CN113698483A - 一种靶向egfr的spect显像剂及其制备方法和应用 - Google Patents
一种靶向egfr的spect显像剂及其制备方法和应用 Download PDFInfo
- Publication number
- CN113698483A CN113698483A CN202111020878.8A CN202111020878A CN113698483A CN 113698483 A CN113698483 A CN 113698483A CN 202111020878 A CN202111020878 A CN 202111020878A CN 113698483 A CN113698483 A CN 113698483A
- Authority
- CN
- China
- Prior art keywords
- cet
- mag
- egfr
- targeting
- imaging agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002603 single-photon emission computed tomography Methods 0.000 title claims abstract description 24
- 239000012216 imaging agent Substances 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 229960005395 cetuximab Drugs 0.000 claims abstract description 25
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 18
- 230000008685 targeting Effects 0.000 claims abstract description 7
- 238000002372 labelling Methods 0.000 claims abstract description 5
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims abstract 4
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims abstract 4
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims abstract 3
- 239000012634 fragment Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000002738 chelating agent Substances 0.000 claims description 4
- 238000006011 modification reaction Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 229940095064 tartrate Drugs 0.000 claims description 3
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 claims description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 claims description 2
- 230000001588 bifunctional effect Effects 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 239000002243 precursor Substances 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 238000003260 vortexing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 claims 4
- 238000003384 imaging method Methods 0.000 abstract description 7
- 239000000523 sample Substances 0.000 abstract description 7
- 238000001262 western blot Methods 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 4
- 238000013170 computed tomography imaging Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 230000037323 metabolic rate Effects 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 102000001301 EGF receptor Human genes 0.000 description 20
- 108060006698 EGF receptor Proteins 0.000 description 20
- 238000002474 experimental method Methods 0.000 description 10
- 230000027455 binding Effects 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 208000029742 colonic neoplasm Diseases 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 2
- 238000011537 Coomassie blue staining Methods 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RXACEEPNTRHYBQ-UHFFFAOYSA-N 2-[[2-[[2-[(2-sulfanylacetyl)amino]acetyl]amino]acetyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)CNC(=O)CNC(=O)CS RXACEEPNTRHYBQ-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000001297 coherence probe microscopy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000003936 denaturing gel electrophoresis Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011470 radical surgery Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000012217 radiopharmaceutical Substances 0.000 description 1
- 229940121896 radiopharmaceutical Drugs 0.000 description 1
- 230000002799 radiopharmaceutical effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/1063—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from stomach or intestines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种靶向EGFR的SPECT显像剂及其制备方法和应用。本发明对cetuximab的结构进行改造，保留功能区F(ab’)2，并通过放射性核素99mTc标记后得到靶向EGFR的SPECT显像剂，探针分子量由150kDa减少到100kDa，加速了探针在体内的代谢速度，有利于临床转化，16h显像效果最好，而完整单抗约为48h；本发明通过Western blots和IF筛选出高表达EGFR细胞系MGC803。通过生物分布和SPECT/CT成像，结果表明99mTc‑MAG3‑Cet‑F(ab')2可以清楚地显示EGFR阳性肿瘤，并且代谢率得到明显提高。
Description
技术领域
本发明涉及一种靶向EGFR的SPECT显像剂及其制备方法和应用，属于放射性药物化学技术领域。
背景技术
消化系统常见肿瘤主要包括胃癌和结肠癌。在所有恶性肿瘤中，胃癌和结肠癌的发病率分别位列第五和第三。胃癌和结肠癌均排在肿瘤相关死亡的前五名。大多数患者在确诊时已处于中晚期，通常不适合进行根治性手术。以EGFR(表皮生长因子受体)为靶点的单克隆抗体(mAbs)在临床上具有较高的治疗效果。
EGFR的表达不仅是评价治疗效果的生物标志物之一，还是指导临床单抗用药的依据。尽管病理结果是金标准，但是对于不适合手术的病人，病理并不适用。ImmunoSPECT是一种非侵入性分子成像方法，它利用放射性标记的抗体来可视化特定的标记物。西妥昔单抗(Cetuximab)是FDA批准的单克隆抗体，广泛用于EGFR过表达的消化道肿瘤。而如何无创性地评估肿瘤的EGFR表达成为了亟待解决的问题。
发明内容
本发明所要解决的技术问题是：如何利用西妥昔单抗(Cetuximab)设计出一种能够无创性地评估肿瘤的EGFR表达的分子探针。
为了解决上述技术问题，本发明提供了一种靶向EGFR的SPECT显像剂的前体化合物，该前体化合物为MAG3-Cet-F(ab')2，所述的MAG3-Cet-F(ab')2的化学结构式如式I所示：
其中，Cet-F(ab')2为西妥昔单抗的功能片段F(ab')2。
本发明还提供了一种靶向EGFR的SPECT显像剂，该SPECT显像剂为99mTc-MAG3-Cet-F(ab')2，所述的99mTc-MAG3-Cet-F(ab')2的化学结构式如式II所示：
其中，Cet-F(ab')2为西妥昔单抗的功能片段F(ab')2。
本发明还提供了上述的靶向EGFR的SPECT显像剂的制备方法，包括以下步骤：
步骤1：将西妥昔单抗与双功能螯合剂NHS-MAG3进行修饰反应，纯化后得到MAG3-Cet；
步骤2：将MAG3-Cet经酶切后去除西妥昔单抗的Fc片段，得到MAG3-Cet-F(ab')2，其保留了西妥昔单抗的功能片段F(ab')2；
步骤3：MAG3-Cet-F(ab')2与99mTcO4 -进行放射性核素标记反应，纯化后得到99mTc-MAG3-Cet-F(ab')2。
优选地，所述步骤1中的修饰反应具体为：将摩尔比为1:5的西妥昔单抗与NHS-MAG3在碳酸氢盐缓冲液中常温反应2小时，所述碳酸氢盐缓冲液的浓度为0.1mol/L，pH值为9.0。
优选地，所述步骤2中的酶切采用的是IdeS蛋白酶，所述IdeS蛋白酶的使用量为：每1mg西妥昔单抗加入50U IdeS酶。
优选地，所述步骤3中的放射性核素标记反应具体为：每100μgMAG3-Cet-F(ab')2中，加入45μL 0.25M的醋酸铵和15μL酒石酸盐标准缓冲液的组合溶液中，然后加入不超过5mCi的99mTcO4 -，混匀后立即加入3μL新鲜制备的1mg/mL SnCl2·2H2O溶液，混匀后在室温下涡旋温育1小时。
本发明还提供了上述的靶向EGFR的SPECT显像剂的应用，包括在制备评估肿瘤的EGFR表达水平的试剂和/或试剂盒中的应用。
本发明的SPECT显像剂的设计原理：
西妥昔单抗(Cetuximab)完整抗体的分子量约为150kDa，这使得抗体在血液中的代谢速度很慢，体内半衰期甚至达到7天以上，一般必须使用长半衰期正电子核素(89Zr，T1/2约3.3天)显像，这无疑增大了患者的总体辐射剂量。而单抗的F(ab')2(约100kDa)片段可以通过酶切产生，它保留了与完整抗体相同的抗原结合位点和免疫结合活性，因此，本发明对cetuximab的结构进行改造，保留功能区F(ab')2，并通过放射性核素99mTc标记后得到靶向EGFR的SPECT显像剂，探针分子量由150kDa减少到100kDa，可以加速探针在体内的代谢速度。
与现有技术相比，本发明的有益效果在于：
1.本发明利用cetuximab结合EGFR的特点，对cetuximab的结构进行改造，保留功能区F(ab')2，减少了探针分子量，由150kDa减少到100kDa，加速了探针在体内的代谢速度，有利于临床转化，16h显像效果最好，而完整单抗约为48h；与其他F(ab')2片段不同的是，IdeS酶的作用位点是特异的，反应时间短(30-60min)；而传统胃蛋白酶切割的F(ab')2片段是通过类似于“削铅笔”的方式切除Fc段，作用位点多，特异性差，且作用时间长(通常10小时以上)；
2.本发明在实验方法上，改进了先制备F(ab')2片段，后连接螯合剂的方式。本发明先连接螯合剂，后切割，减少了连接螯合剂过程中F(ab')2片段发生二聚体的几率，最大程度保留了F(ab')2片段的生物活性。
3.本发明成功合成了99mTc-MAG3-Cet-F(ab')2，并通过Western blots和IF筛选出高表达EGFR细胞系MGC803；通过生物分布和SPECT/CT成像，结果表明99mTc-MAG3-Cet-F(ab')2可以清楚地显示EGFR阳性肿瘤，达到无创评估肿瘤EGFR表达的目的。
附图说明
图1为本发明的SPECT显像剂99mTc-MAG3-Cet-F(ab')2的合成路线图；
图2为通过Western blots和IF筛选出高表达EGFR的细胞系MGC803；其中，2A为Western blots实验结果，2B为IF实验结果；
图3为SDS-PAGE测试Cet-F(ab')2的分子量；
图4为Cet-F(ab')2和Cetuximab的生物亲和性鉴定；
图5为Cet-F(ab')2的稳定性测试结果；
图6为99mTc-MAG3-Cet-F(ab')2的细胞饱和结合曲线；其中，左图为99mTc-MAG3-Cet-F(ab')2与MGC803细胞的饱和结合曲线，右图为99mTc-MAG3-Cet-F(ab')2与HT29细胞的饱和结合曲线；
图7为99mTc-MAG3-Cet-F(ab')2在小鼠体内的显像实验结果，其中，定位线焦点为肿瘤区域；
图8为99mTc-MAG3-Cet-F(ab')2在小鼠体内的生物分布。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
实施例1
靶向EGFR的SPECT显像剂99mTc-MAG3-Cet-F(ab')2制备：
99mTc-MAG3-Cet-F(ab')2的合成路线如图1所示。Cetuximab与NHS-MAG3在0.1mol/L碳酸氢盐缓冲液(pH 9.0)中常温反应2小时，西妥昔单抗：MAG3摩尔比＝1:5；反应产物经Zeba Spin脱盐柱7K MWCO用于纯化除去剩余MAG3；MAG3-Cet与IdeS蛋白酶(用量按照每1mg西妥昔单抗加入50U IdeS酶的比例进行计算)在消化缓冲液(50mM磷酸钠，150mM NaCl，pH6.6)中于37℃孵育30分钟；然后将消化的产物与蛋白A珠温育1小时并离心。附着在珠子上的Fc部分在沉淀物中被去除，而纯化的MAG3-Cet-F(ab')2留在上清液中；通过SDS-PAGE评价产物：SDS-PAGE在4-15％凝胶上在150V下进行1小时后，考马斯亮蓝染色显示产物分子量；99mTc放射性标记：将100μgMAG3-Cet-F(ab')2偶联物加入45μL醋酸铵(0.25M)和15μL酒石酸盐标准缓冲液的组合溶液中，然后加入不超过25μL(约5mCi)99mTcO4 -。混匀后立即加入3μL新鲜制备的1mg/mL SnCl2·2H2O溶液。将混匀溶液在室温下涡旋温育1小时。使用PD-10脱盐柱获得纯化的99mTc-MAG3-Cet-F(ab')2。
实施例2
1、通过Western blots和IF筛选出高表达EGFR细胞系：
Western blots实验：Ripa裂解MGC803细胞株和HT29细胞株蛋白，经加热变性后，每孔上样20微克，进行SDS-PAGE变性凝胶电泳。100V电泳1.5h后，进行转膜、一抗4摄氏度孵育过夜、洗膜、二抗孵育1h后，进行条带曝光，结果如图2A所示，显示EGFR在MGC803细胞株中高表达。
IF实验：MGC803肿瘤和HT29肿瘤组织石蜡切片脱蜡脱水后，0.01M枸橼酸钠缓冲液95℃抗原修复15分钟；降温后5％BSA封闭1小时；甩掉封闭液后，一抗4℃孵育过夜；PBS清洗3次，每次5分钟；0.01mg/ml荧光二抗孵育30分钟，DAPI染色5分钟；PBS清洗3次，每次5分钟，封片后荧光显微镜下观察，结果如图2B所示，MGC803为EGFR高表达细胞系。
2、凝胶电泳实验测Cet-F(ab')2的分子量：采用非还原性Loading Buffer混匀Cet-F(ab')2样品，加热变性后进行SDS-PAGE变性非还原电泳。150V 1.5h电泳结束后，完整取出凝胶，考马斯亮蓝染色，结果如图3所示，Cet-F(ab')2的分子量为100KDa。
3、鉴定生物亲和力：将Cetuximab与NHS-罗丹明在碳酸氢盐缓冲液(pH 9.0)中常温反应2小时，西妥昔单抗：NHS-罗丹明摩尔比＝1:5；反应产物经Zeba Spin脱盐柱7K MWCO用于纯化除去剩余NHS-罗丹明；罗丹明-Cet与IdeS蛋白酶在消化缓冲液(50mM磷酸钠，150mM NaCl，pH 6.6)中于37℃孵育30分钟；然后将消化的产物与蛋白A珠温育1小时并离心。附着在珠子上的Fc部分在沉淀物中被去除，而纯化的罗丹明-Cet-F(ab')2留在上清液中；将罗丹明-Cet-F(ab')2与罗丹明-Cet加入MGC803细胞株的培养液中，使终浓度均为10nmol/L，过夜后荧光显微镜观察，结果显示Cet-F(ab’)2和Cetuximab具有相近的生物亲和力，如图4所示。
4、体外稳定性实验：将20微克Cet-F(ab’)2分别置于PBS和1％BSA中，分别在1h、6h、12h和24h进行SDS-PAGE变性非还原电泳。随后考马斯亮蓝染色，结果如图5所示，由图5可知，Cet-F(ab’)2在PBS及1％BSA中经过24h几乎未分解，具有良好的稳定性。
实施例3
99mTc-MAG3-Cet-F(ab')2的细胞结合试验：
将MGC803细胞和HT29细胞(2×105/孔)接种于24孔板，各孔加入99mTc-MAG3-Cet-F(ab')2使终浓度由0.1nm递增至4nm。敷育2h后，PBS洗三遍，用1M NaOH消化5分钟，测各孔每分钟放射性计数(CPM)，得到细胞饱和结合曲线，如图6所示，由图6可知，99mTc-MAG3-Cet-F(ab')2与EGFR高表达细胞系MGC803细胞具有极佳的亲和力。
实施例4
99mTc-MAG3-Cet-F(ab')2在荷瘤小鼠体内的显像实验及生物学分布实验：
1、显像实验：用PBS重悬MGC803细胞，制备MGC803细胞悬液，浓度为1×107个/ml；将100μl细胞悬液接种在裸鼠右肩，常规饲养14天。肿瘤直径1cm左右时，将99mTc-MAG3-Cet-F(ab')2通过尾静脉注射，16h后进行小动物SPECT/CT显像，结果如图7所示。
2、生物学分布实验：取16只肿瘤模型鼠，随机分成4组，将99mTc-MAG3-Cet-F(ab')2通过尾静脉注射。在注射探针后1h、6h、16h和24h麻醉后处死，取组织和器官进行放射性计数，并称重，得到每克组织的放射性计数，结果如图8所示，由图8可知，显像剂注入小鼠体内后主要分布于血液、肝脏和肾脏中，其余各器官组织分布较少，16h后显像剂浓聚于MGC803细胞。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (7)
1.一种靶向EGFR的SPECT显像剂的前体化合物，其特征在于，该前体化合物为MAG3-Cet-F(ab')2，所述的MAG3-Cet-F(ab')2的化学结构式如式I所示：
其中，Cet-F(ab')2为西妥昔单抗的功能片段F(ab')2。
2.一种靶向EGFR的SPECT显像剂，其特征在于，该SPECT显像剂为99mTc-MAG3-Cet-F(ab')2，所述的99mTc-MAG3-Cet-F(ab')2的化学结构式如式II所示：
其中，Cet-F(ab')2为西妥昔单抗的功能片段F(ab')2。
3.权利要求2所述的靶向EGFR的SPECT显像剂的制备方法，其特征在于，包括以下步骤：
步骤1：将西妥昔单抗与双功能螯合剂NHS-MAG3进行修饰反应，纯化后得到MAG3-Cet；
步骤2：将MAG3-Cet经酶切后去除西妥昔单抗的Fc片段，得到MAG3-Cet-F(ab')2，其保留了西妥昔单抗的功能片段F(ab')2；
步骤3：MAG3-Cet-F(ab')2与99mTcO4 -进行放射性核素标记反应，纯化后得到99mTc-MAG3-Cet-F(ab')2。
4.如权利要求3所述的靶向EGFR的SPECT显像剂的制备方法，其特征在于，所述步骤1中的修饰反应具体为：将摩尔比为1:5的西妥昔单抗与NHS-MAG3在碳酸氢盐缓冲液中常温反应2小时，所述碳酸氢盐缓冲液的的浓度为0.1mol/L，pH值为9.0。
5.如权利要求3所述的靶向EGFR的SPECT显像剂的制备方法，其特征在于，所述步骤2中的酶切采用的是IdeS蛋白酶，所述IdeS蛋白酶的使用量为：每1mg西妥昔单抗加入50U IdeS酶。
6.如权利要求3所述的靶向EGFR的SPECT显像剂的制备方法，其特征在于，所述步骤3中的放射性核素标记反应具体为：每100μg MAG3-Cet-F(ab')2中，加入45μL 0.25M的醋酸铵和15μL酒石酸盐标准缓冲液的组合溶液中，然后加入不超过5mCi的99mTcO4 -，混匀后立即加入3μL新鲜制备的1mg/mL SnCl2·2H2O溶液，混匀后在室温下涡旋温育1小时。
7.权利要求2所述的靶向EGFR的SPECT显像剂的应用，其特征在于，包括在制备评估肿瘤的EGFR表达水平的试剂和/或试剂盒中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111020878.8A CN113698483A (zh) | 2021-09-01 | 2021-09-01 | 一种靶向egfr的spect显像剂及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111020878.8A CN113698483A (zh) | 2021-09-01 | 2021-09-01 | 一种靶向egfr的spect显像剂及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113698483A true CN113698483A (zh) | 2021-11-26 |
Family
ID=78658753
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111020878.8A Pending CN113698483A (zh) | 2021-09-01 | 2021-09-01 | 一种靶向egfr的spect显像剂及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113698483A (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403702A (zh) * | 2015-12-10 | 2016-03-16 | 复旦大学附属华山医院 | 用于检测曲霉菌的锝99标记抗曲霉单抗及其制备和应用 |
-
2021
- 2021-09-01 CN CN202111020878.8A patent/CN113698483A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403702A (zh) * | 2015-12-10 | 2016-03-16 | 复旦大学附属华山医院 | 用于检测曲霉菌的锝99标记抗曲霉单抗及其制备和应用 |
Non-Patent Citations (4)
| Title |
|---|
| 余锋: "西妥昔单抗联合放疗对人胃癌细胞株MGC803及BGC823抑制作用的研究", 万方学位论文数据库, pages 20 * |
| 党亚洁: "锝99m 标记抗曲霉单克隆抗体检测肺内烟曲霉菌的实验研究", 中国优秀硕士学位论文全文数据库 医药卫生科技辑, pages 29 - 30 * |
| 居雪玲等: "抗体药物N-糖表征及质量研究策略探讨", 药物资讯, vol. 10, no. 3, pages 87 * |
| 苑陆杰: "靶向EGFR 的西妥昔单抗在基底样乳腺癌中的放射免疫显像研究", 中国博士学位论文全文数据库 医药卫生科技辑, pages 9 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104159618B (zh) | 治疗剂及其用途 | |
| Luo et al. | Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry | |
| Ege et al. | Lymphoscintigraphy: a comparison of 99Tcm antimony sulphide colloid and 99Tcm stannous phytate | |
| US20030190319A1 (en) | Antibodies specific for CD44v6 | |
| RU2016124229A (ru) | Полипептиды антител и их применения | |
| Sugyo et al. | Evaluation of 89Zr-labeled human anti-CD147 monoclonal antibody as a positron emission tomography probe in a mouse model of pancreatic cancer | |
| Li et al. | ImmunoPET/NIRF/Cerenkov multimodality imaging of ICAM-1 in pancreatic ductal adenocarcinoma | |
| Burianova et al. | Radiotracers for positron emission tomography (PET) targeting tumour-associated carbonic anhydrase isoforms | |
| CN110496233B (zh) | 一种spect显像剂及其标记前体及其制备方法、组合物和用途 | |
| EP1397387A1 (en) | Antibodies specific for cd44v6 | |
| Hagemans et al. | Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents | |
| AU2004205435A1 (en) | Cancerous disease modifying antibodies | |
| Guo et al. | A novel indium-111-labeled gonadotropin-releasing hormone peptide for human prostate cancer imaging | |
| CN113444146A (zh) | 靶向成纤维细胞活化蛋白探针、制备方法及其在制备pet显像剂中的应用 | |
| Cai et al. | Non-invasive monitoring of HER2 expression in breast cancer patients with 99mTc-Affibody SPECT/CT | |
| CN113698483A (zh) | 一种靶向egfr的spect显像剂及其制备方法和应用 | |
| CN116333142A (zh) | Trop2特异性诊疗一体化分子影像探针的制备方法及应用 | |
| CN116444666A (zh) | Cdh17特异性诊疗一体化分子影像探针的制备方法及应用 | |
| CN115925951A (zh) | Cd70特异性诊疗一体化分子影像探针的制备方法及应用 | |
| CN112661812B (zh) | 一种kk-lc-1抗原靶向结合肽及其衍生物、探针与应用 | |
| CN115651063A (zh) | 放射性核素标记的ptp多肽及其应用 | |
| CN108904821B (zh) | 188Re标记的肿瘤靶向环九肽分子探针及其制备方法 | |
| Shi et al. | Molecular imaging of HER2 expression in breast cancer patients using a novel peptide-based tracer 99mTc-HP-Ark2: a pilot study | |
| RU2018115734A (ru) | Полипептиды антител и их применение | |
| Heo et al. | Clinical big-data-based design of GLUT2-targeted carbon nanodots for accurate diagnosis of hepatocellular carcinoma |
Legal Events
| 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 |