A targeted antibody-polyethylene glycol-siRNA drug conjugate

Abstract

This invention discloses a targeted antibody-siRNA conjugate drug, its preparation method, and its application. The ligand drug conjugate has the structure shown in general formula (I). Furthermore, this invention uses a biocompatible polyethylene glycol derivative as a linker to specifically target and deliver a specific CFD-siRNA by utilizing the binding of the antibody to the antigen. It has high biocompatibility, low toxicity, and can effectively inhibit CFD gene expression, providing a new option for the prevention or treatment of CFD-related diseases (I).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antibody-drug conjugate, its preparation method, and its application. Background Technology

[0002] Complement, first discovered by Jules Bordet, is a heat-labile component in normal plasma that plays an opsonizing and bactericidal role. Complement factor D (CFD), also known as factor B convertase, is a component of the complement system and participates in the activation of the complement alternative pathway. Its molecular structure is a single polypeptide chain, and its concentration in plasma is very low. In plasma, factor D exists only in its activated form. This factor D can cleave factor B in the C3bBb complex to form C3bBb, the C3 convertase of the alternative pathway. Activation of the complement alternative pathway plays a crucial anti-infective role in the early stages of infection, killing invading pathogens before corresponding antibodies are produced. Therefore, factor D is a suitable disease-suppressing target in activating the complement alternative pathway.

[0003] Complement dysfunction or overactivation has been linked to certain autoimmune diseases, inflammatory and neurodegenerative diseases, as well as ischemia-reperfusion injury and cancer. For example, activation of the alternative pathway of the complement cascade contributes to the production of C3a and C5a (both potent anaphylatoxins), which also play a role in many inflammatory diseases. Therefore, in some cases, it is desirable to reduce the response of the complement pathway, including the alternative complement pathway.

[0004] Downregulating complement activation can effectively treat several conditions, including systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiopulmonary bypass surgery and hemodialysis, ultrafiltration rejection in organ transplantation, myocardial infarction, tissue damage caused by ischemia-reperfusion injury, and adult respiratory distress syndrome. Other inflammatory conditions and autoimmune diseases are also closely related to complement activation, including heat injury, severe asthma, anaphylactic shock, enteritis, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, psoriasis, dermatomyositis, membranous proliferative glomerulonephritis, and Sjögren's syndrome.

[0005] RNA interference (RNAi) can effectively suppress the expression of CFD genes. RNA interference is a novel gene silencing technology developed in recent years that can specifically inhibit gene expression in mammalian cells. siRNA is the effector molecule of RNAi. However, naked siRNA is unstable in vivo and easily degraded by various enzymes, greatly limiting its application. Furthermore, because naked siRNA carries a strong negative charge, it cannot cross the cell membrane to enter the cytoplasm. Therefore, it is necessary to utilize vectors with good delivery efficiency to more effectively deliver siRNA to appropriate sites within cells or organisms to realize its therapeutic potential. Currently, there are many challenges in delivering siRNA to elicit the desired response in biological systems.

[0006] In existing technologies, cationic liposomes are commonly used as siRNA delivery vectors. The principle is that the positive charge on the surface of the cationic liposome interacts with the phosphate group of nucleic acid molecules via electrostatic attraction, encapsulating the nucleic acid molecules to form a nucleic acid-lipid complex. This complex can be adsorbed by the negatively charged cell membrane, and then delivered into the cell through membrane fusion or endocytosis to exert its effects. However, liposomes themselves participate in cellular physiological activities, causing upregulation or downregulation of gene expression, such as participating in the regulation of the PKC (protein kinase C) pathway, inhibiting ATPase activity, interacting with the mitochondrial membrane, and causing off-target effects when transfecting siRNA. The magnitude of cytotoxicity often reflects the magnitude of its impact on cellular physiological activities, and these effects of liposomes are the root cause of cytotoxicity. Furthermore, the positive charge on the surface of liposomes can activate the complement system, promoting macrophage phagocytosis and subsequent clearance, resulting in a short half-life of liposomes in the bloodstream, making them ineffective in delivering to target tissues. All of these factors make them unsuitable for clinical application.

[0007] Antibody-drug conjugates (ADCs) are a novel class of targeted drugs that effectively deliver small-molecule drugs to their target sites, avoiding many of the problems associated with using cationic liposomes as carriers. ADCs generally consist of three parts: an antibody or antibody-like ligand, a small-molecule drug, and a linker that conjugates the ligand and drug. ADCs utilize the specific recognition of antigens by antibodies, entering cells through endocytosis, thereby transporting drug molecules to the target site and effectively releasing the drug molecules to achieve therapeutic purposes.

[0008] In August 2011, the U.S. Food and Drug Administration (FDA) approved Adecteis, an ADC drug developed by Seattle Genetics, for the treatment of Hodgkin's lymphoma and relapsed large cell lymphoma (ALCL). TM The safety and efficacy of these drugs have been proven through market launch and clinical application.

[0009] Polyethylene glycol (PEG) is a versatile polyether polymer. This polymer exhibits excellent biocompatibility, dissolving in tissue fluid within the body and being rapidly excreted without any toxic side effects. Currently, PEG derivatives are widely used to bind with proteins, peptides, and other therapeutic agents to prolong their physiological half-life and reduce their immunogenicity and toxicity. In clinical use, PEG and its derivatives have been widely applied as carriers in many pharmaceutical formulations.

[0010] However, there is limited research on antibody-polyethylene glycol-siRNA drugs for the prevention or treatment of CFD-related diseases in the current technology, and the technology still needs further optimization. Summary of the Invention

[0011] To address the technical problems existing in the background art, the present invention provides a targeted antibody-polyethylene glycol-siRNA conjugate drug for inhibiting CFD expression, its preparation method and application, using a biocompatible polyethylene glycol derivative as a linker to conjugate an antibody to a selected siRNA. The specific targeting of the siRNA by the antibody-antigen binding mechanism achieves the goal of targeted delivery of the selected siRNA. It exhibits high biocompatibility, low toxicity, and effectively inhibits CFD gene expression, providing a new option for the prevention or treatment of CFD-related diseases.

[0012] The technical solution of this invention:

[0013] This invention provides an antibody-siRNA conjugate drug, the antibody-drug conjugate having the structure shown in general formula I:

[0014]

[0015] Among them, R inhibits the siRNA of CFD;

[0016] x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9.

[0017] More preferably, x1 is an integer from 1 to 3;

[0018] x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.

[0019] Ab represents antibodies;

[0020] L is the connection unit connecting Ab and R.

[0021] The 3′ or 5′ end of the siRNA is connected to a linker unit;

[0022] The siRNA is double-stranded, consisting of a sense strand and an antisense strand;

[0023] The sense strand of the siRNA contains the following nucleotide sequence: 5′-GCAAGAAGCCCGGGAUCUA-3′;

[0024] The antisense strand of the siRNA contains the following nucleotide sequence: 5′-UAGAUCCCGGGCUUCUUGC-3′;

[0025] Preferably, the siRNA further comprises a hanging base;

[0026] Preferably, the number of suspended bases is 1-10;

[0027] More preferably, the number of suspended bases is 2-4;

[0028] More preferably, the suspended base is a deoxynucleoside;

[0029] More preferably, the dangling base is located at the 3′ end of the sense strand and / or antisense strand of the RNA.

[0030] The suspended base is selected from dTdT, dTdC, or dUdU.

[0031] The positive strand of the interfering RNA contains the following nucleotide sequence: 5′-GCAAGAAGCCCGGGAUCUA-dTdT-3′; and / or,

[0032] The antisense strand of the interfering RNA contains the following nucleotide sequence: 5′-UAGAUCCCGGGCUUCUUGC-dTdT-3′.

[0033] The CFD-siRNA used in this invention can effectively inhibit CFD gene expression and downregulate complement activation, and has potential efficacy in treating autoimmune diseases, inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, eye diseases, or cancer.

[0034] L is the connection unit connecting Ab and R;

[0035] The L portion has a general formula (II) structure:

[0036]

[0037] in,

[0038] In Formula II, x3 is selected from integers from 1 to 12; preferably 1 to 3.

[0039] In Formula II, x4 is selected from integers from 1 to 12; preferably 1 to 3.

[0040] P1 and P2 may be the same or different polyethylene glycol residues;

[0041] L1 is the connection unit connecting Ab and P1;

[0042] L2 is the connection unit connecting P2 and R;

[0043] A1 is the connection unit connecting P1 and P2;

[0044] Preferably, P1 and P2 are independently selected from linear, Y-shaped, multi-branched polyethylene glycol residues;

[0045] Preferably, P1 and P2 are single molecular weight polyethylene glycols with a molecular weight of 176-4400 Da; more preferably, the molecular weight of P1 and P2 is 176-1056 Da.

[0046] Preferably, P1 and P2 are non-monolithic polyethylene glycol with a molecular weight of 1000 Da-40 kDa;

[0047] More preferably, the molecular weights of P1 and P2 are 2000 Da to 10 kDa.

[0048] Polyethylene glycol (PEG) is a water-soluble polymer with very low interfacial free energy in water. Its molecular chains are flexible and highly mobile, exhibiting excellent biocompatibility. PEG and its derivatives are among the most widely used synthetic biopolymers, not only possessing good biocompatibility but also helping to evade the recognition and clearance of reticuloepithelial tissue. Appropriate PEG can simultaneously achieve the effects of EPR and cellular bioavailability. Generally, the fusion effect of PEG with cells is directly proportional to the molecular weight and concentration of PEG; however, the higher the molecular weight and concentration of PEG, the greater its toxicity to cells. To balance these two aspects, this invention primarily uses PEG derivatives with specific structures and molecular weights that possess excellent biocompatibility as linkers in the coupling structure. Their specific structures and molecular weights can improve the biocompatibility of the coupling and reduce its toxic side effects.

[0049] L1 is the linker group between the antibody and PEG, selected from linear or branched C16 groups. 1-12 Alkylene, C 6-12 Alpha-aryl, C 3-12 Cycloalkylene, -S-, A combination of one or more groups;

[0050] The C of the straight chain or branched chain 1-12 Alkylene, C 6-12 aryl or C 3-12Any H atom on a cycloalkylene group can be -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, C 1-12 Alkyl chain, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups Group substitution consisting of one or more groups;

[0051] L2 is the linker group between P2 and siRNA, selected from linear or branched C-type RNA. 1-12 Alkylene, C 6-12 Alpha-aryl, C 3-12 Cycloalkylene, -S-, A combination of one or more groups;

[0052] The C of the straight chain or branched chain 1-12 Alkylene, C 6-12 aryl or C 3-12 Any H atom on a cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, or C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups Group substitution consisting of one or more groups;

[0053] Preferably, L1 is an amide bond, a hydrazone bond, and a thiol-maleimide bond;

[0054] More preferably, L1 is

[0055] Preferably, L2 is a disulfide bond and a thiol-maleimide bond;

[0056] More preferably, L2 is -SS-.

[0057] A1 is the connection unit between P1 and P2;

[0058] The A1 has a general formula (Ⅲ) structure.

[0059]

[0060] The L3 is a linking group, selected from straight-chain or branched C-type carbonyl groups. 1-12 Alkylene, C 6-12 Alpha-aryl, C 3-12 Cycloalkylene, -S-, -O-, -SS-、 A combination of one or more groups;

[0061] The C of the straight chain or branched chain 1-12 Alkylene, C 6-12 aryl or C 3-12 Any H atom on a cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, or C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups Group substitution consisting of one or more groups in the group;

[0062] Preferably, L3 is selected from C 1-12 Alkylene;

[0063] More preferably, L3 is ethylene;

[0064] The end base Q has the following structure:

[0065]

[0066] R1, R2, R3, R5, and R7 are independently selected from H, -L4-Q1; or, R1 and R2 together with C atoms 1 and 2 form an A3 ring, and R3 and R5 together with C atoms 3 and 4 form an A2 ring, wherein the A2 or A3 ring is selected from aryl, cycloalkyl, or heterocyclic groups.

[0067] Optionally, any H on A2 or A3 may be replaced by R8;

[0068] Optionally, R8 is selected from -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, straight-chain or branched C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups A group consisting of one or more groups;

[0069] or,

[0070] Wherein, R1, R2, R3, R5, R6, or R7 are independently selected from H, -L4-Q1, or R1 and R2 together with C atoms 1 and 2 to form an A3 ring, R3 and R5 together with C atoms 5 and 6 to form an A2 ring, and R6 and R7 together with C atoms 3 and 4 to form an A4 ring, wherein the A2, A3, or A4 rings are selected from aryl, cycloalkyl, or heterocyclic groups; and one of them is -R4-;

[0071] Alternatively, when R1, R2, R3, R5, R6 or R7 are not -R4-, any H on A2, A3 or A4 is replaced by -R4-;

[0072] Wherein, A2, A3 or A4 may be selected as the same or different aryl, cycloalkyl or heterocyclic groups;

[0073] Optionally, any H on A2, A3, or A4 may be replaced by R8;

[0074] Optionally, R8 is selected from -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, straight-chain or branched C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups A group consisting of one or more groups;

[0075] Among them, R1, R2, R3, R5, R6 or R7 are independently selected from H or —L4—Q1 structure;

[0076] Preferably, R4 is selected from C 1-12 Straight-chain or branched alkylene groups, C 6-12 Straight-chain or branched arylene, C3- 12 Cycloalkylene, -S-, -O-, -SS-、 A combination of one or more groups;

[0077] The C of the straight chain or branched chain 1-12 Alkylene, C 6-12 aryl or C 3-12 Any H atom on a cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, or C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups Group substitution consisting of one or more groups;

[0078] Preferably, L4 is a linking group, selected from straight-chain or branched C4 groups. 1-12 Alkylene, straight-chain or branched C 6-12 aryl, linear or branched C 3-12 Cycloalkylene, -S-, -O- -SS- A combination of one or more groups;

[0079] The C of the straight chain or branched chain 1-12 Alkylene, straight-chain or branched C 6-12 arylene or straight-chain or branched C 3-12 Any H atom on a cycloalkylene group is replaced by -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, C. 1-12 Alkyl, C 3-12 cycloalkyl, C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups Group substitution consisting of one or more groups;

[0080] Preferably, Q1 is selected from: -H, -F, -Cl, -Br, -I, -O-, -S-, -SO2, -NO2, straight-chain or branched C. 1-12 Alkyl, straight-chain or branched C 3-12 Cycloalkyl, straight-chain or branched C 6-12 Aryl groups, substituted or unsubstituted heterocyclic groups, or substituted or unsubstituted heterocyclic alkyl groups A group consisting of one or more functional groups.

[0081] Preferably, the end base Q is:

[0082]

[0083] Among them, R1, R2, R3, R5, R6 or R7 are independently selected from H, —L4—Q1, and one of them is -R4-.

[0084] When ring A4 is a cycloalkyl group, the terminal group Q is:

[0085]

[0086] Where j1 and u1 are the same or different, are integers ≥0 and ≤5, and j1+u1≤5;

[0087] Among them, R1, R2, R3 or R5 are independently selected from H, —L4—Q1, and one of them is -R4-;

[0088] Alternatively, when R1, R2, R3, or R5 are not -R4-, any H on the cycloalkyl group A4 is substituted with -R4-.

[0089] Preferably, the end base Q is:

[0090]

[0091] More preferably, the structure of the end base Q is as follows:

[0092]

[0093] The Ab is an antibody selected from monoclonal antibodies, polyclonal antibodies, proteins, peptides, and oligonucleotides. This invention uses specific antibodies that can specifically bind to antigens, improve targeting, and effectively deliver siRNA to target cells.

[0094] Preferably, Ab is a monoclonal antibody.

[0095] More preferably, the monoclonal antibody is reactive to antigens or epitopes associated with cancer, malignant cells, infectious organisms, or autoimmune diseases.

[0096] More preferably, the monoclonal antibody is selected from: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD30 antibody, anti-CD22 antibody, anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB antibody, anti-CD138 antibody, anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRⅧ antibody, anti-SLC44A4 antibody, anti-mesothelin antibody, anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-CD79b antibody, anti-MUC16 antibody, and anti-Muc1 antibody.

[0097] The antibody-siRNA conjugate has the following structure:

[0098] It has the following structure:

[0099]

[0100] Preferably, the antibody-siRNA conjugate has the following structure:

[0101]

[0102] Preferably, the antibody-siRNA conjugate has the following structure:

[0103]

[0104] Preferably, the antibody-siRNA conjugate has the following structure:

[0105]

[0106] Preferably, the antibody-siRNA conjugate has the following structure:

[0107]

[0108] The n1 and n2 are independent integers selected from 4 to 100, preferably 4 to 24;

[0109] Preferably, the antibody-siRNA conjugate has the following structure:

[0110]

[0111] The n1 and n2 are independent integers selected from 4 to 100, preferably 4 to 24;

[0112] Preferably, the antibody-siRNA conjugate has the following structure:

[0113]

[0114] The n1 and n2 are independent integers selected from 4 to 100, preferably from 4 to 24.

[0115] The present invention also provides a method for preparing the above-mentioned antibody-siRNA conjugate drug composition, the steps of which include:

[0116] (1) Add the antibody to the buffer solution using a desalting column and mix well. Measure the protein concentration. Weigh the polyethylene glycol derivative and add it to the buffer solution to dissolve it and obtain the antibody solution. Take the antibody solution and add the polyethylene glycol solution. React and remove impurities using an ultrafiltration centrifuge tube to obtain conjugate a.

[0117] (2) Mix the conjugate a from step (1) with the aqueous solution of the interfering RNA modified with polyethylene glycol to inhibit the expression of CFD mRNA, centrifuge and ultrafilter the purified component using an ultrafiltration centrifuge tube, and freeze dry to obtain the final product.

[0118] Preferably, in an embodiment of the present invention, in step (1), the antibody is an EGFR antibody (purchased from Eli Lillyand Company);

[0119] Preferably, in one embodiment of the present invention, in step (1), the protein concentration is 3.3 mg / mL;

[0120] Preferably, in one embodiment of the present invention, in step (1), the concentration of the buffer solution is 5 mM;

[0121] Preferably, in an embodiment of the present invention, in step (1), the buffer solution is a phosphate buffer with a pH of 7.0;

[0122] Preferably, in one embodiment of the present invention, in step (1), the polyethylene glycol derivative is N3-PEG12-SPA;

[0123] Preferably, in one embodiment of the present invention, in step (1), the dissolution is a shaking dissolution;

[0124] Preferably, in one embodiment of the present invention, in step (1), the amount of polyethylene glycol solution added is 2.53 μL;

[0125] Preferably, in one embodiment of the present invention, in step (1), the reaction is carried out by shaking for 2 hours at a temperature of 20℃±5℃;

[0126] Preferably, in one embodiment of the present invention, in step (1), the molecular weight cutoff of the ultrafiltration centrifuge tube is 30 kDa;

[0127] Preferably, in one embodiment of the present invention, in step (2), the polyethylene glycol-modified interfering RNA that inhibits CFD mRNA expression is DBCO-PEG3500-CFD-siRNA;

[0128] Preferably, in one embodiment of the present invention, in step (2), the molecular weight cutoff of the ultrafiltration centrifuge tube is 30 kDa;

[0129] The present invention also provides the use of the above-mentioned antibody-siRNA conjugate drug in a drug designed for the prevention and / or treatment of CFD-related diseases.

[0130] The CFD-related diseases are complement overactivation-related diseases.

[0131] Preferably, the complement overactivation-related diseases include autoimmune diseases, inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, eye diseases, or cancer.

[0132] The "autoimmune diseases" described in this invention include, but are not limited to, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, myasthenia gravis, multiple sclerosis, urticaria, psoriasis, dermatomyositis, Sjögren's syndrome, pain, or neurological disorders.

[0133] The "inflammatory diseases" described in this invention include both acute and chronic inflammation. Specifically, they include, but are not limited to, degenerative inflammation, exudative inflammation, proliferative inflammation, and specific inflammation, including but not limited to severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, post-streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2-induced vascular leakage syndrome, or radiographic (contrast) contrast agent allergies.

[0134] The "neurodegenerative diseases" described in this invention include, but are not limited to, Alzheimer's disease, progressive blindness or extraocular muscle paralysis, multiple system atrophy, frontotemporal dementia, Huntington's disease, corticobasal degeneration, spinocerebellar ataxia, motor neuron disease, and hereditary motor-sensory neuropathy.

[0135] The "ischemia-reperfusion injury" described in this invention includes, but is not limited to, ischemia-reperfusion injury following acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiple organ failure, intestinal ischemia, complement activation during cardiopulmonary bypass surgery, or other events that cause ischemia.

[0136] The "eye diseases" described in this invention include, but are not limited to, macular degeneration diseases, such as age-related macular degeneration (AMD) at all stages, diabetic retinopathy and other ischemic retinopathy, choroidal neovascularization (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization. Age-related macular degeneration (AMD) includes non-exudative (e.g., intermediate dry AMD or geographic atrophy (GA)) and exudative (e.g., wet AMD (choroidal neovascularization (CNV)) AMD), diabetic retinopathy (DR), endophthalmitis, and uveitis. Non-exudative AMD may also include hard drusen, soft drusen, geographic atrophy, and / or pigmentary lesions.

[0137] The "cancer" referred to in this invention includes, but is not limited to, lymphoma, B-cell tumors, T-cell tumors, myeloid / monocyte tumors, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. The leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0138] Compared with the prior art, the present invention has the following beneficial effects:

[0139] (1) This invention provides a novel targeted antibody-siRNA conjugate for inhibiting CFD expression. The antibody-siRNA conjugate is used as the delivery carrier for CFD-siRNA. By utilizing the specific binding of the antibody to the antigen, the targeting is improved, and CFD-siRNA is effectively delivered into the target cells. Furthermore, the conjugate structure mainly uses a PEG derivative with excellent biocompatibility as a linker to reduce toxic side effects. It has high biocompatibility, low toxic side effects, and can effectively inhibit CFD gene expression. The inhibition of CFD mRNA expression can reach 71.08%, providing a new option for the prevention or treatment of CFD-related diseases.

[0140] (2) The present invention provides a method for preparing a targeted antibody-siRNA conjugate drug that inhibits CFD expression. The method is simple, efficient and stable, and can efficiently prepare antibody-siRNA conjugate drugs. Attached Figure Description

[0141] Figure 1 It is the relative expression level of CFD mRNA

[0142] Figure 2 The electrophoresis results of the EGFR-Antibody-PEG12-PEG3500-CFD-siRNA conjugate are as follows:

[0143] From left to right: EGFR-Antibody-PEG12-PEG3500-CFD-siRNA, DBCO-PEG3500-CFD-siRNA, and Marker. Detailed Implementation

[0144] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0145] In this invention, the term "CFD" stands for Complement Factor D.

[0146] The term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology, 170:4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system capable of recognizing and binding specific antigens (Janeway, C. et al. (2001) Immunobiology, 5th Ed., Garland Publishing, New York).

[0147] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that, apart from the possible small number of naturally occurring mutations, the antibodies contained within this group are identical. Monoclonal antibodies are highly specific antibodies that target a single antigenic site. Moreover, unlike typical polyclonal antibody products, which include different antibodies targeting different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. In addition to their specificity, a key advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the antibody's characteristic of being derived from a substantially homogeneous group of antibodies and does not imply that the antibody needs to be produced using any particular method.

[0148] The term siRNA refers to the ability of interfering RNA to reduce or inhibit the expression of a target gene or sequence when the interfering RNA is in the same cell as the target gene or sequence (e.g., by mediating the degradation and inhibition of the translation of mRNA complementary to the interfering RNA sequence).

[0149] In one specific embodiment, the siRNA is chemically synthesized. The siRNA molecules of the present invention are capable of silencing the expression of target sequences in vitro and / or in vivo.

[0150] The term "polyethylene glycol" refers to a polymer that includes the (CH2CH2O) repeating group.

[0151] The term "antibody-drug conjugate" refers to a small molecule drug with biological activity linked to an antibody via a chemical link, with the antibody acting as a carrier to target and deliver the small molecule drug to the target cell.

[0152] The term “treatment” includes eradicating, removing, reversing, alleviating, altering or controlling a disease and / or condition after its onset.

[0153] The term "prevention" refers to the ability to avoid, minimize, or prevent the onset or development of a disease and / or condition through treatment before it occurs.

[0154] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0155] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0156] Example

[0157] Several embodiments of the present invention are illustrated by the following examples, but are not intended to limit the invention.

[0158] Example 1

[0159] Synthesis of EGFR-Antibody-PEG12-N3 conjugate

[0160]

[0161] A certain amount of EGFR antibody (purchased from Eli Lilly and Company) was exchanged into 12 mL of 5 mM phosphate buffer (pH = 7.0) using a desalting column to obtain an antibody solution, and the protein concentration was accurately determined to be 3.3 mg / mL. 40 mg of N3-PEG12-SPA was weighed and added to 1 mL of 5 mM phosphate buffer (pH = 7.0), and mixed well to obtain a PEG solution. 3 mL of the antibody solution was taken, and 2.53 μl of PEG12 solution was added. The mixture was incubated at 20℃ ± 5℃ with shaking for 2 h. Unreacted N3-PEG12-SPA was removed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, yielding an aqueous solution of the EGFR-Antibody-PEG12-N3 conjugate, with a protein concentration of 5.3 mg / mL.

[0162] Example 2

[0163] Synthesis of EGFR-Antibody-PEG12-PEG3500-CFD-siRNA conjugate

[0164]

[0165] Take 300 μl of the above-mentioned EGFR-Antibody-PEG12-N3 conjugate aqueous solution, add 100 μl of DBCO-PEG3500-CFD-siRNA aqueous solution (226 μM), and react with shaking at 20℃±5℃ for 1 h. Purify using a receptor column, centrifuge the purified fraction using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, ultrafilter using the same tube, and lyophilize to obtain the EGFR-Antibody-PEG12-PEG3500-CFD-siRNA conjugate. Electrophoresis results are as follows. Figure 2 As shown.

[0166] Experimental Example 1

[0167] CFD-siRNA synthesis:

[0168] First, weigh 1 μmol of a universal solid-phase support, 3'-cholesterol-modified CPG island (Chemgenes product), and dissolve the monomer of RNA phosphorous amide protected by the 2'-O-TBDMS protecting group in anhydrous acetonitrile solution to achieve a concentration of 0.2 M. Prepare a 0.25 M acetonitrile solution of 5-ethylthio-1H-tetrazole (Chemgenes product) as an activator, a 0.02 M pyridine / water solution of iodine as an oxidant, and a 3% trichloroacetic acid-dichloromethane solution as a deprotection reagent. Place these solutions in the designated reagent positions on an ABI 394 DNA / RNA automated synthesizer. Set the synthesis program, input the specified oligonucleotide base sequence, and begin the cyclic oligonucleotide synthesis. Each coupling step takes 6 minutes, and the coupling time for the L and S monomers corresponding to the galactose ligand is 10-20 minutes. After automated cycling, the oligonucleotide solid-phase synthesis is complete. CPG was dried with dry nitrogen gas and transferred to a 5 mL EP tube. 2 mL of ammonia / ethanol solution (3 / 1) was added, and the mixture was heated at 55°C for 16–18 hours. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The concentrated ammonia / ethanol was removed, yielding a white gel-like solid. The solid was dissolved in 200 μL of 1 M TBAF-THF solution and incubated at room temperature for 20 hours with shaking. 0.5 mL of 1 M Tris-HCl buffer (pH 7.4) was added, and the mixture was incubated at room temperature with shaking for 15 minutes. The mixture was then centrifuged until the volume was reduced to half its original volume to remove THF.

[0169] The solution was extracted twice with 0.5 mL of chloroform, and 1 mL of 0.1 M TEAA loading solution was added. The mixture was poured into a solid-phase extraction column, and mass spectrometry analysis was performed on an HTCS LC-MS system (Novatia). After the first-stage scan, the molecular weight of the nucleic acid was calculated using Promass software. Two single strands were synthesized using the above method. After confirmation by mass spectrometry, the two single strands were mixed in an equimolar ratio and annealed to form a double strand, which was the siRNA sequence.

[0170] Experiment Example 2

[0171] Synthesis of DBCO-PEG-CFD-SiRNA

[0172] Dissolve 200 nmol of CFD-siRNA in 1 mL of DEPC-treated water to obtain an aqueous solution of CFD-siRNA. Weigh 2 mg of DBCO-PEG3500-OPSS and dissolve it in 2 mL of DEPC-treated water to obtain an aqueous solution of DBCO-PEG3500-OPSS. Add 1 mL of the CFD-siRNA aqueous solution to 2.1 mL of the DBCO-PEG3500-OPSS aqueous solution and react at 40°C for 1 h. Purify the reaction solution using a DEAE column to obtain DBCO-PEG-CFD-siRNA.

[0173] Experimental Example 3

[0174] Testing the inhibitory effect of antibody-siRNA

[0175] To test the inhibitory effect of the antibody-siRNA mentioned in this invention, anti-EGFR-PEG-siR-CFD was prepared and cell experiments were performed. In the cell experiments, the prepared conjugate was first transfected into cultured cells, then RNA was extracted, and the expression of mRNA was obtained by real-time quantitative PCR. The steps are as follows:

[0176] 1. Cell Culture

[0177] Cell name: 293T

[0178] (1) 293T cells were routinely cultured at 37℃ and 5% CO2. 24 hours before the experiment, 1.2×105 cells were seeded in each well of a 12-well plate and cultured for 18-26 hours.

[0179] (2) Take 100 μL of OPTI-MEM medium to dilute 2.5 μL of the 20 μM stock solution of the conjugate, and allow it to dilute evenly. In the negative control and positive control wells, take 50 μL of OPTI-MEM medium to dilute 2.5 μL of the 20 μM stock solution of siR-NC or siR-CFD, and take 50 μL of OPTI-MEM medium to dilute 3 μL of Lipofectamine 3000™ transfection reagent. Mix the two, shake gently, and let stand for 15 min. In addition, a Blank group and a mock group were set up.

[0180] (3) Change the medium for each well of the cell plate, add DMEM + 10% FBS medium, 900 μL per well, then add 100 μL of the above mixture to each well, for a final total volume of 1000 μL per well. The siRNA (or siRNANC) transfection concentration is 50 nM.

[0181] (4) 48 h after transfection, the 12-well plate was removed from the incubator at 37°C and 5% CO2 and the cells were collected for RNA extraction and subsequent detection.

[0182] 2. RNA extraction

[0183] (1) RNA was extracted using the Promega RNA extraction kit. Cells were washed with PBS, and after washing, 300 μL of lysis buffer was added. Cells were pipetted and lysed thoroughly. Then, 300 μL of diluent was added, and the cells were incubated at 70°C for 3 min. After centrifugation at 14000 rpm for 10 min, the supernatant was transferred to a new 1.5 mL Eppendorf tube, 300 μL of anhydrous ethanol was added, and the mixture was thoroughly mixed before being added to the adsorption column. After centrifugation at 14000 rpm for 1 min, the filtrate was discarded, and 600 μL of washing buffer was added. After centrifugation for 1 min, the filtrate was discarded, and 50 μL of freshly prepared DNase reaction solution was added to each well. The cells were incubated at room temperature for 15 min. After centrifugation, 600 μL of washing buffer was added, and the cells were centrifuged for 1 min. After centrifugation, 600 μL of washing buffer was added, and the cells were centrifuged for 1 min. After centrifugation, the eluted RNA was collected.

[0184] (2) RNA quality control: Nanodrop was used to detect RNA content, and 1% agarose gel electrophoresis was used to detect RNA integrity.

[0185] 3. Q-PCR testing procedure

[0186] (1) RNA reverse transcription

[0187] Using total RNA extracted from the sample as a template, the following reaction system was established:

[0188]

[0189] In the above system, 1 μg of RNA template is first treated at 70℃ for 5 min, then placed on ice for 10 min. Other reagents are then added and mixed. The liquid is collected by centrifugation to the bottom of the tube, and then incubated at 42℃ for 60 min and 72℃ for 10 min. The product is the cDNA template.

[0190] (2) Quantitative real-time PCR detection

[0191] The reaction system is established as follows:

[0192]

[0193] Table 1 Primer sequences

[0194]

[0195] Perform PCR amplification according to the following procedure.

[0196] Pre-denaturation at 95℃ for 10 minutes, followed by the following cycle.

[0197] *95℃ for 10 seconds

[0198] 60℃ 20s

[0199] Reading board

[0200] Return * A total of 40 loops were performed.

[0201] To create a melting curve: between 65℃ and 95℃, read the plate every 0.5℃ and pause for 5 seconds.

[0202] 4. Inhibition effect

[0203] Using GAPDH as an internal control gene, the relative expression level of CFD mRNA was calculated using the ΔΔCt method; the mRNA expression level of each group was standardized with the blank group expression level as 100%. The relative expression levels of cellular mRNA in each group are shown in Table 2 or... Figure 1 .

[0204] Table 2. Relative expression levels of CFD mRNA

[0205]

[0206]

[0207] The results showed that the relative expression level of CFD mRNA in cells treated with Anti-EGFR2-PEG1-PEG2-siR-CFD was 29.92% ± 3.17% compared to the blank group; the relative expression level of CFD mRNA in cells treated with Anti-EGFR2-PEG1-PEG2-siR-CFD was also 29.92% ± 3.17% compared to the blank group. Anti-EGFR2-PEG1-PEG2-siR-CFD showed similar inhibitory effects compared to the lipofectamine 3000 delivery group.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0209] The embodiments and methods described herein may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0210] In this invention, simply listing the steps of the method in a certain order does not constitute any restriction on the order of the method steps. SEQUENCE LISTING <110> Beijing Keyka Technology Co., Ltd. <120> A targeted antibody-polyethylene glycol-siRNA drug conjugate <130> P0102021040269 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <400> 1 gcaagaagcc cgggaucua 19 <210> 2 <211> 19 <212> RNA <213> Artificial sequence <400> 2 uagaucccgg gcuucuugc 19 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 tctgacttca acagcgacac 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 gccaaattcg ttgtcatacc 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 tcacccaagc aacaaagtcc 20 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 gtaggtgctc aataaagacc aacca 25

Claims

1. An antibody-siRNA conjugate, characterized in that, The general formula (I) of the antibody-siRNA conjugate is: （Ⅰ） Where R is a siRNA that inhibits complement factor D; x1 is 1; x2 is 1; Ab represents antibodies; L is the connection unit connecting Ab and R; The L has a general formula (II) structure: ； （Ⅱ） in, In Equation II, x3 is 1; In Equation II, x4 is 1; P1 and P2 are the same or different linear polyethylene glycol residues; L1 is a connecting unit between Ab and P1, wherein L1 is an amide bond, an hydrazone bond, or a mercapto-maleimide bond; L2 is a connecting unit between P2 and R, and L2 is a disulfide bond or a mercapto-maleimide bond; A1 is the connection unit connecting P1 and P2; The A1 has a general formula (Ⅲ) structure. （Ⅲ） L3 is ethylene; The end base Q is: ； R1, R2, and R7 are H, and R4 is selected from C. 1-12 Straight-chain or branched alkylene groups, C 3-12 Cycloalkylene, -S-, A combination of one or more groups; The C 1-12 Straight-chain or branched alkylene or C 3-12 Any H atom on the cycloalkylene group is replaced by a group consisting of one or more groups such as -F, -Cl, -Br, -I, -SO2, and -NO2.

2. The antibody-siRNA conjugate according to claim 1, characterized in that, The 3' or 5' end of the siRNA is connected to a linker unit; The siRNA is double-stranded, consisting of a sense strand and an antisense strand; The nucleotide sequence of the positive strand of the siRNA is: 5´-GCAAGAAGCCCGGGAUCUA-3´.

3. The antibody-siRNA conjugate according to claim 2, characterized in that, The nucleotide sequence of the antisense strand of the siRNA is: 5´-UAGAUCCCGGGCUUCUUGC-3´.

4. The antibody-siRNA conjugate according to claim 2, characterized in that, The siRNA also contains dangling bases.

5. The antibody-siRNA conjugate according to claim 4, characterized in that, The number of suspended bases is 1-10.

6. The antibody-siRNA conjugate according to claim 4, characterized in that, The number of suspended bases is 2-4.

7. The antibody-siRNA conjugate according to claim 4, characterized in that, The suspended base is a deoxynucleoside.

8. The antibody-siRNA conjugate according to claim 4, characterized in that, The dangling base is located at the 3' end of the sense strand and / or antisense strand of the siRNA.

9. The antibody-siRNA conjugate according to claim 4, characterized in that, The suspended base is selected from dTdT, dTdC, or dUdU.

10. The antibody-siRNA conjugate according to claim 9, characterized in that, The nucleotide sequence of the positive strand of the siRNA is: 5´-GCAAGAAGCCCGGGAUCUA-dTdT-3´; and / or, The nucleotide sequence of the antisense strand of the siRNA is: 5´-UAGAUCCCGGGCUUCUUGC-dTdT-3´.

11. The antibody-siRNA conjugate according to claim 1, characterized in that, When P1 and P2 are single molecular weight polyethylene glycols with a molecular weight of 176-4400 Da.

12. The antibody-siRNA conjugate according to claim 11, characterized in that, The molecular weights of P1 and P2 are 176-1056 Da.

13. The antibody-siRNA conjugate according to claim 1, characterized in that, When P1 and P2 are non-monolithic polyethylene glycols with molecular weights ranging from 1000 Da to 40 kDa.

14. The antibody-siRNA conjugate according to claim 13, characterized in that, The molecular weights of P1 and P2 are 2000 Da to 10 kDa.

15. The antibody-siRNA conjugate according to claim 1, characterized in that, L1 is an amide bond.

16. The antibody-siRNA conjugate according to claim 15, characterized in that, The L2 is a disulfide bond.

17. The antibody-siRNA conjugate according to claim 1, characterized in that, The structure of the end base Q is as follows: .

18. The antibody-siRNA conjugate according to claim 1, characterized in that, The Ab is selected from monoclonal antibodies or polyclonal antibodies.

19. The antibody-siRNA conjugate according to claim 18, characterized in that, Ab is a monoclonal antibody.

20. The antibody-siRNA conjugate according to claim 18, characterized in that, The monoclonal antibody is reactive to antigens or epitopes associated with infectious organisms or autoimmune diseases.

21. The antibody-siRNA conjugate according to claim 18, characterized in that, The antibody (Ab) is selected from one or more of the following: anti-HER2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD30 antibody, anti-CD22 antibody, anti-CD56 antibody, anti-CD29 antibody, anti-GPNMB antibody, anti-CD138 antibody, anti-CD74 antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRⅧ antibody, anti-SLC44A4 antibody, anti-mesothelin antibody, anti-ET8R antibody, anti-CD37 antibody, anti-CEACAM5 antibody, anti-CD70 antibody, anti-CD79b antibody, anti-MUC16 antibody, and anti-Muc1 antibody.

22. The antibody-siRNA conjugate according to any one of claims 1-16, 18-21, characterized in that, The antibody-siRNA conjugate has the following structure: 。 23. The antibody-siRNA conjugate according to claim 22, characterized in that, The antibody-siRNA conjugate has the following structure: ； The n1 and n2 are independent integers selected from 4 to 100.

24. The antibody-siRNA conjugate according to claim 23, characterized in that, The n1 and n2 are independent integers selected from 4 to 24.

25. The antibody-siRNA conjugate according to claim 22, characterized in that, The antibody-siRNA conjugate has the following structure: ； The n1 and n2 are independent integers selected from 4 to 100.

26. The antibody-siRNA conjugate according to claim 25, characterized in that, The n1 and n2 are independent integers selected from 4 to 24.

27. The antibody-siRNA conjugate according to claim 22, characterized in that, The antibody-siRNA conjugate has the following structure: The n1 and n2 are independent integers selected from 4 to 100.

28. The antibody-siRNA conjugate according to claim 27, characterized in that, The n1 and n2 are independent integers selected from 4 to 24.

29. An antibody-siRNA pharmaceutical composition comprising the conjugate of any one of claims 1-28 and pharmaceutically acceptable excipients.

30. The use of the antibody-siRNA conjugate according to any one of claims 1-28 in the preparation of a medicament for the prevention and / or treatment of diseases related to complement overactivation; The diseases associated with complement overactivation include autoimmune diseases or inflammatory diseases.

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