Two-site specific modified antibody-functional molecule complex, method for producing the same, and use

Abstract

This invention relates to a two-site specific modification antibody-functional molecule complex, a method for producing the same, and its use. The two-site specific modification antibody-functional molecule complex comprises an antibody, ligation fragment 1, ligation fragment 2, and a functional molecule, wherein the functional molecule is modified via ligation fragment 1 to a conserved glycosylation site in the antibody's Fc region and via ligation fragment 2 to the 246th or 248th lysine site of the antibody. In some embodiments, the two-site specific modification antibody-functional molecule complex is represented by formula I or II. The two-site specific modification antibody-functional molecule complex of the present invention exhibits excellent stability, cytotoxicity, and in vivo activity, and has good pharmacoagulation, making it suitable for use in the production of pharmaceuticals, diagnostic and therapeutic reagents, and reagent kits for tumors, inflammatory diseases, viral infectious diseases, and immunological diseases. Its production method has the advantages of being easy to operate, requiring few purification steps, and possessing excellent pharmacoagulation, making it easy to industrialize. [Formula 1] JPEG2026510582000069.jpg93170

Description

[Technical Field]

[0001] This invention relates to the fields of medicinal chemistry and biotechnology pharmaceuticals, and more particularly to a type of two-site specific modified antibody-functional molecule conjugate, a method for producing the same, and its use. [Background technology]

[0002] Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a cytotoxin, and a linker. The antibody plays a role in transporting the cytotoxin to tumor tissue, thereby achieving targeted toxin delivery and exerting antitumor activity. A large amount of data has shown that site-directed modified antibody-drug conjugates have a superior therapeutic index compared to conventional randomly modified ADCs. The drug-to-antibody ratio (DAR) represents the average number of toxins bound to the antibody. The DAR value is extremely important for the efficacy of ADCs. A low DAR value may affect the therapeutic effect of ADCs, while a high DAR value increases the hydrophobicity of ADCs and negatively impacts pharmacokinetics in the body. Therefore, when designing the structure of ADCs, the antibody, toxin, linker, and modification site should be comprehensively considered to determine the optimal DAR value. For example, for relatively weak cytotoxins, it is possible to produce ADCs compounds with high DAR values ​​by introducing a hydrophilic drug-linker fragment, thereby exhibiting superior lethal effects.

[0003] Furthermore, in recent years, the concept of dual-drug ADCs (ADCs) that are expected to exert a synergistic effect (1+1>2) has emerged. However, whether it is an ADC compound with a specific DAR value or a dual-drug ADC, both require specific methods for production. Stephen Caddick et al. have reported producing a dual-drug ADC compound by introducing linear alkyne and cyclic strained alkyne structures into an antibody using cysteine ​​crosslinking technology, and then introducing two different toxin molecules using a bioorthogonal reaction. Christoph Rader et al. produced a dual-drug ADC compound by combining THIOMAB technology and SELENOMAB technology. Matthew R. Levengood et al. produced a dual-drug ADC compound exhibiting excellent antitumor activity by introducing two thiol groups with different protecting groups into an antibody, deprotecting each, and then sequentially modifying them with different toxin molecules. Nazzareno Dimasi et al. synthesized a trifunctional linker containing maleimide, alkynyl, and ketose groups, and used it to ultimately produce a complex drug ADC compound.

[0004] While the above studies offer various strategies for the production of high-DAR-value ADCs and conjugate-agent ADCs, these strategies typically require the design of highly branched structures, the use of different genetic engineering techniques, complex enzyme combinations, or the formation of protein-protein complexes. Most of these strategies can lead to the introduction of multiple hydrophobic structures and low modification efficiency. This complicates the production of conjugate-agent antibody-functional molecule complexes, hindering industrialization, while also causing increased local hydrophobicity and reduced stability of the resulting drugs.

[0005] Therefore, there remains a strong need to provide two-site-specific modified antibody-functional molecule complexes with novel structures, as well as simple and efficient methods for their production. [Overview of the project]

[0006] Based on the above background, the present inventors have invented a two-site-specific modified antibody-functional molecule complex. By combining site-specific modification technology for glycosylation and site-specific modification technology induced by guider peptides, they have achieved the specific control over the location and amount of the functional molecule introduced to the glycosylation site of the antibody and the 246th or 248th lysine residue (Lys246 / Lys248), thereby producing an antibody-functional molecule complex with a specific DAR value. The two-site-specific modified ADC compound provided in this disclosure has a simple and efficient manufacturing method. Furthermore, the ADC molecule obtained by this method has a novel structure and exhibits excellent in vitro and in vivo stability and activity.

[0007] One objective of the present invention is to provide a bisite-specific modified antibody-functional molecule complex.

[0008] Another object of the present invention is to provide a method for producing the two-site-specific modified antibody-functional molecule complex.

[0009] Another object of the present invention is to provide the use of the two-site-specific modified antibody-functional molecule complex in applications such as antitumor therapy.

[0010] According to one embodiment of the present invention, a two-site specific modification antibody-functional molecule complex is provided, comprising an antibody, a linking fragment 1, a linking fragment 2, and a functional molecule, wherein the functional molecule is modified via linking fragment 1 to a conserved glycosylation site in the antibody's Fc region and via linking fragment 2 to the 246th or 248th lysine site of the antibody.

[0011] According to one embodiment of the present invention, the functional molecule is modified via linking fragment 1 to a conserved glycosylation site in the antibody Fc region, and via linking fragment 2 to the 248th lysine site of the antibody.

[0012] According to one embodiment of the present invention, The antibody is selected from the group consisting of a humanized antibody containing an Fc region, or an antibody derived from another animal containing an Fc region; The functional molecules may be identical or different from each other, and are independently selected from the group consisting of reactive functional groups, fluorescent groups, drugs, toxins, radioactive structures, and lysosome-directed peptides; The linked fragment 1 comprises a linker and a sugar structure, the sugar structure being bound to a conserved glycosylation site in the antibody Fc region, and the linker being bound to a functional molecule; The linked fragment 2 comprises a linker and fragment A, wherein fragment A is bound to the 246th or 248th lysine site of the antibody, and the linker is bound to the functional molecule; The sugar structure represents a monosaccharide, disaccharide, oligosaccharide, or branched sugar structure; fragment A represents an amide structure or a triazole structure; the glycosylation site of the antibody Fc structural domain represents any structure in which a linker can connect the sugar structure and the functional molecule; and the 246th or 248th lysine site of the antibody represents any structure in which a linker can connect fragment A and the functional molecule.

[0013] In this invention, more specifically, The aforementioned antibody is selected from the group consisting of different IgG subtype antibodies, monoclonal antibodies, bifunctional or multifunctional antibodies, polyclonal antibodies, and functional antibodies derived from different species; for example, it is selected from the group consisting of trastuzumab (Herceptin®), rituximab (Mabutera®), pertuzumab, panitumumab, triplilimab, nivolumab, mouse-derived antibodies, rabbit-derived antibodies, and sheep-derived antibodies; In the functional molecule, the reactive functional group is selected from the group consisting of azide group, tetrazine group, TCO group, alkynyl group structure, aldehyde group structure, carbonyl group structure, hydroxylamine structure, isothiocyanate, amino group, carboxyl group, thiol group, alkenyl group, maleimide group, acylhydrazone structure, etc. The aforementioned fluorescent group is selected from the group consisting of biotin, FITC, rhodamine, Cy3, Cy5, etc. The aforementioned pharmaceutical or toxin is selected from the group consisting of MMAE, MMAF, Dxd (deruxtecan), Dx8951, SN38, DM1, DM4, PBD, PBD dimer, eribulin, duocalmycin, and derivatives of these drugs; The aforementioned radioactive structure, 68 Ga, 18 F, 89 Selected from the group consisting of Zr, etc.; The lysosome-targeting peptide is SignalTAC.

[0014] According to one embodiment of the present invention, the two-site-specific modified antibody-functional molecule complex is represented by the following formula I.

[0015] [ka] In formula I, the central "Y" shaped structure represents the antibody; [ka] The core fucose structure is represented; n is 0 or 1; the linker represents a PEG chain, carbon chain, cleavable linker, etc.; the sugar structure represents a monosaccharide, disaccharide, oligosaccharide, or branched sugar structure; fragment A represents an amide structure or triazole structure; [ka] The symbol represents a functional molecule, and the functional molecules of the glycosylation site and the lysine site may be the same or different, and one or more functional molecules may be modified.

[0016] According to one aspect of the present invention, the two-site-specific modified antibody-functional molecule complex of formula I is represented by the following formula II: [ka] In Equation II, the definitions of each part are the same as in the above text.

[0017] According to one aspect of the present invention, the two-site-specific modified antibody-functional molecule complex of formula II is selected from the following specific compounds: [ka] [ka] [ka] Here, the MMAE structure [ka] The MMAF structure is [ka] The Dx8951 structure is [ka] And; the eribulin structure is [ka] The SignalTAC structure is [ka] That is the case.

[0018] According to one embodiment of the present invention, a method for producing the two-site-specific modified antibody-functional molecule complex is provided, which is selected from the following methods 1 to 3 and is represented by the following reaction formula. [ka] Here, the definitions of each part in the above reaction equation are as defined above.

[0019] Method 1: A wild-type antibody or a defucosified antibody is treated with a glycosyltransferase or endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-functional molecule complex in which a functional molecule is modified at the glycosylation site of the antibody; subsequently, the 246th or 248th lysine site of the antibody is modified with a functional molecule-linker using a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to finally obtain a two-site-specific modified antibody-functional molecule complex; or Method 2: Add the enzyme and substrate required for site-specific glycosylation technology to a wild-type antibody or a defucosed antibody, and simultaneously add the substrate required for guider peptide / protein-mediated lysine site-specific modification technology, and after incubation, obtain a two-site-specific modified antibody-functional molecule complex; or Method 3: A wild-type antibody or a defucosified antibody is first treated with a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to modify the 246th or 248th lysine site of the antibody with a functional molecule-linker, and then a glycosyltransferase or endoglycosidase-mediated glycosylation site-specific modification technique is used to modify the glycosylation site of the antibody with a functional molecule, ultimately obtaining a two-site-specific modified antibody-functional molecule complex; Here, the glycosyltransferases include, but are not limited to, fucosyltransferases, galactosyltransferases, sialyltransferases, etc.; the endoglycosidases include, but are not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and also include fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0020] According to one embodiment of the present invention, Method 1 is, Step a1: The antibody is placed in a buffer (e.g., pH 5.5-8.0), a sugar structure-functional molecule complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to overnight (e.g., 20 hours) to obtain a sugar site-specific modified antibody-functional molecule complex in which the glycosylation site of the antibody is modified with a functional molecule. Step a2: Next, the product obtained in step a1 is placed in a buffer (e.g., pH 5.5-8.0), the guider peptide-functional molecule complex is added, and the mixture is reacted at a temperature of 0°C to 40°C for 30 minutes to overnight (e.g., 20 hours) to modify the 246th or 248th lysine site of the antibody with a functional molecule-linker, ultimately obtaining a two-site specific modified antibody-functional molecule complex. Includes.

[0021] According to one embodiment of the present invention, method two is, The antibody is placed in a buffer (pH 5.5-8.0), a glycosyltransferase or endoglycosidase and a sugar structure-functional molecule complex are added, and at the same time, a guide peptide-functional molecule complex is added. The mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to overnight (e.g., 20 hours), and after incubation, a two-site specific modified antibody-functional molecule complex is obtained. Includes.

[0022] According to one embodiment of the present invention, the method third is, Step c1: The antibody is placed in a buffer (pH 5.5-8.0), the guider peptide-functional molecule complex is added, and the mixture is reacted at a temperature of 0°C to 40°C for 30 minutes to overnight (e.g., 20 hours), thereby modifying the 246th or 248th lysine site of the antibody with a functional molecule-linker. Step c2: Next, the product obtained in step c1 is placed in a buffer (pH 5.5-8.0), a glycosyltransferase or endoglycosidase and a sugar structure-functional molecule complex are added, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to overnight (e.g., 20 hours) to finally obtain a two-site specific modified antibody-functional molecule complex. Includes.

[0023] The glycosyltransferases described in methods 1, 2, and 3 above include, but are not limited to, fucosyltransferases, galactosyltransferases, sialyltransferases, etc.; the endoglycosidases include, but are not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and further include fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0024] According to one embodiment of the present invention, the intermediate obtained in step a1 of Method 1, which is a glycosylation site-specific modification antibody-functional molecule complex, can proceed directly to the next lysine-specific modification step without purification, or to the next operation after purification, and / or The intermediate K246 or K248 site-specific modified antibody-functional molecule complex obtained in step c1 of Method 3 can proceed directly to the next glycosylation site-specific modification step without purification, or it can proceed to the next operation after purification.

[0025] According to one embodiment of the present invention, the two-site-specific modified antibody functional molecule complex is produced by Method 1 or Method 3, wherein in step a1 of Method 1 and step c2 of Method 3, an endoglycosidase-mediated site-specific modification technique is used; and in step a2 of Method 1 and step c1 of Method 3, an Fc-binding peptide is used as a guider fragment to the antibody Fc structural domain, and a thioester structure is used as an acyl-transfer fragment. Specifically: Method 1: A wild-type antibody or a defucosified antibody is treated with an endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-functional molecule complex in which a functional molecule is modified at the glycosylation site of the antibody. Subsequently, the 246th or 248th lysine site of the antibody is modified with a functional molecule-linker using a guider peptide-mediated K246 or K248 site-specific modification technique to finally obtain a two-site-specific modified antibody-functional molecule complex. Here, the intermediate site-specific modified antibody-functional molecule complex can be purified, or the reaction system can be directly advanced to the next lysine-specific modification step.

[0026] Method 3: A wild-type antibody or a defucosified antibody is first treated with a guider peptide-mediated K246 or K248 site-specific modification technique to modify the 246th or 248th lysine site of the antibody with a functional molecule linker. Subsequently, an endoglycosidase-mediated glycosylation site-specific modification technique is used to modify the glycosylation site of the antibody with a functional molecule, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. Here, the intermediate K246 or K248 site-specific modified antibody-functional molecule complex can be purified, or the reaction system can be directly advanced to the next glycosylation site-specific modification step.

[0027] According to one embodiment of the present invention, a method for producing the two-site-specific modified antibody-functional molecule complex is provided, selected from the following methods four and five, and represented by the following reaction formula.

[0028] Method four: [ka] Method five: [ka] Here, the definition of each part in the above reaction equation is the same as that defined in claim 5, [ka] " and " [ka] " represents a reactive functional group: Method IV: A wild-type antibody or a defucosified antibody is treated by a glycosyltransferase or endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a reactive functional group-linker is modified at the 246th or 248th lysine site of the antibody by a guider peptide or guider protein-mediated site-specific modification technique to obtain a bisite-specific modified bifunctional antibody. Then, a bioorthogonal reaction is performed to introduce a functional molecule (e.g., a toxin) at the glycosylation site and the K246 or K248 site, respectively, to finally obtain a bisite-specific modified antibody-functional molecule complex; or Method 5: A wild-type antibody or a defucosified antibody is first treated with a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to modify the 246th or 248th lysine site of the antibody with a reactive functional group-linker. Subsequently, a glycosylation site-specific modification technique mediated by a glycosyltransferase or endoglycosidase is used to modify the glycosylation site of the antibody with a reactive functional group to obtain a bisite-specific modified bifunctional antibody. Then, a bioorthogonal reaction is performed to introduce a functional molecule (e.g., a toxin) into the glycosylation site and the K246 or K248 site, respectively, to finally obtain a bisite-specific modified antibody-functional molecule complex.

[0029] Here, the glycosyltransferase includes, but is not limited to, fucosyltransferase, galactosyltransferase, sialyltransferase, etc.; the endoglycosidase includes, but is not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and further includes fusion proteins formed by fusing the functional regions of these different endoglycosidases; and the definition of the reactive functional group is the same as above.

[0030] According to one embodiment of the present invention, The aforementioned method four is, Step b1: The antibody is placed in a buffer, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 15°C to 37°C to obtain a sugar site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a guider peptide-reactive functional group complex is added to the above system, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C to modify the 246th or 248th lysine site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step b2: Subsequently, two types of functional molecule-linkers are added to the buffer system of step b1, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. including, or The aforementioned method five is, Step d1: The antibody is placed in a buffer, a guider peptide-reactive functional group complex is added, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C to obtain a lysine-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the 246th or 248th lysine site of the antibody. Subsequently, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added to the above system, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 15°C to 37°C to modify the glycosylation site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step d2: Subsequently, two types of functional molecule-linkers are added to the buffer system of step d1, and the reaction is carried out at a temperature of 0°C to 40°C for 30 minutes to 20 hours to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. Includes.

[0031] According to one embodiment of the present invention, The intermediate obtained in step b1 of Method 4, the glycosylation site-specific modified antibody-reactive functional group complex and the bisite-specific modified difunctionalized antibody, can proceed directly to the next step without purification or after ultrafiltration purification, and / or The intermediates obtained in step d1 of Method 5, namely the K246 or K248 site-specific modification-reactive functional group complex and the bisite-specific modification bifunctionalized antibody, can be used without purification or after ultrafiltration purification to proceed directly to the next step.

[0032] According to one embodiment of the present invention, the method is carried out by the following method 6: Method six: [ka] Here, the definitions of each part in the above reaction equation are as defined above.

[0033] Method 6: A wild-type antibody or a defucosified antibody is treated by an endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-reactive functional group complex in which an acylhydrazone structure is added to the glycosylation site of the antibody. Subsequently, an azide functional group is added to the 246th or 248th lysine site of the antibody by a guider peptide or guider protein-mediated site-specific modification technique to obtain a bifunctionalized antibody that is bisite-specifically modified with acylhydrazone and azide. Then, functional molecules are introduced to the glycosylation site and the K246 or K248 site, respectively, via an acylhydrazone-oxime exchange reaction and a click chemistry reaction, finally obtaining a bisite-specific modified antibody-functional molecule complex.

[0034] According to one embodiment of the present invention, method six is, Step e1: The antibody is placed in a buffer at pH 6.0-8.0, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to 20 hours to obtain a sugar site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a guide peptide-reactive functional group complex is added to the above system, and different concentrations of organic solvents are used as solubility aids, and the mixture is reacted at a temperature of 0°C to 40°C for 30 minutes to 20 hours to modify the 246th or 248th lysine site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step e2: Subsequently, two types of functional molecule-linkers and a catalyst that catalyzes the acylhydrazone-oxime exchange reaction are added to the buffer system of Step e1, and the reaction is carried out for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. Includes.

[0035] The endoglycosidase described in the above method includes, but is not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and also includes fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0036] In yet another aspect, the present invention provides the use of the above-mentioned two-site-specific modified antibody-functional molecule complex in the manufacture of pharmaceuticals, diagnostic and therapeutic reagents, reagent kits, etc., for treating tumors, inflammatory diseases, viral infectious diseases, and immune diseases.

[0037] In one embodiment of the present invention, the tumor is one selected from the group consisting of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell tumors and germinal carcinomas, esophageal cancer, malignant glioma, Ewing's sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and hematological cancer; The aforementioned inflammatory disease is selected from the group consisting of conjunctivitis, bronchitis, sinusitis, Crohn's disease, etc. The aforementioned viral infectious diseases are selected from the group consisting of influenza, herpes zoster, human papillomavirus infection, AIDS, hepatitis A, hepatitis B, varicella, etc. The aforementioned immune diseases are selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, myeloma, dermatomyositis, psoriasis, etc.

[0038] Beneficial effects This invention combines glycosylation site-specific modification technology with guider peptide / protein-mediated K246 or K248 site-specific modification technology to enable the introduction of functional molecules to two sites on an antibody—the glycosylation site and the 246th or 248th lysine residue—making it applicable to the production of antibody-functional molecule complexes and fluorescent diagnostic reagents. The two-site-specific modified antibody-functional molecule complexes produced by this invention exhibit excellent stability, cytotoxicity, and in vivo activity, and have good drug suitability.

[0039] The two specific sites selected by this invention, namely the glycosylation site and the K246 or K248 site, allow for site-directed modification techniques that, compared to techniques used for other two-site-directed modified antibody-functional molecule complexes, do not require in vivo engineering modification of the antibody and are applicable to a wide variety of antibodies. Furthermore, they have the advantages of being easy to operate, requiring fewer purification steps, and possessing excellent drug suitability, making industrialization easy. In addition, compared to antibody-functional molecule complexes or compound drug antibody-functional molecule complexes modified at a single site with high DAR values, two-site-directed modification disperses the local hydrophobicity of the antibody-functional molecule complex and avoids local spatial congestion, thereby improving stability. [Brief explanation of the drawing]

[0040] [Figure 1] The results of the association stability of the two-site-specific modified antibody-functional molecule complex of this invention are shown. [Figure 2]The results of the association stability of the two-site-specific modified antibody-functional molecule complex of this invention are shown. [Figure 3] The results for the uniformity and hydrophilicity of the two-site-specific modified antibody-functional molecule complex of this invention are shown. The ADC compound produced by the method of this invention exhibits excellent uniformity. [Figure 4] The results for the uniformity and hydrophilicity of the two-site-specific modified antibody-functional molecule complex of this invention are shown. The ADC compound produced by the method of this invention exhibits excellent uniformity. [Figure 5] Figures 5-7 show experimental data on the in vitro activity of the two-site-specific modified antibody-functional molecule complexes of the present invention. Figure 5 shows the inhibition rate of these two-site-specific modified antibody-functional molecule complexes against Her2-positive cells SK-Br-3. [Figure 6] This shows the suppression rate against Her2-positive cells NCI-N87. [Figure 7] This shows the effect on survival rate in Her2-negative cells MDA-MB-231. [Figure 8] The results of experimental data regarding the in vitro activity of the bisite-specific modified antibody-functional molecule complex of this invention are shown. Compared with single-drug ADC compounds, some bisite-specific modified antibody-functional molecule complexes show superior suppression rates in cells with moderate to high Trop2 expression levels. [Figure 9] Figures 9-10 show experimental data regarding the in vivo antitumor activity of the bisite-specific modified antibody-functional molecule complex of this invention. These figures represent the suppression of tumor volume in the body and the effect on nude mouse body weight, respectively. [Figure 10] These figures show the inhibitory effect of the bisite-specific modified antibody-functional molecule complex on in vivo tumor volume and its effect on nude mouse body weight, respectively. [Modes for carrying out the invention]

[0041] The glycosidase used in this invention was expressed in an E. coli system and was expressed in the laboratory. The low molecular weight cytotoxic drugs MMAE and MMAF used in this invention were purchased from Shanghai Lishan Pharmaceutical Technology Co., Ltd. The Fc-binding peptide was manufactured on order from Nanjing Jinsrui Biotechnology Co., Ltd. Protein A was purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd. 2-(triazine thioyl)acetic acid was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Other compounds and reagents were purchased from Sinopharmaceutical Group Chemical Reagents Co., Ltd. unless otherwise specified.

[0042] The instruments and columns used in this invention include Waters Xevo G2 XS QTOF, analytical high-performance liquid chromatography system (Thermo ultimate 3000), analytical high-performance liquid chromatography system (Beijing Chuangxin Tongheng LC3000), preparative high-performance liquid chromatography system (Beijing Chuangxin Tongheng LC3000), C18 analytical column (Thermo, Acclaim™ 120, 5 μm, 4.6 x 250 mm), C18 analytical column (Agilent, SB C18, 4.6 x 150 mm), and C18 chromatography column for mass spectrometry (Waters, ACQUITY UPLC BEH C18, 1.7 mm, 2.1 x 50 mm).

[0043] The antibody molecular weight was measured using a liquid chromatography-mass spectrometer (LC MS), a Waters Xevo G2 XS Q-TOF, and a C4 column (ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm x 50 mm).

[0044] General operations: Method for producing glycosylation site-specific modified antibody-functional molecule complexes using a one-step method based on endoglycosidase The prepared disaccharide-toxin complexes (compounds 2a and 2b), wild-type antibody, and wild-type endoglycosidase Endo S2 were adjusted to concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.0, and the mixture was incubated at 30°C for 0.5 to 12 hours. After confirming conversion to the product by LC-MS, the mixture underwent protein A purification to obtain the predetermined glycan site-specific quantitative modification antibody-functional molecule complexes GsADC 3a - GsADC-3b (see Examples 16-17 below).

[0045] General operation 2: Method for producing K248 site-specific modified antibody-functional molecule complexes using a one-step method based on guider peptides The prepared affinity peptide-toxin complexes (compounds 4a and 4b) and wild-type antibodies were adjusted to concentrations of 0.5 mM and 5 mg / mL, respectively, and added to PB buffer (50 mM, pH 7.4, containing 20% ​​DMF). After adjusting the pH of the reaction system to 7.4, it was incubated at 37°C for 2 to 4 hours. After confirming conversion to the product by LC-MS, the mixture was purified with protein A to obtain the predetermined K248 site-specific quantitative modification antibody-functional molecule complexes KsADC 5a to KsADC 5b (see Examples 22-23 below).

[0046] General operation three: Method for producing bisite-directed modified antibody-functional molecule complexes by combining glycosylation site-directed modification technology and affinity peptide-directed modification technology The prepared disaccharide-toxin complexes (compounds 2a and 2b), wild-type antibody, and wild-type endoglycosidase Endo S2 were adjusted to concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.0, and the system was incubated at 30°C for 0.5 to 12 hours. After confirming the conversion to a homogeneous glycosylation site-specific modified antibody-functional molecule complex by LC-MS, the prepared affinity peptide-toxin complexes (compounds 4a and 4b) were added to the reaction system and adjusted to a concentration of 0.5 mM. After incubation at 37°C for 2 to 4 hours and confirming the conversion to the product by LC-MS, protein A purification was performed to obtain bisite-specific modified antibody-functional molecule complexes ADC-6a to ADC-6d (see Examples 30-33 below).

[0047] General operation four: Method for producing bisite-directed modified antibody-functional molecule complexes by combining glycosylation site-directed modification technology and affinity peptide-directed modification technology The prepared affinity peptide-toxin complex (compound 4a) and wild-type antibody were adjusted to concentrations of 0.5 mM and 5 mg / mL, respectively, and the reaction system was neutralized before incubation at 37°C for 2 to 4 hours. After confirming the conversion to a homogeneous K248 site-specific modified antibody-functional molecule complex by LC-MS, the prepared disaccharide-toxin complex (compound 2a) and wild-type endoglycosidase Endo-S2 were added to the reaction system, and the concentrations were adjusted to 0.5 mM and 0.4 mg / mL, respectively. After incubation at 30°C for 0.5 to 12 hours and confirming the conversion to the product by LC-MS, protein A purification was performed to obtain the bisite-specific modified antibody-functional molecule complex ADC-6a-1 (see Example 34 below).

[0048] General operations five: Method for producing bisite-directed modified antibody-functional molecule complexes by combining glycosylation site-directed modification technology and affinity peptide-directed modification technology The prepared disaccharide-toxin complex (compound 2a), affinity peptide-toxin complex (compound 4a), wild-type antibody, and wild-type endoglycosidase Endo-S2 were adjusted to concentrations of 0.5 mM, 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. After adjusting the reaction system to neutral, the mixture was incubated at 30°C for 0.5 to 12 hours. After confirming conversion to the product by LC-MS, protein A purification was performed to obtain the two-site-specific modified antibody-functional molecule complex ADC-6a-2 (see Example 35 below).

[0049] General operations six: Method for producing bisite-directed modified antibody-functional molecule complexes by combining glycosylation site-directed modification technology and affinity peptide-directed modification technology Step 1: Dissolve the prepared disaccharide-functional molecule complex (compound 2c), wild-type antibody, and wild-type endoglycosidase Endo S2 in a 50 mM His-HCl, pH 6.5 buffer system, adjusting the concentrations to 0.5 mM, 5 mg / mL, and 0.1 mg / mL, respectively. Adjust the pH of the reaction system to 7.0 and incubate at 30°C for 1 hour. After confirming the conversion to a homogeneous glycosylation site-specific modified antibody-functional molecule complex by LC-MS, replace the buffer system with 50 mM PIPES, pH 7.4 by ultrafiltration concentration. Add the affinity peptide-functional molecule complex (compound 4c) to the system, adjust the concentration to 0.34 mM, add 20% DMF as a solubility aid, and incubate at 37°C for 10 hours. After confirming conversion to the product by LC-MS, bifunctional antibodies Ab-1 to Ab-2 were obtained by ultrafiltration concentration (see Examples 28 and 29 below).

[0050] Step 2: The bifunctionalized antibodies prepared above, drug-linkers D1-D3 / D9, drug-linkers D4-D8, and the catalyst meta-aminobenzoic acid are added to His-HCl buffer (50 mM, pH 6.0, containing 20% ​​DMF) and adjusted to concentrations of 5 mg / mL, 0.34 mM, and 0.16 mM, respectively. The mixture is incubated at 37°C for 8 hours, and conversion to homogeneous bisite-specific modified antibody-functional molecule complexes ADC-7 to ADC-19 is confirmed by LC-MS (see Examples 36-48 below).

[0051] In general operation 6, the catalyst may be a catalyst capable of catalyzing the acylhydrazone-oxime exchange reaction, such as meta-aminobenzoic acid or other aniline-based catalysts.

[0052] General operation seven: Method for producing single-drug ADCs by combining glycosylation site-specific modification technology and acylhydrazone-oxime exchange chemistry. The prepared disaccharide-functional molecule complex (compound 2c), wild-type antibody, and wild-type endoglycosidase Endo S2 are dissolved in a 50 mM His-HCl, pH 6.5 buffer system, and the concentrations are adjusted to 0.5 mM, 5 mg / mL, and 0.1 mg / mL, respectively. After adjusting the pH of the reaction system to 7.0, it is incubated at 30°C for 1 hour. After confirming the conversion to a homogeneous glycosylation site-specific modified antibody-functional molecule complex by LC-MS, ultrafiltration concentration is performed. The obtained homogeneous glycosylation site-specific modified antibody-functional molecule complex and drug-linker D1 or D9 are dissolved in His-HCl buffer (50 mM, pH 6.0, containing 136 mM meta-aminobenzoic acid and 20% DMF), and the concentrations are adjusted to 5 mg / mL and 0.34 mM, respectively. After incubation at 30°C for 4-8 hours and confirmation of homogeneous conversion to N297 site antibody-drug conjugates by LC-MS, protein A purification was performed to obtain GsADC-3c to GsADC-3f (see Examples 18-21 below).

[0053] General operation eight: Method for producing a single-drug ADC by combining K-site-specific modification technology and click chemistry The prepared affinity peptide-functional molecule complex (compound 4c) and wild-type antibody are dissolved in a 50 mM PIPES, pH 7.4 buffer system, and the concentrations are adjusted to 0.34 mM and 5 mg / mL, respectively. After incubation at 37°C for 10 hours and confirmation of conversion to a homogeneous K-site-specific modified antibody-functional molecule complex by LC-MS, ultrafiltration concentration is performed. The obtained homogeneous antibody-functional molecule complex and drug-linker D5 or D7 are dissolved in a 50 mM PB, pH 7.4 buffer system, and the concentrations are adjusted to 5 mg / mL and 0.17 mM, respectively. After incubation at 30°C for 4 hours and confirmation of conversion to a homogeneous K248-site antibody-drug complex by LC-MS, protein A purification is performed to obtain KsADC-5c to KsADC-5f (see Examples 24-27 below).

[0054] I. Production of disaccharide-functional molecule complexes (e.g., disaccharide-toxin complexes) Examples 1-2: Synthesis of compounds 2a and 2b The structures and synthesis methods of compounds 2a and 2b were as follows: [ka] Compounds 2a and 2b [ka] compound 2a Step 1: Compound S2 (11 mg, 9.8 mM) was dissolved in 500 mL of DMF, and CHO-LacNAc S1 (14.8 mg, 38.8 mM) was added to the reaction system. DMF was added to make a DMF / 0.2 M PB = 1:1, and the pH of the reaction system was adjusted to 6.0 using NaOH / HCl. Then sodium borohydride cyanohydride (6.3 mg, 100 mM) was added, and the reaction was carried out at room temperature for 2 hours. After confirming the near completion of the reaction by LC-MS, the reaction was separated and purified using a half-size C18 column, and the target fraction was recovered and freeze-dried to obtain compound S4 (yield 81%). HRMS, calculated value C 72 H 117 N 11 O 22 :[M+2H] 2+744.9187, measured value 744.9110.

[0055] Step 2: Dissolve compound S4 (20 mg, 10 mM) in 400 μL of DMF / H2O = 1:1. Add DMC (67.6 mg, 300 mM) and triethylamine (120 mg, 900 mM) to the above reaction system, mix uniformly, then cool on ice and react at 0 °C for 2 hours. After confirming almost complete reaction by LC-MS, separate and purify using a basic C18 column, recover the target product, and then freeze-dry to obtain compound 2a (yield 87%). HRMS, calculated value C 72 H 115 N 11 O 21 : [M + 2H] 2+ 735.9134, measured value 735.8130.

[0056] Compound 2b Step 1: Dissolve compound S3 (11 mg, 9.8 mM) in 500 mL of DMF. Add CHO-LacNAc S1 (14.8 mg, 38.8 mM) to the above reaction system. Add additional DMF to make DMF / 0.2M PB = 1:1, adjust the pH of the reaction system to 6.0 using NaOH / HCl, then add sodium cyanoborohydride (6.3 mg, 100 mM) and react at room temperature for 2 hours. After confirming almost complete reaction by LC-MS, separate and purify using a semi-preparative C18 column, recover the target fraction, and then freeze-dry to obtain compound S5 (yield 75%). HRMS, calculated value C 72 H 115 N 11 O 21 : [M + 2H] 2+ 751.9083, measured value 751.9417.

[0057] Step 2: Compound S5 (20 mg, 10 mM) was dissolved in 400 mL of DMF / H2O = 1:1. DMC (67.6 mg, 300 mM) and triethylamine (120 mg, 900 mM) were added to the reaction system and mixed uniformly. The mixture was then cooled on ice and reacted at 0°C for 2 hours. After confirming the near completion of the reaction by LC-MS, the product was separated and purified using a basic C18 column. The target product was recovered and freeze-dried to obtain compound 2b (yield 88%). HRMS, calculated C 72 H 113 N 11 O 22 :[M+2H] 2+ 742.9031, measured value 742.9319.

[0058] Example 3: Synthesis of Compound 2c [ka] Step 1: Compound CHO-LacNAc S1 (20 mg, 52.5 μmol) was dissolved in 1 mL of 50 mM pH 7.4 phosphate buffer solution. Compound S9 (15.24 mg, 131.3 μmol) was added to the reaction system and the mixture was reacted at 30°C for 4 hours. After confirming the near completion of the reaction by LC-MS, the mixture was separated and purified using a half-size column, and the target fraction was recovered. The fraction was then freeze-dried to obtain compound S10 (yield 90%). HRMS, calculated value C 14 H 33 N3O 11 [M+H] + 480.2193, measured value 480.2424.

[0059] Step 2: Compound S10 (20 mg, 41.8 μmol) was dissolved in 2 mL of 50 mM PB, pH 8.0 buffer solution. 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride (136 mg, 0.627 mmol) and potassium phosphate (265 mg, 1.25 mmol) were added to the reaction system and mixed uniformly. The mixture was then cooled on ice and reacted at 0°C for 2 hours. After confirming the near completion of the reaction by LC-MS, the product was separated and purified using a basic column. The target product was recovered and freeze-dried to obtain compound 2c (yield 87%). HRMS, calculated value C 19 H31 N3O 10 [M+H] + 462.2088, measured value 462.1957.

[0060] II: Production of affinity peptide-functional molecule complexes (e.g., affinity peptide-toxin complexes) Examples 4-5: Synthesis of compounds 4a and 4b The structures and synthesis methods of compounds 4a and 4b were as follows.

[0061] [ka] Compounds 4a and 4b [ka] compound 4a Step 1: Compound S2 (10 mg, 9.8 μmol) was dissolved in DMF (500 μL), bis(succinimidyl)glutarate (DSG, 15.6 mg, 0.048 mM) and triethylamine (4 μL, 0.029 mM) were added, and the mixture was reacted at room temperature for 2 hours. After confirming the completion of the reaction by LC-MS, the mixture was separated and purified using a half-size column, and lyophilized to obtain white powder S6 (yield 91%). HRMS, calculated value: C 67 H 103 N 11 O 17 :[M+H] + 1334.7567, measured value 1334.7655.

[0062] Step 2: Compound S8 (15.7 mg, 0.047 mM) was dissolved in DMF, and HATU (7.14 mg, 0.0188 mM), DIPEA (4.84 μL, 0.028 mM), and compound P1 (20 mg, 0.0094 mM) were added sequentially. The mixture was stirred at room temperature for 1 hour, and the completion of the reaction was confirmed by LC-MS. The mixture was then separated and purified using a half-size column, and lyophilized to obtain compound P2 as a white powder (yield 92%). HRMS, calculated value C 114 H 151 N 31 O 23 S3:[M+H] +2419.0839, [M+3H] 3+ 807.0332, [M+4H] 4+ 605.5269, measured value 807.0342, 605.5232.

[0063] Step 3: Compound P2 (15 mg, 0.0062 mM) was dissolved in 500 μL of dichloromethane, and under ice bath conditions, 450 μL of trifluoroacetic acid and 50 μL of triisopropylsilane were added. After reacting at room temperature for 1 hour, the mixture was dried over N2 to obtain compound P3. HRMS, calculated value C 95 H 137 N 31 O 23 S3:[M+H] + 2176.9744, [M+3H] 3+ 726.3300, [M+4H] 4+ 544.9995, measured value 726.3278, 545.0035.

[0064] Step 4: Compound S6 (13.3 mg, 0.01 mM) was dissolved in a DMF / PB7.4 = 1 / 1 mixed solution, compound P3 was added, and the mixture was reacted at room temperature for 30 minutes. The mixture was then separated and purified using a half-size column, and lyophilized to obtain compound 4a, a white powder (yield 76%). HRMS, calculated value: C 158 H 235 N 41 O 37 S3: [M+H] + 3395.7007, [M+3H] 3+ 1132.5721, [M+4H] 4+ 849.6810, measured value 1133.5587, 849.6596.

[0065] compound 4b Step 1: Compound S3 (10 mg, 9.8 μmol) was dissolved in DMF (500 μL), bis(succinimidyl)glutarate (DSG, 15.6 mg, 0.048 mM) and triethylamine (4 μL, 0.029 mM) were added, and the mixture was reacted at room temperature for 2 hours. After confirming the completion of the reaction by LC-MS, the mixture was separated and purified using a half-size column, and lyophilized to obtain white powder S7 (yield 91%). HRMS, calculated value: C 68H 102 N 10 O 18 :[M+H] + 1346.7374, measured value 1346.7935.

[0066] Step 2: Compound S7 (13.3 mg, 0.01 mM) was dissolved in a DMF / PB7.4 = 1 / 1 mixed solution, compound P3 was added, and the mixture was reacted at room temperature for 30 minutes. The mixture was then separated and purified using a half-size column, and lyophilized to obtain compound 4b, a white powder (yield 76%). HRMS, calculated value: C 159 H 233 N 40 O 38 S3: [M+H] + 3407.6692, [M+3H] 3+ 1136.8897, [M+4H] 4+ 852.6692, measured values ​​1137.9250, 853.6930.

[0067] Example 6: Synthesis of Compound 4c [ka] Step 1: Compound P3 (50 mg, 23 μmol) was dissolved in 500 μL of DMF. Compound S27 (13.4 mg, 34.5 μmol) and triethylamine (7 mg, 69 μmol) were added to the system and mixed uniformly. The mixture was then reacted at 30°C for 4 hours. Separation and purification were performed using a half-size column, and after lyophilization, compound 4c was obtained (36.6 mg, yield 65%). HRMS, calculated value C 106 H 156 N 34 O 28 S3[M+2H] 2+ 1225.5573, measured value 1225.586.

[0068] III: Drug-Linker Synthesis Example 7: Synthesis of Compound D1 [ka] [ka] Step 1: Compound S11 (100 mg, 319.2 μmol) was dissolved in 1 mL of DMF, and NHS (44 mg, 382.6 μmol), HATU (145.8 mg, 382.6 μmol), and DIPEA (123.8 mg, 957.6 μmol) were added sequentially. After reacting at room temperature for 1 hour, H2N-VC-PAB-MMAF S3 (181.3 mg, 159.6 μmol) and DIPEA (41.3 mg, 319.2 μmol) were added to the system and mixed uniformly. The mixture was then reacted at 30°C for 2 hours. After confirming completion of the reaction by LC-MS, 0.2 mL of piperidine was added to the reaction system and reacted at room temperature for 20 minutes. The compound D1 was separated and purified using a half-size column and lyophilized to obtain compound D1 (146.5 mg, yield 76%). HRMS, calculated value C 62 H 99 N 11 O 15 [M+H] + 1238.7400, [M+2H] 2+ 619.8739, measured value 1238.7392, 619.8727.

[0069] Example 8: Synthesis of Compound D2 [ka] [ka] Step 1: Compound Fmoc-VA-PAB-PNP (S12, 40 mg, 59 μmol) was dissolved in 800 μL of DMSO. Dx8951 (31.3 mg, 59 μmol), HOBt (1.6 mg, 12 μmol), and pyridine (370.2 mg, 4.68 mmol) were added to the reaction system and mixed uniformly. The mixture was then allowed to react overnight at 30°C. After confirming the completion of the reaction by LC-MS, an equivalent volume of triethylamine was added, and the mixture was reacted at 30°C for 2 hours. The mixture was separated and purified using a half-size column, and lyophilized to obtain compound H2N-VA-PAB-Dx8951 (S13, 28.5 mg, yield 64%). HRMS, calculated value C 40 H 43 FN6O8[M+H] + 755.3204, measured value 755.3214.

[0070] Step 2: Compound S14 (65.1 mg, 66.3 μmol) was dissolved in 650 μL of DMF. Subsequently, HATU (50.4 mg, 132.6 μmol), S13 (50 mg, 66.3 μmol), and DIPEA (25.7 mg, 198.9 μmol) were added and mixed uniformly. After reacting at room temperature for 2 hours and confirming the completion of the reaction by LC-MS, an equal volume of triethylamine was added, and the reaction was carried out at 30 °C for 2 hours. It was separated and purified using a preparative column and freeze-dried to obtain compound S15 (74.4 mg, yield 百分之75). HRMS, calculated value C 74 H 109 FN8O 23 [M+H] + 1497.7668, [M+2H] 2+ 749.3873, measured value 1497.7222, 749.3452.

[0071] Step 3: Compound S16 (20 mg, 54.2 μmol) was dissolved in 200 μL of DMF. Subsequently, HATU (61.8 mg, 162.6 μmol), S15 (202.7 mg, 135.5 μmol), and DIPEA (20 mg, 16..6 μmol) were added and mixed uniformly. After reacting at 30 °C for 2 hours and confirming the completion of the reaction by LC-MS, an equal volume of triethylamine was added, and the reaction was carried out at 30 °C for 2 hours. It was separated and purified using a preparative column and freeze-dried to obtain compound S17 (72.5 mg, yield 百分之43). HRMS, calculated value C 153 H 222 F2N 17 O 48 [M+2H] 2+ 1553.2827, [M+3H] 3+ 1035.8577, [M+4H] 4+ 777.1452, measured value 1553.2460, 1035.8512, 777.1463.

[0072] It should be noted that there seems to be a small error in the original text where "16..6 μmol" in step 3 should probably be "162.6 μmol". This has been corrected in the translation. Also, the "百分之" in the yield expressions are assumed to be "%" for proper English representation.Step 4: Dissolve compound S11 (0.92 mg, 3 μmol) in 200 μL of DMF, sequentially add HATU (2.3 mg, 6 μmol), S17 (10 mg, 3.2 μmol) and DIPEA (1.1 mg, 9 μmol), react at 30 °C for 2 hours. After confirming the completion of the reaction by LC-MS, add an equal volume of triethylamine, react at 30 °C for 2 hours, separate and purify using a semi-preparative column, and freeze-dry to obtain compound D2 (7.7 mg, yield 81%). HRMS, calculated value C 155 H 226 F2N 18 O 50 [M+2H] 2+ 1589.7909, [M+3H] 3+ 1060.1965, [M+4H] 4+ 795.3993, measured values 1589.7994, 1060.1973, 795.4008.

[0073] Example 9: Synthesis of compound D3

Chem.

Chem.

[0074] Step 2: Compound S19 (80 mg, 74.7 μmol) was dissolved in 800 μL of DMF, N,N'-dimethylethylenediamine (S21, 65.7 mg, 747 μmol) was added, and the mixture was reacted at 30°C for 3 hours. Compound S20 was obtained by separation and purification using a half-size column (67 mg, yield 86%). HRMS, calculated value C 51 H 73 ClN6O 13 S [M+H] + 1045.4723, measured value 1045.4816.

[0075] Step 3: Compound S11 (15 mg, 47.9 μmol) was dissolved in 150 μL of DMF, and HATU (54.7 mg, 144 μmol), S20 (25 mg, 24 μmol), and DIPEA (31 mg, 240 μmol) were added sequentially. The mixture was reacted at 30°C for 2 hours, and the completion of the reaction was confirmed by LC-MS. Then, diethylamine (17.5 mg, 240 μmol) was added, and the mixture was reacted at 30°C for 1 hour. The mixture was separated and purified using a half-size column, and lyophilized to obtain compound D3, H2N-O-MCC-DM1 (18 mg, yield 67%). HRMS, calculated value C 53 H 76 ClN7O 15 S [M+H] + 1118.4887, measured value 1118.4950.

[0076] Example 10: Synthesis of Compound D4 [ka] [ka] Step 1: Compound S22 (50 mg, 113.1 μmol) was dissolved in 500 μL of DMF, NHS (39 mg, 339.4 μmol) and DCC (70 mg, 339.4 μmol) were added, and the mixture was reacted at 30°C for 2 hours. After confirming the completion of the reaction by LC-MS, Fmoc-Lys-OH (41.7 mg, 113.1 μmol) and triethylamine (34.3 mg, 339.4 μmol) were added, and the mixture was reacted at 30°C for 3 hours. Compound S23 was separated and purified using a half-size column to obtain compound S23 (64.5 mg, yield 72%). HRMS, calculated value C 40 H 60 N2O 14 [M+H] + 793.4123, measured value 793.4137.

[0077] Step 2: Compound S23 (28 mg, 35.4 μmol) was dissolved in 280 μL of DMF, an equivalent volume of triethylamine was added, and the reaction was carried out at 30°C for 2 hours. The reaction system was then freeze-dried, and then 200 μL of DMF was added and dissolved. DBCO-PEG4-NHS (22.8 mg, 35.4 μmol) was added to the system and the reaction was carried out at 30°C for 3 hours. The mixture was separated and purified using a half-size column, freeze-dried to obtain compound S28 (22.7 mg, yield 58%). HRMS, calculated value C 55 H 84 N4O 19 [M+H] + 1105.5808, measured value 1105.5289.

[0078] Step 3: Compound S28 (20 mg, 18.1 μmol) was dissolved in 200 μL of DMF, and HATU (13.8 mg, 36.2 μmol), S2 (20.3 mg, 18.1 μmol), and DIPEA (7.1 mg, 54.3 μmol) were added sequentially. The mixture was reacted at 30°C for 2 hours, separated and purified using a half-size column, and lyophilized to obtain compound D4 (30.4 mg, yield 76%). HRMS, calculated value C 113 H 176 N 14 O 30 [M+2H] 2+ 1105.6416, [M+3H] 3+737.4303, measured values ​​1105.6397, 737.4502.

[0079] Example 11: Synthesis of Compound D5 [ka] [ka] Step 1: Compound S28 (20 mg, 18.1 μmol) was dissolved in 200 μL of DMF, and HATU (8.3 mg, 21.72 μmol), H2N-VC-PAB-MMAF S3 (10.8 mg, 9.5 μmol), and DIPEA (7 mg, 54.3 μmol) were added sequentially. After reacting at room temperature for 1 hour, the compounds were separated and purified using a half-size column, and lyophilized to obtain compound D5 (13.7 mg, yield 65%). HRMS, calculated value C 113 H 174 N 14 O 31 [M+2H] 2+ 1112.6312, [M+3H] 3+ 742.0901, measured value 1112.6254, 742.1034.

[0080] Example 12: Synthesis of Compound D6 [ka] [ka] Step 1: Dissolve DBCO-PEG4-NHS (20 mg, 18.1 μmol) in 200 μL of DMF, and sequentially add S17 (56.2 mg, 18.1 μmol) and triethylamine (5.5 mg, 54.3 μmol). React at 30°C for 2 hours, separate and purify using a half-size column, and freeze-dry to obtain compound D6 (29.8 mg, yield 66%). HRMS, calculated value C 183 H 257 F2N 19 O 55 [M+3H] 3+ 1213.9366, [M+4H] 4+910.7044, measured values ​​1213.9476, 910.7107.

[0081] Example 13: Synthesis of Compound D7 [ka] [ka] Step 1: Dissolve Fmoc-VC-PAB-PNP (S24, 126.1 mg, 164.4 μmol) in 1.2 mL of DMF, and sequentially add Eribulin (100 mg, 137 μmol), HOBt (27.4 mg, 27.4 μmol), and pyridine (314 μL, 4.1 mmol). React overnight at room temperature, and after confirming completion of the reaction by LC-MS, add an equivalent volume of triethylamine and react at 30°C for 2 hours. Separate and purify using a half-size column, and freeze-dry to obtain compound S25 (135 mg, yield 87%). HRMS, calculated value C 59 H 86 N6O 16 [M+H] + 1135.6178, [M+2H] 2+ 568.3128, measured value 1135.6298, 568.3045.

[0082] Step 2: Compound S28 (58.4 mg, 52.9 μmol) was dissolved in 200 μL of DMF, and HATU (50.3 mg, 132.3 μmol), S25 (51 mg, 44.1 μmol), and DIPEA (17.1 mg, 132.3 μmol) were added sequentially. The mixture was reacted at 30°C for 2 hours, separated and purified using a half-size column, and lyophilized to obtain compound D7 (57.8 mg, yield 59%). HRMS, calculated value C 114 H 168 N 10 O 34 [M+2H] 2+ 1111.5940, [M+3H] 3+ 741.3986, measured value 1111.5784, 741.3497.

[0083] Example 14: Synthesis of Compound D8 [ka] [ka] Step 1: Compound S28 (20 mg, 18.1 μmol) was dissolved in 200 μL of DMF. NHS (2.5 mg, 21.72 μmol), HATU (8.3 mg, 21.72 μmol), and DIPEA (4.7 mg, 36.2 μmol) were sequentially added to the system and reacted at room temperature for 1 hour. Then, SignalTAC (S26, 23.9 mg, 9.5 μmol) and DIPEA (2.3 mg, 18.1 μmol) were added to the system and reacted at 30°C for 2 hours. Diethylamine (7 mg, 95 μmol) was added to the reaction system and reacted at 30°C for 3 hours. Compound D8 was separated and purified using a half-size column to obtain compound D8 (13.7 mg, yield 54%). HRMS, calculated value C 151 H 237 N 39 O 49 [M+3H] 3+ 1127.9162, [M+4H] 4+ 846.1891, [M+5H] 5+ 677.1528, [M+6H] 6+ 564.4620, measured values ​​1127.9238, 846.2126, 677.1782, 564.4285.

[0084] Example 15: Synthesis of compound D9 [ka] [ka] Step 1: Compound S11 (20 mg, 17.8 μmol) was dissolved in 200 μL of DMF. HATU (13.5 mg, 35.6 μmol), compound S2 (6.7 mg, 21.4 μmol), and DIPEA (93 μL, 53.4 μmol) were sequentially added to the reaction system and mixed uniformly. The mixture was then reacted at 30°C for 3 hours. 124 μL of piperidine was added to the system and mixed uniformly. The mixture was then reacted at room temperature for 10 minutes. After separation and purification using a half-size column, the compound D9 was obtained by lyophilization (16 mg, 75% yield). HRMS, calculated value C 60 H 97 N 11 O 14 [M+H] + 1196.7295, [M+2H] 2+ 598.8686, measured value 1196.7263, 598.8622.

[0085] IV: Synthesis of GsADC-3f from Glycan site-specific modified antibody-functional molecule complex GsADC-3a Example 16: Synthesis of GsADC-3a The antibody-functional molecule conjugate GsADC-3a was obtained from compound 2a and the wild-type antibody Herceptin (trastuzumab) by a standard procedure.

[0086] Example 17: Synthesis of GsADC-3b The antibody-functional molecule conjugate GsADC-3b was obtained from compound 2b and the wild-type antibody Herceptin (trastuzumab) by a standard procedure.

[0087] Example 18: Synthesis of GsADC-3c The antibody-functional molecule conjugate GsADC-3c was obtained from compound 2c, D9 and the wild-type antibody Sacituzumab by general procedure 7. The measured value after HRMS desuperposition calculation was 149025.

[0088] Example 19: Synthesis of GsADC-3d The antibody-functional molecule conjugate GsADC-3d was obtained from compounds 2c, D1 and the wild-type antibody Sacituzumab by general procedure 7. The measured value after HRMS desuperposition calculation was 159047.

[0089] Example 20: Synthesis of GsADC-3e The antibody-functional molecule conjugate GsADC-3e was obtained from compound 2c, D9 and the wild-type antibody hu-Mov-19 by general procedure 7. The measured value after HRMS desuperposition calculation was 149453.

[0090] Example 21: Synthesis of GsADC-3f The antibody-functional molecule conjugate GsADC-3f was obtained from compound 2c, D1 and the wild-type antibody hu-Mov-19 by general procedure 7. The measured value after HRMS desuperposition calculation was 149481.

[0091] V: Synthesis of KsADC-5f from K248 site-directed modified antibody-functional molecule complex KsADC-5a Example 22: Synthesis of KsADC-5a The antibody-functional molecule conjugate KsADC-5a was obtained from compound 4a and the wild-type antibody Herceptin (trastuzumab) by general procedure 2.

[0092] Example 23: Synthesis of KsADC-5b The antibody-functional molecule conjugate KsADC-5b was obtained from compound 4b and the wild-type antibody Herceptin (trastuzumab) by general procedure 2.

[0093] Example 24: Synthesis of KsADC-5c The antibody-functional molecule conjugate KsADC-5c was obtained from compound 4c, D5 and the wild-type antibody Sacituzumab by general procedure 8. The measured value after HRMS desuperposition calculation was 150933.

[0094] Example 25: Synthesis of KsADC-5d The antibody-functional molecule conjugate KsADC-5d was obtained from compounds 4c, D7 and the wild-type antibody Sacituzumab by general procedure 8. The measured value after HRMS desuperposition calculation was 150930.

[0095] Example 26: Synthesis of KsADC-5e The antibody-functional molecule conjugate KsADC-5e was obtained from compound 4c, D5 and the wild-type antibody hu-Mov-19 by general procedure 8. The measured value after HRMS desuperposition calculation was 151372.

[0096] Example 27: Synthesis of KsADC-5f The antibody-functional molecule conjugate KsADC-5f was obtained from compound 4c, D7 and the wild-type antibody hu-Mov-19 by general procedure 8. The measured value after HRMS desuperposition calculation was 151368.

[0097] VI: Production of the bifunctionalized antibody Ab-2 from Ab-1 [ka] As shown in Examples 28 and 29 below, the distinction between Ab-1 and Ab-2 lies in the fact that different "bifunctionalized antibodies" were obtained by using different wild-type antibodies.

[0098] Example 28: Synthesis of Ab-1 The bifunctionalized antibody Ab-1 was obtained from compounds 2c and 4c and the wild-type antibody Sacituzumab by step 1 of general procedure 6. The measured value after HRMS desuperposition calculation was 147182.

[0099] Example 29: Synthesis of Ab-2 The bifunctionalized antibody Ab-2 was obtained from compounds 2c and 4c and the wild-type antibody hu-Mov-19 by step 1 of general procedure 6. The measured value after HRMS desuperposition calculation was 147644.

[0100] VII: Synthesis of bisite-specific modified antibody-functional molecule complexes ADC-6a~ADC-6d, ADC-7~ADC-19 Example 30: Synthesis of ADC-6a The antibody-functional molecule conjugate ADC-6a was obtained from compounds 2a and 4a and the wild-type antibody Herceptin (trastuzumab) by general procedure 3. The measured value after HRMS desuperposition calculation was 151247.

[0101] Example 31: Synthesis of ADC-6b The antibody-functional molecule conjugate ADC-6b was obtained from compounds 2a and 4b and the wild-type antibody Herceptin (trastuzumab) by general procedure 3. The measured value after HRMS desuperposition calculation was 151273.

[0102] Example 32: Synthesis of ADC-6c The antibody-functional molecule conjugate ADC-6c was obtained from compounds 2b and 4a and the wild-type antibody Herceptin (trastuzumab) by general procedure 3. The measured value after HRMS desuperposition calculation was 157215.

[0103] Example 33: Synthesis of ADC-6d The antibody-functional molecule conjugate ADC-6d was obtained from compounds 2b and 4b and the wild-type antibody Herceptin (trastuzumab) by general procedure 3. The measured value after HRMS desuperposition calculation was 151302.

[0104] Example 34: Synthesis of ADC-6a-1 The antibody-functional molecule conjugate ADC-6a-1 was obtained from compounds 2a and 4a and the wild-type antibody Herceptin (trastuzumab) by general procedure IV.

[0105] Example 35: Synthesis of ADC-6a-2 The antibody-functional molecule conjugate ADC-6a-2 was obtained from compounds 2a and 4a and the wild-type antibody Herceptin (trastuzumab) by general procedure 5.

[0106] Example 36: Synthesis of ADC-7 [ka] The antibody-functional molecule conjugate ADC-7 was obtained from compounds D9, D5, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS decoupling was 154020.

[0107] Example 37: Synthesis of ADC-8 [ka] The antibody-functional molecule conjugate ADC-8 was obtained from compounds D9, D6, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 156858.

[0108] Example 38: Synthesis of ADC-9 [ka] The antibody-functional molecule conjugate ADC-9 was obtained from compounds D9, D7, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS decoupling was 154021.

[0109] Example 39: Synthesis of ADC-10 [ka] The antibody-functional molecule conjugate ADC-10 was obtained from compounds D9, D8, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 156341.

[0110] Example 40: Synthesis of ADC-11 [ka] The antibody-functional molecule conjugate ADC-11 was obtained from compounds D1, D4, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 154018.

[0111] Example 41: Synthesis of ADC-12 [ka] The antibody-functional molecule conjugate ADC-12 was obtained from compounds D2, D4, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 157958.

[0112] Example 42: Synthesis of ADC-13 [ka] The antibody-functional molecule conjugate ADC-13 was obtained from compounds D3, D4, and Ab-1 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 153835.

[0113] Example 43: Synthesis of ADC-14 The antibody-functional molecule conjugate ADC-14 was obtained from compounds D9, D5, and Ab-2 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 154454.

[0114] The only difference between ADC-14 and ADC-7 is the antibody itself; the structural formula does not indicate this difference, therefore its structural formula is the same as that of ADC-7.

[0115] Similarly, the structural formulas for ADC-15, ADC-16, ADC-17, ADC-18, and ADC-19 below are the same as those for ADC-8, ADC-9, ADC-11, ADC-12, and ADC-13 above, respectively.

[0116] Example 44: Synthesis of ADC-15 The antibody-functional molecule conjugate ADC-15 was obtained from compounds D9, D6, and Ab-2 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 157282.

[0117] Example 45: Synthesis of ADC-16 The antibody-functional molecule conjugate ADC-16 was obtained from compounds D9, D7, and Ab-2 by step 2 of general procedure 6. The measured value after HRMS decoupling was 154451.

[0118] Example 46: Synthesis of ADC-17 The antibody-functional molecule conjugate ADC-17 was obtained from compounds D1, D4, and Ab-2 by step 2 of general procedure 6. The measured value after HRMS desuperposition calculation was 154455.

[0119] Example 47: Synthesis of ADC-18 The antibody-functional molecule complex ADC-18 was obtained from compounds D2, D4, and Ab-2 by step 2 of General Procedure VI. Measured value 158386 after HRMS deconvolution calculation.

[0120] Example 48: Synthesis of ADC-19 The antibody-functional molecule complex ADC-19 was obtained from compounds D3, D4, and Ab-2 by step 2 of General Procedure VI. Measured value 154273 after HRMS deconvolution calculation.

[0121] Examples of in vitro association stability evaluation Proteins or antibodies themselves may form soluble or insoluble aggregates due to changes in various chemical or physical factors, and most of the antibody associations are irreversible. Furthermore, since the small molecule cytotoxins used in the synthesis of ADCs are highly hydrophobic, hydrophobic interactions may occur between the synthesized ADC molecules, causing drug associations. Specific operation: 200 μg was collected from each obtained ADC sample, dissolved in 100 mL of PBS, and then heated at 37°C for 28 days. Equal amounts were aliquoted at regular intervals and analyzed under the gradient conditions of a BioCore SEC-300 column (particle size 7.8 × 300 mm, 5 μm), 100% mobile phase (150 mM sodium phosphate, pH 6.8), for 15 minutes. Absorbance was detected at 280 nm. The results of size exclusion chromatography (SEC) analysis are shown in Figure 1. The bispecific modified ADC compound produced by the technology of the present invention showed good stability even after 28 days of incubation, and no obvious protein degradation or aggregation was observed.

[0122] In vitro stability evaluation based on FRα target The prepared double-loaded ADC compound targeting FRα was dissolved in a 1×PBS buffer system and stored in an oven at 37°C for 4 weeks. Sampling was performed at the time points of week 0, week 1, week 2, week 3, and week 4, and SEC column analysis was carried out to evaluate the association stability of the double-loaded ADC molecules. The results are shown in Figure 2.

[0123] From the SEC analysis results, the manufactured FRα-targeted dual-loaded ADCs maintained good association stability even after 28 days of incubation, and no obvious protein degradation or association phenomena were observed.

[0124] Examples of Uniformity and Hydrophilicity Evaluation In Vitro Uniformity and Hydrophilicity Evaluation Based on Trop2 Target After diluting the obtained 11 samples of ADC-7 to ADC-13 targeting Trop2, as well as GsADC-3c to GsADC-3d and KsADC-5c to KsADC-5d with 1×PBS, they were analyzed using a reversed-phase chromatography column (PLRP). Each injection volume was approximately 20 μg, and the results are shown in Figure 3.

[0125] From the RP data results, all the dual-loaded ADCs showed good uniformity. Due to more loads being modified, their hydrophobicity was higher than that of single-agent ADCs. Specifically, ADC-7, ADC-9, and ADC-13 modified with three toxins, MMAF, eribulin, and DM1, had similar polarities, and the retention time was approximately 11 minutes. ADC-8 and ADC-12 modified with MMAE and Dx8951 toxins had smaller polarities, and the retention time was approximately 12 - 13 minutes, which might be due to four Dx8951 toxins being modified. ADC-10 modified with MMAE and lysosome sorting peptide (SignalTAC) had slightly larger polarities, and the retention time was approximately 9 minutes.

[0126] In Vitro Uniformity and Stability Evaluation Based on FRα Target After diluting the obtained ADC compounds targeting FRα with 1×PBS, they were analyzed using a reversed-phase chromatography column (PLRP). Each injection volume was approximately 20 μg, and the results are shown in Figure 4.

[0127] ​​​​​​ Experimental process and results analysis of in vitro activity data based on HER2 targets We performed cellular-level activity evaluations on the two site-specific ADCs mentioned above. Three cell lines were selected, of which SK-Br-3 and NCI-N87 cells were Her2-positive, and MDA-MB-231 cells were Her2-negative. The cellular activity and toxicity of the ADC molecule were evaluated using the MTT assay.

[0129] The specific procedure involved adding 100 μL of PBS to the outermost edge of a 96-well plate, leaving three wells with only culture medium, and seeding approximately 6000 corresponding cells in each of the remaining wells. The plates were then incubated overnight at 37°C in a CO2 incubator. Next, 10 μL of each ADC molecule (each ADC molecule was adjusted to 9 concentrations by 5-fold serial dilution starting from the highest concentration of 100 nM, with 3 double-pore containers for each concentration) was added. For the control and blank groups, 10 μL of culture medium was added to the three seeded wells and the three wells with only culture medium. The 96-well plates were incubated in a CO2 incubator at 37°C for 72 hours. 10 μL of 5 mg / mL MTT was added to each well and incubated at 37°C for 4 hours. Then, 90 μL of SDS lysis solution was added to each well and incubated at 37°C for 7 hours to completely lyse the cells. Finally, the absorbance (OD value) at 570 nm was measured for each well, and the data was processed using GraphPad Prism 8. The results are shown in Figure 5-7. The bisite-specific modified antibody-functional molecule complex showed excellent in vitro cell activity, and no toxicity was observed in negative cells.

[0130] Pharmacological Example 2 Experimental process and results analysis of in vitro activity data based on Trop2 targets We performed cellular-level activity evaluations on the two-site-specific ADCs targeting Trop2 described above. A total of four cell lines were selected, of which BxPC-3 cells were used as Trop2-high-expressing cells, Colo-205 and Capan-1 cells as Trop2-moderate-expressing cells, and MDA-MB-231 cells as Trop2-low-expressing cells. The cellular activity and toxicity of the ADC molecule were evaluated using the MTT assay.

[0131] Specifically, 100 μL of PBS was added to the outermost edge of a 96-well plate, three wells were left with only culture medium, and approximately 6000 corresponding cells were seeded in each of the remaining wells. The plates were then incubated overnight at 37°C in a CO2 incubator. Next, 20 μL of each ADC molecule was added (each ADC molecule was adjusted to 9 concentrations by serial dilution 5-fold from the highest concentration of 100 nM, with 3 double-pore containers for each concentration). For the control and blank groups, 20 μL of culture medium was added to the three seeded wells and the three wells with only culture medium, bringing the total volume of each well to 200 μL. The 96-well plates were incubated in a CO2 incubator at 37°C for 144 hours. After replacing the culture medium with 100 μL of fresh medium, 10 μL of 5 mg / mL MTT was added to each well and incubated at 37°C for 4 hours. Then, 90 μL of SDS lysis solution was added to each well and incubated at 37°C for 7 hours to completely lyse the cells. Finally, the absorbance (OD value) at 570 nm was measured for each well, and the data was processed using GraphPad Prism 8. The results are shown in Figure 8.

[0132] The bisite-specific modified antibody-functional molecule complexes showed excellent in vitro cell activity, and no toxicity was observed in negative cells. In the high-expression cell line BxPC-3, the bisite-specific modified ADC compounds and single-drug ADCs showed comparable in vitro inhibitory activity. In the moderate-expression cell line Colo-205, the combinations of MMAE(N)-MMAF(K), MMAE(N)-eribulin(K), and MMAF(N)-MMAE(K) (ADC-7, ADC-9, ADC-11) showed improved cell inhibitory activity. On the other hand, the combinations of MMAE and Dx8951 (ADC-8, ADC-12), MMAE and SignalTAC (ADC-10), and DM1 and MMAE (ADC-13) did not exhibit good inhibitory effects. This may be due to factors such as the low sensitivity of the Dx8951 toxin to the cells in question, insufficient quantity of the lysosomal-targeting peptide SignalTAC, and the fact that DM1 employs a non-cleaving linker.

[0133] Pharmacological Example 3: Experimental process and results analysis of in vivo antitumor activity based on HER2 targeting A BALB / c nude mouse transplant tumor model was constructed using gastric cancer cells NCI-N87. Six mice were assigned to each group according to the principle of dividing mice into large, medium, and small groups based on ear labeling and size.

[0134] One disaccharide ADC compound, Gs-ADC 3a, one affinity fragment-directed synthetic ADC compound, Ks-ADC-5b, and four bisite-specific antibodies, ADC-6a to ADC-6d, were selected, and their activity was evaluated at the animal level. A cysteine ​​randomly modified ADC compound (DAR ≈ 3.8) was used as a positive control, and PBS was used as a negative control. All samples were diluted to 0.1 mg / mL with 1×PBS before administration, and tested after sterilization through a 0.22 μm filter.

[0135] All samples were administered intraperitoneally at a concentration of 1 mg / kg, with a total of three administrations every 7 days. Tumor size and mouse body weight were measured every 3 days after the initial administration using calipers. The experimental process was conducted in accordance with animal ethics requirements. The measured data were plotted and analyzed using GraphPad Prism 8 software. As shown in Figures 9-10, the two-site specific modification ADC compounds produced by the present invention showed excellent in vivo activity, and no apparent toxicity was observed.

Claims

1. The material comprises an antibody, a linking fragment 1, a linking fragment 2, and a functional molecule, wherein the functional molecule is modified via linking fragment 1 to a conserved glycosylation site in the antibody's Fc region, and via linking fragment 2 to the 246th or 248th lysine site of the antibody. Bisite-specifically modified antibody-functional molecule complex.

2. The functional molecule is modified via linking fragment 1 to the conserved glycosylation site of the antibody Fc region, and via linking fragment 2 to the lysine site at position 248 of the antibody. The two-site-specific modified antibody-functional molecule complex according to claim 1.

3. The antibody is selected from the group consisting of a humanized antibody containing an Fc region, or an antibody derived from another animal containing an Fc region; The functional molecules may be identical or different from each other, and are independently selected from the group consisting of reactive functional groups, fluorescent groups, drugs, toxins, radioactive structures, and lysosome-targeting peptides; The linked fragment 1 comprises a linker and a sugar structure, the sugar structure being bound to a conserved glycosylation site in the antibody Fc region, and the linker being bound to a functional molecule; The linked fragment 2 comprises a linker and fragment A, wherein fragment A is bound to the 246th or 248th lysine site of the antibody, and the linker is bound to the functional molecule; The sugar structure represents a monosaccharide, disaccharide, oligosaccharide, or branched sugar structure; the fragment A represents an amide structure or a triazole structure; the glycosylation site of the antibody Fc structural domain represents any structure in which a linker can connect the sugar structure and the functional molecule; the 246th or 248th lysine site of the antibody represents any structure in which a linker can connect fragment A and the functional molecule. The two-site-specific modified antibody-functional molecule complex according to claim 1.

4. The antibody is selected from the group consisting of different IgG subtype antibodies, monoclonal antibodies, bifunctional or multifunctional antibodies, polyclonal antibodies, and functional antibodies derived from different species; In the functional molecule, the reactive functional group is selected from the group consisting of azide group, tetrazine group, TCO group, alkynyl group structure, aldehyde group structure, carbonyl group structure, hydroxylamine structure, isothiocyanate, amino group, carboxyl group, thiol group, alkenyl group, maleimide group, acylhydrazone structure, etc. The aforementioned fluorescent group is selected from the group consisting of biotin, FITC, rhodamine, Cy3, Cy5, etc. The pharmaceutical or toxin is selected from the group consisting of MMAE, MMAF, Dxd, Dx8951, SN38, DM1, DM4, PBD, PBD dimer, eribulin, duocalmycin, and derivatives of these drugs; The aforementioned radioactive structure, ６８ Ga, １８ F, ８９ Selected from the group consisting of Zr, etc.; The lysosome-targeting peptide is SignalTAC. The two-site-specific modified antibody-functional molecule complex according to claim 3.

5. The aforementioned two-site-specific modified antibody-functional molecule complex is represented by the following formula I, 【Chemistry 1】 In formula I, the central "Y" shaped structure represents the antibody; 【Chemistry 2】 The '' represents a core fucose structure; n is 0 or 1; the linker represents a PEG chain, carbon chain, cleavable linker, etc.; the sugar structure represents a monosaccharide, disaccharide, oligosaccharide, or branched sugar structure; fragment A represents an amide structure, triazole structure; 【Transformation 3】 The symbol represents a functional molecule, and the functional molecules of the glycosylation site and the lysine site may be the same or different, and one or more functional molecules may be modified. The two-site-specific modified antibody-functional molecule complex according to claim 1.

6. The two-site-specific modified antibody-functional molecule complex of formula I is represented by the following formula II: 【Chemistry 4】 In formula II, the definition of each part is the same as in claim 5. The two-site-specific modified antibody-functional molecule complex according to claim 5.

7. The two-site-specific modified antibody-functional molecule complex of formula II is selected from the following specific compounds: 【Transformation 5】 【Transformation 6】 【Transformation 7】 Here, the MMAE structure 【Transformation 8】 And; MMAF structure 【Chemistry 9】 And; The Dx8951 structure 【Chemistry 10】 And; The eribulin structure 【Chemistry 11】 The SignalTAC structure is 【Chemistry 12】 That is, The two-site-specific modified antibody-functional molecule complex according to claim 5.

8. A method for producing a two-site specific modified antibody-functional molecule complex, selected from the following methods 1 to 3, and represented by the following reaction formula, 【Chemistry 13】 Here, the definitions of each part in the above reaction equation are those defined in the preceding text. Method 1: A wild-type antibody or a defucosified antibody is treated with a glycosyltransferase or endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-functional molecule complex in which a functional molecule is modified at the glycosylation site of the antibody; subsequently, the 246th or 248th lysine site of the antibody is modified with a functional molecule-linker using a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to finally obtain a two-site-specific modified antibody-functional molecule complex; or Method 2: Add the enzymes and substrates necessary for site-specific glycosylation technology to a wild-type antibody or a defucosed antibody, and simultaneously add the substrates necessary for guider peptide / protein-mediated lysine site-specific modification technology, and after incubation, obtain a two-site-specific modified antibody-functional molecule complex; or Method 3: A wild-type antibody or a defucosified antibody is first treated with a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to modify the lysine site 246 or 248 of the antibody with a functional molecule-linker. Subsequently, a glycosylation site-specific modification technique mediated by a glycosyltransferase or endoglycosidase is used to modify the glycosylation site of the antibody with a functional molecule, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. Here, the glycosyltransferase is selected from the group consisting of fucosyltransferase, galactosyltransferase, sialyltransferase, etc.; the endoglycosidase is selected from the group consisting of Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and further includes fusion proteins formed by fusing the functional regions of these different endoglycosidases. method.

9. The above method 1 is, Step a1: A step in which an antibody is placed in a buffer, a sugar structure-functional molecule complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to 20 hours to obtain a sugar site-specific modified antibody-functional molecule complex in which the glycosylation site of the antibody is modified with a functional molecule, Step a2: Next, the product obtained in step a1 is placed in a buffer, the guider peptide-functional molecule complex is added, and the mixture is reacted for 30 minutes to 20 hours under a temperature of 0°C to 40°C to modify the 246th or 248th lysine site of the antibody with a functional molecule-linker, ultimately obtaining a two-site-specifically modified antibody-functional molecule complex. including, or, The above method two is, The process involves placing the antibody in a buffer, adding a glycosyltransferase or endoglycosidase and a sugar structure-functional molecular complex, simultaneously adding a guider peptide-functional molecular complex, reacting under a temperature of 15°C to 37°C for 30 minutes to 20 hours, and obtaining a two-site-specific modified antibody-functional molecular complex after incubation. including, or, The above method three is, Step c1: The antibody is placed in a buffer, the guide peptide-functional molecule complex is added, and the mixture is reacted for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C, thereby modifying the 246th or 248th lysine moiety of the antibody with a functional molecule-linker. Step c2: Next, the product obtained in step c1 is placed in a buffer, a glycosyltransferase or endoglycosidase and a sugar structure-functional molecular complex are added, and the mixture is reacted for 30 minutes to 20 hours under a temperature of 15°C to 37°C to finally obtain a two-site specific modified antibody-functional molecular complex. including, The method according to claim 8.

10. The two-site-specific modified antibody functional molecule complex is produced by Method 1 or Method 3. In step a1 of Method 1 and step c2 of Method 3, an endoglycosidase-mediated glycosylation site-specific modification technique is used; in step a2 of Method 1 and step c1 of Method 3, the Fc-binding peptide is used as a guider fragment to the antibody Fc structural domain, and the thioester structure is used as an acyl group transfer fragment. The method according to claim 8.

11. A method for producing a two-site specific modified antibody-functional molecule complex, selected from the following methods 4 and 5, and represented by the following reaction formula, Method 4: 【Chemistry 14】 Method five: 【Chemistry 15】 Here, the definition of each part in the above reaction equation is the same as that defined in claim 5, 【Chemistry 16】 " and " 【Chemistry 17】 " represents a reactive functional group, Method 4: A wild-type antibody or a defucosified antibody is treated with a glycosyltransferase or endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a reactive functional group-linker is modified at the 246th or 248th lysine site of the antibody by a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to obtain a bisite-specific modified bifunctional antibody. Then, a functional molecule (e.g., a toxin) is introduced into the glycosylation site and the K246 or K248 site, respectively, via a bioorthogonal reaction, to finally obtain a bisite-specific modified antibody-functional molecule complex; or Method 5: A wild-type antibody or a defucosified antibody is first treated with a guider peptide or guider protein-mediated K246 or K248 site-specific modification technique to modify the lysine site at position 246 or 248 of the antibody with a reactive functional group-linker. Subsequently, a glycosylation site-specific modification technique mediated by a glycosyltransferase or endoglycosidase is used to modify the glycosylation site of the antibody with a reactive functional group to obtain a bisite-specific modified bifunctional antibody. Then, a bioorthogonal reaction is performed to introduce functional molecules into the glycosylation site and the K246 or K248 site, respectively, to finally obtain a bisite-specific modified antibody-functional molecule complex. Here, the glycosyltransferase is selected from the group consisting of fucosyltransferase, galactosyltransferase, sialyltransferase, etc.; the endoglycosidase is selected from the group consisting of Endo-S, Endo-S2, Endo-F3, Endo-M, and variants of these enzymes, and further includes fusion proteins formed by fusing the functional regions of these different endoglycosidases; and the definition of the reactive functional group is the same as in claim 4. method.

12. The above method four is, Step b1: The antibody is placed in a buffer, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to 20 hours to obtain a sugar site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a guider peptide-reactive functional group complex is added to the above system, and the mixture is reacted at a temperature of 0°C to 40°C for 30 minutes to 20 hours to modify the 246th or 248th lysine site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step b2: Subsequently, two types of functional molecule-linkers are added to the buffer system of step b1, and the mixture is reacted for 30 minutes to 20 hours under a temperature of 0°C to 40°C to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specifically modified antibody-functional molecule complex. including, or The above method five is, Step d1: The antibody is placed in a buffer, a guider peptide-reactive functional group complex is added, and the mixture is reacted at a temperature of 0°C to 40°C for 30 minutes to 20 hours to obtain a lysine-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the 246th or 248th lysine site of the antibody. Subsequently, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added to the above system, and the mixture is reacted at a temperature of 15°C to 37°C for 30 minutes to 20 hours to modify the glycosylation site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step d2: Subsequently, two types of functional molecule-linkers are added to the buffer system of step d1, and the mixture is reacted for 30 minutes to 20 hours under a temperature of 0°C to 40°C to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specifically modified antibody-functional molecule complex. including, The method according to claim 11.

13. The method is carried out by the following method six: Method 6: [Chemistry 18] Here, the definitions of each part in the above reaction equation are those defined in claim 5. Method 6: A wild-type antibody or a defucosified antibody is treated by an endoglycosidase-mediated site-specific modification technique to obtain a site-specific modified antibody-reactive functional group complex in which an acylhydrazone structure is added to the glycosylation site of the antibody. Subsequently, an azide functional group is added to the 246th or 248th lysine site of the antibody by a guider peptide or guider protein-mediated site-specific modification technique to obtain a bifunctionalized antibody that is bisite-specifically modified with acylhydrazone and azide. Then, functional molecules are introduced into the glycosylation site and the K246 or K248 site, respectively, via an acylhydrazone-oxime exchange reaction and a click chemistry reaction, finally obtaining a bisite-specific modified antibody-functional molecule complex. The method according to claim 11.

14. The method described above is Step e1: The antibody is placed in a buffer solution with a pH of 6.0–8.0, a sugar structure-reactive functional group complex and a glycosyltransferase or endoglycosidase are added, and the mixture is reacted at a temperature of 15°C–37°C for 30 minutes to 20 hours to obtain a sugar site-specific modified antibody-reactive functional group complex in which a reactive functional group is modified at the glycosylation site of the antibody. Subsequently, a guide peptide-reactive functional group complex is added to the above system, and different concentrations of organic solvents are used as solubility aids, and the mixture is reacted at a temperature of 0°C–40°C for 30 minutes to 20 hours to modify the 246th or 248th lysine site of the antibody with a reactive functional group-linker to obtain a bisite-specific modified bifunctionalized antibody. Step e2: Subsequently, two types of functional molecules-linkers and a catalyst that catalyzes the acylhydrazone-oxime exchange reaction are added to the buffer system of step e1, and the reaction is carried out for 30 minutes to 20 hours under temperature conditions of 0°C to 40°C to introduce different or identical functional molecules to the glycosylation site and the 246th or 248th lysine site of the antibody, ultimately obtaining a two-site-specific modified antibody-functional molecule complex. including, The method according to claim 13.

15. Use of a two-site specific modified antibody-functional molecule complex according to any one of claims 1 to 7 in the manufacture of pharmaceuticals, diagnostic and therapeutic reagents, reagent kits, etc. for treating tumors, inflammatory diseases, viral infectious diseases, and immune diseases.

16. The tumor is one of the following selected from the group consisting of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell tumors and germinal carcinomas, esophageal cancer, malignant glioma, Ewing's sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and hematological cancer; The aforementioned inflammatory disease is selected from the group consisting of conjunctivitis, bronchitis, sinusitis, and Crohn's disease; The aforementioned viral infectious diseases are selected from the group consisting of influenza, herpes zoster, human papillomavirus infection, AIDS, hepatitis A, hepatitis B, and varicella; The aforementioned immune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, myeloma, dermatomyositis, and psoriasis. Use as described in claim 15.

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