Bifunctional molecule targeting glucose transporter and application thereof in preparation of antibody-drug conjugate

Abstract

The present invention provides a glucose transporter (GLUT)-based lysosome targeting chimera. The lysosome targeting chimera can effectively enrich and degrade in lysosomes different types of target proteins on the cell surface and outside the cell. The present invention further provides an application of the lysosome targeting chimera, and an antibody-drug conjugate obtained by conjugating the lysosome targeting chimera with a cytotoxic drug.

Description

Bifunctional molecules targeting glucose transporters and their use in the preparation of antibody conjugated drugs

[0001] Cross-reference to Related Applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN202411343894.4, filed on September 25, 2024, the entire contents of which are incorporated herein for all purposes. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, and in particular, the present application relates to bifunctional molecules targeting glucose transporters and their applications. BACKGROUND

[0004] Targeted Protein Degradation (TPD) technology is a new technology developed in recent years for interfering with protein function and regulating protein expression. The most mature technology in this field is the use of ubiquitin-proteasome degradation pathway to construct targeted chimera (PROTAC). This technology recruits target proteins and E3 ubiquitin ligase ligands, so that the target proteins are tagged with ubiquitination, thereby achieving target protein degradation. In addition to the ubiquitin-proteasome pathway, the endosome-lysosome pathway is another important protein degradation system in eukaryotic cells. This pathway is mainly related to the degradation of membrane proteins and extracellular proteins, and plays an important role in regulating the homeostasis of intracellular and extracellular proteins.

[0005] For the endosome-lysosome pathway, Lysosome Targeting Chimera (LYTAC) technology has been proposed. Lysosome Targeting Chimera is a class of bifunctional molecules, one part of which is a ligand that specifically binds to the cell surface lysosome targeting receptor (LTR), and the other part is an antibody, polypeptide or small molecule that specifically binds to a molecule located on the cell membrane or extracellularly. Currently, two LTRs reported to be used in LYTAC technology are the cation-independent mannose-6-phosphate receptor (CI-M6PR) and the asialoglycoprotein receptor (ASGPR), and their corresponding ligands are mannose-6-phosphate (M6PR) and N-acetylgalactosamine (GalNAc), respectively. However, CI-M6PR is expressed in multiple cells and does not have cell or tissue specificity, which has potential safety problems in its application; while ASGPR is limited to liver tissue specificity, greatly limiting its application range.

[0006] Glucose transporter (GLUT) is a family of transmembrane proteins that regulate the entry of extracellular glucose into cells, involved in processes such as glucose metabolism, inflammatory response and immune response. Glucose is an important source of energy, and the uptake of glucose by tissue cells requires the help of glucose transporters on the cell membrane as carriers to carry glucose into epithelial cells and promote cell metabolism. GLUT is one of the most important transmembrane proteins in the human body. GLUT is very sensitive to changes in the body environment and is regulated by the body environment, signal molecules, small molecule factors and metabolic products, and is widely involved in the "network" of signal pathways and related to the occurrence and development of many diseases. For example, tumor cells must express a large amount of GLUT to meet their glucose uptake and metabolic needs in order to maintain their rapid growth, proliferation and metastasis, so overexpression of GLUT has become one of the important markers of tumor cells. However, there is currently no report on the degradation strategy of membrane proteins and extracellular proteins based on GLUT.

[0007] Antibody-drug conjugate (ADC) is currently considered one of the most promising anti-tumor drugs. Antibody-drug conjugate is usually composed of three parts: an antibody or antibody-like ligand that can recognize and bind to a specific antigen on the target cell, a small molecule drug load, and a linker connecting the two. This immunoconjugate has high killing activity of toxic drugs and high targeting of antibodies, so it can effectively broaden the therapeutic window and reduce adverse reactions.

[0008] Whether ADC can be effectively internalized into the lysosome of the target cell and then release the small molecule drug is an important factor affecting the efficacy of ADC. Referring to the LYTAC technology, by specifically binding to the lysosomal targeting receptor on the cell surface, the lysosome-targeting chimera can internalize the target molecule into the lysosome of the cell, so that the target molecule is degraded in the lysosome. After the development of the degradation strategy of membrane proteins and extracellular proteins based on GLUT, whether this strategy can be further applied to antibody-drug conjugates to improve their internalization efficiency and promote the release and accumulation of small molecule drugs in target cells, thereby ultimately exerting the efficacy of small molecule drugs, remains to be studied. SUMMARY

[0009] In view of the above technical problems, the purpose of the present application is to provide a lysosome-targeting chimera based on glucose transporter (GLUT) which can be used for targeted degradation of membrane proteins or extracellular proteins via GLUT, and further used for the prevention and treatment of diseases such as tumors.

[0010] Further, the present application aims to provide the use of the lysosome-targeting chimera in the preparation of antibody conjugated drugs. In addition, the present application also aims to provide an antibody conjugated drug based on the lysosome-targeting chimera.

[0011] The technical solutions of the present application are as follows.

[0012] In the first aspect, the present application provides a lysosome-targeting chimera based on glucose transporter (GLUT), which comprises two parts:

[0013] (1) The first part is capable of specifically binding to a target molecule, and is one or more selected from polypeptide, protein, nucleic acid, nanoparticle and small molecule compound;

[0014] (2) The second part is a substrate capable of binding to glucose transporter (GLUT) or being transported by GLUT.

[0015] In the lysosome-targeting chimera provided by the present application, the first part can be a protein, and further can be an antibody or an antigen-binding fragment thereof capable of specifically binding to a target molecule. When the first part is an antibody, it can be one or more selected from polyclonal antibody, monoclonal antibody, monospecific antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human source antibody, etc. When the first part is an antigen-binding fragment of an antibody, the antigen-binding fragment can be one or more of Fab, Fab', F(ab')2, Fv, dsFv, scFv, sc(Fv)2 and VHH.

[0016] The first part is capable of specifically binding to a target molecule, which can be located on the cell surface or outside the cell, and can be a cell surface protein, a ligand of a receptor, a cytokine, a hormone, a secreted protein, an antibody, a protein carrier, an enzyme, etc.

[0017] The target molecule can be a target molecule located on the cell surface, which includes but is not limited to growth factor receptor (such as EGFR, HER2, HER3, cMET), immune checkpoint molecule (such as PD-1, PD-L1, CTLA4, TIGHT, TIM3), leukocyte differentiation antigen (such as CD20, CD22, CD24, CD38, CD71, CD80), etc. According to the specific embodiments of the present application, the target molecule is human epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2).

[0018] The target molecule can be an extracellular target molecule, including but not limited to growth factors (e.g., VEGF, EGF), cytokines (e.g., interleukins, interferons, tumor necrosis factor, transforming growth factor), chemokines, and secreted proteins (e.g., alpha-synuclein), protein hormones (e.g., insulin), antibodies (e.g., allergic antibodies, autoantibodies), protein toxins, viruses, and infectious particles, etc. According to the specific embodiments of the present application, the target molecule is streptavidin and tumor necrosis factor-alpha (TNF-alpha).

[0019] In the lysosome-targeting chimera provided by the present application, the first portion comprises a reactive group, which is one or more selected from the group consisting of amino group, carboxyl group, thiol group, halogen group, and bio-orthogonal group; wherein the bio-orthogonal group is one or more selected from the group consisting of azido group, alkyne group, aldehyde group, ketone group, and fluoro-sulfonate group.

[0020] In the lysosome-targeting chimera provided by the present application, the second portion is a substrate capable of binding to or being transported by GLUT. As a substrate of GLUT, the second portion can have a binding affinity (measured by dissociation constant K d dissociation constant K

[0021] The second portion can comprise one or more selected from the group consisting of glucose, fructose, and derivatives thereof. And, as a ligand of GLUT, the second portion can be a monovalent ligand, a bivalent ligand, a trivalent ligand, or even a multivalent ligand.

[0022] When the second portion is a monovalent ligand, its general structure can be as shown in Formula I:

[0023] When the second portion is a bivalent ligand, its general structure can be as shown in Formula II, wherein the structures of Linker1, Linker2, and Linker4 can be the same or different:

[0024] When the second portion is a trivalent ligand, its general structure can be as shown in Formula III, wherein the structures of Linker1, Linker2, Linker3, and Linker4 can be the same or different:

[0025] When the second portion is a multivalent ligand, its general structure can be as shown in Formula IV or Formula V, wherein n = 1-999, preferably n = 1-500, more preferably n = 1-200, further preferably n = 1-50, particularly preferably n = 1-20, for example n = 1-10 (e.g., n = 150, 100, 80, 40, 30, 15, etc.):

[0026] In the present application, X is -O- or -NH-; R is amino, carboxyl, maleimide group, azido group, alkyne group, aldehyde group, ketone group, fluorosulfonate or halogen group.

[0027] In the present application, each Linker (including Linker1, Linker2, Linker3 or Linker4) is a cleavable or non-cleavable linker.

[0028] According to the embodiments of the present application, each Linker can be independently selected from one or more of the following: polypeptide, oligosaccharide, -(CH2) n -(CH2CH2O) n -OCH2(CH2) n CH2O-, -CONH-, -Lys(N3)-, Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Phe-Gly, Val-Cit-PABC, Val-Ala-PABC, Val-Lys(Ac)-PABC, Phe-Lys-PABC, Phe-Lys(Ac)-PABC, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PABC, Ala-PABC, PABC, triazole, piperazine, piperidine. Wherein, Cit is citrulline, Ac is acetyl, and PABC is p-aminobenzyloxy carbonyl.

[0029] According to the embodiments of the present application, in the lysosome-targeting chimera of the present application, the second moiety is a monovalent ligand, and the structure is shown in formula e or g, wherein n is the same as the general formula above:

[0030] Alternatively, the second moiety is a trivalent ligand, and the structure is shown in formula i, wherein n is the same as the general formula above:

[0031] Alternatively, the second moiety is a multivalent ligand, and the structure is shown in formula j, wherein n is the same as the general formula above:

[0032] In the lysosome-targeting chimera of the present application, the first moiety is directly or indirectly linked to the second moiety via its reactive group. The two can be linked together by chemical or enzymatic methods, for example, when the first moiety is an antibody or an antigen-binding fragment, the first moiety and the second moiety can be linked together by site-directed coupling or non-site-directed coupling. For example, the structure of the chimera is shown in formula VI:

[0033] In the formula VI provided by the present application, the three-dimensional structure on the right side is the first part, which can specifically bind to the target molecule and is one or more selected from polypeptide, protein, nucleic acid, nanoparticle and small molecule compound, as described above; further, the first part comprises a functional group and the functional group forms a reactive pair with the R group; the functional group includes but is not limited to amino, carboxyl, sulfydryl and the like. In the formula VI provided by the present application, R, Linker and the second part ligand are derived from any one of the formulae I to V, formula e, formula g, formula i, formula j provided above in the present application.

[0034] According to the specific embodiments of the present application, the first part can comprise sulfydryl, and the lysosome-targeting chimera can have the structure shown as follows:

[0035] According to the specific embodiments of the present application, the first part can comprise amino, and the lysosome-targeting chimera can have the structure shown as follows:

[0036] According to the specific embodiments of the present application, the first part can comprise carboxyl, and the lysosome-targeting chimera can have the structure shown as follows:

[0037] According to the specific embodiments of the present application, when the first part is a monoclonal antibody, the lysosome-targeting chimera has the structure shown as follows:

[0038] wherein a, b = 1-999, preferably a, b = 1-500, more preferably a, b = 1-200, further preferably a, b = 1-50, particularly preferably a, b = 1-20, for example a, b = 1-10 (for example a, b = 150, 100, 80, 40, 30, 15, etc.); m = 1-50, preferably m = 1-25, for example m = 1-10 (for example m = 2, 5, 10, 15, 25, etc.).

[0039] For example, when a = 8, b = 1, m = 25, the lysosome-targeting chimera provided by the present application can have the following structure:

[0040] Alternatively, according to the specific embodiments of the present application, when the first part is an antigen-binding fragment (VHH), the lysosome-targeting chimera has the structure shown as follows:

[0041] In a second aspect, the present application provides use of the lysosome-targeting chimera in the preparation of a medicament for treating a disease associated with a target molecule. As described above, the target molecule can be located on the cell surface or outside the cell, and can be a cell surface protein, a ligand of a receptor, a cytokine, a hormone, a secreted protein, an antibody, a protein carrier, an enzyme, etc. The lysosome-targeting chimera of the present application can bind to any of these target molecules through the first part thereof, and then be transported to the lysosome through the second part thereof, i.e. the part capable of binding to GLUT or a substrate transported by GLUT, thereby degrading the target molecule and achieving the treatment of the disease associated therewith.

[0042] The disease can be selected from one or more of cancer, inflammation-related disease, immune-related disease, viral infection, metabolic disease, and neurodegenerative disease. Preferably, the disease is an inflammation-related disease, such as rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis. Preferably, the disease is a cancer, such as breast cancer, ovarian cancer, breast ductal carcinoma, gastric cancer, epidermal carcinoma, bladder cancer. Preferably, the viral infection is, for example, Epstein-Barr virus infection, SARS-CoV-2 infection, influenza virus infection, human papillomavirus infection, herpes virus infection. Preferably, the metabolic disease is, for example, non-alcoholic fatty liver disease / hepatitis, diabetes, hyperbilirubinemia, bone metabolism disease, cardiovascular disease. Preferably, the neurodegenerative disease is, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia.

[0043] In a third aspect, the present application provides use of the lysosome-targeting chimera in the preparation of an antibody conjugate drug. Through the reactive group of the first part of the lysosome-targeting chimera provided by the present application, the lysosome-targeting chimera can be further conjugated with a cytotoxic drug to form an antibody conjugate drug.

[0044] As described above, according to the specific embodiments of the present application, the first part of the lysosome-targeting chimera provided by the present application can be an antibody (such as a monoclonal antibody) or an antigen-binding fragment of an antibody (such as a nanobody or VHH), and the antibody conjugate drug provided by the present application can be obtained by conjugating the antibody or antigen-binding fragment with a cytotoxic drug via a linker.

[0045] In a fourth aspect, the present application provides an antibody conjugate drug comprising the lysosome-targeting chimera provided by the present application and a cytotoxic drug. In the antibody conjugate drug provided by the present application, the cytotoxic drug can be connected to the lysosome-targeting chimera via the reactive group contained in the first part of the lysosome-targeting chimera.

[0046] As described above, according to the embodiments of the present application, the first moiety in the lysosome-targeting chimera provided by the present application can be an antibody (such as a monoclonal antibody) or an antigen-binding fragment of an antibody (such as a nanobody or VHH), and the antibody-drug conjugate provided by the present application can be obtained by conjugating the antibody or the antigen-binding fragment to a cytotoxic drug via a linker.

[0047] In the antibody-drug conjugate provided by the present application, the cytotoxic drug can be a cytotoxic agent conventionally used in the art for preparing ADCs, such as a microtubulin inhibitor or a prodrug thereof, a DNA-damaging agent or a prodrug thereof, a topoisomerase inhibitor or a prodrug thereof, etc.

[0048] The microtubulin inhibitor can be selected from any one of auristatin or auristatin derivatives and analogs, maytansinoid or maytansinoid derivatives and analogs, paclitaxel or paclitaxel derivatives and analogs, vinca-alkaloid or vinca-alkaloid derivatives and analogs, cryptophycin or cryptophycin derivatives and analogs, tubulysin or tubulysin derivatives and analogs.

[0049] The DNA-damaging agent can be selected from any one of doxorubicin or doxorubicin derivatives and analogs, calicheamicin or calicheamicin derivatives and analogs, pyrrolobenzodiazepines (PBD) or PBD derivatives and analogs, duocarmycin or duocarmycin derivatives and analogs.

[0050] The topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor, such as camptothecin or camptothecin derivatives and analogs, such as Exatecan, Belotecan, SN-38, Topotecan, Irinotecan (CPT-11), Deruxtecan, Dxd, etc.

[0051] According to the embodiments of the present application, the antibody-drug conjugate provided by the present application can have the following structure:

[0052] wherein a, b = 1-999, preferably a, b = 1-500, more preferably a, b = 1-200, further preferably a, b = 1-50, particularly preferably a, b = 1-20, for example a, b = 1-10 (for example a, b = 150, 100, 80, 40, 30, 15, etc.); m = 1-50, preferably m = 1-25, for example m = 1-10 (for example m = 2, 5, 10, 15, 25, etc.).

[0053] For example, when a = 8, b = 1, m = 25, the antibody drug conjugate provided by the present application can have the following structure:

[0054] In a fifth aspect, the present application also provides a method for treating a disease, comprising administering a lysosome-targeting chimera or an antibody drug conjugate provided by the present application to a subject in need thereof.

[0055] The disease is a disease associated with a target molecule, as described in the second aspect of the present application. The subject can be a mammal; preferably, the subject is a human.

[0056] It has been proved that the lysosome-targeting chimera of the present application can effectively achieve the enrichment and efficient degradation of different types of target proteins on the cell surface and outside the cell. Without being limited to any theory or mechanism, the lysosome-targeting chimera of the present application carries a glucose ligand, which can utilize the glucose transporter receptor to achieve the degradation of the target protein in the lysosome through the clathrin-mediated endocytosis pathway. Therefore, depending on the target protein to which the first part of the chimera carrying the glucose ligand is directed, the lysosome-targeting chimera provided by the present application can more effectively inhibit the proliferation of tumor cells and the growth of tumors associated with the target protein, and has significant anti-tumor activity. Animal experiments also show that the tumor targeting ability of the chimera of the present application is significantly enhanced. These results collectively indicate that the glucose transporter receptor-mediated chimera technology of the present application is an effective disease treatment strategy. Moreover, the present application also provides the application of the chimera in the targeted protein degradation technology, and the efficient degradation of the target protein is achieved by constructing the glucose transporter receptor-mediated chimera.

[0057] Moreover, the lysosome-targeting chimera of the present application can be further conjugated with a small molecule toxic drug to obtain an antibody drug conjugate. Compared with the antibody drug conjugates known in the art, the antibody drug conjugate provided by the present application can utilize the glucose transporter receptor to more efficiently internalize into the interior of tumor cells due to the inclusion of the lysosome-targeting chimera of the present application, and has stronger tumor inhibition effect. BRIEF DESCRIPTION OF DRAWINGS

[0058] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0059] Figure 1 is a schematic diagram showing the mechanism of action of the lysosome targeting chimeric body based on glucose transporter (GLUT) of the present application;

[0060] Figure 2 is a synthetic route diagram of monovalent ligand (2A), trivalent ligand (2B), and multivalent ligand (2C) in Example 1 of the present application;

[0061] Figure 3 shows the nuclear magnetic resonance characterization data of compound a, compound b, compound c, and compound d in Example 1 of the present application;

[0062] Figure 4 is a non-site-specific coupling route diagram for preparing the lysosome targeting chimeric body in Example 2 of the present application;

[0063] Figure 5 shows the SDS-PAGE, SEC-HPLC, and MS characterization results of chimeric body Adm-Glc25 in Example 2 of the present application;

[0064] Figure 6 shows the SDS-PAGE, SEC-HPLC, and MS characterization results of chimeric body Tz-Glc25 in Example 2 of the present application;

[0065] Figure 7 is a coupling route diagram of chimeric body 7D12-Glc in Example 2 of the present application.

[0066] Figure 8 is a route diagram showing the labeling of TAMRA by click chemistry of chimeric body 7D12-Glc in Example 2 of the present application.

[0067] Figure 9 is a flow cytometry diagram and quantification diagram showing the internalization of 7D12 and chimeric body 7D12-Glc in Example 3 of the present application;

[0068] Figure 10 is a confocal laser scanning microscope diagram showing the internalization of extracellular protein streptavidin in Example 4 of the present application;

[0069] Figure 11 is a confocal laser scanning microscope diagram showing the internalization of TNF-α in Example 5 of the present application;

[0070] Figure 12 is a confocal laser scanning microscope diagram showing the internalization of Tz or chimeric body Tz-Glc25 in SKBR3 or 293T cells in Example 6 of the present application;

[0071] Figure 13 is a confocal laser scanning microscope diagram of SKBR3 cells after incubation with Tz or chimeric body Tz-Glc25 in Example 7 of the present application;

[0072] Figure 14 is an immunoblot diagram of membrane protein HER2 after incubation of SKBR3 cells with Tz or chimeric bodies Tz-Glc2, Tz-Glc15, and Tz-Glc25 in Example 7 of the present application;

[0073] Figure 15 is an immunoblotting diagram of membrane protein HER2 after Tz or chimera Tz-Glc25 is incubated with SKBR3, SKOV3, NCI-N87, BT474 cells in Example 7 of the present application;

[0074] Figure 16 is an immunoblotting diagram and quantification results of membrane protein HER2 after Tz or chimera Tz-Glc25 is incubated with SKBR3 cells for different time in Example 7 of the present application;

[0075] Figure 17 is an immunoblotting diagram and quantification results of membrane protein HER2 after Tz or chimera Tz-Glc25 is incubated with SKBR3 cells at different concentrations in Example 7 of the present application;

[0076] Figure 18 is an immunoblotting diagram of HER2 after Tz or chimera Tz-Glc25 is co-incubated with cells with different inhibitors in Example 8 of the present application;

[0077] Figure 19 is a confocal laser scanning microscope diagram of cells after Tz or chimera Tz-Glc25 is incubated with SKBR3 cells in Example 9 of the present application;

[0078] Figure 20 shows cell clone staining results after Tz or chimera Tz-Glc25 is incubated with SKBR3 cells in Example 9 of the present application;

[0079] Figure 21 is a survival rate line graph of cells after Tz or chimera Tz-Glc25 is incubated with SKBR3 cells in Example 9 of the present application;

[0080] Figure 22 is a fluorescence image of live mice and an ex vivo organ tissue fluorescence imaging diagram of mice after Tz or chimera Tz-Glc25 is administered in a mouse gastric cancer model in Example 10 of the present application;

[0081] Figure 23 is a construction route diagram of a chimera-based ADC in Example 11 of the present application;

[0082] Figure 24 shows SDS-PAGE, SEC-HPLC, MS characterization results of a chimera-based ADC MMAF-Tz-Glc25 in Example 11 of the present application;

[0083] Figure 25 shows a dose-dependent binding curve of MMAF-Tz and chimera-based ADC MMAF-Tz-Glc25 with HER2 in Example 12 of the present application;

[0084] Figure 26 is a survival rate line graph of cells after 7D12-MMAF or chimera-based ADC 7D12-MMAF-Glc is incubated with A431 cells in Example 13 of the present application;

[0085] Figure 27 is a cell survival rate line graph of MMAF-Tz or chimera-based ADC MMAF-Tz-Glc25 incubated with tumor cells of different HER2 expression levels in Example 13 of the present application;

[0086] Figure 28 shows cell clone staining results of MMAF-Tz and chimera-based ADC MMAF-Tz-Glc25 incubated with SKBR3 cells and 5637 cells in Example 13 of the present application;

[0087] Figure 29 is a size statistics chart and survival rate statistics chart of breast cancer organoids incubated with MMAF-Tz and chimera-based ADC MMAF-Tz-Glc25 in Example 13 of the present application.

[0088] Best Mode for Carrying Out the Invention

[0089] The mechanism of action of the glucose transporter (GLUT)-based lysosome-targeting chimera of the present application is shown in Figure 1.

[0090] The present application will be described with reference to specific examples below. Those skilled in the art will understand that these examples are only used to illustrate the present application, and do not limit the scope of the present application in any way.

[0091] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available unless otherwise specified.

[0092] Example 1 Synthesis of glucose ligand

[0093] In this example, a synthetic route of a glucose ligand is exemplarily provided, as shown in Figure 2; the glucose ligand is subsequently used to establish the lysosome-targeting chimera of the present application.

[0094] For convenience of description, the compounds in Figure 2 are replaced by letters, wherein 2A, 2B and 2C are respectively the synthetic route diagrams of the exemplified monovalent ligand, trivalent ligand and multivalent ligand in this example. The product structures are confirmed by nuclear magnetic resonance.

[0095] Figure 3 shows the nuclear magnetic resonance characterization data of compound a, compound b, compound c and compound d, respectively, wherein 3A and 3B are respectively the hydrogen spectrum and carbon spectrum characterization results of compound a; 3C and 3D are respectively the hydrogen spectrum and carbon spectrum characterization results of compound b; 3E and 3F are respectively the hydrogen spectrum and carbon spectrum characterization results of compound c; 3G and 3H are respectively the hydrogen spectrum and carbon spectrum characterization results of compound d.

[0096] Example 2 Construction of chimera

[0097] In this example, different types and molecular weight range of molecules (monoclonal antibodies, nanobodies (VHH)) were exemplarily selected to conjugate with glucose ligand to construct the lysosome targeting chimera of the present application.

[0098] In this example, anti-TNF-a monoclonal antibody Adalimumab (Adm), anti-HER2 monoclonal antibody Trastuzumab (Tz), and anti-EGFR nanobody (7D12) were selected. The glucose ligand of the present application can be chimerized on the antibody through several different conjugation means.

[0099] 1) Construction and characterization of the chimera based on monoclonal antibody

[0100] The glucose ligand was conjugated to the antibody using site-directed conjugation and non-site-directed conjugation, and the number of glucose ligands was adjusted by controlling the experimental conditions. The conjugation product, i.e. the chimera, was structurally characterized by SDS-PAGE, HPLC, and MS.

[0101] i. Site-directed conjugation

[0102] The route is as follows: first, introduce amino acid mutations (mutated to cysteine) into the antibody by genetic engineering, and then modify the thiol group of cysteine. When conjugating, DTT is used to open the interchain disulfide bond of the antibody and expose the thiol group of the introduced cysteine, then DHAA is added to reconnect the interchain disulfide bond, and finally the free thiol group will undergo Michael addition reaction with the sugar ligand to achieve site-directed conjugation of the antibody ligand.

[0103] Exemplarily, Tz and compound d of Example 1 were subjected to site-directed conjugation as above to obtain conjugation products Tz-Glc2, Tz-Glc6, and Tz-Glc12.

[0104] ii. Non-site-directed conjugation

[0105] The route is as follows: first, introduce an appropriate amount of BCN-PEG8-NHS into the antibody through lysine reaction to obtain an intermediate product, i.e. antibody-BCN, which has a reactive group BCN conjugated; then, the pre-synthesized sugar ligand is conjugated to the intermediate product by click reaction. By controlling the amount of BCN-PEG8-NHS and sugar ligand added, chimeras with different numbers of glucose ligands are obtained. The conjugation route is shown in Figure 4, where m represents the number of sugar ligands, and m = 1-50; and exemplarily, compound d of Example 1 is used as the sugar ligand.

[0106] Exemplarily, non-site specific coupling was performed as above with Adm, Tz and compound d of Example 1 to obtain the coupling products, named Adm-GTAC, Tz-GTAC; followed by structural characterization of both by SDS-PAGE, SEC-HPLC, MS, respectively.

[0107] FIG. 5 shows SDS-PAGE (5A), SEC-HPLC (5B) and MS (5C) characterization results of the coupling product Adm-GTAC, respectively; FIG. 6 shows SDS-PAGE (6A), SEC-HPLC (6B) and MS (6C) characterization results of the coupling product Tz-GTAC, respectively. As shown in 5A and 6A, the light chain and the heavy chain of Adm-GTAC, Tz-GTAC have obvious upward shift compared with Adm, Tz after introducing the glucose ligand, which indicates that the light chain and the heavy chain of Adm, Tz are coupled with the glucose ligand; as shown in 5B and 6B, the retention time of Adm-GTAC, Tz-GTAC is shortened compared with Adm, Tz after introducing the glucose ligand and no obvious aggregation peak is observed, which indicates that Adm, Tz are coupled with the glucose ligand, the molecular weight is increased and no obvious aggregation occurs; as shown in 5C and 6C, the molecular weight of Adm-GTAC, Tz-GTAC is obviously increased compared with Adm, Tz after introducing the glucose ligand, and the number of glucose ligand coupling is calculated to be 25.

[0108] In summary, it is determined that both coupling products are coupled with 25 sugar ligands (compound d), which are named Adm-Glc25, Tz-Glc25, and the structures are as follows:

[0109] 2) Construction and characterization of chimeras based on antigen-binding fragments (nanobodies; VHH)

[0110] The route is as follows: first, introduce cysteine mutation into nanobody for subsequent thiol labeling coupling; after antibody expression, add azido group that can be used for bio-orthogonal reaction to the C-terminus of the antibody through transpeptidation reaction; when modifying, first couple glucose ligand carrying maleimide linker to the cysteine thiol of the antibody, and finally optionally couple fluorescent small molecule to the antibody through click chemistry. The constructed chimeras are structurally characterized by SDS-PAGE, HPLC and MS.

[0111] i. Transpeptidation reaction

[0112] This embodiment uses the polypeptide GGGGGKN3 obtained by pre-solid-phase synthesis to carry out transpeptidation reaction. Exemplarily, the nanobody 7D12 with a cysteine mutation S85C introduced is used as a substrate, and the substrate 7D12-S85C, the transpeptidase SrtA and the polypeptide GGGGGKN3 are reacted in a certain ratio, and after the reaction is completed, the product 7D12-S85C-(G)5KN3 is obtained after repeated purification.

[0113] ii. Site-directed coupling

[0114] A glucose ligand composed of glucose and a thiol-reactive group maleimide linker is used. When coupling, TCEP is used to reduce the nanobody so that the thiol group on the cysteine is fully exposed. After the liquid is changed to remove TCEP, the glucose ligand is added to couple to the nanobody to obtain a nanobody containing the reaction group. The experimental results of this step can be analyzed by SDS-PAGE and ESI-MS.

[0115] Exemplarily, the compound e of Example 1 is subjected to site-directed coupling as described above to obtain a coupling product, which is named 7D12-Glc.

[0116] FIG. 7 shows a coupling route diagram and a structure diagram of the coupling product 7D12-Glc obtained by site-directed coupling of the nanobody 7D12 with a cysteine mutation S85C reduced by TCEP with the compound e of Example 1 as described above.

[0117] It is determined that one sugar ligand (compound e) is coupled to the coupling product, and the structure is as follows:

[0118] iii. Click chemistry

[0119] DBCO-PEG4-TAMRA is dissolved in PBS buffer (pH 7.4) with the nanobody 7D12-Glc containing an azido group, mixed at a molar ratio of 1:3, and reacted at room temperature for 2 hours. After the reaction, 10kD ultrafiltration tube centrifugation is added for purification to obtain the nanobody TAMRA-7D12-Glc labeled with TAMRA.

[0120] FIG. 8 shows a coupling diagram and a structure diagram of TAMRA-7D12-Glc obtained by click chemistry coupling of 7D12-Glc with the compound DBCO-PEG4-TAMRA.

[0121] Example 3: Chimeric body induces EGFR internalization

[0122] Epidermal Growth Factor Receptor (EGFR) is another important member of the epidermal growth factor receptor family, and is also an important tumor treatment target. In this embodiment, EGFR is selected as the target protein to verify whether the chimer provided by the present application can induce the internalization and degradation of membrane proteins.

[0123] Illustratively, in this embodiment, the A431 cell line with high expression of EGFR is selected for the experiment. The cells are plated one day before administration, and the 7D12 and 7D12-Glc are diluted with complete culture medium and incubated with the cells for 28 h; then flow cytometry is performed.

[0124] Figure 9 shows the flow cytometry results (fluorescence intensity) and quantification (relative mean fluorescence intensity) of the internalization of 7D12 and 7D12-Glc. As can be seen from the figure, 7D12-Glc with a glucose ligand moiety has a stronger internalization ability than 7D12. Thus, it is proved that the chimer provided by the present application has a significant internalization ability.

[0125] Example 4: Receptor-mediated streptavidin (SA) endocytosis and degradation

[0126] In this embodiment, confocal laser scanning microscopy is used to observe whether streptavidin (SA) as an extracellular protein can be taken up by a glucose-labeled biotin compound, glucose-4-ethylene glycol-biotin (Glc-Biotin; purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.), and transported to lysosomes. The inventors predict that glucose-biotin can bind both extracellular SA and cell surface GLUT, thereby triggering GLUT-mediated SA endocytosis and lysosomal transport.

[0127] In this embodiment, human breast cancer cells SKBR3 are cultured with 500 nM of fluorescently labeled streptavidin (SA-647): In the first experiment, the cells are co-cultured with 2 μM of Glc-Biotin (shown as "Bio-Glc2" or "Bio-Glc" in the figure) in the presence or absence of chloroquine (CQ) for 12 h; in the second experiment, the cells are co-cultured with 0, 0.5, 2 and 10 μM of Glc-Biotin, respectively, for 6 h to observe the effect of the dose; in the third experiment, the cells are co-cultured with 2 μM of Glc-Biotin for 0 h, 1.5 h, 6 h and 20 h, respectively, to observe the effect of time.

[0128] The results show that from Figure 10A, it can be seen that Glc-Biotin can induce the internalization of SA-647, and the degradation of Glc-Biotin is mediated by CQ; the NC group is untreated cells. From Figure 10B, it can be seen that Glc-Biotin can induce the internalization of SA-647, and the cellular uptake of SA-647 is dose-dependent. From Figure 10C, it can be seen that Glc-Biotin-mediated SA-647 uptake occurs in a time-dependent manner, and strong internalization begins at 6 hours.

[0129] As known above, glucose (Glc) is combined with biotin (Biotin) to form Glc-Biotin, which retains the structural characteristics of glucose, so that it can be recognized by glucose transporter receptors (GLUT). Glc-Biotin forms a complex with SA-647 through specific binding between biotin and streptavidin. The glucose transporter receptor (GLUT) on the surface of the cell can recognize and bind Glc-Biotin, thereby initiating the transport process, and when the Glc-Biotin complex is transported into the cell, SA-647 also enters the cell and is further processed to the lysosome by endocytosis in the cell. Thus, the experiment of the present embodiment demonstrates the feasibility of promoting the uptake of extracellular proteins by targeting GLUT.

[0130] Example 5: Chimeric body induces extracellular target TNF-α endocytosis and lysosomal targeting

[0131] Tumor necrosis factor-α (TNF-α) is an important inflammatory mediator involved in regulating immune responses and inflammatory processes, and its excessive activation is closely related to the occurrence and development of various autoimmune diseases. Adalimumab blocks the binding of TNF-α to its receptor by binding to TNF-α, thereby inhibiting the inflammatory response mediated by TNF-α. In the present embodiment, TNF-α is selected as the target protein to verify whether the chimeric body can be used for the degradation of extracellular targets.

[0132] In this embodiment, confocal laser scanning microscopy was used to observe the co-localization of TNF-α with the cell nucleus. SKBR3 cell lines were used for the experiment. Adm and Adm-Glc25 were diluted with complete culture medium and co-incubated with AF-647-labeled TNF-α at a final concentration of 50 nM and 25 nM for 24 h. The chloroquine-treated group was first co-incubated with SKBR3 cells at a final concentration of 20 μM for 30 min, then co-incubated with Adm, Adm-Glc25, AF-647-labeled TNF-α at a final concentration of 25 nM, and chloroquine at a final concentration of 20 μM in complete culture medium for another 16 h. Afterwards, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and the cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI). The localization of AF-647-labeled TNF-α (TNF-α-647) in the cell nucleus was observed using confocal laser scanning microscopy, and the results are shown in Figure 8.

[0133] Figure 11 shows, from top to bottom, the nuclear signal, the TNF-α-647 signal, and the combined signal of the two. The NC group represents untreated cells. The signal results show that the Adm-Glc25 group exhibited significantly enhanced purple fluorescence compared to the Adm group, indicating that, under the same incubation time, the chimeric administration resulted in a greater amount of TNF-α entering the cells. This demonstrates that the chimeric pathway promotes the entry of extracellular proteins into intracellular lysosomes: the CQ (chloroquine) group showed significantly enhanced purple fluorescence compared to all groups without CQ, suggesting that the degradation of TNF-α after entry into cells, facilitated by the chimeric pathway, may be achieved through lysosomes.

[0134] Example 6: Chimera internalization via HER2 binding

[0135] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family. Its overexpression is associated with tumor occurrence, development, increased invasiveness, and increased mortality, making it one of the important targets for current cancer therapy. In this embodiment, HER2 was selected as the target to verify whether the chimeric formulation of the present invention has better internalization and lysosomal localization capabilities.

[0136] This example uses SKBR3, 293T cell lines (negative control) for experiment. Site-specific labeling of Tz or Tz-Glc25 is performed with TAMRA. Fluorescently labeled Tz or Tz-Glc25 (100 nM) is incubated with HER2 high expression cell line SKBR3 (200,000 cells / well), 293T cells (200,000 cells / well) for 16 h. Subsequently, lysosomes are stained with LysoTracker Green. After staining, cells are fixed with 4% paraformaldehyde at room temperature, and the nucleus is stained with DAPI. The internalization of Tz or Tz-Glc25 and lysosome localization after incubation with cells are observed by confocal laser scanning microscopy, and the results are shown in Figure 12.

[0137] In Figure 12, 12A (SKBR3 cells) and 12B (293T cells) from left to right are the nucleus signal, TAMRA signal, lysosome signal, and the combined signal of the three; the combined color of TAMRA signal and lysosome is yellow. From the TAMRA signal in 12A, it can be seen that the TAMRA signal after Tz-Glc25 treatment is stronger than that of Tz, indicating that Tz-Glc25 is more internalized into lysosomes. Thus, it is proved that the chimera of the application can be internalized and targeted to lysosomes.

[0138] Example 7: Chimeric body induces HER2 degradation

[0139] This example uses HER2 as a degradation target to verify whether the chimeric body of the application can induce the internalization of HER2 on the surface of tumor cells into the intracellular to be degraded. The expression amount of HER2 on the surface of cell membrane after chimeric body treatment is detected by Western Blot and laser confocal detection, so as to evaluate the ability of chimeric body to induce HER2 degradation.

[0140] 1) Laser confocal experiment to observe HER2 on the surface of cell membrane

[0141] This example uses SKBR3 cell line for experiment. First, Tz or Tz-Glc25 is diluted with complete culture medium to a final concentration of 100 nM and incubated with SKBR3 cells (200,000 cells / well). Subsequently, the cell membrane is stained with a cell membrane staining kit (purchased from Biyun Technology Co., Ltd.). After staining, the cells are fixed with 4% paraformaldehyde at room temperature, and 1% BSA is used to incubate the cells at room temperature for 1 h; the BSA-treated cells are first incubated with the primary antibody (anti-HER2 antibody) at 37°C for 1 h, and then incubated with the fluorescently labeled secondary antibody at room temperature for 30 min, and the nucleus is stained with DAPI. The fluorescence intensity of HER2 on the surface of SKBR3 cells after incubation with Tz or Tz-Glc25 is observed by confocal laser scanning microscopy, and the results are shown in Figure 13.

[0142] Figure 13 is a confocal laser scanning microscope image of cells after incubation with Tz or chimera Tz-Glc25, wherein from left to right are the nuclear signal, cell membrane, cell membrane surface HER2 signal, and the combined signal of the three, and the combined color of cell membrane surface HER2 signal and cell membrane signal is yellow. As can be seen from the cell membrane surface HER2 signal, the cell surface HER2 fluorescence intensity after treatment with chimera Tz-Glc25 is lower than that after treatment with Tz, indicating that the expression of HER2 is less. Thus, it is proved that the chimera of the application can effectively promote the internalization of HER2 and be degraded in the cell.

[0143] 2) Western Blot detection of HER2 expression

[0144] In this example, four HER2 high expression cell lines (SKBR3, SKOV3, BT474, 300,000 cells / well) were selected for experiments, and the cells were plated one day before drug administration. The antibodies or constructed chimeras were diluted with complete culture medium and incubated with the three cell lines for 48 h, respectively; then the cells were lysed with lysis buffer, and the total protein was extracted and quantified. Then the protein was separated by 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer; the protein-containing membrane was blocked in 5% skim milk at room temperature for 1 h, then the milk was removed, and the membrane was incubated with the primary antibody (anti-HER2 antibody) at 4°C overnight. The next day, the membrane was quickly immersed in TBST and washed 3 times, then the secondary antibody was incubated with the membrane at room temperature for 1 h. Finally, the protein was detected and recorded by chemiluminescence reagent and Tanon chemiluminescence instrument, and the protein was quantitatively analyzed by Image J.

[0145] Figure 14 is the immunoblotting result of membrane protein HER2 on the surface of SKBR3 cells after incubation with Tz or different chimeras. As can be seen from the figure, the expression of HER2 is significantly reduced under the treatment of Tz modified with monovalent sugar or multivalent sugar (Tz-Glc2, Tz-Glc15, Tz-Glc25 in the figure), and there is a significant difference compared with Tz. Thus, it is proved that the chimeras carrying glucose ligands have a significant degradation induction effect on HER2, and as shown in the figure, the effect increases with the increase of the number of glucose ligands, thus indicating that the chimeras of the application can promote the degradation of cell membrane surface antigen HER2.

[0146] Figure 15 shows the results of western blotting of surface membrane protein HER2 after incubation of Tz or chimera Tz-Glc25 with different cells. As can be seen from the figure, in SKBR3 cells, BT474 cells, SKOV3 cells, NCI-N87 cells, the expression of HER2 was significantly reduced after treatment with Tz-Glc25, and there was a significant difference compared with Tz. This proves that the chimera of the application can promote the degradation of different cell membrane surface antigen HER2.

[0147] 3) Time course study of HER2 degradation

[0148] In this example, the time course of HER2 degradation induced by the chimera was studied by evaluating the change in the expression of HER2 in SKBR3 cells treated with Tz or chimera Tz-Glc25 over time. The cell treatment process of Tz or chimera Tz-Glc25 in this example is described in the above "2) Western Blot Detection of HER2 Expression" section, and the incubation time with the cells is shown in Figure 13. The content of HER2 was also detected by western blot.

[0149] Figure 16 shows the results of western blotting of surface membrane protein HER2 after incubation of Tz or chimera Tz-Glc25 with cells for different times and the quantification statistics. As shown in the figure, the expression of HER2 was significantly reduced after 12 hours, proving that the chimera can achieve most of the degradation of HER2 at this time. This data further proves that the chimera of the application can effectively achieve the degradation of the target molecule.

[0150] 4) Dose effect study of HER2 degradation

[0151] In this example, the dose effect of HER2 degradation induced by the chimera was studied by evaluating the change in the expression of HER2 in SKBR3 cells treated with different concentrations of Tz-Glc25. The cell treatment process of Tz or chimera Tz-Glc25 in this example is described in the above "2) Western Blot Detection of HER2 Expression" section, and the concentration of Tz or chimera Tz-Glc25 in the incubation system is shown in Figure 17. The content of HER2 was also detected by western blot.

[0152] Figure 17A shows the results of western blotting of surface membrane protein HER2 after incubation of Tz or chimera Tz-Glc25 with cells at different concentrations and the quantification statistics. As shown in the figure, the expression of HER2 was significantly reduced after 48 hours of treatment of cells with 50 nM Tz-Glc25, proving that the chimera can achieve most of the degradation of HER2 at this concentration. This data further proves that the chimera of the application can effectively achieve the degradation of the target molecule.

[0153] Figure 17B in Figure 17 shows the results of the immunoblotting of HER2 after incubation of the chimera Tz-Glc25 with cells at a high dose concentration. As shown, the expression of HER2 was still significantly reduced after 48h of treatment of cells with 1000nM of Tz-Glc25, demonstrating that the chimera at this dose can still achieve most of the degradation of HER2. This data thus demonstrates that there is no hook effect in the function of the chimeras of the present application to achieve degradation of the target molecule.

[0154] Example 8: Mechanism study of inducing protein degradation

[0155] This example is mainly to explore the specific mechanism of protein degradation by Western Blot. In this example, lysosome inhibitor chloroquine, proteasome inhibitor MG132 are selected to observe whether the expression of HER2 changes after inhibiting the function of lysosome or proteasome; phloretin and phlorizin, glucose transport receptor, are selected to observe whether the expression of HER2 changes after inhibiting the function of glucose transport receptor; chlorpromazine, amiloride, clathrin inhibitor, are selected to observe whether the expression of HER2 changes after inhibiting clathrin-mediated endocytosis and pinocytosis, to explore the possible mechanism of HER2 degradation.

[0156] This example is carried out in SKBR3 cell line. First, chloroquine (20μM), MG132 (20μM), phloretin (1mM), phlorizin (1mM), chlorpromazine (10μg / mL), amiloride (10μg / mL) are respectively incubated with cells for 30min, then the antibody or the chimera is incubated with cells at a final concentration of 100nM, and the inhibitors chloroquine (20μM), MG132 (20μM), phloretin (1mM), phlorizin (1mM), chlorpromazine (10μg / mL), amiloride (10μg / mL) are incubated with cells for 48h; then the cells are lysed with lysis solution, the total protein of the cells is extracted and quantified by BCA kit. Then the protein is separated by 10% SDS-PAGE gel, and transferred to polyvinylidene fluoride (PVDF) membrane by electrotransfer; the membrane with protein is blocked in 5% skim milk at room temperature for 1h, then the milk is removed, and the membrane is incubated with the primary antibody (anti-HER2 antibody) at 4°C overnight. The next day, the membrane is quickly immersed in TBST and washed 3 times, then the secondary antibody is incubated with the membrane at room temperature for 1h. Finally, the protein is detected and recorded by chemiluminescence reagent and Tanon chemiluminescence instrument, and the protein is quantitatively analyzed by Image J.

[0157] Figure 18 shows the results of immunoblotting of HER2 after co-incubation of Tz or chimera Tz-Glc25 with inhibitors in cells. It is shown that HER2 expression is higher in the presence of chloroquine than in the control (PBS), i.e. less degradation of HER2, while MG132 does not show a significant effect, indicating that lysosomes are involved in the degradation of HER2 mediated by the chimera; in the presence of any GLUT inhibitor, the expression of HER2 is relatively high, i.e. the degradation is significantly inhibited, indicating that HER2 is degraded in a GLUT-dependent manner; finally, the possible endocytosis mechanism of the degradation of HER2 mediated by the chimera is studied, and the results show that the expression of HER2 does not change significantly after the addition of chlorpromazine, i.e. no significant degradation, while the expression of HER2 is significantly reduced after the addition of amiloride, indicating that even after the addition of amiloride, the protein is still significantly degraded, which indicates that the chimera of the application mainly induces the degradation of HER2 through the clathrin-mediated endocytosis mechanism.

[0158] In summary, the results show that the chimera of the application can trigger the degradation of HER2 in a lysosome- and GLUT-dependent manner, and clathrin-mediated endocytosis may play a key role in the internalization and lysosomal transport of the target protein.

[0159] Example 9: Anti-tumor activity in vitro

[0160] 1) EdU experiment to analyze the effect of the chimera on cell proliferation

[0161] Detection of cell proliferation capacity is a basic method for evaluating cell viability, anti-tumor effect of drugs, etc. 5-ethynyl-2'-deoxyuridine (5-ethynyl-2'-deoxyuridine, abbreviated as EdU) is a new type of thymidine analog that can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis, and is commonly used for cell proliferation capacity detection.

[0162] This example uses SKBR3 cells, Tz or Tz-Glc25 is diluted with complete medium to a final concentration of 100 nM and incubated with SKBR3 cells. Prepare 2X EdU working solution (purchased from Biyun Technology Co., Ltd.) (20 mM), and add an equal volume of 37°C preheated EdU working solution to the 6-well plate. Continue to incubate the cells for 2 h, and after the EdU labeling of the cells is complete, remove the culture medium and add 1 mL of 4% paraformaldehyde for 15 min of room temperature fixation. Remove the fixation solution and wash the cells with 1 mL of washing solution per well for 3 min each time. Remove the washing solution and permeabilize the cells with 1 mL of PBS containing 0.3% Triton X-100 per well for 10 min at room temperature. Remove the permeabilization solution and wash the cells with 1 mL of washing solution per well for 3 min each time. Prepare Click reaction solution and add 0.5 mL of Click reaction solution per well, and incubate at room temperature for 30 min in the dark, then wash with washing solution for 3 min each time. Use DAPI to stain the cell nuclei for 15 min, and then observe under a fluorescence microscope.

[0163] Figure 19 is a confocal laser scanning microscope image of cells after incubation with Tz or chimeric Tz-Glc25 and SKBR3 cells, in which the nuclear signal, EdU signal, and combined signal of the two are shown from left to right. The stronger the EdU signal, the more normal cells there are; and as can be seen from the figure, the EdU signal of Tz-Glc25 is weaker than that of Tz. This shows that the glucose ligand-coupled chimeric body of the application can significantly inhibit the proliferation of tumor cells, and therefore has in vitro anti-tumor activity.

[0164] 2) Cell colony formation experiment to analyze the effect of chimeric bodies on cell proliferation

[0165] Cell colony formation experiments can directly test the ability of a reagent to inhibit tumor cell proliferation. Crystal violet is a basic dye that can bind to DNA in the cell nucleus, producing cell nucleus staining for observation, and is commonly used in this experiment.

[0166] This example uses SKBR3 cells, and the cells (1000 cells per well, 12-well plate) are plated 3 days before the experiment. Dilute Tz or Tz-Glc25 with complete medium to different concentrations, then incubate the cells with 1 mL of complete medium containing different concentrations of Tz or Tz-Glc25 at 37°C for 15 days or more. Stop the culture and perform staining after obvious colony formation. Before staining, wash twice with PBS and fix with 4% paraformaldehyde for 15 min. After washing twice with PBS, add 1 mL of crystal violet reagent and incubate for about 20 min, then wash slowly with water until the background is clean, and then take a photo using a camera.

[0167] Figure 20 shows the cell clone staining results after Tz or chimeric Tz-Glc25 was co-incubated with SKBR3 cells. It can be found from the figure that the cell clone treated with Tz-Glc25 is less than that treated with Tz, so it is known that Tz-Glc25 has a stronger tumor cell proliferation inhibition effect, and the effect increases with the increase of the concentration. Therefore, it is shown that the glucose ligand-coupled chimeric body of the application can significantly inhibit the proliferation of tumor cells, and therefore has in vitro anti-tumor activity.

[0168] 3) CCK-8 experiment analyzes the effect of chimeric body on cell proliferation

[0169] CCK-8 reagent contains WST-8, which is reduced to highly water-soluble yellow formazan dye under the action of electron carrier (1-MethoxyPMS) in the mitochondria of cells by dehydrogenase in cells. The amount of formazan produced is proportional to the number of living cells, so this property can be used to directly analyze cell proliferation and toxicity.

[0170] In this embodiment, SKBR3 cells are used, and the cells in good growth state are plated in a 96-well plate (8000 cells / well). Tz or Tz-Glc25 is diluted to different concentrations using complete culture medium; after the cells are attached, different concentrations of Tz or Tz-Glc25 are added for co-incubation and culture, and after the culture is completed, the culture medium is aspirated and washed with PBS, then 1:10 mixed CCK-8 reagent and culture medium mixture is added, incubated at 37°C for 1-4h, and detected when the OD value reaches 1.0-1.5, and the detection wavelength is 450nm.

[0171] Figure 21A is a cell survival rate line chart after Tz or Tz-Glc25 of different concentrations was co-incubated with SKBR3 cells for 96h. It can be found from the figure that the cell survival rate treated with Tz-Glc25 is significantly lower than that treated with Tz when co-incubated for the same time, so it is known that Tz-Glc25 has a stronger tumor cell proliferation inhibition effect, and the effect increases with the increase of the concentration.

[0172] Figure 21B is a cell survival rate line chart after 10nM Tz or Tz-Glc25 was co-incubated with SKBR3 cells for different times. It can be found from the figure that the cell survival rate treated with Tz-Glc25 is significantly lower than that treated with Tz when the co-incubation time is prolonged, so it is known that Tz-Glc25 has a stronger tumor cell proliferation inhibition effect.

[0173] Figure 21, 21C is a survival rate fold line graph of 50nM of Tz or Tz-Glc25 co-incubated with SKBR3 cells for different time. It can be found from the graph that compared with Tz, the cell survival rate using higher concentration of Tz-Glc25 is still significantly lower as the co-incubation time is prolonged, thus it can be known that Tz-Glc25 has stronger inhibitory effect on tumor cell proliferation.

[0174] Therefore, it can be illustrated that the glucose ligand conjugated chimera of the application can significantly inhibit the proliferation of tumor cells, thus having in vitro anti-tumor activity.

[0175] Example 10: In vivo tumor targeting ability

[0176] The experimental animals used in this example are 9 SPF BALB / c female nude mice purchased from Beijing Vantoll Life Sciences, the mice are 6-8 weeks old and weigh 20-25g.

[0177] Grouping and modeling: after adaptive feeding for 1 week, the mice in each group were randomly divided into Tz group and Tz-Glc25 group, 6 mice in each group. 1x10 7 NCI-N87 cell suspension was inoculated on the back of the nude mice, the cell suspension inoculation amount was 0.2mL, and the tumor was considered to be successfully modeled when the tumor grew to 100-150mm 3 .

[0178] Dosing method: according to the grouping, each mouse was injected with 3mg / kg of drug (Tz, Tz-Glc25 was labeled with TAMRA) via the tail vein, and the model group was injected with PBS.

[0179] Specimen collection and detection: the dosing time was recorded as 0h, at 7h, 24h, 48h, 72h and 96h after the dosing, the mice were photographed using the live imaging system, and the mice were sacrificed at 96h and the organs were dissected for ex vivo photography.

[0180] Figure 22, 22A is a live imaging photograph of the mice at 0h, 7h, 24h, 48h, 72h and 96h. The fluorescence enrichment results show that compared with Tz, Tz-Glc25 has higher uptake and localization level in the tumor, and Tz-Glc25 shows significantly stronger fluorescence signal at 96h after injection, and no rapid or large amount of clearance is observed, which indicates that Tz-Glc25 has metabolic stability.

[0181] Figure 22, 22B is a photograph of the tumor tissue after the mouse is taken. The fluorescence enrichment results show that in the main organs and tumor tissues harvested at 96h after injection, Tz-Glc25 is mainly accumulated in the tumor.

[0182] These data demonstrate that the chimeras of the present application can act in a significantly tumor-selective manner, overcoming toxic side effects and increasing the efficacy of the drug.

[0183] Example 11 Construction of chimeric-based ADCs

[0184] In this example, different types and molecular weight ranges of molecules (monoclonal antibodies, nanobodies), glucose ligands, linkers and toxic small molecules were exemplarily selected for conjugation to construct lysosome-targeting chimeric-based ADCs of the present application.

[0185] As mentioned above, the glucose ligands of the present application can be chimerized on antibodies through several different conjugation means in Example 2; the resulting lysosome-targeting chimeras can be further conjugated with toxic small molecules via linkers to obtain ADCs. In addition, the present application provides a route to introduce toxic small molecules into antibodies according to the Thiomab technology of site-specific conjugation first, and then introduce glucose ligands by a two-step method according to the lysine conjugation procedure to obtain chimeric-based ADCs.

[0186] FIG. 23 is an exemplary construction flowchart of chimeric-based ADCs of the present application, in which compound d is exemplified as a glucose ligand, and non-cleavable linker maleimidocaproyl (hereinafter referred to as mc) and tubulin inhibitor monomethyl auristatin F (hereinafter referred to as MMAF) are exemplified as a linker and a toxic small molecule, respectively; m represents the number of sugar ligands, wherein m = 1-50.

[0187] According to this exemplary flowchart, exemplified with antibody Tz, MMAF groups were introduced into Tz to obtain MMAF-Tz according to the Thiomab technology of site-specific conjugation using the sequence of thiol conjugation followed by lysine conjugation, and then glucose ligands (compound d, FIG. 2) were introduced by a two-step method according to the lysine conjugation procedure to obtain MMAF-Tz-Glc25. The prepared MMAF-Tz-Glc25 was added to an ultrafiltration tube (Amicon® Ultra centrifugal ultrafiltration tube, 30 kDa MWCO) and diluted with an appropriate amount of buffer (PBS, pH 7.5) for centrifugal concentration and buffer exchange. After purification, structure characterization was performed by SDS-PAGE, HPLC and MS, respectively.

[0188] FIG. 24 shows the SDS-PAGE (24A), HPLC (24B) and MS (24C) characterization results of the construction process of the conjugation product MMAF-Tz-Glc25.

[0189] The results show that, as shown in 24A, the light chain and heavy chain of MMAF-Tz-Glc25 are obviously up-shifted compared with MMAF-Tz after introducing the glucose ligand, which indicates that the light chain and heavy chain of MMAF-Tz are coupled with the glucose ligand; as shown in 34B, the retention time of MMAF-Tz-Glc25 is shortened compared with MMAF-Tz after introducing the glucose ligand, and no obvious aggregation peak is observed, which indicates that the glucose ligand is coupled to MMAF-Tz, the molecular weight is increased, and no obvious aggregation occurs; as shown in 24C, the molecular weight of MMAF-Tz-Glc25 is increased compared with MMAF-Tz after introducing the glucose ligand, and the number of glucose ligand coupling is calculated to be 25.

[0190] The structure of MMAF-Tz-Glc25 is as follows:

[0191] In addition, the MMAF in the above process is replaced by TAMRA to obtain TAMRA-labeled Tz-Glc25.

[0192] Example 12: Affinity of ADC to HER2

[0193] In this embodiment, the affinity of MMAF-Tz and MMAF-Tz-Glc25 to HER2 target molecules is detected by ELISA method.

[0194] After diluting the HER2 antigen to an appropriate concentration, it is coated on a microplate and incubated overnight; then, different concentrations (0.001-1000 nM) of MMAF-Tz and MMAF-Tz-Glc25 are added to the microplate, and incubated at room temperature; after incubation, washing is performed, then HRP-labeled goat anti-human IgG Fab fragment antibody is added, incubation is performed again, then color developing reagent is added, and the absorbance value is determined by an enzyme marker.

[0195] Figure 25 shows the dose-dependent binding curves of MMAF-Tz and MMAF-Tz-Glc25 to HER2. The results show that the corresponding maximum binding rates and half effective concentrations (EC50) of the two are almost identical, indicating that there is no significant difference in the affinity of the two reagents to HER2 in the tested concentration range, suggesting that the modification of Glc25 does not significantly affect the recognition and binding of the chimeric ADC to the HER2 target molecule.

[0196] Therefore, it is proved that the GLUT-based lysosome targeting chimeric body of the present application can maintain high affinity binding ability to target molecules after being coupled with a cytotoxic drug, which lays a foundation for subsequent targeting and effectiveness in tumor treatment.

[0197] Example 13: In vitro anti-tumor activity of ADC

[0198] In this embodiment, different conjugation methods were used to construct ADCs based on monoclonal antibodies and antigen-binding fragments, and loaded with glucose ligands, respectively, to explore the in vitro anti-tumor activity of the ADCs constructed by chimeras.

[0199] 1) In vitro anti-tumor activity of ADCs based on antigen-binding fragments

[0200] In this embodiment, A431 cells were used, and the well-grown cells were plated in a 96-well plate (7000 / well). 7D12 conjugated with MMAF (7D12-MMAF) or 7D12-Glc (7D12-MMAF-Glc) was diluted to different concentrations using complete culture medium; after the cells adhered, different concentrations of 7D12-MMAF or 7D12-MMAF-Glc were added for co-incubation and culture, after the culture was completed, the culture medium was aspirated, washed with PBS, and 1:10 mixed CCK-8 reagent and culture medium mixture was added, incubated at 37°C for 1-4h, and detected when the OD value reached 1.0-1.5, with a detection wavelength of 450nm.

[0201] Figure 26 is a line graph of cell survival rate after 7D12-MMAF or 7D12-MMAF-Glc of different concentrations was co-incubated with A431 cells for 96h. It can be found from the figure that compared with 7D12-MMAF, the cell survival rate of 7D12-MMAF-Glc treated cells was significantly lower under the same co-incubation time, indicating that 7D12-MMAF-Glc had a stronger tumor cell proliferation inhibition effect. Therefore, it can be shown that the ADC based on the glucose ligand conjugated chimeras of the application can significantly inhibit the proliferation of tumor cells, and thus has in vitro anti-tumor activity.

[0202] 2) In vitro anti-tumor activity of ADCs based on monoclonal antibodies

[0203] a) Anti-tumor activity in 2D cancer cell models with different HER2 expression levels

[0204] In this embodiment, HER2 high expression (SKOV3 cells, NCI-N87 cells), HER2 medium expression (5637 cells) and HER2 low expression (MDA-MB-231 cells) cancer cells were used, and the well-grown cells were plated in a 96-well plate (5000 / well). MMAF-Tz or MMAF-Tz-Glc25 was diluted to different concentrations using complete culture medium; after the cells adhered, different concentrations of MMAF-Tz or MMAF-Tz-Glc25 were added for co-incubation and culture, after the culture was completed, the culture medium was aspirated, washed with PBS, and 1:10 mixed CCK-8 reagent and culture medium mixture was added, incubated at 37°C for 1-4h, and detected when the OD value reached 1.0-1.5, with a detection wavelength of 450nm.

[0205] Fig. 27, 27A-27D are cell survival rate line graphs of SKOV3 cells (27A), NCI-N87 cells (27B), 5637 cells (27C), MDA-MB-231 cells (27D) after incubation with different concentrations of MMAF-Tz or MMAF-Tz-Glc25 for 96h, respectively. It can be found from the figure that for HER2 high expression SKOV3 cells and NCI-N87 cells and HER2 medium expression 5637 cells, the cell survival rate is significantly lower when MMAF-Tz-Glc25 is used than when MMAF-Tz is used, so it can be known that MMAF-Tz-Glc25 has a stronger tumor cell proliferation inhibition effect on HER2 high expression and medium expression tumor cells; and for HER2 low expression MDA-MB-231 cells, MMAF-Tz-Glc25 and MMAF-Tz have no inhibition and killing effect.

[0206] Therefore, it is proved that the chimera-based ADC of the present application can significantly inhibit the proliferation of tumor cells in HER2 high expression and HER2 medium expression cancer cell lines, and therefore has in vitro anti-tumor activity.

[0207] Fig. 28, 28A is a cell clone crystal violet staining graph of HER2 high expression SKBR3 cells after incubation with 0.5nM concentration of MMAF-Tz or MMAF-Tz-Glc25 for 15 days, from which it can be found that the cell clones treated with MMAF-Tz-Glc25 are less than those treated with MMAF-Tz, so it can be known that it has a stronger tumor cell proliferation inhibition effect.

[0208] Fig. 28, 28B is a cell clone crystal violet staining graph of HER2 medium expression 5637 cells after incubation with different concentrations (50nM, 10nM, 1nM) of MMAF-Tz or MMAF-Tz-Glc25 for 15 days, from which it can be found that the cell clones treated with MMAF-Tz-Glc25 are less than those treated with MMAF-Tz, so it has a stronger tumor cell proliferation inhibition effect.

[0209] Therefore, it can be explained that the ADC based on the glucose ligand-coupled chimera of the present application can significantly inhibit the proliferation of tumor cells, and therefore has in vitro anti-tumor activity.

[0210] b) Anti-tumor activity in HER2 high expression level breast cancer organoid model

[0211] This example evaluates the anti-tumor activity of MMAF-Tz and MMAF-Tz-Glc25 by a HER2 high expression breast cancer organoid model. The breast cancer organoid is purchased from Guangzhou Jingke Medical Inspection Co., Ltd.

[0212] The breast cancer organoids were randomly divided into three groups: one group was administered with MMAF-Tz treatment, one group was administered with MMAF-Tz-Glc25 treatment, and the other group was used as a blank control (complete medium was added without drug treatment). The concentration of each group was 2 μM, and the incubation time was 120 hours. Then, the activity of the organoids was detected by the LDH kit to evaluate the killing effect of the drugs on tumor cells; at the same time, the morphological changes and proliferation of the organoids were observed by using the high-content imaging system.

[0213] Figure 29A is a statistical diagram of the size of the organoids after MMAF-Tz or MMAF-Tz-Glc25 was co-incubated with breast cancer organoids for 120 hours. It can be found from the figure that, compared with MMAF-Tz, MMAF-Tz-Glc25 has a stronger inhibitory effect on the growth and proliferation of organoids.

[0214] Figure 29B is a statistical diagram of the survival rate of organoids by comparing different drug groups through LDH experiments after MMAF-Tz or MMAF-Tz-Glc25 was co-incubated with breast cancer organoids for 120 hours. It can be found from the figure that, compared with MMAF-Tz, MMAF-Tz-Glc25 has a stronger inhibitory activity on the growth and killing of organoids.

[0215] Therefore, it is proved that the ADC (MMAF-Tz-Glc25) based on the GLUT-targeted lysosomal chimera of the present application exhibits superior anti-tumor effect in the breast cancer organoid model, which may be related to the efficient internalization and lysosome-targeted degradation mechanism mediated by GLUT.

[0216] The above description of the specific embodiments of the present application does not limit the present application, and those skilled in the art can make various changes or modifications to the present application without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of the appended claims of the present application.

Claims

1. A lysosome-targeting chimera based on glucose transporter (GLUT) comprising two moieties: (1) a first moiety capable of specifically binding to a target molecule and is one or more selected from the group consisting of a polypeptide, a protein, a nucleic acid, a nanoparticle and a small molecule compound; (2) a second moiety capable of binding to a glucose transporter (GLUT) or a substrate transported by GLUT.

2. The lysosome-targeting chimera of claim 1, wherein, the first moiety is a protein; preferably, the first moiety is an antibody or an antigen-binding fragment thereof capable of specifically binding to a target molecule; preferably, the first moiety is an antibody and is one or more selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and the like; or, the first moiety is an antigen-binding fragment of an antibody and is one or more selected from the group consisting of a Fab, a Fab', a F(ab')2, a Fv, a dsFv, a scFv, a sc(Fv)2, and a VHH.

3. The lysosome-targeting chimera of claim 1 or 2, wherein, the target molecule is located on the cell surface or extracellularly; preferably, the target molecule is a cell surface protein, a ligand of a receptor, a cytokine, a hormone, a secreted protein, an antibody, a protein carrier, an enzyme; further preferably, the target molecule is located on the cell surface and includes but is not limited to a growth factor receptor, an immune checkpoint molecule, a leukocyte differentiation antigen; or, the target molecule is located extracellularly and includes but is not limited to a growth factor, a cytokine, a chemokine and a secreted protein, a protein hormone, an antibody, a protein toxin, a virus and an infectious particle.

4. The lysosome-targeting chimera of any one of claims 1 to 3, wherein, the first moiety comprises a reactive group selected from one or more of an amino group, a carboxyl group, a thiol group, a halogen group and a bio-orthogonal group; wherein the bio-orthogonal group is selected from one or more of an azido group, an alkyne group, an aldehyde group, a ketone group and a fluoro-sulfonate group.

5. The lysosome-targeting chimera of any one of claims 1 to 4, wherein, the second moiety comprises one or more selected from the group consisting of glucose, fructose and derivatives thereof; preferably, the second moiety can be a monovalent ligand, a bivalent ligand, a trivalent ligand or even a ligand of more valence.

6. The lysosome-targeting chimera of any one of claims 1 to 5, wherein, The structure of the second part is shown in any one of Formula I, Formula II, Formula III, Formula IV, and Formula V: Formula II (wherein Linker1, Linker2, and Linker4 are the same or different in structure) Formula III (structure of Linker1, Linker2, Linker3 and Linker4 is the same or different) wherein n = 1-999, preferably n = 1-500, more preferably n = 1-200, further preferably n = 1-50, particularly preferably n = 1-20, for example n = 1-10; in formula I to V, X is -O- or -NH-; R is an amino group, a carboxyl group, a maleimide group, an azido group, an alkyne group, an aldehyde group, a ketone group, a fluoro-sulfonate or a halogen group; In Formulae I to V, each Linker is a cleavable or non-cleavable linker; preferably, each Linker is independently selected from one or more of: a polypeptide, an oligosaccharide, -(CH2) n - -(CH2CH20) n - -OCH2(CH2) n CH20-, -CONH-, -Lys(N3)-, Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Phe-Gly, Val-Cit-PABC, Val-Ala-PABC, Val-Lys(Ac)-PABC, Phe-Lys-PABC, Phe-Lys(Ac)-PABC, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PABC, Ala-PABC, PABC, triazole, piperazine, piperidine.

7. [Amended according to Rule 26 20.10.2025] The lysosome-targeting chimera according to any one of claims 1 to 6, characterized in that, The second part is a monovalent ligand, having a structure according to formula e or g, wherein n = 1-999, preferably n = 1-500, more preferably n = 1-200, further preferably n = 1-50, particularly preferably n = 1-20, for example n = 1-10: Alternatively, the second moiety is a trivalent ligand having the structure of formula I, wherein n = 1 to 999, preferably n = 1 to 500, more preferably n = 1 to 200, further preferably n = 1 to 50, particularly preferably n = 1 to 20, for example n = 1 to 10: Alternatively, the second moiety is a multivalent ligand, having the structure of formula j, wherein n = 1-999, preferably n = 1-500, more preferably n = 1-200, further preferably n = 1-50, particularly preferably n = 1-20, for example n = 1-10:

8. The lysosome-targeting chimera of any one of claims 1 to 7, wherein, in the lysosome-targeting chimera, the first moiety is directly or indirectly linked to the second moiety via its reactive group; Preferably, the structure of the chimera is as shown in formula VI: in formula VI, the three-dimensional structure on the right side is the first moiety; further, the first moiety comprises a functional group and the functional group forms a reactive pair with the R group; preferably, the functional group includes but is not limited to an amino group, a carboxyl group, a thiol group; R, Linker and the second moiety ligand are derived from formula I to V as defined in claim 6 or from formula e, formula g, formula i or formula j as defined in claim 7.

9. The lysosome-targeting chimera of any one of claims 1 to 8, wherein, In case the first moiety is a monoclonal antibody, the lysosome targeting chimera has the following structure: wherein a, b = 1-999, preferably a, b = 1-500, more preferably a, b = 1-200, further preferably a, b = 1-50, particularly preferably a, b = 1-20, for example a, b = 1-10 (e.g. a, b = 150, 100, 80, 40, 30, 15, etc.); m = 1-50, preferably m = 1-25, for example m = 1-10; Preferably, when a = 8, b = 1, m = 25, the lysosome-targeting chimera provided by the application can have the following structure: Alternatively, when the first moiety is an antigen binding fragment (VHH), the lysosome targeting chimera has the structure shown below:

10. Use of the lysosome targeting chimera of any one of claims 1 to 9 for the manufacture of a medicament for the treatment of a disease selected from one or more of cancer, inflammation related disease, immune related disease, viral infection, metabolic disease and neurodegenerative disease.

11. Use according to claim 10, characterized in that, The disease is selected from one or more of cancer, inflammation related disease, immune related disease, viral infection, metabolic disease and neurodegenerative disease; Preferably, the disease is an inflammation related disease, for example rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, ulcerative colitis, psoriatic arthritis; Preferably, the disease is a cancer, for example breast cancer, ovarian cancer, breast ductal carcinoma, gastric cancer, epidermal carcinoma, bladder cancer; Preferably, the viral infection is for example Epstein-Barr virus infection, SARS-CoV-2 infection, influenza virus infection, human papillomavirus infection, herpes virus infection; Preferably, the metabolic disease is for example non-alcoholic fatty liver disease / hepatitis, diabetes, hyperbilirubinemia, bone metabolism disease, cardiovascular disease; Preferably, the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, frontotemporal dementia.

12. Use of the lysosome targeting chimera of any one of claims 1 to 9 for the manufacture of an antibody conjugate.

13. An antibody conjugate comprising the lysosome targeting chimera of any one of claims 1 to 9 and a cytotoxic drug.

14. The antibody-drug conjugate of claim 13, wherein, The cytotoxic drug is selected from a drug or a prodrug of the class of microtubulin inhibitors, a drug or a prodrug of the class of DNA damaging agents, a drug or a prodrug of the class of topoisomerase inhibitors; Preferably, the microtubulin inhibitor is selected from any one of auristatin or auristatin derivatives and analogs, maytansinoid or maytansinoid derivatives and analogs, paclitaxel or paclitaxel derivatives and analogs, vinca-alkaloid or vinca-alkaloid derivatives and analogs, cryptophycin or cryptophycin derivatives and analogs, tubulysin or tubulysin derivatives and analogs. Preferably, the DNA damaging agent is selected from any one of: doxorubicin or doxorubicin derivatives and analogues, calicheamicin or calicheamicin derivatives and analogues, pyrrolobenzodiazepines (PBDs) or PBD derivatives and analogues, duocarmycin or duocarmycin derivatives and analogues. Preferably, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor, for example a camptothecin or a camptothecin derivative and analogue, for example Exatecan, Belotecan, SN-38, Topotecan, Irinotecan (cpt-11), Deruxtecan, Dxd, etc.

15. A method of treating a disease, the method comprising administering to a subject in need thereof the lysosome targeting chimera of any one of claims 1 to 9 or the antibody conjugate drug of claim 13 or 14; The subject is a mammal; preferably, the subject is a human.

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