CN113476619A - A kind of18F-labeled nano antibody probe and preparation method and application thereof - Google Patents
A kind of18F-labeled nano antibody probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN113476619A CN113476619A CN202110771591.2A CN202110771591A CN113476619A CN 113476619 A CN113476619 A CN 113476619A CN 202110771591 A CN202110771591 A CN 202110771591A CN 113476619 A CN113476619 A CN 113476619A
- Authority
- CN
- China
- Prior art keywords
- nano antibody
- probe
- preparation
- click chemistry
- dbco
- 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.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
- A61K51/041—Heterocyclic compounds
- A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
- A61K51/0455—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
-
- 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
- A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/63—One oxygen atom
- C07D213/65—One oxygen atom attached in position 3 or 5
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Optics & Photonics (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a18An F-labeled nano antibody probe, a preparation method and an application thereof relate to the field of nuclear medicine and molecular imaging, a targeting carrier of the probe comprises a derivative of a nano antibody MM01, a nano antibody MM02 or a nano antibody MM02, and the preparation method comprises the following steps: synthesizing a small molecule compound precursor RJDJ 01; preparation of18F labelling precursor18F[F]-RJDJ 01; preparation of fluoro-18 by click chemistry reaction(s) ((R))18F) Labeled nano antibody probeA needle; the probe is a CD38 specific nano antibody for detecting multiple myeloma. The invention creates a CD38 specific novel nano antibody probe18F[F]MM01, enabling non-invasive visualization of CD38 expression, non-invasive detection of multiple myeloma. The probe has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like.
Description
Technical Field
The invention relates to the field of nuclear medicine and molecular imaging, in particular to a nuclear magnetic resonance imaging system18F-labeled nano antibody probe and preparation method and application thereof.
Background
The presence of a naturally light chain-deficient antibody in the peripheral blood of an alpaca was first reported in 1993 by the scientist Hamers et al in Nature journal (Nature.1993; 363(6428):446-8.) and this antibody with a specific domain is a Heavy chain antibody (HCAbs). Through molecular biology means, the Variable region of the Heavy Chain Antibody is cloned to obtain an antigen binding fragment only with the Heavy Chain Variable region, namely a nano Antibody (VHH). VHH crystals 2.5nm wide and 4nm long with a molecular weight of only 15KDa, and are therefore also called Nanobodies ((R))
Registered trade names of Ablynx corporation). The nano antibody is the minimum antibody unit which can be combined with a target antigen and is known at present, and has the advantages of high affinity, small molecular weight, low preparation cost (the expression can be carried out by using escherichia coli, and the expression can also be carried out by using eukaryotic expression systems such as yeast and Chinese hamster ovary cells), easy clinical transformation and popularization and application.
The nano antibody is a hot targeting carrier (Theranostics.2014; 4(4):386-98.) for constructing a molecular imaging probe in recent years. At present, various nuclides with short half-life periods are used for marking nano antibodies and preparing nano antibody molecular imaging probes. Technetium-99 m (99 mTc; T1/2 ═ 6.02h) labeled nanobody probes targeting programmed death ligand 1(PD-L1) have been successfully transformed into the clinic for noninvasive diagnosis of patients with non-small cell lung cancer (J Nucl Med.2019; 60(9): 1213-; gallium-68 (68 Ga; T1/2 ═ 1.1h) labeled Nanobody probes targeting human epidermal growth factor receptor (HER2) have also been successfully transformed into clinics for noninvasive diagnosis of breast cancer (J Nucl Med.2016; 57(1): 27-33.). The above examples show that the radionuclide labeled nano antibody probe has great clinical transformation application prospect, and can be used for early noninvasive diagnosis of human malignant tumor and key pathogenic targetVisualization, screening of monoclonal antibody (mAb) treated patients, and evaluation of efficacy following monoclonal antibody treatment. However, 99mTc belongs to a single photon emission radionuclide, and the imaging performance of the labeled nano antibody probe is not good enough; 68Ga generally needs to be prepared by a germanium-gallium generator or a medical cyclotron provided with a solid target, the preparation cost is high, the half-life period is short, and the marked nano antibody probe is not suitable for transportation, popularization and application. Fluorine-18 (18F; t1/2-109.8 min h) is the longest used radionuclide for clinical Positron Emission Tomography (PET), the positron emission rate is as high as 97%, the positron range is 0.5mm, no gamma ray is emitted, and the radionuclide is one of the best nuclides for creating PET imaging probes. However, due to limitations such as low radiochemical yield (RCY), harsh labeling conditions (high temperature, organic solvent required), and the like,18f has not been widely used for the nuclide labeling of nanobodies (Chem Rev. 2020; 120(8): 3787-3851.).
Multiple Myeloma (MM) is a B cell-derived hematological malignancy, and no effective clinical treatment is available at present. CD38 is a biomarker specific for multiple myeloma. The monoclonal antibody daratumumab (daratumumab) targeting CD38 has already been clinically approved in Europe, America and China, and is used for treating new-onset or relapsed refractory multiple myeloma. Many factors mediate the therapeutic efficacy of daratuzumab, the most significant of which is the level of CD38 protein expression. Currently, the clinical judgment of CD38 expression levels in multiple myeloma cells is heavily dependent on flow cytometric detection of bone marrow punctures. However, this method is very traumatic and has poor reproducibility. Therefore, there is a need for a molecular imaging method for non-invasive visualization of CD38 expression level and early diagnosis of multiple myeloma.
The immuno-PET imaging organically combining the high sensitivity of PET imaging and the high affinity of antibodies is a novel molecular imaging mode. The preparation of the immune PET imaging probe is mainly based on a monoclonal antibody, an antibody fragment or a nano antibody randomly or site-specifically marked by radionuclide. Wherein the content of the first and second substances,18the F-labeled nano antibody probe is most clinically usefulAnd the application prospect is good. Currently, a variety of precursors have been used to label biological macromolecules with 18F. [18F]P-fluorobenzaldehyde ([ alpha ])18F]FBA) is the most commonly used18F labeling the precursor; N-Succinimidyl-4-, [ solution of a salt of N-Succinimidyl-4 ], [ solution of a carboxylic acid18F]-fluorobenzoate([18F]SFB) are also more common18F labeling a precursor which can react with an amino group of lysine (Lys) of the antibody to form a stable amide bond; n- [2- (4- [ 18F)]-Fluorobenzamido)-ethyl]maleimide([18F]FBEM) is a labeled precursor with thiol reactivity, can be used for site-directed labeling of nano-antibodies, but has complex synthetic steps and low radiochemical yield; 2,3,5,6-tetrafluorophenyl6- [18F [ ]]-fluoronicotinate([18F]TFPFN) although the conditions for labeling the nano-antibody are mild (37-40 ℃,15min, pH 8.5-9.0), the radiochemical yield is only about 5 percent, and the application is severely limited. In addition, other 18F labeling methods often require an organic solvent such as acetonitrile, and require reaction in an acidic environment (pH 2.0 to 2.5). However, the antibody is easy to be changed or inactivated in extreme environment, so that the common labeling method is not suitable for labeling the nano antibody. Clinically, an 18F labeling nano antibody method with mild labeling conditions, good repeatability and high radiochemical yield is urgently needed to realize the room-temperature and high-efficiency 18F labeling of the target specific nano antibody.
In terms of MM diagnosis, the current means of assessing CD38 expression levels are primarily flow cytometric examination of the penetrating bone marrow or immunohistochemical staining (IHC). However, the bone marrow puncture is not only large in trauma and poor in repeatability, but also causes false negative or false positive of biopsy puncture results due to factors such as sampling errors and tumor heterogeneity.
Clinically, a molecular imaging method for noninvasive visualization of CD38 expression level and early accurate detection of multiple myeloma antibodies is urgently needed, a nano antibody probe which is low in price, low in radiation dose, short in imaging period and easy to clinically transform and apply is needed, and an 18F labeled nano antibody method which is mild in labeling condition, good in repeatability and high in radiochemical yield is needed, so that room-temperature and efficient target-specific nano antibodies can be realized18And F, marking.
Therefore, those skilled in the art are working to develop a priceCD38 specific nano antibody probe with low cost, low radiation dose, short imaging period and easy clinical transformation application18F[F]MM01, enabling noninvasive visualization of CD38 and MM target-specific noninvasive diagnosis.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides an automated synthesis module18F, a method for efficiently labeling the nano antibody; the technical method is innovative to prepare the CD38 specific nano antibody probe which has low price, low radiation dose, short imaging period and easy clinical transformation application18F[F]MM01, enabling noninvasive visualization of CD38 and MM target-specific noninvasive diagnosis.
To achieve the above object, the present invention provides a method for producing a light emitting diode18The F-labeled nano antibody probe comprises a targeting carrier of a nano antibody MM01, a nano antibody MM02 or a derivative of a nano antibody MM 02.
Further, the derivatives of the nanobody MM02 include derivatives of the MM02 nanobody modified by a fixed-point PEG.
The invention also provides a18The preparation method of the F-labeled nano antibody probe comprises the following steps:
Further, the chemical structural formula of the small molecule compound precursor RJDJ01 in the step 1 is as follows:
further, the chemical synthesis route of the small molecule compound precursor RJDJ01 in step 1 is as follows:
firstly, reacting tetraethylene glycol, p-toluenesulfonyl chloride and potassium hydroxide to prepare a crude product; dissolving the crude product in dichloromethane, adding 3-hydroxy-2-nitropyridine to react with sodium chloride, and performing column chromatography separation to obtain 2- (2- (2- (2- ((2-nitropyridine-3-yl) oxy) ethoxy) 4-methyl benzene sulfonic acid ethyl ester; reacting ethyl 2- (2- (2- (2- ((2-nitropyridine-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate with sodium azide, extracting by using dichloromethane, and performing chromatography to obtain 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine, namely a small molecular compound precursor RJDJ 01.
Further, the precursor in the above step 218F[F]The chemical structural formula of-RJDJ 01 is:
further, the preparation in the step 3 is performed by click chemistry reaction18The F-labeled nano antibody probe comprises18F random labeling nano antibody preparation method or18F, preparing a fixed-point labeled nano antibody.
Further, the air conditioner is provided with a fan,18the click chemical substrate 2 in the preparation mode of the F random labeling nano-antibody is a DBCO random coupling CD38, BCMA, TROP-2, HER2, EGFR, VEGFR, HER2, CD47, CD146, ICAM-1, Nectin-4, CAIX, GPC3, GPA33, Claudin18.2, GD2, MM01 or PD-L1 specific nano-antibody.
Further, the air conditioner is provided with a fan,18the click chemical substrate 2 in the preparation mode of the F site-specific labeled nano antibody is a DBCO site-specific coupled CD38, BCMA, TROP-2, HER2, EGFR, VEGFR, HER2, CD47, CD146, ICAM-1, Nectin-4, CAIX, GPC3, GPA33, Claudin18.2, GD2, MM01, MM02 or PD-L1 specific nano antibodyAn antibody; wherein, MM02 is prepared by the site-specific reaction of Cysteine (Cystein, Cys, C) in a linker GGGGSCGSGSGSLLQS and diphenylcyclooctyne-maleimide (DBCO-Mal, CAS #:1395786-30-7, MeloPEG) to prepare DBCO-MM 02; or through an enzymatic reaction mediated by microbial Glutamine transaminase (mTGase), realizing the site-specific reaction of diphenyl cyclooctyne-Amine (DBCO-Amine, CAS #:1255942-06-3, MeloPEG) and Glutamine (Glutamine, Gln, Q) in the MM01 or MM02 linker GGGGSCGSGSGSLLQS, thereby preparing DBCO-MM01 or DBCO-MM 02; or DBCO-PEG-MM01 or DBCO-PEG-MM02 is prepared by the microorganism Glutamine transaminase (mTGase) mediated diphenyl cyclooctyne-polyethylene glycol-Amine (DBCO-PEG-Amine, MeloPEG) and Glutamine (Glutamine, Gln, Q) in MM01 or MM02 linker GGGGSCGSGSGSLLQS in a site-directed reaction.
Further, the CD38 specific nano antibody MM01 has an amino acid sequence shown as SEQ ID No.1 in a sequence table and a gene sequence shown as SEQ ID No.2 in the sequence table.
Further, the CD38 specific nano antibody MM02 has an amino acid sequence shown as SEQ ID No.3 in a sequence table and a gene sequence shown as SEQ ID No.4 in the sequence table.
Further, the probe prepared by random labeling18F[F]MM01 human CD38 specific nanobody probe.
Further, the PEG may be of different molecular weights, i.e., 100Da, 200Da, 500Da, 1KDa, 5KDa, 10KDa or 20 KDa.
The invention also provides a18An application method of the F-labeled nano antibody probe, which is applied to CD38 specific nano antibodies for detecting multiple myeloma.
In the preferred embodiment of the present invention, the path of synthesizing RJDJ01 is detailed;
in another preferred embodiment of the present invention, the click chemistry reaction substrate 1 is specified18F[F]-automated preparation of RJDJ 01;
in another preferred embodiment of the present invention, the preparation of a substrate DBCO-MM01 for click chemistry reaction for preparing a novel substance by click chemistry reaction is described in detailCD38 specific nano antibody probe18F[F]-MM01；
In another preferred embodiment of the present invention, the detailed description is given18F[F]-MM01 quality control method;
in another preferred embodiment of the present invention, the detailed description is given18F[F]-MM01 immuno-PET imaging detection of disseminated multiple myeloma;
in another preferred embodiment of the invention, mTGase enzymatic reactions are specified to mediate nanobody site-directed PEG modification.
Currently, examination items for diagnosis of multiple myeloma include: blood tests, urine tests, bone marrow tests, imaging tests, and the like. Among them, bone marrow aspiration examination is more traumatic, less reproducible, and has a lower detection rate for heterogeneous multiple myeloma. Conventional molecular imaging examination methods such as X-ray plain film, Computed Tomography (CT), and Magnetic Resonance Imaging (MRI) all reflect structural changes in disease and fail to reflect changes in disease function or molecular level, and therefore have a low detection rate and lack specificity for early stage multiple myeloma. Fluorodeoxyglucose (A)18F-FDG) PET/CT examination is also used for diagnosing multiple myeloma, but still has the defects of low sensitivity, low specificity and the like. CD38 is one of the most specific markers for multiple myeloma, and monoclonal antibodies targeting CD38, such as daratouzumab, have been approved in the United states and China for clinical treatment of multiple myeloma (blood.2018; 131(1): 13-29.). The development of a CD38 specific molecular imaging probe is expected to realize early accurate diagnosis of multiple marrow, effective screening of patients treated by the daratuzumab and noninvasive dynamic evaluation of CD38 expression before and after treatment.
At present, no CD38 specific molecular imaging probe exists at home and abroad, and the probe is based on68Ga[Ga]The immune PET imaging of NOTA-MMO1 achieves better detection effect, but the defects are shown as follows:68ga needs to be prepared by a germanium-gallium generator, so that the probe68Ga[Ga]The cost of preparing NOTA-MMO1 is high;68ga half-life (T1/2 ═ 1.1h) results in a limited range of application of the probe.
The invention creates a CD38 specific novel nano antibody probe18F[F]MM01, enabling noninvasive visualization of CD38 expression, further enabling noninvasive diagnosis of multiple myeloma. Novel probe18F[F]the-MM 01 has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like, and has the following beneficial technical effects:
1. the invention provides18F is a new method for rapidly and efficiently labeling the nano antibody, and the method effectively avoids a two-step method mediated by click chemistry18The influence of factors such as high temperature and organic solvent on the activity and structure of the nano antibody in the process of F labeling the precursor compound reduces the preparation cost of the nano antibody.
2. Prepare a18F-labeled CD38 specific nano antibody probe18F[F]MM01, realizes the express delivery and accurate detection of multiple myeloma. Fluorine-18 (18F; t1/2 ═ 109.8min h) half-life ratio68Ga is long, and the application range of the labeled probe is wide.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of the RJDJ01 chemical synthesis path in accordance with a preferred embodiment 1 of the present invention;
FIG. 2 is a chromatogram of the detection products by high performance liquid chromatography (Agilent 1260 HPLC and 6120 MSD) and Mass Spectrometry Detector (MSD) Total Ion Chromatogram (TIC) according to a preferred embodiment 1 of the present invention;
FIG. 3 is a mass spectrum of the product detected by mass spectrometry according to a preferred embodiment of the present invention 1;
FIG. 4 is a graph showing the results of detecting the product by Nuclear Magnetic Resonance (NMR) method according to a preferred embodiment 1 of the present invention;
FIG. 5 shows another preferred embodiment 3 of the present invention18F, randomly labeling a nano antibody;
FIG. 6 shows another preferred embodiment 5 of the present invention18F[F]Imaging of MM01 immunoPET/CT for in situ multiple myeloma;
FIG. 7 shows another preferred embodiment of the present invention, namely, after blocking of the daratumumab monoclonal antibody (daratumumab) of example 518F[F]MM01 immunoPET/CT imaging;
FIG. 8 shows another preferred embodiment 6 of the present invention18F, site-specific labeling of the nano antibody.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1 Synthesis of RJDJ01
The synthesis path shown in fig. 1 autonomously synthesizes RJDJ 01:
1.1 g of tetraethylene glycol (about 5.1mmol) was precisely weighed, dissolved in 15mL of methylene chloride, and then 2g (about 10.5mmol) of p-toluenesulfonyl chloride and 0.59g (about 10.5mmol) of potassium hydroxide were slowly added to the above mixture to react overnight at room temperature. After washing with water, the mixture was separated to obtain 2g of a crude product (yield: about 78%).
2. The above crude product was dissolved in methylene chloride, followed by addition of 0.56g (about 4mmol) of 3-hydroxy-2-nitropyridine and 96mg of sodium chloride (about 4mmol), reaction overnight at room temperature, and column chromatography to give 1g (about 2.1mmol) of ethyl 2- (2- (2- (2- ((2-nitropyridin-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate in about 51% yield.
3.940mg (about 2mmol) of ethyl 2- (2- (2- (2- ((2-nitropyridin-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate was dissolved in 10mL of dichloromethane, and then 130mg (about 2mmol) of a 2mL aqueous solution of sodium azide was added to the reaction solution, and after 5 hours at room temperature, dichloromethane was extracted and column chromatography was carried out to give 477mg of 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine as described in this patent, namely RJDJ01, in a yield of about 70%.
Verifying the product by using a liquid chromatography, a mass spectrometry and a nuclear magnetic resonance detection method, wherein the liquid chromatography uses Agilent 1260 HPLC to configure an ultraviolet detector, the detection result is shown in figure 2, the upper figure is a chromatogram of the product with the wavelength of 220nm, the retention time is 2.711min, the lower figure is a Total Ion Chromatogram (TIC) of a mass spectrometry detector 6120MSD, and the retention time is 2.724 min; FIG. 3 is a mass spectrum of mass spectrometric detection with the strongest peak at 341.9 m/z; FIG. 4 is a Nuclear Magnetic Resonance (NMR) chart of RJDJ01, which shows that the product has the correct molecular weight and chemical structure of RJDJ 01.
Example 2 click chemistry reaction substrate 118F[F]Automated preparation of RJDJ01
The specific experimental scheme is as follows: weighing 2-2.5 mg RJDJ01, dissolving in 1mL dimethyl sulfoxide (DMSO); 18F-fluoride (33.49-39.39 GBq) was transferred using a pre-equilibrated anion exchange column, and was treated with potassium carbonate (3mg) and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8 ] cyclohexane in 1.4mL of acetonitrile]Eluting the anion exchange column with hexacosane (15mg) as a mobile phase; after the solvent was evaporated to dryness, RJDJ01 dissolved in 1mL DMSO was added; heating the reaction system at 120 ℃ for 14 minutes; adding 3mL of sterile water for injection into the reaction system, transferring the reaction system to a C18 column, setting the mobile phase to be 0.1% trifluoroacetic acid (trifluoroacetic acid) containing 32% acetonitrile, setting the flow rate to be 4.6mL/min, purifying by HPLC, and collecting18F-RJDJ01 to pear; adding 10mL of sterilized water for injection to dilute in pear-shaped bottle18F[F]RJDJ01, and transferred to C18 column and extracted with 3mL of ethanol18F[F]RJDJ01, heated to 100 ℃ to evaporate ethanol and then resuspended in 100uL of Phosphate Buffer (PBS)18F[F]-RJDJ01。
Example 3 preparation of a substrate for click chemistry reaction DBCO-MM01, preparation of a novel CD 38-specific Nanobody probe by click chemistry reaction18F[F]-MM01
The reaction principle is shown in figure 518The F random labeling nano antibody schematic diagram shows that a click chemistry reaction substrate DBCO-MM01 is prepared, and a novel CD38 specific nano antibody probe is prepared through click chemistry reaction18F[F]-MM01。
DBCO-NHS ester (CAS #:1353016-71-3, MeloPEG) was randomly coupled to MM01 to prepare a substrate for click chemistry reaction DBCO-MM 01. The method comprises the following specific steps: 1mg of CD 38-specific nanobody MM01 was dissolved in Phosphate Buffered Saline (PBS) in a volume of about 1mL, and 80-100. mu.L of 0.1M sodium carbonate (Na2CO3) buffer was added to adjust the pH of the nanobody solution to 9.0-10. DBCO-NHS ester freshly dissolved in dimethyl sulfoxide (DMSO) was added to the nanobody solution at a DBCO-NHS ester/MM01 molar ratio of 10:1, i.e., 670nM/0.27 mg. Placing the reaction system at room temperature for reacting for 2 hours, then using PBS as a mobile phase, purifying the nano antibody randomly coupled with DBCO by using a pre-balanced PD-10 desalting column (GE Healthcare), and collecting DBCO-MM 01; the nanobody sample was concentrated using an ultrafiltration tube (Merck Millipore) with a cut-off of 10kDa, and finally the concentration of DBCO-MM01 was determined using NanoDrop, and DBCO-MM01 was placed in a refrigerator at 4 ℃ for future use.
Preparation of novel CD38 specific nano antibody probe by click chemical reaction18F[F]The specific experimental protocol for MM01 is as follows: adding 20mL of DBCO-MM 01332 uL (320ug)18F[F]-MM01 to DBCO-MM01 nanobody solution; placing the reaction system in a constant temperature oscillator, and reacting for 45 minutes at 45 ℃; after the reaction, the unreacted reaction mixture was separated again by using a pre-equilibrated PD-10 desalting column using PBS as a mobile phase18F-RJDJ01, purifying and collecting final product18F[F]-MM01。
Example 418F[F]-MM01 quality control
Example 518F[F]MM01 immune PET imaging detection of disseminated multiple myeloma
The method comprises the following steps: the PET/CT Imaging collection of the small animals involved in the study was performed using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems), and each tumor-bearing NCG mouse was injected via tail vein with 3.7-7.4 MBq18F[F]MM01 (3 per group), 1 hour after injection, tumor-bearing nude mice were anesthetized with isoflurane (2% concentration) mixed with oxygen, and the nude mice entering deep anesthesia were placed on a PET/CT scanning bed in a supine position, PET and CT images were continuously acquired, and image reconstruction was completed with IRIS system equipped with software, as shown in FIG. 6, CD38 specific nanobody probe18F[F]MM01 has higher uptake in tumor tissues, and the imaging result shows that the bilateral femurs (1, 2) and tibias (3, 4) have obvious uptake of the imaging agent,18F[F]MM01 immune PET imaging can accurately detect disseminated multiple myeloma, including lesions in bilateral femurs, tibiae and the like. Delineating regions of interest such as tumors, hearts and major tissues and organs (liver, spleen, lung, kidney, pancreas and muscle) on the reconstructed PET image by using an OsiriX Lite image processing workstation (Pixmeo SARL), calculating the radioactive uptake values of the tumor tissues and the important tissues and organs by taking% ID/g (percent of injected dose gram) as a unit, and obtaining high non-specific uptake in major excretory (kidney) and metabolic (liver) tissues; after the imaging is finished, taking tumor tissues and main tissues and organs to perform in-vitro biodistribution experiment, and obtaining CD38 specific nano antibody probe18F[F]MM01 also has a higher uptake.
CD38 specific immune PET imaging probe18F[F]MM01 specifically detects multiple myeloma and the daratheon monoclonal antibody can effectively block the pair of skeletal tissues18Based on that the daratuzumab can effectively reduce the ingestion of a bone tissue imaging agent for the involvement of multiple myeloma, the ingestion of F-MM01 is realized by injecting 1mg of daratuzumab into each tumor-bearing mouse in the experiment 24 hours in advance, and then injecting 3.7-7.4 MBq 18F [ F ] into each tumor-bearing NCG mouse through tail vein according to the steps]MM01 (3 mice per group), 1 hour after injection, tumor-bearing nude mice were anesthetized with isoflurane (2% concentration) mixed with oxygen, nude mice under deep anesthesia were placed on a PET/CT scanning bed in a supine position, PET and CT images were continuously acquired, image reconstruction was completed with IRIS system equipped with software, and the results are shown in FIG. 7, after occlusion with Daratoumab (daratumumab)18F[F]MM01 immunoPET/CT imaging, the imaging results showing the daradeur monoclonal antibodyAfter body occlusion, the uptake of imaging agents in bilateral femurs (1, 2) and tibias (3, 4) was significantly reduced, indicating that the daratuoyou monoclonal antibody effectively occluded18F[F]Binding site of MM01 on MM.1S tumor cells, demonstrating a probe18F[F]MM01 is highly specific and its antigen binding site coincides with that of daratumumab. Sealing parts of bilateral humerus, bilateral scapula, sternum, vertebral body, bilateral ilium and bilateral femur after daratuzumab18F[F]Significant decrease in MM01 uptake, suggesting18F[F]The MM01 probe is highly specific for human CD38, and18F[F]the MM01 probe and daratuzumab have the same antigen binding site.
Example 6mTGase enzymatic reaction mediated Nanobody site-directed PEG modification
The reaction principle is shown in figure 818The schematic diagram of the F fixed-point labeled nano antibody is shown. The specific implementation route is as follows: mixing 3mg MM01 with LLQS tag at C terminal, 1mg PEG-NH2(5kDa) or 2mg PEG-NH2(10KDa) and 1mg mTGase are dissolved in 1mL PBS solution, and the reaction system is placed in a constant temperature oscillator to react for 1 hour at room temperature; the sample volume was concentrated to 300. mu.L with an ultrafiltration tube (Merck Millipore) with a cut-off of 10 kDa; by means of a device equipped with SuperdexTMOf 75 Increatase column
pure protein purification instrument (Cytiva, for merly GE Healthcare Life Science) purified and collected MM01 derivatives site-directed modified with PEG of different molecular weights.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Shanghai university of traffic medical college affiliated renji hospital
<120>A kind of18F-labeled nano antibody probe and preparation method and application thereof
<130> 2020
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 145
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met His His His His His His Asp Val Gln Leu Gln Glu Ser Gly Gly
1 5 10 15
Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Thr Gly Ser
20 25 30
Gly Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg Gln Ala Pro
35 40 45
Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Thr Trp Val Gly Ala Ser
50 55 60
Thr Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75 80
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
85 90 95
Asp Thr Ala Val Tyr Ser Cys Ala Ala Gly Arg Gly Ile Val Ala Gly
100 105 110
Arg Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Leu Gln
130 135 140
Ser
145
<210> 2
<211> 450
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
catatgcacc atcatcatca tcacgacgtc caactgcaag aatcgggcgg cggtctggtc 60
caagcgggcg gttccctgcg tctgtcatgc accggcagcg gtcgtacgtt tcgcaactat 120
ccgatggcat ggttccgtca ggctccgggc aaagaacgcg aatttgtggc gggcattacc 180
tgggttggtg ccagtacgct gtacgcagat tttgctaaag gtcgtttcac catctcccgc 240
gacaacgcga aaaatacggt ttatctgcag atgaatagcc tgaaaccgga agataccgca 300
gtctactctt gtgccgcggg tcgtggtatt gttgccggtc gtatcccggc cgaatatgca 360
gactggggcc aaggtacgca ggtgacggtt tcttctggtg gtggcggctc tggtggtggc 420
ggttctctgc tgcaaagtta atgaaagctt 450
<210> 3
<211> 147
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met His His His His His His Asp Val Gln Leu Gln Glu Ser Gly Gly
1 5 10 15
Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Thr Gly Ser
20 25 30
Gly Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg Gln Ala Pro
35 40 45
Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Thr Trp Val Gly Ala Ser
50 55 60
Thr Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75 80
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
85 90 95
Asp Thr Ala Val Tyr Ser Cys Ala Ala Gly Arg Gly Ile Val Ala Gly
100 105 110
Arg Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Gly Gly Gly Gly Ser Cys Gly Ser Gly Ser Gly Ser Leu
130 135 140
Leu Gln Ser
145
<210> 4
<211> 456
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
catatgcatc atcatcatca tcacgacgtc caactgcaag aatctggcgg cggtctggtt 60
caagcgggcg gtagcctgcg tctgtcatgt accggcagcg gtcgtacgtt tcgcaactat 120
ccgatggcat ggttccgtca ggctccgggc aaagaacgcg aatttgtggc gggcattacc 180
tgggttggtg ccagtacgct gtacgcagat tttgctaaag gtcgtttcac catctcccgc 240
gacaacgcga aaaatacggt ttatctgcaa atgaatagcc tgaaaccgga agataccgca 300
gtctactctt gtgccgcggg tcgtggtatt gttgccggtc gtattccggc cgaatatgca 360
gactggggtc agggtacgca agtcacggtc tcttcaggcg gtggcggttc gtgtggctcg 420
ggctcgggct ctctgctgca atcgtaatga aagctt 456
Claims (10)
1. A kind of18The F-labeled nano antibody probe is characterized in that a targeting carrier of the probe comprises a nano antibody MM01, a nano antibody MM02 or a derivative of the nano antibody MM 02.
2. A process as claimed in claim 118The F-labeled nanobody probe is characterized in that the derivative of the nanobody MM02 comprises a derivative of the MM02 nanobody modified by site-directed PEG.
3. A process as claimed in claim 1 or 218The preparation method of the F-labeled nano antibody probe is characterized by comprising the following steps of:
step 1, synthesizing a small molecular compound precursor RJDJ 01;
step 2,18F labeling the small molecule compound precursor RJDJ01 obtained in step 1, and preparing the precursor for the rapid click chemistry labeling of the nano antibody18F labels the precursor, i.e.18F[F]-RJDJ01；
Step 3, preparing the compound by click chemistry reaction18Labeling a nano antibody probe by F; the reactants of the click chemistry reaction comprise a click chemistry substrate 1 and a click chemistry substrate 2, wherein the click chemistry substrate 1 is the click chemistry substrate obtained in the step 218F[F]-RJDJ01, said click chemistry substrate 2 being said nanobody and the product of said click chemistry reaction being said18And F, labeling the nano antibody probe.
4. The preparation method according to claim 3, wherein the chemical structural formula of the small molecule compound precursor RJDJ01 in step 1 is as follows:
5. the preparation method according to claim 3, wherein the chemical synthesis route of the small molecule compound precursor RJDJ01 in step 1 is as follows: firstly, reacting tetraethylene glycol, p-toluenesulfonyl chloride and potassium hydroxide to prepare a crude product; dissolving the crude product in dichloromethane, adding 3-hydroxy-2-nitropyridine to react with sodium chloride, and performing column chromatography separation to obtain 2- (2- (2- (2- ((2-nitropyridine-3-yl) oxy) ethoxy) 4-methyl benzene sulfonic acid ethyl ester; reacting the ethyl 2- (2- (2- (2- ((2-nitropyridine-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate with sodium azide, extracting with dichloromethane, and performing chromatography to obtain 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine, namely the small molecule compound precursor RJDJ 01.
6. The method of claim 3, wherein the precursor in step 2 is18F[F]The chemical structural formula of-RJDJ 01 is:
7. the method according to claim 3, wherein the preparation in step 3 is performed by a click chemistry reaction18The F-labeled nano antibody probe comprises18F random labeling nano antibody preparation method or18F, preparing a fixed-point labeled nano antibody.
8. The method of claim 7, wherein the step of preparing the composition is carried out in the presence of a catalyst18The click chemistry substrate 2 in the preparation mode of the F random labeling nano-antibody is CD38, BCMA, TROP-2, HER2, EGFR, VEGFR, HER2, CD47, CD146, ICAM-1, Nectin-4, CAIX, GPC3, GPA33, Claudin18.2, GD2, MM01 or PD-L1 specific nano-antibody randomly coupled with DBCO.
9. The method of claim 7, wherein the step of preparing the composition is carried out in the presence of a catalyst18(ii) said click chemistry substrate 2 in a F site-directed labeling nanobody preparation is said CD38, said BCMA, said TROP-2, said HER2, said EGFR, said VEGFR, said HER2, said CD47, said CD146, said ICAM-1, said Nectin-4, said CAIX, said GPC3, said GPA33, said claudin18.2, said GD2, said MM01, MM02, or said PD-L1 specific nanobody of said DBCO site-directed coupling; wherein, the MM02 prepares DBCO-MM02 by the site-specific reaction of Cysteine (Cystein, Cys, C) in a linker GGGGSCGSGSGSLLQS and diphenylcyclooctyne-maleimide (DBCO-Mal, CAS #:1395786-30-7, MeloPEG); or through a microbial Glutamine transaminase (mTGase) -mediated enzymatic reaction, realizing the site-directed reaction of diphenyl cyclooctyne-Amine (DBCO-Amine, CAS #:1255942-06-3, MeloPEG) and Glutamine (Glutamine, Gln, Q) in the MM01 or the MM02 linker GGGGSCGSGSGSLLQS, thereby preparing DBCO-MM01 or the DBCO-MM 02; or through the microorganism Glutamine transaminase (mTGase) mediated diphenyl cyclooctyne-polyethylene glycol-Amine (DBCO-PEG-Amine, MeloPEG) and the MM01 or Glutamine (Glutamine, Gln, Q) in the MM02 linker GGGGSCGSGSGSLLQS to perform site-directed reaction, preparing DBCO-PEG-MM01 or DBCO-PEG-MM 02.
10. A method as claimed in claim 1 or 218The F-labeled nano-antibody probe is characterized by being applied to a CD38 specific nano-antibody for detecting multiple myeloma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110771591.2A CN113476619B (en) | 2021-07-08 | 2021-07-08 | A kind of 18 F-labeled nano antibody probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110771591.2A CN113476619B (en) | 2021-07-08 | 2021-07-08 | A kind of 18 F-labeled nano antibody probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113476619A true CN113476619A (en) | 2021-10-08 |
| CN113476619B CN113476619B (en) | 2022-11-29 |
Family
ID=77937914
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110771591.2A Active CN113476619B (en) | 2021-07-08 | 2021-07-08 | A kind of 18 F-labeled nano antibody probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113476619B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114617983A (en) * | 2022-05-16 | 2022-06-14 | 中山大学附属第五医院 | Fluorine-18 labeled CEA molecular targeting compound and preparation method and application thereof |
| CN117327182A (en) * | 2023-09-19 | 2024-01-02 | 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103221072A (en) * | 2010-12-02 | 2013-07-24 | 免疫医疗公司 | In vivo copper-free click chemistry for delivery of therapeutic and/or diagnostic agents |
| CN107889489A (en) * | 2015-06-25 | 2018-04-06 | 伊玛提克斯生物技术有限公司 | For myeloma and the novel cell epitope and cell epitope composition of other cancer immunotherapies |
| CN112457401A (en) * | 2020-10-21 | 2021-03-09 | 上海交通大学医学院附属仁济医院 | Novel molecular imaging probe for diagnosing multiple myeloma |
-
2021
- 2021-07-08 CN CN202110771591.2A patent/CN113476619B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103221072A (en) * | 2010-12-02 | 2013-07-24 | 免疫医疗公司 | In vivo copper-free click chemistry for delivery of therapeutic and/or diagnostic agents |
| CN107889489A (en) * | 2015-06-25 | 2018-04-06 | 伊玛提克斯生物技术有限公司 | For myeloma and the novel cell epitope and cell epitope composition of other cancer immunotherapies |
| CN112457401A (en) * | 2020-10-21 | 2021-03-09 | 上海交通大学医学院附属仁济医院 | Novel molecular imaging probe for diagnosing multiple myeloma |
Non-Patent Citations (2)
| Title |
|---|
| DAVID J. DONNELLY ET AL.: ""Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression"", 《J NUCL MED》 * |
| 袁志斌: "核医学影像诊断多发性骨髓瘤的临床价值", 《国外医学.放射医学核医学分册》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114617983A (en) * | 2022-05-16 | 2022-06-14 | 中山大学附属第五医院 | Fluorine-18 labeled CEA molecular targeting compound and preparation method and application thereof |
| CN117327182A (en) * | 2023-09-19 | 2024-01-02 | 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113476619B (en) | 2022-11-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2017217347A1 (en) | SITE-SPECIFIC RADIOISOTOPE-LABELED ANTIBODY USING IgG-BINDING PEPTIDE | |
| CN109414514A (en) | Immunomodulator is imaged in PET | |
| CN109824765B (en) | 68Ga-labeled AEEA modified c-Met molecular imaging probe, preparation and application thereof | |
| CN112028916A (en) | Programmed cell death protein receptor-1 targeted molecular probe and preparation | |
| CN109942687B (en) | 68Ga-marked EACA modified c-Met molecular imaging probe, preparation and application | |
| CN113583089B (en) | Tumor PD-L1 targeted PET imaging agent, labeling precursor, preparation method and application thereof | |
| CN114773433B (en) | CD25 targeted polypeptide, molecular probe and application | |
| CN112043839A (en) | Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof | |
| CN113476619B (en) | A kind of 18 F-labeled nano antibody probe and preparation method and application thereof | |
| CN112933249A (en) | PD-L1 targeted bimodal molecular probe and preparation method and application thereof | |
| CN110496233B (en) | SPECT imaging agent, marked precursor thereof, preparation method, composition and application thereof | |
| CN113880917B (en) | Tumor high affinity peptides and application thereof | |
| Postupalenko et al. | Template directed synthesis of antibody Fc conjugates with concomitant ligand release | |
| CN114262362A (en) | EphA2 receptor targeting68Ga-NODAGA-cyclic polypeptide FG01, preparation method and application | |
| CN107586317B (en) | Activatable tumor apoptosis PET imaging agent and preparation method and application thereof | |
| CN113444146A (en) | Targeted fibroblast activation protein probe, preparation method and application thereof in preparation of PET (polyethylene terephthalate) imaging agent | |
| CN108434469A (en) | A kind of HER2 affinities body68Ga markers and preparation method thereof, application | |
| CN113912607B (en) | SNAP-tag probe and preparation method and application thereof | |
| CN112999369B (en) | HER2 affinity radionuclide marker composition and application thereof | |
| CN105611948B (en) | For the diagnosticum for the treatment of of cancer effect | |
| CN116212058A (en) | Immune PET molecular imaging probe for targeting apoptosis | |
| CN109350751B (en) | EGFR-targeting polypeptide PET imaging agent and preparation method and application thereof | |
| CN114853851B (en) | Targeting PD-L1 polypeptide probe and application thereof in preparation of PET imaging agent | |
| CN115282299B (en) | TGF beta tracer, antibody probe preparation method and application | |
| CN117752824A (en) | Pretargeted tumor immune probe, bioorthogonal preparation, kit and application |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |