CN113476619B

CN113476619B - A kind of 18 F-labeled nano antibody probe and preparation method and application thereof

Info

Publication number: CN113476619B
Application number: CN202110771591.2A
Authority: CN
Inventors: 魏伟军; 王成; 刘建军
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2022-11-29
Anticipated expiration: 2041-07-08
Also published as: CN113476619A

Abstract

The invention discloses a ¹⁸ An 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 nano antibody MM01, a nano antibody MM02 or a derivative of the nano antibody MM02, and the preparation method comprises the following steps: synthesizing a small molecular compound precursor RJDJ01; preparation of ¹⁸ F labelling precursor ¹⁸ F[F]-RJDJ01; preparation of fluoro-18 by click chemistry reaction(s) ((R)) ¹⁸ F) Labeling the nano antibody probe; the probe is a CD38 specific nano antibody for detecting multiple myeloma. The invention creates a CD38 specific novel nano antibody probe ¹⁸ F[F]MM01, enabling noninvasive visualization of CD38 expression, noninvasive 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

A kind of 18 F-labeled nano antibody probe and preparation method and application thereof

Technical Field

The invention relates to the field of nuclear medicine and molecular imaging, in particular to a nuclear magnetic resonance imaging system ¹⁸ F-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 referred to as Nanobodies: (

Registered trade name of Ablynx corporation). Nanobodies are the smallest antibody units currently known to bind to the antigen of interestHas high affinity, small molecular weight and low preparation cost (can be expressed by using escherichia coli, can also be expressed by using eukaryotic expression systems such as yeast, chinese hamster ovary cells and the like), and is easy to clinically transform, popularize and apply.

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 (99mtc, t1/2= 6.02h) labeled nanobody probes targeting programmed death ligand 1 (PD-L1) have been successfully transformed into a clinical for noninvasive diagnosis of non-small cell lung cancer patients (J nuclear med 2019 (9): 1213-1220.); gallium-68 (68ga; t1/2=1.1 h) labeled nanobody probes targeting the human epidermal growth factor receptor (HER 2) have also been successfully transformed into the clinic for noninvasive diagnosis of breast cancer (J nuclear med. 2016 (1): 27-33.. The above examples illustrate 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, visualization of key pathogenic targets, screening of monoclonal antibody (mAb) treatment patients, and therapeutic effect evaluation after 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 ( ¹⁸ F; t1/2=109.8min h) is the longest used radionuclide for clinical Positron Emission Tomography (PET), the positron emission rate is up to 97%, the positron range is 0.5mm, no gamma ray is emitted, and the radionuclide is one of the best nuclides for creating a PET imaging probe. However, due to limitations such as low radiochemical yield (RCY), harsh labeling conditions (high temperature, organic solvent required), and the like, ¹⁸ f 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 prominent of which is the level of CD38 protein expression. Currently, the clinical judgment of CD38 expression levels in multiple myeloma cells relies heavily on flow cytometric detection of bone marrow punctures. However, this method is very invasive and less reproducible. Therefore, there is a need for a molecular imaging method for non-invasive visualization of CD38 expression level and early and accurate 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, ¹⁸ the F-labeled nano antibody probe has the best clinical application prospect. Currently, a variety of precursors have been used to label biological macromolecules with 18F. [ ¹⁸ F]Para-fluorobenzaldehyde (,) ¹⁸ F]FBA) is the most commonly used ¹⁸ F labeling the precursor; N-Succinimidyl-4-, [ solution of a salt of N-Succinimidyl-4 ], [ solution of a carboxylic acid ¹⁸ F]-fluorobenzoate([ ¹⁸ F]SFB) are also more common ¹⁸ F 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 = 2.0-2.5). 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. The clinical urgent need of a medicine with mild and mild labeling conditionsThe 18F labeling nano antibody method with good repeatability and high radiochemical yield is used for realizing 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 mainly 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 antibody is urgently needed, a nano antibody probe which is low in price, low in radiation dose, short in imaging period and easier for clinical transformation application 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 to realize room-temperature and efficient target-specific nano antibody ¹⁸ And F, marking.

Therefore, those skilled in the art are working on developing a CD 38-specific nanobody probe that is inexpensive, has a low radiation dose, has a short imaging period, and is more easily applied for clinical transformation ¹⁸ F[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 module ¹⁸ F, 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 easier clinical transformation application ¹⁸ F[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 diode ¹⁸ The F-labeled nano antibody probe comprises a targeting carrier of nano antibody MM01, nano antibody MM02 or a derivative of nano antibody MM02.

Further, the derivative of the nanobody MM02 includes a derivative of the MM02 nanobody modified with PEG at a fixed point.

The invention also provides a ¹⁸ The preparation method of the F-labeled nano antibody probe comprises the following steps:

step 1, synthesizing a small molecular compound precursor RJDJ01;

step 2, ¹⁸ F labeling the small molecule compound precursor RJDJ01 obtained in step 1, and preparing the precursor for the rapid click chemical labeling of the nano antibody ¹⁸ F labels the precursor, i.e. ¹⁸ F[F]-RJDJ01；

Step 3, preparation by click chemistry reaction ¹⁸ Labeling 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 obtained in the step 2 ¹⁸ F[F]RJDJ01, click chemistry substrate 2 is a nanobody, and the product of the click chemistry reaction is ¹⁸ And F, labeling the nano antibody probe.

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 the step 1 is as follows:

firstly, reacting tetraethylene glycol with paratoluensulfonyl chloride and potassium hydroxide to prepare a crude product; dissolving the crude product in dichloromethane, adding 3-hydroxy-2-nitropyridine and sodium chloride for reaction, 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 2- (2- (2- (2- ((2-nitropyridine-3-yl) oxy) ethoxy) 4-methylbenzenesulfonic acid ethyl ester with sodium azide, extracting by dichloromethane, and performing chromatography to obtain 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine, namely a small molecular compound precursor RJDJ01.

Further, the precursor in the above step 2 ¹⁸ F[F]-the chemical structural formula of RJDJ01 is:

further, the preparation in the above step 3 is performed by click chemistry ¹⁸ The F-labeled nano antibody probe comprises ¹⁸ F random labeling nano antibody preparation method or ¹⁸ F, preparing a fixed-point labeled nano antibody.

Further, the air conditioner is provided with a fan, ¹⁸ the click chemical 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.

Further, the air conditioner is provided with a fan, ¹⁸ the 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 antibody; wherein, the MM02 prepares DBCO-MM02 through the fixed-point reaction of Cysteine (Cysteine, cys, C) in a linker GGGGSCGSGSGSGSLLQS and diphenyl cyclooctyne-maleimide (DBCO-Mal, CAS #:1395786-30-7, meloPEG); or the site-directed reaction of diphenyl cyclooctyne-Amine (DBCO-Amine, CAS #:1255942-06-3, meloPEG) and Glutamine (Glutamine, gln, Q) in MM01 or MM02 linker GGGGSCGSGSGSLLQS is realized through the enzymatic reaction mediated by microbial Glutamine transaminase (mTGase), thereby preparing DBCO-MM01 or DBCO-MM02; 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 GGGGGGSCGSGSGSLLQS in a fixed-point reaction mode.

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 the sequence table and a gene sequence shown as SEQ ID No.4 in the sequence table.

Further, the probe prepared by random labeling ¹⁸ F[F]MM01 is a human CD 38-specific nanobody probe.

Further, the PEG can be of different molecular weights, i.e., 100Da,200Da,500Da,1KDa,5KDa, 10KDa, or 20KDa.

The invention also provides a ¹⁸ An application method of the F-labeled nano antibody probe is applied to detecting CD38 specific nano antibodies of multiple myeloma.

In the preferred embodiment of the present invention, the path for synthesizing RJDJ01 is described in detail;

in another preferred embodiment of the present invention, the click chemistry reaction substrate 1 is specified ¹⁸ F[F]-automated preparation of RJDJ01;

in another preferred embodiment of the present invention, the preparation of a substrate DBCO-MM01 for click chemistry reaction and the preparation of a novel CD 38-specific nanobody probe by click chemistry reaction are described in detail ¹⁸ F[F]-MM01；

In another preferred embodiment of the present invention, the detailed description is given ¹⁸ F[F]-MM01 quality control method;

in another preferred embodiment of the present invention, the detailed description is given ¹⁸ F[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 of disease and fail to reflect changes in function or molecular level of disease, and thus are not suitable for early diagnosisStage multiple myeloma has a low detection rate and lacks specificity. Fluorodeoxyglucose (F) ¹⁸ F-FDG) PET/CT examination has also been used for diagnosing multiple myeloma, but still faces 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 daratuzumab, have been approved in the United states and China for clinical treatment of multiple myeloma (blood.2018; 131 (1): 13-29.). The research and development of the 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 is based on the probe ⁶⁸ Ga[Ga]The immune PET imaging of NOTA-MMO1 achieves better detection effect, but the defects are shown as follows: ⁶⁸ ga needs to be prepared by a germanium-gallium generator, so that the probe ⁶⁸ Ga[Ga]The cost of preparing NOTA-MMO1 is high; ⁶⁸ the half-life of Ga (T1/2 = 1.1h) results in a limited range of application of the probe.

The invention creates a CD38 specific novel nano antibody probe ¹⁸ F[F]MM01, enabling noninvasive visualization of CD38 expression, further enabling noninvasive diagnosis of multiple myeloma. Novel probe ¹⁸ F[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 provides ¹⁸ F is a new method for rapidly and efficiently labeling the nano antibody, and the method effectively avoids a two-step method mediated by click chemistry ¹⁸ The 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 a ¹⁸ F-labeled CD38 specific nano antibody probe ¹⁸ F[F]And the MM01 is used for realizing express delivery and accurate detection of the multiple myeloma. Fluorine-18 ( ¹⁸ F; t1/2=109.8min h) half-life ratio ⁶⁸ Ga-length, probe application range marked by itWide in scope.

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 route of the 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 of the present invention;

FIG. 5 shows another preferred embodiment 3 of the present invention ¹⁸ F, randomly labeling a nano antibody;

FIG. 6 shows another preferred embodiment 5 of the present invention ¹⁸ F[F]Imaging of MM01 immunoPET/CT detection of multiple myeloma in situ;

FIG. 7 shows another preferred embodiment of the present invention, namely, after blocking of the daratumumab monoclonal antibody (daratumumab) of example 5 ¹⁸ F[F]MM01 immunoPET/CT imaging;

FIG. 8 shows another preferred embodiment 6 of the present invention ¹⁸ F, site-specific labeling of the nano antibody.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be made clear 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 RJDJ01:

1.1 g of tetraethylene glycol (about 5.1 mmol) was precisely weighed, dissolved in 15mL of methylene chloride, and then 2g (about 10.5 mmol) of p-toluenesulfonyl chloride and 0.59g (about 10.5 mmol) 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 4 mmol) of 3-hydroxy-2-nitropyridine and 96mg of sodium chloride (about 4 mmol), reaction overnight at room temperature, and column chromatography to give 1g (about 2.1 mmol) of ethyl 2- (2- (2- (2- ((2-nitropyridin-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate in about 51% yield.

3.940mg (about 2 mmol) of ethyl 2- (2- (2- ((2-nitropyridin-3-yl) oxy) ethoxy) 4-methylbenzenesulfonate was dissolved in 10mL of dichloromethane, and then 130mg (about 2 mmol) 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 obtain 477mg of 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine, which is RJDJ01 described in the patent, 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 2.724min; FIG. 3 is a mass spectrum measured by mass spectrometry, having the strongest peak at 341.9 m/z; FIG. 4 is a Nuclear Magnetic Resonance (NMR) chart for the characterization of RJDJ01, which indicates that the product has the correct molecular weight and chemical structure of RJDJ01.

Example 2 click chemistry reaction substrate 1 ¹⁸ F[F]Automated preparation of-RJDJ 01

The specific experimental scheme is as follows: weighing 2-2.5mg RJDJ01, dissolving in 1mL dimethyl sulfoxide (DMSO); 18F-fluoride (33.49-39.39 GBq) was transferred using a pre-equilibrated anion exchange column with potassium carbonate (3 mg) and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8 ] in 1.4mL acetonitrile]Elution of anion exchange resin (15 mg) as the Mobile phaseColumn replacement; 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 collecting ¹⁸ F-RJDJ01 to pear-shaped bottles; adding 10mL of sterilized water for injection to dilute in pear-shaped bottle ¹⁸ F[F]RJDJ01 and transfer to C18 column, extract with 3mL ethanol ¹⁸ F[F]RJDJ01, heated to 100 ℃ to evaporate ethanol, then 100uL of Phosphate Buffer (PBS) was added to resuspend ¹⁸ F[F]-RJDJ01。

Example 3 preparation of substrate for click chemistry reaction DBCO-MM01, preparation of novel CD 38-specific Nanobody Probe by click chemistry reaction ¹⁸ F[F]-MM01

The reaction principle is shown in figure 5 ¹⁸ F is shown in a schematic diagram of randomly labeling the nano antibody, preparing a click chemistry reaction substrate DBCO-MM01, and preparing a novel CD38 specific nano antibody probe through a click chemistry reaction ¹⁸ F[F]-MM01。

DBCO-NHS ester (CAS #:1353016-71-3, meloPEG) was randomly coupled to MM01 to prepare a substrate DBCO-MM01 for a click chemistry reaction. The method comprises the following specific steps: 1mg of CD38-specific nanobody MM01 was dissolved in Phosphate Buffer Solution (PBS) in a volume of about 1mL, and the pH of the nanobody solution was adjusted to 9.0-10 by adding 80-100. Mu.L of 0.1M sodium carbonate (Na 2CO 3) buffer. 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, i.e. 670nM/0.27mg. 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-MM01; the nanobody sample was concentrated using an ultrafiltration tube (Merck Millipore) with a cut-off of 10kDa, and finally DBCO-MM01 concentration was determined using NanoDrop and DBCO-MM01 was kept in a refrigerator at 4 ℃ for further use.

Preparation of novel CD38 specific nano antibody probe by click chemical reaction ¹⁸ F[F]The specific experimental protocol for MM01 is as follows: adding 20mL of DBCO-MM01 uL (320 ug) into the mixture ¹⁸ F[F]-MM01 to DBCO-MM01 nanobodiesA 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 phase ¹⁸ F-RJDJ01, purifying and collecting final product ¹⁸ F[F]-MM01。

Example 4 ¹⁸ F[F]-MM01 quality control

Aspirate 2. Mu.L ¹⁸ F[F]-MM01 was spotted on silica gel plates using 0.1M sodium citrate solution (pH = 5) as mobile phase and using a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) for the Radiochemical purity (RCP) of the probes; the integrity, RCP and immunoreactivity of the prepared nano antibody probe are further determined by High Performance Liquid Chromatography (HPLC).

Example 5 ¹⁸ F[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 3.7-7.4MBq ¹⁸ F[F]MM01 (3 per group), 1 hour after injection, anesthetizing tumor-bearing nude mice with isoflurane (2% concentration) mixed with oxygen, placing the nude mice in deep anesthesia on a PET/CT scanning bed in a supine position, continuously acquiring PET and CT images, and performing image reconstruction with IRIS system self-contained software, as shown in FIG. 6, CD38 specific nanobody probe ¹⁸ F[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, ¹⁸ F[F]MM01 immune PET imaging can accurately detect disseminated multiple myeloma, including lesions at bilateral thighbones, shines and the like. Delineating the interested regions such as tumor, heart, and major tissues and organs (liver, spleen, lung, kidney, pancreas, muscle) on the reconstructed PET image by using OsiriX Lite image processing workstation (Pixmeo SARL), calculating the radioactivity uptake value of tumor tissue and important tissues and organs by taking% ID/g (percent of injected dose gram) as unit, and obtaining high nonspecific uptake value in major excretory (kidney) and metabolic (liver) tissuesTaking; after the development is finished, taking tumor tissues and main tissues and organs to perform in-vitro biodistribution experiment, and obtaining CD38 specific nano antibody probe ¹⁸ F[F]MM01 likewise has a higher uptake.

CD38 specific immune PET imaging probe ¹⁸ F[F]MM01 specifically detects multiple myeloma, and the daratou monoclonal antibody can effectively block the pair of skeletal tissues ¹⁸ Based on that the daratuzumab can effectively reduce the uptake of a bone tissue imaging agent for the involvement of multiple myeloma, 1mg of daratuzumab is injected into each tumor-bearing mouse in the experiment 24 hours in advance, and then 3.7-7.4MBq 18F [ F ], [ F ] is injected into each tumor-bearing NCG mouse through the 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, the 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 Daratou monoclonal antibody (daratumumab) ¹⁸ F[F]The imaging result shows that after the darathey monoclonal antibody is blocked, the imaging agent uptake of the bilateral thighbones (1, 2) and the shinbones (3, 4) is obviously reduced, which indicates that the darathey monoclonal antibody effectively blocks ¹⁸ F[F]Binding site of MM01 on MM.1S tumor cells, proving that the probe ¹⁸ F[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 daratuzumab ¹⁸ F[F]A significant decrease in MM01 uptake, indicating ¹⁸ F[F]The MM01 probe is highly specific for human CD38, and ¹⁸ F[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 8 ¹⁸ The schematic diagram of the F fixed-point labeled nano antibody is shown. The specific implementation route is as follows: adding MM01 with LLQS tag at C end of 3mg and PEG-NH of 1mg ₂ (5 kDa) or 2mg PEG-NH ₂ (10 kDa) and 1mg mDissolving TGase in 1mL of PBS solution, and placing the reaction system 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 provided with Superdex ^TM Of 75 Increatase column

pure protein purification instrument (Cytiva, formerly GE Healthcare Life Science) purified and collected MM01 derivatives site-specifically 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 that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention should be within the scope of protection determined by the claims.

Sequence listing

<110> Shanghai university of traffic medical college affiliated renji hospital

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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

1. Multiple myeloma treating medicine ¹⁸ The 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 RJDJ01;

step 2, ¹⁸ F labeling the small molecule compound precursor RJDJ01 obtained in step 1, and preparing the small molecule compound precursor RJDJ01 for the rapid click chemical labeling of the nano antibody ¹⁸ F labels the precursor, i.e. ¹⁸ F[F]-RJDJ01；

Step 3, preparing the compound by click chemistry reaction ¹⁸ Labeling 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 2 ¹⁸ F[F]-RJDJ01, said click chemistry substrate 2 beingThe nano antibody, the product of the click chemistry reaction is ¹⁸ Labeling the nano antibody probe;

the chemical structural formula of the small molecule compound precursor RJDJ01 in the step 1 is as follows:

the chemical synthesis path of the small molecule compound precursor RJDJ01 in the 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 by using dichloromethane, and performing chromatography to obtain 3- (2- (2- (2- (2-azidoethoxy) ethoxy) -2-nitropyridine, namely the small molecular compound precursor RJDJ01;

the precursor in the step 2 ¹⁸ F[F]-the chemical structural formula of RJDJ01 is:

the preparation by click chemistry reaction in the step 3 ¹⁸ The F-labeled nano antibody probe comprises ¹⁸ F random labeling nano antibody preparation method or ¹⁸ F, preparing a fixed-point labeled nano antibody;

the above-mentioned ¹⁸ In the preparation method of the F random labeling nano antibody, the click chemistry substrate 2 is an MM01 specific nano antibody randomly coupled with DBCO;

the described ¹⁸ In the preparation method of the F site-specific labeled nano antibody, the click chemistry substrate 2 is the MM01 specific nano antibody coupled with the DBCO site-specific; wherein, the statorThe point coupling mode is that the point coupling reaction of diphenyl cyclooctyne-amine and glutamine in the MM01 linker GGGGSCGSGSGSLLQS is realized through the enzymatic reaction mediated by microbial glutamine transaminase (mTGase), so as to prepare DBCO-MM01; or DBCO-PEG-MM01 is prepared by microorganism glutamine transaminase (mTGase) mediated diphenyl cyclooctyne-polyethylene glycol-Amine (DBCO-PEG-Amine, meloPEG) and glutamine site-specific reaction in MM01 linker GGGGSCGSGSGSLLQS.

2. The method of claim 1 ¹⁸ The preparation method of the F-labeled nano antibody probe is characterized in that the probe is applied to the CD38 specific nano antibody for detecting multiple myeloma.