CN115814111A - Near-infrared fluorescence ADC immune preparation and preparation method and application thereof - Google Patents

Abstract

The invention discloses a near-infrared fluorescence ADC immune preparation, a preparation method and application thereof. 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 polymethine cyanine dye with absorption spectrum and emission spectrum within 600-900 nm. The near-infrared fluorescence ADC immune preparation provided by the invention is the first near-infrared fluorescence ADC immune preparation based on a single-domain antibody, the single-domain antibody/near-infrared fluorescence dye/cytotoxic drug is 1mol/1mol/1mol, the maximum absorption is in a 600-1000nm region, the maximum emission wavelength is in a 600-1700nm region, and the preparation has a larger molar absorption coefficient and is suitable for fluorescence living body imaging (near-infrared one-window living body imaging and near-infrared two-window living body imaging).

Description

Near-infrared fluorescence ADC immune preparation and preparation method and application thereof

Technical Field

The invention relates to a near-infrared fluorescence ADC immune preparation, a preparation method and application thereof, and belongs to the technical field of fluorescence imaging.

Background

The main measures for treating cancer at present stage comprise chemotherapy, immunotherapy, radiotherapy, stem cell therapy, laser therapy, thermotherapy, surgery, photodynamic therapy and the like. Of these, chemotherapy is the primary intervention in the treatment of cancer ¹ . The precondition of the above treatment is that the adopted medicine does not harm normal cells, but preferentially stops the division process of tumor cells, and efficiently and rapidly kills the tumor cells. Nitrogen mustards, the first chemotherapeutic agent used in humans, exert their apoptotic effects through DNA alkylation, ultimately leading to bone marrow damage ² . Thereafter, methotrexate, thioguanine, 5-fluorouracil and cytosine arabinoside (ara-C), cisplatin, actinomycin D, anthracyclines and vinca alkaloids have in turn entered the chemotherapy drug market for cancer. Despite the progress of anticancer chemotherapy based on small molecule anticancer drugs ³ Limited selectivity for cancer cells, pantoxicity to biological systems, and significantly enhanced resistance, leading to a narrowing of the therapeutic window. At the present stage, researchers have focused on the efficacy of cytotoxic drugs, reducing the effective dose required for treatment, and increasing the selectivity to tumor tissues. Antibody-drug conjugates (ADCs) are produced as appropriate. Antibody-drug conjugates (ADCs) are one of the fastest growing anticancer drugs ⁴ . The method comprises covalently coupling a cytotoxic drug to a monoclonal antibodyAnd efficiently deliver it to cancer cells expressing the antigen of interest. The obviously improved targeting property greatly reduces the probability of systemic exposure to cytotoxic drugs, thereby reducing the toxicity of the cytotoxic drugs. ADCs are complex molecules that require precise assembly of parts. The choice of target and monoclonal antibody, 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 the german physician and scientist, paulo erlichi, 100 years ago. After 45 years, methotrexate was attached to an antibody that targets leukemia cell surfaces, as envisaged by erlichia paulo, and constitutes the first example of an ADC drug. In the 80 s of the 20 th century, one example of ADC drugs based on mouse IgG molecules entered clinical trials. The first chimeric and humanized based monoclonal antibodies were generated in 1990. Based on highly specific monoclonal antibodies and drugs with extremely strong cytotoxicity, the ADC can selectively deliver the strong-effect drugs to the periphery of tumor cells, so that healthy cells are reserved, the main clinical obstacles of traditional chemotherapy are greatly reduced, and a wider treatment window is provided. Since 2013, with the rapid development of the biomedical industry, the research and development technology of fully human monoclonal antibodies is becoming mature day by day, which directly causes the ADC field to become very active. To date, there have been 12 cases of ADC drugs approved by FDA for clinical use ^5，6 . However, the fully human monoclonal antibody is generally in the form of IgG, has relatively large molecular weight (150 kDa), and has low tissue or tumor penetration rate, and will be an important aspect to be considered for ADC drugs. The presence of the single domain antibody will greatly reduce the hydrated particle size of its final ADC, resulting in better solid tumor permeability. It has the characteristics of small molecular weight (15 kDa), high stability, good solubility, easy expression, low immunogenicity and the like, and is a small molecular antibody which has simple source and low price and is suitable for batch production ^7，8 。

Linker (linker) plays a key role in ADC design. The linker must ensure that the ADC is stable in the blood to avoid release of cytotoxic drugs in off-target tissues, must bind to the antibody in an inactive, non-toxic state, and does not interfere with the normal function of the antibodyAnd physicochemical properties ⁹ . At the same time, the linker should release the cytotoxic drug when the ADC is endocytosed by the cell ¹⁰ . The linkers used at this stage can be classified into cleavable ones and non-cleavable ones. The cleavable linker is the main type of ADC linker. It is mainly characterized by fragmentation through differences in internal environment (e.g., redox potential, pH, etc.) and specific recognition by intracellular lysosomal enzymes. For example, an acid-sensitive linker is a group of linkers that are sensitive to an acidic environment, but stable in a basic environment near the systemic circulation. When targeted tumor cells are endocytosed, acid-sensitive linkers, such as hydrazone groups, are hydrolyzed in lysosomes (pH 4.8) and endosomes, and the acidic tumor microenvironment (pH 5-6) also causes a certain degree of release, so that the targeted tumor cells have a certain marginal killing effect ^3，8 . Lysosomal protease-sensitive linkers, also known as peptide-based linkers, are the most commonly used designs in ADCs ⁹ . Such as a valine-citrulline based linker, are among such designs. Yet another common linker is glutathione-sensitive disulfide bond. Glutathione is a short peptide chain containing thiol with low molecular weight, and has normal intracellular content of 0.5-10 mmol/L and extracellular environment of 2-20 mmol/L ¹¹ . Glutathione concentrations are higher in cancer cells than in normal cells. Furthermore, tumor cells also contain sulfides from enzyme proteins from the isomerase family, which can assist in disulfide bond cleavage. However, to date, research and development and application of ADC mainly focus on animal experiments and tumor treatment, and the research and development cost is huge, but the mechanism of cell level of different ADC drugs, monitoring of real-time effect of living body, and summarization of final curative effect and physicochemical property thereof are still lacking, and a real-time quantification means is needed to evaluate the ADC.

Optical imaging can provide visual images and quantitative dynamic biological analysis results, and has gained wide acceptance in biological experiments and clinical applications. At present, for the fluorescent signal-based detection means mainly applied to the non-invasive monitoring and detection of clinically relevant species, the accuracy of the detection means can be improved by improving the targeting accuracy and the selection of biological imaging window ¹² . Eyes of a userThere have been many reports on the fact that organic dyes approved for clinical use by the traditional FDA, such as Methylene Blue (MB), fluorescein sodium (FITC) and indocyanine green (ICG), can be used for imaging specific regions or antigens with high specificity, such as FITC13 for folate receptor alpha targeting, GE-13714 for c-MET targeting, and tuximab-IRdye 800cw for epidermal growth factor receptor targeting ¹³ . The emission fluorescence of the immunoprobe is concentrated in a Near Infrared (NIR) region, and the immunoprobe shows good fluorescence imaging effect.

Reference documents:

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3.Khongorzul，P.；Ling，C.J.；Khan，F.U.；Ihsan，A.U.；Zhang，J.，Antibody-Drug Conjugates:A Comprehensive Review.Mol Cancer Res 2020，18(1)，3-19.

4.Lambert，J.M.；Morris，C.Q.，Antibody-Drug Conjugates(ADCs)for Personalized Treatment of Solid Tumors:A Review.Adv Ther 2017，34(5)，1015-1035.

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，in patients with solid tumors.Cancer 2017，123(16)，3080-3087.

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

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.

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-Domain Antibodies against SARS-CoV-2.Cell Host Microbe 2020，27(6)，891-898e5.

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 of antibody-drug conjugates in mice.Nat Commun 2018，9(1)，2512.

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-drug conjugate leads to potent antitumour activity in mice.Nat Commun 2018，9(1)，1484.

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disclosure of Invention

The purpose of the invention is: provided is a near-infrared fluorescent ADC immune preparation based on a single-domain antibody, wherein the ADC immune preparation has high specificity and can be stably expressed.

In order to achieve the above object, the present invention provides a near-infrared fluorescent ADC immune preparation, 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 polymethine cyanine dye with absorption spectrum and emission spectrum within 600-900nm, and the chemical structural formula of the near-infrared fluorescent dye is shown as formula I:

wherein n is ₁ 、n ₂ 、n ₃ Each independently selected from 0, 1, 2 or 3;

R ₁ 、R ₂ each independently selected from a hydrogen atom, an alkyl group, a phenyl group, an alkylsulfonic group or a phenyl-substituted alkyl group; r ₃ An alkylene group selected from C1-12;

X ₁ 、X ₂ each independently selected from S, O, hydrogen atom, alkyl, alkane sulfonic group or phenyl substituted alkyl; x ₃ Selected from O, S or Se.

Preferably, n in the formula I ₁ 、n ₂ 、n ₃ Are all 1,R ₁ 、R ₂ Are each phenyl, R ₃ Is ethylene-CH ₂ -CH ₂ -，X ₁ 、X ₂ Are all-CH (CH) ₃ ) ₂ ，X ₃ Is S, namely the specific chemical structural formula of the near-infrared fluorescent dye is shown as a formula II:

preferably, the ADC immune preparation is a covalent conjugate of a near-infrared fluorescent dye and a carboxyl-containing cytotoxic drug and a single domain antibody in a molar ratio of 1.

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

Preferably, the single domain antibody is an anti-5T 4 antibody.

Preferably, the anti-5T 4 antibody is n501 (n 501 is the application number "201611040981.8", invention name "fully human single domain antibody specifically binding to human 5T4 antigen and its application" a fully human single domain antibody specifically binding to h5T4 disclosed in the patent).

Preferably, the carboxyl group-containing cytotoxic drug includes auristatin peptide drugs (Auristatins); the auristatin peptide drug comprises monomethyylaauristatin F (MMAF).

The invention also provides a preparation method of the near-infrared fluorescence ADC immune preparation, which comprises the following steps:

step 1): reacting the single domain antibody with a reducing agent to reduce a disulfide bond in the single domain antibody, adding a near-infrared fluorescent dye dissolved in an organic solvent, and reacting to obtain a mixture;

step 2): adding the mixture obtained in the step 1) into a desalting column, removing residual near-infrared fluorescent dye, centrifuging and collecting supernatant to obtain the near-infrared two-window fluorescent immune probe.

Preferably, the reducing agent in the 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-dimethylacetamide and dimethyl sulfoxide.

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

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

More preferably, the reaction conditions in step 1) are: the reaction was shaken at 37 ℃ for 60min.

Preferably, the conditions of the centrifugation in the step 2) are: the temperature is 3-5 ℃, the time is 0.5-1.5min, and the rotating speed is 7000-12000r/min.

More preferably, the conditions of centrifugation in step 2) are: the temperature is 4 ℃, the time is 1min, and the rotating speed is 8000r/min.

The invention also provides application of the near-infrared fluorescence ADC immune preparation in preparation of a near-infrared fluorescence probe or a fluorescence detection kit. The near-infrared fluorescent probe or the fluorescent detection kit can be used for fluorescent living body imaging, and specifically comprises tumor targeted imaging, blood vessel imaging and in vivo circulation tracing.

The invention also provides application of the near-infrared fluorescence ADC immune preparation in preparation of a medicine for targeted treatment of tumors. The tumor includes a pancreatic tumor.

In the present invention, "Near Infrared" means Infrared light (NIR), which is an electromagnetic wave between visible light (VIS) and mid-Infrared light (MIR), and conventionally divides the Near Infrared region into two regions, i.e., a first Near Infrared region (750 to 900 nm) and a second Near Infrared region (1000 to 1700 nm). The near infrared region is the first non-visible region of light to be found.

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 is used as a carrier to target and transport the small molecule to a target cell.

By "antibody" in the context of the present invention is meant a protein consisting of one or more polypeptides encoded by substantially all or part of a recognized immunoglobulin gene. The recognized immunoglobulin genes, for example in humans, include kappa (. Kappa.), lambda (. Lamda.), and heavy chain loci, which contain a myriad of variable region genes, as well as constant region genes mu (. Mu.), delta (. Delta.), gamma (. Gamma.), epsilon., alpha (. Alpha.), which encode 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 recombinantly produced antibodies for testing, therapeutic purposes, or other purposes as further specified below. The term "antibody" includes antibody fragments, as known in the art, such as Fab, fab ', F (ab') 2, fv, scFv or other subsequences for antigen binding of an antibody, or antibody fragments produced by modification of whole antibodies or those antibodies synthesized de novo using recombinant DNA techniques. The term "antibody" includes monoclonal as well as polyclonal antibodies. The antibody may be an antagonist, agonist, neutralizing antibody, or inhibitory antibody, or stimulatory antibody. The antibodies of the invention may be non-human, chimeric, humanized or fully human antibodies.

"antigen" as used herein means a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into the animal, which may be proteins, carbohydrates, lipids or other pathogens.

"amino acid" as used herein means one of the 20 naturally occurring amino acids or any non-natural analog, which may be located at a specifically defined position. By "protein" is meant in the present invention at least two covalently linked amino acids, which include proteins, polypeptides, oligopeptides and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds, or can be composed of synthetic peptidomimetic structures, i.e., "analogs". Thus "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 the (R) or (S) configuration. In preferred embodiments, the amino acids are present in the (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 in vivo degradation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. 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 case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Compared with the prior art, the invention has the beneficial effects that:

1. the near-infrared fluorescence ADC immune preparation provided by the invention is a covalent conjugate of a near-infrared fluorescence dye, an antibody and a cytotoxic drug, the maximum absorption of the preparation is in a 600-1000nm region, the maximum emission wavelength is in a 600-1700nm region, the preparation has a larger molar absorption coefficient, and the preparation is suitable for fluorescence living body imaging (near-infrared one-window living body imaging and near-infrared two-window living body imaging);

2. the fluorescent immune probe is the first near-infrared fluorescent ADC immune preparation based on a single-domain antibody; the single domain antibody/near infrared fluorescent dye/cytotoxic drug is 1mol/1mol/1mol, and is very suitable for controlled release of the drug and mechanism research in a complex biological environment system; the maximum absorption and emission of the polypeptide can specifically release cytotoxic drugs in a near-infrared window (650-900 nm) region, an environment with acidic pH and high glutathione content (such as a tumor microenvironment), and the fluorescence intensity of the polypeptide in the near-infrared region is greatly reduced, so that the polypeptide has the advantages of real-time observation of drug controlled release, timely adjustment of administration dosage, capability of providing ADC mechanism research on a cell level and the like, and can be applied to the treatment of tumors and the exploration and research of the tumor microenvironment;

3. the fluorescent immune probe has the advantages of single component, low cost, biological safety, suitability for patent medicine and the like.

Drawings

FIG. 1 is a schematic diagram of the general formula of the near-infrared fluorescent dye and the preparation process of covalent coupling of the near-infrared fluorescent dye with a cytotoxic drug having carboxyl;

FIG. 2 is a schematic diagram showing the preparation process of the near-infrared fluorescent dye CYMMAF in example 1;

FIG. 3 is a nuclear magnetic hydrogen spectrum characterization of intermediate 4 prepared in example 1;

FIG. 4 is a nuclear magnetic carbon spectrum characterization of intermediate 4 prepared in example 1;

FIG. 5 is a nuclear magnetic hydrogen spectrum characterization of intermediate 8 prepared in example 1;

FIG. 6 is a nuclear magnetic carbon spectrum characterization of intermediate 8 prepared in example 1;

FIG. 7 is a nuclear magnetic hydrogen spectrum characterization of intermediate 9 prepared in example 1;

FIG. 8 is a nuclear magnetic hydrogen spectrum characterization of intermediate 9 prepared in example 1;

FIG. 9 is a nuclear magnetic hydrogen spectrum characterization of intermediate 10 prepared in example 1;

FIG. 10 is a nuclear magnetic carbon spectrum characterization of intermediate 10 prepared in example 1;

FIG. 11 is a nuclear magnetic hydrogen spectrum characterization of intermediate 11 prepared in example 1;

FIG. 12 is a nuclear magnetic carbon spectrum characterization of intermediate 11 prepared in example 1;

FIG. 13 is a nuclear magnetic hydrogen spectrum characterization plot of CYMMAF prepared in example 1;

FIG. 14 is a nuclear magnetic carbon spectral characterization of CYMMAF prepared in example 1;

FIG. 15 shows the synthesis and characterization of the NIR fluorescent ADC immune preparation of 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 n501, B is a high-efficiency liquid chromatogram of the prepared near-infrared fluorescence ADC immune preparation, C is an SDS-PAGE gel diagram of n501-CYMMAF and target antibody n501 obtained after covalently coupling a target antibody, and D is a summary diagram of absorption spectra and color changes of CYMMAF in different pH values;

fig. 16 is an ultraviolet-visible absorption spectrum of n501-CYMMAF and ultraviolet-visible absorption spectra of CYMMAF at different concentrations, obtained after covalent coupling of a near-infrared fluorescent dye CYMMAF and a target antibody in example 3, and a concentration curve obtained based on concentration and absorbance fitting of the CYMMAF is used for calibrating the concentration of n 501-CYMMAF;

FIG. 17 is a condition screen for controlled drug release of n501-CYMMAF in example 3; a is the sum of ultraviolet-visible absorption spectra after cysteine with different concentrations is added for 20min in a buffer solution with a pH value of 7.4, and is calculated based on a fitting curve of glutathione concentration and the absorbance of n501-CYMMAF (10 mu M), in an environment with the pH value of 7.4, cysteine with 14-fold concentration is needed to quench n501-CYMMAF (10 mu M) to cause drug release, B is the sum of the ultraviolet absorption spectra of n501-CYMMAF (10 mu M) in the buffer solution with the pH value of 7.4 for 18 hours and the maximum absorbance of the ultraviolet absorption spectra at 670nm, C is the sum of the ultraviolet absorption spectra of n501-CYMMAF (10 mu M) in the buffer solution with the pH value of 5.0, and D is the sum of the fluorescence spectra corresponding to A; e is the summary of the fluorescence spectra corresponding to B; f is the summary of the fluorescence spectrum corresponding to C;

FIG. 18 is a test for selectivity of n501-CYMMAF (10. Mu.M) for amino acids in example 3; wherein A is a spectrum of ultraviolet-visible absorption spectrum of n501-CYMMAF (10. Mu.M) in a buffer solution to which 14-fold equivalent of the different kind of amino acid is added, B is a spectrum of fluorescence spectrum of n501-CYMMAF (10. Mu.M) in a buffer solution to which 14-fold equivalent of the different kind of amino acid is added, and C is a photograph of a solution of n501-CYMMAF (10. Mu.M) to which 14-fold equivalent of the different kind of amino acid is added;

FIG. 19 is a graph of the affinity test of N501-CYMMAF, N501, CYMMAF and the unrelated antibody N118 for the target antigen 5T4 in example 3;

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

FIG. 21 is a tumor targeting assay for n501-CYMMAF in example 3; wherein, A is an imaging graph of a 4T1 in-situ breast cancer model mouse constructed at the age of 10 weeks at an imaging window of 710 +/-30 nm after intraperitoneal injection of n501-CYMMAF (0.3 mu mol/Kg) for 1 hour, and B is an imaging graph of the imaging window of 790 +/-30 nm; c is an imaging image of a 4T1 in-situ breast cancer model mouse at the age of 10 weeks at an imaging window of 710 +/-30 nm after intraperitoneal injection of CYMMAF (0.3 mu mol/Kg) for 1 hour, and D is an imaging image of the mouse at an imaging window of 790 +/-30 nm;

FIG. 22 is a summary of the therapeutic effects of n501-CYMMAF on tumors in example 3; wherein, A is a diagram of the seed tumor and the administration process of the mouse model of n501-CYMMAF, CYMMAF and PBS to BXPC-3 subcutaneous tumor, B is a summary of the body weight of three groups of mice (three groups) receiving n501-CYMMAF (0.3 mu mol/Kg), CYMMAF (0.3 mu mol/Kg) and PBS (500 mu L), and C is a summary of the tumor volume of three groups of mice (three groups) receiving n501-CYMMAF (0.3 mu mol/Kg), CYMMAF (0.3 mu mol/Kg) and PBS (500 mu L).

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Standard recombinant DNA techniques and Molecular cloning techniques used in the following examples are well known in the art (published by Ausubel, F.M et al, current Protocols in Molecular Biology, greene Publishing Assoc. And Wiley-Interscience), and materials and methods suitable for the growth of microorganisms are well known in the art. The primary chemical, biological reagents are purchased from KAPA Biosystems, new England Biolabs, transGen Biotech, thermo Fisher Scientific, OMEGA bio-tek, etc., or may be prepared by methods known in the art (a) A.L.Antaris, H.Chen, K.Cheng, Y.Sun, G.hong, C.Qu, S.Diao, Z.Deng, X.Hu, B.Zhang, X.Zhang, O.K.Yaghi, Z.R.Alamparambil, X.hong, Z.ChengH.Dai, nat.Mater.2016, 15, 235-242.; (b) K.k.maiti, a.samanta, m.vendrell, k.s.soh, m.olivoy.t.chang, chem.commun.2011, 47, 3514-3516.

The preparation method of the near-infrared fluorescent dye for covalently coupling the cytotoxic drug and the single-domain antibody in the following embodiment of the invention is shown in fig. 1, and specifically comprises the following steps:

(1) Preparation of intermediate 2

Phosphorus oxychloride (37mL, 39.7 mol) was mixed with dichloromethane (35 mL) and slowly added dropwise to a mixture of N, N' -dimethylformamide (20 mL) and dichloromethane (20 mL) in an ice-water bath. After the addition was completed, cyclohexanone (10g, 100mmol) was rapidly added to the above system by means of a syringe. Heated to 120 ℃ and refluxed for 2 hours. Poured into a 400mL beaker, cooled in an ice bath, and a volume of approximately 500mL of ice was added and the layers separated. The mixture was allowed to stand overnight in the frozen layer (-4 ℃ C.) of a refrigerator. A yellow solid (compound 1) was obtained. Aniline and ethanol are mixed in ice water (1/1, v/v), after stirring for 1 hour, a compound 1 is added, the molar ratio of aniline to the compound 1 is 2.

(2) Preparation of intermediate 4

Dissolving the compound 3 in a solvent 1 (one or more of toluene, benzene, dimethyl sulfoxide, N-dimethylformamide and N, N-dimethylacetamide), dropwise adding the solution into an eggplant-shaped bottle containing sultone, and reacting at 40-120 ℃ (preferably 110 ℃) for 1-3 hours (preferably 2 hours) to obtain an intermediate 4.

(2) Preparation of intermediate 5

Dissolving the intermediate 2 and alkali in the solvent 2, adding the mixture into a eggplant-shaped bottle containing the intermediate 4 in batches, and stirring the mixture at a reaction temperature of 40-90 ℃ (preferably 80 ℃) for 40-100 minutes (preferably 60 minutes) to obtain an 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 at least one selected from acetic anhydride, acetic acid, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide.

(3) Preparation of intermediate 7

Grinding the compound 6 and the compound b in a solid state until the mixture is uniformly mixed, wherein the reaction temperature is 100-200 ℃ (preferably 140 ℃) and the reaction time is 1-3 hours (preferably 2 hours), and obtaining an intermediate 7.

(4) Preparation of intermediate 8

Dissolving Compound 7 in a solution containing (BOC) ₂ Reaction temperature of O and ammonium salt in solvent 3 is 40-90 deg.c (preferably 60 deg.c) and reaction time is 4-8 hr (preferably 6 hr) to obtain intermediate 8. The ammonium salt is selected from triethylamine and diethylAt least one of amine and DIPEA, preferably triethylamine, and the solvent 3 is at least one selected from the group consisting of toluene, benzene, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably chloroform.

(5) Preparation of intermediate 9

Dissolving the intermediate 8 and the base in the solvent 4, adding the mixture in portions into a eggplant-shaped bottle containing the intermediate 5, and stirring the mixture at a reaction temperature of 40 to 90 ℃ (preferably 80 ℃) for 40 to 100 minutes (preferably 60 minutes) to obtain a compound 9. 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 at least one selected from acetic anhydride, acetic acid, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide.

(6) Preparation of intermediate 10

Dissolving the intermediate 9 in the solvent 5, adding into an acid-containing bottle, and reacting under stirring at 40-90 deg.C (preferably 60 deg.C) for 4-8 hr (preferably 6 hr) to obtain 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-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably chloroform.

(7) Preparation of Compound 11

Dissolving the intermediate 10 in a solvent 6, adding the solution into a bottle in the shape of eggplant containing the compound c and a base, and reacting the mixture with stirring at a reaction temperature of 25 to 60 ℃ (preferably 37 ℃) for 4 to 8 hours (preferably 6 hours) to obtain a compound 11. The alkali is selected from various organic 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 at least one selected from toluene, benzene, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, and chloroform is preferred.

(8) Preparation of Compound 12

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 carried out with stirring at a reaction temperature of 40 to 100 deg.C (preferably 80 deg.C) for 4 to 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 at least one selected from toluene, benzene, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane, acetonitrile and ethyl acetate, preferably dichloromethane.

The preparation method of the Conjugate (CYMMAF) of the near-infrared fluorescent dye and the cytotoxic drug in the embodiment of the invention comprises the following steps:

dissolving near infrared fluorescent dye in solvent, adding condensing agent and cytotoxic drug MMAF, and reacting for 1-6 hr, preferably two hr, at 0-100 deg.C, preferably room temperature. The solvent is at least one selected from toluene, benzene, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, trichloromethane, dichloromethane, acetonitrile and ethyl acetate, and is preferably DMF; the condensing agent is at least one selected from EDC, DCC and DIC, preferably EDC.

The preparation method of the near-infrared fluorescence ADC immune preparation (covalent conjugate of near-infrared fluorescence dye, cytotoxic drug and single-domain antibody) in the embodiment of the invention comprises the following steps:

(1) After the single-domain antibody and the reducing agent are mixed, the mixture is oscillated and reacted for a period of time, the conjugate of the near-infrared fluorescent dye and the cytotoxic drug is dissolved in the organic solvent, and the solution is added into the mixed solution containing the antibody and the reducing agent in batches to obtain the mixture. A reducing agent for disrupting 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 at least one of N, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide, the reaction temperature is 4-60 ℃, the reaction time is 40-100 minutes, and the reaction time is 60 minutes.

(2) Adding the mixture obtained in the step (1) into a desalting column. Collecting the fractions with the corresponding colors of the micromolecular dye, centrifuging for a period of time on a centrifuge, and collecting the supernatant. The centrifugation temperature is preferably 4 ℃, the centrifugation time is preferably 1 minute, and the centrifugation speed is preferably 12000rpm. Obtaining the conjugate of the target near-infrared fluorescent dye, the cytotoxic drug and the single-domain antibody.

Example 1

The present embodiment provides a method for preparing a conjugate CYMMAF of a near-infrared fluorescent dye and a cytotoxic drug, as shown in fig. 2, specifically including the following steps:

(1) Preparation of intermediate 4

Compound 3 (2.09g, 10mmol) was dissolved in 5mL of toluene, added dropwise to a eggplant type bottle containing 1, 3-propanesultone, reacted at 110 ℃ for 8 hours, and a dark gray solid was collected by filtration and washed with 20mL of chloroform three times to give intermediate 4. Fig. 3 and 4 are nuclear magnetic hydrogen and carbon spectra representations of intermediate 4, 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)：δ196.69，138.49，137.30，133.82，131.07，129.67，128.27，127.72，127.27，123.01，112.59，69.44，55.94，43.72，43.68，23.48，23.41，21.01。

(2) Preparation of intermediate 5

Intermediate 4 (3.31g, 10 mmol) and Compound 2 (3.22g, 10 mmol) were added to a 50mL round-bottomed flask containing acetic anhydride (3 mL) and potassium acetate (0.98g, 10 mmol). The whole mixture was heated to 70 ℃ and held for 1 hour, cooled to room temperature and poured into saturated sodium bicarbonate solution. The red precipitate was washed with diethyl ether.

(3) Preparation of intermediate 6

Compound 3 (4.18g, 20mmol) and 2-bromoethylamine hydrobromide (2.04g, 10mmol) were milled and charged into a round-bottomed flask, and heated to 130 ℃ to react for 8 hours. The mixture was cooled to room temperature and washed with 30mL of chloroform to give a white precipitate.

(4) Preparation of intermediate 7

Intermediate 6 (3.48g, 10mmol) and di-tert-butyl dicarbonate (2.18g, 10mmol) were added to 30mL of chloroform containing N, N' -diisopropylethylamine (3.23g, 25mmol). The mixture was heated to 60 ℃ for 6 hours. The final solution was washed with water and then extracted with ether. The final product is eluted with CH ₂ Cl ₂ Column chromatography on MeOH (50/1 v/v) afforded intermediate 7.

(5) Preparation of intermediate 8

Intermediate 5 (0.602g, 1mmol) and intermediate 7 (0.353g, 1mmol) were charged into a 50mL round-bottomed flask containing acetic anhydride (3 mL) and potassium acetate (0.098g, 10mmol). The mixture was heated to 80 ℃ for 10 minutes, turned green, cooled to room temperature and washed with ether. Purifying the green precipitate by chromatography, eluting with CH ₂ Cl ₂ MeOH (20/1 v/v) to afford intermediate 8. FIGS. 5 and 6 are respectively intermediate 8 (C) ₄₉ H ₅₆ ClN ₃ O ₅ S) nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum. ¹ H NMR(400MHz，CDCl ₃ )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)，5.86(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)；

¹³ C NMR(101MHz，CDCl ₃ )δ207.16，174.35，172.87，156.60，149.97，144.48，142.84，139.68，139.56，134.05，133.46，132.06，131.82，131.03，130.80，130.13，128.11，128.04，128.01，127.69，127.61，127.13，125.13，124.87，122.02，111.23，110.89，102.05，100.09，79.08，77.48，77.16，76.85，58.09，53.52，51.19，50.89，47.78，43.84，37.97，30.93，28.50，27.83，27.69，27.66，26.47，23.97，20.95，18.40。

(6) Preparation of intermediate 9

Add intermediate 8 to TFA/CH ₂ Cl ₂ (1/20, v/v), and stirring at room temperature for 2 hours to obtain a green mixed solution. The green mixture was evaporated using a rotary evaporator to remove excess TFA and CH ₂ Cl ₂ Washing the obtained solid with water, filtering, and filtering the obtained solid with eluent CH ₂ Cl ₂ Purification by chromatography with MeOH (4/1 v/v). FIGS. 7 and 8 are intermediate 9 (C) ₄₄ H ₄₈ ClN ₃ O ₃ S) nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum. ¹ H 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)；

¹³ C NMR(101MHz，DMSO)δ175.66，172.09，158.71，147.86，144.28，140.99，140.29，139.92，135.07，133.12，132.29，131.65，131.11，130.80，130.42，130.35，128.37，128.17，128.09，127.80，127.73，126.53，125.87，125.09，122.88，122.59，118.34，115.41，112.51，111.75，103.96，99.95，51.64，50.70，48.13，43.93，41.27，36.98，27.57，27.41，26.52，25.82，24.42，21.05。

(7) Preparation of intermediate 10

Intermediate 9 (0.360g, 0.5 mmol) and DIPEA (0.064g, 0.5 mmol) were added to a solution containing TSTU (0.150g, 0.5 mmol), 3-maleimidopropionic acid (0.085g, 0.5 mmol) and 30mL CH ₂ Cl ₂ The round-bottomed bottle of (1). All stirred for 6 hours and followed by TLC thin plate chromatography. Then using eluent CH ₂ Cl ₂ MeOH (15/1 v/v). FIGS. 9 and 10 are respectively intermediate 10 (C) ₅₁ H ₅₃ ClN ₄ O ₆ S) nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum. 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)；

¹³ C NMR(101MHz，DMSO)δ175.01，172.63，171.25，170.03，147.85，143.72，141.45，140.31，140.06，135.06，134.71，133.46，132.16，131.73，131.05，130.43，128.30，128.19，128.03，127.86，127.46，126.48，125.70，122.83，122.66，112.45，111.90，103.28，100.54，53.96，51.46，50.85，49.06，48.16，40.62，40.41，40.20，39.99，39.78，39.57，39.36，36.49，34.49，27.64，27.53，27.46，26.47，25.70，24.39，18.49，17.17。

(7) Preparation of intermediate 11

Intermediate 10 (0.435g, 0.5mmol) and TEA (0.01g, 0.1mmol) were added to a mixture containing cystamine dihydrochloride (0.115g, 0.5mmol) and 30mL CH ₂ Cl ₂ In a round-bottomed bottle. The reaction was stirred for 6 hours and followed by TLC thin plate chromatography. Then using eluent CH ₂ Cl ₂ MeOH (15/1, v/v) to give a blue solid. FIGS. 11 and 12 are respectively intermediate 11 (C) ₅₅ H ₆₄ N ₆ O ₆ S ₃ ) And (3) the nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the obtained product are used for representing results. ¹ H NMR(400MHz，DMSO)δ8.34(d，J＝14.3Hz，1H)，8.26(d，J＝8.7Hz，1H)，8.20(d，J＝9.5Hz，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)；

¹³ C NMR(101MHz，DMSO)δ173.86，169.21，147.86，145.07，142.68，141.09，140.26，134.08，131.93，131.43，130.94，130.83，130.41，130.33，130.30，130.13，128.40，128.22，127.95，127.76，127.07，125.43，124.00，122.21，112.30，111.40，102.03，70.24，51.14，49.59，48.64，48.25，45.78，38.30，34.56，29.52，29.48，29.46，29.27，29.14，29.03，28.74，28.32，27.52，27.09，27.06，27.02，26.52，25.28，24.20，23.45，22.60，21.87，14.40，8.88。

(8) Preparation of CYMMAF:

cytotoxic drug MMAF (10mg, 0.014mmol) and EDC & HCl (2.62mg, 0.014mmol) were dissolved in anhydrous DMF, and after stirring and reacting at room temperature for 1 hour, near infrared fluorescent dye intermediate 11 (20mg, 0.2mmol) was added, and the reaction was carried out at room temperature for 2 hours, followed by column chromatography to obtain CYMMAF. FIGS. 13 and 14 are CYMMAF (C) ₉₄ H ₁₂₇ N ₁₁ O ₁₃ S ₃ ) And (3) the nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the obtained product are used for representing results. ¹ H 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)；

¹³ C NMR(126MHz，DMSO)δ173.63，173.25，169.92，169.84，169.14，168.63，147.10，142.40，142.08，141.55，141.44，140.50，139.83，139.61，138.17，133.69，133.21，131.41，131.26，130.76，130.39，130.25，130.06，129.71，129.53，127.77，127.67，127.60，127.36，127.33，127.17，126.54，126.04，124.93，124.75，123.41，122.15，121.62，120.11，111.76，111.47，110.83，110.57，108.81，101.99，101.45，100.90，94.75，69.65，54.83，50.65，50.55，49.03，48.46，48.04，47.60，45.16，43.40，42.99，41.97，41.51，41.09，37.68，37.61，36.44，35.87，34.00，33.74，33.54，31.16，30.66，28.95，28.87，28.70，28.57，28.42，27.73，27.52，26.90，26.70，26.44，25.92，25.83，24.62，24.45，24.37，23.67，22.87，22.43，22.33，21.34，20.48，13.85，8.29。

Example 2

The embodiment provides synthesis, purification and characterization of a near-infrared fluorescence ADC immune preparation n 501-CYMMAF:

preparation scheme As shown in FIG. 15A, a reducing agent TCEP (available from Merck Sigma-Aldrich chemical reagent) was mixed with an anti-5T 4 antibody n501 (n 501 is application No. "201611040981.8", title of the invention "fully human Single Domain antibody specifically binding to human 5T4 antigen and antibody disclosed in the application" patent ") at a molar ratio of 3. The resulting n501-CYMMAF was centrifuged at 12000rpm for 1 minute, and a blue supernatant was obtained. The purity was confirmed by HPLC and SDS-PAGE protein electrophoresis (FIG. 15B-C), and the affinity parameters were measured by ELISA (FIG. 19) (see the literature Cell Host Microbe2020, 27, 891-898e895 nat. Mater.2016, 15, 235-242).

FIG. 15B-C shows the SDS-PAGE gel (C) and high performance liquid chromatogram (B) of ICGM-n501 and target antibody n501 obtained by covalently coupling near infrared fluorescent dye CYMMAF with the target antibody, and the results show that the obtained product n501-CYMMAF is stable and uniform. Figure 15D is a graph of the change in absorption spectra of CYMMAF, containing a pH sensitive secondary amine group, over a range of pH values and the corresponding change in solution color, that gradually oxidized in a more acidic and basic environment, thereby promoting release of the loaded drug.

Example 3

This example provides a performance test of the near-infrared fluorescent ADC immune formulation n501-CYMMAF synthesized in example 2:

(1) Concentration calibration of n501-CYMMAF

FIG. 16 is an ultraviolet-visible spectrum and a near-infrared two-window fluorescence emission spectrum of n501-CYMMAF obtained after covalent coupling of a fluorescent dye CYMMAF and a target antibody; the concentration of n501-CYMMAF is 2.86 μ M, and the excitation light source is xenon lamp excitation (PAO photoelectricity technologies, inc. of Beijing Pin). The absorption peak of CYMMAF in PBS is 673nm, the fluorescence peak is 730nm, and the absorption peak of n501-CYMMAF is red-shifted to 685nm. The concentration curve obtained based on the fitting of the concentration and absorbance of CYMMAF was used to calibrate the concentration of n 501-CYMMAF. The n501-CYMMAF used herein to measure the UV-visible absorption was 0.35mg/mL based on the absorption of the antibody at 260nm and 280 nm.

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

FIG. 17A shows that in the buffer at pH 7.4, with the addition of glutathione (0-14 equiv), the absorbance at 673nm of n501-CYMMAF (10. Mu.M) is gradually decreased, the whole quenching process takes 10 hours, and 14 times equivalent cysteine (140. Mu.M) is required, and since n501-CYMMAF is a single-domain antibody-based ADC immune preparation, the half-life is short, only 1-2 hours, the release amount of n501-CYMMAF at normal blood pH is relatively small, and the fluorescence spectrum (FIG. 17D) can be concluded similarly. FIG. 17B shows a summary of the UV absorption spectra of n501-CYMMAF (10 μ M) in pH 7.4 buffer for 18 hours, wherein the absorbance at 673nm is substantially unchanged within 18 hours, so that n501-CYMMAF releases substantially no cytotoxic drug at blood pH, and the fluorescence spectra (FIG. 17E) shows the same conclusion; FIG. 17C is a summary of the UV absorption spectra of n501-CYMMAF (10 μ M) in a buffer at pH5.0, which has a secondary amine group that is more sensitive to pH changes in addition to a disulfide bond that is reductively cleavable by glutathione (cysteine) (FIG. 17D). The group retains the property of being more sensitive to pH after covalent coupling of CYMMAF to n501. Thus n501-CYMMAF in pH5.0 buffer showed a significant decrease in absorbance at 673nm, a new absorption peak at 543nm (FIG. 17C), which just corresponded to the peak of the oxidation product of heptamethine cyanine dye, with a concomitant decrease in fluorescence intensity at 730nm, and a small amplitude blue-shift (FIG. 17F).

(3) n501-CYMMAF Selectivity test for amino acids:

FIG. 18 shows the selectivity test of n501-CYMMAF (10. Mu.M) on amino acids in example 3, wherein 15 amino acids were sequentially added to n501-CYMMAF at a concentration of 14 times equivalent, and the UV-visible absorption spectrum and the corresponding fluorescence spectrum were shown in FIGS. 18A-B, and n501-CYMMAF exhibited high selectivity for thiol-containing cysteine and glutathione, and the absorbance at 673nm exhibited only small fluctuations with the addition of the remaining 13 amino acids (proline, threonine, sarcosine, alanine, pyroglutamic acid, serine, tryptophan, valine, histidine, phenylalanine, leucine, isoleucine, methionine). FIG. C is a photograph of n501-CYMMAF (10. Mu.M) solutions to which 14-fold equivalent of each of different kinds of amino acids (proline, threonine, sarcosine, alanine, pyroglutamic acid, serine, tryptophan, valine, histidine, phenylalanine, leucine, isoleucine, methionine) was added, and they were all still a brilliant blue solution, and only the n501-CYMMAF (10. Mu.M) solution to which cysteine was added became light blue, while the n501-CYMMAF (10. Mu.M) solution to which glutathione was added became transparent colorless solution.

(4) Affinity detection of n501-CYMMAF for antigen:

N501-CYMMAF, N501, CYMMAF and the irrelevant antibody N118, and FIG. 19 is an affinity assay based on enzyme-linked immunosorbent assay (ELISA) for the antigen of interest (5T 4 antigen, purchased from Boolpek, inc.) (see Cell Host Microbe2020 for specific detection methods and steps). The affinity test results show that the covalent coupling of antibody n501 and the fluorescent dye ICGM has no effect on affinity.

(5) n501-CYMMAF cytotoxicity assay

FIG. 20 shows the results of the cytotoxicity test of n501-CYMMAF in example 3, seeded with SKOV3 cells (1X 10) ⁴ ) Different concentrations of n501-CYMMAF, n501, CYMMAF, MMAF (15 nM,30nM,60nM,120nM,240nM,480 nM) were added to the 96-well plate and incubated for 24 hours. A group of control groups are reserved on two sides of a 96-well plate, only culture solution is added, supernatant is sucked after culture is carried out under the same condition, 10 mu L of CCK8 reagent (per 100 mu L of cell culture solution) is added into each well, the OD value at 450nm is measured after incubation for 4 hours and is used as an experimental value, the OD value at 600nm is used as a reference value, and the cell survival rate is calculated as follows:

the results are shown in fig. 20, where MMAF and CYMMAF both showed significant killing effect in the 24-hour experimental group (fig. 20A), with higher cell survival in the n501-CYMMAF and n501 groups, confirming lower drug release in the first 24 hours n 501-CYMMAF. In the 48-hour experimental group (fig. 20B), n501-CYMMAF, CYMMAF and MMAF all showed significant killing effect, confirming successful release of MMAF drug.

(6) Tumor targeting test for n501-CYMMAF

FIG. 21 shows the results of a tumor targeting assay for n501-CYMMAF with CYMMAF as a control. The abdominal cavity of a 4T1 in-situ breast cancer model mouse which is constructed at the age of 10 weeks is injected with n501-CYMMAF (0.3 mu mol/Kg), the imaging graph at 710 +/-30 nm imaging window after 1 hour is shown in figure 21A, and the imaging graph at 790 +/-30 nm imaging window is shown in figure 21B. In both imaging windows, the fluorescence signal was concentrated around the tumor at its left abdomen, and the single dose could be adjusted higher due to intraperitoneal administration, but the complicated peritoneal delivery system of peritoneal cavity would cause the fluorescence signal to be distributed throughout the peritoneum, resulting in higher background signal, although it was observed that the CYMMAF was concentrated essentially only in the abdomen and not selectively around the tumor, compared to the control group of fig. 21C, in which only CYMMAF was injected. FIG. 21D is an image of the 790. + -.30 nm imaging window, which may be similarly concluded.

(7) Summary of therapeutic effects of n501-CYMMAF on tumors

BXPC-3(1×10 ⁶ ) Pancreatic cancer cells were subcutaneously implanted into SPF grade 7-week old balb/c female mice, and 1 week later, the mice were divided into three groups of n501-CYMMAF, CYMMAF and PBS, as shown in FIG. 22A, and the mice were administered once every three days at doses of n501-CYMMAF (0.3. Mu. Mol/Kg), CYMMAF (0.3. Mu. Mol/Kg) and an equal volume of PBS (500. Mu.L). The body weights of the mice were followed and summarized (fig. 22B), and the volume of mice receiving n501-CYMMAF showed a significant decrease, while the mice given CYMMAF and PBS showed a small amplitude shift. Accordingly, tumor volumes of mice were measured with a vernier caliper and summarized as shown in fig. 22C, the tumor volumes of the PBS group showed small amplitude increases (from 99.21 mm) over the time of three doses ³ Rise to 111.10mm ³ ) While the CYMMAF group was leveled after a significant drop (from 128.03 mm) ³ Reduced to 59.71mm ³ And finally 44.37mm ³ ) The tumor volume in the n501-CYMMAF group decreased to 7.8% of the original volume (from 118.47mm ³ Reduced to 9.13mm ³ ) Showing significant therapeutic effect.

The above-described embodiments are intended to be preferred embodiments of the present invention only, and not to limit the invention in any way and in any way, it being noted that those skilled in the art will be able to make modifications and additions without departing from the scope of the invention, which shall be deemed to also encompass the scope of the invention.

Claims (13)

1. A near-infrared fluorescence ADC immune preparation is characterized in that the ADC immune preparation is a covalent conjugate of a near-infrared fluorescence dye, a carboxyl-containing cytotoxic drug and a single-domain antibody; the near-infrared fluorescent dye is polymethine cyanine dye with an absorption spectrum and an emission spectrum within a range of 600-900nm, and the chemical structural formula of the near-infrared fluorescent dye is shown as a formula I:

wherein n is ₁ 、n ₂ 、n ₃ Each independently selected from 0, 1, 2 or 3;

R ₁ 、R ₂ each independently selected from a hydrogen atom, an alkyl group, a phenyl group, an alkylsulfonic group or a phenyl-substituted alkyl group; r ₃ An alkylene group selected from C1-12;

X ₁ 、X ₂ each independently selected from S, O, hydrogen atom, alkyl, alkane sulfonic group or phenyl substituted alkyl; x ₃ Selected from O, S or Se.

2. The near-infrared fluorescent ADC immunity formulation of claim 1, wherein n in formula I is ₁ 、n ₂ 、n ₃ Are all 1,R ₁ 、R ₂ Are each phenyl, R ₃ Is ethylene-CH ₂ -CH ₂ -，X ₁ 、X ₂ Are all-CH (CH) ₃ ) ₂ ，X ₃ Is S; namely, the specific chemical structural formula of the near-infrared fluorescent dye is shown as formula II:

3. the near-infrared fluorescent ADC immune formulation of claim 1, wherein said ADC immune formulation is a covalent conjugate of a near-infrared fluorescent dye with a carboxyl-containing cytotoxic drug and a single domain antibody at a molar ratio of 1.

4. The near-infrared fluorescent ADC immunizing agent according to claim 1, wherein said single domain antibody is a type of single domain antibody in which serine in the FR3 region is specifically mutated to cysteine.

5. The near-infrared fluorescent ADC immune formulation of claim 4, wherein said single domain antibody is an anti-5T 4 antibody.

6. The near-infrared fluorescent ADC immune formulation of claim 5, wherein said anti-5T 4 antibody is n501.

7. The near-infrared fluorescent ADC biologics of claim 5 wherein said carboxy-containing cytotoxic drug comprises a auristatin peptide drug.

8. The method of preparing the near-infrared fluorescent ADC immunity preparation of any one of claims 1 to 7, comprising the steps of:

1) Reacting the single domain antibody with a reducing agent to reduce a disulfide bond in the single domain antibody, adding a near-infrared fluorescent dye-cytotoxic drug conjugate dissolved in an organic solvent, and reacting to obtain a mixture;

2) Adding the mixture obtained in the step 1) into a desalting column, removing residual near-infrared fluorescent dye, and then centrifuging and collecting supernatant to obtain the near-infrared two-window fluorescent immune probe.

9. The method of claim 8, wherein 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-dimethylacetamide, and dimethylsulfoxide.

10. The method for preparing the near-infrared fluorescent ADC immunizing agent according to claim 8, wherein the reaction conditions in step 1) are: shaking at 4-60 deg.C for reaction for 50-70min.

11. The method of claim 8, wherein the centrifugation conditions in step 1) are: the temperature is 3-5 ℃, the time is 0.5-1.5min, and the rotating speed is 7000-12000r/min.

12. Use of the near-infrared fluorescent ADC immunizing agent of any one of claims 1-7 in preparing a near-infrared fluorescent probe or a fluorescence detection kit.

13. Use of the near-infrared fluorescent ADC immunizing formulation of any one of claims 1-7 for preparing a medicament for targeted treatment of a tumor.

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