A near-infrared fluorescent ADC immune preparation and its preparation method and application

Abstract

The present invention discloses a near-infrared fluorescent ADC immune preparation and its preparation method and application. The ADC immune preparation of the present invention is a covalent conjugate of a near-infrared fluorescent dye, a carboxyl-containing cytotoxic drug and a single-domain antibody; the near-infrared fluorescent dye is a polymethyl cyanine dye with an absorption spectrum and an emission spectrum within the range of 600-900nm. The near-infrared fluorescent ADC immune preparation provided by the present invention is the first near-infrared fluorescent ADC immune preparation based on a single-domain antibody, and its single-domain antibody / near-infrared fluorescent dye / cytotoxic drug is 1mol / 1mol / 1mol, its maximum absorption is in the 600-1000nm region, and the maximum emission wavelength is in the 600-1700nm region. It has a large molar absorption coefficient and is suitable for fluorescent in vivo imaging (near-infrared one-window in vivo imaging and near-infrared two-window in vivo imaging).

Description

Technical Field

[0001] The invention relates to a near-infrared fluorescent ADC immune preparation and a preparation method and application thereof, belonging to the technical field of fluorescent imaging. Background Art

[0002] The main measures for treating cancer at this stage include chemotherapy, immunotherapy, radiotherapy, stem cell therapy, laser therapy, hyperthermia, surgery, photodynamic therapy, etc. Among them, chemotherapy is the main way to intervene in the treatment of cancer. 1 The premise of the above treatment methods is that the drugs used will not harm normal cells, but will first prevent the division process of tumor cells and kill tumor cells efficiently and quickly. Nitrogen mustard, as the first chemotherapy drug used in humans, exerts its apoptotic effect through DNA alkylation, which ultimately leads to bone marrow damage. 2 Since then, methotrexate, thioguanine, 5-fluorouracil and cytosine arabinoside (ara-C), cisplatin, actinomycin D, anthracyclines and vinca alkaloids have successively entered the cancer chemotherapy drug market. Although anticancer chemotherapy based on small molecule anticancer drugs has made progress 3 , its limited selectivity for cancer cells, pan-toxicity to biological systems, and significantly enhanced drug resistance have led to a narrowing therapeutic window. At present, researchers are focusing on improving the efficacy of cytotoxic drugs, reducing the effective dose required for treatment, and improving the selectivity for tumor tissue. Antibody-drug conjugates (ADCs) came into being. Antibody-drug conjugates (ADCs) are one of the fastest growing anticancer drugs. 4. This method effectively delivers cytotoxic drugs to cancer cells expressing the target antigen by covalently coupling them to a monoclonal antibody. The significantly improved targeting greatly reduces the probability of systemic exposure to cytotoxic drugs, thereby reducing their toxicity. ADCs are complex molecules that require precise assembly of various parts. The choice of target and monoclonal antibody, the cytotoxic drug payload, and the efficiency of the payload all play a crucial role in the safety and efficacy of ADCs. The concept of ADC was first proposed by German physician and scientist Paul Ehrlich 100 years ago. 45 years later, as Paul Ehrlich envisioned, methotrexate attached to an antibody targeted to the surface of leukemia cells constituted the first ADC drug. In the 1980s, an ADC drug based on mouse IgG molecules entered the clinical trial stage. The first chimeric and humanized monoclonal antibodies were produced in 1990. Based on highly specific monoclonal antibodies and highly cytotoxic drugs, ADC can selectively deliver powerful drugs to the vicinity of tumor cells, retaining healthy cells, greatly reducing the main clinical obstacles of traditional chemotherapy and providing a wider treatment window. Since 2013, with the rapid development of the biopharmaceutical industry, the research and development technology of fully human monoclonal antibodies has become increasingly mature, which has directly led to the ADC field becoming very active. So far, 12 ADC drugs have been approved by the FDA for clinical use. 5，6 . However, fully human monoclonal antibodies are generally in the form of IgG, with a relatively large molecular weight (150kDa) and a relatively low penetration rate into tissues or tumors, which will be an important aspect that ADC drugs need to consider. The emergence of single-domain antibodies will significantly reduce the hydrated particle size of the final ADC, thereby having better solid tumor penetration. It has the characteristics of small molecular weight (15kDa), high stability, good solubility, easy expression and low immunogenicity. It is a small molecule antibody with a simple source, low price and suitable for mass production. 7，8 .

[0003] Linkers play a key role in ADC design. Linkers must ensure that ADCs are stable in the blood to avoid releasing cytotoxic drugs in off-target tissues, must be bound to antibodies in an inactive and non-toxic state, and must not affect the normal function and physicochemical properties of the antibodies. 9 At the same time, the linker should release the cytotoxic drug when the ADC is internalized by the cell. 10. The linkers currently used can be divided into two types: cleavable and non-cleavable. Cleavable linkers are the main type of ADC linkers. Their main feature is that they are broken by differences in the internal environment (such as redox potential, pH value, etc.) and specific recognition by intracellular lysosomal enzymes). For example, acid-sensitive linkers are a group of linkers that are sensitive to acidic environments but stable in alkaline environments close to systemic circulation. When internalized by targeted tumor cells, acid-sensitive linkers, such as hydrazone groups, are hydrolyzed in lysosomes (pH 4.8) and endosomes, and the acidic tumor microenvironment (pH 5-6) will also cause them to be released to a certain extent, thereby having a certain marginal killing effect. 3，8 Lysosomal protease-sensitive linkers, also known as peptide-based linkers, are the most commonly adopted design in ADCs. 9 . For example, the valine-citrulline based linker belongs to this type of design. Another common linker is the glutathione sensitive disulfide bond. Glutathione is a low molecular weight short peptide chain containing thiol. Its normal content in the cell is (0.5-10mmol / L) and the extracellular environment is (2-20mmol / L). 11 . The concentration of glutathione in cancer cells is higher than that in normal cells. In addition, tumor cells also contain sulfides from enzyme proteins of the isomerase family, which can assist in the breaking of disulfide bonds. However, to date, the research and development and application of ADCs have mainly focused on animal experiments and tumor treatment. Its research and development costs are huge, but there is still a lack of information on the cellular mechanism of different ADC drugs, the monitoring of real-time effects in vivo, and the summary of the final efficacy and its physical and chemical properties. A real-time quantitative method is needed to evaluate them.

[0004] Optical imaging can provide intuitive visual images and quantitative dynamic biological analysis results, and has been widely recognized in biological experiments and clinical applications. At present, the detection method based on fluorescence signals is mainly used for non-invasive monitoring and detection of clinically relevant species. The accuracy of this detection method can be improved by improving the targeting accuracy and the selection of biological imaging windows. 12 At present, there are many reports confirming that the new fluorescent immunoprobes obtained by modifying antibodies with traditional FDA-approved organic dyes for clinical use, such as methylene blue (MB), fluorescein isothiocyanate (FITC) and indocyanine green (ICG), can be used for imaging of specific areas or antigens with high specificity, such as FITC13 for folate receptor α targeting, GE-13714 for c-MET targeting, and cetuximab-IRdye800cw for epidermal growth factor receptor targeting. 13The fluorescence emission of the above-mentioned immune probes is concentrated in the near infrared (NIR) region, and all show good fluorescence imaging effects.

[0005] References:

[0006] 1.Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N., Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov 2017, 16(5), 315-337.

[0007] 2.Andrew Mastal; Gray, PJ; Phillipsl, DR, Nitrogen mustard inhibits transcription and translation in a cell free system. Nucleic Acids Research1995, 23, 3508-3515.

[0008] 3.Khongorzul, P.; Ling, CJ; Khan, FU; Ihsan, AU; Zhang, J., Antibody-DrugConjugates: A Comprehensive Review. Mol Cancer Res 2020, 18(1), 3-19.

[0009] 4. Lambert, JM; Morris, CQ, Antibody-Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review. Adv Ther 2017, 34(5), 1015-1035.

[0010] 5.Moore,K.N.;Borghaei,H.;O'Malley,D.M.;Jeong,W.;Seward,S.M.;Bauer,T.M.;Perez,R.P.;Matulonis,U.A.;Running,K.L.;Zhang,X.;Ponte,J.F.;Ruiz-Soto,R.;Birrer,M.J.,Phase 1dose-escalation study of mirvetuximab soravtansine(IMGN853),a folate receptor alpha-targeting antibody-drug conjugate,inpatients with solid tumors.Cancer 2017,123(16),3080-3087.

[0011] 6.Tong,J.T.W.;Harris,P.W.R.;Brimble,M.A.;Kavianinia,I.,An Insightinto FDA Approved Antibody-Drug Conjugates for Cancer Therapy.Molecules 2021,26(19).

[0012] 7.Wu,T.;Liu,M.;Huang,H.;Sheng,Y.;Xiao,H.;Liu,Y.,Clustered nanobody-drug conjugates for targeted cancer therapy.Chem Commun(Camb)2020,56(65),9344-9347.

[0013] 8.Wu,Y.;Li,C.;Xia,S.;Tian,X.;Kong,Y.;Wang,Z.;Gu,C.;Zhang,R.;Tu,C.;Xie,Y.;Yang,Z.;Lu,L.;Jiang,S.;Ying,T.,Identification of Human Single-DomainAntibodies against SARS-CoV-2.Cell Host Microbe 2020,27(6),891-898e5.

[0014] 9.Anami,Y.;Yamazaki,C.M.;Xiong,W.;Gui,X.;Zhang,N.;An,Z.;Tsuchikama,K.,Glutamic acid-valine-citrulline linkers ensure stability and efficacy ofantibody-drug conjugates in mice.Nat Commun 2018,9(1),2512.

[0015] 10.Rossin,R.;Versteegen,R.M.;Wu,J.;Khasanov,A.;Wessels,H.J.;Steenbergen,E.J.;Ten Hoeve,W.;Janssen,H.M.;van Onzen,A.;Hudson,P.J.;Robillard,M.S.,Chemically triggered drug release from an antibody-drugconjugate leads to potent antitumour activity in mice.Nat Commun 2018,9(1),1484.

[0016] 11.Bansal,A.;Simon,M.C.,Glutathione metabolism in cancer progressionand treatment resistance.J Cell Biol 2018,217(7),2291-2298.

[0017] 12.Nani,R.R.;Gorka,A.P.;Nagaya,T.;Kobayashi,H.;Schnermann,M.J.,Near-IR Light-Mediated Cleavage of Antibody-Drug Conjugates Using CyaninePhotocages.Angew Chem Int Ed Engl 2015,54(46),13635-8.

[0018] 13.aJ.Huang, K.Pu, Angewandte Chemie 2020, 59, 11717; bG.M.van Dam, G.Themelis, LMCrane, NJHarlaar, RGPleijhuis, W.Kelder, A.Sarantopoulos, JSde Jong, HJArts, AGvan der Zee, J. Bart, PSLow, V. Ntziachristos, Naturemedicine 2011, 17, 1315. Summary of the invention

[0019] The purpose of the present invention is to provide a near-infrared fluorescent ADC immune preparation based on a single domain antibody, which has high specificity and can be stably expressed.

[0020] In order to achieve the above object, the present invention provides a near-infrared fluorescent ADC immunogen, which is a covalent conjugate of a near-infrared fluorescent dye, a carboxyl-containing cytotoxic drug and a single-domain antibody; the near-infrared fluorescent dye is a polymethyl cyanine dye with an absorption spectrum and an emission spectrum in the range of 600-900nm, and its chemical structure is shown in Formula I:

[0021]

[0022] Wherein, n1, n2, and n3 are independently selected from 0, 1, 2, or 3;

[0023] R1 and R2 are independently selected from hydrogen, alkyl, phenyl, alkanesulfonic acid or phenyl-substituted alkyl; R3 is selected from C1-12 alkylene;

[0024] X1 and X2 are independently selected from S, O, a hydrogen atom, an alkyl group, an alkylsulfonic acid group or an alkyl group substituted with a phenyl group; X3 is selected from O, S or Se.

[0025] Preferably, n1, n2, and n3 in the formula I are all 1, R1 and R2 are both phenyl, R3 is ethylene -CH2-CH2-, X1 and X2 are both -CH(CH3)2, and X3 is S, that is, the specific chemical structure of the near-infrared fluorescent dye is as shown in formula II:

[0026]

[0027] Preferably, the ADC immunopreparation is a covalent conjugate formed by a near-infrared fluorescent dye, a carboxyl-containing cytotoxic drug and a single domain antibody in a molar ratio of 1:1:1.

[0028] Preferably, the single-domain antibody is a type of single-domain antibody in which the serine in the FR3 region is specifically mutated to cysteine; the single-domain antibody is a camel-derived nanobody VHH (or nanobody), a shark-derived VNAR or a human-derived fully human nanobody, all of which have very high stability and expression yield.

[0029] Preferably, the single domain antibody is an anti-5T4 antibody.

[0030] Preferably, the anti-5T4 antibody is n501 (n501 is the application number "201611040981.8", and the invention name is a fully human single-domain antibody that specifically binds to h5T4 disclosed in the patent "Fully human single-domain antibody that specifically binds to human 5T4 antigen and its application").

[0031] Preferably, the carboxyl-containing cytotoxic drug includes auristatins peptide drugs (Auristatins); the auristatins peptide drugs include Monomethylauristatin F (MMAF).

[0032] The present invention also provides a method for preparing the above-mentioned near-infrared fluorescent ADC immune preparation, comprising the following steps:

[0033] Step 1): reacting the single domain antibody with a reducing agent to reduce the disulfide bonds in the single domain antibody, and then adding a near-infrared fluorescent dye dissolved in an organic solvent to obtain a mixture after the reaction;

[0034] Step 2): Add the mixture obtained in step 1) to a desalting column to remove residual near-infrared fluorescent dye, and then collect the supernatant by centrifugation to obtain the near-infrared two-window fluorescent immunoprobe.

[0035] Preferably, the reducing agent in step 1) is at least one of tris(2-chloroethyl) phosphate, diethyltriaminepentaacetic acid or 5,5'-dithiobis(2-nitrobenzoic acid) and tris(2-chloroethyl) phosphate, and the organic solvent is at least one of N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0036] More preferably, the reducing agent is tris(2-chloroethyl) phosphate.

[0037] Preferably, the reaction conditions in step 1) are: shaking reaction at 4-60° C. for 50-70 min.

[0038] More preferably, the reaction conditions in step 1) are: shaking reaction at 37° C. for 60 min.

[0039] Preferably, the centrifugal conditions in step 2) are: temperature 3-5°C, time 0.5-1.5 min, and rotation speed 7000-12000 r / min.

[0040] More preferably, the centrifugal conditions in step 2) are: temperature 4°C, time 1 min, and rotation speed 8000 r / min.

[0041] The present invention also provides the use of the above-mentioned near-infrared fluorescent ADC immune preparation in the preparation of near-infrared fluorescent probes or fluorescent detection kits. The near-infrared fluorescent probes or fluorescent detection kits can be used for fluorescent in vivo imaging, specifically including tumor targeted imaging, vascular imaging and in vivo circulation tracing.

[0042] The present invention also provides the use of the above-mentioned near-infrared fluorescent ADC immune preparation in the preparation of drugs for targeted treatment of tumors. The tumors include pancreatic tumors.

[0043] In the present invention, "near infrared" means infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-infrared light (MIR). The near infrared region is usually divided into two regions: near infrared region 1 (750-900nm) and near infrared region 2 (1000-1700nm). The near infrared region is the first non-visible light region discovered by humans.

[0044] In the present invention, "antibody conjugate" means that a small molecule with biological activity is connected to an antibody through a chemical link, and the antibody acts as a carrier to transport the small molecule into the target cell.

[0045] In the present invention, "antibody" means a protein composed of one or more polypeptides encoded by all or part of a generally recognized immunoglobulin gene. The recognized immunoglobulin gene, for example, in humans, includes kappa (κ), lambda (λ) and heavy chain loci, which contain numerous variable region genes, and constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. Antibodies herein are meant to include full-length antibodies and antibody fragments, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic purposes or other purposes as further specified below. The term "antibody" includes antibody fragments, which are well known in the art, such as Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, or antibody fragments produced by modifying complete antibodies or resynthesizing those antibodies using recombinant DNA technology. The term "antibody" includes monoclonal and polyclonal antibodies. The antibody may be an antagonist, an agonist, a neutralizing antibody, or an inhibitory antibody, or a stimulatory antibody. The antibody of the present invention may be a non-human antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0046] The "antigen" used in the present invention refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into an animal, which can be proteins, carbohydrates, lipids or other pathogens.

[0047] As used herein, "amino acid" means one of the 20 naturally occurring amino acids or any non-natural analogs, which may be located at a specifically defined position. In the present invention, "protein" means at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of naturally occurring amino acids and peptide bonds, or of synthetic peptide mimetic structures, which peptide mimics are "analogs". Therefore, "amino acid" or "peptide residue" as used herein means naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in (R) or (S) configuration. In a preferred embodiment, the amino acid is present in (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example to prevent or delay degradation in vivo.

[0048] Unless otherwise stated, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural referents. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, materials, methods and embodiments are illustrative only and not restrictive.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. The near-infrared fluorescent ADC immunopreparation provided by the present invention is a covalent conjugate of a near-infrared fluorescent dye, an antibody and a cytotoxic drug, with a maximum absorption in the 600-1000nm region, a maximum emission wavelength in the 600-1700nm region, a large molar absorption coefficient, and is suitable for fluorescent in vivo imaging (near-infrared one-window in vivo imaging and near-infrared two-window in vivo imaging);

[0051] 2. The fluorescent immunoprobe of the present invention is the first near-infrared fluorescent ADC immunopreparation based on a single-domain antibody; its single-domain antibody / near-infrared fluorescent dye / cytotoxic drug is 1 mol / 1 mol / 1 mol, which is very suitable for drug controlled release and mechanism research in complex biological environment systems; its maximum absorption and emission are in the near-infrared window (650-900nm) region, and it can specifically release cytotoxic drugs in an environment with acidic pH and high glutathione content (such as a tumor microenvironment), and its fluorescence intensity in the near-infrared region is greatly reduced, so that it has the advantages of real-time observation of drug controlled release, timely adjustment of drug dosage, and can provide ADC mechanism research at the cellular level, etc., and can be applied to the treatment of tumors and the exploration and research of tumor microenvironment;

[0052] 3. The fluorescent immunoprobe of the present invention has the advantages of single component, low cost, biosafety, and suitability for drug formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the general formula of the near-infrared fluorescent dye in the present invention and the final preparation process of covalent coupling with a cytotoxic drug with a carboxyl group;

[0054] Figure 2 Schematic diagram of the preparation process of the near-infrared fluorescent dye CYMMAF in Example 1;

[0055] Figure 3 This is the H NMR spectrum characterization diagram of the intermediate 4 prepared in Example 1;

[0056] Figure 4 The NMR carbon spectrum characterization diagram of the intermediate 4 prepared in Example 1;

[0057] Figure 5 This is the H NMR spectrum characterization of the intermediate 8 prepared in Example 1;

[0058] Figure 6 The NMR carbon spectrum characterization diagram of the intermediate 8 prepared in Example 1;

[0059] Figure 7 This is the H NMR spectrum characterization of the intermediate 9 prepared in Example 1;

[0060] Figure 8 This is the H NMR spectrum characterization of the intermediate 9 prepared in Example 1;

[0061] Fig. 9 This is the H NMR spectrum characterization diagram of the intermediate 10 prepared in Example 1;

[0062] Fig.10 The NMR carbon spectrum characterization diagram of the intermediate 10 prepared in Example 1;

[0063] Fig.11 This is the H NMR spectrum characterization of the intermediate 11 prepared in Example 1;

[0064] Fig.12 The NMR carbon spectrum characterization diagram of the intermediate 11 prepared in Example 1;

[0065] Fig.13 This is the H NMR spectrum characterization diagram of CYMMAF prepared in Example 1;

[0066] Fig.14 This is the carbon NMR spectrum characterization diagram of CYMMAF prepared in Example 1;

[0067] Fig.15 The synthesis process and characterization of the near-infrared fluorescent ADC immunogen in Example 2; wherein A is a schematic diagram of the process of coupling CYMMAF intermediate 10 with carboxyl-containing MMAF to obtain CYMMAF and finally covalently coupling it with n501, B is a high-performance liquid chromatogram of the prepared near-infrared fluorescent ADC immunogen, C is an SDS-PAGE gel image of n501-CYMMAF and the target antibody n501 obtained after covalent coupling of the target antibody, and D is a summary of the absorption spectra and color changes of CYMMAF at different pH values;

[0068] Fig.16The UV-visible absorption spectrum of n501-CYMMAF obtained after the near-infrared fluorescent dye CYMMAF is covalently coupled to the target antibody in Example 3 and the UV-visible absorption spectrum of CYMMAF at different concentrations. The concentration curve obtained by fitting the concentration and absorbance of CYMMAF is used to calibrate the concentration of n501-CYMMAF;

[0069] Fig.17 The controlled release conditions of n501-CYMMAF in Example 3 are screened; wherein A is a summary of ultraviolet-visible absorption spectra after adding different concentrations of cysteine ​​for 20 minutes in a buffer solution at a pH of 7.4, and it is calculated based on the fitting curve of glutathione concentration and the absorbance of n501-CYMMAF (10 μM) that, in an environment of pH 7.4, 14 times the concentration of cysteine ​​is required to quench n501-CYMMAF (10 μM) to cause drug release, B is a summary of ultraviolet absorption spectra of n501-CYMMAF (10 μM) in a buffer solution at a pH of 7.4 for 18 hours, and its maximum absorbance at 670 nm, C is a summary of ultraviolet absorption spectra of n501-CYMMAF (10 μM) in a buffer solution at a pH of 5.0, D is a summary of fluorescence spectra corresponding to A; E is a summary of fluorescence spectra corresponding to B; and F is a summary of fluorescence spectra corresponding to C;

[0070] Fig.18 The selectivity test of n501-CYMMAF (10 μM) for amino acids in Example 3; wherein A is a UV-visible absorption spectrum of n501-CYMMAF (10 μM) in a buffer solution with 14 times the equivalent of different kinds of amino acids added, B is a fluorescence spectrum of n501-CYMMAF (10 μM) in a buffer solution with 14 times the equivalent of different kinds of amino acids added, and C is a photograph of a solution of n501-CYMMAF (10 μM) with 14 times the equivalent of different kinds of amino acids added;

[0071] Fig.19 This is a graph showing the affinity test of n501-CYMMAF, n501, CYMMAF and the unrelated antibody N118 for the target antigen 5T4 in Example 3;

[0072] Fig. 20The cytotoxicity test of n501-CYMMAF in Example 3; wherein A is the summary of cell survival rates of n501-CYMMAF, n501, CYMMAF, and MMAF at different concentrations (15nM, 30nM, 60nM, 120nM, 240nM, and 480nM) after co-incubation with tumor cells for 24 hours, and B is the summary of cell survival rates of n501-CYMMAF, n501, CYMMAF, and MMAF at different concentrations (15nM, 30nM, 60nM, 120nM, 240nM, and 480nM) after co-incubation with tumor cells for 48 hours. The cell line used was the SKOV3 ovarian cancer cell line (1×10 4 ), the cell detection solution used was CCK8 solution;

[0073] Fig.21 The tumor targeting test of n501-CYMMAF in Example 3; wherein A is an imaging image of a 10-week-old mouse with a 4T1 orthotopic breast cancer model constructed with n501-CYMMAF (0.3 μmol / Kg) injected intraperitoneally for 1 hour at a 710±30 nm imaging window, and B is an imaging image of a 10-week-old mouse with a 4T1 orthotopic breast cancer model constructed with CYMMAF (0.3 μmol / Kg) injected intraperitoneally for 1 hour at a 710±30 nm imaging window, and D is an imaging image of a 10-week-old mouse with a 4T1 orthotopic breast cancer model constructed with CYMMAF (0.3 μmol / Kg) injected intraperitoneally for 1 hour at a 790±30 nm imaging window;

[0074] Fig. 22 The therapeutic effect of n501-CYMMAF on tumors in Example 3 is summarized; wherein, A is a diagram of the tumor inoculation and administration process of n501-CYMMAF, CYMMAF and PBS in the BXPC-3 subcutaneous tumor mouse model, B is a weight summary graph of three groups of mice (three mice in each group) receiving n501-CYMMAF (0.3 μmol / Kg), CYMMAF (0.3 μmol / Kg) and PBS (500 μL), and C is a tumor volume summary graph of three groups of mice (three mice in each group) receiving n501-CYMMAF (0.3 μmol / Kg), CYMMAF (0.3 μmol / Kg) and PBS (500 μL). DETAILED DESCRIPTION

[0075] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0076] Standard recombinant DNA techniques and molecular cloning techniques used in the following examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and materials and methods suitable for growing microorganisms are well known in the art. The main chemical and biological reagents were purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc., or can be prepared according to methods known in the art (a) ALA Antaris, H. Chen, K. Cheng, Y. Sun, G. Hong, C. Qu, S. Diao, Z. Deng, X. Hu, B. Zhang, X. Zhang, OK Yaghi, ZR Alamprambil, X. Hong, Z. Cheng H. Dai, Nat. Mater. 2016, 15, 235-242.; (b) KK Maiti, A. Samanta, M. Vendrell, KS Oh, M. Olivo Y. T. Chang, Chem. Commun. 2011, 47, 3514-3516.

[0077] The preparation method of the near-infrared fluorescent dye for covalently coupling cytotoxic drugs and single-domain antibodies in the following embodiments of the present invention is as follows: Figure 1 As shown, the specific steps include:

[0078] (1) Preparation of intermediate 2

[0079] Phosphorus oxychloride (37mL, 39.7mol) was mixed with dichloromethane (35mL), and slowly added dropwise to a mixture of N,N'-dimethylformamide (20mL) and dichloromethane (20mL) in an ice-water bath. After the addition, cyclohexanone (10g, 100mmol) was quickly added to the above system using a syringe. Heat to 120°C and reflux for 2 hours. Pour into a 400mL beaker, cool in an ice bath, add ice cubes with a volume of about 500mL, and separate into layers. Let stand overnight in the freezer layer (-4°C). A yellow solid (i.e., compound 1) was obtained. Aniline and ethanol were mixed in ice water (1 / 1; v / v), and compound 1 was added after stirring for 1 hour. The molar ratio of aniline to compound 1 was 2:1. Hydrochloric acid solution mixed with ice water was poured into an ice bath environment. After mixing and reacting for 1 hour, the mixture was allowed to stand in a refrigerator at 4°C overnight. A purple-red solid was obtained by filtration, and washed with petroleum ether (30 mL×3) to obtain intermediate 2.

[0080] (2) Preparation of intermediate 4

[0081] Compound 3 is dissolved in solvent 1 (one or more of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide and N,N-dimethylacetamide), and added dropwise into an eggplant-shaped bottle containing sultone. The reaction temperature is 40-120° C. (preferably 110° C.) and the reaction time is 1-3 hours (preferably 2 hours) to obtain intermediate 4.

[0082] (2) Preparation of Intermediate 5

[0083] The intermediate 2 and the base are dissolved in the solvent 2, and added in batches to the eggplant-shaped bottle containing the intermediate 4 and stirred. The reaction temperature is 40-90°C (preferably 80°C) and the reaction time is 40-100 minutes (preferably 60 minutes) to obtain the intermediate 5. The base is selected from various organic bases or inorganic bases, preferably at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium acetate, DIPEA, DEA and TEA. The solvent 2 is selected from at least one of acetic anhydride, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0084] (3) Preparation of intermediate 7

[0085] Compound 6 and compound b are ground in a solid state until they are uniformly mixed. The reaction temperature is 100-200° C. (preferably 140° C.) and the reaction time is 1-3 hours (preferably 2 hours) to obtain intermediate 7.

[0086] (4) Preparation of Intermediate 8

[0087] Compound 7 is dissolved in a solvent 3 containing (BOC)2O and an ammonium salt, the reaction temperature is 40-90°C (preferably 60°C), and the reaction time is 4-8 hours (preferably 6 hours) to obtain an intermediate 8. The ammonium salt is selected from at least one of triethylamine, diethylamine and DIPEA, preferably triethylamine, and the solvent 3 is selected from at least one of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably chloroform.

[0088] (5) Preparation of Intermediate 9

[0089] The intermediate 8 and the base are dissolved in the solvent 4, added in batches to the eggplant-shaped bottle containing the intermediate 5 and stirred, the reaction temperature is 40-90°C (preferably 80°C), the reaction time is 40-100 minutes (preferably 60 minutes), and the compound 9 is obtained. The base is selected from various organic bases or inorganic bases, preferably at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium acetate, DIPEA, DEA and TEA. The solvent 4 is selected from at least one of acetic anhydride, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0090] (6) Preparation of Intermediate 10

[0091] After the intermediate 9 is dissolved in the solvent 5, it is added to the eggplant-shaped bottle containing the acid, and the reaction is stirred at a temperature of 40-90° C. (preferably 60° C.) and a reaction time of 4-8 hours (preferably 6 hours) to obtain the intermediate 10. The acid is selected from various organic acids or inorganic acids, preferably at least one of trifluoroacetic acid, acetic acid, formic acid and hydrochloric acid, and the solvent 5 is selected from at least one of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably chloroform.

[0092] (7) Preparation of Compound 11

[0093] After dissolving the intermediate 10 in the solvent 6, the mixture is added to the eggplant-shaped bottle containing the compound c and the base, and the reaction is stirred. The reaction temperature is 25-60°C (preferably 37°C) and the reaction time is 4-8 hours (preferably 6 hours) to obtain the compound 11. The base is selected from various organic bases or inorganic bases, preferably at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium acetate, DIPEA, DEA and TEA. The solvent 6 is selected from at least one of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably chloroform.

[0094] (8) Preparation of Compound 12

[0095] The intermediate 11 and the base are added to a round-bottomed flask containing a diprimary amine compound and a solvent 7, and the reaction is stirred. The reaction temperature is 40-100° C. (preferably 80° C.) and the reaction time is 4-8 hours (preferably 6 hours). The base is selected from various organic bases or inorganic bases, preferably at least one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium acetate, DIPEA, DEA and TEA. The solvent 7 is selected from at least one of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably dichloromethane.

[0096] In an embodiment of the present invention, the preparation method of the conjugate of near-infrared fluorescent dye and cytotoxic drug (CYMMAF) comprises the following steps:

[0097] The near-infrared fluorescent dye is dissolved in a solvent, and a condensing agent and a cytotoxic drug MMAF are added. The reaction time is 1 to 6 hours, preferably two hours, and the reaction temperature is 0-100°C, preferably room temperature. The solvent is selected from at least one of toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably DMF; the condensing agent is selected from at least one of EDC, DCC and DIC, preferably EDC.

[0098] The preparation method of the near-infrared fluorescent ADC immunopreparation (covalent conjugate of near-infrared fluorescent dye, cytotoxic drug and single domain antibody) in the embodiment of the present invention comprises the following steps:

[0099] (1) After the single domain antibody and the reducing agent are mixed, the reaction is shaken for a period of time, and the conjugate of the near-infrared fluorescent dye and the cytotoxic drug is dissolved in an organic solvent and added in batches to the mixed solution containing the antibody and the reducing agent to obtain a mixture. The reducing agent is used to destroy the disulfide bond of the antibody, wherein the reducing agent is selected from various organic and inorganic reducing agents, preferably at least one of tris(2-chloroethyl) phosphate, diethyltriaminepentaacetic acid and 5,5'-dithiobis(2-nitrobenzoic acid). The organic solvent is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide, the reaction temperature is 4-60°C, preferably 4°C, and the reaction time is 40-100 minutes, preferably 60 minutes.

[0100] (2) Add the mixture obtained in step (1) to a desalting column. Collect the fraction corresponding to the color of the small molecule dye, centrifuge for a period of time, and collect the supernatant. The centrifugation temperature is preferably 4°C, the centrifugation time is preferably 1 minute, and the centrifugation speed is preferably 12000 rpm. Obtain a conjugate of the target near-infrared fluorescent dye with the cytotoxic drug and the single-domain antibody.

[0101] Example 1

[0102] This embodiment provides a method for preparing a conjugate of a near-infrared fluorescent dye and a cytotoxic drug, CYMMAF. Figure 2 As shown, the specific steps include:

[0103] (1) Preparation of intermediate 4

[0104] Compound 3 (2.09 g, 10 mmol) was dissolved in 5 mL of toluene and added dropwise into an eggplant-shaped bottle containing 1,3-propane sultone. The mixture was reacted at 110° C. for 8 hours. The dark grey solid was collected by filtration and washed three times with 20 mL of chloroform to obtain intermediate 4. Figure 3 and Figure 4 The H-NMR and C-NMR spectra of intermediate 4 are shown respectively. Compound 4: 1 H NMR (400MHz, MeOD) δ8.30 (d, J=8.5Hz, 1H), 8.21 (d, J=9.0Hz, 1H), 8.12 (dd, J=8.5, 3.6Hz, 2H), 7.78 (t, J=7 .4Hz, 1H), 7.69 (t, J=7.6Hz, 1H), 4.88–4.83 (m, 5H), 3.05 (t, J=6.5Hz, 2H), 2.47–2.38 (m, 2H), 1.83 (s, 6H); 13 C NMR (101MHz, MeOD): δ196.69, 138.49, 137.30, 133.82, 131.07, 129.67, 128.27, 1 27.72, 127.27, 123.01, 112.59, 69.44, 55.94, 43.72, 43.68, 23.48, 23.41, 21.01.

[0105] (2) Preparation of Intermediate 5

[0106] Intermediate 4 (3.31 g, 10 mmol) and compound 2 (3.22 g, 10 mmol) were added to a 50 mL round bottom bottle containing acetic anhydride (3 mL) and potassium acetate (0.98 g, 10 mmol). The entire mixture was heated to 70 ° C and maintained for 1 hour, cooled to room temperature, and poured into a saturated sodium bicarbonate solution. The red precipitate was washed with ether.

[0107] (3) Preparation of intermediate 6

[0108] Compound 3 (4.18 g, 20 mmol) and 2-bromoethylamine hydrobromide (2.04 g, 10 mmol) were ground and added to a round-bottom flask, heated to 130°C for 8 hours, cooled to room temperature, and washed with 30 mL of chloroform to obtain a white precipitate.

[0109] (4) Preparation of intermediate 7

[0110] Intermediate 6 (3.48 g, 10 mmol) and di-tert-butyl dicarbonate (2.18 g, 10 mmol) were added to 30 mL of chloroform containing N, N'-diisopropylethylamine (3.23 g, 25 mmol). The mixture was heated to 60°C for 6 hours. The final solution was washed with water and then extracted with ether. The final product was separated by column chromatography with eluent CH2Cl2 / MeOH (50 / 1; v / v) to obtain intermediate 7.

[0111] (5) Preparation of Intermediate 8

[0112] Intermediate 5 (0.602 g, 1 mmol) and intermediate 7 (0.353 g, 1 mmol) were added to a 50 mL round bottom flask containing acetic anhydride (3 mL) and potassium acetate (0.098 g, 10 mmol). The mixture was heated to 80°C for 10 minutes, turned green, cooled to room temperature, and washed with ether. The green precipitate was purified by chromatography with CH2Cl2 / MeOH (20 / 1; v / v) as the eluent to obtain intermediate 8. Figure 5 and Figure 6 The intermediates 8(C 49 H 56 The H-NMR and C-NMR characterization results of ClN3O5S). 1 H NMR (400MHz, CDCl3) δ8.45 (dd, J=46.0, 14.0Hz, 3H), 8.11 (t, J=8.3Hz, 2H), 7.93 (dd, J=19.1 , 8.2Hz, 4H), 7.64–7.55 (m, 3H), 7.51–7.40 (m, 3H), 6.72 (d, J=14.5Hz, 1H), 6.21 (s, 1H), 5.8 6(s, 1H), 4.68-4.58(m, 2H), 4.36–4.26(m, 2H), 3.37(s, 3H), 3.12–3.06(m, 2H), 2.92–2.85( m, 2H), 2.73 (t, J=9.6Hz, 2H), 2.40 (s, 2H), 2.16 (s, 2H), 2.00 (d, J=4.5Hz, 12H), 1.42 (s, 9H);

[0113] 13C NMR (101MHz, CDCl3) δ207.16, 174.35, 172.87, 156.60, 149.97, 144.48, 142.84, 139.68, 139.56, 134. 05, 133.46, 132.06, 131.82, 131.03, 130.80, 130.13, 128.11, 128.04, 128.01, 127.69, 127.61, 127.1 3, 125.13, 124.87, 122.02, 111.23, 110.89, 102.05, 100.09, 79.08, 77.48, 77.16, 76.85, 58.09, 53.52, 51.19, 50.89, 47.78, 43.84, 37.97, 30.93, 28.50, 27.83, 27.69, 27.66, 26.47, 23.97, 20.95, 18.40.

[0114] (6) Preparation of Intermediate 9

[0115] Intermediate 8 was added to TFA / CH2Cl2 (1 / 20; v / v) and stirred at room temperature for another 2 hours to obtain a green mixed solution. The green mixed solution was removed by rotary evaporation to remove excess TFA and CH2Cl2, and the obtained solid was washed with water and filtered. The filtered solid was purified by chromatography using an eluent of CH2Cl2 / MeOH (4 / 1; v / v). Figure 7 and Figure 8 It is intermediate 9 (C 44 H 48 ClN3O3S) was characterized by H-NMR and C-NMR. 1H NMR (400MHz, DMSO) δ8.47 (d, J=14.3Hz, 1H), 8.37–8.23 (m, 3H), 8.18–8.01 (m, 4H), 7.94 (d, J=8.6Hz, 2H), 7.83–7. 77 (m, 2H), 7.74 (d, J = 9.3Hz, 1H), 7.72–7.67 (m, 1H), 7.66–7.61 (m, 1H), 7.57 (t, J = 8.2Hz, 1H), 7.48 (t, J = 8.7Hz, 1H ), 6.81(d, J=14.4Hz, 1H), 6.24(d, J=13.4Hz, 1H), 4.66–4.58(m, 2H), 4.39–4.29(m, 2H), 2.99(s, 2H), 2.88–2.81( m, 2H), 2.80–2.72 (m, 2H), 2.63 (t, J=6.1Hz, 2H), 2.16–2.01 (m, 4H), 1.98 (d, J=7.5Hz, 10H), 1.90 (d, J=6.4Hz, 3H);

[0116] 13 C NMR (101MHz, DMSO) δ175.66, 172.09, 158.71, 147.86, 144.28, 140.99, 140.29, 139.92, 135.0 7, 133.12, 132.29, 131.65, 131.11, 130.80, 130.42, 130.35, 128.37, 128.17, 128.09, 127.80 , 127.73, 126.53, 125.87, 125.09, 122.88, 122.59, 118.34, 115.41, 112.51, 111.75, 103.96, 99.95, 51.64, 50.70, 48.13, 43.93, 41.27, 36.98, 27.57, 27.41, 26.52, 25.82, 24.42, 21.05.

[0117] (7) Preparation of Intermediate 10

[0118] Intermediate 9 (0.360 g, 0.5 mmol) and DIPEA (0.064 g, 0.5 mmol) were added to a round bottom bottle containing TSTU (0.150 g, 0.5 mmol), 3-maleimidopropionic acid (0.085 g, 0.5 mmol) and 30 mL CH2Cl2. All stirred for 6 hours and followed by TLC thin plate chromatography. Then use eluent CH2Cl2 / MeOH (15 / 1; v / v). Fig. 9 and Fig.10 The intermediate 10(C51 H 53 ClN4O6S) was characterized by H-NMR and C-NMR. 1 H NMR (400MHz, DMSO) δ8.40 (d, J=14.2Hz, 1H), 8.27 (dd, J=14.4, 9.6Hz, 2H), 8.14–7.99 (m, 4H), 7.88 (d, J=9.0Hz, 1H), 7.69 ( d, J=8.1Hz, 1H), 7.67–7.58 (m, 2H), 7.55–7.49 (m, 1H), 7.49–7.44 (m, 1H), 6.67 (d, J=14.8Hz, 1H), 6.23 (d, J=14.7Hz, 1H), 4.59–4.50 (m, 2H), 4.31–4.21 (m, 2H), 3.59 (t, J=7.1Hz, 2H), 3.13 (s, 7H), 2.81–2.75 (m, 2H), 2.74–2.68 (m, 2H), 2.58 (dd, J=12.3, 6.4Hz, 2H), 2.34 (t, J=8.0Hz, 2H), 2.12–2.02 (m, 2H), 1.93 (d, J=5.0Hz, 7H), 1.90–1.80 (m, 4H), 1.26–1.18 (m, 4H);

[0119] 13 C NMR (101MHz, DMSO) δ175.01, 172.63, 171.25, 170.03, 147.85, 143.72, 141.45, 140.31, 140.06, 135.06, 1 34.71, 133.46, 132.16, 131.73, 131.05, 130.43, 128.30, 128.19, 128.03, 127.86, 127.46, 126.48, 125.7 0, 122.83, 122.66, 112.45, 111.90, 103.28, 100.54, 53.96, 51.46, 50.85, 49.06, 48.16, 40.62, 40.41, 40.20, 39.99, 39.78, 39.57, 39.36, 36.49, 34.49, 27.64, 27.53, 27.46, 26.47, 25.70, 24.39, 18.49, 17.17.

[0120] (7) Preparation of Intermediate 11

[0121] Intermediate 10 (0.435 g, 0.5 mmol) and TEA (0.01 g, 0.1 mmol) were added to a round-bottomed bottle containing cystamine dihydrochloride (0.115 g, 0.5 mmol) and 30 mL CH2Cl2. The reaction was stirred for 6 hours and followed by TLC thin plate chromatography. Then, the eluent CH2Cl2 / MeOH (15 / 1; v / v) was used to obtain a blue solid. Fig.11 and Fig.12 The intermediates 11(C 55 H 64 N6O6S3) was characterized by H-NMR and C-NMR. 1 H NMR (400MHz, DMSO) δ8.34 (d, J=14.3Hz, 1H), 8.26 (d, J=8.7Hz, 1H), 8.20 (d, J=9.5 Hz, 1H), 8.12 (d, J=8.9Hz, 3H), 8.04 (dd, J=11.1, 8.7Hz, 1H), 7.93 (d, J=8.6Hz, 3H ), 7.83 (d, J=8.8Hz, 1H), 7.77 (d, J=12.2Hz, 1H), 7.61 (d, J=8.9Hz, 2H), 7.52 (dd, J=13.9, 5.6Hz, 2H), 7.47(d, J=7.4Hz, 1H), 7.38–7.32(m, 2H), 6.52(d, J=14.9Hz, 1H), 5.97(s, 2H), 4.51–4.44(m, 1H), 4.23(s, 3H), 4.04(d, J=3.7Hz, 2H), 3.15(dd , J=12.4, 6.9Hz, 2H), 3.01 (dd, J=14.2, 7.1Hz, 3H), 2.95–2.89 (m, 2H), 2.79–2.72 (m, 1H), 2.64–2.57 (m, 3H), 2.56–2.50 (m, 3H), 2.05–1.96 (m, 4H), 1.92 (s, 3H), 1. 87(s, 7H), 1.81(d, J=5.6Hz, 2H), 1.75–1.69(m, 2H), 1.17(dd, J=12.9, 5.5Hz, 4H);

[0122] 13C NMR (101MHz, DMSO) δ173.86, 169.21, 147.86, 145.07, 142.68, 141.09, 140.26, 134.08, 131.93, 131.43, 130.94 , 130.83, 130.41, 130.33, 130.30, 130.13, 128.40, 128.22, 127.95, 127.76, 127.07, 125.43, 124.00, 122.21, 1 12.30, 111.40, 102.03, 70.24, 51.14, 49.59, 48.64, 48.25, 45.78, 38.30, 34.56, 29.52, 29.48, 29.46, 29.27, 29.14, 29.03, 28.74, 28.32, 27.52, 27.09, 27.06, 27.02, 26.52, 25.28, 24.20, 23.45, 22.60, 21.87, 14.40, 8.88.

[0123] (8) Preparation of CYMMAF:

[0124] Cytotoxic drug MMAF (10 mg, 0.014 mmol) and EDC·HCl (2.62 mg, 0.014 mmol) were dissolved in anhydrous DMF and stirred at room temperature for 1 hour. Near-infrared fluorescent dye intermediate 11 (20 mg, 0.2 mmol) was added and reacted at room temperature for 2 hours. CYMMAF was separated by column chromatography. Fig.13 and Fig.14 They are CYMMAF(C 94 H 127 N 11 O 13 S3) were characterized by H-NMR and C-NMR spectra. 1H NMR(400MHz,dmso)δ8.38(d,J=15.1Hz,1H),8.30(d,J=8.1Hz,1H),8.15(d,J=8.9Hz,2H),8.11–8.03(m,2H),7.96(d,J=9.4Hz,3H),7.91(d,J=10.5Hz,1H),7.89–7.76(m,3H),7.64(d,J=9.0Hz,2H),7.59–7.49(m,3H),7.42–7.35(m,2H),7.15(t,J=15.8Hz,5H),6.56(d,J=14.3Hz,1H),5.99(s,2H),4.73(s,1H),4.69–4.63(m,1H),4.62–4.56(m,1H),4.55–4.48(m,1H),4.36(s,1H),4.25(s,3H),4.07(s,2H),3.98(s,2H),3.76(d,J=9.3Hz,1H),3.59–3.56(m,1H),3.52(d,J=8.7Hz,1H),3.19(dd,J=21.0,8.7Hz,9H),3.08(s,2H),2.97(t,J=5.7Hz,2H),2.93–2.83(m,3H),2.82–2.76(m,1H),2.73(d,J=6.6Hz,1H),2.66–2.60(m,3H),2.58(d,J=9.6Hz,3H),2.42(dd,J=19.4,8.2Hz,3H),2.35(s,1H),2.31–2.20(m,3H),2.16(d,J=7.4Hz,2H),2.04(dd,J=18.7,10.8Hz,5H),1.96(s,3H),1.87(dd,J=21.9,6.8Hz,9H),1.80–1.71(m,4H),1.62–1.55(m,2H),1.47(d,J=5.8Hz,1H),1.39(d,J=3.8Hz,1H),1.33(s,1H),1.23(s,6H),1.14(d,J=6.3Hz,1H),1.02(dd,J=10.6,6.7Hz,3H),0.92(d,J=6.5Hz,1H),0.90–0.81(m,9H),0.80–0.71(m,5H);

[0125] 13C NMR (126MHz, DMSO) δ173.63, 173.25, 169.92, 169.84, 169.14, 168.63, 147.10, 142.40, 142.0 8, 141.55, 141.44, 140.50, 139.83, 139.61, 138.17, 133.69, 133.21, 131.41, 131.26, 130.76 , 130.39, 130.25, 130.06, 129.71, 129.53, 127.77, 127.67, 127.60, 127.36, 127.33, 127.17, 126.54, 126.04, 124.93, 124.75, 123.41, 122.15, 121.62, 120.11, 111.76, 111.47, 110.83, 11 0.57, 108.81, 101.99, 101.45, 100.90, 94.75, 69.65, 54.83, 50.65, 50.55, 49.03, 48.46, 48.04, 47.60, 45.16, 43.40, 42.99, 41.97, 41.51, 41.09, 37.68, 37.61, 36.44, 35.87, 34.00, 33.7 4, 33.54, 31.16, 30.66, 28.95, 28.87, 28.70, 28.57, 28.42, 27.73, 27.52, 26.90, 26.70, 26.44, 25.92, 25.83, 24.62, 24.45, 24.37, 23.67, 22.87, 22.43, 22.33, 21.34, 20.48, 13.85, 8.29.

[0126] Example 2

[0127] This example provides the synthesis, purification and characterization of a near-infrared fluorescent ADC immunogen n501-CYMMAF:

[0128] Preparation process Fig.15As shown in A, the reducing agent TCEP (purchased from Merck Sigma-Aldrich Chemical Reagents) and the anti-5T4 antibody n501 (n501 is the antibody disclosed in the patent with application number "201611040981.8" and invention name "Fully humanized single-domain antibody specifically binding to human 5T4 antigen and its application") were mixed in a molar ratio of 3:1, reacted at 4°C for 1 hour to open the disulfide bonds between the n501 bonds, and CYMMAF (2 mg) dissolved in 30 μL DMSO was added in batches, mixed and shaken at 4°C, reacted for 1 hour, and the residual CYMMAF was removed by a Thermo Scientific Zeba desalting column. The obtained n501-CYMMAF was centrifuged at 12000rpm for 1 minute, and the blue supernatant was taken. Its purity was confirmed by HPLC and SDS-PAGE protein electrophoresis (see attached Fig.15 BC), and the affinity parameters were obtained by ELISA (see Appendix Fig.19 ) (For specific detection methods, see the literature Cell Host Microbe 2020, 27, 891-898e895; Nat. Mater. 2016, 15, 235-242.).

[0129] Attached Fig.15 BC is the SDS-PAGE gel image (C) and HPLC image (B) of ICGM-n501 and target antibody n501 obtained by covalently coupling the near-infrared fluorescent dye CYMMAF with the target antibody. The results show that the obtained product n501-CYMMAF is stable and uniform. Fig.15 D is the change in the absorption spectrum of CYMMAF at different pH values ​​and the corresponding change in solution color. CYMMAF contains a secondary amine group that is sensitive to pH and will gradually be oxidized in acidic and alkaline environments, thereby promoting the release of loaded drugs.

[0130] Example 3

[0131] This example provides a performance test of the near-infrared fluorescent ADC immunopreparation n501-CYMMAF synthesized in Example 2:

[0132] (1) Concentration calibration of n501-CYMMAF

[0133] Fig.16The UV-visible spectrum and near-infrared two-window fluorescence emission spectrum of n501-CYMMAF obtained after the fluorescent dye CYMMAF was covalently coupled to the target antibody; the concentration of n501-CYMMAF was 2.86 μM, and the excitation light source was xenon lamp excitation (Beijing Pinao Optoelectronics Technology Co., Ltd.). The absorption peak of CYMMAF in PBS is 673 nm, the fluorescence peak is 730 nm, and the absorption peak of n501-CYMMAF is red-shifted to 685 nm. The concentration curve obtained based on the concentration and absorbance fitting of CYMMAF is used to calibrate the concentration of n501-CYMMAF. The n501-CYMMAF used here to detect UV-visible absorption is 0.35 mg / mL based on the absorption of the antibody at 260 nm and 280 nm.

[0134] (2) Screening of drug controlled release conditions for n501-CYMMAF

[0135] Fig.17 A is in a buffer solution with a pH value of 7.4. With the addition of glutathione (0-14 equiv), the absorbance of n501-CYMMAF (10 μM) at 673 nm gradually decreases. The entire quenching process takes 10 hours and requires 14 times the equivalent of cysteine ​​(140 μM). Since n501-CYMMAF is an ADC immunopreparation based on a single domain antibody, its half-life is relatively short, only 1-2 hours. The release amount of n501-CYMMAF at normal blood pH is relatively small, and its fluorescence spectrum ( Fig.17 D) The same conclusion can be reached. Fig.17 B shows the summary of the UV absorption spectra of n501-CYMMAF (10 μM) in a buffer solution with a pH value of 7.4 for 18 hours. Its absorbance at 673 nm remains basically unchanged within 18 hours. Therefore, under the condition of blood pH, n501-CYMMAF basically does not release cytotoxic drugs. Its fluorescence spectrum ( Fig.17 E) The same conclusion can be reached; Fig.17 C is the summary of the UV absorption spectrum of n501-CYMMAF (10 μM) in a buffer solution with a pH of 5.0. CYMMAF has a disulfide bond that can be cleaved by glutathione (cysteine) reduction, and a secondary amine group that is sensitive to pH changes ( Fig.17 D). After CYMMAF was covalently coupled to n501, the group still maintained its sensitive property to pH value. Therefore, in the buffer solution of pH 5.0, the absorbance of n501-CYMMAF at 673nm decreased significantly, and a new absorption peak appeared at 543nm ( Fig.17 C), which corresponds to the absorption peak of the oxidation product of the heptamethine cyanine dye. Its fluorescence intensity at 730 nm also decreases and a small blue shift occurs ( Fig.17 F).

[0136] (3) Selective test of n501-CYMMAF for amino acids:

[0137] Fig.18 In order to test the selectivity of n501-CYMMAF (10 μM) for amino acids in Example 3, 15 kinds of amino acids were added to n501-CYMMAF in sequence at a concentration of 14 times the equivalent. The UV-visible absorption spectrum and the corresponding fluorescence spectrum are shown in FIG. Fig.18 As shown in AB, n501-CYMMAF exhibits high selectivity for thiol-containing cysteine ​​and glutathione, and its absorbance at 673 nm only fluctuates slightly with the addition of the remaining 13 amino acids (proline, threonine, sarcosine, alanine, pyroglutamic acid, serine, tryptophan, valine, histidine, phenylalanine, leucine, isoleucine, and methionine). Figure C is a photograph of n501-CYMMAF (10 μM) solutions to which 14 times the equivalent of different types of amino acids (proline, threonine, sarcosine, alanine, pyroglutamic acid, serine, tryptophan, valine, histidine, phenylalanine, leucine, isoleucine, and methionine) were added, and all of them were still bright blue solutions, except for the n501-CYMMAF (10 μM) solution with cysteine ​​added, which turned into light blue, while the n501-CYMMAF (10 μM) solution with glutathione added turned into a transparent colorless solution.

[0138] (4) Detection of affinity of n501-CYMMAF for antigen:

[0139] Affinity test of n501-CYMMAF, n501, CYMMAF and irrelevant antibody N118 to target antigen (5T4 antigen, purchased from Biopac Biotech, Inc.), Fig.19 This is an affinity test chart based on enzyme-linked immunosorbent assay (ELISA) (for specific detection methods and steps, see the reference Cell Host Microbe 2020). The affinity test results show that the covalent coupling of antibody n501 and fluorescent dye ICGM has no effect on affinity.

[0140] (5) n501-CYMMAF cytotoxicity test

[0141] Fig. 20 The cytotoxicity test results of n501-CYMMAF in Example 3 were obtained by seeding SKOV3 cells (1×10 4) were added to a 96-well plate with different concentrations of n501-CYMMAF, n501, CYMMAF, and MMAF (15nM, 30nM, 60nM, 120nM, 240nM, and 480nM) and incubated for 24 hours. A control group was left on both sides of the 96-well plate and only culture medium was added. After culture under the same conditions, the supernatant was aspirated, and 10μL CCK8 reagent was added to each well (per 100μL cell culture medium). After incubation for 4 hours, the OD value at 450nm was measured as the experimental value, and the OD value at 600nm was measured as the reference value. The cell survival rate was calculated as follows:

[0142]

[0143] The results are as follows Fig. 20 As shown, in the 24-hour experimental group ( Fig. 20 A), MMAF and CYMMAF showed significant killing effects, and the cell survival rates of n501-CYMMAF and n501 groups were higher, confirming that the drug release in n501-CYMMAF was lower in the first 24 hours. Fig. 20 B), n501-CYMMAF, CYMMAF and MMAF all showed significant killing effects, confirming the successful release of MMAF drug.

[0144] (6) Tumor targeting test of n501-CYMMAF

[0145] Fig.21 The results of the tumor targeting test of n501-CYMMAF are shown below, with CYMMAF as the control. n501-CYMMAF (0.3 μmol / Kg) was injected intraperitoneally into 10-week-old mice with 4T1 orthotopic breast cancer model. The images at 710±30 nm were taken 1 hour later. Fig.21 As shown in A, the imaging image in the 790±30nm imaging window is as follows Fig.21 As shown in B. In these two imaging windows, the fluorescence signal is concentrated around the tumor on the left side of the abdomen. Since it is intraperitoneal injection, the single dose can be adjusted to a higher level, but the complex peritoneal delivery system in the abdominal cavity will cause the fluorescence signal to be distributed throughout the peritoneum, resulting in a higher background signal. Fig.21 Compared with the control group injected with CYMMAF only in C, it can be observed that CYMMAF is basically concentrated only in the abdomen and cannot be selectively concentrated around the tumor. Fig.21 Figure D is its imaging image in the 790±30nm imaging window, and a similar conclusion can be drawn.

[0146] (7) Summary of the therapeutic effects of n501-CYMMAF on tumors

[0147] BXPC-3(1×106 ) Pancreatic cancer cells were implanted subcutaneously into 7-week-old SPF female balb / c mice. One week later, the mice were divided into three groups: n501-CYMMAF, CYMMAF, and PBS. Fig. 22 As shown in A, mice were dosed once every three days, and each dose consisted of n501-CYMMAF (0.3 μmol / Kg), CYMMAF (0.3 μmol / Kg) and an equal volume of PBS (500 μL). The body weights of the mice were tracked and summarized ( Fig. 22 B), the volume of mice receiving n501-CYMMAF showed a significant downward trend, while mice given CYMMAF and PBS showed a small fluctuation. Accordingly, the tumor volume of mice was measured with a vernier caliper and summarized as follows Fig. 22 As shown in C, the tumor volume of the PBS group showed a slight increase (from 99.21 mm 3 Increased to 111.10mm 3 ), while the CYMMAF group decreased significantly and then remained the same (from 128.03mm 3 Down to 59.71mm 3 , the final value is 44.37mm 3 ), the tumor volume of the n501-CYMMAF group decreased to 7.8% of the initial volume (from 118.47 mm 3 Down to 9.13mm 3 ), showing significant therapeutic effect.

[0148] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent ADC immune preparation, characterized in that: The ADC immune preparation is a covalent conjugate of a near-infrared fluorescent dye, a carboxyl-containing cytotoxic drug and a single-domain antibody; the near-infrared fluorescent dye is a polymethylcyanine dye with an absorption spectrum and an emission spectrum within the range of 600-900nm, and its chemical structure is shown in Formula I: Wherein, n1, n2, n3 are independently selected from 0, 1, 2 or 3; R1 and R2 are both phenyl, R3 is ethylene -CH2-CH2-, X1 and X2 are both -CH(CH3)2, and X3 is S.

2. The near-infrared fluorescent ADC immune preparation according to claim 1, characterized in that In the formula I, n1, n2, and n3 are all 1, that is, the specific chemical structure of the near-infrared fluorescent dye is as shown in formula II:

3. The near-infrared fluorescent ADC immune preparation according to claim 1, characterized in that: The ADC immune preparation is a covalent conjugate formed by a near-infrared fluorescent dye, a carboxyl-containing cytotoxic drug and a single-domain antibody in a molar ratio of 1:1:

1.

4. The near-infrared fluorescent ADC immune preparation according to claim 1, characterized in that The single domain antibody is a type of single domain antibody in which the serine in the FR3 region is specifically mutated to cysteine.

5. The near-infrared fluorescent ADC immune preparation according to claim 4, characterized in that: The single domain antibody is an anti-5T4 antibody.

6. The near-infrared fluorescent ADC immune preparation according to claim 5, characterized in that: The anti-5T4 antibody is n501.

7. The near-infrared fluorescent ADC immune preparation according to claim 5, characterized in that: The carboxyl-containing cytotoxic drugs include auristatin peptide drugs.

8. The method for preparing the near-infrared fluorescent ADC immune preparation according to any one of claims 1 to 7, characterized in that: The following steps are involved: 1) reacting the single domain antibody with a reducing agent to reduce the disulfide bonds in the single domain antibody, and then adding a near-infrared fluorescent dye-cytotoxic drug conjugate dissolved in an organic solvent to obtain a mixture after the reaction; 2) adding the mixture obtained in step 1) to a desalting column to remove residual near-infrared fluorescent dye, and then collecting the supernatant by centrifugation to obtain the near-infrared two-window fluorescent immunoprobe.

9. The method for preparing the near-infrared fluorescent ADC immune preparation according to claim 8, characterized in that: The reducing agent in step 1) is at least one of tris(2-chloroethyl) phosphate, diethyltriaminepentaacetic acid or 5,5'-dithiobis(2-nitrobenzoic acid) and tris(2-chloroethyl) phosphate, and the organic solvent is at least one of N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

10. The method for preparing the near-infrared fluorescent ADC immune preparation according to claim 8, characterized in that: The reaction conditions in step 1) are: shaking reaction at 4-60° C. for 50-70 min.

11. The method for preparing the near-infrared fluorescent ADC immune preparation according to claim 8, characterized in that: The centrifugal conditions in step 1) are: temperature 3-5°C, time 0.5-1.5 min, and rotation speed 7000-12000 r / min.

12. Use of the near-infrared fluorescent ADC immune preparation according to any one of claims 1 to 7 in the preparation of a near-infrared fluorescent probe or a fluorescent detection kit.

13. Use of the near-infrared fluorescent ADC immunogenic preparation according to any one of claims 1 to 7 in the preparation of drugs for targeted treatment of tumors.

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