WO2022183689A1 - 一种her2亲合体放射性核素标记物组合物及其应用 - Google Patents

一种her2亲合体放射性核素标记物组合物及其应用 Download PDF

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WO2022183689A1
WO2022183689A1 PCT/CN2021/112782 CN2021112782W WO2022183689A1 WO 2022183689 A1 WO2022183689 A1 WO 2022183689A1 CN 2021112782 W CN2021112782 W CN 2021112782W WO 2022183689 A1 WO2022183689 A1 WO 2022183689A1
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mmol
concentration
her2
affinity
composition
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蔡炯
刘毅
高大林
宋旭
肖凯
梁晨
丁国中
刘明霞
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江苏元本生物科技有限公司
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Definitions

  • the invention relates to the fields of radiopharmaceuticals and nuclear medicine, in particular to a HER2 affinity radionuclide 99m Tc marker composition and application thereof.
  • HER2 Human Epidermal Growth Factor Receptor 2
  • HER2+BC HER2-positive breast cancer
  • HER2+BC is the most aggressive subtype of breast cancer, and its prognosis is often poor and prone to recurrence, and the probability of HER2+BC resistance to chemotherapy and endocrine therapy is also greatly increased. Therefore, HER2 is an important molecular imaging marker for HER2+BC and an ideal target for targeted therapy.
  • HER2-targeted therapy There are two important challenges for HER2-targeted therapy: First, the screening of patients with high HER2 expression: only patients with high HER2 expression may benefit from targeted therapy, while patients with no or low expression of HER2 may be over-treated. It may increase the side effects caused by targeted therapy and miss more suitable treatment methods; the second is the monitoring of therapeutic effects: for targeted therapy, a dynamic, non-invasive and overall evaluation method needs to be found to achieve the best therapeutic effect.
  • the clinical detection of HER2 expression in tumors generally adopts biochemical and histopathological examination methods of biopsy specimens, including the use of real-time quantitative PCR to determine the content of HER2 gene, the use of Western blotting electrophoresis to determine the expression of HER2 protein, or the use of immunohistochemical staining to detect pathology.
  • pathological biopsy is the gold indicator for evaluating HER2 expression, this invasive test cannot be performed multiple times as a routine test for tumor efficacy evaluation, and the positive rate depends on the operator's clinical experience and level.
  • the local conditions of biopsy cannot Reflecting the overall tumor, it lacks timeliness and comprehensiveness.
  • CT and MRI often analyze tumor characteristics and treatment response based on anatomical and morphological features, but lack the expression analysis of the underlying biological and physiological information of tumors, especially the lack of sensitivity to changes in biological and physiological information at the initial stage of treatment.
  • CT and MRI also face difficulties, because molecularly targeted drugs usually inhibit tumor cells, and the morphological characteristics of tumors do not change significantly.
  • HER2 status For the degree of survival benefit, toxicity effect, and drug-effect ratio of molecular targeted therapy, there is a need for a more accurate and rapid method for evaluating HER2 status. Therefore, finding an in vivo, non-invasive, holistic and dynamic evaluation method for HER2 expression detection is the focus and difficulty of HER2+BC diagnosis and treatment.
  • HER2 imaging has the following meanings: first, to screen the most suitable treatment plan through imaging; second, to make early judgment and prediction of treatment effect through imaging; third, to combine diagnosis and treatment drugs through carriers.
  • the integration of diagnosis and treatment is a new trend in the development of nuclear medicine research in recent years.
  • nuclide imaging treatment is guided according to the individual conditions of patients, reducing adverse reactions and improving treatment effects.
  • Radionuclide imaging has become an important means of integration of diagnosis and treatment by detecting changes in organ function in the body. With the help of molecular probes integrated in diagnosis and treatment, it can detect the changes in physiological functions of diseases and simultaneously implement therapeutic effects.
  • Affibody is a new type of scaffold protein, originally derived from Staphylococcus aureus protein A, composed of 58 amino acid residues, with a molecular weight between antibodies and polypeptides (about 6.5kDa), which can be specific to targeting proteins. Binding with high affinity and targeting specificity. Affibodies and antigen-binding domains are comparable in size to antibodies. Affibodies have no activating effect and can be injected without too many restrictions. Due to the advantages of small relative molecular weight, high binding capacity, strong specificity, rapid target concentration, and rapid blood clearance, radiolabeled HER2-targeted affibodies are used in the in vivo imaging of HER2-positive tumors. Has good application value.
  • Li Xin et al. successfully prepared 99 Tc m -human epidermal growth factor receptor 2 (HER2) affinity body (ABH2), and showed that 99 Tc m -ABH2 can specifically image HER2-positive breast cancer through nuclear tumor mouse experiments .
  • HER2 human epidermal growth factor receptor 2
  • ABS2 affinity body
  • the present invention provides a HER2 affinity radionuclide 99m Tc marker composition.
  • the HER2 affinity 99m Tc marker composition of the present invention includes HEPES, sodium glucoheptonate, vitamin C, cysteine, stannous chloride and HER2 affinity, which is not easy to produce dimers and has good stability , which can reduce the storage cost and transportation cost, and has a better application prospect.
  • the present invention provides a HER2 affinity 99m Tc marker composition comprising HEPES, sodium glucoheptonate, vitamin C, cysteine, stannous chloride and HER2 affinity.
  • the HEPES concentration is 2-30 mmol/L
  • the sodium glucoheptonate concentration is 5-40 mmol/L
  • the vitamin C concentration is 2-30 mmol/L
  • the cysteine concentration is 5 -40 mmol/L
  • the concentration of stannous chloride is 0.1-5 mmol/L
  • the concentration of the HER2 affinity is 0.01-0.6 mmol/L
  • the pH of the composition is 5.0-7.5.
  • the HEPES concentration is 5-20mmol/L
  • the sodium glucoheptonate concentration is 10-30mmol/L
  • the vitamin C concentration is 5-20mmol/L
  • the cysteine concentration is 10-30 mmol/L
  • the concentration of the stannous chloride is 0.2-2 mmol/L
  • the concentration of the HER2 affinity is 0.03-0.3 mmol/L
  • the pH of the composition is 6.0-7.0.
  • the HEPES concentration is 10mmol/L
  • the sodium glucoheptonate concentration is 20mmol/L
  • the vitamin C concentration is 10mmol/L
  • the cysteine concentration is 20mmol/L
  • the The concentration of stannous chloride was 0.4 mmol/L
  • the concentration of the HER2 affinity was 0.05 mmol/L
  • the pH of the composition was 6.6.
  • the carboxyl terminus of the HER2 affinity is designed with a GGGC sequence to facilitate isotopic labeling
  • the amino terminus of the HEHEHE sequence is designed to facilitate affinity column purification.
  • the present invention provides the application of the above-mentioned HER2 affinity 99m Tc marker composition in the preparation of an imaging agent product.
  • the present invention provides an imaging agent product comprising the above-mentioned HER2 affinity 99m Tc marker composition.
  • the imaging agent is a PET or SPECT imaging agent
  • the product is an independent reagent or a kit.
  • the present invention provides a method for preparing a HER2 affinity 99m Tc marker, the preparation method comprising the following steps: adding 500-20000 ⁇ Ci of 99m Tc to the above HER2 affinity 99m Tc marker composition; The reaction was carried out at room temperature for 5-60 min under airtight conditions.
  • the added amount of 99m Tc is 2000-10000 ⁇ Ci, preferably 5000 ⁇ Ci.
  • reaction time is 10-15 min.
  • the HER2 affinity 99m Tc marker composition of the present invention has a high labeling rate, is not easy to produce dimers, has good stability, can be stored at room temperature for more than 60 days, reduces storage costs and transportation costs, and has better clinical applications prospect.
  • Figure 1 is a graph showing the identification results of SDS-PAGE dimers after storage at low temperature and room temperature for 60 days.
  • Figure 2 is a graph showing the results of storage time of HER2 affinity in different formulations.
  • FIG. 3 is a graph showing the results of the stability detection of E. coli supernatant HER2 affinities and E. coli cells HER2 affinities.
  • Figure 4 is the ITLC diagram of Example 2-10 99m Tc-ABH2.
  • the GGGC sequence was designed at the carboxyl terminus of the affinity body to facilitate isotopic labeling, and the amino terminus was designed with the HEHEHE sequence for affinity column purification.
  • the amino acid sequence is shown in SEQ ID NO: 2.
  • DNAstar software was used to design the HER2 affimer gene (SEQ ID NO: 1) with the expression preference of E. coli.
  • the HER2 affimer gene was synthesized by oligonucleotide synthesis and overlapping PCR method, and the HindIII enzyme digestion was used for identification. Synthetic gene length, gene sequencing determines the sequence of the synthetic gene.
  • the HER2 affinity body gene was double digested with NcoI and EcoRI, it was inserted into the pET22b(+) vector digested in the same way, and transformed into DH5 ⁇ competent Escherichia coli.
  • the plasmid was transformed into BL21(DE3) competent E. coli. Expression was induced with IPTG, and bacterial clones expressing the nascent protein were identified by SDS-PAGE.
  • Escherichia coli expressing HER2 affibodies were centrifuged, and the cells were washed with washing buffer (50 mM Na 2 HPO 4 , 300 mM NaCl, pH 7.0), and resuspended in purification buffer (20 mM Tris ⁇ HCl, 500 mM NaCl, pH 7.0). 9) Ultrasonic fragmentation. Cell debris was removed by centrifugation, and the supernatant was treated at 60°C for 10 min. After centrifugation, the supernatant was filtered through a membrane filter and applied to a nickel column pre-equilibrated with disruption buffer (20 mM Tris ⁇ HCl, 500 mM NaCl, 1 mM EDTA, pH 7.9).
  • the purification was eluted with purification buffer containing 60 mM imidazole. Then, it was purified by ion exchange through the Q column, eluted and purified under the condition of 0.5M NaCl, the purified product was subjected to ultrafiltration to remove the buffer, and replaced with an aqueous solution to obtain the Escherichia coli HER2 affinity body.
  • Example 2 The difference from Example 1 is that after centrifugation of the Escherichia coli expressing the HER2 affinity body in this example, the supernatant was directly collected and dialyzed with a purification buffer (20 mM Tris HCl, 500 mM NaCl, pH 7.9) overnight, and then centrifuged. The supernatant was filtered through a membrane filter and applied to a nickel column pre-equilibrated with disruption buffer. After washing with purification buffer containing 5 mM imidazole, the purification was eluted with purification buffer containing 60 mM imidazole.
  • a purification buffer (20 mM Tris HCl, 500 mM NaCl, pH 7.9
  • the supernatant was filtered through a membrane filter and applied to a nickel column pre-equilibrated with disruption buffer. After washing with purification buffer containing 5 mM imidazole, the purification was eluted with purification buffer containing 60 mM imidazole.
  • the HER2 affinity was labeled with 99m Tc, and the specific steps were as follows: vitamin C was added to the affinity prepared in Example 1 or 2, and the concentration of the affinity was adjusted to 1 mg/mL. Add 100 ⁇ L of the concentration-adjusted affinity into the labeling bottle, add deoxygenated labeling buffer (10 mmol/L HEPES, 20 mmol/L sodium glucoheptonate, 20 mmol/L cysteine, 0.4 mmol/L chlorinated chloride) tin) 200 ⁇ L, add 5000 ⁇ Ci 99m Tc, and react at room temperature for 10 min to complete the reaction.
  • deoxygenated labeling buffer (10 mmol/L HEPES, 20 mmol/L sodium glucoheptonate, 20 mmol/L cysteine, 0.4 mmol/L chlorinated chloride
  • Example 1 The HER2 affinity purified in Example 1 was added to different formulations, stored at room temperature and 4°C, respectively, and the production of dimers was identified by SDS-PAGE every 2-3 days.
  • the composition formulation is shown in Table 1 below.
  • Figure 1 shows the results of SDS-PAGE dimer identification after 60 days of storage at low temperature and room temperature
  • Figure 2 shows the storage time limit of HER2 affinity in different formulations.
  • the formulas that are better preserved at low temperature include adding vitamin C, SnCl 2 , and argon gas, etc.; while adding cysteine, vitamin C and SnCl 2 at room temperature, the HER2 affinity can be stored for more than 60 days. .
  • Example 2 Store the purified HER2 affinity in Example 2 at room temperature, 4°C low temperature and -20°C freezing conditions, respectively, and the results are shown in Figure 3 . It can be seen from Figure 3 that the stability of the E. coli supernatant HER2 affibody is lower than that of the E. coli bacterial HER2 affibody.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 1mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 35mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity 0.05mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 4mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 45mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.005mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.65mmol/L; pH 6.6.
  • Isotope 5000 ⁇ Ci (463GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 300 ⁇ Ci (28GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Composition formula HEPES 10mmol/L; sodium glucoheptonate 20mmol/L; vitamin C 10mmol/L; cysteine 20mmol/L; stannous chloride 0.4mmol/L; HER2 affinity body 0.05mmol/L; pH 6.6.
  • Isotope 25000 ⁇ Ci (2313GBq/L).
  • HER2 affinity was prepared according to Example 1
  • 99m Tc-ABH2 was prepared according to Example 3.
  • Detection of labeling rate 0.5 ⁇ L of the reaction mixture was spotted on the origin of ITLC-SG chromatography paper, and 0.1M citric acid was used as the mobile phase for upward chromatography, and the radioactive count was measured by a radiation scanner to calculate the labeling rate.
  • Radiochemical purity detection Purify 99m Tc-ABH2 with a Waters Sep-pek solid phase extraction column C18 reversed-phase column, take 0.5 ⁇ L of the reaction mixture and spot it on the origin of ITLC-SG chromatography paper, and use 0.1M citric acid as the The mobile phase was chromatographed, and the radioactive count was measured by a radiation scanner, and the radiochemical purity was calculated.
  • Ovarian cancer cell line SKOV-3 was cultured in DMEM high glucose medium plus 10% FBS, 100U/mL penicillin and 100 ⁇ g/mL streptomycin. SKOV-3 cells were seeded in a 24-well plate 1 d before the activity assay, with 1 mL per well (5 ⁇ 10 5 /mL). On the 2nd day, use the currently labeled 99m Tc-ABH2, and add different concentrations (26, 56, 112, 225, 450, 900, 1800, 36000 nmol/L, 3 parallel wells for each concentration) according to 1:2 fold dilution. 99m Tc-ABH2 (specific activity 12.9 GBq/ ⁇ mol).
  • ABH2 with a concentration of 50 times the amount of 99m Tc-ABH2 in the non-blocking group was added, and then the same 99m Tc-ABH2 as in the non-blocking group was added.
  • the 24-well plate was placed at 37°C for 1 h and then cooled with ice. Cells were washed with PBS, digested with 0.1N NaOH, and the cell-bound radioactivity was measured by multichannel gamma spectrometer: the specifically bound radioactivity count was obtained by subtracting the radioactivity count bound by the blocking group from the nonblocking group. Kd values were determined by GraphPad Prism5 software.
  • Impurities IPTG, ampicillin, DTT, TCEP and EDTA were detected by HPLC.

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Abstract

本发明提供了一种HER2亲合体 99mTc标记物组合物及其应用，属于放射性药物及核医学领域。本发明所述的HER2亲合体 99mTc标记物组合物包括HEPES、葡庚糖酸钠、维生素C、半胱氨酸、氯化亚锡及HER2亲合体。

Description

一种HER2亲合体放射性核素标记物组合物及其应用 技术领域
本发明涉及放射性药物及核医学领域，具体涉及一种HER2亲合体放射性核素 99mTc标记物组合物及其应用。
背景技术
人表皮生长因子受体2(Human Epidermal Growth Factor Receptor，HER2)是一种跨膜蛋白，在细胞生长、生存及分化中具有重要作用。大概1/3乳腺癌患者HER2表达显著增高，HER2高表达的细胞增殖异常，最终导致肿瘤的发生。HER2表达阳性乳腺癌(HER2+BC)是乳腺癌中最具侵袭性亚型，其预后往往较差且容易复发，且HER2+BC对化疗及内分泌治疗的抵抗概率也大大增加。因此，HER2是HER2+BC重要的分子显像标识物及理想的靶向治疗目标。对于HER2靶向治疗具有两个重要难题：一是HER2高表达患者的筛选：只有高表达HER2患者才有可能从靶向治疗中受益，而不表达或少表达HER2患者则有可能过度治疗，有可能增加靶向治疗带来的副反应及错过更适合的治疗方式；二是治疗效果的监测：针对靶向治疗需要寻找一种动态、无创、整体的评估方法，以达到最佳的治疗效果。
临床上对肿瘤HER2表达检测一般采用取活检标本进行生化和组织病理学检查的方法，包括使用实时定量PCR测定HER2基因的含量，使用蛋白印迹电泳测定HER2蛋白表达量，或使用免疫组织染色检测病理组织切片的HER2蛋白表达。虽然病理活检是评价HER2表达的金指标，但是这种有创性的检查不能作为肿瘤疗效评价的常规检查而多次进行，且阳性率依赖操作者的临床经验和水平，其次活检的局部情况不能反映肿瘤的整体，缺乏时效性和全面性。CT和MRI往往基于解剖形态学特征分析肿瘤特性及治疗反应，缺乏对肿瘤内在基础生物及生理信息表达分析，尤其对治疗初始时期生物及生理信息的改变缺乏敏感性。对于分子靶向药物疗效监测，CT与MRI同样面临困境，因为分子靶向药物通常对肿瘤细胞起到抑制作用，肿瘤形态特征改变不明显。对于分子靶向治疗生存收益程度与毒性效应、药效比迫使临床需要一种更为准确及迅速的评估HER2状态的方法。因此寻找一种对HER2的表达检测可活体、无创、整体、动态的评价方法是HER2+BC诊治的重点及难点。
HER2显像具有以下意义：一是通过显像筛选最适合的治疗方案；二是通过显像对治疗 效果进行早期判定及预测；三是通过载体将诊断和治疗药物相结合。诊疗一体化是近年来核医学研究发展的新趋势，在核素显像的基础上，根据患者的个体情况指导实施治疗，减少不良反应，提高治疗效果。核素显像通过检测体内器官功能的改变因而成为诊疗一体化的重要手段，借助诊疗一体化的分子探针在探测疾病生理功能改变的同时实施治疗作用。由于HER2+BC高表达HER2受体的特性及现阶段分子靶向治疗疗效预测及耐药性抵抗仍然是临床面临的重点难题，因此，基于HER2受体开发诊疗一体化分子探针是实现乳腺癌精准诊治的重要策略。
亲和体是一类新型支架蛋白，最初来源于金黄色葡萄球菌蛋白A，由58个氨基酸残基组成，分子量介于抗体和多肽之间(约6.5kDa)，它可以与靶向蛋白特异性的结合，具有很高的亲和力及靶向特异性。亲和体和抗原结合区域大小和抗体相当。亲和体没有激活作用，可以在注射剂量上不受太多限制。由于亲和体具有相对分子量小、结合力高、特异性强、靶向浓聚迅速、血液清除快等优点，因此放射性标记的HER2靶向的亲和体在HER2表达阳性的肿瘤体内显像中具有很好的应用价值。
李欣等人成功制备了 99Tc m-人表皮生长因子受体2(HER2)亲和体(ABH2)，通过核瘤鼠实验表明 99Tc m-ABH2能够特异地对HER2阳性乳腺癌进行显像。在保证肿瘤靶向性的前提下，尽可能减少对非肿瘤组织的辐射损坏以及通过减少肝脏的非特异性摄取以提高对病灶的探测效能，加速基础研究向临床应用的转化，推进个体化诊疗，实现HER2+BC早发现早治疗的重要策略(李欣,蔡炯,朱朝晖,李方.HER2放射性配体99Tcm-ABH2的制备及荷乳腺癌裸鼠显像.中华核医学与分子影像杂志.2015,35(3):222-226)。Ahlgrena等进行了小分子HER2靶向单光子显像亲和体放射性药物的临床前药物组方研究，其药盒组分为葡庚糖酸、EDTA和氯化亚锡，但该配方存在标记率低的问题，标记率不足10％。相比之下，葡庚糖酸、HEPES和氯化亚锡组方的标记率为90％左右(Tait JF,Brown DS,Gibson DF,Blankenberg FG,Strauss HW.Development and characterization of annexin V mutants with endogenous chelation sites for(99m)Tc.Bioconjug Chem.2000,11(6):918-925)。即便如此，药盒一般保存在-20℃，温度要求低，有效期在1个月左右，在-20℃条件保存3个月的药盒，标记率只有80％左右，存在有效期短的问题。
因此，亟需开发一种稳定性更高的HER2亲合体 99mTc标记物组合物，用于HER2的显像。
发明内容
针对上述不足，本发明提供了一种HER2亲合体放射性核素 99mTc标记物组合物。本发 明所述的HER2亲合体 99mTc标记物组合物包括HEPES、葡庚糖酸钠、维生素C、半胱氨酸、氯化亚锡及HER2亲合体，其不易产生二聚体，稳定性好，可降低保存成本和运输成本，具有更好的应用前景。
为了实现上述发明目的，本发明的技术方案如下：
一方面，本发明提供了一种HER2亲合体 99mTc标记物组合物，所述的组合物包括HEPES、葡庚糖酸钠、维生素C、半胱氨酸、氯化亚锡及HER2亲合体。
具体地，所述HEPES浓度为2-30mmol/L，所述葡庚糖酸钠浓度为5-40mmol/L，所述维生素C浓度为2-30mmol/L，所述半胱氨酸浓度为5-40mmol/L，所述氯化亚锡浓度为0.1-5mmol/L，所述HER2亲合体浓度为0.01-0.6mmol/L，所述组合物pH为5.0-7.5。
进一步具体地，所述HEPES浓度为5-20mmol/L，所述葡庚糖酸钠浓度为10-30mmol/L，所述维生素C浓度为5-20mmol/L，所述半胱氨酸浓度为10-30mmol/L，所述氯化亚锡浓度为0.2-2mmol/L，所述HER2亲合体浓度为0.03-0.3mmol/L，所述组合物pH为6.0-7.0。
进一步具体地，所述HEPES浓度为10mmol/L，所述葡庚糖酸钠浓度为20mmol/L，所述维生素C浓度为10mmol/L，所述半胱氨酸浓度为20mmol/L，所述氯化亚锡浓度为0.4mmol/L，所述HER2亲合体浓度为0.05mmol/L，所述组合物pH为6.6。
具体地，所述的HER2亲合体羧基端设计GGGC序列便于同位素标记，氨基端设计HEHEHE序列便于亲和柱纯化。
另一方面，本发明提供了上述HER2亲合体 99mTc标记物组合物在制备显像剂产品中的应用。
又一方面，本发明提供了一种显像剂产品，所述的显像剂产品包括上述HER2亲合体 99mTc标记物组合物。
具体地，所述的显像剂为PET或SPECT显像剂，所述的产品为独立试剂或试剂盒。
又一方面，本发明提供了一种HER2亲合体 99mTc标记物制备方法，所述的制备方法包括以下步骤：在上述HER2亲合体 99mTc标记物组合物中加入500-20000μCi的 99mTc，在密闭条件下室温反应5-60min。
具体地，所述的 99mTc的加入量为2000-10000μCi，优选为5000μCi。
具体地，反应时间为10-15min。
与现有技术相比，本发明的积极和有益效果在于：
本发明所述的HER2亲合体 99mTc标记物组合物标记率高，且不易产生二聚体，稳定性好，室温下可保存60天以上，降低保存成本和运输成本，具有更好的临床应用前景。
附图说明
图1为低温及室温保存60天后SDS-PAGE二聚体鉴定结果图。
图2为HER2亲合体在不同配方中的保存时限结果图。
图3为大肠杆菌上清液HER2亲和体和大肠杆菌菌体HER2亲和体稳定性检测结果图。
图4为实施例2-10  99mTc-ABH2 ITLC图。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
除非另外定义，否则本文中所用的全部技术与科学用语均具有本领域技术人员通常理解的含义。
实施例1 HER2亲合体(ABH2)制备
亲和体羧基端设计GGGC序列便于同位素标记，氨基端设计HEHEHE序列便于亲和柱纯化，其氨基酸序列如SEQ ID NO:2所示。按照三联密码，使用DNAstar软件，设计出大肠杆菌表达偏好的HER2亲和体基因(SEQ ID NO:1)，通过寡核苷酸合成，重叠PCR的方法合成HER2亲和体基因，HindIII酶切鉴定合成基因长度，基因测序确定合成基因的序列。
将HER2亲和体基因用NcoI和EcoRI双酶切后，插入经过同样方式酶切的pET22b(+)载体，转化入DH5α感受态大肠杆菌中，经抗生素筛选后送菌液进行基因测序鉴定，提取质粒转化BL21(DE3)感受态大肠杆菌。用IPTG诱导表达，经过SDS-PAGE鉴定表达新生蛋白的细菌克隆。
表达HER2亲和体的大肠杆菌经离心，菌体用洗涤缓冲液(50mM Na 2HPO 4，300mM NaCl，pH7.0)洗涤，重悬于纯化缓冲液(20mM Tris·HCl，500mM NaCl，pH7.9)中超声破碎。通过离心去除细胞碎片，上清液60℃处理10min。然后离心，上清液经过滤膜过滤后上预先用破碎缓冲液(20mM Tris·HCl，500mM NaCl，1mM EDTA，pH7.9)平衡的镍柱。经含5mM咪唑的纯化缓冲液洗涤后，用含60mM咪唑的纯化缓冲液洗脱纯化。再通过Q柱进行离子交换纯化，在0.5M NaCl条件下洗脱纯化，纯化产物经过超滤去除缓冲液，更换为水溶液得到大肠杆菌菌体HER2亲和体。
实施例2
与实施例1的区别在于，本实施例表达HER2亲和体的大肠杆菌经离心后，直接取上清 经纯化缓冲液(20mM Tris·HCl，500mM NaCl，pH7.9)透析过夜后离心，上清液经过滤膜过滤后上预先用破碎缓冲液平衡的镍柱。经含5mM咪唑的纯化缓冲液洗涤后，用含60mM咪唑的纯化缓冲液洗脱纯化。再通过Q柱进行离子交换纯化，在0.5M NaCl条件下洗脱纯化，纯化产物经过超滤去除缓冲液，更换为水溶液，得到大肠杆菌上清液HER2亲和体。
实施例3
HER2亲合体 99mTc标记物
采用 99mTc标记HER2亲合体，具体步骤为：将实施例1或2制备的亲合体中加入维生素C，亲合体浓度调整到1mg/mL。取浓度调整后的亲合体100μL加入到标记瓶中，加入脱氧的标记缓冲液(10mmol/L HEPES，20mmol/L葡庚糖酸钠，20mmol/L半胱氨酸，0.4mmol/L氯化亚锡)200μL，加入5000μCi  99mTc，室温反应10min完成反应。
经ITLC检测可知， 99mTc-ABH2和 99mTc有显著区别， 99mTc-ABH2的Rf＝0， 99mTc的Rf＝1。
实验例1 稳定性检测
1.将实施例1纯化后的HER2亲合体加入不同的组合物配方中，分别在室温及4℃低温下进行保存，每2-3天通过SDS-PAGE鉴定二聚体的产生。组合物配方如下表1所示。
表1 组合物配方
Figure PCTCN2021112782-appb-000001
Figure PCTCN2021112782-appb-000002
图1为低温及室温保存60天后SDS-PAGE二聚体鉴定结果图，图2为HER2亲合体在不同配方中的保存时限。
由图1可知，HER2亲合体在不加入维生素C和半胱氨酸条件下，稳定性差，易产生二聚体；加半胱氨酸的稳定性相比于加维生素C的稳定性较差；而同时加入维生素C和半胱氨酸，稳定性优于单独加维生素C或半胱氨酸。
由图2可知，在低温下保存较好的配方包括添加维生素C、SnCl 2、通氩气等；而室温下同时添加半胱氨酸、维生素C和SnCl 2，HER2亲合体可以保存60天以上。
2.将实施例2纯化后的HER2亲合体分别在室温、4℃低温和-20℃冷冻条件下保存，结果如图3所示。由图3可知，大肠杆菌上清液HER2亲和体的稳定性低于大肠杆菌菌体HER2亲和体稳定性。
实验例2
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例3
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 1mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例4
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 35mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例5
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸4mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例6
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸45mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例7
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.005mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例8
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.65mmol/L；pH 6.6。
同位素：5000μCi(463GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例9
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：300μCi(28GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例10
组合物配方：HEPES 10mmol/L；葡庚糖酸钠20mmol/L；维生素C 10mmol/L；半胱氨酸20mmol/L；氯化亚锡0.4mmol/L；HER2亲合体0.05mmol/L；pH 6.6。
同位素：25000μCi(2313GBq/L)。
其中，HER2亲合体按实施例1制备得到，按实施例3制备 99mTc-ABH2。
实验例11
1.标记率检测：取0.5μL反应混合物点样于ITLC-SG层析纸原点，以0.1M柠檬酸为流动相上行层析，放射扫描仪测量放射性计数，计算标记率。
2.放化纯检测：以沃特世Sep-pek固相萃取柱C18反相柱纯化 99mTc-ABH2，取0.5μL反应混合物点样于ITLC-SG层析纸原点，以0.1M柠檬酸为流动相上行层析，放射扫描仪测量放射性计数，计算放化纯。
3.体外稳定性检测：将上述实验例2-10制备的 99mTc-ABH2与0.1mol/L PBS(pH7.4)或健康人血清按照1:10体积比混合，37℃保温，于6h测定 99mTc-ABH2的放化纯。
4.生物结合活性检测：卵巢癌细胞株SKOV-3用DMEM高糖培养基加体积分数10％FBS、100U/mL青霉素、100μg/mL链霉素培养。活性测定前1d将SKOV-3细胞接种于24孔板，每孔1mL(5×10 5/mL)。第2天现用现标记 99mTc-ABH2，按照1:2倍比稀释加入不同浓度(26、56、112、225、450、900、1800、36000nmol/L，每个浓度3个平行孔)的 99mTc-ABH2(比活度为12.9GBq/μmol)。阻断组第2天先加入50倍非阻断组 99mTc-ABH2物质的量浓度的ABH2，然后加入与非阻断组同样的 99mTc-ABH2。24孔板37℃下放置1h后用冰冷的PBS洗涤，0.1N NaOH消化细胞后用多道γ谱仪测量细胞结合的放射性：用非阻断组减去阻断组结合的放射性计数得到特异性结合的放射性计数。通过GraphPad Prism5软件测定Kd值。
标记率、放化率、体外稳定性、生物结合活性检测如下表2所示。
表2
Figure PCTCN2021112782-appb-000003
Figure PCTCN2021112782-appb-000004
杂质IPTG、氨苄青霉素、DTT、TCEP、EDTA采用HPLC检测。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种HER2亲合体 99mTc标记物组合物，其特征在于，所述的组合物包括HEPES、葡庚糖酸钠、维生素C、半胱氨酸、氯化亚锡及HER2亲合体。
  2. 根据权利要求1所述的组合物，其特征在于，所述HEPES浓度为2-30mmol/L，所述葡庚糖酸钠浓度为5-40mmol/L，所述维生素C浓度为2-30mmol/L，所述半胱氨酸浓度为5-40mmol/L，所述氯化亚锡浓度为0.1-5mmol/L，所述HER2亲合体浓度为0.01-0.6mmol/L，所述组合物pH为5.0-7.5。
  3. 根据权利要求2所述的组合物，其特征在于，所述HEPES浓度为5-20mmol/L，所述葡庚糖酸钠浓度为10-30mmol/L，所述维生素C浓度为5-20mmol/L，所述半胱氨酸浓度为10-30mmol/L，所述氯化亚锡浓度为0.2-2mmol/L，所述HER2亲合体浓度为0.03-0.3mmol/L，所述组合物pH为6.0-7.0。
  4. 根据权利要求3所述的组合物，其特征在于，所述HEPES浓度为10mmol/L，所述葡庚糖酸钠浓度为20mmol/L，所述维生素C浓度为10mmol/L，所述半胱氨酸浓度为20mmol/L，所述氯化亚锡浓度为0.4mmol/L，所述HER2亲合体浓度为0.05mmol/L，所述组合物pH为6.6。
  5. 根据权利要求4所述的组合物，其特征在于，所述的HER2亲合体羧基端包括GGGC序列，氨基端包括HEHEHE序列。
  6. 权利要求1-5任一项所述的HER2亲合体 99mTc标记物组合物在制备显像剂产品中的应用。
  7. 一种显像剂产品，其特征在于，所述的显像剂产品包括权利要求1-5任一项所述的HER2亲合体 99mTc标记物组合物。
  8. 根据权利要求7所述的显像剂产品，其特征在于，所述的显像剂为PET或SPECT显像剂，所述的产品为独立试剂或试剂盒。
  9. 一种HER2亲合体 99mTc标记物制备方法，其特征在于，所述制备方法包括以下步骤：在上述HER2亲合体 99mTc标记物组合物中加入500-20000μCi的 99mTc，在密闭条件下室温反应5-60min。
  10. 根据权利要求9所述的制备方法，其特征在于，所述的 99mTc的加入量为2000-10000μCi，加入量为5000μCi；反应时间为10-15min。
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