CN116370651A - anti-PSMA antibody-drug conjugate, and preparation method and application thereof - Google Patents
anti-PSMA antibody-drug conjugate, and preparation method and application thereof Download PDFInfo
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- CN116370651A CN116370651A CN202310350727.1A CN202310350727A CN116370651A CN 116370651 A CN116370651 A CN 116370651A CN 202310350727 A CN202310350727 A CN 202310350727A CN 116370651 A CN116370651 A CN 116370651A
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- antibody
- drug conjugate
- psma
- linker
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Abstract
本发明属于生物医药技术领域，具体涉及一种抗PSMA抗体‑药物缀合物及其制备方法和应用。本发明提供了一种抗PSMA抗体‑药物缀合物，具有式1所示结构。本发明采用能够特异性结合PSMA且具有高内化活性的人源化单克隆抗体J591，基于该抗体偶联活性药物获得的抗体‑药物缀合物，所述抗体‑药物缀合物包含具有对肿瘤的治疗作用的活性药物，并且具有对PSMA阳性肿瘤细胞较高的杀伤活性，可应用于PSMA阳性肿瘤治疗。
The invention belongs to the technical field of biomedicine, and in particular relates to an anti-PSMA antibody-drug conjugate and its preparation method and application. The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in formula 1. The present invention adopts the humanized monoclonal antibody J591 that can specifically bind to PSMA and has high internalization activity, and obtains an antibody-drug conjugate based on the antibody coupled with an active drug, the antibody-drug conjugate comprising The active drug for the treatment of tumors has high killing activity on PSMA-positive tumor cells, and can be applied to the treatment of PSMA-positive tumors.
Description
技术领域technical field
本发明属于生物医药技术领域，具体涉及一种抗PSMA抗体-药物缀合物及其制备方法和应用。The invention belongs to the technical field of biomedicine, and specifically relates to an anti-PSMA antibody-drug conjugate and a preparation method and application thereof.
背景技术Background technique
前列腺癌是指发生在前列腺的上皮性恶性肿瘤，是男性泌尿生殖系统最常见的恶性肿瘤之一。由于前列腺癌具有显著的肿瘤异质性，因此如何对前列腺癌患者进行精准化和个体化治疗一直是领域内研究重点。Prostate cancer is an epithelial malignant tumor that occurs in the prostate gland, and is one of the most common malignant tumors in the male genitourinary system. Due to the significant tumor heterogeneity of prostate cancer, how to carry out precise and individualized treatment for prostate cancer patients has always been the focus of research in the field.
目前，前列腺癌的治疗以主动监测、前列腺癌根治术、化疗等为主。研究表明随着内分泌治疗的进行，多数患者出现对内分泌药物抵抗的现象，进入去势抵抗性前列腺癌（castration resistant prostate cancer, CRPC）阶段，最终会发展为转移性去势抵抗性前列腺癌（metastatic castration resis tant prostate cancer, mCRPC）。At present, the treatment of prostate cancer is mainly based on active surveillance, radical prostatectomy, and chemotherapy. Studies have shown that with the progress of endocrine therapy, most patients develop resistance to endocrine drugs, enter the stage of castration-resistant prostate cancer (CRPC), and eventually develop into metastatic castration-resistant prostate cancer (metastatic). castration resistance tant prostate cancer, mCRPC).
前列腺特异性膜抗原（Prostate specific membrane antigen，PSMA）是一种与前列腺癌相关的标志物，是前列腺癌靶向治疗的理想靶标。PSMA在去势抵抗性前列腺癌中显著过表达，其过表达与高肿瘤分级、疾病进展和复发、临床预后较差和患者生存期缩短相关。Prostate specific membrane antigen (PSMA) is a marker associated with prostate cancer and is an ideal target for targeted therapy of prostate cancer. PSMA is significantly overexpressed in castration-resistant prostate cancer, and its overexpression is associated with high tumor grade, disease progression and recurrence, poor clinical prognosis and shortened patient survival.
抗体药物缀合物（Antibody-Drug Conjugates）是一种新型的肿瘤靶向治疗方法，通过肿瘤标志物特异性抗体将治疗剂递送到肿瘤组织或肿瘤细胞以杀死肿瘤。鉴于PSMA的物理特性及其表达模式与前列腺癌进展相关，PSMA是开发抗体偶联药物（Antibody -drugconjugates，ADCs）的理想靶点。Antibody-Drug Conjugates (Antibody-Drug Conjugates) is a new type of tumor-targeted therapy, which uses tumor marker-specific antibodies to deliver therapeutic agents to tumor tissues or tumor cells to kill tumors. Given that the physical properties of PSMA and its expression patterns are associated with prostate cancer progression, PSMA is an ideal target for the development of antibody-drug conjugates (ADCs).
但是，目前已经报道的常规抗PSMA抗体-药物缀合物抗体药物缀合物的抗肿瘤活性仍然处于较低水平。However, the anti-tumor activity of conventional anti-PSMA antibody-drug conjugate antibody-drug conjugates reported so far is still at a low level.
发明内容Contents of the invention
本发明的目的在于提供一种抗PSMA抗体-药物缀合物及其制备方法和应用，本发明提供的抗PSMA抗体-药物缀合物基于特异性抗PSMA的人源化单抗J591通过连接子接头与在细胞中发挥毒性作用的活性药物缀合，实现了比常规PSMA抗体-药物缀合物的抗肿瘤活性更强的抗肿瘤活性。The object of the present invention is to provide an anti-PSMA antibody-drug conjugate and its preparation method and application. The anti-PSMA antibody-drug conjugate provided by the present invention is based on the specific anti-PSMA humanized monoclonal antibody J591 through a linker The linker is conjugated to an active drug that exerts toxic effects in cells, achieving stronger antitumor activity than that of conventional PSMA antibody-drug conjugates.
本发明提供了一种抗PSMA抗体-药物缀合物，具有式1所示结构：The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in formula 1:
式1中：Ab为人源化抗体J591或其功能性片段；In formula 1: Ab is humanized antibody J591 or its functional fragment;
S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；S is the sulfur atom in the sulfhydryl residue formed after the interchain disulfide bond on the Ab is opened;
L为连接所述Ab与活性药物单元的连接子接头；L is a linker joint connecting the Ab and the active drug unit;
D为活性药物单元；D is an active drug unit;
p为1~16的任意整数。p is any integer from 1 to 16.
本发明提供了一种抗PSMA抗体-药物缀合物，具有式2所示结构：The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in formula 2:
式2中：Ab为人源化抗体J591或其功能性片段；In formula 2: Ab is humanized antibody J591 or its functional fragment;
S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；S is the sulfur atom in the sulfhydryl residue formed after the interchain disulfide bond on the Ab is opened;
D1为第一活性药物；D1 is the first active drug;
D2为第二活性药物；D2 is the second active drug;
Aa为包含一个或多个氨基酸的氨基酸单元；Aa is an amino acid unit comprising one or more amino acids;
G为可裂解单元；G is a cleavable unit;
p为1~16的任意整数。p is any integer from 1 to 16.
优选的，所述L包括可切割的连接子接头或者不可切割的连接子接头；Preferably, said L comprises a cleavable linker adapter or a non-cleavable linker adapter;
所述可切割的连接子接头包括肽、腙或二硫化物连接子接头；The cleavable linker linker comprises a peptide, hydrazone or disulfide linker linker;
所述不可切割的连接子接头包括马来酰亚氨基己酰基。The non-cleavable linker linker comprises a maleimidocaproyl group.
优选的，所述活性药物单元D包括细胞毒性药物、免疫增强剂或放射性同位素；Preferably, the active drug unit D includes cytotoxic drugs, immune enhancers or radioactive isotopes;
所述细胞毒性药物包括抗代谢药、抗肿瘤药、抗生素和生物碱中的一种或多种。The cytotoxic drugs include one or more of antimetabolites, antineoplastics, antibiotics and alkaloids.
优选的，所述活性药物单元D为单甲基耳抑素肽E、单甲基耳抑素肽F、SN-38或MK4827。Preferably, the active drug unit D is monomethyl auristatin peptide E, monomethyl auristatin peptide F, SN-38 or MK4827.
优选的，所述D1包括式3所示结构的的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐或溶剂化物中的一种或多种；Preferably, the D1 includes the compound of the structure shown in
优选的，所述D2包括细胞毒性分子、免疫增强剂和放射性同位素中的一种或多种；Preferably, the D2 includes one or more of cytotoxic molecules, immune enhancers and radioactive isotopes;
所述细胞毒性分子包括微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂和PARP抑制剂中的一种或多种；所述微管蛋白抑制剂包括海兔毒素类细胞毒分子、澳瑞他汀类细胞毒分子和美登素类细胞毒分子中的一种或多种；所述DNA损伤剂包括卡奇霉素类衍生物、倍癌霉素类衍生物和安曲霉素类衍生物中的一种或多种；所述拓扑异构酶抑制剂包括喜树碱及喜树碱类衍生物。The cytotoxic molecules include one or more of tubulin inhibitors, DNA damage agents, topoisomerase inhibitors, ALK inhibitors and PARP inhibitors; the tubulin inhibitors include dolastatins One or more of cytotoxic molecules, auristatin cytotoxic molecules, and maytansinoid cytotoxic molecules; the DNA damage agents include calicheamicin derivatives, duocarmycin derivatives, and antradine One or more of mycin derivatives; the topoisomerase inhibitors include camptothecin and camptothecin derivatives.
优选的，所述人源化抗体J591包含重链和轻链；Preferably, the humanized antibody J591 comprises a heavy chain and a light chain;
所述轻链包含可变区和恒定区；所述轻链的可变区具有如SEQ ID NO.1所示的氨基酸序列，或者具有与SEQ ID NO.1至少80％序列同一性的序列；The light chain comprises a variable region and a constant region; the variable region of the light chain has an amino acid sequence as shown in SEQ ID NO.1, or a sequence with at least 80% sequence identity to SEQ ID NO.1;
所述重链包含可变区和恒定区；所述重链的可变区具有如SEQ ID NO.3所示的氨基酸序列，或者具有与SEQ ID NO.3至少80％序列同一性的序列；The heavy chain comprises a variable region and a constant region; the variable region of the heavy chain has an amino acid sequence as shown in SEQ ID NO.3, or a sequence with at least 80% sequence identity to SEQ ID NO.3;
所述轻链的恒定区和重链的恒定区为人源化的或人源的。The constant region of the light chain and the constant region of the heavy chain are humanized or humanized.
本发明提供了上述技术方案所述的PSMA抗体-药物缀合物的制备方法，包括以下步骤：The present invention provides a method for preparing the PSMA antibody-drug conjugate described in the above technical scheme, comprising the following steps:
将还原剂、保护剂和人源化抗体J591或其功能性片段混合，进行还原，得到还原后的人源化J591抗体或其功能性片段；mixing the reducing agent, the protecting agent and the humanized antibody J591 or its functional fragments, and performing reduction to obtain the reduced humanized J591 antibody or its functional fragments;
将连接底物、活性药物、偶联试剂和溶剂混合，进行偶联反应，得到linker-活性药物偶联物；Mix the linking substrate, active drug, coupling reagent and solvent, and perform a coupling reaction to obtain a linker-active drug conjugate;
将所述还原后的人源化J591抗体、所述linker-活性药物偶联物和溶剂混合，进行偶联反应，得到PSMA抗体-药物缀合物。The reduced humanized J591 antibody, the linker-active drug conjugate and a solvent are mixed for a coupling reaction to obtain a PSMA antibody-drug conjugate.
本发明提供了上述技术方案所述的PSMA抗体-药物缀合物或上述技术方案所述的制备方法制备得到的PSMA抗体-药物缀合物在制备治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。The present invention provides the PSMA antibody-drug conjugate described in the above technical scheme or the PSMA antibody-drug conjugate prepared by the preparation method described in the above technical scheme can be used in the preparation of treatment or prevention of cancer, infectious diseases or autoimmunity Application in the medicine of disease.
本发明提供了一种抗PSMA抗体-药物缀合物，具有式1所示结构。本发明采用能够特异性结合PSMA且具有高内化活性的人源化单克隆抗体J591，基于该抗体偶联活性药物获得的抗体-药物缀合物，所述抗体-药物缀合物包含具有对肿瘤的治疗作用的活性药物，并且具有对PSMA阳性肿瘤细胞较高的杀伤活性，可应用于PSMA阳性肿瘤治疗。The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in
本发明提供了一种抗PSMA抗体-药物缀合物，具有式2所示结构。在本发明中，所述活性药物单元中包括第一活性药物和第二活性药物；所述抗PSMA抗体-药物缀合物具有式2所示结构。本发明采用第一活性药物和第二活性药物复配，相较于常规PSMA抗体-药物缀合物，本发明提供的抗PSMA抗体-药物缀合物的抗肿瘤活性更强。The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in
进一步的，在本发明中，所述L包括可切割的连接子接头或者不可切割的连接子接头；所述可切割的连接子接头包括肽、腙或二硫化物连接子接头；所述不可切割的连接子接头包括马来酰亚氨基己酰基。本发明选择特定的连接子接头能够实现人源化抗体J591和活性药物的有效结合，提高抗PSMA抗体-药物缀合物的抗肿瘤活性，具有良好的肿瘤治疗应用前景。Further, in the present invention, the L includes a cleavable linker linker or a non-cleavable linker linker; the cleavable linker linker includes a peptide, hydrazone or disulfide linker linker; the non-cleavable The linker linker includes maleimidocaproyl. In the present invention, selecting a specific linker linker can realize the effective combination of the humanized antibody J591 and the active drug, improve the anti-tumor activity of the anti-PSMA antibody-drug conjugate, and has a good application prospect for tumor treatment.
附图说明Description of drawings
图1为本发明实施例中MC-VC-PAB-MMAE的制备路线图；Fig. 1 is the preparation roadmap of MC-VC-PAB-MMAE in the embodiment of the present invention;
图2为本发明实施例中MC-VC-PAB-DMEDA-PEG(N3)-SN38的制备路线图；Fig. 2 is the preparation roadmap of MC-VC-PAB-DMEDA-PEG(N3)-SN38 in the embodiment of the present invention;
图3为本发明实施例中MP2-VC-PAB-DMEDA-PEG-SN38的制备路线图；Fig. 3 is the preparation roadmap of MP2-VC-PAB-DMEDA-PEG-SN38 in the embodiment of the present invention;
图4为本发明实施例中MC-MMAF-VC-PAB-MK4827的制备路线图；Fig. 4 is the preparation roadmap of MC-MMAF-VC-PAB-MK4827 in the embodiment of the present invention;
图5为本发明实施例中MC-MMAF-VC-PAB-DMEDA-PEG-SN38的制备路线图；Fig. 5 is the preparation roadmap of MC-MMAF-VC-PAB-DMEDA-PEG-SN38 in the embodiment of the present invention;
图6为本发明实施例制备的人源化J591单抗在还原和非还原条件下SDS-PAGE电泳图；Fig. 6 is the SDS-PAGE electrophoresis diagram of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention under reducing and non-reducing conditions;
图7为本发明实施例制备的人源化J591单抗在214 nm波长条件下的尺寸排阻色谱分析结果；Fig. 7 is the size exclusion chromatography analysis result of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention at a wavelength of 214 nm;
图8为本发明实施例制备的人源化J591单抗在280 nm波长条件下的尺寸排阻色谱分析结果；Fig. 8 is the size exclusion chromatography analysis result of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention at a wavelength of 280 nm;
图9为吸光度值与Biotin-PSMA重组蛋白浓度曲线图；Fig. 9 is a graph of absorbance value and Biotin-PSMA recombinant protein concentration;
图10为人源化单抗J591对不同PSMA阳性肿瘤细胞系结合后荧光强度对比图；Figure 10 is a comparison of the fluorescence intensity of the humanized monoclonal antibody J591 after binding to different PSMA-positive tumor cell lines;
图11为人源化单抗J591在不同PSMA阳性肿瘤细胞中的内化活性测定图；Figure 11 is a graph showing the internalization activity assay of humanized monoclonal antibody J591 in different PSMA-positive tumor cells;
图12为本发明实施例制备的不同种类的抗PSMA抗体-药物缀合物对前列腺癌细胞系C4-2B的生长抑制结果；Figure 12 shows the growth inhibition results of different types of anti-PSMA antibody-drug conjugates prepared in the examples of the present invention on the prostate cancer cell line C4-2B;
图13为本发明实施例制备的不同种类的抗PSMA抗体-药物缀合物对前列腺癌细胞系LNCap的生长抑制结果；Figure 13 shows the growth inhibition results of different types of anti-PSMA antibody-drug conjugates prepared in the examples of the present invention on the prostate cancer cell line LNCap;
图14为本发明实施例制备的不同种类的抗PSMA抗体-药物缀合物对前列腺癌细胞系22RV1的生长抑制结果；Figure 14 shows the growth inhibition results of different types of anti-PSMA antibody-drug conjugates prepared in the examples of the present invention on the prostate cancer cell line 22RV1;
图15为本发明实施例制备的不同种类的抗PSMA抗体-药物缀合物对前列腺癌细胞系PC-3的生长抑制结果。Figure 15 shows the growth inhibition results of different types of anti-PSMA antibody-drug conjugates prepared in the examples of the present invention on the prostate cancer cell line PC-3.
实施方式Implementation
本发明提供了一种抗PSMA抗体-药物缀合物，具有式1所示结构：The present invention provides an anti-PSMA antibody-drug conjugate, which has the structure shown in formula 1:
式1中：Ab为人源化抗体J591或其功能性片段；In formula 1: Ab is humanized antibody J591 or its functional fragment;
S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；S is the sulfur atom in the sulfhydryl residue formed after the interchain disulfide bond on the Ab is opened;
L为连接所述Ab与活性药物单元的连接子接头；L is a linker joint connecting the Ab and the active drug unit;
D为活性药物单元；D is an active drug unit;
p为1~16的任意整数。p is any integer from 1 to 16.
在本发明中，若无特殊说明，所有制备原料/组分均为本领域技术人员熟知的市售产品。In the present invention, unless otherwise specified, all preparation raw materials/components are commercially available products well known to those skilled in the art.
本发明提供的式1所示结构抗PSMA抗体-药物缀合物中，Ab为人源化抗体J591或其功能性片段。In the anti-PSMA antibody-drug conjugate with the structure shown in
在本发明中，所述人源化抗体J591优选包含重链和轻链。In the present invention, the humanized antibody J591 preferably comprises a heavy chain and a light chain.
在本发明中，所述轻链优选包含可变区和恒定区；所述轻链的可变区具有如SEQID NO.1所示的氨基酸序列，或者具有与SEQ ID NO.1至少80％序列同一性的序列，优选为85~99%，具体优选为85％、90％、95％、98％或者99％。In the present invention, the light chain preferably comprises a variable region and a constant region; the variable region of the light chain has an amino acid sequence as shown in SEQ ID NO.1, or has at least 80% of the sequence of SEQ ID NO.1 The sequence identity is preferably 85-99%, specifically preferably 85%, 90%, 95%, 98% or 99%.
在本发明中，SEQ ID NO.1所示的氨基酸序列为：In the present invention, the amino acid sequence shown in SEQ ID NO.1 is:
DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK。DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK.
在本发明中，SEQ ID NO.1所示的氨基酸序列由SEQ ID NO.2所示的核苷酸序列获得，SEQ ID NO.2所示的核苷酸序列为：In the present invention, the amino acid sequence shown in SEQ ID NO.1 is obtained from the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence shown in SEQ ID NO.2 is:
GACATTGTGATGACCCAGAGCCACAAATTTATGAGCACCAGCGTGGGAGATAGAGTGAGTATTATCTGCAAGGCCAGCCAGGACGTGGGAACCGCCGTGGACTGGTATCAGCAGAAACCTGGCCAGAGCCCAAAACTGCTGATCTACTGGGCCAGCACCAGACATACCGGAGTGCCCGACAGGTTCACAGGATCAGGCAGCGGCACAGACTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTATTTCTGCCAGCAGTACAACAGCTACCCCCTGACATTTGGAGCCGGCACCATGCTGGACCTGAAG。GACATTGTGTGACCCAGAGCCACAAATTTATGAGCACCAGCGTGGGAGATAGAGTGAGTATTATCTGCAAGGCCAGCCAGGACGTGGGAACCGCCGTGGACTGGTATCAGCAGAAACCTGGCCAGAGCCCAAAACTGCTGATCTACTGGGCCAGCACCAGACATACCGGAGTGCCCGACAGGTTCACAGGATCAGGCAGCGGCACAGACTTCACCCCTG ACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTATTTCTGCCAGCAGTACAACAGCTACCCCCCTGACATTTGGAGCCGGCACCATGCTGGACCTGAAG.
在本发明中，所述轻链的恒定区优选为人源化的或人源的，更优选为人源的。In the present invention, the constant region of the light chain is preferably humanized or human, more preferably human.
在本发明中，所述重链优选包含可变区和恒定区；所述重链的可变区具有如SEQID NO.3所示的氨基酸序列，或者具有与SEQ ID NO.3至少80％序列同一性的序列，优选为85~99%，具体优选为85％、90％、95％、98％或者99％。In the present invention, the heavy chain preferably comprises a variable region and a constant region; the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.3, or has at least 80% of the sequence of SEQ ID NO.3 The sequence identity is preferably 85-99%, specifically preferably 85%, 90%, 95%, 98% or 99%.
在本发明中，SEQ ID NO.3所示的氨基酸序列为：In the present invention, the amino acid sequence shown in SEQ ID NO.3 is:
EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS。EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS.
在本发明中，SEQ ID NO.3所示的氨基酸序列由SEQ ID NO.4所示的核苷酸序列获得，SEQ ID NO.4所示的核苷酸序列为：In the present invention, the amino acid sequence shown in SEQ ID NO.3 is obtained from the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.4 is:
GAGGTGCAGCTGCAGCAGAGCGGGCCAGAGCTGAAGAAGCCAGGGACAAGCGTGAGAATTAGCTGCAAGACAAGCGGGTACACATTTACAGAGTATACCATTCACTGGGTGAAACAGAGCCACGGTAAGAGCCTGGAATGGATTGGAAACATCAACCCCAATAACGGAGGAACAACCTACAACCAGAAATTCGAGGACAAAGCCACCCTGACCGTGGACAAAAGCAGTAGCACCGCCTACATGGAGCTGAGAAGCCTGACCAGCGAAGACAGCGCCGTGTACTACTGCGCCGCCGGCTGGAACTTCGACTATTGGGGACAGGGCACCACCCTGACCGTGAGCAGC。GAGGTGCAGCTGCAGCAGAGCGGGCCAGAGCTGAAGAAGCCAGGGACAAGCGTGAGAATTAGCTGCAAGACAAGCGGGTACACATTTACAGAGTATACCATTCACTGGGTGAAACAGAGCCACGGTAAGAGCCTGGAATGGATTGGAAACATCAACCCCAATAACGGAGGAACAACCTACAACCAGAAATTCGAGGACAAAGCCACCCTGACCGTGGACA AAAGCAGTAGCACCGCCTACATGGAGCTGAGAAGCCTGACCAGCGAAGACAGCGCCGTGTACTACTGCGCCGCCGGCTGGAACTTCGACTATTGGGGACAGGGCACCACCCTGACCGTGAGCAGC.
在本发明中，所述重链的恒定区优选为人源化的或人源的，更优选为人源的。In the present invention, the constant region of the heavy chain is preferably humanized or human, more preferably human.
在本发明中，所述人源化抗体J591的亚型优选包括IgM、IgG1、IgG2、IgG3和IgG4中的一种或多种，更优选为IgG1。In the present invention, the subtype of the humanized antibody J591 preferably includes one or more of IgM, IgG1, IgG2, IgG3 and IgG4, more preferably IgG1.
本发明提供的式1所示结构抗PSMA抗体-药物缀合物中，L为连接所述Ab与活性药物单元的连接子接头。In the anti-PSMA antibody-drug conjugate with the structure shown in
本发明提供的抗PSMA抗体-药物缀合物中包括连接子。The anti-PSMA antibody-drug conjugate provided by the present invention includes a linker.
在本发明中，所述连接子接头优选通过巯基和/或氨基与所述人源化抗体J591连接。In the present invention, the linker linker is preferably connected to the humanized antibody J591 through a sulfhydryl group and/or an amino group.
作为本发明的一个或多个实施例，所述连接子接头优选通过巯基与人源化抗体J591连接。As one or more embodiments of the present invention, the linker linker is preferably connected to the humanized antibody J591 through a sulfhydryl group.
在本发明中，所述L优选包括可切割的连接子接头或者不可切割的连接子接头。在本发明中，所述可切割的连接子接头为在体内环境下发生断裂的连接子接头。In the present invention, said L preferably includes a cleavable linker adapter or a non-cleavable linker adapter. In the present invention, the cleavable linker linker is a linker linker that breaks under the in vivo environment.
在本发明中，所述可切割的连接子接头优选包括肽、腙或二硫化物连接子接头，更优选包括马来酰亚氨基己酰基-缬氨酸-瓜氨酸-p-氨基苯甲氧羰基(maleimidocaproyl -valine -citrulline -p- aminobenzyloxycarbonyl，简称为：mc-vc-pAB或者vc)。In the present invention, the cleavable linker linker preferably comprises a peptide, hydrazone or disulfide linker linker, more preferably maleimidocaproyl-valine-citrulline-p-aminobenzidine Oxycarbonyl (maleimidocaproyl-valine-citrulline-p-aminobenzoxyloxycarbonyl, abbreviated as: mc-vc-pAB or vc).
在本发明中，所述不可切割的连接子接头优选包括马来酰亚氨基己酰基(maleimidocaproyl，简称为mc)。In the present invention, the non-cleavable linker linker preferably includes maleimidocaproyl (maleimidocaproyl, mc for short).
本发明提供的式1所示结构抗PSMA抗体-药物缀合物中，D为活性药物单元。In the anti-PSMA antibody-drug conjugate with the structure shown in
在本发明中，所述活性药物单元D优选包括细胞毒性药物、免疫增强剂或放射性同位素。In the present invention, the active drug unit D preferably includes cytotoxic drugs, immunopotentiators or radioactive isotopes.
在本发明中，所述细胞毒性药物包括抗代谢药、抗肿瘤药、抗生素和生物碱中的一种或多种。In the present invention, the cytotoxic drugs include one or more of antimetabolites, antitumor drugs, antibiotics and alkaloids.
在本发明中，所述活性药物单元D更优选包括微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂、PARP抑制剂；进一步优选的，所述微管蛋白抑制剂包括海兔毒素(dolastatin)及澳瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂优选包括卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物（PBD）；所述拓扑异构酶抑制剂优选包括喜树碱(camptothecins)及喜树碱类衍生物；所述PARP抑制剂优选包括尼拉帕尼（Niraparib，简称为MK4827）；进一步优选的，所述澳瑞他汀(auristatin)类细胞素分子包括但不限于单甲基耳抑素肽E（Monomethylauristatin E，简称为MMAE）及其衍生物或单甲基耳抑素肽F（Monomethyl auristatin F，简称为MMAF）及其洐生物，所述喜树碱及喜树碱类衍生物包括但不限于7-乙基-10-羟基喜树碱（SN-38）或DXd，所述美登素类细胞毒分子优选包括DM1及其衍生物和DM4及其衍生物。In the present invention, the active drug unit D more preferably includes tubulin inhibitors, DNA damage agents, topoisomerase inhibitors, ALK inhibitors, PARP inhibitors; further preferably, the tubulin inhibitors Including dolastatin (dolastatin) and auristatin (auristatin) class of cytotoxic molecules, maytansine (maytansine) class of cytotoxic molecules; the DNA damage agent preferably includes calicheamicin (calicheamicin) class, duocarmycin (duocarmycin) class, antramycin derivatives (PBD); the topoisomerase inhibitor preferably includes camptothecin (camptothecins) and camptothecin derivatives; the PARP inhibitor preferably includes nirapag Niraparib (abbreviated as MK4827); further preferably, the auristatin-like cytokine molecules include but not limited to monomethylauristin peptide E (Monomethylauristatin E, abbreviated as MMAE) and its derivatives or Monomethyl auristatin F (Monomethyl auristatin F, referred to as MMAF) and its derivatives, the camptothecin and camptothecin derivatives include but not limited to 7-ethyl-10-hydroxycamptothecin ( SN-38) or DXd, the maytansinoid cytotoxic molecules preferably include DM1 and its derivatives and DM4 and its derivatives.
在本发明中，所述活性药物单元D进一步优选为MMAE、MMAF、SN-38或MK4827，最优选为MMAE。In the present invention, the active drug unit D is further preferably MMAE, MMAF, SN-38 or MK4827, most preferably MMAE.
在本发明中，所述活性药物单元中优选包括第一活性药物和第二活性药物；本发明提供的所述抗PSMA抗体-药物缀合物优选具有式2所示结构：In the present invention, the active drug unit preferably includes the first active drug and the second active drug; the anti-PSMA antibody-drug conjugate provided by the present invention preferably has the structure shown in formula 2:
式2中：Ab为人源化抗体J591或其功能性片段；In formula 2: Ab is humanized antibody J591 or its functional fragment;
S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；S is the sulfur atom in the sulfhydryl residue formed after the interchain disulfide bond on the Ab is opened;
D1为第一活性药物；D1 is the first active drug;
D2为第二活性药物；D2 is the second active drug;
Aa为包含一个或多个氨基酸的氨基酸单元；Aa is an amino acid unit comprising one or more amino acids;
G为可裂解单元；G is a cleavable unit;
p为1~16的任意整数。p is any integer from 1 to 16.
在本发明中，所述活性药物单元优选包括第一活性药物D1、第二活性药物D2、包含一个或多个氨基酸的氨基酸单元Aa和可裂解单元G。In the present invention, the active drug unit preferably includes a first active drug D1, a second active drug D2, an amino acid unit Aa comprising one or more amino acids, and a cleavable unit G.
在本发明中，所述D1优选为澳瑞他汀(auristatin)类细胞毒性剂。In the present invention, the D1 is preferably an auristatin-like cytotoxic agent.
在本发明中，所述D1更优选包括式3所示结构的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其药学上可接受的盐或溶剂化物中的一种或多种；In the present invention, the D1 more preferably includes the compound of the structure shown in
在本发明中，所述D1具体优选为MMAF。In the present invention, the D1 is specifically preferably MMAF.
在本发明中，所述D2优选包括细胞毒性分子、免疫增强剂和放射性同位素中的一种或多种。In the present invention, the D2 preferably includes one or more of cytotoxic molecules, immune enhancers and radioactive isotopes.
在本发明中，所述细胞毒性分子优选包括微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂和PARP抑制剂中的一种或多种。In the present invention, the cytotoxic molecule preferably includes one or more of tubulin inhibitors, DNA damage agents, topoisomerase inhibitors, ALK inhibitors and PARP inhibitors.
在本发明中，所述微管蛋白抑制剂优选包括海兔毒素(dolastatin)类细胞毒分子、澳瑞他汀(auristatin)类细胞毒分子和美登素(maytansine)类细胞毒分子中的一种或多种。在本发明中，所述澳瑞他汀类细胞毒分子优选包括MMAE及其衍生物和/或MMAF及其洐生物；所述美登素类细胞毒分子优选包括DM1及其衍生物和/或DM4及其洐生物。In the present invention, the tubulin inhibitor preferably includes one or more of dolastatin-like cytotoxic molecules, auristatin-like cytotoxic molecules and maytansine-like cytotoxic molecules. Various. In the present invention, the auristatin cytotoxic molecules preferably include MMAE and its derivatives and/or MMAF and its derivatives; the maytansinoid cytotoxic molecules preferably include DM1 and its derivatives and/or DM4 and its living organisms.
在本发明中，所述DNA损伤剂优选包括卡奇霉素(calicheamicin)类衍生物、倍癌霉素(duocarmycin)类衍生物和安曲霉素类衍生物中的一种或多种。In the present invention, the DNA damaging agent preferably includes one or more of calicheamicin derivatives, duocarmycin derivatives and antramycin derivatives.
在本发明中，所述拓扑异构酶抑制剂优选包括喜树碱(camptothecins)及喜树碱类衍生物。In the present invention, the topoisomerase inhibitors preferably include camptothecins and camptothecin derivatives.
在本发明中，所述D2更优选包括ALK抑制剂、PARP抑制剂和拓扑异构酶抑制剂中的一种或多种，进一步优选为SN-38或MK4827。In the present invention, the D2 more preferably includes one or more of ALK inhibitors, PARP inhibitors and topoisomerase inhibitors, more preferably SN-38 or MK4827.
作为本发明得以一个或多个具体实施例，所述D1和D2分别为MMAF和SN38。As one or more specific embodiments of the present invention, the D1 and D2 are MMAF and SN38 respectively.
作为本发明的一个或多个实施例，所述D1和D2分别为MMAF和MK4827。As one or more embodiments of the present invention, the D1 and D2 are MMAF and MK4827 respectively.
本发明提供了上述技术方案所述的PSMA抗体-药物缀合物的制备方法，包括以下步骤：The present invention provides a method for preparing the PSMA antibody-drug conjugate described in the above technical scheme, comprising the following steps:
将还原剂、保护剂和人源化抗体J591混合，进行还原，得到还原后的人源化J591抗体；Mix the reducing agent, the protecting agent and the humanized antibody J591, and perform reduction to obtain the reduced humanized J591 antibody;
将连接底物、活性药物、偶联试剂和溶剂混合，进行偶联反应，得到linker-活性药物偶联物；Mix the linking substrate, active drug, coupling reagent and solvent, and perform a coupling reaction to obtain a linker-active drug conjugate;
将所述还原后的人源化J591抗体、所述linker-活性药物偶联物和溶剂混合，进行偶联反应，得到PSMA抗体-药物缀合物。The reduced humanized J591 antibody, the linker-active drug conjugate and a solvent are mixed for a coupling reaction to obtain a PSMA antibody-drug conjugate.
本发明将还原剂、保护剂和人源化抗体J591混合，进行还原，得到还原后的人源化J591抗体。In the present invention, the reducing agent, the protecting agent and the humanized antibody J591 are mixed and reduced to obtain the reduced humanized J591 antibody.
在本发明中，所述人源化抗体J591的制备方法优选包括以下步骤：In the present invention, the preparation method of the humanized antibody J591 preferably includes the following steps:
将鼠源单抗J591的可变区与人IgG1κ恒定区通过重叠延伸PCR的方法连接成完整片段，将所述完整片段亚克隆到表达载体上，将构建的质粒转染到悬浮的宿主细胞中表达产生所述人源化抗体J591。The variable region of the mouse monoclonal antibody J591 and the constant region of human IgG1κ were ligated into a complete fragment by overlapping extension PCR, the complete fragment was subcloned into an expression vector, and the constructed plasmid was transfected into suspended host cells Expression produced the humanized antibody J591.
在本发明中，所述表达载体具体优选为表达载体pcDNA3.0。In the present invention, the expression vector is specifically preferably the expression vector pcDNA3.0.
在本发明中，所述宿主细胞具体优选为CHO细胞（Invitrogen）。In the present invention, the host cells are specifically preferably CHO cells (Invitrogen).
本发明优选从所述转染的悬浮的宿主细胞培养上清液中收集表达的人源化抗体J591。在本发明中，所述转染的悬浮的宿主细胞培养上清液中收集的为人源化抗体J591的粗品，本发明优选对所述人源化抗体J591的粗品进行纯化，得到人源化抗体J591的纯品。在本发明中，所述纯化优选采用Protein A进行纯化。In the present invention, the expressed humanized antibody J591 is preferably collected from the culture supernatant of the transfected suspended host cells. In the present invention, the crude product of the humanized antibody J591 is collected from the culture supernatant of the transfected suspended host cells. In the present invention, the crude product of the humanized antibody J591 is preferably purified to obtain the humanized antibody Pure product of J591. In the present invention, the purification is preferably performed using Protein A.
本发明优选通过所述SDS-PAGE电泳及SEC分析所述人源化抗体J591的纯度。In the present invention, the purity of the humanized antibody J591 is preferably analyzed by the SDS-PAGE electrophoresis and SEC.
在本发明中，所述人源化抗体J591的纯品的纯度优选≥95%。In the present invention, the purity of the pure humanized antibody J591 is preferably ≥95%.
在本发明中，所述还原剂优选为TCEP还原剂(Tris-2-ca rboxyethyl-phosphine)。In the present invention, the reducing agent is preferably TCEP reducing agent (Tris-2-carboxyethyl-phosphine).
在本发明中，所述保护剂优选为二乙基三胺五乙酸（Diethylenetriaminepentacetate acid，简称为DTPA）。In the present invention, the protective agent is preferably diethylenetriaminepentacetate acid (DTPA for short).
在本发明中，所述还原剂和所述保护剂的摩尔比优选为1~20:1~20。In the present invention, the molar ratio of the reducing agent to the protecting agent is preferably 1-20:1-20.
在本发明中，所述还原剂和所述人源化抗体J591的摩尔比优选为0.5~6.0:1。In the present invention, the molar ratio of the reducing agent to the humanized antibody J591 is preferably 0.5-6.0:1.
在本发明中，所述还原反应优选在溶液中进行。所述还原反应时的溶剂优选为PBS缓冲液和纯化水。In the present invention, the reduction reaction is preferably carried out in a solution. The solvent during the reduction reaction is preferably PBS buffer and purified water.
在本发明中，所述还原剂、保护剂和人源化抗体J591的优选包括以下步骤：将还原剂和保护剂溶解于超纯水中，得到混合母液；将人源化抗体J591用30KD膜包超滤透析到PBS缓冲液中（即用PBS置换J591抗体中的溶剂），浓缩后得到人源化抗体J591溶液；将所述混合母液和所述人源化抗体J591溶液混合。在本发明中，所述混合母液中，所述还原剂的摩尔浓度优选为1~20mM，所述保护剂的摩尔浓度优选为1~20mM；所述人源化抗体J591溶液的质量浓度优选为5~30mg/mL；所述混合母液和所述人源化抗体J591溶液的体积比优选为1:1。In the present invention, the reducing agent, protective agent and humanized antibody J591 preferably include the following steps: dissolving the reducing agent and protective agent in ultrapure water to obtain a mixed mother solution; Include ultrafiltration and dialysis into PBS buffer (that is, replace the solvent in the J591 antibody with PBS), and concentrate to obtain a humanized antibody J591 solution; mix the mixed mother solution and the humanized antibody J591 solution. In the present invention, in the mixed mother liquor, the molar concentration of the reducing agent is preferably 1-20mM, and the molar concentration of the protective agent is preferably 1-20mM; the mass concentration of the humanized antibody J591 solution is preferably 5 ~ 30mg/mL; the volume ratio of the mixed mother solution and the humanized antibody J591 solution is preferably 1:1.
在本发明中，所述还原反应的温度优选为25℃，所述还原反应的时间优选为1h。In the present invention, the temperature of the reduction reaction is preferably 25° C., and the time of the reduction reaction is preferably 1 h.
本发明将连接底物、活性药物、偶联试剂和溶剂混合，进行偶联反应，得到linker-活性药物偶联物。In the present invention, the linking substrate, the active drug, the coupling reagent and the solvent are mixed, and the coupling reaction is carried out to obtain the linker-active drug conjugate.
在本发明中，所述连接底物优选为Fmoc-Val-Cit-PAB-PNP、MC-VC-PAB-DMEDA-PEG、Mal-PEG2-Val-Cit-PABA-PNP或Fmoc-VC-PAB-PNP。In the present invention, the connection substrate is preferably Fmoc-Val-Cit-PAB-PNP, MC-VC-PAB-DMEDA-PEG, Mal-PEG2-Val-Cit-PABA-PNP or Fmoc-VC-PAB- PNP.
在本发明中，MC-VC-PAB-DMEDA-PEG购买于烟台迈百瑞国际生物医药股份有限公司。In the present invention, MC-VC-PAB-DMEDA-PEG was purchased from Yantai Medberry International Biomedicine Co., Ltd.
在本发明中，所述Fmoc-VC-PAB-PNP购买于烟台迈百瑞国际生物医药股份有限公司。In the present invention, the Fmoc-VC-PAB-PNP was purchased from Yantai Mileberry International Biomedicine Co., Ltd.
在本发明中，所述活性药物优选为MMAE、MMAF、SN-38和MK4827中的一种或两种。In the present invention, the active drug is preferably one or two of MMAE, MMAF, SN-38 and MK4827.
在本发明中，所述linker-活性药物偶联物具体优选为MC-VC-PAB-MMAE、MC-VC-PAB-DMEDA-PEG(N3)-SN38、MP2-VC-PAB-DMEDA-PEG-SN38、MC-MMAF-VC-PAB-MK4827、MC-MMAF-VC-PAB-DMEDA-PEG-SN38。In the present invention, the linker-active drug conjugate is specifically preferably MC-VC-PAB-MMAE, MC-VC-PAB-DMEDA-PEG(N3)-SN38, MP2-VC-PAB-DMEDA-PEG- SN38, MC-MMAF-VC-PAB-MK4827, MC-MMAF-VC-PAB-DMEDA-PEG-SN38.
在本发明中，所述MC-VC-PAB-MMAE的制备方法优选包括以下步骤：In the present invention, the preparation method of described MC-VC-PAB-MMAE preferably comprises the following steps:
将Fmoc-Val-Cit-PAB-PNP和MMAE溶于N,N-二甲基甲酰胺（DMF）中，依次加入1-羟基苯并三唑（HoBt）和吡啶，室温下搅拌反应过夜；反应产物使用高效液相色谱进行分离纯化，收集纯品，冻干反应产物，得到所述MC-VC-PAB-MMAE的纯品。在本发明中，所述Fmoc-Val-Cit-PAB-PNP和MMAE的摩尔比优选为0.125:0.083；所述Fmoc-Val-Cit-PAB-PNP和HoBt的摩尔比优选为0.125:0.074；所述Fmoc-Val-Cit-PAB-PNP的物质的量和所述吡啶的体积之比优选为0.125mmol:0.5mL。Dissolve Fmoc-Val-Cit-PAB-PNP and MMAE in N,N-dimethylformamide (DMF), add 1-hydroxybenzotriazole (HoBt) and pyridine in turn, and stir overnight at room temperature; The product is separated and purified by high performance liquid chromatography, the pure product is collected, and the reaction product is freeze-dried to obtain the pure product of MC-VC-PAB-MMAE. In the present invention, the molar ratio of the Fmoc-Val-Cit-PAB-PNP and MMAE is preferably 0.125:0.083; the molar ratio of the Fmoc-Val-Cit-PAB-PNP and HoBt is preferably 0.125:0.074; The ratio of the amount of the substance of the Fmoc-Val-Cit-PAB-PNP to the volume of the pyridine is preferably 0.125mmol:0.5mL.
在本发明中，所述MC-VC-PAB-DMEDA-PEG(N3)-SN-38的制备方法优选与所述MC-VC-PAB-MMAE的制备方法基本相同，不同之处在于：将所述Fmoc-Val-Cit-PAB-PNP替换为MC-VC-PAB-DMEDA-PEG；将所述MMAE替换为SN-38。In the present invention, the preparation method of the MC-VC-PAB-DMEDA-PEG(N3)-SN-38 is preferably basically the same as the preparation method of the MC-VC-PAB-MMAE, except that the The Fmoc-Val-Cit-PAB-PNP is replaced by MC-VC-PAB-DMEDA-PEG; the MMAE is replaced by SN-38.
在本发明中，所述MP2-VC-PAB-DMEDA-PEG-SN-38的制备方法优选包括以下步骤：将Mal-PEG2-Val-Cit-PABA-PNP与DMEDA和PEG2反应制得MP2-VC-PAB-DMEDA-PEG2；将所述MP2-VC-PAB-DMEDA-PEG2与SN38反应，分离纯化得所述MP2-VC-PAB-DMEDA-PEG-SN38的纯品。In the present invention, the preparation method of MP2-VC-PAB-DMEDA-PEG-SN-38 preferably comprises the following steps: reacting Mal-PEG2-Val-Cit-PABA-PNP with DMEDA and PEG2 to prepare MP2-VC -PAB-DMEDA-PEG2: react the MP2-VC-PAB-DMEDA-PEG2 with SN38, separate and purify to obtain the pure product of MP2-VC-PAB-DMEDA-PEG-SN38.
在本发明中，所述MC-MMAF-VC-PAB-MK4827的制备方法优选包括以下步骤：取Fmoc-VC-PAB-PNP和MK4827，加入第一N,N-二甲基甲酰胺和第一N,N-二异丙基乙胺中，室温至反应结束，旋干溶剂，Flash法纯化后得Fmoc-VC-PAB-MK4827粗品；向Fmoc-VC-PAB-MK4827粗品中加入第二N,N-二甲基甲酰胺和第二N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，制备液相法纯化，得VC-PAB-MK4827纯品；取MMAF和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯（TSTU），加入第三N,N-二异丙基乙胺和VC-PAB-MK4827纯品，室温至反应结束，旋干溶剂，制备液相法纯化，得Mc-MMAF-VC-PAB-MK4827纯品。在本发明中，所述Fmoc-VC-PAB-PNP和MK4827的质量比优选为131.7:50；所述第一N,N-二甲基甲酰胺和第一N,N-二异丙基乙胺的体积比优选为5mL:78μL；所述第二N,N-二甲基甲酰胺和第二N,N-二异丙基乙胺的体积比优选为4:1；所述Mc-MMAF和TSTU的质量比优选为20:9.8；所述MMAF和VC-PAB-MK4827纯品的质量比优选为20:18.8。In the present invention, the preparation method of MC-MMAF-VC-PAB-MK4827 preferably includes the following steps: take Fmoc-VC-PAB-PNP and MK4827, add the first N,N-dimethylformamide and the first In N,N-diisopropylethylamine, room temperature to the end of the reaction, the solvent was spin-dried, and the crude product of Fmoc-VC-PAB-MK4827 was obtained after purification by Flash method; the second N, N-dimethylformamide and the second N,N-diisopropylethylamine, room temperature to the end of the reaction, spin to dry the solvent, and purify by preparative liquid phase method to obtain pure VC-PAB-MK4827; take MMAF and 2- Succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), add third N,N-diisopropylethylamine and VC-PAB-MK4827 pure, room temperature After the reaction was completed, the solvent was spin-dried and purified by preparative liquid phase to obtain the pure product of Mc-MMAF-VC-PAB-MK4827. In the present invention, the mass ratio of the Fmoc-VC-PAB-PNP and MK4827 is preferably 131.7:50; the first N,N-dimethylformamide and the first N,N-diisopropylethyl The volume ratio of the amine is preferably 5mL:78μL; the volume ratio of the second N,N-dimethylformamide and the second N,N-diisopropylethylamine is preferably 4:1; the Mc-MMAF The mass ratio of MMAF and TSTU is preferably 20:9.8; the mass ratio of the pure MMAF and VC-PAB-MK4827 is preferably 20:18.8.
在本发明中，所述MC-MMAF-VC-PAB-DMEDA-PEG-SN-38的制备方法优选包括以下步骤：取Fmoc-VC-PAB-PNP和色瑞替尼（Ceritinib），加入第一N,N-二甲基甲酰胺和第一N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，Flash法纯化后得到Fmoc-VC-PAB-Ceritinib粗品；向Fmoc-VC-PAB-Ceritinib粗品中加入第二N,N-二甲基甲酰胺和第二N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，高效液相法纯化后得到VC-PAB-Ceritinib纯品；取Mc-MMAF和TSTU，加入第三N,N-二异丙基乙胺和VC-PAB-Ceritinib纯品，室温至反应结束，旋干溶剂，高效液相法纯化，得MC-MMAF-VC-PAB-Ceritinib纯品；取DMEDA-PEG-SN38和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯（TSTU），加入第三N,N-二异丙基乙胺和MC-MMAF-VC-PAB-Ceritinib纯品，室温至反应结束，旋干溶剂，高效液相法纯化，得MC-MMAF-VC-PAB-DMEDA-PEG-SN38。在本发明中，所述Fmoc-VC-PAB-PNP和色瑞替尼的质量比优选为75.6:50；所述第一N,N-二甲基甲酰胺和第一N,N-二异丙基乙胺的体积比优选为5mL:49μL；所述第二N,N-二甲基甲酰胺和第二N,N-二异丙基乙胺的体积比优选为4:1；所述Mc-MMAF和TSTU的质量比优选为20:9.8；所述Mc-MMAF和VC-PAB-Ceritinib纯品的质量比优选为20:18.8。In the present invention, the preparation method of MC-MMAF-VC-PAB-DMEDA-PEG-SN-38 preferably includes the following steps: take Fmoc-VC-PAB-PNP and Ceritinib, add the first N,N-dimethylformamide and the first N,N-diisopropylethylamine, room temperature to the end of the reaction, spin-dry the solvent, and obtain the crude product of Fmoc-VC-PAB-Ceritinib after purification by Flash method; to Fmoc-VC -Add the second N,N-dimethylformamide and the second N,N-diisopropylethylamine to the crude product of PAB-Ceritinib, wait until the end of the reaction at room temperature, spin to dry the solvent, and obtain VC- Pure PAB-Ceritinib; take Mc-MMAF and TSTU, add the third N,N-diisopropylethylamine and VC-PAB-Ceritinib pure, room temperature to the end of the reaction, spin to dry the solvent, and purify by HPLC, Obtain pure MC-MMAF-VC-PAB-Ceritinib; take DMEDA-PEG-SN38 and 2-succinimidyl-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU), add The third pure product of N,N-diisopropylethylamine and MC-MMAF-VC-PAB-Ceritinib, from room temperature to the end of the reaction, spin-dried the solvent, and purified by high performance liquid phase to obtain MC-MMAF-VC-PAB-DMEDA -PEG-SN38. In the present invention, the mass ratio of Fmoc-VC-PAB-PNP and ceritinib is preferably 75.6:50; the first N,N-dimethylformamide and the first N,N-diiso The volume ratio of propylethylamine is preferably 5mL:49μL; the volume ratio of the second N,N-dimethylformamide and the second N,N-diisopropylethylamine is preferably 4:1; the The mass ratio of Mc-MMAF and TSTU is preferably 20:9.8; the mass ratio of Mc-MMAF and VC-PAB-Ceritinib pure product is preferably 20:18.8.
得到还原后的人源化J591抗体和所述linker-活性药物偶联物后，本发明将所述还原后的人源化J591抗体、所述linker-活性药物偶联物和溶剂混合，进行偶联反应，得到PSMA抗体-药物缀合物。在本发明中，所述溶剂优选为二甲基亚砜（DMSO）。在本发明中，所述linker-活性药物偶联物与所述还原后的人源化J591抗体的巯基的摩尔比优选为0.3～2.8:1；所述偶联反应的温度优选为25℃，所述偶联反应的时间优选为1~4h；所述偶联反应在搅拌的条件下进行。在本发明中，所述偶联反应得到的反应液，本发明优选采用PBS缓冲液对所述偶联反应得到的反应液离心超滤3次，纯化去除残余未反应linker-活性药物偶联物以及DMSO等游离小分子。在本发明中，本发明优选利用SDS-PAGE电泳和疏水高效液相(HIC-HPLC)法检测偶联情况。After the reduced humanized J591 antibody and the linker-active drug conjugate are obtained, the present invention mixes the reduced humanized J591 antibody, the linker-active drug conjugate and a solvent for coupling Combined reaction to obtain PSMA antibody-drug conjugates. In the present invention, the solvent is preferably dimethylsulfoxide (DMSO). In the present invention, the molar ratio of the linker-active drug conjugate to the sulfhydryl group of the reduced humanized J591 antibody is preferably 0.3-2.8:1; the temperature of the coupling reaction is preferably 25°C, The time of the coupling reaction is preferably 1~4h; the coupling reaction is carried out under the condition of stirring. In the present invention, the reaction solution obtained from the coupling reaction, the present invention preferably adopts PBS buffer solution to centrifuge and ultrafilter the reaction solution obtained from the
在本发明中，所述PSMA抗体-药物缀合物具体优选为Ab-MC-VC-PAB-DMEDA-PEG(N3)SN38（记为YC1613）、Ab-MP2-VC-PAB-DMEDA-PEG(N3)-SN38（记为YC1628）、Ab-MC-VC-PAB-MMAE（记为YC1605）、Ab-MC-MMAF-VC-PAB-MK4827（记为YC1667）或Ab-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38（记为YC1663）。In the present invention, the PSMA antibody-drug conjugate is specifically preferably Ab-MC-VC-PAB-DMEDA-PEG (N3) SN38 (denoted as YC1613), Ab-MP2-VC-PAB-DMEDA-PEG ( N3)-SN38 (denoted as YC1628), Ab-MC-VC-PAB-MMAE (denoted as YC1605), Ab-MC-MMAF-VC-PAB-MK4827 (denoted as YC1667) or Ab-Mc-MMAF-VC- PAB-DMEDA-PEG(N3)-SN38 (marked as YC1663).
本发明提供了上述技术方案所述的PSMA抗体-药物缀合物或上述技术方案所述的制备方法制备得到的PSMA抗体-药物缀合物在制备治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。The present invention provides the PSMA antibody-drug conjugate described in the above technical scheme or the PSMA antibody-drug conjugate prepared by the preparation method described in the above technical scheme can be used in the preparation of treatment or prevention of cancer, infectious diseases or autoimmunity Application in the medicine of disease.
在本发明中，所述癌症优选为前列腺癌、结直肠癌、胃癌、透明细胞肾癌、膀胱癌或肺癌，进一步优选为前列腺癌。所述前列腺癌具体优选为去势难治性前列腺癌。In the present invention, the cancer is preferably prostate cancer, colorectal cancer, gastric cancer, clear cell renal cancer, bladder cancer or lung cancer, more preferably prostate cancer. The prostate cancer is particularly preferably castration-resistant prostate cancer.
为了进一步说明本发明，下面结合附图和实施例对本发明提供的技术方案进行详细地描述，但不能将它们理解为对本发明保护范围的限定。In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.
在本发明实施例中：In the embodiment of the present invention:
原料种类及来源为：The types and sources of raw materials are:
1.细胞系：1. Cell line:
LNCap、C4-2B、PC-3和22RV1购自武汉尚恩生物，PC-3细胞用含10 FBS（Gibco）的F-12（Gibco）培养基培养，其余细胞均用含10 FBS（Gibco）的1640（Gibco）培养基培养。细胞在5％CO2的37℃的恒温培养箱（Thermo）中培养。LNCap, C4-2B, PC-3 and 22RV1 were purchased from Wuhan Shangen Biotechnology Co., Ltd., PC-3 cells were cultured in F-12 (Gibco) medium containing 10 FBS (Gibco), and other cells were cultured in F-12 (Gibco) medium containing 10 FBS (Gibco) 1640 (Gibco) medium for culture. Cells were cultured in an incubator (Thermo) at 37°C with 5% CO2.
2.试剂：2. Reagents:
Cell Counting Kit-8(CCK8)购自碧云天，人PSMA重组蛋白购自ACRO。Protein A抗体纯化填料购自GE公司；MC-VC-PAB-PNP、MMAE、mc-MMAF、SN38、MK4827等细胞毒性药物购自上海皓元化学科技有限公司。Cell Counting Kit-8 (CCK8) was purchased from Beyontien, and human PSMA recombinant protein was purchased from ACRO. Protein A antibody purification packing was purchased from GE; MC-VC-PAB-PNP, MMAE, mc-MMAF, SN38, MK4827 and other cytotoxic drugs were purchased from Shanghai Haoyuan Chemical Technology Co., Ltd.
实施例Example
人源化抗体J591的重组表达和纯化：Recombinant expression and purification of humanized antibody J591:
将鼠源单抗J591的可变区与人IgG1κ恒定区通过重叠延伸PCR的方法连接成完整片段，其中鼠源单抗J591的具有如SEQ ID NO.1所示的氨基酸序列；所述重链的可变区具有如SEQ ID NO.3所示的氨基酸序列。The variable region of the mouse monoclonal antibody J591 and the constant region of human IgG1κ were connected into a complete fragment by overlapping extension PCR, wherein the mouse monoclonal antibody J591 has the amino acid sequence shown in SEQ ID NO.1; the heavy chain The variable region of has the amino acid sequence shown in SEQ ID NO.3.
将上述扩增片段亚克隆到表达载体pcDNA3.0上，将构建的质粒转染到悬浮的宿主细胞（Invitrogen）中以产生人源化抗体J591。从宿主细胞培养上清液中收集表达的抗体，经Protein A进行纯化。通过尺寸排阻色谱法和还原和非还原条件下SDS-PAGE分析抗体纯度，结果表明表达纯化的人源化抗体J591纯度大于95%，经0.22 μm滤膜过滤后内毒素含量小于1 EU/mg。纯化后的人源化抗体J591的SDS-PAGE分析结果如图6所示，尺寸排阻色谱分析结果见图7和图8所示。The amplified fragment above was subcloned into the expression vector pcDNA3.0, and the constructed plasmid was transfected into suspended host cells (Invitrogen) to produce humanized antibody J591. The expressed antibody was collected from the host cell culture supernatant and purified by Protein A. The purity of the antibody was analyzed by size exclusion chromatography and SDS-PAGE under reducing and non-reducing conditions. The results showed that the purified humanized antibody J591 was more than 95% pure, and the endotoxin content was less than 1 EU/mg after filtration through a 0.22 μm filter membrane . The SDS-PAGE analysis results of the purified humanized antibody J591 are shown in Figure 6, and the size exclusion chromatography analysis results are shown in Figures 7 and 8.
图6为人源化抗体J591在还原和非还原条件下SDS-PAGE电泳图，其中：Lane M：Marker；Lane R：还原电泳；LaneN-R：非还原电泳。图7为：214 nm波长条件下SEC-HPLC图谱；图8为：280 nm波长条件下SEC-HPLC图谱。Fig. 6 is the SDS-PAGE electrophoresis image of the humanized antibody J591 under reducing and non-reducing conditions, wherein: Lane M: Marker; Lane R: reducing electrophoresis; Lane N-R: non-reducing electrophoresis. Figure 7 is: SEC-HPLC spectrum under the condition of 214 nm wavelength; Figure 8 is: SEC-HPLC spectrum under the condition of 280 nm wavelength.
实施例Example
（1）人源化单抗J591的生物活性分析：(1) Bioactivity analysis of humanized monoclonal antibody J591:
ELISA分析实施例1制备的人源化抗体J591对PSMA重组蛋白的结合活性：采用购自ACRO公司的PSMA重组蛋白验证表达的人源化单抗J591的结合活性。将人源化单抗J591稀释至2 μg/mL，100 μL/孔包被酶标板，4°C包被过夜。2%BSA 37 °C封闭2 h。将Biotin-PSMA重组蛋白稀释至1 μg/mL，2倍梯度稀释7个点，100 μL/孔加入酶标板，37 °C孵育1 h。100 μL/孔加入1:10000倍稀释的HRP-Streptavidin酶标二抗，37 °C孵育1 h后加入TMB显色液进行显色反应，2 M H2SO4终止显色后测定450 nm波长处的吸光度值。绘制吸光度值与Biotin-PSMA重组蛋白浓度曲线图，分析人源化单抗J591的生物活性。结果如图9，图9表明实施例1制备的人源化单抗J591可结合PSMA重组蛋白，具有与PSMA蛋白结合活性。ELISA analysis of the binding activity of the humanized antibody J591 prepared in Example 1 to the PSMA recombinant protein: the binding activity of the expressed humanized monoclonal antibody J591 was verified by using the PSMA recombinant protein purchased from ACRO. Humanized monoclonal antibody J591 was diluted to 2 μg/mL, 100 μL/well was coated on the microtiter plate, and coated overnight at 4°C. Block with 2% BSA at 37 °C for 2 h. Dilute Biotin-PSMA recombinant protein to 1 μg/mL, 2-fold serial dilution to 7 points, add 100 μL/well to the microtiter plate, and incubate at 37 °C for 1 h. Add 1:10000 times diluted HRP-Streptavidin enzyme-labeled secondary antibody to 100 μL/well, incubate at 37 °C for 1 h, then add TMB chromogenic solution for color reaction, stop color development with 2 M H2SO4, measure absorbance at 450 nm wavelength value. Draw the absorbance value and Biotin-PSMA recombinant protein concentration curve to analyze the biological activity of humanized monoclonal antibody J591. The results are shown in Figure 9, which shows that the humanized monoclonal antibody J591 prepared in Example 1 can bind to PSMA recombinant protein and has the activity of binding to PSMA protein.
（2）流式细胞术分析J591对PSMA阳性肿瘤细胞系的结合活性：(2) Flow cytometry analysis of the binding activity of J591 to PSMA-positive tumor cell lines:
检测人源化单抗J591对PSMA阳性前列腺癌细胞系LNCap细胞、C4-2B细胞和22RV1细胞，以PSMA阴性前列腺癌细胞系PC-3的结合活性。取对数生长期的上述四种细胞，消化后收集细胞，离心收集细胞沉淀，PBS重悬细胞后离心清洗细胞一次。去除离心后上清，PBS重悬细胞，计数后分别取上述细胞每种5×105个在4℃下与5 μg/mL的J591单抗孵育1 h，用PBS洗涤细胞2次。PE标记的羊抗人IgG 1:100稀释加入各管内，4℃避光孵育30 min，PBS溶液润洗2遍，用流式细胞仪检测各细胞系的荧光强度。结果如图10，实施例1制备的人源化单抗J591与PSMA高表达细胞系C4-2B细胞和LNCap细胞表现出强结合，与低表达细胞系22RV1细胞结合较弱，与PSMA阴性细胞系PC-3不结合。The binding activity of humanized monoclonal antibody J591 to PSMA-positive prostate cancer cell line LNCap cells, C4-2B cells and 22RV1 cells, and PSMA-negative prostate cancer cell line PC-3 was detected. Take the above four kinds of cells in the logarithmic growth phase, collect the cells after digestion, collect the cell pellet by centrifugation, resuspend the cells in PBS, and wash the cells by centrifugation once. The supernatant after centrifugation was removed, and the cells were resuspended in PBS. After counting, 5×105 of each of the above cells were taken and incubated with 5 μg/mL J591 monoclonal antibody for 1 h at 4°C, and the cells were washed twice with PBS. PE-labeled goat anti-human IgG was diluted 1:100 and added to each tube, incubated at 4°C in the dark for 30 min, washed twice with PBS solution, and the fluorescence intensity of each cell line was detected by flow cytometry. The results are shown in Figure 10. The humanized monoclonal antibody J591 prepared in Example 1 showed strong binding to PSMA high-expressing cell lines C4-2B cells and LNCap cells, weakly binding to low-expressing cell line 22RV1 cells, and binding to PSMA-negative cell lines PC-3 does not bind.
实施例Example
Biacore测定人源化单抗J591亲和性：Biacore determined the affinity of humanized monoclonal antibody J591:
为进一步表征抗PSMA的人源化单抗J591，测定了人源化单抗J591对人PSMA蛋白的亲和性。将重组PSMA蛋白固定在CM5Biacore芯片上，将分析物以0.125、0.25、0.5、1.0、2.0、4.0 μg/mL；浓度在流动相中与蛋白结合，流动相流速：30μL/min；结合时间：180 s；解离时间：300 s。测得实施例1制备的人源化单抗J591对人PSMA的结合亲和力Kd为1.612 nM。In order to further characterize the anti-PSMA humanized monoclonal antibody J591, the affinity of humanized monoclonal antibody J591 to human PSMA protein was determined. The recombinant PSMA protein was immobilized on the CM5Biacore chip, and the analyte was bound to the protein in the mobile phase at a concentration of 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 μg/mL; the flow rate of the mobile phase: 30 μL/min; the binding time: 180 s; dissociation time: 300 s. The binding affinity Kd of the humanized monoclonal antibody J591 prepared in Example 1 to human PSMA was measured to be 1.612 nM.
实施例Example
流式细胞术检测人源化单抗J591在PSMA阳性肿瘤细胞中的内化活性测定：Determination of the internalization activity of humanized monoclonal antibody J591 in PSMA-positive tumor cells by flow cytometry:
将LNCap细胞使用5 μg/mL，人源化单抗J591和对照抗体anti-hPSMA AF488在4℃下孵育30 min，洗涤后，将细胞在37℃孵育使抗体内化。分别在放入37℃环境中第0 min、30min、60 min、120 min时间点通过流式检测各组细胞的荧光强度，分析J591的内化活性。结果如图11，人源化单抗J591在PSMA阳性肿瘤细胞LNCap中具有较高内化活性，在第30 min、60 min、120 min内化率分别为：18.71%、27.37%和45.67%。LNCap cells were incubated with 5 μg/mL, humanized monoclonal antibody J591 and control antibody anti-hPSMA AF488 at 4°C for 30 min. After washing, the cells were incubated at 37°C to internalize the antibodies. The fluorescence intensity of the cells in each group was detected by flow cytometry at the time points of 0 min, 30 min, 60 min, and 120 min when placed in a 37°C environment, and the internalization activity of J591 was analyzed. The results are shown in Figure 11. Humanized monoclonal antibody J591 has high internalization activity in PSMA-positive tumor cells LNCap, and the internalization rates at 30 min, 60 min, and 120 min were 18.71%, 27.37%, and 45.67%, respectively.
实施例Example
抗PSMA抗体-药物缀合物中化合物的制备：Preparation of compounds in anti-PSMA antibody-drug conjugates:
（1）MC-VC-PAB-MMAE的制备：(1) Preparation of MC-VC-PAB-MMAE:
将Fmoc-Val-Cit-PAB-PNP (93mg,0.125mmol)和MMAE (60mg,0.083mmol)溶于4mLDMF中，依次加入HoBt(10mg，0.074mmol)和0 .5mL吡啶，室温下搅拌反应过夜。反应产物使用高效液相法进行分离纯化，收集纯品，冻干反应产物，制得MC-VC-PAB-MMAE；Fmoc-Val-Cit-PAB-PNP (93mg, 0.125mmol) and MMAE (60mg, 0.083mmol) were dissolved in 4mL DMF, HoBt (10mg, 0.074mmol) and 0.5mL pyridine were added successively, and the reaction was stirred overnight at room temperature. The reaction product was separated and purified by high performance liquid phase method, the pure product was collected, and the reaction product was freeze-dried to obtain MC-VC-PAB-MMAE;
（2）MC-VC-PAB-DMEDA-PEG(N3)-SN38的制备：(2) Preparation of MC-VC-PAB-DMEDA-PEG(N3)-SN38:
将0.15mmol MC-VC-PAB-DMEDA-PEG连接子（由烟台迈百瑞公司提供）和0.1mmolSN38溶解于4mL无水DMF中，向溶液中加入0.082mmol DIEA，混合后在室温22℃下搅拌反应2h，通过高效液相法纯化收集纯品，制得MC-VC-PAB-DMEDA-PEG(N3)-SN38；Dissolve 0.15mmol MC-VC-PAB-DMEDA-PEG linker (provided by Yantai Medberry Co.) and 0.1mmol SN38 in 4mL anhydrous DMF, add 0.082mmol DIEA to the solution, mix and stir at room temperature 22°C After reacting for 2 hours, the pure product was collected by high performance liquid phase purification to obtain MC-VC-PAB-DMEDA-PEG(N3)-SN38;
（3）MP2-VC-PAB-DMEDA-PEG-SN38的制备：(3) Preparation of MP2-VC-PAB-DMEDA-PEG-SN38:
将化合物Mal-PEG2-Val-Cit-PABA-PNP(0.3mmol)、DMEDA(0.25mmol)和PEG2(0.25mmol)溶于4mL无水DMF，加入0.16mmol DIEA，于室温下搅拌反应30分钟，通过高效液相法纯化出MP2-VC-PAB-DMEDA-(PEG2)。将0.15mmol MP2-VC-PAB-DMEDA-(PEG2)和0.1mmolSN38溶于4mL无水DMF，向溶液中加入0.082mmol DIEA，混匀后在室温22℃下搅拌反应2小时，并通过高效液相法直接纯化出目的产物MP2-VC-PAB-DMEDA-PEG-SN38。The compound Mal-PEG2-Val-Cit-PABA-PNP (0.3mmol), DMEDA (0.25mmol) and PEG2 (0.25mmol) were dissolved in 4mL anhydrous DMF, 0.16mmol DIEA was added, and the reaction was stirred at room temperature for 30 minutes, passed MP2-VC-PAB-DMEDA-(PEG2) was purified by HPLC. Dissolve 0.15mmol MP2-VC-PAB-DMEDA-(PEG2) and 0.1mmol SN38 in 4mL of anhydrous DMF, add 0.082mmol DIEA to the solution, mix well and stir the reaction at room temperature 22°C for 2 hours, and pass through the high performance liquid phase The target product MP2-VC-PAB-DMEDA-PEG-SN38 was directly purified by the method.
（4）MC-MMAF-VC-PAB-MK4827的制备：(4) Preparation of MC-MMAF-VC-PAB-MK4827:
取Fmoc-VC-PAB-PNP 131.7mg和MK4827(即Niraparib)50.0mg，加入5mL N,N-二甲基甲酰胺和78μL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，Flash法纯化后得Fmoc-VC-PAB-MK4827粗品98.0mg。向上述Fmoc-VC-PAB-MK4827粗品中加入8mL N,N-二甲基甲酰胺和2mL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，制备液相法纯化，得VC-PAB-MK4827纯品60 .0mg。取Mc-MMAF 20 .0mg和TSTU 9.8mg，加入14.2μL N,N-二异丙基乙胺和VC-PAB-MK4827 18.8mg，室温至反应结束16h，旋干溶剂，高效液相法纯化，得Mc-MMAF-VC-PAB-MK4827纯品7.0mg。Take Fmoc-VC-PAB-PNP 131.7mg and MK4827 (Niraparib) 50.0mg, add 5mL N,N-dimethylformamide and 78μL N,N-diisopropylethylamine, room temperature to the end of the reaction, spin dry Solvent, after purification by Flash method, 98.0 mg of crude product Fmoc-VC-PAB-MK4827 was obtained. Add 8mL of N,N-dimethylformamide and 2mL of N,N-diisopropylethylamine to the above crude product of Fmoc-VC-PAB-MK4827, wait until the end of the reaction at room temperature, spin to dry the solvent, and purify by preparative liquid phase method, 60.0 mg of pure VC-PAB-MK4827 was obtained. Take Mc-MMAF 20.0mg and TSTU 9.8mg, add 14.2μL N,N-diisopropylethylamine and VC-PAB-MK4827 18.8mg, room temperature to the end of the reaction for 16h, spin to dry the solvent, and purify by HPLC. 7.0 mg of pure Mc-MMAF-VC-PAB-MK4827 was obtained.
（5）MC-MMAF-VC-PAB-DMEDA-PEG-SN38的制备：(5) Preparation of MC-MMAF-VC-PAB-DMEDA-PEG-SN38:
取Fmoc-VC-PAB-PNP 75.6mg和Ceritinib(即色瑞替尼)50.0mg，加入5mLN,N-二甲基甲酰胺和49μL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，Flash法纯化后得到Fmoc-VC-PAB-Ceritinib粗品80.0mg。向Fmoc-VC-PAB-Ceritinib粗品中加入8mL N,N-二甲基甲酰胺和2mL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，制备液相法纯化后得到VC-PAB-Ceritinib纯品50.0mg。取Mc-MMAF 20.0mg和TSTU 9.8mg，加入14.2μL N,N-二异丙基乙胺和18.8mg VC-PAB-Ceritinib，室温至反应结束，旋干溶剂，制备液相法纯化，得MC-MMAF-VC-PAB-Ceritinib纯品12.0mg；取DMEDA-PEG-SN38和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯（TSTU），加入第三N,N-二异丙基乙胺和MC-MMAF-VC-PAB-Ceritinib纯品，室温至反应结束，旋干溶剂，高效液相法纯化，得MC-MMAF-VC-PAB-DMEDA-PEG-SN-38。Take Fmoc-VC-PAB-PNP 75.6mg and Ceritinib (ceritinib) 50.0mg, add 5mL N,N-dimethylformamide and 49μL N,N-diisopropylethylamine, room temperature until the end of the reaction, The solvent was spin-dried, and 80.0 mg of the crude product of Fmoc-VC-PAB-Ceritinib was obtained after purification by Flash method. Add 8mL N,N-dimethylformamide and 2mL N,N-diisopropylethylamine to the crude product of Fmoc-VC-PAB-Ceritinib, wait until the end of the reaction at room temperature, spin the solvent to dry, and obtain after purification by preparative liquid phase Pure VC-PAB-Ceritinib 50.0mg. Take 20.0 mg of Mc-MMAF and 9.8 mg of TSTU, add 14.2 μL of N,N-diisopropylethylamine and 18.8 mg of VC-PAB-Ceritinib, wait until the end of the reaction at room temperature, spin to dry the solvent, and purify by preparative liquid phase to obtain MC - MMAF-VC-PAB-Ceritinib pure 12.0mg; take DMEDA-PEG-SN38 and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), add The third pure product of N,N-diisopropylethylamine and MC-MMAF-VC-PAB-Ceritinib, from room temperature to the end of the reaction, spin-dried the solvent, and purified by high performance liquid phase to obtain MC-MMAF-VC-PAB-DMEDA - PEG-SN-38.
实施例Example
抗PSMA抗体-药物缀合物的制备：Preparation of anti-PSMA antibody-drug conjugates:
（1）人源化抗体J591的纯化及处理：(1) Purification and treatment of humanized antibody J591:
经Protein A从宿主细胞培养上清液中捕获的人源化抗体J591，通过SDS-PAGE电泳及SEC分析抗体纯度达到95%以上。用30KD膜包将所获抗体蛋白超滤透析到PBS缓冲液中，浓缩后用紫外分光光度计标定浓度，用于后续偶联反应；The humanized antibody J591 captured from the host cell culture supernatant by Protein A has a purity of over 95% through SDS-PAGE electrophoresis and SEC analysis. Ultrafiltration dialyzed the obtained antibody protein into PBS buffer with 30KD membrane bag, concentrated and calibrated the concentration with UV spectrophotometer for subsequent coupling reaction;
（2）抗PSMA抗体-药物缀合物的制备(2) Preparation of anti-PSMA antibody-drug conjugate
抗体-药物缀合物的制备采用通用的偶联方法：用纯化水分别配制还原剂和保护剂如下：1mM TCEP (Tris-2-ca rboxyethyl-phos phine)、1mM DTPA（Diethylenetriaminepentacetate acid）母液；与质量浓度为5~30mg/mL单克隆抗体按照体积比(1:1)混合，TCEP与抗体的终浓度摩尔比0.5:1，于25℃搅拌反应1h。TCEP还原后的抗体可直接进行偶联；The preparation of the antibody-drug conjugate adopts a common coupling method: prepare the reducing agent and the protecting agent with purified water as follows: 1mM TCEP (Tris-2-carboxyethyl-phosphine), 1mM DTPA (Diethylenetriaminepentacetate acid) mother solution; The mass concentration is 5~30mg/mL monoclonal antibody is mixed according to the volume ratio (1:1), the final concentration molar ratio of TCEP and antibody is 0.5:1, and the reaction is stirred at 25°C for 1h. Antibodies after TCEP reduction can be directly conjugated;
配制摩尔浓度为5mM的linker-活性药物偶联物（分别为实施例5制备的MC-VC-PAB-MMAE、MC-VC-PAB-DMEDA-PEG(N3)-SN38、MP2-VC-PAB-DMEDA-PEG-SN38、MC-MMAF-VC-PAB-MK4827、MC-MMAF-VC-PAB-DMEDA-PEG-SN38）溶于25％的DMSO (dimethyl sulfoxide，二甲亚砜)，按照linker-活性药物偶联物的活性药物与还原后的抗体中的巯基的摩尔比2.8:1缓慢加药，于25℃搅拌反应-4h。反应结束后，用PBS缓冲液离心超滤3次，纯化去除残余未反应药物以及DMSO等游离小分子，并且利用SDS-PAGE电泳和疏水高效液相(HIC-HPLC)法检测偶联情况；The linker-active drug conjugate (respectively MC-VC-PAB-MMAE, MC-VC-PAB-DMEDA-PEG(N3)-SN38, MP2-VC-PAB-MMAE, MC-VC-PAB-DMEDA-PEG(N3)-SN38, MP2-VC-PAB- DMEDA-PEG-SN38, MC-MMAF-VC-PAB-MK4827, MC-MMAF-VC-PAB-DMEDA-PEG-SN38) dissolved in 25% DMSO (dimethyl sulfoxide, dimethyl sulfoxide), according to linker-activity The molar ratio of the active drug of the drug conjugate to the sulfhydryl group in the reduced antibody is 2.8:1, slowly add the drug, and react with stirring at 25°C for -4h. After the reaction, centrifuge and ultrafilter three times with PBS buffer, purify and remove residual unreacted drugs and free small molecules such as DMSO, and use SDS-PAGE electrophoresis and hydrophobic high-performance liquid phase (HIC-HPLC) to detect the coupling situation;
通过本实施例提供的方法，分别制备了抗体-药物缀合物Ab-MC-VC-PAB-DMEDA-PEG(N3)SN38(即YC1613)、Ab-MP2-VC-PAB-DMEDA-PEG(N3)-SN38(即YC1628)、Ab-MC-VC-PAB-MMAE(即YC1605)、Ab-MC-MMAF-VC-PAB-MK4827(即YC1667)、Ab-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38(即YC1663)。Through the method provided in this example, antibody-drug conjugates Ab-MC-VC-PAB-DMEDA-PEG(N3)SN38 (ie YC1613), Ab-MP2-VC-PAB-DMEDA-PEG(N3 )-SN38 (ie YC1628), Ab-MC-VC-PAB-MMAE (ie YC1605), Ab-MC-MMAF-VC-PAB-MK4827 (ie YC1667), Ab-Mc-MMAF-VC-PAB-DMEDA- PEG(N3)-SN38 (ie YC1663).
实施例Example
不同有效载荷的抗PSMA抗体-药物缀合物对PSMA阳性肿瘤细胞系的抗肿瘤活性测定：Anti-tumor activity assay of anti-PSMA antibody-drug conjugates with different payloads against PSMA-positive tumor cell lines:
（1）使用细胞计数试剂盒8(CCK-8)测定细胞活力。将前列腺癌细胞系LNCap、C4-2B、22RV1和PC-3细胞以3000-10000细胞/孔接种到96孔板中，孵育过夜。将待测抗体-药物缀合物进行10倍浓度梯度稀释后加入各细胞孔，以J591裸抗体作为对照，同时设置空白对照组和阴性对照组。将细胞板在37℃，5％CO2的潮湿箱中孵育72小时。加入CCK8试剂并放回培养箱中，孵育1h后检测450nm波长处的吸光度值。根据各组细胞的吸光度值，按式1所示公式计算细胞成活率，使用Prism GraphPad软件分析数据绘制剂量反应曲线，分析抗体-药物缀合物对四个细胞系的杀伤活性，并计算各药物的IC50值。结果如图12~15。(1) Cell viability was measured using Cell Counting Kit 8 (CCK-8). Prostate cancer cell lines LNCap, C4-2B, 22RV1 and PC-3 were seeded into 96-well plates at 3000-10000 cells/well and incubated overnight. The antibody-drug conjugate to be tested was diluted in a 10-fold concentration gradient and added to each cell well. The J591 naked antibody was used as a control, and a blank control group and a negative control group were set at the same time. The cell plates were incubated for 72 hours at 37°C in a humidified chamber with 5% CO2. Add the CCK8 reagent and put it back into the incubator, and detect the absorbance value at the wavelength of 450nm after incubation for 1h. According to the absorbance value of each group of cells, the cell survival rate was calculated according to the formula shown in
细胞成活率=（加药组A450值-空白组A450值）/（对照组A450值-空白组A450值）×100% 式1Cell survival rate=(A450 value of drug-dosed group-A450 value of blank group)/(A450 value of control group-A450 value of blank group)×100
（2）图12~15结果表明，制备的抗PSMA抗体-药物缀合物中，与J591裸抗（YC1691）相比，偶联毒素后均能提高对肿瘤细胞的杀伤活性；其中，YC1605、YC1663和YC1667对PSMA阳性肿瘤细胞的杀伤活性较高。计算得YC1605对C4-2B、LNCap细胞的IC50值分别为85.38ng/mL和50.53ng/mL；YC1663对C4-2B、LNCap细胞的IC50值分别为16.45ng/mL和9.269ng/mL；YC1667对C4-2B、LNCap细胞的IC50值分别为11.16ng/mL和5.623ng/mL。在PSMA不同表达水平的肿瘤细胞系中，药物SN38的对细胞的杀伤活性弱于MMAE，且MMAF/MK4827和MMAF/SN38两个双毒素分子的杀伤活性明显强于MMAE和SN38单毒素分子。(2) The results shown in Figures 12 to 15 show that compared with the J591 naked antibody (YC1691), the prepared anti-PSMA antibody-drug conjugates can increase the killing activity on tumor cells after coupling toxins; among them, YC1605, YC1663 and YC1667 have higher killing activity on PSMA-positive tumor cells. The calculated IC50 values of YC1605 on C4-2B and LNCap cells were 85.38ng/mL and 50.53ng/mL; the IC50 values of YC1663 on C4-2B and LNCap cells were 16.45ng/mL and 9.269ng/mL; The IC50 values of C4-2B and LNCap cells were 11.16ng/mL and 5.623ng/mL, respectively. In tumor cell lines with different expression levels of PSMA, the killing activity of the drug SN38 on cells was weaker than that of MMAE, and the killing activity of the two dual toxin molecules of MMAF/MK4827 and MMAF/SN38 was significantly stronger than that of the single toxin molecule of MMAE and SN38.
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and other embodiments can also be obtained according to the present embodiment without inventive step, and these embodiments are all Belong to the protection scope of the present invention.
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