsive antibody-drug complex of the present invention
(1)将6-8周的雄性BALB/c小鼠随机分为对照组(n＝5)和EAM组(n＝25)。除对照组外，所有EAM小鼠分别于第0天和第7天，用200μg小鼠α-心肌肌球蛋白重链(MyHC-α614-629：乙酰-SLKLMATLFSTYAS，上海GL生物化工(GL Biochem Shanghai))溶液按描述用弗式完全佐剂进行乳化(西格玛(Sigma)，USA)，之后对小鼠进行皮下注射引发免疫性心肌炎。(1) Male BALB/c mice at 6-8 weeks were randomly divided into control group (n=5) and EAM group (n=25). Except for the control group, all EAM mice were injected with 200 μg mouse α-cardiac myosin heavy chain (MyHC-α614-629: acetyl-SLKLMATLFSTYAS, Shanghai GL Biochemical (GL Biochem Shanghai) on day 0 and day 7, respectively. )) The solution was emulsified with Freund's complete adjuvant (Sigma, USA) as described and injected subcutaneously into mice to induce immune myocarditis.
(2)从第7天开始，EAM小鼠随机分为5组(每组n＝5)。除EAM组外，其余4组均为免疫检查点抑制剂相关的心肌炎组(ICIs，模型组)，每2天腹腔注射PD-1抗体、CTLA4抗体、抗PD1和CTLA4复合物或游离的抗PD1和CTLA4，剂量为5mg/kg，连续5次注射诱导ICIs相关心肌炎。如图6a所示。(2) From day 7, EAM mice were randomly divided into 5 groups (n=5 in each group). Except for the EAM group, the other 4 groups were immune checkpoint inhibitor-related myocarditis group (ICIs, model group), and PD-1 antibody, CTLA4 antibody, anti-PD1 and CTLA4 complex or free anti-PD1 were injected intraperitoneally every 2 days and CTLA4 at a dose of 5 mg/kg, five consecutive injections induced ICIs-associated myocarditis. As shown in Figure 6a.
(3)第21天，通过超声心动评估各组小鼠左心室的功能及左心室前后壁的厚度。如图6b-图6c所示，与抗PD1和CTLA4抗体药物复合体组相比，游离PD1和CTLA4抗体组小鼠的左心室射血分数和缩短分数明显低于其他组，证明游离PD1和CTLA4抗体的使用能够抑制小鼠心肌炎的左心室收缩功能，而抗体药物复合体组的小鼠并未加剧该现象。(3) On the 21st day, the function of the left ventricle and the thickness of the front and rear walls of the left ventricle of the mice in each group were evaluated by echocardiography. As shown in Figure 6b-6c, compared with the anti-PD1 and CTLA4 antibody-drug complex group, the left ventricular ejection fraction and fractional shortening of the mice in the free PD1 and CTLA4 antibody group were significantly lower than those in the other groups, proving that the free PD1 and CTLA4 The use of antibodies can inhibit the left ventricular systolic function of myocarditis in mice, while the mice in the antibody-drug complex group did not exacerbate this phenomenon.
(4)取出小鼠的心脏组织，用预冷的磷酸盐缓冲盐溶液进行冲洗，后续将心脏组织进行石蜡包埋和组织切片，对其进行HE染色和Masson染色分析炎症表现和心肌纤维化情况。图7a-图7b所示，心肌组织呈现不同程度的炎症浸润和纤维化，其中游离PD1和CTLA4组炎症浸润程度和心肌纤维化现象最为严重，而PD1和CTLA4抗体药物复合体组与EAM组小鼠差异不明显，证明其未加重心肌炎小鼠的炎症表现和心肌纤维化情况。(4) Take out the heart tissue of the mouse, wash it with pre-cooled phosphate-buffered saline solution, then embedding the heart tissue in paraffin and sectioning it, and then perform HE staining and Masson staining to analyze the inflammation and myocardial fibrosis . As shown in Figure 7a-7b, myocardial tissue showed different degrees of inflammatory infiltration and fibrosis, among which the degree of inflammatory infiltration and myocardial fibrosis in the free PD1 and CTLA4 group was the most serious, while the PD1 and CTLA4 antibody-drug complex group was less than that in the EAM group. There was no obvious difference between mice, which proved that it did not aggravate the inflammatory manifestations and myocardial fibrosis of myocarditis mice.
(5)对心肌损伤进行评估：采用试剂盒(武汉赛沛生物技术有限公司，中国)检测血清标志物肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)和乳酸脱氢酶-1(LDH-1)的含量，使用商业检测试剂盒(江莱生物，中国)定量检测血清cTnI和cTnT水平。如图7c-图g所示，心肌炎小鼠能够诱发CK，CK-MB、LDH-1、cTnI和cTnT等心肌炎指标的升高，其中游离PD1和CTLA4组小鼠增高幅度最为显著，而与EAM组相比，PD1和CTLA4抗体药物复合体对此影响甚微，进一步证明其不会加剧心肌炎诱发的心肌损伤程度。(5) Assess myocardial injury: Serum markers creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase were detected using a kit (Wuhan Saipei Biotechnology Co., Ltd., China) -1 (LDH-1), using a commercial detection kit (Jianglai Bio, China) to quantitatively detect serum cTnI and cTnT levels. As shown in Figure 7c-figure g, myocarditis mice can induce the increase of myocarditis indicators such as CK, CK-MB, LDH-1, cTnI and cTnT, among which the mice in the free PD1 and CTLA4 group have the most significant increase, while those in the EAM Compared with the control group, the PD1 and CTLA4 antibody-drug complex had little effect on this, which further proved that it would not aggravate the degree of myocardial injury induced by myocarditis.
综合以上结果，本发明的ROS响应抗体药物复合体能够明显规避免疫检查点抑制剂的使用所造成的免疫不良反应，毒副作用低，具有良好的临床应用前景。Based on the above results, the ROS-responsive antibody-drug complex of the present invention can obviously avoid adverse immune reactions caused by the use of immune checkpoint inhibitors, has low toxic and side effects, and has good clinical application prospects.
上述实施例仅供说明本发明之用，而并非是对本发明的限制，有关技术领域的普通技术人员，在不脱离本发明范围的情况下，还可以做出各种变化和变型，因此，所有等同的技术方案也应属于本发明公开的范畴。The above-described embodiments are only for illustrating the present invention, rather than limiting the present invention. Those of ordinary skill in the relevant technical field can also make various changes and modifications without departing from the scope of the present invention. Therefore, all Equivalent technical solutions should also belong to the scope of the disclosure of the present invention.
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