技术领域Technical Field
本发明涉及肿瘤诊断与治疗的分子影像、核医学和单域抗体技术领域，尤其是涉及一种CLDN18.2单域抗体探针的制备方法及应用。The present invention relates to the fields of molecular imaging, nuclear medicine and single-domain antibody technology for tumor diagnosis and treatment, and in particular to a preparation method and application of a CLDN18.2 single-domain antibody probe.
背景技术Background technique
胃癌是我国最常见的恶性肿瘤之一，发病率居恶性肿瘤中的第二位，死亡率仅次于肝癌和肺癌。手术为最常见的治疗方法，但多数患者确诊时已处于晚期，整体预后不佳。进展期胃癌一线治疗方式为化疗，随着肿瘤耐药性的增加，患者无法获得较好的远期生存。在肿瘤诊疗策略中，分子靶向治疗如小分子抑制剂和单克隆抗体以及免疫治疗(如免疫检查点抑制剂)等正在改变多种实体肿瘤及血液系统肿瘤的治疗现状。寻找合适的胃癌诊疗靶点对于胃癌患者群体获益具有重要意义。Gastric cancer is one of the most common malignant tumors in my country, with the second highest incidence among malignant tumors and a mortality rate second only to liver cancer and lung cancer. Surgery is the most common treatment method, but most patients are already in the advanced stage when diagnosed, and the overall prognosis is poor. The first-line treatment for advanced gastric cancer is chemotherapy, but as tumor resistance increases, patients cannot achieve good long-term survival. In tumor diagnosis and treatment strategies, molecular targeted therapies such as small molecule inhibitors and monoclonal antibodies, as well as immunotherapy (such as immune checkpoint inhibitors), are changing the treatment status of a variety of solid tumors and hematological tumors. Finding suitable diagnostic and therapeutic targets for gastric cancer is of great significance for the benefit of gastric cancer patients.
紧密连接蛋白18(Claudin18,CLDN18)是位于上皮和内皮的紧密连接中的跨膜蛋白，是构成细胞间紧密连接重要的组成及功能结构。人类CLDN18基因第一个外显子存在两个等位基因，其差异形成了两种不同的剪切突变体，分别是CLDN18.1蛋白和CLDN18.2蛋白。CLDN18在正常组织中高度保守，CLDN18.2蛋白主要分布于分化周期短、更新速度快的胃黏膜(胃正常腺体、主细胞、壁细胞、内分泌细胞)，以及十二指肠的潘氏细胞内。目前普遍认为肿瘤在恶性转化中细胞极性发生改变，导致CLDN18.2广泛分布于细胞膜表面。胃癌中CLDN18.2阳性率，以及CLDN18.2阳性胃癌在胃癌中占比，在不同研究中存在较大差异，目前的部分研究将胃癌CLDN18.2表达率限定42％～86％，CLDN18.2阳性胃癌约占胃癌人群中的16％～73％，CLDN18.2表达具备一定的分子病理特点：弥漫型胃癌高于肠型胃癌，EBV病毒(Epstein-Barr virus)阳性胃癌高于阴性胃癌(81.0％vs.40.2％，P<0.001)，原发灶、周围淋巴结转移以及远处转移病灶表达基本一致。由于在消化系统恶性肿瘤中的异常激活且高表达，CLDN18.2被认为是消化系统恶性肿瘤治疗中的一个极具潜力的靶点。Claudin18 (CLDN18) is a transmembrane protein located in the tight junctions between epithelium and endothelium. It is an important component and functional structure of tight junctions between cells. There are two alleles in the first exon of the human CLDN18 gene, and their differences form two different splice mutants, namely CLDN18.1 protein and CLDN18.2 protein. CLDN18 is highly conserved in normal tissues. CLDN18.2 protein is mainly distributed in the gastric mucosa (normal gastric glands, chief cells, parietal cells, endocrine cells) with a short differentiation cycle and fast renewal rate, as well as Paneth cells in the duodenum. It is generally believed that the cell polarity of tumors changes during malignant transformation, resulting in the widespread distribution of CLDN18.2 on the cell membrane surface. The CLDN18.2 positive rate in gastric cancer and the proportion of CLDN18.2 positive gastric cancer in gastric cancer vary greatly in different studies. Some current studies limit the CLDN18.2 expression rate in gastric cancer to 42% to 86%, and CLDN18.2 positive gastric cancer accounts for about 16% to 73% of the gastric cancer population. CLDN18.2 expression has certain molecular pathological characteristics: diffuse gastric cancer is higher than intestinal gastric cancer, EBV virus (Epstein-Barr virus) positive gastric cancer is higher than negative gastric cancer (81.0% vs. 40.2%, P < 0.001), and the expression of primary lesions, peripheral lymph node metastasis and distant metastatic lesions is basically the same. Due to its abnormal activation and high expression in digestive system malignancies, CLDN18.2 is considered to be a very potential target in the treatment of digestive system malignancies.
已有研究表明CLDN18.2是实体瘤治疗的良好靶点。Zolbetuximab(IMAB362，claudixmab)是嵌合的IgG1单克隆抗体，在肿瘤细胞表面与CLDN18.2特异结合，从而引发抗体依赖性细胞毒性(antibody-dependent cellular cytotoxicity,ADCC)、补体依赖性细胞毒性(complement dependent cytotoxicity,CDC)，凋亡和抑制细胞增殖。目前多个I/II期试验评估了其临床疗效和安全性。2022年11月17日安斯泰来公司(Astellas)宣布Claudin18.2抗体Zolbetuximab+化疗联合治疗Claudin18.2阳性、HER2阴性的复发性转移性胃癌的三期临床SPOTLIGHT达到主要终点。研究结果显示与安慰剂+mFOLFOX6相比，zolbetuximab+mFOLFOX6治疗的患者的无进展生存期(PFS)及总生存期(OS)具有统计学意义。人源化抗CLDN18.2自体CAR-T疗法也显示了抗肿瘤活性和安全性，正在进行的多项临床试验也有望为CLDN18.2阳性肿瘤患者提供更多选择。CLDN18.2涉及靶向药、小分子抑制剂、以及CAR-T、免疫细胞治疗、抗体耦联药、肿瘤疫苗等，癌种涉及胃癌，胃-食管交界部癌以及胰腺癌，CLDN18.2的个体化免疫治疗或将是下一个消化系统恶性肿瘤研究的热点。Studies have shown that CLDN18.2 is a good target for the treatment of solid tumors. Zolbetuximab (IMAB362, claudixmab) is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the surface of tumor cells, thereby inducing antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), apoptosis and inhibiting cell proliferation. Currently, multiple phase I/II trials have evaluated its clinical efficacy and safety. On November 17, 2022, Astellas announced that the Phase III clinical SPOTLIGHT of Claudin18.2 antibody Zolbetuximab + chemotherapy combined with Claudin18.2-positive, HER2-negative recurrent metastatic gastric cancer reached the primary endpoint. The results showed that compared with placebo + mFOLFOX6, patients treated with zolbetuximab + mFOLFOX6 had statistically significant progression-free survival (PFS) and overall survival (OS). Humanized anti-CLDN18.2 autologous CAR-T therapy has also shown anti-tumor activity and safety, and the ongoing clinical trials are expected to provide more options for patients with CLDN18.2-positive tumors. CLDN18.2 involves targeted drugs, small molecule inhibitors, CAR-T, immune cell therapy, antibody-drug conjugates, tumor vaccines, etc. Cancer types include gastric cancer, gastroesophageal junction cancer and pancreatic cancer. Individualized immunotherapy of CLDN18.2 may be the next hot spot in the study of digestive system malignancies.
抗体因其明确的结构、相对的稳定性、高特异性和高亲和力在生物医学研究中受到特别关注。基于抗体构建的探针所开展的PET显像被称之为免疫PET显像，属于分子影像的一个分支，采用靶向肿瘤细胞及肿瘤微环境受体的探针，可以实时有效地反映肿瘤发生发展。基于单克隆抗体构建的免疫PET探针具有稳定性高、特异性强的特点，临床应用相对广泛，但单克隆抗体(约150kD)分子量过大，组织渗透能力低，显像靶本比较差，非靶器官易产生非特异性摄取。单抗空间结构复杂，表达制备成本较高，具有很高的免疫原性，而改造的抗体很难达到原来的亲和力。诸多因素限制其在临床中的应用及普及。Antibodies have received special attention in biomedical research due to their clear structure, relative stability, high specificity and high affinity. PET imaging based on antibody-based probes is called immune PET imaging, which is a branch of molecular imaging. It uses probes targeting tumor cells and tumor microenvironment receptors to effectively reflect the occurrence and development of tumors in real time. ImmunoPET probes based on monoclonal antibodies have the characteristics of high stability and strong specificity, and are relatively widely used in clinical practice. However, monoclonal antibodies (about 150kD) have a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake in non-target organs. Monoclonal antibodies have complex spatial structures, high expression and preparation costs, and high immunogenicity, but modified antibodies are difficult to achieve the original affinity. Many factors limit their application and popularization in clinical practice.
纳米抗体来源于成年骆驼体内重链抗体的最小的功能性抗原结合片段，分子量仅为15kDa。纳米抗体具有高度稳定性和与抗原结合的高亲合力，和常规抗体相比具有许多独特的性质：1)纳米抗体编码的序列与人VH家族3和4同源性高，免疫原性弱；2)纳米抗体分子量小，结构简单，可在微生物体系中大量表达，易于纯化；3)纳米抗体可以识别大量的抗原表位，包括一些藏在分子裂缝中的表位都能识别；4)由于分子量小，使得它们易于穿透组织，到达常规抗体难以到达的部位；5)可与短半衰期核素匹配，实现当日显像。纳米抗体的种种特性使其成为很有前途的疾病诊断和治疗工具：作为显像示踪剂，纳米抗体可以及早获取高质量图像；作为治疗剂，纳米抗体可以通过与细胞毒性药物偶联，通过特异性传送至靶点实现精准的靶向治疗。近年来，我们专注于纳米抗体衍生示踪剂的开发和临床转化，以发挥其优越的分子成像特性。基于上述证据和我们之前的发现，我们假设靶向CLDN18.2的免疫PET显像探针可以无创地显示胃癌肿瘤细胞CLDN18.2表达，服务于胃癌系统的诊疗体系，推进精准医学个体化治疗的发展。Nanobodies are derived from the smallest functional antigen-binding fragment of heavy chain antibodies in adult camels, with a molecular weight of only 15kDa. Nanobodies are highly stable and have high affinity for binding to antigens. Compared with conventional antibodies, they have many unique properties: 1) The sequences encoded by nanobodies are highly homologous to human VH families 3 and 4, and have weak immunogenicity; 2) Nanobodies have a small molecular weight and a simple structure, can be expressed in large quantities in microbial systems, and are easy to purify; 3) Nanobodies can recognize a large number of antigenic epitopes, including some epitopes hidden in molecular cracks; 4) Due to their small molecular weight, they can easily penetrate tissues and reach sites that are difficult for conventional antibodies to reach; 5) They can be matched with short-half-life nuclides to achieve same-day imaging. The various characteristics of nanobodies make them promising tools for disease diagnosis and treatment: as imaging tracers, nanobodies can obtain high-quality images early; as therapeutic agents, nanobodies can be coupled with cytotoxic drugs and delivered to targets specifically to achieve precise targeted therapy. In recent years, we have focused on the development and clinical transformation of nanobody-derived tracers to give full play to their superior molecular imaging properties. Based on the above evidence and our previous findings, we hypothesized that the immunoPET imaging probe targeting CLDN18.2 could noninvasively display CLDN18.2 expression in gastric cancer tumor cells, serve the diagnosis and treatment system of gastric cancer, and promote the development of personalized treatment in precision medicine.
目前CLDN18.2分子影像探针领域尚处于待开发阶段，本领域的技术人员致力于开发一种制备成本低廉、分子量小、体内循环时间短、显像周期短、辐射剂量低、易于临床转化应用的纳米抗体免疫PET显像探针。At present, the field of CLDN18.2 molecular imaging probes is still in the development stage. Technical personnel in this field are committed to developing a nano-antibody immune PET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation and application.
发明内容Summary of the invention
为了填补该领域的空白，我们在这里描述了纳米抗体衍生的CLDN18.2靶向诊断探针的构建，并表征了其在正常Balb/c小鼠中的生物分布情况，以期探索其对胃癌的诊断及潜在治疗价值。具体而言，本发明提供了一种CLDN18.2纳米抗体探针的制备方法及应用。To fill the gap in this field, we describe here the construction of a nanobody-derived CLDN18.2-targeted diagnostic probe and characterize its biodistribution in normal Balb/c mice, in order to explore its diagnostic and potential therapeutic value for gastric cancer. Specifically, the present invention provides a method for preparing a CLDN18.2 nanobody probe and its application.
CLDN18.2特异性纳米抗体CLDN18.2-specific nanobody
在一个方面，本发明提供了一种CLDN18.2特异性纳米抗体，其包含：In one aspect, the present invention provides a CLDN18.2-specific Nanobody comprising:
(1)具有SEQ ID No.1所示的氨基酸序列的CDR1、具有SEQ ID No.2所示的氨基酸序列的CDR2和具有SEQ ID No.3所示的氨基酸序列的CDR3，(1) a CDR1 having the amino acid sequence shown in SEQ ID No. 1, a CDR2 having the amino acid sequence shown in SEQ ID No. 2, and a CDR3 having the amino acid sequence shown in SEQ ID No. 3,
(2)具有SEQ ID No.6所示的氨基酸序列的CDR1、具有SEQ ID No.7所示的氨基酸序列的CDR2和具有SEQ ID No.8所示的氨基酸序列的CDR3，(2) a CDR1 having the amino acid sequence shown in SEQ ID No. 6, a CDR2 having the amino acid sequence shown in SEQ ID No. 7, and a CDR3 having the amino acid sequence shown in SEQ ID No. 8,
(3)具有SEQ ID No.11所示的氨基酸序列的CDR1、具有SEQ ID No.12所示的氨基酸序列的CDR2和具有SEQ ID No.13所示的氨基酸序列的CDR3。(3) CDR1 having the amino acid sequence shown in SEQ ID No.11, CDR2 having the amino acid sequence shown in SEQ ID No.12, and CDR3 having the amino acid sequence shown in SEQ ID No.13.
更具体地，本发明的CLDN18.2特异性纳米抗体具有SEQ ID No.4、9或14所示的氨基酸序列。More specifically, the CLDN18.2-specific Nanobody of the present invention has the amino acid sequence shown in SEQ ID No. 4, 9 or 14.
在本发明中，为了简便起见，将具有SEQ ID No.4、9或14所示的氨基酸序列的CLDN18.2特异性纳米抗体分别称为3E10、3E11或3A12。In the present invention, for the sake of simplicity, the CLDN18.2-specific Nanobodies having the amino acid sequence shown in SEQ ID No. 4, 9 or 14 are referred to as 3E10, 3E11 or 3A12, respectively.
如本文所用，术语“纳米抗体(nanobody)”也称为“单域抗体(single-domainantibody,sdAb)”或VHH(Variable Domain of Heavy Chain of Heavy Chain Antibody)，它们可互换使用。纳米抗体具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，通常来源于重链抗体(例如骆驼科动物抗体或鲨鱼抗体)的可变区。典型地，纳米抗体由4个构架区和3个互补性决定区组成，具有FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。纳米抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。As used herein, the term "nanobody" is also referred to as "single-domain antibody (sdAb)" or VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody), which are used interchangeably. Nanobody has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomer variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus so that they contain only part of FR1 and/or FR4, or lack one or two of those framework regions, as long as they substantially maintain antigen binding and specificity.
在一些实施方案中，本发明还涵盖如本文所述的CLDN18.2特异性纳米抗体的抗原结合片段。In some embodiments, the invention also encompasses antigen-binding fragments of the CLDN18.2-specific Nanobodies as described herein.
如本文中所使用的，术语“抗原结合片段”是指包含纳米抗体的片段的多肽，其保持特异性结合纳米抗体所结合的相同抗原的能力，和/或与纳米抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文以用于所有目的。可通过重组DNA技术或通过本发明纳米抗体的酶促或化学断裂产生本发明抗体的抗原结合片段。在一些实施方案中，所述纳米抗体的“抗原结合片段”与全长纳米抗体相比可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。As used herein, the term "antigen-binding fragment" refers to a polypeptide comprising a fragment of a Nanobody that retains the ability to specifically bind to the same antigen to which the Nanobody binds, and/or competes with the Nanobody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the Nanobody may be truncated at the N-terminus or C-terminus compared to the full-length Nanobody so that it comprises only part of FR1 and/or FR4, or lacks one or two of those framework regions, as long as it substantially retains antigen binding and specificity.
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的纳米抗体(例如本发明提供的纳米抗体)获得纳米抗体的抗原结合片段，并且以与用于完整纳米抗体的方式相同的方式就特异性筛选纳米抗体的抗原结合片段。Antigen-binding fragments of Nanobodies can be obtained from a given Nanobody (e.g., the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of Nanobodies can be screened for specificity in the same manner as for intact Nanobodies.
在本文中，除非上下文明确指出，否则当提及术语“纳米抗体”时，其不仅包括完整纳米抗体，而且包括纳米抗体的抗原结合片段。In this document, unless the context clearly indicates otherwise, when referring to the term "Nanobody", it includes not only complete Nanobodies but also antigen-binding fragments of Nanobodies.
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在纳米抗体中含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的纳米抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
如本文中所使用的，术语“CLDN18.2特异性”是指特异性结合CLDN18.2。As used herein, the term "CLDN18.2 specific" refers to specific binding to CLDN18.2.
如本文中所使用的，术语“特异性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以由该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD ) of the interaction. In the present invention, the term "KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见MalmqvistM,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,AnnualRev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site/antigen complex. Both the "association rate constant" (ka orkon ) and the "dissociation rate constant" (kdis orkoff ) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio ofkdis /kon is equal to the dissociation constantKD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473).KD ,kon andkdis values can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured by bioluminescence interferometry or Kinexa.
在一些实施方案中，本发明还提供了如本文所述的CLDN18.2特异性纳米抗体的变体，其与SEQ ID No.4、9或14所示的氨基酸序列的具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性，并且基本保留了其所源自的纳米抗体的生物学功能(例如特异性结合CLDN18.2的生物活性)。In some embodiments, the present invention also provides a variant of a CLDN18.2-specific Nanobody as described herein, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID No. 4, 9 or 14, and substantially retains the biological function of the Nanobody from which it is derived (e.g., biological activity that specifically binds to CLDN18.2).
更具体地，所述变体与如本文所述的CLDN18.2特异性纳米抗体相比差异仅在于一个或多个(例如，至多20个、至多15个、至多10个、至多5个或至多1个氨基酸的保守置换)氨基酸残基的保守置换。More specifically, the variants differ from the CLDN18.2-specific Nanobodies as described herein only in conservative substitutions of one or more (for example, conservative substitutions of up to 20, up to 15, up to 10, up to 5 or up to 1 amino acid residues).
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residuetable)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.NatlAcad.Set USA 94:412-417(1997)，其通过引用并入本文)。As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).
多核苷酸Polynucleotide
在另一个方面，本发明还提供了编码上述纳米抗体或其抗原结合片段的多核苷酸。In another aspect, the present invention also provides a polynucleotide encoding the above-mentioned Nanobody or its antigen-binding fragment.
更具体地，所述多核苷酸具有SEQ ID No.5、10或15所示的核苷酸序列。More specifically, the polynucleotide has a nucleotide sequence shown in SEQ ID No. 5, 10 or 15.
更具体地，编码如本文所述的CLDN18.2特异性纳米抗体的多核苷酸具有SEQ IDNo.5、10或15所示的核苷酸序列。More specifically, the polynucleotide encoding the CLDN18.2-specific Nanobody as described herein has the nucleotide sequence shown in SEQ ID No. 5, 10 or 15.
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.
术语“编码多肽/蛋白/抗体的多核苷酸”可以是包括编码此多肽/蛋白/抗体的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。The term "polynucleotide encoding a polypeptide/protein/antibody" may include a polynucleotide encoding the polypeptide/protein/antibody, or may include additional coding and/or non-coding sequences.
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，优选地至少70％，更优选地至少80％，最优选至少90％同一性的多核苷酸，并且它们编码的多肽/蛋白/抗体具有基本上相同的功能和活性。本发明特别涉及可在严格条件下与本发明所述多核苷酸杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的同一性至少在90％以上,更优选95％以上时才发生杂交。The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences, and the polypeptides/proteins/antibodies encoded by them have substantially the same functions and activities. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably 95%.
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
载体Carrier
在另一个方面，本发明还提供了包含编码上述CLDN18.2抗体或其抗原结合片段的多核苷酸的载体。In another aspect, the present invention also provides a vector comprising a polynucleotide encoding the above-mentioned CLDN18.2 antibody or an antigen-binding fragment thereof.
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.
宿主细胞Host cells
在另一个方面，本发明还提供了包含如本文所述的载体的宿主细胞。In another aspect, the present invention also provides a host cell comprising the vector as described herein.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或其他人细胞等的动物细胞。宿主细胞可以包括单个细胞或细胞群体。As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include a single cell or a cell population.
将载体导入宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。The vector can be introduced into the host cell by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
本发明的纳米抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。The Nanobodies of the invention can be used alone or in combination or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety or any combination of these.
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。优选的可检测标记物为放射性核素。Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing a detectable product. Preferred detectable labels are radionuclides.
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.
可检测标记物或治疗剂与抗体的结合或偶联可以通过本领域技术人员熟知的常规方法进行。例如，可检测标记物可以直接或间接结合至纳米抗体，例如通过可切割或不可切割的接头肽，或掺入纳米抗体中。可检测标记物尤其可以通过替换(例如通过用酪氨酸残基水平的I替换H)，通过复合或通过螯合与纳米抗体结合。例如治疗剂可以经由可切割接头(例如肽基、二硫化物或腙接头)缀合至纳米抗体。The binding or coupling of a detectable marker or therapeutic agent to an antibody can be carried out by conventional methods well known to those skilled in the art. For example, a detectable marker can be directly or indirectly bound to a nanobody, for example, by a cleavable or non-cleavable linker peptide, or incorporated into a nanobody. The detectable marker can be bound to a nanobody, in particular, by replacement (for example, by replacing H with I at the level of tyrosine residues), by complexing or by chelation. For example, a therapeutic agent can be conjugated to a nanobody via a cleavable linker (for example, a peptidyl, a disulfide or a hydrazone linker).
在优选的实施方案中，如下文更详细描述的，本发明的纳米抗体与放射性核素偶联以用作CLDN18.2特异性分子影像探针。In a preferred embodiment, as described in more detail below, the Nanobodies of the invention are conjugated to a radionuclide for use as CLDN18.2-specific molecular imaging probes.
CLDN18.2特异性分子影像探针CLDN18.2-specific molecular imaging probe
在另一个方面，本发明提供了一种CLDN18.2特异性18F标记单价纳米抗体探针，其包含经放射性核素标记的如本文所述的CLDN18.2特异性纳米抗体。In another aspect, the present invention provides a CLDN18.2-specific18 F-labeled monovalent Nanobody probe, which comprises a CLDN18.2-specific Nanobody as described herein labeled with a radionuclide.
更具体地，如本文所述的CLDN18.2特异性纳米抗体经由双功能螯合剂被放射性核素标记。More specifically, the CLDN18.2-specific Nanobodies as described herein are radionuclide-labeled via a bifunctional chelator.
如本文所用，双功能螯合剂是同时具有金属螯合端和蛋白锚定端的一类螯合剂。双功能螯合剂可以选自(±)-H3RESCA-TFP、(±)H3RESCA-Mal中的至少一种。As used herein, a bifunctional chelator is a type of chelator having both a metal chelating end and a protein anchoring end. The bifunctional chelator can be selected from at least one of (±)-H3RESCA-TFP and (±)H3RESCA-Mal.
如本文所用，所述(±)-H3RESCA-TFP为一种约束络合剂(RESCA)的四氟苯基酯衍生物，可用于通过胺偶联(例如，N末端和/或赖氨酸的ε-氨基)使螯合剂与生物分子偶联；As used herein, the (±)-H3RESCA-TFP is a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA) that can be used to couple the chelator to biomolecules via amine coupling (e.g., the N-terminus and/or the ε-amino group of lysine);
所述(±)H3RESCA-Mal为(±)H3RESCA-马来酰亚胺，是一种约束络合剂(RESCA)的四氟苯基酯衍生物，可用于通过胺偶联(例如，N末端和/或赖氨酸的ε-氨基)使螯合剂与生物分子偶联。The (±)H3RESCA-Mal is (±)H3RESCA-maleimide, a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA), which can be used to couple the chelator to biomolecules via amine coupling (e.g., N-terminus and/or ε-amino group of lysine).
更具体地，如本文所述的CLDN18.2特异性纳米抗体经由(±)-H3RESCA-TFP或(±)H3RESCA-Mal被放射性核素标记。More specifically, the CLDN18.2-specific Nanobodies as described herein were radionuclide-labeled via (±)-H3RESCA-TFP or (±)H3RESCA-Mal.
更具体地，放射性核素选自F-18。More particularly, the radionuclide is selected from F-18.
更具体地，如本文所述的[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11或[18F]F-H3RESCA-3A12。More specifically, [18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11 or [18 F]F-H3RESCA-3A12 as described herein.
更具体地，所述CLDN18.2特异性18F标记单价纳米抗体探针为[18F]F-H3RESCA-3A12，根据具有SEQ ID No.11所示的氨基酸序列的CDR1、具有SEQ ID No.12所示的氨基酸序列的CDR2和具有SEQ ID No.13所示的氨基酸序列的CDR3，或具有SEQ ID No.14所示的氨基酸序列的CLDN18.2特异性纳米抗体经由(±)-H3RESCA-TFP或(±)H3RESCA-Mal被F-18标记。其在正常胃组织中的显像效果更佳。More specifically, the CLDN18.2-specific18 F-labeled monovalent nanobody probe is [18 F]F-H3RESCA-3A12, according to CDR1 having the amino acid sequence shown in SEQ ID No. 11, CDR2 having the amino acid sequence shown in SEQ ID No. 12, and CDR3 having the amino acid sequence shown in SEQ ID No. 13, or the CLDN18.2-specific nanobody having the amino acid sequence shown in SEQ ID No. 14 is labeled with F-18 via (±)-H3RESCA-TFP or (±)H3RESCA-Mal. Its imaging effect in normal gastric tissue is better.
在另一个方面，本发明还提供了制备CLDN18.2特异性18F标记单价纳米抗体探针的方法，包括通过双功能螯合剂修饰CLDN18.2特异性纳米抗体，得到放射性核素标记前体；和使用放射性核素标记所述放射性核素标记前体，得到CLDN18.2特异性18F标记单价纳米抗体探针。In another aspect, the present invention also provides a method for preparing a CLDN18.2-specific18 F-labeled monovalent Nanobody probe, comprising modifying a CLDN18.2-specific Nanobody by a bifunctional chelator to obtain a radionuclide-labeled precursor; and labeling the radionuclide-labeled precursor with a radionuclide to obtain a CLDN18.2-specific18 F-labeled monovalent Nanobody probe.
更具体地，当所述双功能螯合剂为(±)H3RESCA-Mal时，在进行放射性核素标记前体制备前，需在CLDN18.2特异性纳米抗体的末端表达含有半胱氨酸的肽段(GGGGS)nC，n＝1-10；具体可为n＝1、2、3、4、5、6、7、8、9或10。More specifically, when the bifunctional chelator is (±)H3RESCA-Mal, before preparing the radionuclide labeled precursor, a cysteine-containing peptide (GGGGS)nC is required to be expressed at the end of the CLDN18.2-specific nanobody, where n=1-10; specifically, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
组合物combination
在另一个方面，本发明提供了一种组合物，其包含如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针。所述组合物可用于检测CLDN18.2的表达水平、诊断CLDN18.2相关的肿瘤、预测CLDN18.2相关肿瘤的治疗效果或治疗CLDN18.2相关肿瘤。In another aspect, the present invention provides a composition comprising a CLDN18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein. The composition can be used to detect the expression level of CLDN18.2, diagnose CLDN18.2-related tumors, predict the treatment effect of CLDN18.2-related tumors, or treat CLDN18.2-related tumors.
在一些实施方案中，所述组合物可以是药物组合物。In some embodiments, the composition may be a pharmaceutical composition.
在一些实施方案中，所述药物组合物还可包含药学上可接受的载体和/或赋形剂。In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or excipient.
在一些实施方案中，所述药物组合物还可以包含另外的药学活性剂。In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.
在一些实施方案中，所述另外的药学活性剂是抗炎药物或免疫抑制剂。In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.
在一些实施方案中，在所述药物组合物中，如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针与所述另外的药学活性剂可以作为分离的组分或作为混合的组分提供。因此，如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针与所述另外的药学活性剂可以同时、分开或相继施用。In some embodiments, in the pharmaceutical composition, the CLDN18.2-specific nanobodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein and the additional pharmaceutically active agent may be provided as separate components or as mixed components. Thus, the CLDN18.2-specific nanobodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.
在一些实施方案中，所述药学上可接受的载体和/或赋形剂可以包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。In some embodiments, the pharmaceutically acceptable carrier and/or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针。“预防有效量”是指足以预防、阻止或延迟疾病的发生的量。“治疗有效量”是指足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a CLDN18.2-specific Nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein. "Prophylactically effective amount" refers to an amount sufficient to prevent, prevent or delay the occurrence of a disease. "Therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.
试剂盒Reagent test kit
本发明还提供了一种试剂盒，其包含如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针。The present invention also provides a kit comprising the CLDN18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein.
所述试剂盒可用于检测CLDN18.2的表达水平、诊断CLDN18.2相关的肿瘤、预测CLDN18.2相关肿瘤的治疗效果或治疗CLDN18.2相关肿瘤。The kit can be used to detect the expression level of CLDN18.2, diagnose CLDN18.2-related tumors, predict the treatment effect of CLDN18.2-related tumors, or treat CLDN18.2-related tumors.
所述试剂盒还可包含还包括容器、使用说明书、以及实际应用所需的其他试剂和缓冲液，例如用于溶解样本的裂解介质，各种缓冲液，检测标记，检测底物等。The kit may also include a container, instructions for use, and other reagents and buffers required for practical applications, such as a lysis medium for dissolving the sample, various buffers, detection labels, detection substrates, etc.
诊断和治疗应用Diagnostic and therapeutic applications
本发明的CLDN18.2特异性纳米抗体对CLDN18.2具有极高的亲和力，因而可以用于检测CLDN18.2的表达水平、诊断CLDN18.2相关的肿瘤、预测CLDN18.2相关肿瘤的治疗效果或治疗CLDN18.2相关肿瘤。The CLDN18.2-specific nanoantibodies of the present invention have extremely high affinity for CLDN18.2 and can thus be used to detect the expression level of CLDN18.2, diagnose CLDN18.2-related tumors, predict the therapeutic effect of CLDN18.2-related tumors, or treat CLDN18.2-related tumors.
特别地，由本发明的CLDN18.2特异性纳米抗体制备的CLDN18.2特异性分子影像探针具有亲和力显著提高、正常组织摄取非特异性摄取明显降低、且图像质量明显提高的特点，可用于无创、精准、高效探测人CLDN18.2的表达，因此特别适合用于诊断CLDN18.2相关的肿瘤和预测CLDN18.2相关肿瘤的治疗效果。在选择合适的放射性核素进行偶联后，也可以用于精准治疗CLDN18.2相关肿瘤。In particular, the CLDN18.2-specific molecular imaging probe prepared by the CLDN18.2-specific nanoantibody of the present invention has the characteristics of significantly improved affinity, significantly reduced nonspecific uptake of normal tissue uptake, and significantly improved image quality, and can be used for non-invasive, accurate, and efficient detection of human CLDN18.2 expression, and is therefore particularly suitable for diagnosing CLDN18.2-related tumors and predicting the therapeutic effect of CLDN18.2-related tumors. After selecting a suitable radionuclide for coupling, it can also be used for precise treatment of CLDN18.2-related tumors.
因此，在另一个方面，本发明还涉及如本文所述的CLDN18.2特异性纳米抗体、多核苷酸、载体、宿主细胞或分子影像探针在制备用于检测CLDN18.2的表达水平、诊断CLDN18.2相关的肿瘤、预测CLDN18.2相关肿瘤的治疗效果或治疗CLDN18.2相关肿瘤的试剂盒或药物中的用途。Therefore, in another aspect, the present invention also relates to the use of the CLDN18.2-specific nanoantibodies, polynucleotides, vectors, host cells or molecular imaging probes described herein in the preparation of a kit or drug for detecting the expression level of CLDN18.2, diagnosing CLDN18.2-related tumors, predicting the therapeutic effect of CLDN18.2-related tumors, or treating CLDN18.2-related tumors.
如本文所用，CLDN18.2相关的肿瘤可以包括本领域熟知的各种肿瘤或癌症。例如，CLDN18.2相关的肿瘤可以包括消化道肿瘤，例如胃癌、胰腺癌、食管癌、胆管癌、胆囊癌等；还可以包括乳腺癌、结肠癌、肝癌、头颈癌、支气管癌以及非小细胞肺癌等。As used herein, CLDN18.2-related tumors may include various tumors or cancers well known in the art. For example, CLDN18.2-related tumors may include digestive tract tumors, such as gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, gallbladder cancer, etc.; and may also include breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.
本发明的有益效果Beneficial Effects of the Invention
1)本发明制备的CLDN18.2特异性分子探针[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11及[18F]F-H3RESCA-3A12为正电子核素发射型探针，用于免疫PET显像；通过开展基于上述探针的免疫PET显像，可实现CLDN18.2在肿瘤组织及正常组织器官表达的无创可视化，进一步用于特定类型肿瘤的无创靶点特异性诊断。1) The CLDN18.2-specific molecular probes [18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11 and [18 F]F-H3RESCA-3A12 prepared by the present invention are positron nuclide emission probes used for immuno-PET imaging; by carrying out immuno-PET imaging based on the above probes, non-invasive visualization of CLDN18.2 expression in tumor tissues and normal tissues and organs can be achieved, and further used for non-invasive target-specific diagnosis of specific types of tumors.
2)本发明制备的探针还具有制备工艺简单、成本低廉、特异性高、稳定性高、显像周期短、辐射剂量低，并且易于临床转化等优点。2) The probe prepared by the present invention also has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical transformation.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示了SDS-PAGE测定纳米抗体3E10、3E11和3A12表达情况的结果；Figure 1 shows the results of SDS-PAGE assay for the expression of Nanobodies 3E10, 3E11, and 3A12;
图2显示了CLDN18.2的组织化学染色结果；Figure 2 shows the histochemical staining results of CLDN18.2;
图3显示了[18F]F-H3RESCA-3E10的放射化学纯度测定结果；FIG3 shows the results of the radiochemical purity determination of [18 F]F-H3RESCA-3E10;
图4显示了[18F]F-H3RESCA-3E11的放射化学纯度测定结果；FIG4 shows the results of the radiochemical purity determination of [18 F]F-H3RESCA-3E11;
图5显示了[18F]F-H3RESCA-3A12的放射化学纯度测定结果；FIG5 shows the results of the radiochemical purity determination of [18 F]F-H3RESCA-3A12;
图6显示了[18F]F-H3RESCA-3E10在正常Balb/c鼠注射45分钟后PET/CT显像结果；FIG6 shows the PET/CT imaging results of [18 F]F-H3RESCA-3E10 in normal Balb/c mice 45 minutes after injection;
图7显示了[18F]F-H3RESCA-3E10在正常Balb/c鼠注射45分钟后ROI分析结果；FIG7 shows the results of ROI analysis 45 minutes after injection of [18 F]F-H3RESCA-3E10 in normal Balb/c mice;
图8显示了[18F]F-H3RESCA-3E10在正常Balb/c鼠的体外生物分布结果；FIG8 shows the in vitro biodistribution results of [18 F]F-H3RESCA-3E10 in normal Balb/c mice;
图9显示了[18F]F-H3RESCA-3E11在正常Balb/c鼠的PET/CT显像结果；FIG9 shows the PET/CT imaging results of [18 F]F-H3RESCA-3E11 in normal Balb/c mice;
图10显示了[18F]F-H3RESCA-3E11在正常Balb/c鼠的ROI图结果；FIG10 shows the ROI map results of [18 F]F-H3RESCA-3E11 in normal Balb/c mice;
图11显示了[18F]F-H3RESCA-3E11在正常Balb/c鼠的体外生物分布结果；FIG11 shows the in vitro biodistribution results of [18 F]F-H3RESCA-3E11 in normal Balb/c mice;
图12显示了[18F]F-H3RESCA-3A12在正常Balb/c鼠的PET/CT显像结果；FIG12 shows the PET/CT imaging results of [18 F]F-H3RESCA-3A12 in normal Balb/c mice;
图13显示了[18F]F-H3RESCA-3A12在正常Balb/c鼠的ROI图结果；FIG13 shows the ROI map results of [18 F]F-H3RESCA-3A12 in normal Balb/c mice;
图14显示了[18F]F-H3RESCA-3A12在正常Balb/c鼠的体外生物分布结果；FIG14 shows the in vitro biodistribution results of [18 F]F-H3RESCA-3A12 in normal Balb/c mice;
图15显示了螯合剂(±)H3RESCA-TFP的化学结构；Figure 15 shows the chemical structure of the chelating agent (±)H3RESCA-TFP;
图16显示了螯合剂(±)H3RESCA-Mal化学结构。FIG. 16 shows the chemical structure of the chelator (±)H3RESCA-Mal.
具体实施方式Detailed ways
为了便于理解本发明，下面结合附图和具体实施例，对本发明进行更详细的说明。需要说明的是，本发明并不限于本文中所述的特定方法、方案、细胞系、构筑体和试剂，并且同样可改变。除非另有定义，本说明书所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本说明书中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是用于限制本发明。For ease of understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the ad hoc methods, schemes, cell lines, constructs and reagents described herein, and can be changed equally. Unless otherwise defined, all technical and scientific terms used in this specification are identical with the meanings generally understood by those skilled in the art belonging to the technical field of the present invention. The terms used in this specification in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.
传统筛选CLDN18.2阳性患者群体依赖于活检或手术标本的病理结果，创伤性较大、可重复性差，且由于取样的误差及肿瘤的异质性可能导致染色结果的差异。常用的CLAUDETECTTM18.2免疫组化试剂盒无法区分CLDN18的两种剪切突变体，可能存在假阳性结果。常用的非特异性显像剂18F-FDG PET/CT在胃癌远处转移即M分期方面的价值已经得到公认,而在原发肿瘤和局部淋巴结的诊断、评价方面与比其他影像方法并无令人信服的优势,尤其是对早期胃癌、印戒细胞癌或粘液腺癌的诊断价值差强人意。目前亟需能无创早期诊断胃癌及筛选合适的靶向治疗患者群体的工具。Traditional screening of CLDN18.2-positive patient groups relies on pathological results of biopsy or surgical specimens, which is highly invasive and has poor reproducibility. In addition, sampling errors and tumor heterogeneity may lead to differences in staining results. The commonly used CLAUDETECTTM 18.2 immunohistochemistry kit cannot distinguish between the two splice mutants of CLDN18, and false positive results may occur. The commonly used nonspecific imaging agent18 F-FDG PET/CT has been recognized for its value in distant metastasis of gastric cancer, i.e., M staging, but it has no convincing advantages over other imaging methods in the diagnosis and evaluation of primary tumors and local lymph nodes, especially for early gastric cancer, signet ring cell carcinoma, or mucinous adenocarcinoma. At present, there is an urgent need for tools that can non-invasively diagnose gastric cancer at an early stage and screen suitable patients for targeted treatment.
分子影像学在协助进行疾病筛查、鉴别诊断、患者分层及临床分期、疗效评估及预后方面发挥着不可忽视的作用。开发新型探针实现胃癌特异性靶点无创可视化对于早期诊断和精准治疗对降低胃癌患者死亡率和延长生存期至关重要。Molecular imaging plays an important role in assisting disease screening, differential diagnosis, patient stratification and clinical staging, efficacy evaluation and prognosis. The development of new probes to achieve non-invasive visualization of gastric cancer-specific targets is crucial for early diagnosis and precise treatment to reduce mortality and prolong survival of gastric cancer patients.
已有研究表明免疫PET相较于免疫组织化学染色或其他传统预测标志物可以更好的显示体内感兴趣靶点的分布情况及分布丰度，并更好的预测对于靶向治疗的应答反应。传统的基于单克隆抗体构建的免疫PET探针具有稳定性高、特异性强的特点，临床应用相对广泛，但单克隆抗体(约150kDa)分子量过大，组织渗透能力低，显像靶本比较差，非靶器官易产生非特异性摄取。单抗空间结构复杂，表达制备成本较高，具有很高的免疫原性，而改造的抗体很难达到原来的亲和力。诸多因素限制其在临床中的应用及普及。目前CLDN18.2分子影像探针领域尚处于待开发阶段。Existing studies have shown that immuno-PET can better display the distribution and abundance of targets of interest in the body, and better predict the response to targeted therapy, compared with immunohistochemical staining or other traditional predictive markers. Traditional immuno-PET probes based on monoclonal antibodies have the characteristics of high stability and strong specificity, and are relatively widely used in clinical applications. However, monoclonal antibodies (about 150 kDa) have a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake in non-target organs. Monoclonal antibodies have complex spatial structures, high expression and preparation costs, and high immunogenicity, but modified antibodies are difficult to achieve the original affinity. Many factors limit its application and popularization in clinical practice. At present, the field of CLDN18.2 molecular imaging probes is still in the development stage.
基于此，本发明研究构建了一种新型Claudin 18.2(CLDN18.2)特异性分子影像探针，以实现恶性肿瘤细胞表面CLDN18.2的无创可视化，在体监测表达水平差异及动态演变，并在此基础上进一步提高探针的效能，推进实现诊疗一体化。Based on this, the present invention studies and constructs a novel Claudin 18.2 (CLDN18.2)-specific molecular imaging probe to achieve non-invasive visualization of CLDN18.2 on the surface of malignant tumor cells, monitor the differences in expression levels and dynamic evolution in vivo, and on this basis further improve the effectiveness of the probe to promote the realization of integrated diagnosis and treatment.
实施例1：CLDN18.2特异性纳米抗体的制备Example 1: Preparation of CLDN18.2-specific Nanobodies
按照发明人前期公布的方法(参考发明创造名称“一种诊断多发性骨髓瘤的分子影像探针”的专利号ZL202011131233.7的专利，其通过引用整体并入本文)，制备新型CLDN18.2特异性纳米抗体3E10、3E11、3A12。所述纳米抗体3E10的氨基酸序列如SEQ IDNO.4所示(其中CDR1序列为GNIVSINY(SEQ ID NO.1)，CDR2序列为ITNGGSA(SEQ ID NO.2)，CDR3序列为HASSVITTASLWGTDY(SEQ ID NO.3))、基因序列如SEQ ID NO.5所示；纳米抗体3E11的氨基酸序列如SEQ ID NO.9所示(其中CDR1序列为GRIFMINN(SEQ ID NO.6)，CDR2序列为ITRGGST(SEQ ID NO.7)，CDR3序列为NVNDTMPWRLQNDY(SEQ ID NO.8))、基因序列如SEQID NO.10所示；纳米抗体3A12的氨基酸序列如SEQ ID NO.14所示(其中CDR1序列为GRTFSDYN(SEQ ID NO.11)，CDR2序列为ITWSGSIR(SEQ ID NO.12)，CDR3序列为AANRLAMHRGLNYDY(SEQ ID NO.13))、基因序列如SEQ ID NO.15所示。According to the method previously published by the inventor (refer to the invention entitled "A molecular imaging probe for diagnosing multiple myeloma", patent No. ZL202011131233.7, which is incorporated herein by reference in its entirety), novel CLDN18.2-specific nanoantibodies 3E10, 3E11, and 3A12 were prepared. The amino acid sequence of the nanobody 3E10 is shown in SEQ ID NO.4 (wherein the CDR1 sequence is GNIVSINY (SEQ ID NO.1), the CDR2 sequence is ITNGGSA (SEQ ID NO.2), and the CDR3 sequence is HASSVITTASLWGTDY (SEQ ID NO.3)), and the gene sequence is shown in SEQ ID NO.5; the amino acid sequence of the nanobody 3E11 is shown in SEQ ID NO.9 (wherein the CDR1 sequence is GRIFMINN (SEQ ID NO.6), the CDR2 sequence is ITRGGST (SEQ ID NO.7), and the CDR3 sequence is NVNDTMPWRLQNDY (SEQ ID NO.8)), and the gene sequence is shown in SEQ ID NO.10; the amino acid sequence of the nanobody 3A12 is shown in SEQ ID NO.14 (wherein the CDR1 sequence is GRTFSDYN (SEQ ID NO.11), the CDR2 sequence is ITWSGSIR (SEQ ID NO.12), and the CDR3 sequence is AANRLAMHRGLNYDY (SEQ ID NO.13). NO.13)), the gene sequence is shown in SEQ ID NO.15.
SDS-PAGE测定纳米抗体3E10、3E11、3A12表达情况，具体步骤如下：首先按照SDS-PAGE凝胶试剂盒的方法配制1.5mm厚，15well凝胶，预热金属浴至100℃，加热含有loadingbuffer(5X)的蛋白样品5min；将SDS-PAGE凝胶组装完毕后加入1x SDS-PAGE buffer500ml，将蛋白样品缓慢点样至上样孔中，80V恒压电浴约30min，待溴酚蓝指示剂越过浓缩胶后调整电压至120V，电泳至凝胶底部，将凝胶取下，于考马斯蓝染液中加热染色50min后取出，脱色液脱色至背景干净、条带清晰时进行拍照。如图1所示，可见纳米抗体3E10、3E11、3A12分子量约为14KDa。SDS-PAGE was used to determine the expression of nanoantibodies 3E10, 3E11, and 3A12. The specific steps are as follows: first, a 1.5 mm thick, 15 well gel was prepared according to the method of the SDS-PAGE gel kit, a metal bath was preheated to 100°C, and a protein sample containing loading buffer (5X) was heated for 5 minutes; after the SDS-PAGE gel was assembled, 1x SDS-PAGE buffer 500 ml was added, and the protein sample was slowly spotted into the loading well, and the 80V constant voltage electric bath was used for about 30 minutes. After the bromophenol blue indicator passed through the concentrated gel, the voltage was adjusted to 120V, and electrophoresis was performed to the bottom of the gel, and the gel was removed, and the gel was heated and stained in Coomassie blue dye for 50 minutes, and then taken out, and the decolorization solution was decolorized until the background was clean and the bands were clear, and then the photo was taken. As shown in Figure 1, the molecular weight of nanoantibodies 3E10, 3E11, and 3A12 was about 14KDa.
验证CLDN18.2在正常裸鼠主要组织器官的表达情况，具体包括以下步骤：获取并使用组织固定液固定正常裸鼠的胃、肝脏、肾脏、肺脏、脾脏及胰腺，以重组CLDN18.2单克隆抗体(EPR19202,ab222512,Abcam)作为一抗，以辣根过氧化物酶偶连的兔抗人IgG H&L(HRP-labeled rabbit anti-human IgG H&L；ab6759；Abcam)作为二抗开展免疫组织化学染色。结果如图2所示，通过免疫组织化学实验发现小鼠的胃腺上皮表达阳性，其他器官诸如肝脏、肺脏、肾脏、胰腺、脾脏表达为阴性。Verification of the expression of CLDN18.2 in the main tissues and organs of normal nude mice specifically includes the following steps: obtaining and fixing the stomach, liver, kidney, lung, spleen and pancreas of normal nude mice using tissue fixative, using recombinant CLDN18.2 monoclonal antibody (EPR19202, ab222512, Abcam) as the primary antibody, and using horseradish peroxidase-conjugated rabbit anti-human IgG H&L (HRP-labeled rabbit anti-human IgG H&L;ab6759; Abcam) as the secondary antibody for immunohistochemical staining. The results are shown in Figure 2. Immunohistochemical experiments found that the gastric glandular epithelium of mice was positively expressed, while other organs such as liver, lung, kidney, pancreas, and spleen were negative.
实施例2：CLDN18.2特异性纳米抗体探针的制备Example 2: Preparation of CLDN18.2-specific nanobody probe
1)中间体H3RESCA-3E10、H3RESCA-3E11或H3RESCA-3A12的制备，具体步骤如下：用4×5mL 0.05M NaHCO3溶液预平衡PD-10柱。将1mg纳米抗体(3E10、3E11或3A12)溶液加入PD-10柱，加入0.05M NaHCO3溶液补充体积至2.5mL。使用0.05M NaHCO3溶液洗脱，收集2.5mL洗脱液。称取12倍纳米抗体摩尔量的(±)-H3RESCA-TFP(CAS number:1919794-40-3；其具有如图15所示的化学结构式)或(±)H3RESCA-Mal(其具有如图16所示的化学结构式；当用该双功能螯合剂时，需在纳米抗体末端表达含有半胱氨酸的肽段(GGGGS)nC，n＝1-10,本实施例中采用n＝3,即肽段为GGGGSGGGGSGGGGSC,该末端含有半胱氨酸的肽段(GGGGS)nC的纳米抗体参考实施例1所述纳米抗体的制备方法得到)，溶于40μL DMSO中。向以上纳米抗体溶液中分别加入(±)-H3RESCA-TFP或(±)H3RESCA-Mal溶液后，充分打匀。在室温下置于摇床上反应2小时。用4×5mL 0.1M CH3COONH4溶液预平衡PD-10柱。将反应液加入PD-10柱，使用0.1M CH3COONH4溶液洗脱，收集3mL洗脱液。使用DD水润洗离心过滤器。并使用Amicon超速离心过滤器进行浓缩，使用NanoDrop测定产物(H3RESCA-3E10、H3RESCA-3E11或H3RESCA-3A12)浓度。1) Preparation of intermediate H3RESCA-3E10, H3RESCA-3E11 or H3RESCA-3A12, specifically, the steps are as follows: pre-equilibrate the PD-10 column with 4×5 mL 0.05 M NaHCO3 solution. Add 1 mg of the nanobody (3E10, 3E11 or 3A12) solution to the PD-10 column, add 0.05 M NaHCO3 solution to make up the volume to 2.5 mL. Elute with 0.05 M NaHCO3 solution, and collect 2.5 mL of the eluate. Weigh 12 times the molar amount of the nanobody (±)-H3RESCA-TFP (CAS number: 1919794-40-3; it has the chemical structure shown in FIG. 15) or (±)H3RESCA-Mal (it has the chemical structure shown in FIG. 16; when using the bifunctional chelator, a peptide segment (GGGGS)n C containing cysteine needs to be expressed at the end of the nanobody, n = 1-10, in this embodiment, n = 3, that is, the peptide segment is GGGGSGGGGSGGGGSC, and the nanobody with a peptide segment (GGGGS)n C containing cysteine at the end is obtained by the preparation method of the nanobody described in Reference Example 1), and dissolve in 40 μL DMSO. After adding (±)-H3RESCA-TFP or (±)H3RESCA-Mal solution to the above nanobody solution, mix well. Place on a shaker at room temperature for 2 hours. Pre-equilibrate the PD-10 column with 4×5mL 0.1M CH3 COONH4 solution. The reaction solution was added to a PD-10 column and eluted with 0.1 M CH3 COONH4 solution, and 3 mL of the eluate was collected. The centrifugal filter was rinsed with DD water, and concentrated using an Amicon ultracentrifugal filter. The concentration of the product (H3RESCA-3E10, H3RESCA-3E11 or H3RESCA-3A12) was determined using NanoDrop.
2)18F标记H3RESCA-3E10、H3RESCA-3E11、H3RESCA-3A12的制备，具体步骤如下：依次使用5mL灭菌注射用水-5mL空气-0.9％5mL生理盐水-5mL空气活化QMA柱子。从加速器中取500μL含有18F离子的富[18O]水通过QMA柱子，弃过滤液，取500μL0.9％生理盐水过QMA柱子，收集过滤液，测活度为24mCi。加入8μL 2mM AlCl3(in 0.1M CH3COONH4)溶液静置5分钟。将160μL(7.6mCi)18+19F–/Al3+溶液，150μL(200)μg，690μL 0.1M CH3COONH4溶液混合，室温反应12分钟。期间，使用4×5mL含有5mg/mL抗坏血酸的0.9％NaCl洗脱液(pH 5.9-6.1)预平衡PD-10柱子。将反应液加入PD-10，使用洗脱液补充至2.5mL，然后每次加入0.5mL进行洗脱，共5管，分别测量产物([18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11、[18F]F-H3RESCA-3A12)的活度。2) Preparation of18 F labeled H3RESCA-3E10, H3RESCA-3E11, and H3RESCA-3A12, the specific steps are as follows: 5 mL sterile water for injection-5 mL air-0.9% 5 mL saline-5 mL air were used to activate the QMA column. 500 μL of [18 O] water containing 18 F ions was taken from the accelerator and passed through the QMA column, the filtrate was discarded, 500 μL of 0.9% saline was taken and passed through the QMA column, and the filtrate was collected. The activity was measured to be 24 mCi. 8 μL of 2 mM AlCl3 (in 0.1 M CH3 COONH4 ) solution was added and allowed to stand for 5 minutes. 160 μL (7.6 mCi)18+19 F–/Al3+ solution, 150 μL (200) μg, and 690 μL 0.1 M CH3 COONH4 solution were mixed and reacted at room temperature for 12 minutes. During this time, the PD-10 column was pre-equilibrated with 4×5 mL of 0.9% NaCl eluent (pH 5.9-6.1) containing 5 mg/mL ascorbic acid. The reaction solution was added to the PD-10 column and the eluent was added to 2.5 mL. Then, 0.5 mL was added each time for elution. A total of 5 tubes were added and the activities of the products ([18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11, [18 F]F-H3RESCA-3A12) were measured respectively.
3)[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11、[18F]F-H3RESCA-3A12的质量控制。具体步骤如下：使用毛细玻璃管吸取少量[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11或[18F]F-H3RESCA-3A12在硅胶板上点样，用生理盐水作为流动相，用放射性薄层色谱仪(Radio-TLC，Eckert&Ziegler Radiopharma Inc)测定探针的放射化学纯度(Radiochemical purity，RCP)。如图3、图4、图5所示，新鲜制备的[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11、[18F]F-H3RESCA-3A12 RCP大于99％。3) Quality control of [18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11, and [18 F]F-H3RESCA-3A12. The specific steps are as follows: Use a capillary glass tube to draw a small amount of [18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11, or [18 F]F-H3RESCA-3A12 and spot it on a silica gel plate, use physiological saline as the mobile phase, and use a radioactive thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc) to determine the radiochemical purity (RCP) of the probe. As shown in Figures 3, 4 and 5, the RCP of freshly prepared [18F ]F-H3RESCA-3E10, [18F ]F-H3RESCA-3E11 and [18F ]F-H3RESCA-3A12 was greater than 99%.
4)[18F]F-H3RESCA-3E10在正常Balb/c鼠中的显像。具体步骤如下：本研究所涉及的小动物PET/CT显像采集均使用IRIS小动物PET/CT扫描仪(Inviscan Imaging Systems)完成。每只小鼠经尾静脉注射3.7-7.4MBq[18F]F-H3RESCA-3E10(每组3只)，在注射后的0.5小时使用异氟烷(浓度为2％)麻醉小鼠，并将进入深度麻醉状态的小鼠以仰卧位姿势置于PET/CT扫描床上，续惯采集PET和CT图像，用IRIS系统自带软件完成图像重建，用OsiriXLite图像处理工作站(Pixmeo SARL)在重建后的PET图像上勾画心脏及主要组织脏器(肝脏、肺脏、肾脏、肌肉)等感兴趣区(Region of interest，ROI)，以％ID/g(percent ofinjected dose per gram)为单位计算重要组织器官的放射性摄取值，并绘制主要组织器官摄取值随时间变化曲线。如图6、图7所示，45分钟时胃有明显摄取，探针([18F]F-H3RESCA-3E10)主要经由泌尿系统排泄。体外实验结果如图8所示，[18F]F-H3RESCA-3E10在肾脏中的摄取最高，为213.5±33.3％ID/g，这是由于示踪剂需在肾脏中的清除所致。同时可以看到，在正常胃组织中，[18F]F-H3RESCA-3E10有明显摄取，为26.8±4.4％ID/g。4) Imaging of [18 F]F-H3RESCA-3E10 in normal Balb/c mice. The specific steps are as follows: The small animal PET/CT imaging acquisition involved in this study was completed using the IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Each mouse was injected with 3.7-7.4MBq [18F ]F-H3RESCA-3E10 via the tail vein (3 mice per group). The mice were anesthetized with isoflurane (concentration of 2%) 0.5 hours after injection, and the mice in deep anesthesia were placed in a supine position on the PET/CT scanning bed. PET and CT images were acquired continuously, and image reconstruction was completed using the IRIS system's own software. The OsiriXLite image processing workstation (Pixmeo SARL) was used to outline the heart and major tissues and organs (liver, lung, kidney, muscle) and other regions of interest (ROI) on the reconstructed PET images. The radioactive uptake values of important tissues and organs were calculated in %ID/g (percent of injected dose per gram), and the uptake values of major tissues and organs over time were plotted. As shown in Figures 6 and 7, there was obvious uptake in the stomach at 45 minutes, and the probe ([18F ]F-H3RESCA-3E10) was mainly excreted through the urinary system. The results of the in vitro experiment are shown in Figure 8. The highest uptake of [18 F]F-H3RESCA-3E10 in the kidney is 213.5±33.3%ID/g, which is due to the clearance of the tracer in the kidney. At the same time, it can be seen that in normal gastric tissue, [18 F]F-H3RESCA-3E10 has a significant uptake of 26.8±4.4%ID/g.
5)[18F]F-H3RESCA-3E11在正常Balb/c鼠中的显像。显像步骤如上述步骤4)中所述。PET/CT显像结果如图9所示；通过勾画ROI得到主要器官的摄取与显像结果相匹配，结果如图10所示。体外实验结果如图11所示，[18F]F-H3RESCA-3E11在肾脏中的摄取最高，为251.3±65.2％ID/g。在正常胃组织中[18F]F-H3RESCA-3E11摄取为84.8±50.3％ID/g。5) Imaging of [18 F]F-H3RESCA-3E11 in normal Balb/c mice. The imaging steps were as described in step 4). The PET/CT imaging results are shown in FIG9 ; the uptake of the main organs was matched with the imaging results by outlining the ROI, and the results are shown in FIG10 . The in vitro experimental results are shown in FIG11 , and the uptake of [18 F]F-H3RESCA-3E11 was the highest in the kidney, which was 251.3±65.2%ID/g. The uptake of [18 F]F-H3RESCA-3E11 in normal gastric tissue was 84.8±50.3%ID/g.
6)[18F]F-H3RESCA-3A12在正常Balb/c鼠中的显像。显像步骤如上述步骤4)中所述。PET/CT显像结果如图12所示；通过勾画ROI得到主要器官的摄取与显像结果相匹配，结果如图13所示。体外实验结果如图14所示，与[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11不同，[18F]F-H3RESCA-3A12在胃组织中的摄取最高，为269.8±162.9％ID/g。在肾脏中的摄取为204.7±107.4％ID/g，较在胃组织中的摄取低。6) Imaging of [18 F]F-H3RESCA-3A12 in normal Balb/c mice. The imaging steps were as described in step 4). The PET/CT imaging results are shown in FIG12 ; the uptake of the main organs obtained by delineating the ROI matched the imaging results, and the results are shown in FIG13 . The in vitro experimental results are shown in FIG14 . Unlike [18 F]F-H3RESCA-3E10 and [18 F]F-H3RESCA-3E11, the uptake of [18 F]F-H3RESCA-3A12 in gastric tissue was the highest, at 269.8±162.9%ID/g. The uptake in the kidney was 204.7±107.4%ID/g, which was lower than that in gastric tissue.
以上结果表明，18F标记抗CLDN18.2纳米抗体探针[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11、[18F]F-H3RESCA-3A12均可与分布于正常胃上皮细胞的CLDN18.2特异性结合，其中，[18F]F-H3RESCA-3A12探针在正常胃组织中的显像效果较[18F]F-H3RESCA-3E10、[18F]F-H3RESCA-3E11更佳。以上数据表明18F标记抗CLDN18.2纳米抗体探针是具有一定潜能的靶向CLDN18.2的在体无创诊疗工具。The above results show that18 F-labeled anti-CLDN18.2 nanoantibody probes [18 F]F-H3RESCA-3E10, [18 F]F-H3RESCA-3E11, and [18 F]F-H3RESCA-3A12 can all specifically bind to CLDN18.2 distributed in normal gastric epithelial cells, among which the imaging effect of [18 F]F-H3RESCA-3A12 probe in normal gastric tissue is better than that of [18 F]F-H3RESCA-3E10 and [18 F]F-H3RESCA-3E11. The above data show that18 F-labeled anti-CLDN18.2 nanoantibody probes are potential in vivo non-invasive diagnostic and therapeutic tools targeting CLDN18.2.
需要说明的是，本发明的说明书及其附图中给出了本发明的较佳的实施例，但是，本发明可以通过许多不同的形式来实现，并不限于本说明书所描述的实施例，这些实施例不作为对本发明内容的额外限制，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。并且，上述各技术特征继续相互组合，形成未在上面列举的各种实施例，均视为本发明说明书记载的范围；进一步地，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the scope of protection of the claims attached to the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311210180.1ACN117327182B (en) | 2023-09-19 | 2023-09-19 | Preparation method and application of CLDN18.2 single domain antibody probe |
| PCT/CN2024/107813WO2025060679A1 (en) | 2023-09-19 | 2024-07-26 | Preparation method for and use of cldn18.2 single-domain antibody probe |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311210180.1ACN117327182B (en) | 2023-09-19 | 2023-09-19 | Preparation method and application of CLDN18.2 single domain antibody probe |
| Publication Number | Publication Date |
|---|---|
| CN117327182A CN117327182A (en) | 2024-01-02 |
| CN117327182Btrue CN117327182B (en) | 2024-06-04 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311210180.1AActiveCN117327182B (en) | 2023-09-19 | 2023-09-19 | Preparation method and application of CLDN18.2 single domain antibody probe |
| Country | Link |
|---|---|
| CN (1) | CN117327182B (en) |
| WO (1) | WO2025060679A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120271707A (en)* | 2023-08-25 | 2025-07-08 | 上海交通大学医学院附属仁济医院 | Claudin18.2 specific molecular imaging probe and preparation method and application thereof |
| CN117327182B (en)* | 2023-09-19 | 2024-06-04 | 上海交通大学医学院附属仁济医院 | Preparation method and application of CLDN18.2 single domain antibody probe |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111978402A (en)* | 2019-05-24 | 2020-11-24 | 三优生物医药(上海)有限公司 | Novel CLDN18.2 binding molecules |
| CN113476619A (en)* | 2021-07-08 | 2021-10-08 | 上海交通大学医学院附属仁济医院 | A kind of18F-labeled nano antibody probe and preparation method and application thereof |
| CN113754780A (en)* | 2020-06-04 | 2021-12-07 | 四川科伦博泰生物医药股份有限公司 | Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof |
| CN113788894A (en)* | 2021-09-03 | 2021-12-14 | 深圳市先康达生命科学有限公司 | Monoclonal antibody of targeted human Claudin18.2 protein and application thereof |
| WO2022161282A1 (en)* | 2021-01-28 | 2022-08-04 | 北京免疫方舟医药科技有限公司 | Anti-cldn18.2 antibody and application thereof |
| CN114904015A (en)* | 2021-02-09 | 2022-08-16 | 江苏迈威康新药研发有限公司 | Antibody drug conjugates comprising an antibody against CLDN18.2 or antigen binding fragment thereof and uses thereof |
| CN115461372A (en)* | 2020-04-27 | 2022-12-09 | 启愈生物技术(上海)有限公司 | A bispecific antibody targeting human claudin and human PDL1 protein and its application |
| CN115715202A (en)* | 2020-05-15 | 2023-02-24 | 四川科伦博泰生物医药股份有限公司 | Antibody drug conjugate, preparation method and application thereof |
| CN116082523A (en)* | 2022-12-30 | 2023-05-09 | 邦恩泰(山东)生物医药科技集团股份有限公司 | Chimeric antigen receptor targeting Claudin18.2 and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4105237A4 (en)* | 2020-02-10 | 2024-03-27 | Shanghai Escugen Biotechnology Co., Ltd. | Cldn18.2 antibody and use thereof |
| CN112457401B (en)* | 2020-10-21 | 2023-02-24 | 上海交通大学医学院附属仁济医院 | A molecular imaging probe for the diagnosis of multiple myeloma |
| WO2022100590A1 (en)* | 2020-11-10 | 2022-05-19 | 齐鲁制药有限公司 | Adcc-enhanced humanized antibody for claudin 18a2 and application thereof |
| CN112480248B (en)* | 2020-11-24 | 2023-05-05 | 三优生物医药(上海)有限公司 | Molecules that specifically bind to CLD18A2 |
| CN117327182B (en)* | 2023-09-19 | 2024-06-04 | 上海交通大学医学院附属仁济医院 | Preparation method and application of CLDN18.2 single domain antibody probe |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111978402A (en)* | 2019-05-24 | 2020-11-24 | 三优生物医药(上海)有限公司 | Novel CLDN18.2 binding molecules |
| CN115461372A (en)* | 2020-04-27 | 2022-12-09 | 启愈生物技术(上海)有限公司 | A bispecific antibody targeting human claudin and human PDL1 protein and its application |
| CN115715202A (en)* | 2020-05-15 | 2023-02-24 | 四川科伦博泰生物医药股份有限公司 | Antibody drug conjugate, preparation method and application thereof |
| CN113754780A (en)* | 2020-06-04 | 2021-12-07 | 四川科伦博泰生物医药股份有限公司 | Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof |
| WO2022161282A1 (en)* | 2021-01-28 | 2022-08-04 | 北京免疫方舟医药科技有限公司 | Anti-cldn18.2 antibody and application thereof |
| CN114904015A (en)* | 2021-02-09 | 2022-08-16 | 江苏迈威康新药研发有限公司 | Antibody drug conjugates comprising an antibody against CLDN18.2 or antigen binding fragment thereof and uses thereof |
| CN113476619A (en)* | 2021-07-08 | 2021-10-08 | 上海交通大学医学院附属仁济医院 | A kind of18F-labeled nano antibody probe and preparation method and application thereof |
| CN113788894A (en)* | 2021-09-03 | 2021-12-14 | 深圳市先康达生命科学有限公司 | Monoclonal antibody of targeted human Claudin18.2 protein and application thereof |
| CN116082523A (en)* | 2022-12-30 | 2023-05-09 | 邦恩泰(山东)生物医药科技集团股份有限公司 | Chimeric antigen receptor targeting Claudin18.2 and application thereof |
| Title |
|---|
| CLDN18.2在消化系统恶性肿瘤中作用的研究进展;王俏丽等;中国肿瘤生物治疗杂志;20220722;第29卷(第7期);681-685* |
| Development and comparison of three 89Zr-labeled anti-CLDN18.2 antibodies to noninvasively evaluate CLDN18.2 expression in gastric cancer: a preclinical study;Guilan Hu等;European Journal of Nuclear Medicine and Molecular Imaging;20220326;第49卷(第12期);2634-2644* |
| immunoglobulin heavy chain variable region, partial [Vicugna pacos];GenBank: UVT36869.1;NCBI;20220903;1-4* |
| 放射免疫治疗在肿瘤诊疗中的最新研究进展;徐东升等;药学进展;20230525;第47卷(第5期);370-378* |
| 放射性核素标记的凋亡显像剂的研究进展;安淑娴等;国际放射医学核医学杂志;20160114;第22卷(第12期);470-478* |
| Publication number | Publication date |
|---|---|
| CN117327182A (en) | 2024-01-02 |
| WO2025060679A1 (en) | 2025-03-27 |
| Publication | Publication Date | Title |
|---|---|---|
| US11466085B2 (en) | Anti-PD-L1 nanobody, coding sequence and use thereof | |
| JP7097636B2 (en) | Anti-Her2 Nanobodies and their Code Orders and Applications | |
| CN117327182B (en) | Preparation method and application of CLDN18.2 single domain antibody probe | |
| JP3492373B2 (en) | Monoclonal antibody | |
| RU2204606C2 (en) | Antibody vff-18 and its analogues | |
| CN109485726B (en) | Application of radiolabeled anti-nanobodies in cancer prognosis and diagnosis | |
| CN117024591A (en) | Monoclonal antibody against human B7-H3 and application thereof | |
| Sharifi et al. | Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors | |
| EP4389769A1 (en) | Anti-ptk7 single-domain antibody and application thereof | |
| CN117281928A (en) | 18 F-labeled nano antibody probe and preparation method and application thereof | |
| CN116444666A (en) | Preparation method and application of CDH17 specific diagnosis and treatment integrated molecular imaging probe | |
| WO2020015687A1 (en) | Anti-her3 humanized monoclonal antibody | |
| AU2020366846A1 (en) | Humanized antibody and method for using the same | |
| CN120383679A (en) | Anti-FAP single-domain antibodies, Fc fusion proteins, immunoconjugates and their applications | |
| CN116514978A (en) | Preparation method and application of PD-L1 specific nano antibody molecule image probe | |
| US20240368302A1 (en) | Anti-trop2 single-domain antibody and use thereof | |
| WO2025119257A1 (en) | Protein targeting and binding to mesothelin, and encoding nucleic acid and use thereof | |
| CN117304318B (en) | Claudin18.2 specific molecular imaging probe and its preparation method and application | |
| CN117327183B (en) | Preparation method and application of nuclide-labeled Trop2 specific single-domain antibody probe | |
| JP7378088B2 (en) | Monospecific and bispecific antibodies that bind hERG1 and hERG1/integrin β1 | |
| CN114989270B (en) | Polypeptide with binding affinity to humanized CEA and application thereof | |
| CN117384292A (en) | Preparation method and application of 68Ga labeled Trop2 immunoPET imaging probe | |
| CN116655793A (en) | Preparation method and application of BCMA (bcmA-specific diagnosis and treatment) integrated molecular imaging probe | |
| WO2024216505A1 (en) | Anti-notch2 nanoantibody and use thereof | |
| WO2024093147A1 (en) | Antibody specifically binding to v5 exon of cd44, and use thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |