Labeling method based on cyclic amplification and composition for antibody labeling
By employing a cyclic amplification labeling method and a solid-liquid dual-phase reaction system, the problem of limited antibody label loading was solved, achieving efficient and uniform label loading and improving detection and treatment efficacy.
Patent Information
- Application Number
- CN202510888870.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-14
AI Technical Summary
Existing antibody labeling technologies have limited label loading capacity, resulting in uneven labeling sites and affecting detection and treatment efficacy.
A labeling method based on cyclic amplification is adopted, which forms a dendritic topology by alternately coupling multiple compounds with the active groups at the ends of the antibody, thereby increasing the number of active reaction sites at the ends of the antibody, and the purification steps are simplified by using a solid-liquid two-phase reaction system.
It significantly increased the label loading capacity, simplified the purification process, improved the efficiency and yield of antibody labeling, and enhanced the detection and treatment effects.
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Figure CN120943881A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a labeling method based on cyclic amplification and a composition for antibody labeling. Background Technology
[0002] Antibody labeling technology, as a key means of functionally modifying antibodies through chemical or biological conjugation, essentially aims to achieve covalent linkage between antibody molecules and labels, thereby expanding their applications in medical testing, bioimaging, and disease treatment. For example, fluorescent dye-labeled antibodies are widely used in flow cytometry and immunohistochemistry to achieve highly sensitive detection of specific antigens; radioisotope-labeled antibodies are used for molecular imaging diagnosis of diseases; and antibody-drug conjugates (ADCs) combine the high specificity of antibodies with the potent killing activity of cytotoxic drugs to achieve targeted therapy of cancer cells. Theoretically, loading more labels will significantly enhance signal intensity and lower the detection limit in imaging applications, while in therapeutic applications it is expected to improve drug delivery efficiency and thus improve treatment outcomes. However, the number of reaction sites suitable for labeling on the surface of natural antibodies is very limited. Using traditional chemical random conjugation methods will lead to heterogeneous products with uneven labeling positions and quantities, and may even cause the loss of antibody targeting function. Achieving high-load, high-precision antibody labeling still faces significant technical challenges. Summary of the Invention
[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a labeling method based on cyclic amplification and a composition for antibody labeling, aiming to solve the problem of limited label loading in traditional antibody labeling technology.
[0004] The technical solution of the present invention is as follows:
[0005] In a first aspect, a labeling method based on cyclic amplification is provided, comprising the steps of: alternatingly coupling the target compound with two or more compounds at least once, and then coupling it with the labeling compound to obtain a labeled product;
[0006] The terminant and the marker each have at least one active group at their ends;
[0007] Each compound has two or more active groups at its end, one of which can be coupled to the active group at the end of the target compound and / or another compound, and the other active group can be coupled to the active group at the end of the target compound and / or another compound.
[0008] The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine.
[0009] The number of at least one active group in at least one compound is greater than 2.
[0010] In a preferred embodiment, the labeling agent is selected from one or more of antibodies, nucleic acid aptamers, peptides, nanoparticles, and carbon-based materials.
[0011] In a preferred embodiment, the antibody is selected from one or more of monoclonal antibodies, single-domain antibodies, multivalent antibodies, and antibody fragments.
[0012] In a preferred embodiment, the marker is an imaging probe or a drug.
[0013] In a preferred embodiment, the imaging probe is selected from one or more of fluorescent molecules, magnetic resonance imaging contrast agents, and radionuclides.
[0014] And / or, the drug is selected from one or more small molecule compounds, nucleic acids, polypeptides, and proteins.
[0015] In a second aspect, a composition for antibody labeling is provided, the composition comprising: an antibody conjugate, two or more compounds, and a labeler;
[0016] The antibody has at least one active group at its terminal.
[0017] The antibody conjugate includes two or more active groups, one of which can be coupled to the active group of the antibody, and the other active group can be coupled to the active group at the end of at least one compound.
[0018] The marker has at least one active group at its end;
[0019] Each compound comprises two or more active groups at its end, wherein one active group is capable of coupling with the active group at the end of the antibody conjugate and / or another compound, and the other active group is capable of coupling with the active group at the end of the label and / or another compound.
[0020] The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine.
[0021] The number of at least one active group in the antibody conjugate or at least one compound is greater than 2.
[0022] In a preferred embodiment, the compound comprises a first compound and a second compound, i.e., the composition comprises an antibody conjugate, a first compound, a second compound, and a labeler;
[0023] The antibody has an active A group at its terminal;
[0024] The antibody conjugate has B active groups and C active groups at its ends;
[0025] The first compound has a D-active group and an E-active group at its end;
[0026] The second compound has an F active group and a C active group at its end;
[0027] The marker has a D active group at its end;
[0028] The active groups A and B, C and D, and E and F are complementary active groups.
[0029] The number of at least one of the C active group of the antibody conjugate, the E active group of the first compound, and the C active group of the second compound is greater than 2.
[0030] Thirdly, an antibody labeling method is provided, including the following steps:
[0031] Antibodies or intermediates are immobilized on a solid support for coupling and / or elution.
[0032] The intermediate is selected from any one of the following: a first complex obtained by conjugating an antibody and an antibody conjugate; an intermediate complex obtained by conjugating a first complex and at least one or two compounds; a final complex obtained by conjugating a first complex or an intermediate complex and a marker.
[0033] The antibody conjugates, compounds, and markers are as described in the second aspect.
[0034] In a preferred embodiment, the solid support is selected from one or more of magnetic particles, silica gel, organic polymer materials, carbon nanotubes, and microfluidic chips.
[0035] The preferred technical solution, the method for immobilizing antibodies or intermediates on a solid support, is selected from one or more of electrostatic adsorption, hydrophobic interaction, affinity immobilization, covalent coupling, chemical crosslinking, and photocrosslinking.
[0036] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:
[0037] (1) When using existing technologies for antibody labeling, the amount of label loaded is limited by the number of reaction sites on the antibody itself, as well as the structure and number of links and branches. Typically, the ratio of antibody to label is in the range of 1:1 to 1:4. The labeling method based on cyclic amplification provided by this invention can construct links with a "tree-like topology" at specific sites on the antibody step by step through alternating coupling and cyclic amplification, thereby multiplying the number of active reaction sites at the antibody ends and thus loading more labels.
[0038] (2) Multi-step modification inevitably leads to complex purification steps. Existing antibody purification techniques, such as column purification and dialysis, are not only time-consuming and labor-intensive, but also often result in significant loss of the target product. This invention utilizes reversible binding to immobilize the antibody onto a solid-phase support, developing a solid-liquid two-phase reaction system. After each modification step, the product can be collected quickly and efficiently by means of magnetic response, centrifugation, or filtration, thereby greatly simplifying the purification steps and significantly improving the final yield of the labeled antibody. Attached Figure Description
[0039] Figure 1 This is a schematic diagram of the labeling strategy for cyclic amplification in this invention.
[0040] Figure 2 This is a schematic diagram illustrating the process of immobilizing antibodies on a solid-phase support for amplification and labeling through reversible binding.
[0041] Figure 3 This is a graph showing the test results of the cell-targeting ability of single-domain antibody conjugates.
[0042] Figure 4 This is a graph showing the test results of the absorbance to concentration ratio of single-domain antibody-dye conjugates.
[0043] Figure 5 It is the fluorescence emission spectrum of a single-domain antibody-dye conjugate.
[0044] Figure 6 This is a graph showing the test results of the average fluorescence intensity of cells labeled with single-domain antibody-dye conjugates.
[0045] Figure 7 This is a graph showing the test results of the in vitro tumor cell killing effect of single-domain antibody-drug conjugates.
[0046] Figure 8 This is a graph showing the test results of single-domain antibody-drug conjugates in vivo regarding tumor targeting and metabolism.
[0047] Figure 9 This is a statistical chart showing the distribution of single-domain antibody-drug conjugates in muscle and tumor tissues.
[0048] Figure 10 This is a graph showing the test results of the distribution of single-domain antibody-drug conjugates in tumors and major organs.
[0049] Figure 11 This is a statistical chart showing the distribution of single-domain antibody-drug conjugates in tumors and major organs.
[0050] Figure 12 This is a graph showing the test results of subcutaneous tumor growth in mice treated with single-domain antibody-drug conjugates.
[0051] Figure 13 This is a graph showing the test results of weight changes in mice during treatment.
[0052] Figure 14 These are images of subcutaneous tumors in mice after treatment monitoring has concluded.
[0053] Figure 15 This is a graph showing the statistical results of subcutaneous tumor weight in mice after the treatment monitoring period.
[0054] Figure 16 This is a diagram showing the H&E staining results of major organ sections from mice after the treatment monitoring period. Detailed Implementation
[0055] This invention provides a labeling method based on cyclic amplification and a composition for antibody labeling. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. Furthermore, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for clearly describing a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed. The component numbers used herein, such as "first," "second," etc., are only used to distinguish the described objects and do not have any sequential or technical meaning. All instruments and reagents used are commercially available products.
[0056] This invention provides a labeling method based on cyclic amplification, comprising the steps of: alternatingly coupling the target compound with two or more compounds at least once, and then coupling it with the labeling compound to obtain a labeled product;
[0057] The object to be labeled and the terminal of the labeled object each have at least one active group;
[0058] Each compound has two or more active groups at its end, one of which can be coupled to the active group at the end of the target compound and / or another compound, and the other active group can be coupled to the active group at the end of the target compound and / or another compound.
[0059] The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine.
[0060] The number of at least one active group in at least one compound is greater than 2.
[0061] Specifically, primary amino groups (-NH2) and N-hydroxysuccinimide esters (-NHS ester), maleimide (-Mal) and thiol groups (-SH), carboxyl groups (-COOH) and amino groups (-NH2), glutamine residues (-Gln) and primary amino groups (-NH2), azide groups (-N3) and cyclooctyne groups (-DBCO), 1,2,4,5-tetraazine groups (-Tz) and trans-cyclooctene groups (-TCO), thioesters and hydroxylamines can undergo covalent coupling reactions. This invention provides at least one active group at the ends of the target compound and the labeled compound, and provides two or more active groups at the ends of each compound. It also specifies that one active group in each compound can couple with the active group at the end of the target compound and / or another compound, and that the other active group in each compound can couple with the active group at the end of the labeled compound and / or another compound, thereby achieving alternating coupling cycles. Furthermore, the number of at least one active group in at least one compound is greater than two, causing the method to amplify during the alternating coupling cycle, thus forming a "tree-like topology," thereby multiplying the number of active reaction sites at the ends of the target compound and achieving a greater loading of labeled compounds (e.g., ...). Figure 1 ).
[0062] In one embodiment of the labeling method, the substance to be labeled is selected from one or more of antibodies, nucleic acid aptamers, peptides, nanoparticles, and carbon-based materials.
[0063] In a more specific embodiment of the labeling method, the antibody is selected from one or more of monoclonal antibodies, single-domain antibodies, multivalent antibodies, and antibody fragments. Preferably, the amino acid sequence of the antibody to be labeled may contain LLQS, which is a specific functional group and a protein tag that can be specifically recognized by transglutaminase. Transglutaminase is an enzyme that can be used for protein modification in enzyme-mediated strategies.
[0064] In one embodiment of the labeling method, the label is an imaging probe or a drug.
[0065] In a more specific embodiment of the labeling method, the imaging probe is selected from one or more of fluorescent molecules, magnetic resonance imaging contrast agents, and radionuclides; the drug is selected from one or more of small molecule compounds, nucleic acids, peptides, and proteins. Specifically, the imaging probe can be DBCO-FAM (CAS No.: 1384485-04-4) or DBCO-PEG4-Val-Cit-PAB-MMAE (CAS No.: 2129164-91-4).
[0066] Based on the same inventive concept, embodiments of the present invention provide a composition for antibody labeling, the composition comprising: an antibody conjugate, two or more compounds, and a labeler;
[0067] The antibody has at least one active group at its terminal.
[0068] The antibody conjugate includes two or more active groups, one of which can be coupled to the active group of the antibody, and the other active group can be coupled to the active group at the end of at least one compound.
[0069] The marker has at least one active group at its end;
[0070] Each compound comprises two or more active groups at its end, wherein one active group is capable of coupling with the active group at the end of the antibody conjugate and / or another compound, and the other active group is capable of coupling with the active group at the end of the label and / or another compound.
[0071] The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine.
[0072] The number of at least one active group in the antibody conjugate or at least one compound is greater than 2.
[0073] In one embodiment of the composition, the compound comprises: a first compound and a second compound, i.e., the composition comprises: an antibody conjugate, a first compound, a second compound, and a labeler;
[0074] The antibody has an active A group at its terminal;
[0075] The antibody conjugate has B active groups and C active groups at its ends;
[0076] The first compound has a D-active group and an E-active group at its end;
[0077] The second compound has an F active group and a C active group at its end;
[0078] The marker has a D active group at its end;
[0079] The active groups A and B, C and D, and E and F are complementary active groups.
[0080] The number of at least one of the C active group of the antibody conjugate, the E active group of the first compound, and the C active group of the second compound is greater than 2.
[0081] Specifically, the A and B active groups are complementary active groups, the C and D active groups are complementary active groups, and the E and F active groups are complementary active groups. After the A active group of the antibody binds to the B active group of the antibody conjugate, a first complex with a C active group at its end is formed. This first complex can either couple with the D active group of the first compound to form a first intermediate complex with an E active group at its end, or couple with a label containing the D active group to form a final complex. The first intermediate complex can couple with the E active group and the F active group of the second compound to form a second intermediate complex with a C active group at its end. The second intermediate complex can then be coupled with a label containing the D active group; alternatively, a new cycle can be performed, where the second intermediate complex is sequentially coupled with the first compound, the second compound, and the label. The number of cycles can be selected according to actual needs, and will not be elaborated further here. Since the number of at least one of the C active groups of the antibody conjugate, the E active group of the first compound, and the C active group of the second compound is greater than 2, the composition of this embodiment can multiply the number of active groups at the antibody end reacting with the label through cyclic amplification.
[0082] In a more specific embodiment of the composition, the antibody conjugate is The first compound is The second compound is The active group at the end of the marker is in, The positions representing the connection points of the active groups are n, m, x, y, and z, which are natural numbers, preferably 3-50.
[0083] In one embodiment of the composition, the antibody is selected from one or more of monoclonal antibodies, single-domain antibodies, multivalent antibodies, and antibody fragments. Preferably, the amino acid sequence of the antibody to be labeled may contain LLQS, which is a specific functional group and a protein tag that can be specifically recognized by transglutaminase. Transglutaminase is an enzyme that can be used for protein modification in enzyme-mediated strategies.
[0084] In one embodiment of the composition, the marker is an imaging probe or a drug.
[0085] In a more specific embodiment of the composition, the imaging probe is selected from one or more of fluorescent molecules, magnetic resonance imaging contrast agents, and radionuclides; the drug is selected from one or more of small molecule compounds, nucleic acids, peptides, and proteins. Specifically, the imaging probe may be DBCO-FAM (CAS No.: 1384485-04-4) or DBCO-PEG4-Val-Cit-PAB-MMAE (CAS No.: 2129164-91-4).
[0086] In one embodiment of the composition, the antibody conjugate may bind to the antibody via complementary active groups, but is not limited thereto; the complementary active groups are as described above. For example, an amino group may be provided at the end of the antibody conjugate, and the glutamine residue on the antibody and the amino group on the antibody conjugate may be linked together by forming an isopeptide bond, which is recognized by glutamine transaminase.
[0087] Based on the same inventive concept, embodiments of the present invention provide an antibody labeling method, including the following steps:
[0088] Antibody labeling was performed using the composition described above;
[0089] Antibodies or intermediates are immobilized on a solid support for coupling and / or elution.
[0090] The intermediate is selected from any one of the following: a first complex obtained by conjugating an antibody and an antibody conjugate, an intermediate complex obtained by conjugating a first complex and at least one compound, or a final complex obtained by conjugating a first complex or an intermediate complex and a marker.
[0091] Specifically, to overcome the purification difficulties and antibody loss caused by multi-step coupling in traditional liquid-phase systems, this invention proposes a solid-liquid two-phase reaction system that immobilizes antibodies or intermediates on a solid-phase support (such as...) through reversible binding. Figure 2After each reaction step, the coupled or labeled complex can be separated and collected by means of magnetic response, centrifugation and filtration. This enables multi-step modification or separation of antibodies on the surface of a solid-phase carrier, which significantly enhances the controllability and stability of antibodies, while reducing the risk of cross-contamination between multi-step reactions.
[0092] In one embodiment of the antibody labeling method, a first complex obtained by coupling an antibody and an antibody conjugate is immobilized on a solid support, and then sequentially coupled with at least two compounds at least once in an alternating manner. Finally, after coupling with a label, the complex is eluted to obtain a labeled antibody; wherein the antibody conjugate, the compound, and the label are as described above.
[0093] In one embodiment of the antibody labeling method, the solid support is selected from one or more of magnetic particles, silica gel, organic polymer materials, carbon nanotubes, and microfluidic chips. Preferably, the magnetic particles are Ni-NTA magnetic beads, specifically HisSep Ni-NTA MagBeads His-tagged protein purification magnetic beads (Yisheng, 20561ES08). Further, the Ni-NTA magnetic beads are activated in an activation solution before being mixed with the first reaction solution. The activation solution comprises 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, and 10 mM imidazole, and the pH of the activation solution is 8.0.
[0094] In one embodiment of the antibody labeling method, the method of immobilizing the antibody or intermediate on a solid support is selected from one or more of electrostatic adsorption, hydrophobic interaction, affinity immobilization, covalent coupling, chemical crosslinking, and photocrosslinking.
[0095] In one embodiment of the antibody labeling method, the antibody labeling method specifically includes the following steps:
[0096] The antibody and antibody conjugate are mixed in the first buffer solution to carry out the first coupling reaction and obtain the first reaction solution.
[0097] The first reaction solution was mixed with the solid support and incubated for the first time. After incubation, the supernatant was discarded to obtain the first intermediate.
[0098] The first intermediate and the first compound were mixed in the second buffer solution to carry out the second coupling reaction. After the reaction, the supernatant was discarded to obtain the second intermediate.
[0099] The second intermediate and the second compound were mixed in a third buffer solution and subjected to a third coupling reaction. After the reaction, the supernatant was discarded to obtain the third intermediate.
[0100] The third intermediate and the label were mixed in the fourth buffer solution to carry out the fourth coupling reaction. After the reaction, the supernatant was discarded to obtain the fourth intermediate.
[0101] The fourth intermediate was mixed with the elution buffer and incubated for the second time. After incubation, the supernatant was collected to obtain the labeled antibody.
[0102] The antibody to be labeled, antibody conjugate, first compound, second compound, and label are as described above.
[0103] In a more specific embodiment of the antibody labeling method, the molar ratio of the antibody, antibody conjugate, first compound, second compound and label is 1:(20-80):(15-30):(15-30):(45-90).
[0104] In a more specific embodiment of the antibody labeling method, the first buffer is tris(hydroxymethyl)aminomethane buffer; the second, third, and fourth buffers are phosphate buffer solutions; and the elution buffer is imidazole buffer.
[0105] Specifically, the first buffer comprises 50 mM tris(hydroxymethyl)aminomethane (Tris) and 400 mM sodium chloride (NaCl); the pH of the first buffer is 8.0. Preferably, the first buffer further comprises 1-3 U of transglutaminase (TGase); when the first group of the first compound is amino, TGase can directly link it to the glutamine residue of the antibody to be labeled via an acyl transfer reaction.
[0106] The second, third, and fourth buffer solutions comprise: 8 mmol / L sodium dihydrogen phosphate (NaH2PO4), 6 mmol / L disodium hydrogen phosphate (Na2HPO4), 136 mmol / L sodium chloride (NaCl), 2.6 mmol / L potassium chloride (KCl), and 0-30% (v / v) dimethyl sulfoxide (DMSO), with a pH of 7.2-7.4.
[0107] The eluent comprises: 50 mM sodium dihydrogen phosphate (NaH2PO4), 300 mM sodium chloride (NaCl), and 250 mM imidazole; the pH of the eluent is 8.0.
[0108] In a more specific embodiment of the antibody labeling method, the specific conditions for the first coupling reaction include: temperature 10-40℃, time 2-24h.
[0109] The specific conditions for the first incubation include: temperature 10-40℃, time 0.5-4h;
[0110] The specific conditions for the second coupling reaction include: temperature 10-40℃, time 4-24h;
[0111] The specific conditions for the third coupling reaction include: temperature 10-40℃, time 2-12h;
[0112] The specific conditions for the second incubation include: temperature 10-40℃, time 10-120min.
[0113] The present invention will be further described below through specific embodiments.
[0114] Example 1
[0115] This embodiment provides compounds for antibody labeling, as follows:
[0116] (1) Synthesis method of NH2-2N3
[0117]
[0118] Methyl 3,5-dihydroxybenzoate (1.15 g, 1 equivalent) and potassium carbonate (5.67 g, 6 equivalent) were dissolved in 200 mL of acetonitrile. Then, TsO-PEG-N3 (4.97 g, 2.2 equivalent, CAS No.: 178685-33-1) was added under argon protection. The mixture was stirred and heated to 80 °C for 12 h. After cooling, the precipitate was discarded by filtration. The residue was purified by column chromatography using a methanol / dichloromethane mixture (1:200 v / v) as the eluent to give 3.17 g of molecule 1 (colorless oily liquid), with a yield of 96%.
[0119] Molecular 1 (0.33 g, 1 equivalent) and potassium hydroxide (0.08 g, 2 equivalents) were dissolved in 10 mL of ethanol and stirred overnight at 80 °C. After cooling, the reaction mixture was evaporated under reduced pressure. The residue was added to 2 mL of hydrochloric acid (1 M), and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The residue was purified by column chromatography using a methanol / dichloromethane mixture (v / v 1:40) as the eluent. 0.07 g of molecule 2 (oily liquid) was given in 94% yield.
[0120] Molecular 2 (0.24 g, 1 equivalent) and 2-[2-(2-T-BOC-aminoethoxy)ethoxy]ethanol (0.19 g, 1.5 equivalent) were dissolved in 100 mL of dry dichloromethane. The solution was cooled to 0 °C, and then EDC·HCl (0.49 g, 5 equivalent) was added under nitrogen protection. The mixture was stirred at room temperature for 12 h, washed with saturated brine and water, and dried over anhydrous sodium sulfate. The crude product was purified by simple column chromatography using a methanol / dichloromethane mixture (1:40 v / v) as the eluent. The product was dissolved in 10 mL of dry dichloromethane, and 2 mL of trifluoroacetic acid was added at 0 °C. The mixture was then stirred at room temperature for 6 h. The solvent was removed, and the residue was extracted three times with ethyl acetate. The combined organic layers were washed with saturated sodium carbonate solution and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane mixture (1:15 v / v) as the eluent. 0.21 g of a yellow oily liquid, NH₂⁻₂N₃, was obtained, with a yield of 68%.
[0121] (2) Synthesis method of Tz-3N3
[0122]
[0123] Methyl gallate (0.34 g, 1 equivalent), potassium carbonate (2.29 g, 9 equivalents), and TsO-PEG-N3 (1.21 g, 3.9 equivalents) were dissolved in 200 mL of acetonitrile. The mixture was heated to 80 °C and stirred for 12 h. After cooling, the precipitate was discarded by filtration. The residue was purified by column chromatography using a methanol / dichloromethane mixture (1:100 v / v) as the eluent. 0.71 g of a colorless oily liquid molecule 3 was given, in a yield of 59%.
[0124] Molecular 3 (0.71 g, 1 equivalent) and potassium hydroxide (0.36 g, 6 equivalent) were dissolved in 10 mL of ethanol and stirred overnight at 80 °C. After cooling, the reaction solution was evaporated under reduced pressure. The residue was added to 2 mL of hydrochloric acid (1 M) and extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The residue was purified by column chromatography using a methanol / dichloromethane mixture (1:40 v / v) as the eluent. 0.64 g of oily liquid molecule 4 was given, in 92% yield.
[0125] Molecular 4 (0.29 g, 1 equivalent) and Tz-PEG-OH (0.24 g, 1.5 equivalent) were dissolved in 100 mL of dry dichloromethane, and the solution was cooled to 0 °C. Then, EDC·HCl (0.43 g, 5 equivalent) was added under nitrogen protection. The mixture was stirred at room temperature for 12 h. The solution was washed with saturated brine and water, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography using a methanol / dichloromethane mixture (v / v 1:80) as the eluent. 0.25 g of a pink oily liquid, Tz-3N3, was obtained, with a yield of 56%.
[0126] (3) Synthesis method of NH2-N3
[0127] The synthesis method of NH2-2N3 is basically the same as that of (1), except that the raw material methyl 3,5-dihydroxybenzoate is replaced with methyl 3-hydroxybenzoate. The structure of NH2-N3 is as follows:
[0128] (4) Synthesis method of NH2-3N3
[0129] The synthesis method of NH2-2N3 is basically the same as that of (1), except that the raw material methyl 3,5-dihydroxybenzoate is replaced with methyl gallate. The structure of NH2-3N3 is as follows:
[0130] Example 2
[0131] This embodiment provides an antibody labeling method based on cyclic amplification. A single-domain antibody is used as the research object, and NH2-3N3, DBCO-TCO, and Tz-3N3 are sequentially conjugated to its ends. The antibody is then loaded with the fluorescent dye DBCO-FAM and the toxic drug DBCO-PEG4-Val-Cit-PAB-MMAE to verify the effectiveness of the cyclic amplification-based antibody labeling method. During the conjugation process, the single-domain antibody is immobilized on the surface of Ni-NTA magnetic beads through affinity binding, and separation is achieved through magnetic response after each reaction step. The specific operation steps are as follows:
[0132] (1) 0.2 mg of single-domain antibody (containing LLQS in the sequence) and 40 times the molar equivalent of NH2-3N3 synthesized in Example 1 were mixed in Tris buffer (containing 50 mM Tris, 400 mM sodium chloride, pH 8.0), 1-3 U of glutamine transaminase solution was added, and the volume was adjusted to 1 mL. The mixture was then incubated by rotation at room temperature for 2 h.
[0133] (2) Take 25-50 μL of Ni-NTA magnetic beads (Yisheng, 20561ES08) suspension in a 2 mL centrifuge tube, perform magnetic separation, and discard the storage solution. Add 200 μL of activation solution (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times. Mix the mixed solution from (1) with the activated magnetic beads and incubate on a rotary mixer for 30 min. After incubation, collect the magnetic beads, add 500 μL of washing solution (PBS buffer containing 0.1 wt% PEG) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times.
[0134] (3) Continue adding 15-30 molar equivalents of DBCO-TCO (CAS No.: 1801863-88-6) relative to the single-domain antibody. The reaction system is 500 μL of PBS buffer containing an appropriate amount of DMSO (0-30% (v / v)). Incubate with the magnetic beads at room temperature for 12 h by rotation. After the reaction is complete, magnetically separate, discard the supernatant, and wash the magnetic beads 3 times.
[0135] (4) Continue to add 15-30 times the molar equivalent of the Tz-3N3 synthesized in Example 1 relative to the single-domain antibody. The reaction system is 500 μL of PBS buffer containing an appropriate amount of DMSO (0-30% (v / v)). After vortexing the mixture with the magnetic beads, incubate it on a rotary mixer for 4 h. After the reaction is complete, wash the magnetic beads 3 times.
[0136] (5) Continue to add 45-90 times the amount of the label DBCO-FAM (CAS No.: 1384485-04-4) or DBCO-PEG4-Val-Cit-PAB-MMAE (CAS No.: 2129164-91-4) relative to the single-domain antibody. The reaction system is PBS buffer containing an appropriate amount of DMSO (0-30% (v / v)). React in the dark for 12 hours on a rotary mixer.
[0137] (6) After the reaction is complete, collect the magnetic beads, wash them three times, add 200 μL of elution buffer (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride and 250 mM imidazole, pH 8.0) to resuspend the magnetic beads, vortex to mix, and incubate for 10 min. After incubation, perform magnetic separation, collect the supernatant, and repeat 2-3 times;
[0138] (7) Transfer the collected supernatant to a 3kDa ultrafiltration centrifuge tube, add the same volume of PBS buffer to dilute the solution, centrifuge at 7500g for 5-10 min, concentrate to the original volume, and repeat the operation 5 times to obtain the labeled single-domain antibody. The unlabeled single-domain antibody conjugate is designated as Nb-L9, the DBCO-FAM-loaded single-domain antibody-dye conjugate is designated as Nb-L9-F, and the DBCO-PEG4-Val-Cit-PAB-MMAE-loaded single-domain antibody-drug conjugate is designated as Nb-L9-D.
[0139] Comparative Example 1
[0140] This comparative example provides an antibody labeling method, and the specific operation steps are as follows:
[0141] (1) 0.2 mg of single-domain antibody (containing LLQS in the sequence) and 40 times the molar equivalent of NH2-N3 synthesized in Example 1 were mixed in Tris buffer (containing 50 mM Tris, 400 mM sodium chloride, pH 8.0), 1-3 U of glutamine transaminase solution was added, and the volume was adjusted to 1 mL. The mixture was then incubated by rotation at room temperature for 2 h.
[0142] (2) Take 25-50 μL of Ni-NTA magnetic beads (Yisheng, 20561ES08) suspension in a 2 mL centrifuge tube, perform magnetic separation, and discard the storage solution. Add 200 μL of activation solution (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times. Mix the mixed solution from (1) with the activated magnetic beads and incubate on a rotary mixer for 30 min. After incubation, collect the magnetic beads, add 500 μL of washing solution (PBS buffer containing 0.1 wt% PEG) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times.
[0143] (3) Continue to add 5-10 times the amount of the label DBCO-FAM (CAS No.: 1384485-04-4) or DBCO-PEG4-Val-Cit-PAB-MMAE (CAS No.: 2129164-91-4) relative to the single-domain antibody. The reaction system is PBS buffer containing an appropriate amount of DMSO (0-30% (v / v)). React in the dark for 12 hours on a rotary mixer.
[0144] (4) After the reaction is complete, collect the magnetic beads, wash them three times, add 200 μL of elution buffer (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride and 250 mM imidazole, pH 8.0) to resuspend the magnetic beads, vortex to mix, and incubate for 10 min. After incubation, perform magnetic separation, collect the supernatant, and repeat 2-3 times;
[0145] (5) Transfer the collected supernatant to a 3kDa ultrafiltration centrifuge tube, add the same volume of PBS buffer to dilute the solution, centrifuge at 7500g for 5-10 min, concentrate to the original volume, and repeat the operation 5 times to obtain the labeled single-domain antibody. The unlabeled single-domain antibody conjugate is designated as Nb-L1, the DBCO-FAM-loaded single-domain antibody-dye conjugate is designated as Nb-L1-F, and the DBCO-PEG4-Val-Cit-PAB-MMAE-loaded single-domain antibody-drug conjugate is designated as Nb-L1-D.
[0146] Comparative Example 2
[0147] This comparative example provides an antibody labeling method, and the specific operation steps are as follows:
[0148] The specific steps are as follows:
[0149] (1) 0.2 mg of single-domain antibody (containing LLQS in the sequence) and 40 times the molar equivalent of NH2-3N3 synthesized in Example 1 were mixed in Tris buffer (containing 50 mM Tris, 400 mM sodium chloride, pH 8.0), 1-3 U of glutamine transaminase solution was added, and the volume was adjusted to 1 mL. The mixture was then incubated by rotation at room temperature for 2 h.
[0150] (2) Take 25-50 μL of Ni-NTA magnetic beads (Yisheng, 20561ES08) suspension in a 2 mL centrifuge tube, perform magnetic separation, and discard the storage solution. Add 200 μL of activation solution (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times. Mix the mixed solution from (1) with the activated magnetic beads and incubate on a rotary mixer for 30 min. After incubation, collect the magnetic beads, add 500 μL of washing solution (PBS buffer containing 0.1 wt% PEG) to resuspend the magnetic beads, perform magnetic separation, and discard the supernatant. Repeat 2-3 times.
[0151] (3) Continue to add 15-30 times the amount of the label DBCO-FAM (CAS No.: 1384485-04-4) or DBCO-PEG4-Val-Cit-PAB-MMAE (CAS No.: 2129164-91-4) relative to the single-domain antibody. The reaction system is PBS buffer containing an appropriate amount of DMSO (0-30% (v / v)). React in the dark for 12 hours on a rotary mixer.
[0152] (4) After the reaction is complete, collect the magnetic beads, wash them three times, add 200 μL of elution buffer (containing 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride and 250 mM imidazole, pH 8.0) to resuspend the magnetic beads, vortex to mix, and incubate for 10 min. After incubation, perform magnetic separation, collect the supernatant, and repeat 2-3 times;
[0153] (5) Transfer the collected supernatant to a 3kDa ultrafiltration centrifuge tube, add the same volume of PBS buffer to dilute the solution, centrifuge at 7500g for 5-10 min, concentrate to the original volume, and repeat the operation 5 times to obtain the labeled single-domain antibody. The unlabeled single-domain antibody conjugate is designated as Nb-L3, the DBCO-FAM-loaded single-domain antibody-dye conjugate is designated as Nb-L3-F, and the DBCO-PEG4-Val-Cit-PAB-MMAE-loaded single-domain antibody-drug conjugate is designated as Nb-L3-D.
[0154] Example 3
[0155] This embodiment describes and tests the labeled single-domain antibodies of Example 2 and Comparative Examples 1-2, as follows:
[0156] (1) Target function: Single-domain antibody conjugates Nb-L1, Nb-L3, and Nb-L9 were labeled with the fluorescent dye FITC, and U87MG cells were subjected to immunofluorescence staining. The flow cytometry analysis results are shown in […]. Figure 3 This result demonstrates that the aforementioned enzyme-mediated and click chemistry-based cyclic amplification labeling and affinity-based antibody immobilization strategies do not affect the antigen recognition and binding ability of the single-domain antibody itself.
[0157] (2) Loading capacity: The antibody concentrations and absorbance at 495 nm of Nb-L1-F, Nb-L3-F, and Nb-L9-F were tested in vitro, and the ratios are shown in the table below. Figure 4 The fluorescence emission results are shown in Figure 5 The average fluorescence intensity results of labeled U87MG cells are shown in the figure. Figure 6 The above results fully demonstrate the successful construction of the dendritic topology and the effectiveness of the cyclic amplification method, increasing the number of terminal reactive groups of single-domain antibodies by 9-fold. The fluorescence emission intensity of the high-load single-domain antibody-dye conjugate Nb-L9-F and the average fluorescence brightness of its labeled tumor cells were both significantly improved.
[0158] (3) In vitro antitumor activity: Different concentrations of single-domain antibody-drug conjugates Nb-L1-D, Nb-L3-D, and Nb-L9-D were co-incubated with U87MG tumor cells for 72 h. After incubation, cell viability was analyzed using the MTT assay. The results are as follows: Figure 7As shown in the figure, these results demonstrate that the cytotoxicity of single-domain antibody-drug conjugates increases with increasing concentration. At the same antibody concentration, the high-load single-domain antibody-drug conjugate (Nb-L9-D) exhibited the best anticancer effect compared to low-load (Nb-L1-D) and medium-load (Nb-L3-D) single-domain antibody-drug conjugates, with less than 20% of tumor cells co-incubated with it surviving.
[0159] (4) In vivo targeting and metabolism: Cy7-labeled Nb-L1-D, Nb-L3-D, and Nb-L9-D were injected into tumor-bearing mice via tail vein at the same antibody dose. Whole-body fluorescence images of the mice were acquired and analyzed at different time points. The in vivo imaging results are as follows: Figure 8 As shown, the statistical analysis results are as follows: Figure 9 As shown, due to the rapid clearance characteristics of the toxic drug MMAE, the metabolic rate of Nb-L9-D loaded with more drug was accelerated in vivo, but its targeted accumulation at the tumor site was not significantly different from the other two groups. Figure 10 The biodistribution of Nb-L1-D, Nb-L3-D, and Nb-L9-D in major organs of mice was shown 24 hours after drug administration. Figure 11 The results show statistical findings on the distribution. It can be observed that Nb-L9-D accumulates most in metabolic organs such as the liver and kidneys, but its specific accumulation in tumor tissue is not significantly different from the other two groups. These results collectively confirm that Nb-L9-D retains excellent tumor targeting and penetration capabilities in vivo.
[0160] (5) In vivo tumor treatment efficacy: Nb-L1-D, Nb-L3-D, and Nb-L9-D were injected into tumor-bearing mice at a dose of 0.5 mg / kg body weight. Mouse body weight and subcutaneous tumor size were measured every 3 days. The results are as follows: Figure 12 and Figure 13 As shown in the figure. Mice were euthanized on day 21 after drug administration, and their subcutaneous tumors were removed, photographed, and weighed. The results are as follows. Figure 14 and Figure 15 As shown. In addition, paraffin sections of major organs from mice were prepared and stained with H&E; the results are shown in [Figure 1]. Figure 16 The above results collectively confirm that Nb-L9-D exhibits the best therapeutic effect in inhibiting tumor growth. At the current dosage, Nb-L1-D, Nb-L3-D, and Nb-L9-D did not cause toxic damage to other normal organs and tissues, demonstrating good biocompatibility.
[0161] In summary, this invention provides a labeling method based on cyclic amplification, comprising the steps of: alternatingly coupling a target compound with two or more compounds at least once, followed by coupling with a labeling compound to obtain a labeled product; wherein the target compound and the labeling compound have at least one active group at their ends; each compound comprises two or more active groups at its ends, wherein one active group can couple with the active group at the end of the target compound and / or another compound, and the other active group can couple with the active group at the end of the labeling compound and / or another compound; the coupling is carried out through complementary active groups, the complementary active groups being selected from one of the following combinations: primary amino and N-hydroxysuccinimide ester, maleimide and thiol, carboxyl and amino, glutamine residue and primary amino, azide and alkyne, tetraazine and transcyclooctene, thioester and hydroxylamine; the number of at least one active group in at least one compound is greater than 2. The compounds in this labeling method can be alternately coupled through terminal active groups to form a "tree-like topology," thereby multiplying the number of terminal active groups of the target compound and achieving a greater loading of the labeling compound. Furthermore, this invention provides a composition for antibody labeling, which enables the aforementioned cyclic amplification-based labeling method in antibody labeling. In addition, to overcome the purification difficulties and antibody loss caused by multi-step coupling in traditional liquid-phase systems, this invention proposes a solid-liquid two-phase reaction system. This system immobilizes antibodies on a solid-phase support through reversible binding. After each reaction step, the labeled antibodies can be separated and collected by magnetic response, centrifugation, and filtration, improving antibody product recovery while simplifying purification steps and reducing the risk of cross-contamination between multi-step reactions.
[0162] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. A labeling method based on cyclic amplification, characterized in that, The steps include: alternating coupling of the target compound with two or more compounds at least once, followed by coupling with the labeled compound to obtain the labeled product; The terminant and the marker each have at least one active group at their ends; Each compound has two or more active groups at its end, one of which can be coupled to the active group at the end of the target compound and / or another compound, and the other active group can be coupled to the active group at the end of the target compound and / or another compound. The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine. The number of at least one active group in at least one compound is greater than 2.
2. The marking method according to claim 1, characterized in that, The labeling agent is selected from one or more of antibodies, nucleic acid aptamers, peptides, nanoparticles, and carbon-based materials.
3. The marking method according to claim 2, characterized in that, The antibody is selected from one or more of monoclonal antibodies, single-domain antibodies, multivalent antibodies, and antibody fragments.
4. The marking method according to claim 1, characterized in that, The marker is an imaging probe or a drug.
5. The marking method according to claim 4, characterized in that, The imaging probe is selected from one or more of fluorescent molecules, magnetic resonance imaging contrast agents, and radionuclides. And / or, the drug is selected from one or more small molecule compounds, nucleic acids, polypeptides, and proteins.
6. A composition for antibody labeling, characterized in that, The composition comprises: an antibody conjugate, two or more compounds, and a labeler; The antibody has at least one active group at its terminal; The antibody conjugate includes two or more active groups, one of which can be coupled to the active group of the antibody, and the other active group can be coupled to the active group at the end of at least one compound. The marker has at least one active group at its end; Each compound comprises two or more active groups at its end, wherein one active group is capable of coupling with the active group at the end of the antibody conjugate and / or another compound, and the other active group is capable of coupling with the active group at the end of the label and / or another compound. The coupling is carried out through complementary active groups selected from one of the following combinations: primary amino and N-hydroxysuccinimide esters, maleimide and thiol, carboxyl and amino, glutamine residues and primary amino, azide and alkyne, tetrazine and transcyclooctene, thioester and hydroxylamine. The number of at least one active group in the antibody conjugate or at least one compound is greater than 2.
7. The composition according to claim 6, characterized in that, The compounds include: a first compound and a second compound; The antibody has an active A group at its terminal; The antibody conjugate has B active groups and C active groups at its ends; The first compound has a D-active group and an E-active group at its end; The second compound has an F active group and a C active group at its end; The marker has a D active group at its end; The active groups A and B, C and D, and E and F are complementary active groups. The number of at least one of the C active group of the antibody conjugate, the E active group of the first compound, and the C active group of the second compound is greater than 2.
8. An antibody labeling method, characterized in that, Including the following steps: Antibody labeling is performed using the composition as described in claim 6 or 7; Antibodies or intermediates are immobilized on a solid support for coupling and / or elution. The intermediate is selected from any one of the following: a first complex obtained by conjugating an antibody and an antibody conjugate, an intermediate complex obtained by conjugating a first complex and at least one compound, or a final complex obtained by conjugating a first complex or an intermediate complex and a marker.
9. The antibody labeling method according to claim 8, characterized in that, The solid support is selected from one or more of magnetic particles, silica gel, organic polymer materials, carbon nanotubes, and microfluidic chips.
10. The antibody labeling method according to claim 8, characterized in that, The method for immobilizing antibodies or intermediates on a solid support is selected from one or more of electrostatic adsorption, hydrophobic interaction, affinity immobilization, covalent coupling, chemical crosslinking, and photocrosslinking.