Dendritic multi-targeting antibody coupling carrier, antibody coupling medicine as well as preparation method and application of dendritic multi-targeting antibody coupling carrier and antibody coupling medicine

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a dendritic multi-targeting antibody coupling carrier, an antibody coupling medicine as well as a preparation method and application of the dendritic multi-targeting antibody coupling carrier and the antibody coupling medicine. The dendritic multi-targeting antibody coupling carrier is composed of a targeting module and a drug coupling module, wherein the targeting module has multispecificity and a multivalent binding receptor function; the drug coupling module can realize fixed-point coupling and high drug loading capacity of drugs. The fusion proteins of the two modules can be specifically bound by a spy reaction through spontaneously formed covalent bonds. According to the present invention, the dendritic multi-targeting antibody coupling carrier is further utilized to construct the antibody coupling drug, such that the targeting property of the drug is improved, the relative drug loading capacity of the drug is improved, and the tumor treatment effect of the antibody coupling drug is finally improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a dendritic multi-targeting antibody conjugate carrier, an antibody conjugate drug and a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major threat to human health, ranking first in the cause of death of Chinese residents. The three major clinical tumor treatments include surgery, radiotherapy and chemotherapy. Surgery and radiotherapy are suitable for local cancer, and the effect is best for early cancer. Chemotherapy is generally administered systemically, which can target all lesions, including primary and metastatic lesions, but has greater toxic side effects. Compared with chemotherapy drugs, biological macromolecular drugs have higher efficacy and safety, and smaller toxic side effects, such as common monoclonal antibody drugs such as cetuximab and herceptin. Further, the connection of small molecule chemotherapy drugs on monoclonal antibodies generates antibody-drug conjugates (Antibody-Drug Conjugate, ADC), which embodies better therapeutic effect. However, there are some problems in the development of current ADC drugs, including: 1) the molecular weight of traditional monoclonal antibodies is generally about 150000 Da, and the penetration in solid tumors is poor; 2) the currently marketed ADC drugs are basically single-targeted, and the preparation of multi-targeted antibodies is more difficult; 3) the molecular size of traditional monoclonal antibodies is large, the in vivo circulation time is too long, the immunogenicity is large, and the drug loading under the same quality condition is less; 4) it is difficult to prepare multi-targets with special structure.

[0003] Peptide-drug conjugate (PDC) is a kind of conjugate drug developed in recent years. Compared with antibodies, polypeptide molecular weight is smaller, generally less than 3000 Da, tumor penetration is strong, has lower immunogenicity and is easy to modify and modify. However, the affinity of polypeptide is low, the in vivo half-life is short, and the polypeptide related drugs are more susceptible to protease-mediated degradation. Therefore, it is necessary to develop antibody conjugate drugs with high targeting, high tumor penetration, appropriate in vivo circulation time and high drug loading, so as to realize precise and efficient anti-tumor treatment. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies of the prior art, to provide a dendritic multi-targeting antibody conjugate carrier through modular design, so as to improve the effect of anti-tumor targeted therapy.

[0005] The technical problem to be solved by the present application is to provide a preparation method of the multi-targeting antibody conjugate carrier.

[0006] The technical problem to be solved by the present application is to provide an antibody conjugate drug.

[0007] The present application also aims to provide a preparation method of the antibody conjugated drug.

[0008] The present application finally aims to provide an application of the multiple targeting antibody conjugated carrier or the antibody conjugated drug in preparing an anti-tumor drug.

[0009] To solve the above technical problems, the present application adopts the technical solutions as follows:

[0010] In a first aspect, the present application provides a dendritic multiple targeting antibody conjugated carrier, comprising a first targeting module and a drug conjugated module.

[0011] The first targeting module comprises three functional regions: a first antibody, a first spy tag and a second antibody; and the drug conjugated module comprises three functional regions: a first spy catcher, a drug conjugated polypeptide and a long circulating polypeptide.

[0012] The first spy tag and the first spy catcher are a fast and reliable irreversible peptide-protein connection coupling tool, including but not limited to any one of SpyTag and SpyCatcher, SnoopTag and SnoopCatcher.

[0013] The amino acid sequences of the SpyTag and the SpyCatcher are shown in SEQ ID No: 9 and SEQ ID No: 10, respectively, and the amino acid sequences of the SnoopTag and the SnoopCatcher are shown in SEQ ID No: 11 and SEQ ID No: 12, respectively.

[0014] The first targeting module and the drug conjugated module can be specifically combined by a covalent bond formed by a spy reaction of the first spy tag and the first spy catcher.

[0015] The first antibody or the second antibody includes but is not limited to any one of EGFR antibody, HER2 antibody, PD-L1 antibody, EGFR affibody, HER2 affibody, PD-L1 affibody, EGFR antigen binding polypeptide, HER2 antigen binding polypeptide, PD-L1 antigen binding polypeptide, EGFR ligand, HER2 ligand, PD-L1 ligand, and any antibody in the prior art that can specifically bind to an antigen, a novel alternative antibody, a tumor related receptor, an immune cell receptor, and a tissue / vessel related receptor, etc. can be used to construct a dendritic multiple targeting antibody conjugated carrier structure containing the first targeting module and the drug conjugated module to prepare a corresponding multiple targeting antibody conjugated carrier.

[0016] Specifically, the first antibody and the second antibody are the same or different.

[0017] In some embodiments of the present invention, the first antibody is a HER2 antibody and the second antibody is an EGFR antibody; or the first antibody is a HER2 antibody and the second antibody is a PD-L1 antibody; or the first antibody is an EGFR antibody and the second antibody is a HER2 antibody; or the first antibody is an EGFR antibody and the second antibody is a PD-L1 antibody; or the first antibody is a PD-L1 antibody and the second antibody is a HER2 antibody; or the first antibody is a PD-L1 antibody and the second antibody is an EGFR antibody.

[0018] In some embodiments of the present invention, the HER2 antibody can be any HER2 antibody obtained by existing technology, including but not limited to conventional Fab-type HER2 antibodies, HER2 single-chain antibodies, HER2 nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the HER2 antibody is shown in SEQ ID No:13.

[0019] In some embodiments of the present invention, the EGFR antibody can be any EGFR antibody obtained by existing technology, including but not limited to traditional Fab-type EGFR antibodies, EGFR single-chain antibodies, EGFR nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the EGFR antibody is shown in SEQ ID No:14.

[0020] In some embodiments of the present invention, the PD-L1 antibody can be any PD-L1 antibody obtained by existing technology, including but not limited to conventional Fab-type PD-L1 antibodies, PD-L1 single-chain antibodies, PD-L1 nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the PD-L1 antibody is shown in SEQ ID No:15.

[0021] The drug-conjugated polypeptide is an amino acid polypeptide that is site-specifically conjugated to a small molecule drug.

[0022] Specifically, the drug-conjugated polypeptide refers to a small drug molecule that is conjugated to a drug through functional groups such as thiol, amino, or azide groups to achieve site-specific conjugation of the drug. It includes, but is not limited to, natural and non-natural amino acids rich in cysteine, lysine, or other reactive functional groups, with a repeating number between 2 and 20.

[0023] In some embodiments of the present invention, the drug-conjugated polypeptide is (GGC)8.

[0024] Among them, the long-circulating polypeptide, after fusion expression, can maintain the activity of the therapeutic protein in vivo for a longer period of time, improve its therapeutic window and efficacy, including but not limited to 1) albumin-binding domain (ABD): by binding to albumin in the blood, it can reduce the rate at which the therapeutic protein is filtered out by the glomerulus, thereby prolonging its half-life in plasma; 2) anti-neonatal Fc receptor (FcRn) affinity (AFF): by binding to FcRn with high affinity, it can improve the circulation efficiency of the protein; 3) anti-albumin single-domain antibody; 4) hydrophilic polypeptide.

[0025] The long-circulating polypeptide is an albumin-binding polypeptide, an Fc-binding polypeptide, or a hydrophilic polypeptide.

[0026] In some embodiments of the present invention, the long circulating polypeptide is an albumin-binding domain (ABD), the amino acid sequence of which is shown in SEQ ID No:16.

[0027] In this context, each functional region in the first targeting module and the drug conjugation module may have one or more repetitions, and the order of arrangement between the functional regions is arbitrary.

[0028] For example, when the first antibody in the first targeting module is an EGFR antibody (abbreviated as E), the first tag is SpyTag (abbreviated as S), and the second antibody is a HER2 antibody (abbreviated as H), the combination and connection order can be HSE and ESH; when the first antibody in the first targeting module is an EGFR antibody (abbreviated as E), the first tag is SnoopTag (abbreviated as T), and the second antibody is a HER2 antibody (abbreviated as H), the combination and connection order can be HTE and ETH.

[0029] For example, when the first spy catcher in the drug conjugation module is SpyCatcher (S), drug conjugated peptide (G), and long circulating peptide (L), the combination and connection order can be SLG, SGL, SGGL, GLS, GLLS, etc.

[0030] In some embodiments of the present invention, the first targeting module is a fusion protein that is HSE, HTE, HSP, ESH, ESP, PSH, or PSE.

[0031] In some embodiments of the present invention, the drug conjugation module is a fusion protein called SGL.

[0032] In this context, the functional regions of the first targeting module and the drug conjugation module are connected by short peptides composed of Gly and Ser residues.

[0033] In some embodiments of the present invention, the short peptide is (GGGGS). n (GGGS)n n is an integer from 1 to 50, most commonly 1 to 5. In some embodiments of the present invention, the short peptide is GGGGS, (GGGGS)3, or GGGS.

[0034] In some embodiments of the present invention, the dendritic multitarget antibody-conjugated vector is a first-generation dendritic dual-target antibody-conjugated vector HSE-SGL, such as... Figure 1 As shown.

[0035] Secondly, the present invention provides a method for preparing the multiple target antibody-conjugated vector described in the first aspect.

[0036] That is, the first targeting module and the drug conjugation module are mixed at equal molar concentrations and reacted to obtain the multi-targeting antibody-conjugated vector.

[0037] The first target module or drug-conjugated module is obtained by introducing the coding gene of the first target module or drug-conjugated module into an expression vector to construct a recombinant expression vector, then introducing the recombinant expression vector into a host bacterium / cell for culture, and then inducing expression, extraction, and purification.

[0038] The reaction is carried out under the following conditions: 20~40 ℃ and 50~200 rpm for 1~24 h.

[0039] The expression vector includes, but is not limited to, any one of pET-28a, pET25b, and pET22b.

[0040] In some embodiments of the present invention, the expression vector is pET-28a.

[0041] Thirdly, the present invention provides another dendritic multiplex antibody-drug conjugate vector, comprising a second targeting module and a drug conjugate module.

[0042] The second targeting module includes a first targeting module and a spy system module, and the drug conjugate module includes three functional regions: a second spy catcher, a drug-conjugate peptide, and a long-circulating peptide.

[0043] The first targeting module includes three functional areas: the first antibody, the first spy tag, and the second antibody. The spy system module includes two functional areas: the first spy catcher and the second spy tag.

[0044] The first spy tag and the first spy catcher, or the second spy tag and the second spy catcher, is a fast and reliable irreversible peptide-protein linking conjugation tool, including but not limited to any one of SpyTag and SpyCatcher, SnoopTag and SnoopCatcher.

[0045] The labels for First Spy and First Spy Catcher, and Second Spy and Second Spy Catcher are different.

[0046] The amino acid sequences of SpyTag and SpyCatcher are shown in SEQ ID No:9 and SEQ ID No:10, respectively, and the amino acid sequences of SnoopTag and SnoopCatcher are shown in SEQ ID No:11 and SEQ ID No:12, respectively.

[0047] The first targeting module and the spy system module can be specifically bound by a covalent bond spontaneously formed by the spy reaction through the first spy tag and the first butterfly catcher.

[0048] The second targeting module and the drug conjugation module can be specifically bound by a covalent bond spontaneously formed by the spy reaction through the first spy tag and the first butterfly catcher.

[0049] The first antibody or the second antibody includes, but is not limited to, any one of EGFR antibody, HER2 antibody, PD-L1 antibody, EGFR affinity, HER2 affinity, PD-L1 affinity, EGFR antigen-binding peptide, HER2 antigen-binding peptide, PD-L1 antigen-binding peptide, EGFR ligand, HER2 ligand, and PD-L1 ligand. Any antibody that can specifically bind to an antigen, novel alternative antibody, or target tumor-related receptors, immune cell receptors, and tissue / vascular receptors can be used to prepare corresponding multiple-target antibody-conjugated vectors using the dendritic multiple-target antibody-conjugated vector structure containing the first targeting module and the drug conjugation module constructed in this invention.

[0050] Specifically, the first antibody and the second antibody may be the same or different.

[0051] In some embodiments of the present invention, the first antibody is a HER2 antibody and the second antibody is an EGFR antibody; or the first antibody is a HER2 antibody and the second antibody is a PD-L1 antibody; or the first antibody is an EGFR antibody and the second antibody is a HER2 antibody; or the first antibody is an EGFR antibody and the second antibody is a PD-L1 antibody; or the first antibody is a PD-L1 antibody and the second antibody is a HER2 antibody; or the first antibody is a PD-L1 antibody and the second antibody is an EGFR antibody.

[0052] In some embodiments of the present invention, the HER2 antibody can be any HER2 antibody obtained by existing technology, including but not limited to conventional Fab-type HER2 antibodies, HER2 single-chain antibodies, HER2 nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the HER2 antibody is shown in SEQ ID No:13.

[0053] In some embodiments of the present invention, the EGFR antibody can be any EGFR antibody obtained by existing technology, including but not limited to traditional Fab-type EGFR antibodies, EGFR single-chain antibodies, EGFR nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the EGFR antibody is shown in SEQ ID No:14.

[0054] In some embodiments of the present invention, the PD-L1 antibody can be any PD-L1 antibody obtained by existing technology, including but not limited to conventional Fab-type PD-L1 antibodies, PD-L1 single-chain antibodies, PD-L1 nanobodies, etc. In a specific example of the present invention, the amino acid sequence of the PD-L1 antibody is shown in SEQ ID No:15.

[0055] The drug-conjugated polypeptide is an amino acid polypeptide that is site-specifically conjugated to a small molecule drug.

[0056] Specifically, the drug-conjugated polypeptide refers to a drug molecule that is conjugated to a drug molecule through functional groups such as thiol, amino, or azide groups to achieve site-specific conjugation of the drug. It includes, but is not limited to, natural and non-natural amino acids rich in cysteine, lysine, or other reactive functional groups, with a repeating number between 2 and 20.

[0057] In some embodiments of the present invention, the drug-conjugated polypeptide is (GGC)8.

[0058] Among them, the long-circulating polypeptide, after fusion expression, can maintain the activity of the therapeutic protein in vivo for a longer period of time, improve its therapeutic window and efficacy, including but not limited to 1) albumin-binding domain (ABD): by binding to albumin in the blood, it can reduce the rate at which the therapeutic protein is filtered out by the glomerulus, thereby prolonging its half-life in plasma; 2) anti-neonatal Fc receptor (FcRn) affinity (AFF): by binding to FcRn with high affinity, it can improve the circulation efficiency of the protein; 3) anti-albumin single-domain antibody; 4) hydrophilic polypeptide.

[0059] The long-circulating polypeptide is an albumin-binding polypeptide, an Fc-binding polypeptide, or a hydrophilic polypeptide.

[0060] In some embodiments of the present invention, the long circulating polypeptide is an albumin-binding domain (ABD), the amino acid sequence of which is shown in SEQ ID No:16.

[0061] In this module, the functional regions of the second targeting module (including the first targeting module and the spy system module) and the drug conjugation module are connected by short peptides composed of Gly and Ser residues.

[0062] In some embodiments of the present invention, the short peptide is (GGGGS). n (GGGS) n n is an integer from 1 to 50, most commonly 1 to 5. In some embodiments of the present invention, the short peptide is GGGGS, (GGGGS)3, or GGGS.

[0063] In some embodiments of the present invention, the other dendritic multi-target antibody-conjugated vector is the second-generation dendritic dual-target antibody-conjugated vector HC-SGL, such as... Figure 3 As shown.

[0064] Fourthly, the present invention provides a method for preparing the multiple targeting antibody-conjugated vector described in the third aspect.

[0065] That is, the first targeting module and the spy system module are thoroughly mixed and reacted, and the unreacted sample is removed to obtain the second targeting module. Then, the second targeting module and the drug conjugation module are mixed and reacted at the same molar concentration to obtain the multi-target antibody conjugation vector.

[0066] The first target module, or drug-conjugated module, or spy system module is obtained by introducing the coding gene of the first target module, or drug-conjugated module, or spy system module into an expression vector to construct a recombinant expression vector, then introducing the recombinant expression vector into a host bacterium / cell for culture, and then inducing expression, extraction, and purification to obtain the first target module, or drug-conjugated module, or spy system module.

[0067] The reaction is carried out under the following conditions: 20~40℃ and 50~200 rpm for 1~24 h.

[0068] Furthermore, by designing the structure of the targeting module as a dendritic structure, this invention promotes the internalization of molecules within cells and improves drug delivery efficiency. On the other hand, through multivalent interactions, it can enhance the binding ability of the targeting molecule to the receptor and improve the targeting of the molecule.

[0069] Furthermore, in this invention, the drug conjugation module and the targeting module are separate modules. Therefore, drug conjugation does not affect the receptor binding ability of the targeting module, and it is a site-specific conjugation. Thus, the number of drug conjugates can be increased or decreased as needed to meet the requirements of drug conjugation with different pharmacodynamic effects.

[0070] Fifthly, the present invention provides a dendritic antibody-drug conjugate, wherein the antibody-drug conjugate contains the multi-targeting antibody-drug conjugate vector and small molecule drug as described in the first aspect, or contains the multi-targeting antibody-drug conjugate vector and small molecule drug as described in the second aspect.

[0071] The small molecule drug can be any small molecule drug conjugated to the antibody using existing technology, including but not limited to cytotoxic drugs (microtubule inhibitors, topozyme inhibitors) Dxd, MMAE, SN38, DM1, cisplatin, oxaliplatin, doxorubicin and paclitaxel and their derivatives, as well as immune-activating drugs R848, MSA2, diABZI, etc.

[0072] In some embodiments of the present invention, the small molecule drug is Deruxtecan.

[0073] In a sixth aspect, the present invention provides a method for preparing the antibody-drug conjugate described in the fifth aspect. After adding TCEP solution and a drug conjugation module to a buffer solution and reacting for 15-45 min, a small molecule drug dissolved in DMSO is added, and the reaction continues for 1-2 h. Then, a first or second targeting module in an equimolar ratio with the drug conjugation module is added, and the reaction continues. After the reaction is complete, unreacted small molecule drug is removed by dialysis, thus obtaining the antibody-drug conjugate.

[0074] The buffer solution is a 0.1-0.5 M PB buffer solution containing 1-10 mM EDTA, with a pH of 7.4.

[0075] In some embodiments of the present invention, the buffer solution is a 0.1 M PB buffer solution containing 5 mM EDTA, pH 7.4.

[0076] The molar ratio of the small molecule drug to the drug conjugation module is 5~30:1.

[0077] In some embodiments of the present invention, the molar ratio of the small molecule drug to the drug conjugation module is 10:1.

[0078] The sustained reaction is carried out under the following conditions: 20~40°C. o React at C for 5-20 h.

[0079] In some embodiments of the present invention, the continuous reaction is carried out under the following conditions: reaction at 25 °C for 12 h.

[0080] In a seventh aspect, the present invention provides the use of the multi-targeting antibody-drug conjugate vector described in the first aspect, or the multi-targeting antibody-drug conjugate vector described in the fourth aspect, or the antibody-drug conjugate drug described in the fifth aspect in the preparation of antitumor drugs.

[0081] In some embodiments of the present invention, the tumor is a cervical cancer tumor obtained by the invasive growth and endless division of human cervical cancer cells—HeLa cells.

[0082] The dendritic antibody-drug conjugate provided by this invention can effectively degrade the corresponding receptor, which has potential significance for improving drug resistance to small molecule drugs.

[0083] The dendritic antibody-drug conjugate provided by this invention can enhance the drug's ability to enter cells, improve the drug's pharmacokinetic behavior in vivo, and ultimately achieve complete tumor clearance.

[0084] Beneficial Effects: This invention provides a dendritic, multi-targeting antibody-drug conjugate vector, composed of a targeting module and a drug conjugate module. The targeting module possesses multispecific and multivalent receptor binding functions, while the drug conjugate module enables site-specific drug conjugation and high drug loading. The fusion protein of the two modules can specifically bind via a spontaneously formed covalent bond through a reaction.

[0085] This invention further utilizes a dendritic, multi-targeting antibody-drug conjugate (ADC) vector to construct an antibody-drug conjugate (ADC) with the following advantages: 1) The drug conjugation module and the targeting module are separate, ensuring that drug conjugation does not affect the targeting function, and the ADC exhibits good tumor targeting and enrichment capabilities; 2) The dendritic structure promotes drug internalization and tissue penetration; 3) Drug conjugation follows a site-specific conjugation strategy, and the number of drug conjugates is adjustable; 4) The albumin-binding polypeptide fragment in the drug conjugation module enhances molecular stability and improves pharmacokinetics; 5) The targeting group in the targeting module can be designed as a bispecific antibody, which is significant in overcoming tumor receptor expression heterogeneity and improving efficacy. This design enhances drug targeting and relative drug loading, ultimately improving the efficacy of the ADC in treating tumors. Attached Figure Description

[0086] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0087] Figure 1This is a schematic diagram illustrating the construction and structure of the first-generation dendritic dual-target antibody-coupled vector HSE-SGL (D1-RP).

[0088] Figure 2 These are SDS-PAGE diagrams of HSE-SGL, HSE, and SGL.

[0089] Figure 3 This is a schematic diagram of the construction and structure of the second-generation dendritic dual-target antibody-coupled vector HC-SGL (D2-RP).

[0090] Figure 4 This is the SDS-PAGE diagram of HTE-CSC-HTE (HC).

[0091] Figure 5 This is the SDS-PAGE image of HC-SGL (D2-RP).

[0092] Figure 6 This is the BLI diagram showing the binding affinity of D1-RP, D2-RP, and human EGFR (hEGFR).

[0093] Figure 7 This is the BLI diagram showing the binding affinity curves between D1-RP, D2-RP, and human HER2.

[0094] Figure 8 This is a flow cytometry graph showing the entry of D1-RP and D2-RP into HeLa cells.

[0095] Figure 9 This is a schematic diagram of the construction and structure of D1-RPDC.

[0096] Figure 10 This is a schematic diagram of the construction and structure of D2-RPDC.

[0097] Figure 11 This refers to the cytotoxicity of dendritic antibody-drug conjugates D1-RPDC and D2-RPDC in HeLa cells.

[0098] Figure 12 These are the antitumor curves of dendritic antibody-drug conjugates D1-RPDC and D2-RPDC against HeLa tumors. Detailed Implementation

[0099] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0100] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0101] In the following examples, the anti-HER2 antibody, anti-EGFR antibody, PD-L1 antibody, SpyTag protein and Spycatcher protein, albumin-binding domain (ABD), SnoopTag protein and SnoopCatcher protein, human EGFR and human HER2, and HeLa cells were purchased from Jiangsu Kaiji Biotechnology Co., Ltd.

[0102] Example 1: Preparation of the fusion protein anti-HER2-SpyTag-anti-EGFR (HSE)

[0103] 1. Construction of the fusion protein anti-HER2-SpyTag-anti-EGFR (HSE) and recombinant plasmid.

[0104] The three domains of anti-HER2 antibody, SpyTag protein and anti-EGFR antibody were sequentially linked using the (GGGGS)3 linker, and a 6*His tag was fused to the C-terminus to obtain the fusion protein anti-HER2-SpyTag-anti-EGFR (HSE) with the amino acid sequence shown in SEQ ID No:1.

[0105] Using pET-28a plasmid as a vector, a recombinant plasmid containing the HSE gene sequence (as shown in SEQ ID No:2) encoding the fusion protein was designed. The synthesis of the recombinant plasmid was commissioned to Nanjing Genscript Biotech Co., Ltd.

[0106] 2. Expression of the fusion protein HSE

[0107] The recombinant plasmid synthesized in step 1 was transformed into BL21 competent cells using a heat shock method. The cells were then plated on LB agar plates containing kanamycin resistance and incubated overnight at 37°C inverted mode. Positive clones were screened. The selected positive clones were transferred to LB shake tubes and incubated overnight at 37°C and 200 rpm. The overnight culture was then transferred to shake flasks containing 400 mL of TB medium at a 1% v / v inoculation rate and incubated at 37°C with shaking for approximately 3 hours with the addition of 0.1% kanamycin. When OD... 600 When the bacterial cell density was between 0.6 and 0.8, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction, and the cells were cultured on a shaker at 18 °C for 20 h. After the induction expression was completed, the bacterial cell pellet was collected by centrifugation at 8000 rpm and 4 °C.

[0108] 3. Extraction and purification of fusion protein HSE

[0109] The bacterial pellet collected in step 2 was resuspended in 10 mL of buffer (composed of 500 mM sodium chloride and 20 mM PBS, adjusted to pH 7.4). The resuspended bacterial cells were sonicated in an ice-water bath at 400 W, with a 5-second pause after every 2 seconds to avoid overheating, ensuring a total sonication time of 30 min to effectively lyse the cells and release proteins. The sonicated lysate was centrifuged at 12000 g at 4°C to separate the pellet and supernatant. The supernatant was filtered through a 0.45 μM filter to remove any small particles and broken cell debris.

[0110] The filtered supernatant was loaded onto an AKTA Purifier 900 FPLC system equipped with a 5 mL His Trap affinity chromatography column. Elution was performed using elution buffers containing 500 mM sodium chloride and 20 mM PBS (pH=7.4) and 500 mM sodium chloride, 20 mM PBS, and 800 mM imidazole (pH=7.4), starting from 0% and gradually increasing the elution buffer to 30%, performing a linear gradient elution of 20 column volumes. The collected target protein was dialyzed to remove small molecules such as imidazole, and then displaced into PBS buffer to obtain the purified fusion protein HSE. The fusion protein HSE can be used as a first-line targeting module.

[0111] Example 2: Preparation of a fusion protein that can also serve as the first targeting module

[0112] By arbitrarily replacing the anti-EGFR antibody and anti-HER2 antibody in Example 1 with PD-L1 antibody, or changing the binding order, and referring to the preparation method described in Example 1, a fusion protein that can also be used as the first targeting module was prepared, as shown in Table 1.

[0113] Table 1 First Targeting Module

[0114]

[0115] Example 3: Preparation of the fusion protein Spycatcher-(GGC)8-ABD (SGL for short)

[0116] 1. Construction of the recombinant plasmid Spycatcher-(GGC)8-ABD (SGL) fusion protein

[0117] The three functional regions of Spycatcher protein, drug-conjugated polypeptide (GGC)8, and albumin-binding domain (ABD) were sequentially linked using GGGGS and GGGS linkers, and a 6*His tag was fused to the N-terminus to obtain the fusion protein Spycatcher-(GGC)8-ABD (abbreviated as SGL) with the amino acid sequence shown in SEQ ID No:3.

[0118] Using pET-28a plasmid as a vector, a recombinant plasmid containing the SGL gene sequence encoding the fusion protein (as shown in SEQ ID No:4) was designed. The SGL gene sequence encodes the fusion protein. The synthesis of the recombinant plasmid was commissioned to Nanjing GenScript Biotech Co., Ltd.

[0119] 2. Expression, extraction and purification of fusion protein SGL

[0120] The subsequent expression, extraction, and purification processes of the fusion protein SGL were performed as described in Example 1, ultimately yielding the purified fusion protein SGL. The fusion protein SGL can be used as a drug conjugation module.

[0121] Example 4: Preparation of a first-generation dendritic dual-target antibody-conjugated vector HSE-SGL (D1-RP)

[0122] Using the fusion protein HSE prepared in Example 1 as the first targeting module and the fusion protein SGL prepared in Example 3 as the drug conjugation module, the concentrations of fusion proteins HSE and SGL were calculated. The two fusion proteins were then mixed at equimolar concentrations and reacted at 25°C and 100 rpm for 3 h to prepare the first-generation dendritic dual-target antibody-conjugated vector HSE-SGL, denoted as D1-RP. The reaction pathway and structural schematic diagram of HSE-SGL are shown below. Figure 1 As shown. The reaction product HSE-SGL was characterized by SDS-PAGE, and the results are as follows. Figure 2 As shown, the molecular weight of HSE-SGL (D1-RP) is the sum of HSE and SGL.

[0123] Example 5: Preparation of the fusion protein anti-HER2-SnoopTag-anti-EGFR (HTE)

[0124] 1. Construction of the fusion protein anti-HER2-SnoopTag-anti-EGFR (HTE) and recombinant plasmid.

[0125] The three domains of anti-HER2 antibody, SnoopTag protein and anti-EGFR antibody were sequentially linked using the (GGGGS)3 linker, and a 6*His tag was fused to the C-terminus to obtain the fusion protein anti-HER2-SnoopTag-anti-EGFR (HTE) with the amino acid sequence shown in SEQ ID No:5.

[0126] Using pET-28a plasmid as a vector, a recombinant plasmid containing the HTE gene sequence (as shown in SEQ ID No:6) encoding the fusion protein was designed. The synthesis of the recombinant plasmid was commissioned to Nanjing Genscript Biotech Co., Ltd.

[0127] 2. Expression, extraction and purification of fusion protein HTE

[0128] The expression, extraction, and purification process of the fusion protein HTE was as described in Example 1, ultimately yielding the purified fusion protein HTE. Similarly, the fusion protein HTE can be used as a first targeting module.

[0129] Example 6: Preparation of SnoopCatcher-SpyTag-SnoopCatcher (CSC)

[0130] 1. Construction of the fusion protein SnoopCatcher-SpyTag-SnoopCatcher (CSC) and recombinant plasmid.

[0131] The three functional regions of SnoopCatcher protein, SpyTag protein, and SnoopCatcher protein were sequentially linked using the (GGGGS)3 linker, and a 6*His tag was fused to the C-terminus to obtain the fusion protein SnoopCatcher-SpyTag-SnoopCatcher (CSC) with the amino acid sequence shown in SEQ ID No:7.

[0132] Using pET-28a plasmid as a vector, a recombinant plasmid containing the CSC-encoding gene sequence of the fusion protein (as shown in SEQ ID No:8) was designed. The CSC-encoding gene sequence of the fusion protein encodes the fusion protein. The synthesis of the recombinant plasmid was commissioned to Nanjing GenScript Biotech Co., Ltd.

[0133] 2. Expression, extraction and purification of fusion protein CSC

[0134] The expression, extraction, and purification process of the fusion protein CSC was performed as described in Example 1, ultimately yielding purified fusion protein CSC. The fusion protein CSC can be used as a module in a spy system.

[0135] Example 7: Preparation of second-generation dendritic dual-target antibody-conjugated vector HC-SGL (D2-RP)

[0136] The preparation of HC-SGL(D2-RP) is achieved through a two-step reaction, and the reaction route and structural schematic diagram are shown below. Figure 3 As shown, the fusion protein HTE constructed in Example 5 was first used as the first targeting module, and the fusion protein CSC constructed in Example 6 was used as the spy system module to prepare the second targeting module; then the second targeting module and the fusion protein SGL, which is used as the drug conjugation module, were used together to prepare the second-generation dendritic dual-target antibody-conjugated vector HC-SGL (D2-RP).

[0137] Specifically, the fusion proteins HTE and CSC were first mixed at a molar ratio of 2:1 and incubated at 25 °C with shaking for 5 h. The fusion protein HTE-CSC-THE (HC) was then prepared using the spoofing reaction between SnoopTag and SnoopCatcher proteins. After the reaction was complete, unreacted samples were removed by ion chromatography, and the product was purified. The obtained product was characterized by SDS-PAGE, and the results are shown below. Figure 4 As shown, the molecular weight of the fusion protein HC matches the target, confirming the success of the reaction.

[0138] Using fusion protein HC as the second targeting module and fusion protein SGL as the drug conjugation module, HC and SGL were mixed at a 1:1 molar ratio and incubated at 25 °C with shaking for 5 h to prepare the second-generation dendritic dual-target antibody-conjugated vector HC-SGL, denoted as D2-RP. After the reaction, the product was characterized by SDS-PAGE, and the results are as follows. Figure 5 As shown, the molecular weight of HC-SGL(D2-RP) matches the target, confirming the success of the reaction.

[0139] Example 8: Affinity Testing Experiment

[0140] The binding affinity of dendritic dual-target antibody-conjugate vectors D1-RP and D2-RP to human EGFR and human HER2, respectively, was evaluated using biolayer interference (BLI) technology. Human EGFR and HER2 were captured using anti-human Fc antibodies on an AHC microarray (Octet BLI platform, Sartorius), and the binding kinetics of D1-RP and D2-RP to human EGFR and HER2 were determined. The specific procedure is as follows:

[0141] Human EGFR at concentrations of 40–50 μg / mL was prepared in PBST and loaded onto an anti-human IgG Fc capture (AHC) biosensor. Subsequently, the loaded biosensor was placed in D1-RP and D2-RP solutions of different concentrations (6.25, 12.5, 25, 50, 100, and 200 nM, dissolved in PBST) for a 180-second binding reaction, followed by a 180-second dissociation reaction in PBST. The equilibrium constant (KD), binding rate constant (ka), and dissociation rate constant (kdis) were obtained through 1:1 global curve fitting analysis. The binding curves of D1-RP and D2-RP with human EGFR are shown below. Figure 6 As shown.

[0142] Human HER2 at concentrations of 40–50 μg / mL was prepared in PBST and loaded onto an anti-human IgG Fc capture (AHC) biosensor. Subsequently, the loaded biosensor was placed in D1-RP and D2-RP solutions of different concentrations (25, 50, 100, and 200 nM, dissolved in PBST) for a 180-second binding reaction, followed by a 180-second dissociation reaction in PBST. The equilibrium constant (KD), binding rate constant (ka), and dissociation rate constant (kdis) were obtained through 1:1 global curve fitting analysis. The binding curves of D1-RP and D2-RP with human HER2 are shown below. Figure 7 As shown.

[0143] Depend on Figure 6 and Figure 7 It can be seen that both D1-RP and D2-RP can bind to human EGFR and human HER2. Specifically, the binding constants of D1-RP with EGFR and HER2 are 7.24 nM and 13.2 nM, respectively, while the binding constants of D2-RP with human EGFR and human HER2 are 2.47 nM and 1.91 nM, respectively.

[0144] Example 9: Cell Uptake Experiment

[0145] First, SGL molecules were labeled with fluorescein isothiocyanate (FITC) to obtain the fluorescently labeled SGL. FITC The free dye was removed by G-25 gel column chromatography. Subsequently, the SGL was transferred using a SpyTag / SpyCatcher linker system. FITCFITC-labeled first- and second-generation dendritic probe molecules were generated by conjugating HSE or HTE-CSC-HTE, respectively. HeLa cells were seeded in 12-well plates containing DMEM at a density of 10,000 cells per well. When the cell count reached approximately 80%, the medium was aspirated, and each well was washed once with 1 mL of PBS. Then, 0.9 mL of fresh medium (free of fetal bovine serum, penicillin, and streptomycin) and 0.1 mL of FITC-labeled first- and second-generation dendritic probe molecules with consistent fluorescence intensity were added. The control group received 0.9 mL of fresh medium (free of fetal bovine serum, penicillin, and streptomycin) and 0.1 mL of 20 mM Tris (pH 8.0). After culturing for another 1 h, the culture medium was aspirated, and each well was washed three times with 1 mL of PBS. 200 μL of 0.25% trypsin solution was added. When the cytoplasm retracted, the cells became rounded, and a small number of floating cells or cell clusters appeared, the trypsin solution was aspirated, and 1 mL of PBS was added. The cells were collected into 1.5 mL centrifuge tubes (processing cells on ice), centrifuged at 1000 rpm for 5 min, the supernatant was aspirated, and the cells were washed 1-2 times with PBS. Finally, the cells were resuspended in 200 μL of PBS and placed on ice. Flow cytometry was then used for analysis. The experimental results are as follows: Figure 8 As shown, the dendritic structure has superior cell internalization ability, and the second-generation D2-RP has even better cell internalization ability.

[0146] Example 10: Preparation of a first-generation dendritic antibody-drug conjugate, D1-RPDC

[0147] Add 6 mL of buffer solution (0.1 M PB, 5 mM EDTA, pH 7.4), 20 μM TCEP solution, and 3 mg of SGL to a 10 mL centrifuge tube. React at 25 °C and 100 rpm for 30 min. Then add Deruxtecan dissolved in DMSO (molar amount 10 times that of SGL) and continue the reaction for 1 h. Subsequently, add HSE at a molar ratio of 1:1 to SGL and continue the reaction at 25 °C for 12 h. Dialyze the completely reacted sample to 20 mM PBS solution to remove excess small molecules, thus preparing the first-generation dendritic antibody-drug conjugate D1-RPDC. The construction and structural diagram of D1-RPDC are shown below. Figure 9 As shown.

[0148] Example 11: Preparation of the second-generation dendritic antibody-drug conjugate D2-RPDC

[0149] Add 6 mL of buffer solution (0.1 M PB, 5 mM EDTA, pH 7.4), 20 μM TCEP solution, and 3 mg of SGL to a 10 mL centrifuge tube. React at 25 °C and 100 rpm for 30 min. Then add Deruxtecan dissolved in DMSO (molar amount 10 times that of SGL) and continue the reaction for 1 h. Subsequently, add HTE-CSC-HTE (HC) in an equimolar ratio with SGL and continue the reaction at 25 °C for 12 h. Dialyze the completely reacted sample to 20 mM PBS solution to remove excess small molecules, thus preparing the second-generation dendritic antibody-drug conjugate D2-RPDC. The construction and structural diagram of D2-RPDC are shown below. Figure 10 As shown.

[0150] Example 12: Cytotoxicity Verification Experiment

[0151] HeLa cells were seeded at a density of 1000 cells per well in opaque white 96-well plates containing DMEM medium, with a final volume of 100 µL per well. The plates were incubated overnight at 37°C with 5% CO2. The medium was then discarded, and fresh medium solutions containing different concentrations of Deruxtecan, D1-RPDC, and D2-RPDC were prepared. Serial dilutions were performed to ensure a consistent final concentration gradient of each drug in the wells, resulting in final drug concentrations of 50, 100, 200, 450, 900, 1800, and 3600 nM. The plates were then incubated for another 6 days. DMEM medium without the drugs served as a control group.

[0152] After incubation is complete, use CellTiter-Lumi TM Cell viability was detected using a luminescent assay. 0.1 mL of the assay reagent was taken in advance and equilibrated to room temperature for each well of a 96-well plate. Spark assay was then used. ® The multifunctional microplate reader was oscillated at room temperature for 2 min to promote cell lysis, followed by 10 min of incubation at room temperature to stabilize the luminescence signal. Finally, chemiluminescence detection was performed using the reader. The parameters were set according to the instrument requirements, with a detection time of 1 s per well. The average RLU value of the control group was used as 100% cell viability, and the relative cell viability was directly calculated based on the chemiluminescence readings.

[0153] The results are as follows Figure 11As shown, within the tested concentration range, both D1-RPDC and D2-RPDC exhibited stronger inhibitory effects on HeLa cell proliferation than Deruxtecan, indicating that both D2-RPDC and D1-RPDC possess strong cytotoxicity. Under the same concentration conditions, D2-RPDC killed more cells, demonstrating slightly stronger cytotoxicity.

[0154] Example 13: Evaluation of Antitumor Treatment

[0155] 1×10 7 HeLa cells were inoculated into the flank region of the back of 18-22 g BALB / c nude mice, and the tumor tissue was allowed to grow to 100 mm. 3 Tumor-bearing mice were prepared by measuring the size of the tumor. The tumor-bearing mice were randomly divided into four groups of four mice each, and injected with PBS, naked Deruxtecan, D1-RPDC, and D2-RPDC, respectively. The injection dose was uniformly 1.5 mg / kg of the drug, administered on days 0, 5, 10, 17, and 24, for a total of five administrations. Tumor volume was measured every two days during this period.

[0156] Tumor growth curves of mice in each treatment group are as follows: Figure 12 As shown, compared with the naked drug Deruxtecan, at the same drug dosage, the dendritic antibody-drug conjugate (ADC) showed superior antitumor effects, with a cure rate of 25% in the D1-RPDC group and 100% in the D2-RPDC group.

[0157] This invention provides a dendritic multi-targeting antibody-drug conjugate carrier, antibody-drug conjugates, their preparation methods, and applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A dendritic, multi-targeting antibody-conjugated vector, characterized in that, Includes a primary targeting module and a drug conjugation module; The first targeting module includes three functional regions: a first antibody, a first tag, and a second antibody; the drug conjugation module includes three functional regions: a first tag, a drug-conjugated peptide, and a long-circulating peptide.

2. The multiple targeting antibody-conjugated vector according to claim 1, characterized in that, The first spy tag and the first spy catcher are any one of SpyTag and SpyCatcher, SnoopTag and SnoopCatcher; The amino acid sequences of SpyTag and SpyCatcher are shown in SEQ ID No:9 and SEQ ID No:10, respectively, and the amino acid sequences of SnoopTag and SnoopCatcher are shown in SEQ ID No:11 and SEQ ID No:12, respectively.

3. The multiple targeting antibody-conjugated vector according to claim 1, characterized in that, The first antibody or the second antibody includes any one of EGFR antibody, HER2 antibody, PD-L1 antibody, EGFR affinity, HER2 affinity, PD-L1 affinity, EGFR antigen-binding peptide, HER2 antigen-binding peptide, PD-L1 antigen-binding peptide, EGFR ligand, HER2 ligand, and PD-L1 ligand; wherein the first antibody and the second antibody may be the same or different.

4. The multiple targeting antibody-conjugated vector according to claim 1, characterized in that, The drug-conjugated polypeptide is an amino acid polypeptide that is site-conjugated to a small molecule drug; the long-circulating polypeptide is an albumin-binding polypeptide, an Fc-binding polypeptide, or a hydrophilic polypeptide; the functional regions in the first targeting module and the drug-conjugated module are connected by short peptides composed of Gly and Ser residues.

5. The method for preparing the multiple target antibody-conjugated vector according to any one of claims 1 to 4, characterized in that, The first targeting module and the drug conjugation module are mixed at equal molar concentrations and reacted to obtain the multi-targeting antibody conjugation vector; wherein the reaction conditions are: 20~40 ℃, 50~200 rpm for 1~24 h.

6. A dendritic, multi-targeting antibody-conjugated vector, characterized in that, Includes a second targeting module and a drug conjugation module; The second targeting module includes a first targeting module and a spy system module, and the drug conjugate module includes three functional regions: a second spy catcher, a drug-conjugate peptide, and a long-circulating peptide. The first targeting module includes three functional areas: a first antibody, a first spy tag, and a second antibody. The spy system module includes two functional areas: a first spy catcher and a second spy tag.

7. The multiple targeting antibody-conjugated vector according to claim 6, characterized in that, The first spy tag and the first spy catcher, or the second spy tag and the second spy catcher, are any one of SpyTag and SpyCatcher, SnoopTag and SnoopCatcher; Among them, the labels for First Spy and First Spy Catcher, and the labels for Second Spy and Second Spy Catcher are different; The amino acid sequences of SpyTag and SpyCatcher are shown in SEQ ID No:9 and SEQ ID No:10, respectively, and the amino acid sequences of SnoopTag and SnoopCatcher are shown in SEQ ID No:11 and SEQ ID No:12, respectively.

8. The multiple targeting antibody-conjugated vector according to claim 6, characterized in that, The first antibody or the second antibody includes any one of EGFR antibody, HER2 antibody, PD-L1 antibody, EGFR affinity, HER2 affinity, PD-L1 affinity, EGFR antigen-binding peptide, HER2 antigen-binding peptide, PD-L1 antigen-binding peptide, EGFR ligand, HER2 ligand, and PD-L1 ligand; wherein the first antibody and the second antibody may be the same or different.

9. The multiple targeting antibody-conjugated vector according to claim 6, characterized in that, The drug-conjugated polypeptide is an amino acid polypeptide that is site-conjugated to a small molecule drug; the long-circulating polypeptide is an albumin-binding polypeptide, an Fc-binding polypeptide, or a hydrophilic polypeptide; the functional regions in the first targeting module, the spy system module, and the drug-conjugated module are connected by short peptides composed of Gly and Ser residues.

10. The method for preparing the multiple target antibody-conjugated vector according to any one of claims 6 to 9, characterized in that, First, the first targeting module and the spy system module are thoroughly mixed and reacted. Then, the unreacted sample is removed to obtain the second targeting module. Next, the second targeting module and the drug conjugation module are mixed and reacted at the same molar concentration to obtain the multi-target antibody conjugation vector. The reaction is carried out under the following conditions: 22~40 ℃ and 50~200 rpm for 1~24 h.

11. A dendritic antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains the multi-target antibody-drug conjugate vector and small molecule drug as described in any one of claims 1 to 4, or contains the multi-target antibody-drug conjugate vector and small molecule drug as described in any one of claims 6 to 9.

12. The method for preparing the antibody-drug conjugate according to claim 11, characterized in that, After adding TCEP solution and drug conjugation module to buffer solution and reacting for 15-45 min, add small molecule drug dissolved in DMSO and continue the reaction for 1-2 h; then add first or second target module in the same molar ratio as drug conjugation module and continue the reaction; after the reaction is completed, dialyze to remove unreacted small molecule drug, thus preparing antibody-drug conjugate.

13. The preparation method according to claim 12, characterized in that, The buffer solution is a 0.1-0.5 M PB buffer solution containing 1-10 mM EDTA, pH 7.4; the molar ratio of the small molecule drug to the drug conjugation module is 5-30:1; the continuous reaction is carried out under the following conditions: 20-40 °C. o React at C for 5-20 h.

14. The use of the multi-targeting antibody-drug conjugate according to any one of claims 1 to 4, or the multi-targeting antibody-drug conjugate according to any one of claims 6 to 9, or the antibody-drug conjugate according to claim 11 in the preparation of antitumor drugs.

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